SFRP2 in skin and we revealed that in the epidermis, this protein is secreted by Langerhans cells (LCs). Similar to well‐known mechanosensitive genes, the increase in SFRP2 expression was proportional to the magnitude of tension, showing a spike at the apex of the expanded skin. This implies that SFRP2 might be a newly discovered effector of mechanotransduction pathways. In addition, we found that acute stretching induces accumulation of b‐catenin in the nuclei of basal keratinocytes (KCs) and LCs, indicating Wnt signalling activation, followed by cell proliferation. Moreover, TE‐activated LCs proliferate and migrate into the suprabasal layer of skin, suggesting that LCs rebuild their steady network within the growing epidermis. We demonstrated that in vitro hrSFRP2 treatment on KCs inhibits Wnt/b‐catenin signalling and stimulates KC differentiation. In parallel, we observed an accumulation of KRT10 in vivo in the expanded skin, pointing to TE‐induced, SFRP2‐augmented KC maturation. Overall, our results reveal that a network of LCs delivers SFRP2 across the epidermis to fine‐tune Wnt/b‐catenin signalling to restore epidermal homeostasis disrupted by TE.Skin can be mechanically stimulated to grow through a clinical procedure called tissue expansion (TE). Using a porcine TE model, we determined that expansion promptly activates transcription of  It belongs to a family of proteins that can bind directly to Wnt ligands to prevent receptor binding and activation of Wnt signalling.Langerhans cells (LCs), dendritic cells of the epidermis, are involved in maintaining epidermal health and tolerance to commensal microorganisms, and in adaptive immune response to pathogens invading the epidermis.SFRP2 as a new target gene of mechanotransduction which modulates Wnt/beta‐catenin signalling and the behaviour of keratinocytes (KCs). We show that secretion of SFRP2 by LCs plays a key role in maintaining epidermal homeostasis. While a typical TE procedure takes several weeks before reconstructive surgery, we learned that stretching activates regenerative mechanisms within hours.Here, we describe 22.1(7+3)*, Ex‐10d(7+3)) or posteriorly over the abdomen *, Ex‐14d(7+7)) , and NIH standards provided in the ‘Guide for the Care and Use of Laboratory Animals’. We have performed eight protocols of tissue expansion (TE) using hairless 5–6 weeks old females Yucatan minipigs,) Figure a,b. The ) Figure c. The bi2.2TM).The full‐thickness skin punch biopsies were preserved in All Protect Tissue Reagent (Qiagen) according to manufacturer's recommendations, and RNA was extracted as described previously.2.32‐ΔΔCT method.B2M expression and for in vitro experiments with human cells results were normalized to the normalization factor, which was calculated as a geometric mean of three reference genes: GAPDH, HPRT and HMBS1.Synthesis of cDNA was performed using the High‐capacity cDNA Reverse Transcription kit (Life Technologies) and 200 ng of total RNA according to the manufacturer's recommendations. The Real‐time PCR analysis (qRT‐PCR) was performed on StepOnePlus Real‐Time PCR system (Applied Biosystems) using TaqMan Assay (Applied Biosystems) in a 10 μl reaction mixture per manufacturer recommendations. Cycling conditions included an initial denaturation at 95°C for 15 min, then 45 cycles at 95°C for 15 s and 60°C for 1 min. The cycle quantification values were calculated using the threshold cycle method. Fold change of gene expression was calculated with the 2.4The tissue biopsies were fixed in 10% neutral buffer formalin for 24 hours at room temperature. The post‐fixation processing, performed with Spin Tissue Processor (Thermo Fisher), included 1 hour fixation in 10% neutral buffer formalin, 30 min incubation in Pen‐Fix (Richard‐Allan Scientific), dehydration in ethanol series  30 min each, then incubation in two changes of xylene for 1 hour each, and in three changes of paraffin for 60 min each. Samples were embedded using paraffin embedding centre and cut at 5 μm using Microtome . The sections were mounted on Superfrost Plus Microscope Slides (Thermo Fisher) using Eco‐Mount medium  and evaluated by haematoxylin and eosin.2.5Immunofluorescence staining (IF) and immunohistochemistry (IHC) staining was performed on paraffin sections deparaffinized in CitriSolv Hybrid solution (Decon) and gradually rehydrated. Permeabilization was done with 0.1% Tween‐20 or 0.05% Triton X‐100 in TBS (TBST). For antigen retrieval, the Target Retrieval Solution (Dako) or Tris‐EDTA buffer  for KRT10 was used at 95°C for 20–30 min, then cooled at room temperature for 30 min and washed in PBS and PBST. Blocking was done with 10% normal serum in PBS at room temperature for 1.5 h. The sections were incubated overnight at 4°C with the primary antibodies: anti‐SFRP2  anti‐CD207 , anti‐beta‐catenin , anti‐KRT10  or 1 hour at room temperature with anti‐KRT5 . For IF of CD207, the biotin‐SP AffiniPure donkey anti‐rat IgG antibody  and Vectastain Elite ABC kit (Vector Labs) were used according to manufacturer recommendations. Following secondary antibodies were used: goat anti‐mouse IgG Alexa Fluor Plus 488 , donkey anti‐rabbit Alexa Fluor Plus 488 , Cy‐3‐conjugated Streptavidin (1:1000), goat anti‐rabbit IgG HRP  for 1 hour at room temperature. Nuclei were counterstained using DAPI in SlowFade Gold antifade reagent (Invitrogen). To reduce auto‐fluorescence Autofluorescence Reducing Reagent was used (MaxVision). The IF/IHC staining of SFRP2, SFRP2/CD207 and Ki‐67 was performed by Histology Core at Northwestern University. Staining without the primary antibodies was performed as a negative control.2.6The slides were imaged on a Zeiss LSM880 confocal microscope using 40x or 63x oil objective. The exposure time was identical between controls and experimental slides. The Blue Zen software was used to measure fluorescence intensity.2.7The accumulation and distribution of beta‐catenin in individual cells were calculated based on Alexa 488 fluorescence intensity using Blue Zen software (Zeiss). The numerical values were extracted from each histogram attached to the collected image. The optical sections were selected based on similar intensity of DAPI, and the mean value of signal intensity (sum of pixels intensity) was calculated from at least four sections, from each Z‐stack. Minimum six sections were tested for each tissue sample.2.82 at 37°C in DMEM GlutaMax medium, supplemented with 10% FBS (Gibco), sodium pyruvate and high glucose. Cells were synchronized in cell cycle by serum starvation for 16 hours, grown to 60% or 100% confluency, and then treated with recombinant protein hrSFRP2 (R&D system) at concentration 20 nM or 40 nM for 6 h. Untreated cells were used as control. Each experiment was performed in triplicates, and collected cells were used for qRT‐PCR analysis and cell cycle assay.The cells were purchased from AddexBio and cultured in 5% CO2.95 cells in single cell suspension were fixed in 66% ethanol, then rehydrated in PBS and stained with Propidium iodide for 30 min at 37°C in the dark with presence of RNaseA. The signal intensity was measured on flow cytometer  at 488 nm laser illumination and analysed at Northwestern University Flow Cytometry Core Facility.Cell cycle analysis was performed using Propidium Iodide Flow Cytometry Kit (Abcam) according to manufacturer's protocol. Briefly, 5x102.10At least 10 microscopic fields per each section (n ≥ 3) were photographed and analysed; the number of LCs was assessed based on expression of CD207 (Novus).2.11The normal human skin tissue was purchased from the Northwestern University Mouse Histology and Phenotyping Laboratory.2.12p values ≤0.05 were considered as significant.All statistics were performed using GraphPad Prism 7 software . The significance of obtained results was calculated with unpaired Student t‐test. All experimental results are presented as mean ±SD. 33.1SFRP2 among the top of the differentially expressed genes in TE. Therefore, we evaluated SFRP2 transcription levels in skin samples collected at eight timepoints of expansion, from three locations on the expanded skin, by qRT‐PCR. Subcutaneously placed expanders were filled with saline to induce acute stretch for one hour (Ex‐1h), 24 h (Ex‐24h), 3 days (Ex‐3d) and 7 days (Ex‐7d). A second experimental group received two injections of saline over either 10 days or 14 days , where the highest stretching force was applied. The fold change in the ‘a’ biopsies over the control level was as follows: 37 ± 17 (p = 0.0057), 59 ± 16 (p < 0.0001), 28 ± 7 (p = 0.001) and 16 ± 3 (p < 0.0001) for Ex‐1h, Ex‐24h, Ex‐3d and Ex‐7d model, respectively. Type ‘d’ biopsies, collected from the periphery of the expanded skin, revealed no statistically significant changes  for Ex‐10d(7+3)*, corresponding to injections of 30 ml at 10 days and 3 days before the tissue was harvested , for Ex‐10d(7+3) was observed when the expander was filled twice with 60 ml. Similar results were obtained for double‐filled 14‐day TE models: transcription increased by FC = 12 ± 8 (p = 0.0149) in Ex‐14d(7+7)* (twice 30 ml) and by 5 ± 3 (p = 0.0469) in Ex‐14d(7+7) (twice 60 ml). These results suggest that in prolonged and repeated stretching, smaller volume/lower stress favours higher transcriptional activation of SFRP2.To parallel clinical practice, in which subcutaneous expanders are refilled weekly to sustain stretching, we continued TE in four models for a total expansion time of 10 or 14 days. These expanders were filled twice, 7 days apart, two models with 30 ml and two with 60 ml each time. Again, the highest d Figure . Much smSFRP2 expression with the expression pattern of known mechanosensitive genes such as TNC (Tenascin‐C), CCN2 (Connective Tissue Growth Factor 2), MMP1 , TGFB1 (Transforming Growth Factor Beta 1) and WNT4 (Wnt Family Member 4).SFRP2 was one of the three most activated genes in the apical biopsies (‘a’) in both Ex‐1h and Ex‐24h models, and its expression presented a similar pattern to other tested genes. This similarity suggests that SFRP2 may also be a mechanosensitive gene.Next, we compared 3.2+ cells by immunohistochemistry staining (IHC), which revealed evenly distributed SFRP2+ cells, morphologically similar to dendritic cells, along the epidermis  with an antibody against CD207 (Langerin)+) as the main source of SFRP2. SFRP2 and CD207 proteins were visible along the LCs dendrites in both control and expanded skin sections, which indicates that SFRP2 is also expressed by LCs in steady‐state conditions. A small number of melanocytes were also positive for SFRP2, as identified by the residual presence of melanin on tissue sections. In addition, we noticed the presence of SFRP2 in dermal fibroblasts, as previously described.To explore the role of SFRP2 in TE, we first examined the localization of SFRP2s Figure . SFRP2+,To examine if LCs secrete SFRP2 in human epidermis, we performed analogous double IF staining on normal human skin samples Figure d. Simila3.3+SFRP2+ cells, we calculated their quantity and location under normal and experimental conditions. We found that the number of CD207+SFRP2+ cells, calculated per image, was two times higher at 1 hour , 24 h  and 7 days of TE   Figure . The incs Figure .+SFRP2+ cells also gradually migrated upward during TE, reaching statistically significant difference in the number of cells located in basal and suprabasal layers by the 7th day , and LC density was reduced by 32% ± 14% (p = 0.0041), suggesting that the network of LCs was not fully rebuilt by the end of the first week of stretching  and Ex‐24h , and normalized at the 7th day of TE  and by 20% ± 1% (p = 0.0388), for each respective hrSFRP2 concentration as compared to control, consistent with cell growth and differentiation  as an in vitro model of normal unperturbed epidermis.n Figure .KRT5 to KRT10,KRT10 and KRT5 transcription showed an increase in KRT10 expression by 2.7 ± 0.4 times (p = 0.0035) and 2.8 ± 0.3 times (p = 0.0011) after treatment with 20 nM and 40 nM of rhSFRP2, respectively, and no significant changes in KRT5 expression, indicating augmented cell maturation  TCF3 (Transcription Factor 3), TCF7 (Transcription Factor 7) and LEF1 (Lymphoid Enhancer Binding Factor 1), using the cell culture conditions described above. The transcription of tested genes was reduced in a dose‐dependent manner  and 37% ± 2% (p = 0.0051), TCF3 by 13% ± 1% (p = 0.0814) and 26% ± 2% (p = 0.0042), TCF7 by 46% ± 17% (p = 0.0387) and 56% ± 8% (p = 0.0016) for 20 nM and 40 nM, respectively, without significant change in LEF1 transcription. These data indicate the inhibitory effect of rhSFRP2 on Wnt signalling.Since SFRP2 is well recognized as a modifier of Wnt signalling, we evaluated the transcription of Wnt target and effector genes, including r Figure . Specifi3.6p ≤ 0.0002) in nuclear beta‐catenin localization in basal KCs  and in KCs adjacent to LCs  after one hour of expansion (Ex‐1h)  and 24 h (Ex‐24h) of expansion Figure . We obse) Figure ′,D,E. In) Figure . These rp = 0.0094) at 24 h of TE ; Data curation (lead); Formal analysis (lead); Investigation (lead); Methodology (lead); Visualization (lead); Writing – original draft . Elbert E. Vaca: Investigation (supporting); Writing – review & editing (supporting). Chiang C. Huang: Resources ; Writing – review & editing (supporting). Lauren J Kelsey: Investigation (supporting); Writing – review & editing . Jennifer L. McGrath: Investigation (supporting); Writing – review & editing (supporting). Jacek Topczewski: Resources ; Writing – review & editing (supporting). Arun K. Gosain: Conceptualization (supporting); Funding acquisition (lead); Supervision ; Writing – review & editing (supporting). Jolanta M. Topczewska: Conceptualization ; Formal analysis (supporting); Funding acquisition (supporting); Investigation (supporting); Methodology ; Supervision (lead); Visualization (supporting); Writing – original draft (lead).Fig S1Click here for additional data file.Fig S2Click here for additional data file.Supplementary MaterialClick here for additional data file.


Protein arginine methyltransferase 5 (PRMT5), a histone methyltransferase responsible for the symmetric dimethylation of histone H4 on Arg 3 (H4R3me2s), is an enzyme that participates in tumor cell progression in a variety of hematological malignancies. However, the biological functions of PRMT5 in multiple myeloma (MM) and the underlying molecular mechanisms remain unclear. In this study, we conducted a bioinformatics analysis and found that PRMT5 expression was significantly upregulated in MM. In vitro and in vivo phenotypic experiments revealed that knockdown of PRMT5 expression enhanced cell pyroptosis in MM. Moreover, we found that CASP1 expression was negatively correlated with PRMT5 expression, and repressing PRMT5 expression rescued both the phenotype and expression markers . Inhibition of PRMT5 activity increased CASP1 expression and promoted MM cell pyroptosis. Finally, high expression of PRMT5 or low expression of CASP1 was correlated with poor overall survival in MM. Collectively, our results provide a mechanism by which PRMT5 regulates cell pyroptosis by silencing CASP1 in MM. Multiple myeloma (MM) is a malignancy that originates from the extensive proliferation of pathological antibody-producing plasma cells in the bone marrow and accounts for ~10% of hematological cancers . Many paProtein arginine methyltransferase 5 (PRMT5) is the main type II methyltransferase that catalyzes the symmetric addition of dimethylarginine to histone proteins, nonhistone proteins, and cytoplasmic proteins and is involved in numerous cellular processes, including transcription, DNA repair, RNA processing, proliferation, and metabolism –11. SeveIn this study, in addition to our cohort, we extracted clinical and RNA sequencing data from the Multiple Myeloma Research Foundation (MMRF) CoMMpass study to assess the clinical significance of PRMT5. We also explored the biological function of PRMT5 in MM cells and investigated the mechanism by which PRMT5 regulates MM cell pyroptosis by repressing CASP1.P = 0.020, Fig. n = 859) and Genotype-Tissue Expression (GTEx) databases (n = 62) was conducted, which revealed that PRMT5 expression was higher in MM tissues than in noncancerous tissues  and successfully knocked down the relative levels of PRMT5 expression in two MM cell lines (NCI-H929 and U266) compared to that in the negative control group as assessed by western blotting and qRT-PCR Fig. .These reP < 0.05, Supplemental Fig. Cell Counting Kit-8 (CCK-8) assays indicated that downregulation of PRMT5 promoted cell viability in the two individual MM cell lines (We conducted a genome array on three NCI-H929 cell samples and three PRMT5-knockdown (PRMT5-kd) NCI-H929 cell samples using Affymetrix Clariom S array chips to determine the function of PRMT5 in MM pathogenesis Fig. . DatasetTo investigate whether PRMT5 regulates cell pyroptosis by repressing CASP1 expression, we transiently overexpressed CASP1 in NCI-H929 and U266 cells Fig. . Our resWe designed six pairs of walking primers across the core promoter regions of the CASP1 gene to test whether PRMT5 directly regulates CASP1 expression and analyzed the enrichment of PRMT5-mediated H4R3me2s around the CASP1 gene promoter using chromatin immunoprecipitation (ChIP) assays and qRT-PCR. Our results demonstrated that, in PRMT5-depleted NCI-H929 and U266 cells, the levels of H4R3me2s were significantly reduced at the promoter region of CASP1 compared to the control group Fig. .Fig. 5TrTo inhibit H4R3me2s at the CASP1 gene promoter, we treated NCI-H929 and U266 cells with GSK591 , which is widely acknowledged as a specific inhibitor of PRMT5 , 15. We P = 0.022) and low CASP1 expression (P = 0.00063) were associated with poor overall survival in MM. We merged these two variants and found that patients with both low PRMT5 and high CASP1 expression exhibited significantly worse survival compared with to those carrying high PRMT5 and low CASP1 expression . Moreover, both the univariate (P < 0.001) and multivariate (P = 0.007) Cox proportional hazards regression analyses demonstrated that PRMT5 upregulation was an independent prognostic risk factor for poor survival in patients with MM  family which primarily involves the inflammatory process . HoweverOur results implied the possibility that the PRMT5 inhibitor GSK591 may represent a therapeutic approach for MM. In fact, PRMT5 inhibitors have shown strong therapeutic potential in oncotherapy, with some of them already commercialized. Traditional inhibitors of PRMT5 mainly consist of SAM analogs , We explored the biological function of PRMT5 in MM and then focused on its mechanism of regulating the pyroptosis pathway both in vitro and in vivo. Taken together, our findings demonstrate that PRMT5 regulates cell pyroptosis by silencing CASP1 in MM, and the PRMT5 inhibitor GSK591 may serve as a potential therapeutic agent for MM.2.MM cell lines  were purchased from American Type Culture Collection (ATCC). All cells were cultured in RPMI 1640 medium  supplemented with 10% fetal bovine serum (Gibco) in an atmosphere at 37 °C with 5% CO® QuantStudio™ 6 Flex Real-Time PCR System. The relative quantitative value was calculated using the 2−ΔΔCt method. The final result was recorded as fold change. Every sample was run using with three replicates. The primers used for qRT-PCR are described in Supplemental Table Total RNA was extracted using RNA-easy Isolation Reagent  and quantified using an ultramicro spectrophotometer . Reverse transcription was performed using ABScript II RT Mix for qPCR with gDNA Remover  according to the manufacturer’s protocol. Quantitative real-time PCR analysis was performed using qPCR SYBR Green Master mix  with an Applied BiosystemsBone marrow samples of newly diagnosed MM patients were collected at Nanjing Drum Tower Hospital, Jiangsu Province, China. MM patients with a distinctive pathologic diagnosis, no preoperative systemic or local treatment, and complete follow-up data were selected for inclusion in this study. The paraffin-embedded tissue were deparaffinized, rehydrated, and then subjected to antigen retrieval. The tissue slides were, respectively, incubated with PRMT5 antibodies  and CASP1 antibodies  overnight at 4 °C. Subsequently, the slides were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody. Each sample was independently scored by two pathologists who were blinded to all the patient clinical data. Based on the percentage of PRMT5- or CASP1-positive tumor cells, the extent of staining was scored 0 (negative), 1 (1–25%), 2 (26–50%), 3 (51–75%), or 4 (76–100%). The final score for each slide was assessed by multiplying the scores for intensity and extent of staining. PRMT5 or CASP1 expression on slides was considered low if the score was <3 and high if the score was >3.PRMT5 shRNA lentivirus was prepared by the co-transfection of 293T cells with pLKO.1 and the packaging plasmids pMD2G and psPAX2. At 48 h after transfection, the viral supernatants were collected. NCI-H929 or U266 cells were incubated with viral supernatants in the presence of polybrene. Positive cells were selected using 1 μg/ml puromycin. The target sequences were PRMT5 shRNA#1: 5ʹ-CCCATCCTCTT CCCTATTAAG-3ʹ and PRMT5 shRNA#2: 5ʹ-GCCCAGTT TGAGATGCCTTAT-3ʹ. A scrambled sequence was used as a negative control.CASP1 siRNAs were synthesized by SyngenTech Inc.  and transfected using Lipofectamine 2000  according to the manufacturer’s protocol. SiRNA sequences targeting CASP1 were siRNA#1: 5ʹ-GGUGUGGUUUAAAGAUUCATT-3ʹ; siRNA#2: 5ʹ-GAAGACUCAUUGAACAUAUTT-3ʹ; and siRNA#3: 5ʹ-CUCUCAAGGAGUACUUUCUTT-3ʹ.CASP1 overexpression plasmids and the control plasmid were purchased from SyngenTech Inc.  and were transfected into cells using Lipofectamine 2000  according to the manufacturer’s protocol.3VO4, leupeptin). The concentration of the protein was quantified using the BCA protein assay . Western blotting was implemented in accordance with the standard methods [Cells were washed with PBS and lysed in lysis buffer . Following fragmentation, 10 μg of cRNA was hybridized for 16 h at 45 °C on GeneChip Rat Gene 2.0. GeneChip were washed and stained in the Affymetrix Fluidics Station 400. GeneChip were scanned using the Hewlett-Packard GeneArray Scanner G3000 7G. The data were analyzed with RMA using Affymetrix default analysis settings and global scaling as normalization method. The trimmed mean target intensity of each array was arbitrarily set to 100.Cells were cultured in RPMI-1640 with 10% FBS and then cross-linked with 1% formaldehyde at room temperature. Then, 10–15 min later, the reaction was quenched with glycine for 10 min at a final concentration of 125 mM. Subsequently, the chromatin was sonicated to produce DNA fragments (200–500 bp). For this, 100 μg of chromatin was incubated overnight with rotation at 4 °C with H4R3me2s (Abcam) antibodies or IgG (2 μg) followed by 1 h incubation with protein A Sepharose beads. After washing with low-salt buffer and high-salt buffer, the beads were incubated with elution buffer and proteinase K at 65 °C overnight to immunoprecipitate the DNA. Total DNA fragments were isolated by phenol/chloroform extraction and ethanol precipitation. After isolation, the DNA was diluted in water and subjected to real-time PCR analysis. The primers designed for qRT-PCR are shown in Supplemental Table NCI-H929 cells and U266 cells were cultured on glass coverslips, fixed in 4% paraformaldehyde (Sigma) for 30 min and permeabilized using 0.3% Triton X-100 for 30 min at room temperature. After blocking with 5% goat serum in PBS, cells were incubated with PRMT5 antibodies (Sigma) for 60 min and secondary antibodies for 45 min, respectively, in a dark and humid chamber. Then, the cells were washed with PBS, and the nuclei were stained with 4,6,diarnidino-2-phenylindole (DAPI) for 5–10 min. Immunofluorescence images were captured under a fluorescence microscope .The indicated cells were seeded into each well at a density of 1000 cells in 96-well plates. We examined cell growth every 24 h using a Cell Counting Kit-8 kit  after incubation for 1 h at 37 °C. According to the reference wavelength, the absorbance value was detected at 450 nm.5 cells were collected and washed twice with PBS. A total of 50 µl binding buffer, 5 µl Annexin V-FITC, and 5 µl PI were added to the cell suspension and mixed at room temperature in the dark for 10 min. Apoptosis was measured using a flow cytometer (BD Accuri C6 Plus) within 1 h. The flow cytometry data were analyzed using FlowJo 10.An Annexin V-FITC/PI kit (BD Biosciences) was used to measure apoptosis according to the manufacturer’s protocol. Briefly, 107 NCI-H929 cells transfected with scr shRNA , PRMT5 shRNA , or PRMT5 shRNA plus CASP1 siRNA . All mice were weighed, and xenografts were measured using Vernier calipers every week after injection. Five weeks later, all the mice were euthanized by cervical dislocation, and the subcutaneous tumor xenografts of the mice were collected. The maximum diameter of the tumor xenografts was measured using Vernier calipers.In all, 18 female BALB/c nude mice were fed sterilized feed and pure water in a specified pathogen-free environment in an air laminar flow chamber. Feed, bedding, and cages for indoor use were autoclaved and transported through a sterile inlet chamber to exclude any microorganisms. Then, the mice were divided into three groups and subcutaneously inoculated with 1 × 10t-test for comparing two groups using GraphPad Prism software. Data are shown as mean ± SD. Differences in the mean values were considered to be significant at P < 0.05.Statistical analysis was performed using Student’s Detailed Author Contribution.Supplemental Figure legends.Supplemental Fig1.Supplemental Fig2.Supplemental Table 1.Supplemental Table 2.


Canonical Wnt signaling pathway plays a crucial role in cancer cell proliferation, which links by the growth of β-catenin in cell due to inactivation of glycogen synthetase kinase-3. Therefore, it is of interest to design novel candidates to bind withβ-catenin. Hence, we document the molecular docking analysis data of aspirin analogues with β-catenin for further consideration. Colorectal cancer (CRC) is the most common form of cancer in oncologic pathology, and it is ranked as second most recurrent cause of death associated to cancer, it affecting the both men as well as women in the same manner worldwide, developed and underdevelopedCountries. It is also predicted to overcome the death ratio of chronic diseases in the upcoming years . Almost Crystal structure of Beta-catenin was retrieved from PDB ((PDB Id: 1JDH)  is used Structure data for aspirin and its analogues were downloaded from pubchem database. All compounds were converted as PDB file format using the Online Smile Translator. Energy minimizations were done using ChemBio 3D Ultra 12.0 as per the standard method.Patch dock ,10 was uThe drug capability and pharmacokinetic estimation of the compounds were carried out by Lipinski filter (http://www.scfbio-iitd.res.in/software/drugdesign/lipinski.jsp), according to which an orally active drug must follow at least of four of the five laiddown condition for drug likeness namely: molecular mass, cLogP, hydrogen donor and acceptor and molar refractive index .The molecular docking analysis data of aspirin analogues with β-catenin is given in Table 1 to 3 (see PDF). The interaction of aspirin analogues with β-catenin is given in Absorption, Distribution, Metabolism and Excretion (ADME) is simple and essential analysis tool. Now days, it is usually accepted in the primary stage of drug development process, as of its exclusive feature nature. In the present, Drug-likeness properties ofthese five compounds was calculated using Lipinski rule of five and shown in Table 2 (see PDF). Results of ADME studies showed that selected compounds have good gastrointestinal (GI) absorption effect. Hence, the results of PatchDock and ADME analysis evidentlyproved that selected five analogues compounds have the ability to inhibit the β-catenin protein and act as potent anti cancer agents.We document the molecular docking analysis data of aspirin analogues with β-catenin for further consideration.


SOD1G85R mutant and corrected SOD1G85G isogenic-induced pluripotent stem cell (iPSC) lines were established. Two SOD1 mutant ALS (G85RSOD1 and SOD1D90A), two SOD1 mutant corrected (SOD1G85G and SOD1D90D), and one sporadic ALS iPSC lines were directed toward MNs. After receiving ~90% purity for MNs, we first demonstrated that SOD1G85R mutant ALS MNs recapitulated ALS-specific nerve fiber aggregates, similar to SOD1D90A ALS MNs in a previous study. Moreover, we found that both SOD1 mutant MNs showed ALS-specific neurite degenerations and neurotransmitter-induced calcium hyperresponsiveness. In a small compound test using these MNs, we demonstrated that gastrodin, a major ingredient of Gastrodia elata, showed therapeutic effects that decreased nerve fiber cytopathies and reverse neurotransmitter-induced hyperresponsiveness. The therapeutic effects of gastrodin applied not only to SOD1 ALS MNs but also to sporadic ALS MNs and SOD1G93A ALS mice. Moreover, we found that coactivation of the GSK3β and IGF-1 pathways was a mechanism involved in the therapeutic effects of gastrodin. Thus, the coordination of compounds that activate these two mechanisms could reduce nerve fiber cytopathies in SOD1 ALS MNs. Interestingly, the therapeutic role of GSK3β activation on SOD1 ALS MNs in the present study was in contrast to the role previously reported in research using cell line- or transgenic animal-based models. In conclusion, we identified in vitro ALS-specific nerve fiber and neurofunctional markers in MNs, which will be useful for drug screening, and we used an iPSC-based model to reveal novel therapeutic mechanisms (including GSK3β and IGF-1 activation) that may serve as potential targets for ALS therapy.Amyotrophic lateral sclerosis (ALS) is a progressive nervous system disease that causes motor neuron (MN) degeneration and results in patient death within a few years. To recapitulate the cytopathies of ALS patients’ MNs,  Amyotrophic lateral sclerosis (ALS) is one of the most serious motor neuron (MN) degenerative diseases: It has an incidence of five to six cases per million people and an effective treatment is not currently available. ALS patients suffer from motor function degeneration from the body terminals; within 3–5 years, patients lose most of their motor function and finally die from respiratory failure . CurrentSOD1 [ALS2 [TARDBP [C9orf72 [FUS [OPTN [VAPB [SOD1 mutations [Most ALS cases are sporadic with unknown causes, whereas 10–20% are from pathogenic genetic mutations. Known mutations of genes such as SOD1 ,6, ALS2 D1 [ALS2 , TARDBP  [TARDBP ,9, C9orff72 [FUS ,12, OPTNUS [OPTN , and VAPTN [VAPB  induce futations ,6.The clinical pathology of ALS includes mutated pathogenic protein accumulation, nerve fiber degeneration, and MN functional loss ,16. OtheSOD1 ALS MNs [SOD1 mutation MNs express SOD1 aggregates and neurofilament dysregulation phenotypes [SOD1-type ALS MNs [SOD1 and sporadic ALS must still be elucidated.Although transgenic cell lines and animals have been used in ALS research for decades, huge gaps still exist between current laboratory models and human patients. Most models have been generated from immortalized cell lines or animals with high-level expression of human ALS pathogenic transgenes ,21. Thus ALS MNs ,31,32,33enotypes . Until nenotypes ,32. HoweSOD1 ALS MNs. The novel paradigmatic nerve fibers and functional cytopathology of ALS were observed in SOD1 ALS iPSCs but not in corrected healthy iPSC-derived MNs within 28–38 days of differentiation. Gastrodin, a natural small compound screened from this model, was able to reduce nerve fiber and neurofunctional cytopathies, while also providing therapeutic effects on sporadic ALS MNs and transgenic mice. IGF-1 and GSK3β activation were the major therapeutic mechanisms of gastrodin treatment and also significantly reduced ALS phenotypes. Interestingly, the role of GSK3β in ALS reported here is opposed to that previously reported in earlier transgenic model-based studies.In the present study, we applied a CHSF-based differentiation method  for efficient generation of high-purity SOD1D90A and SOD1D90D iPSC lines were purchased from WiCell . SOD1G85R and sporadic ALS iPSC lines were generated from patients’ PBMCs with the approval of the Ethic Institutional Review Board in Hualien Tzu Chi Hospital (IRB105-131-A). Informed consent was obtained from patients. Patients’ PBMCs were cultured in StemPro 34 SFM  supplemented with SCF, FLT-3, IL-3, and IL-6 . Reprogramming was processed with a CytoTune iPS 2.0 Sendai Reprogramming Kit [The SOD1G85R mutation correction was processed by Applied StemCell . Briefly, gRNAs were designed near exon 4 of SOD1 and the site-specific cutting efficiency was identified with a surveyor nuclease assay. A 200-nt single-stranded DNA oligonucleotide homologous arm around the SOD1 G256C mutated site was delivered together with Cas9/sgRNA plasmid by nucleofection. Emerging colonies were individually collected and the SOD1 G256 site was analyzed by DNA sequencing. Two clones, A7 and A9, were identified with corrected G256G sites. The SOD1G85G iPSC line used in this study was the A7 clone.While the iPSCs reached 80% confluence in a culture dish, the cells were treated with 1 mg/mL of Dispase II  for 5–15 min until the periphery of colonies started to become rounded up; they were then lifted by scraping. The cell clumps were dissociated into 200–300 μm clusters and transferred into ultra-low attachment dishes in TeSR-E6 medium (StemCell Technologies) for 1 day to form embryoid bodies (EBs). Additionally, 5 μM of Y27632  was added during the first 4 h to avoid iPSC blebbing. The EBs were subjected to the CHSF neural epithelial induction method. The neural induction medium comprised DMEM-F12 basal medium supplied with 1% N2 supplement, 1 mM of NEAAs, and 2 mM of glutamate . CHSF factors, including 3-μM CHIR99021 , 10-μM SB431542 (Sigma-Aldrich), and 10-ng/mL recombinant human FGF-2  were added on days 1–4. On day 4, the neural induction medium was changed to neurobasal medium (ThermoFisher Scientific) with 1% N2 supplement, 1-mM NEAAs, 2-mM glutamate, and MN patterning factors, including 3 μM of CHIR99021, 2 μM of SB431542, 200 nM of LDN193189 , 0.5 μM of SAG , and 0.5 μM of retinoic acid (Sigma-Aldrich), until day 14 of differentiation. Both 0.5 μM SAG and 0.5 μM retinoic acid were continually added until day 25 of differentiation. According to EB condition, 2% B27 supplement (ThermoFisher Scientific) was potentially added to the culture medium and the EBs were moved to cell culture flasks or dishes on day 15. Before experimental assays, the EBs were dissociated into small clumps using Accutase and seeded on 1% Geltrex-coated cell culture dishes supplemented with 0.2 μM of compound E  but without BDNF, GDNF, or dbcAMP. The addition of neurotrophic factors can delay or eliminate the ALS nerve fiber cytopathies in iPSC-derived MNs. In the first 4 h of cell adhesion, 1% RevitaCell supplement (ThermoFisher Scientific) was added to the cells.First, iPSCs and differentiated cells were seeded in 24-well plates and fixed with 4% paraformaldehyde (Sigma-Aldrich) at room temperature; then, they were washed twice with phosphate-buffered saline (PBS). Cells were permeabilized with 0.1% Triton-100 for 15 min at 4 °C and then blocked with 5% horse serum. Primary antibodies were incubated with cells at 4 °C for 16 h, washed twice, and then conjugated with secondary antibodies. The primary antibodies used in this study included those against Oct-4 (1:200), Brachyury (1:50), N-cadherin (1:500), and Sox-1 (1:200) ; Nanog (1:30) and Sox-17 (1:20) (both R&D systems); Pax-6 (1:100), Nestin (1:500), and βIII-tubulin (1:500) ; NF (1:500), phosphorylated neurofilament H & M (1:500), and choline acetyltransferase (1:100) ; and Sox-2 , SSEA-4 , Islet-1 , Oligo-2 , and HB9/MNX1 . Cell nuclei were stained with diamidino-2-phenylindole (DAPI). Fluorescence images were captured using an upright microscope . Neurite density and nerve fiber beads were calculated with Olympus CellSens Dimension Desktop 3.1  using the images from three independent experiments.2, 2 mM of MgCl2, 10 of mM glucose, and 10 mM of HEPES) for 20 min. The cells were then moved to a calcium imaging chamber, washed with physiological buffer for 30 s, and treated with 60 mM of KCl for 60 s. After a 5 min wash in physiological buffer, the cells were treated with 1 mM of l-glutamate for 60 s. Images were taken with an ECLIPSE Ti2-E microscope  and analyzed with NIS-Elements AR (Nikon).Cells were seeded on Geltrex-coated 10-mm cover slides and cultured in physiological buffer with 1 μM of Fluo-4 at 37 °C for 40 min. Subsequently, they were incubated in physiological buffer  were purchased from Jackson Laboratory  and used in this study. All animal-related procedures were performed in accordance with the guidelines of the US National Institutes of Health and Hualien Tzu Chi Hospital (106-46), Taiwan. Mice were separated into three groups at 60 days of age: (1) vehicle control, (2) 50 mg/kg body weight of gastrodin, and (3) 200 mg/kg body weight of gastrodin. They were treated with vehicle or gastrodin by intraperitoneal injection five times per 30 days within the individual animal’s lifespan.Transgenic mice overexpressing the human Motor function of ALS mice was measured using the BBB scale, grip strength test, and rotarod test. BBB scale measurements began at 90 days of age and continued within the individual animal’s lifespan. Briefly, mice were free to walk around their cage and were observed for ~3–5 min. The hindlimb score was based on a 21 to 0-point scoring range from normal to no movement as measured by tail elevation, paw position, sweeping without weight bearing, movement of the hindlimb joints, forelimb–hindlimb step, consistent weight support, and frequent plantar stepping . In the Cells were lysed in a 200-μL lysis buffer composed of M-PER, 1% phosphatase protease inhibitor, and 50 mM of EDTA; subsequently, they were centrifuged at 14,000 rpm and 4 °C for 15 min. Protein concentrations were determined using the Bradford assay . Purified proteins (15 mg) were subjected to SDS-PAGE, transferred to PVDF membranes, blocked with 5% skim milk for 1 h, and then probed with primary antibodies against GAPDH (1:10000), p44/42 MAPK (1:1000), phospho-p44/42 MAPK (Thr202/Tyr204) (1:1000), MEK1/2 (1:1000), phospho-MEK1/2 (Ser217/221) (1:1000), Akt (pan) (1:1000), phospho-Akt (Ser473) (1:2000) , GSK3β , and phospho-GSK3 (Tyr279/Tyr216)  at 4 °C overnight. Membranes were then washed with PBS containing 0.1% Tween-20 before they were incubated with HRP-conjugated secondary antibody at room temperature for 1 h. All proteins were detected using Immobilon Crescendo Western HRP substrate (Millipore) and recorded with FUSION Solo S . The band intensity of Western blotting was quantified with image J  then applied to statistical analysis.An amount of 0.2 μg of total RNA was amplified by a Low Input Quick-Amp Labeling kit  and labeled with Cy3  during the in vitro transcription process. Then 0.6 μg of Cy3-labled cRNA was fragmented by incubation with fragmentation buffer at 60 °C for 30 min. Correspondingly fragmented labeled cRNA was then pooled and hybridized to Agilent SurePrint Microarray (Agilent Technologies) at 65 °C for 17 h. After washing and drying by nitrogen gun blowing, microarrays were scanned with an Agilent microarray scanner (Agilent Technologies). Scanned images were analyzed by Feature extraction10.7.3.1 software (Agilent Technologies), an image analysis and normalization software that is used to quantify signal and background intensity for each feature. Raw signal data were normalized by quantile normalization for differential expressed genes discovering. For functional assay, we provided an enrichment test for differential expressed genes (For most model organisms). Welgen Biotech used clusterProfiler for enrichment test for gene ontology (GO) and pathway (KEGG).p values < 0.05 were considered statistically significant.Data were collected and analyzed from no less than three independent experimental results and are shown as means ± SD. Student’s t-test or one-way ANOVA with Tukey’s post hoc test were used to test for differences between groups. SOD1D90A- and SOD1D90D-corrected isogenic iPSC lines from WiCell, which were previously reported by Chen et al. [SOD1G85R iPSC lines from the peripheral blood mononuclear cells (PBMCs) of an early-onset ALS patient. G85R was one of the earliest discovered SOD1 mutant sites in familial ALS [SOD1G85R) was subjected to iPSC characterization. SOD1G85R iPSC exhibited classical pluripotent stem cell morphology  with a mutant site correction  and 86%–91% of total cells   differentiation method was modified from the previously reported BiSF method ,39. Spec± 2.04%) n in eachSOD1D90A and SOD1A4V MNs were first discovered by Chen et al. [SOD1G85R MNs in somas and bead-like aggregates on neurites (211.4 ± 47.11 beads/5 × 105 pixels2 NF) (SOD1G85G control (18.23 ± 1.13 beads/5 × 105 pixels2 NF)  (SOD1G85R and SOD1D90A non-MNs (data not shown).NF inclusions in the MNs of ALS patient tissue have previously been identified; NF inclusions in n et al. . In the els2 NF) a but notels2 NF) e, similaels2 NF) g,i,k. Fo+ neurite density of SOD1G85R MNs was reduced 36%      b relativth area) d. These th area) h relativth area) j. The daGastrodia eleta previously reported to benefit spinal cord injury [SOD1G85R MNs   k after gcontrol: h to 13.7control: l. These SOD1G85R MNs showed significantly higher calcium flux (ΔF/F0: 3.68 ± 0.21) than did SOD1G85G MNs (ΔF/F0: 3.07 ± 0.14) with potassium chloride treatment but not with glutamate stimulation  than did SOD1D90D MNs (ΔF/F0: 4.26 ± 0.23) with potassium chloride treatment. After glutamate triggering, SOD1D90A MNs showed higher calcium influx (ΔF/F0: 2.68 ± 0.28) than did SOD1D90D MNs (ΔF/F0: 1.63 ± 0.09)  f–h. Bothpatients , but thiSOD1D90A MNs decreased from 2.74 ± 0.32 to 1.53 ± 0.13 after glutamate stimulation and reversed the calcium flux back to a level similar to that of SOD1D90D (ΔF/F0: 1.63 ± 0.09)  j. In conSOD1G85R d,e, or tD90A MNs i.SOD1-mutated ALS MNs, PBMCs were collected from a sporadic ALS patient (sALS), i.e., without known ALS mutations on SOD1, TARDBP, c9orf72, VAPB, and FUS, and reprogrammed to iPSCs. sALS iPSCs presented classical pluripotent stem cell morphology  and MN progenitor markers (Islet1 and Oligo2) were expressed at day 15 of differentiation, whereas MN markers (HB9 and NF) were expressed at day 28 j–l. Nervxels2 NF o, whereaxels2 NF p, i.e., SOD1G93A ALS mice were used to evaluate the in vivo therapeutic effects of gastrodin. Gastrodin was delivered from 60 days-of-age at 50 or 200 mg/kg by intraperitoneal injection five times per month. Results of the rotarod and paw force experiments showed that the motor function of ALS mice was significantly increased after gastrodin treatment    d, consis < 0.05) d–h. The SOD1G85R iPSC-derived MNs were treated with ligands or inhibitors of candidate signaling pathways, including Wnt3a , CHIR99021 (GSK3β inhibitor), IWP-2 , and IGF-1. Immunofluorescence of neurite intensity showed that compared with control  triggered 488-nm fluorescence signals, which were followed by cell death immediately after glutamate treatment. Thus, hyperexcitability might worsen and finally cause MN death after normal levels of neurotransmitter stimulation in aging SOD1 ALS MNs.In neurofunctional tests of ALS MNs lasting > 60 days, we found that the hyperexcitation similar properties of SOD1G85R and SOD1D90A ALS MNs but also in one sALS MN, demonstrating that nerve fiber cytopathies were not only restricted to SOD1-type ALS. Gastrodin treatment rescued nerve fiber beads and promoted neurite growth in sALS MNs, indicating the therapeutic potential of gastrodin on sALS.Nerve fiber beads not only presented in In ALS patients and experimental animals, riluzole can prolong lifespan at the extremely late stage of disease, but it has minor effects on motor function and life quality maintenance ,45. ConvSOD1E100G iPSC-based ALS model: Bhinge et al. (2017) found that XAV-939  treatment postponed the death of SOD1E100G MNs [GSK3β is a potential target for ALS therapy; indeed, MN samples from transgenic ALS animals express higher levels of GSK3β activation than normal . Inhibit100G MNs . In anot100G MNs . Thus, wSOD1 mutations influence nerve fiber dynamics in ALS MNs is not yet fully clear. A previous report [NF-L) mRNA to reduce protein production, which then influences NF assembly, and finally causes ALS nerve fiber cytopathies. The shortage of NF-L protein expression also alters the stability and phosphorylation of NF heavy chain (NF-H) and NF medium chain (NF-M). After supplying NF-L with a Tet-on inducible knock-in, the researchers demonstrated a reduction in nerve fiber cytopathies. This work provided evidence to link SOD1 mutations with ALS NF cytopathy. Here, we identified misfolded SOD1 aggregates in SOD1G85R MNs (data not shown) but they did not colocalize with NF protein aggregates or inclusions, suggesting that mutant SOD1 protein may not directly interact with NF protein. The phosphorylation of NFs, including NF-H, NF-M, and NF-L, plays important roles in NF stability, transport, and assembly, especially at phosphorylation sites on the C-terminus. MAPK and GSK3β are phosphate donors for essential NF phosphorylation [SOD1 ALS MN nerve fiber cytopathies in our experiment. Thus, apart from NF-L resupply, we speculate that activation of MAPK and GSK3β may rescue SOD1-induced nerve fiber phenotypes by providing sufficient phosphorylation activities at specific essential sites, which benefits NF protein transport and assembly to eliminate nerve fiber inclusions and promote neurite extension. However, the specific GSK3β-dependent phosphorylation sites and their antibodies on NFs must still be identified and developed.How s report  indicaterylation ,56. For rylation ; this isSOD1 mutant MNs had NF inclusions and degeneration cytopathies, the level of these cytopathies differed between the two lines. Compared with D90A MNs, G85R SOD1 MNs expressed heavier nerve fiber beads, milder neurite degeneration, and less hyperexcitability, suggesting that the SOD1 mutation site may alter the individual cytopathy levels. Evidently, further research is required to identify the mechanisms underlying the SOD1 mutant site’s effects on ALS MN cytopathies.Although both G85R and D90A In addition, how gastrodin functions with the IGF-1 pathway and GSK3β activity is not yet clear. Indeed, the role of gastrodin in these pathways has not previously been reported. In living cells or animals, gastrodin would be converted into the secondary metabolite p-hydroxybenzyl alcohol within hours ; howeverSOD1 MNs that matched several clinical pathologies of patients. Although the nerve fiber beads of SOD1D90A MNs were previously reported by Chen et al. [SOD1G85R MNs. Moreover, both the nerve fiber degeneration and neurotransmitter hyperexcitability of SOD1G85R and SOD1D90A MNs were reported for the first time here. Gastrodin had therapeutic effects that rescued nerve fiber and neurofunctional cytopathies in ALS MNs while also improving the motor function of ALS animals. IGF-1 and GSK3β, both activated by gastrodin, showed therapeutic effects on nerve fiber cytopathies, demonstrating the potential of their coactivation as an ALS therapy.In conclusion, we established a robust method by which to differentiate iPSCs into MNs (~90%). Consequently, within 38 days of differentiation, we were able to identify ALS-specific cytopathies including neurite degeneration, nerve fiber inclusions, and hyperexcitability in n et al. , we are


For decades, scientists have been doing a lot of research and exploration to find effective long-term analgesic and/or disease-modifying treatments. Microneedles (MNs) are a simple, effective, and painless transdermal drug delivery technology that has emerged in recent years, and exhibits great promise for realizing intelligent drug delivery. With the development of materials science and fabrication technology, the MN transdermal drug delivery technology has been applied and popularized in more and more fields, including chronic illnesses such as arthritis or diabetes, cancer, dermatocosmetology, family planning, and epidemic disease prevention, and has made fruitful achievements. This paper mainly reviews the latest research status of MNs and their fabrication methodology, and summarizes the application of MNs in the treatment of various diseases, as well as the potential to use nanotechnology to develop more intelligent MNs-based drug delivery systems. Syringes and hypodermic needles have been used to deliver drugs to patients for more than 160 years and are still the most routine and effective means . In addi3). Due to the dense structure, it is almost impossible for drugs with molecular weight >500 Da and Log P in the 1–3 range to penetrate the cuticle [The skin is the largest organ, and is a strong biological barrier, preventing infectious diseases and harmful substances from entering the body. However, the skin is also a major barrier to the effective delivery of drugs to lesions via percutaneous administration, while meanwhile also being susceptible to pain . The ski cuticle . Drugs w cuticle ,11. Belo cuticle ,12,13. TMicroneedles (MNs), as the name implies, are a drug delivery tool with a pointer size in the micron scale, and consists of an MN patch in the form of many MN arrays with a needle length of 25–1000 μm that can only penetrate the active cuticle and live epidermis without reaching the nerve endings and blood vessels. Because of the microscopic size of MNs, the use of MNs does not stimulate nerves in the dermis or damage blood vessels, and thus patients do not feel intense pain during the process ,15. MNs The development of a safe, fast, effective, and convenient drug delivery method is the main purpose of MNs research. According to different drug delivery methods, MNs can be divided into solid MNs, coated MNs, hollow MNs, degradable or soluble MNs, swelling MNs, and porous MNs ,20,21. FSolid MNs are made of silicon or metal without carrying drugs, which can be used for skin pretreatment. The MNs are removed after the punctured epidermis forms microchannels, and the drug preparation is applied to the puncture site and then diffuses into the body through the channels ,28. The Coated MNs are surface drug-loaded MNs. The drug is attached to the surface of the MNs by dipping, spraying, or coating. The drug dissolves when the needle is inserted into the skin and is absorbed by the body. This method is mainly applicable to water-soluble drugs, which have the advantages of rapid drug release, high use rate, and easy dose control, as shown in With hollow MNs, the drug is preloaded into the needle body with a hole in the tip and then injected into the skin using a syringe or micropump to pressurize it C. Among Soluble or degradable MNs are a kind of MN that is being studied a lot at present. The MN body can be made of biodegradable or water-soluble polymers . After entering the skin, the needle body is degraded or dissolved within minutes or hours, whereby the needle body separates from the substrate and releases the internal drug after degradation or dissolution . The molSwellable MNs are a relatively new kind of MN, and are prepared from a cross-linked hydrogel and can expand but not dissolve after absorbing interstitial fluid (IF). This kind of hydrogel MNs can be loaded in two ways: one is to pre-position the drug at the base of the MNs, and after piercing into the skin, the hydrogel absorbs the intercellular fluid to expand, forming a gel channel . The othPorous MNs have recently been researched owing to their distinctive and unique characteristics, where porous structures inside MNs with continuous nano- or micro-sized pores can transport drugs or biofluids by capillary action . HoweverCompared with traditional syringe and needle transdermal delivery, the requirements of MNs are higher in terms of material selection and design, depending on its purpose. The materials from which MNs are constructed mainly include silicon, glass, ceramic, metal, hydrogel, polymer, sugar, etc. Common matrix materials for degradable or soluble MNs include sodium carboxymethyl cellulose (CMC), polyvinylpyrrolidone (PVP), hyaluronic acid (HA), polylactic acid (PLA), chitosan, etc. Advanced biodegradable metals have excellent biocompatibility and sufficient mechanical properties, and are also a potential kind of matrix material ,41. The Ideal products must meet the requirements of good mechanical performance and insertion performance, which are closely linked to their materials and design. The transdermal properties of some typical recent MNs are listed in MNs have been investigated for decades; in this review, the construction idea of MNs is divided into two main categories on the basis of MN design concept with respect to their precursors: either the substrate material is formed by physically mixed stacking casting , or by fManufacturing technology is one of the important factors affecting the development and application of MN transdermal drug delivery technology. Based on the substrate material and the characteristics of the loaded drug (such as the stability of the sensitivity to temperature and light), different manufacturing processes need to be selected. In general, MN production can take place through either a mold-based process or a mold-free process. The mold-based process is currently the simplest and most popular one. A mold made by copying the metal or stainless-steel master structures is required first, and the mold usually used is polydimethylsiloxane (PDMS) mold . TypicalWith the rapid development of manufacturing technology, the application of MNs has been extended to many fields, such as immunobiology, disease diagnosis, and cosmetology. By searching the keyword “microneedle” on PubMed, 3,179 results were retrieved (as shown in Osteoarthritis (OA) is the most common degenerative joint disease, affecting more than 250 million people worldwide . Since oMulti-functional bionic MNs allow drugs to be delivered to parts of the body with high flexibility sustainably and over a long period. After MNs loaded with glucocorticoid were used to treat knee osteoarthritis in rats, the degree of swelling and inflammation in the knee joints of rats was greatly reduced  is a unique alkaline polysaccharide in nature that has prominent characteristics of biodegradation, non-toxicity, biocompatibility, natural antibacterial, hemostasis, etc., and is widely used to develop multi-functional smart micron array (CSMNA) patches for wound healing . FurtherMNs used in vaccine delivery comprise an earlier field of study, making it a more mature subject. In particular, MN vaccine can be stored at room temperature and transported in solid form, and can be used for the packaging or encapsulation of DNA vaccine, subunit antigen, or inactivated or live virus vaccine . It alsoMNs have been employed to deliver anticancer drugs (chemotherapy), as well as photodynamic therapy (PDT) and photothermal therapy (PTT) agents, to skin tumor sites in a minimally invasive fashion and with improved therapeutic efficacy. In mice with subcutaneous melanoma tumors, the delivery of ovalbumin-pulsed dendritic cells via cryomicroneedles elicited higher antigen-specific immune responses and led to slower tumor growth than intravenous and subcutaneous injections of the cells. Cryomicroneedles used for delivery cells could retain their viability and proliferative capability. Biocompatible cryomicroneedles may facilitate minimally invasive cell delivery for a range of cell therapies . The comSince MN patch technology was proposed, it has been applied in many biomedical fields, showing great application prospects. Li et al.  extendedInsulin is a natural hormone secreted by pancreatic islet cells in the pancreas to help the body regulate blood sugar. Long-term insulin injections are often necessary for diabetics, whether they are type 1 (which cannot produce insulin itself) or type 2 . However, there is difficulty in controlling blood sugar with insulin. When the dose is wrong, there may be a risk of hypoglycemia, which in severe cases can lead to seizures, coma, and even death. As a result, diabetics need to constantly monitor their blood sugar levels while taking insulin . InsulinInitially, MNs were used to break barriers and enhance transdermal efficiency. Now, the use of microneedles has expanded to many other fields. The prominent studies of biomedical application of MNs were list in MN drug delivery technology is a new transdermal drug delivery method based on the dual drug release characteristics of subcutaneous injection and transdermal patch technology. Research into the drug treatment of chronic illnesses such as arthritis or diabetes, cancer, dermatocosmetology, family planning, genes, and epidemic disease prevention demonstrates a diversification of development, including the use of small molecule chemistry medicine, biological macromolecule polypeptides, protein and gene medicine, insulin, and so on. Local or systemic administration is also carried out according to disease type and drug performance, and drugs with long-term or short-term release have been designed for use with MNs.On the basis of the previous studies, it can be seen that the functional design of MN drug delivery systems using nano-engineering technology could make it possible to realize intelligent control of drug release amount, release kinetics, or stimuli-responsive delivery. The main advantage of nanomaterials is that they can be designed for specific functions, and are responsive to specific microenvironments . There aSince the emergence of MNs as a promising drug delivery technology, MNs have developed into a broad and rapidly growing research field ,114. The


Oxysterol-binding protein-like 2 (OSBPL2), also known as oxysterol-binding protein-related protein (ORP) 2, is a member of lipid transfer protein well-known for its role in regulating cholesterol homeostasis. A recent study reported that OSBPL2/ORP2 localizes to lipid droplets (LDs) and is associated with energy metabolism and obesity. However, the function of OSBPL2/ORP2 in adipocyte differentiation is poorly understood. Here, we report that OSBPL2/ORP2 contributes to the developmental progression of preadipocytes. We found that OSBPL2/ORP2 binds to β-catenin, a key effector in the Wnt signaling pathway that inhibits adipogenesis. This complex plays a role in regulating the protein level of β-catenin only in preadipocytes, not in mature adipocytes. Our data further indicated that OSBPL2/ORP2 mediates the transport of β-catenin into the nucleus and thus regulates target genes related to adipocyte differentiation. Deletion of OSBPL2/ORP2 markedly reduces β-catenin both in the cytoplasm and in the nucleus, promotes preadipocytes maturation, and ultimately leads to obesity-related characteristics. Altogether, we provide novel insight into the function of OSBPL2/ORP2 in the developmental progression of preadipocytes and suggest OSBPL2/ORP2 may be a potential therapeutic target for the treatment of obesity-related diseases. Several members of the OSBP/ORPs family are linked to obesity-related diseases: ORP8 has previously been reported to play a key role in the development of type II diabetes mellitus (T2DM)3; ORP11 is suggested to be closely associated with cardiovascular disease risk factors and is expressed at a high level in visceral adipose tissue (VAT) in obese population with metabolic syndrome4; and ORP5 controls the synthesis of neutral lipid and thus regulates the lipid droplet (LD) size5.Oxysterol-binding protein (OSBP) and OSBP-related protein (ORP) or OSBP-like (OSBPL) proteins have been found to contain a conserved OSBP-related domain (ORD) with oxysterol-binding activity and constitute a large family that regulates lipid homeostasis6. Similar to other members of the OSBP/ORPs family, OSBPL2/ORP2 is a lipid transfer protein (LTP)7 and has a function in lipid transport and regulation of sterol and phospholipid metabolism11. OSBPL2/ORP2 also regulates the actin cytoskeleton and affects cell adhesion12. In addition, OSBPL2/ORP2 is associated with LD formation13 and is considered as an important effector in energy metabolism14. Recent studies have showed a linkage between OSBPL2/ORP2 and obesity-related disease: the regulation of LD lipolysis required OSBPL2/ORP215, and the OSBPL2-disruption resulted in hypercholesterolemia in miniature pig models16. However, the role of OSBPL2/ORP2 in the development of obesity-related disease and the underlying regulatory mechanism of adipocyte differentiation have yet to be fully elucidated.Oxysterol-binding protein-like 2 (OSBPL2/ORP2) is an important ORP that contains an FFAT  motif for ER targeting and a functional ORD for lipid binding18. During this period, pro-adipogenic transcription factors such as PPAR-γ (a preadipocyte marker), together with C/EBPα, commit preadipocytes to differentiate into mature adipose tissue19. The Wnt signaling pathway is one of the previously reported regulators of preadipocyte development and adipocyte differentiation. β-Catenin, a component of adherens junctions, plays a key role in cell adhesion20 and is an effector in the canonical Wnt signaling pathway. β-Catenin has three distinct domains: the N-terminal fraction, armadillo (ARM) domain, and C-terminal fraction22. The ARM domain contains 12 armadillo repeats (40 aa) is an important site for the interaction of its partners. The ARM domain also contains the SRP fraction, which is a conserved domain in the SRP1 superfamily that appears to bind to the nuclear envelope and is associated with the nuclear trans-localization24. It is reported that β-catenin negative regulates adipocyte differentiation by suppressing PPAR-γ and C/EBPα activation, and thus maintains preadipocytes in an undifferentiated state25. In the progression of preadipocyte development, β-catenin is phosphorylated upon forming a destruction-bound complex modified with ubiquitin and is ultimately degraded by the proteasome26.Preadipocyte development is strongly linked to the onset of obesity upon its committed differentiation into an adipocyte. During the developmental process, the morphology of preadipocytes changes, and the function of preadipocytes become similar to that of mature adipocytes, including gaining the capacity for adipogenesis and lipid droplet (LD) storageOSBPL2 might be a novel target for the treatment of obesity-related disease in humans.Here, we demonstrate that OSBPL2/ORP2 is required for maintaining preadipocytes in the undifferentiated stage. Loss of OSBPL2/ORP2 facilitates 3T3-L1 preadipocyte maturation, and the absence of Osbpl2b leads to the adipose tissue (AT) presenting with much larger LDs and the acquisition of an obesity phenotype by the zebrafish model. Overall, our study provides important insights into the function of OSBPL2/ORP2, and the results strongly suggest that BODIPY 493/503 (#D3922) and DAPI (#D1306) were purchased from Invitrogen . SKL2001 was purchased from Selleckchem . Dexamethasone (#D4902), 3-isobutyl-1-methylxanthine (#I5879), insulin (#I2643), Oil red O (#O9755), MS-222 (#E10521), and Triton X-100 (#T8787) were purchased from Sigma-Aldrich . Antibodies against P42/44 (#9102), β-catenin (#9562), Phospho-β-catenin (#9561), PPAR-γ (#2443), Ubiquitin (#3936), GAPDH (#5174), HA-tag (#3724) as well as secondary HRP-conjugated antibodies against mouse (#7076) and rabbit (#7074) were purchased from CST  and used at 1:1,000 dilution for western blotting. Anti-FLAG-tag antibody (#F1804) and anti-FLAG-tag M2 affinity gel (#A2220) were from Sigma-Aldrich . Antibodies against Plin1 (#A16294), OSBPL2 (#A14199), α-Tubulin (#AC012), β-catenin (#A11512) as well as secondary HRP-conjugated Mouse Anti-Rabbit IgG Light Chain (#AS061) were from ABclonal . Antibodies against β-catenin (#BF0319) for zebrafish were from Affinity Biosciences . Alexa Fluor 488 donkey anti-mouse IgG and Alexa Fluor 546 donkey anti-rabbit IgG were purchased from Life Technologies .osbpl2b-knockout zebrafish were previously generated in our laboratory using the CRISPR/Cas9 gene-editing technique. The guide RNA targeting exon 6 of osbpl2b was designed using ZiFiT software (http://zifit.partners.org/ZiFiT/), and sgRNA was co-injected into one-cell stage zebrafish embryos of the Tü wild-type strain, which caused a 5-nucleotide (GAGCT) deletion in exon 6 of osbpl2b.Zebrafish were grown in buffered reverse osmosis (RO) water with a standard light/dark cycle of 14 h/10 h at 28 °C. Adult zebrafish were fed shrimp twice a day and larvae were fed the same amount of paramecium and egg yolk. Three months post fertilization (mpf), the zebrafish were used to mate and breed. All zebrafish were subjected to standard husbandry procedures and used in accordance with standard procedures and guidelines of the Institutional Animal Care and Use Committee (IACUC) of Nanjing Medical University. The 2 at 37 °C. For the transient plasmid transfections, the 3T3-L1 preadipocytes were grown on coverslips in 24-well plates overnight. The 3T3-L1 preadipocytes were then transfected with 1 μg plasmid using Lipofectamine 3000 assay  and incubated for 48 h.The 3T3-L1 preadipocytes and HEK293T cells were cultured in Dulbecco’s modified Eagle’s medium  supplemented with 10% fetal bovine serum  in a humidified atmosphere containing 5% COOsbpl2-knockout 3T3-L1 preadipocytes were generated using the CRISPR/Cas9 gene-editing technique. The guide RNA (CAGCGGCTGGGACTGACTTG) targeting exon 4 of osbpl2 was designed using CHOPCHOP software (https://chopchop.rc.fas.harvard.edu/), and was packaged into an all-in-one CRISPR lentivirus vector . WT 3T3-L1 preadipocytes were seeded into 24-well plates at a density of 0.5 × 105 cells/well and after 18 h, they were incubated with lentivirus for 48 h. Monoclonal cells were obtained by selection in puromycin conditioned culture, and sequencing was performed on the genomic DNA, which revealed a 2-nucleotide (AA) deletion in exon 4 of Osbpl2. In addition, the effectiveness of osbpl2-deficiency was analyzed by western blotting with an anti-OSBPL2 antibody .27 to induce preadipocytes into mature adipocytes, 3T3-L1 preadipocytes were grown in 6-well plates for 48 h before treatment. The 3T3-L1 preadipocytes were treated with MDI  or DI (1 μM dexamethasone and 1 μg/ml insulin) for 48 h, and then the 3T3-L1 cells were maintained in DMEM containing DI and 10% FBS until mature.Following a classical methodThe 3T3-L1 preadipocytes were grown on coverslips and followed by treatment according to the experimental plan. After being treated for the appropriate times, the cells were immobilized with 4% paraformaldehyde (PFA) for 15 min and then permeabilized with 0.1% Triton X-100  for 10 min at room temperature. Then, cells were stained with BODIPY 493/503  for 10 min to and with DAPI  for 5 min. Confocal microscopy was used with ZEN software to acquire high-quality images. The larvae were maintained in RO water containing BODIPY 493/503  for 30 min and then anesthetized with MS-222 . Images were acquired by fluorescence stereo microscope .The 3T3-L1 preadipocytes were immobilized with 4% PFA for 15 min, and then washed with 60% isopropyl alcohol for 5 min. Then the cells were soaked in 0.3% oil red O solution  for 15 min and washed with 60% isopropyl alcohol for 15 s. After cells were washed with PBS, images were acquired by fluorescence stereo microscope .For the analysis of nucleus fractionation and cytosolic proteins, a nucleus and cytoplasmic extraction assay  was used according to the protocol. The 3T3-L1 preadipocytes were first washed with cold PBS and harvested with trypsin-EDTA. After cells were centrifugated, the supernatant was discarded to ensure that the pellet was as dry as possible, and then, ice-cold CER I and CER II was added stepwise. The mixture was centrifugated and the supernatant containing the cytosolic fraction was poured into a new tube. Then, the pellets were suspended with ice-cold NER incubated on ice for 40 min with vigorous vortexing for 15 s every 10 min. After centrifugation, the supernatant containing the nucleus proteins was poured into a new tube. Successful separation of the cytosolic and nucleus fractions was routinely validated by western blotting with GAPDH  and HISTONE H3 , respectively.The samples obtained from the cells or tissues lysed with RIPA buffer  were separated by 10% SDS-PAGE  and transferred onto PVDF membranes . The membranes were blocked with TBS-T containing 5% milk powder  for 2 h and then incubated with primary antibody overnight at 4 °C. The next day, the membranes were incubated with secondary antibodies for 2 h. The bands were visualized with an Odyssey® CLx Imaging System (LI-COR) or by a chemiluminescence method.Total RNA was extracted by TRIzol reagent , and cDNA was synthesized using a HiScript II one step RT-PCR kit . The quantitative real-time PCR (qRT-PCR) was performed on a StepOne Plus system  with ChamQ SYBR qPCR Master Mix . The primers used to amplify the specific genes are listed in Supplementary Table β-catenin plasmid and FLAG-tagged OSBPL2 or truncated proteins plasmid for 48 h. Then samples were washed three times with ice-cold PBS and lysed for 30 min with RIPA lysis buffer  containing 0.2 mM PMSF. The lysate was centrifuged for 30 min at 13,500 rpm and 4 °C. The supernatant was collected and incubated overnight with anti-FLAG-tag M2 affinity gel  at 4 °C. The lysate from the cells transfected with HA-tagged β-catenin was extracted and immunoprecipitated using anti-FLAG-tag M2 affinity gel  as a negative control. The beads were washed more than five times, added to 6×SDS loading buffer, and boiled at 100 °C for 10 min. Then, the samples were assayed by western blotting. For the proteomics experiments, the lysate was extracted from the HEK293T cells transfected with FLAG-tagged OSBPL2 and was immunoprecipitated with anti-FLAG-tag M2 antibody or immunoprecipitated with normal IgG as a negative control. The protein bands were retrieved and subjected to LC-MS/MS analysis .HEK293T cells were co-transfected with HA-tagged The 3T3-L1 preadipocytes were grown on coverslips and followed by a treatment according to the experimental plan. After being treated for the appropriate times, the cells were immobilized with 4% PFA for 15 min, permeabilized with 0.1% Triton X-100  for 10 min at room temperature, and then blocked with 10% goat serum  for 1 h at room temperature. The coverslips were incubated overnight with primary antibody at 4 °C: rabbit anti-HA , and mouse anti-FLAG . Secondary antibodies were incubated for 1 h at 37 °C: Alexa Fluor 488 donkey anti-mouse IgG , and Alexa Fluor 546 donkey anti-rabbit IgG . The cells were incubated with BODIPY 493/503  for 10 min at room temperature and with DAPI  for 5 min. Images were captured by confocal microscopy and visualized with ZEN software.Zebrafish brain and liver paraffin sections obtained from zebrafish at 4 mpf were subjected to gradient ethanol dewaxing and followed by antigen repair. Then, the samples were blocked with 10% goat serum  for 1 h at room temperature, and incubated overnight with primary antibodies at 4 °C: anti-β-catenin  and anti-OSBPL2 . The next day, the samples were incubated with secondary antibodies for 1 h at 37 °C: HRP-conjugated goat anti-rabbit IgG . Images were acquired by microscopy.https://www.rcsb.org/). Protein-protein docking was performed using GRAMM-X software (http://vakser.compbio.ku.edu/resources/gramm/grammx/) and the macromolecular structures were visualized with PyMOL software (https://pymol.org/2/).Biological macromolecular structures of OSBPL2 (ID: 5ZM8) and β-catenin (ID: 1QZ7) were obtained from the RCSB PDB website  was used for single-factor comparisons among multiple groups, and two-way ANOVA was used for two-factor comparisons among multiple groups; P < 0.05 was regarded as significant.The number and size of LDs were analyzed with Image J software. All data are presented as the mean ± SD. Student’s Osbpl2-deficient 3T3-L1 preadipocytes by using the CRISPR–Cas9 gene-editing technique, through which amino acid sequences were truncated and altered  cells for as long as 8 d without any treatment. A confocal microscope was used, and the LDs were fluorescently stained with BODIPY 493/503 to delineate LD size and distribution patterns as previously described28. We confirmed that the size of the LDs increased in the KO cells , 3T3-L1 preadipocytes were induced and to differentiate into mature adipocytesTo seek further evidence to verify the development of preadipocytes and adipocyte differentiation in WT and KO cells, inducers with different constituents were used. We found that the LDs accumulated to a similar extent on day 8 in the MDI group  experiments showed specific interaction of OSBPL2 with β-catenin in 3T3-L1 preadipocytes  of β-catenin.32. The level of β-catenin was detected to be decreased in the KO cells, while the level of PPAR-γ was increased  in KO cells, the intensity of β-catenin was increased in the cytoplasm and the ratio of the intensity of β-catenin within the nucleus versus the entire cell was decreased compared with that in the KO control cells, but transfecting Osbpl2 plasmid or ORD plasmid rescued this phenomenon  decreased, and some of the obesity-related genes  increased. We also found that β-catenin mRNA expression level was increased in the KO cells despite lower protein levels, which is supposed to be a compensatory effect. Further study would be performed to explain this point. We also found that treating with SKL2001 in the KO cells partly rescued the phenotypes of cholesterol accumulation and adipose differentiation  in zebrafish are homologous with human OSBPL2, and osbpl2b is an orthologous gene that shares 71.2% homology with human OSBPL2 amino acid sequences37. We previously generated osbpl2b−/− zebrafish using the CRISPR/Cas9 gene-editing technique, which caused a 5-nucleotide (GAGCT) deletion in exon 6 of osbpl2b and resulted in a truncated protein  group compared with that in the WT group, indicating that the deletion of osbpl2b caused a reduction in β-catenin in multiple organs  and triggering preadipocytes to mature into adipocytes, finally leading to obesity-related characteristics and metabolic disorders. The above findings link OSBPL2/ORP2 to adipocyte differentiation and demonstrate that OSBPL2/ORP2 controls the progression of preadipocyte development, which provides a new molecular model for further understanding preadipocyte development and adipocyte differentiation. It has been reported that the ORP11 level increases in differentiated SGBS adipocytes, while ORP3 and ORP8 levels decrease40. The ORP11 level decreases in adipocyte tissue under caloric restriction, which may be due to fat mobilization41. However, few studies of OSBPL2/ORP2 in adipocytes or adipocyte tissue have been reported. In this study, we show that Osbpl2 deficiency increases the number of LDs in 3T3-L1 preadipocytes when the cells are cultured for 8 d.Our study indicates a novel function of OSBPL2/ORP2 in the developmental progression of preadipocytes: (i) OSBPL2/ORP2 binds to β-catenin and maintains β-catenin in the cytoplasm, (ii) the complex mediates β-catenin transfer into the nucleus and thus regulates the transcription and activation of adipogenic genes. (iii) Deletion of 42, and the stabilization of β-catenin results in a failure of PPAR-γ and C/EBPα activation in preadipocytes44. A recent study showed that raloxifene could reduce fat accumulation and suppress the activation of adipogenic factors by preserving the expression of canonical Wnt10b/β-catenin45. Moreover, carbamazepine inhibits Wnt/β-catenin expression and enhances adipogenesis46. In addition to the function of β-catenin in adipogenesis, a disruption of OCT4 and β-catenin recruitment to target gene promoters may affect differentiation, pluripotency, and cell cycle genes47. A previous study showed that elevating the ORP8 level could significantly reduce the expression levels of Wnt3a and β-catenin48. Our findings show that, in contrast to ORP8, OSBPL2/ORP2 interacts with β-catenin, and a lack of OSBPL2/ORP2 leads to a decrease in β-catenin.The interaction between OSBPL2/ORP2 and β-catenin also provides a clue for follow-up research on preadipocyte development. This finding supports the function of β-catenin in suppressing adipogenesis35. In addition, ORP8 is reported to interact with USP5, and this interaction causes ORP8 deubiquitination and accumulation and leads to growth inhibition49. Interestingly, by mass spectrometric analysis, we also identified several members of the USP family  among the binding partners of OSBPL2/ORP2, suggesting an underlying relationship among OSBPL2/ORP2, β-catenin, and member of the USP family.In this study, we found that OSBPL2/ORP2 maintained β-catenin in the cytoplasm and promoted β-catenin translocation into the nucleus, indicating the underlying molecular mechanism by which OSBPL2/ORP2 and preadipocyte development are related to obesity. It has been reported that USP20 controls the stability of β-catenin and prevents it from degrading by deubiquitinating β-cateninOSBPL2 mutations with autosomal dominant nonsyndromic hearing loss in a large affected Chinese family50, which were then verified in other affected families.52. To confirm the effect of OSBPL2 disruption on hearing loss, we established OSBPL2-disrupted Bama mini pig models. In addition to the expected phenotype of hearing loss, we found the obesity phenotypes with hypercholesterolemia in the OSBPL2 mutation Bama mini pig16. Using HepG2 cells and zebrafish, we have reported that deletion of OSBPL2 affected LD lipolysis15. All these studies suggest that OSBPL2 is a gene/protein with multiple functions. We showed that the absence of OSBPL2/ORP2 caused a decrease in β-catenin, but the mechanism by which β-catenin is reduced was still needed to be further investigated in future work.In addition, we demonstrate that OSBPL2/ORP2 contributes to the developmental progression of preadipocytes using the 3T3-L1 cell line and a zebrafish model. In the previous study, we described the association of Overall, this work revealed an underlying molecular mechanism by which OSBPL2/ORP2 regulates preadipocytes development by maintaining β-catenin in the cytoplasm and participating in the translocation of β-catenin into the nucleus. Our work presented a novel insight into the function of OSBPL2/ORP2, which may provide the potential target and therapeutic approach in the treatment of obesity-related metabolic disease.Supplementary Figure LegendsFigure S1Figure S2Figure S3Figure S4Table S1


Wnt/β-catenin signaling is crucially involved in many biological processes, from embryogenesis to cancer development. Hence, the complete understanding of its molecular mechanism has been the biggest challenge in the Wnt research field. Here, we identified ubiquitin C-terminal hydrolase like 5 (UCHL5), a deubiquitinating enzyme, as a novel negative regulator of Wnt signaling, upstream of β-catenin. The study further revealed that UCHL5 plays an important role in the β-catenin destruction complex, as it physically interacts with multiple domains of Axin1 protein. Our functional analyses also elucidated that UCHL5 is required for both the stabilization and the polymerization of Axin1 proteins. Interestingly, although these events are governed by deubiquitination in the DIX domain of Axin1 protein, they do not require the deubiquitinating activity of UCHL5. The study proposes a novel molecular mechanism of UCHL5 potentiating the functional activity of Axin1, a scaffolder of the β-catenin destruction complex. The transduction of Wnt/β-catenin signaling pathway starts when a ternary complex called the Wnt signalosome forms at the cell surface2. The Wnt signalosome is comprised of a Wnt ligand, a receptor (Frizzled), and a co-receptor (LRP5/6)2. The Wnt signalosome amplifies signal activity by recruiting downstream effectors, including Dishevelled (Dvl), Axin, Casein kinase1 (CK1), and Glycogen synthase kinase3β (GSK3ß) to the plasma membrane3. Within the Wnt signalosome, the intracellular domain of LRP5/6 is phosphorylated by GSK3ß. This phosphorylated intracellular domain functions as a docking site for Axin, and inhibits the kinase activity of GSK3ß2. Therefore, the Wnt signalosome not only inhibits the β-catenin destruction complex, but also sequesters it from β-catenin3. Consequently, the free cytosolic β-catenin moves into the nucleus, and forms a transcriptional complex with T-cell factor/lymphoid enhancer factor (TCF/LEF) to induce Wnt target genes4.Wnt signaling has a myriad of pivotal roles in numerous biological processes, such as embryogenesis, human congenital disorders, and cancer development3. APC recognizes and sets the phosphorylated β-catenin free from Axin for subsequent ubiquitination and proteasome-dependent β-catenin degradation3. This process of β-catenin turnover is shut down upon Wnt signaling activation. Specifically, Axin is most affected by Wnt signaling activation, as it is recruited to the plasma membrane after losing its binding affinity to β-catenin5. Furthermore, Axin is destabilized under long-term activation of Wnt signaling6. Therefore, Axin is a key molecule determining the functional activity of the β-catenin destruction complex. Indeed, Axin is the concentration-limiting factor in the β-catenin destruction complex3. Therefore, Axin must be tightly and sophisticatedly regulated to prevent abnormal activation of Wnt signaling.β-catenin stabilization in the cytoplasm is predominantly determined by the β-catenin destruction complex. The β-catenin destruction complex consists of multiple proteins, including the scaffold protein Axin, Adenomatous polyposis coli (APC), and two serine/threonine Kinases, CK1 and GSK3. Cytoplasmic β-catenin is normally recruited to Axin in the inactive state of Wnt signaling, and is subsequently phosphorylated at the serine/threonine residues  of β-catenin by CK1 and GSK38. Another RING-domain E3 ligase, SIAH(SIAH1/2), outcompetes GSK3ß for Axin binding and mediates Axin degradation under the Wnt active condition6. Lastly, Smurf2, a HECT-type E3 ligase, has been reported to attenuate Axin stability9. As for deubiquitinating enzymes, proteomic analysis revealed that USP34, a member of the ubiquitin-specific protease family (USP), has a role in reversing the Tankyrase/RNF146-dependent ubiquitination of Axin10. Intriguingly, this USP34 acts as a positive regulator downstream of ß-catenin by accumulating Axin in the nucleus10. Recently, genome-wide CRISPR/CAS9 screening and chemical analyses newly identified another USP member, called USP7, that stabilizes Axin under the Wnt active condition11. Nevertheless, the molecular basis of Axin ubiquitination and regulation remains elusive.One of the representative regulatory mechanisms determining Axin activity is ubiquitin-mediated Axin regulation. Many E3 ligases and deubiquitinating enzymes have been reported. RNF146, a RING-domain E3 ligase, recognizes ADP-ribosylated Axin and mediates its protein degradation14. Specifically, the homo-polymeric state of Axin represents its functional activity as a scaffolder of the ß-catenin destruction complex14. The importance of the DIX-mediated Axin polymerization can be demonstrated by the complete abolishment of Axin polymerization and its suppressive activity simply by DIX deficiency or through point mutations within the ß -strands of the DIX domain14. However, how this DIX-mediated Axin polymerization is stably maintained, and what molecule governs the polymerization, are still enigmatic.The functional activity of Axin is also determined by its oligomeric state. The C-terminal DIX domain of Axin is essential for its hetero- and homo-polymerization15. UCHL5 plays multiple roles in diverse cellular processes by interacting with various protein complexes, such as the proteasome, the chromatin remodeling complex (INO80), and transcription factors (E2F1)18. With this molecular basis, UCHL5 has been known not only to rescue the substrate proteins from proteasome-dependent degradation, but also to regulate gene transcription18. In this study, we discovered that UCHL5 plays a negative regulator role in Wnt signaling in various cancer cells. UCHL5 also plays part in the ß-catenin destruction complex by interacting with Axin1. More importantly, UCHL5 stabilizes Axin1 protein by blocking ubiquitination of Axin1 DIX domain, thereby sustaining the DIX-dependent Axin1 polymerization.Here, we suggest Ubiquitin C-terminal hydrolase like 5 (UCHL5) as a key molecule maintaining the functional activity of the ß-catenin destruction complex. UCHL5 is a deubiquitinating enzyme (DUB) belonging to ubiquitin C-terminal hydrolase (UCH) subfamilyTo determine the effect of UCHL5 on the Wnt signaling pathway, we employed a TOPFlash reporter containing repeated Wnt response elements (WREs) Fig. a. We obsGiven that UCHL5 negatively regulates Wnt/ß-catenin signaling pathway by destabilizing ß-catenin, we hypothesized that UCHL5 is functionally involved in the ß-catenin destruction complex. GSK3ß is a key component of the ß-catenin destruction complex, as it mediates ß-catenin turnover by promoting the integrity of the ß-catenin destruction complex and directly phosphorylating ß-catenin. Thus, we speculated that if UCHL5 regulates the ß-catenin destruction complex, the depletion of UCHL5 would further promote the activity of the Wnt signaling pathway. In order to inactivate the ß-catenin destruction complex, we inhibited GSK3ß by treating either BIO or LiCl, both GSK3ß inhibitors. As expected, UCHL5 KD led to a synergistic increase in TOPFlash activity with BIO Fig. a. ConsisUCHL5 is a member of the Ubiquitin C-terminal hydrolase family, and it mediates deubiquitination of its substrate, thereby stabilizing target proteins. Therefore, we examined whether Axin1 is a substrate of UCHL5. On the one hand, the introduction of either Flag-UCHL5 or non-tagged UCHL5 into HeLa cells remarkably enhanced myc-Axin1 and endogenous Axin1 Fig. a,b. On tNext, we determined the involvement of UCHL5 deubiquitinating activity in the regulation of Axin1 stability. To do this, we employed an enzymatically inactive mutant of UCHL5 (Mut.UCHL5). After the introduction of either wild-type UCHL5 (WT.UCHL5) or Mut.UCHL5 into UCHL5 KD cells, the effects of WT. UCHL5 and Mut.UCHL5 on Axin1 stabilization were compared. Interestingly, the protein level of HA-Axin1 was remarkably increased by both WT.UCHL5 and Mut.UCHL5 in a dose-dependent manner Fig. g. ConsisIn order to understand the mechanism of Axin1 stabilization by UCHL5, we first determined the responsive element of Axin1 to be degraded when UCHL5 is depleted. For this analysis, we constructed various fragments of Axin1 protein Fig. . Depleti13. Along the same lines, two DIX proteins in the Wnt signaling cascade, Axin and Dvl, can also form protein complex through either the hetero- or homo-interaction, and regulates the Wnt signaling pathway differently according to its interacting partners19. For signaling activation, Dvl forms a hetero-complex with Axin through their DIX domains. This complex is a prerequisite for Axin recruitment to the plasma membrane13. In contrast, the homo-polymerization of Axin is essential for efficient assembly of the ß-catenin destruction complex and ß-catenin turnover. This Axin polymerization is also accomplished through DIX-mediated head to tail interaction19. Therefore, we verified the involvement of UCHL5 in this DIX-mediated Axin polymerization through an immunostaining analysis. As previously reported, Axin1 formed polymers in the transfected cells, called puncta, which are more visible in cells where UCHL5 is co-expressed 21. These facts strongly suggest the existence of a protective mechanism to maintain Axin’s physiological level by blocking protein degradation caused by either Wnt-dependent or independent degrative mechanism. Thus, deubiquitinating enzymes would be more important determinants of Axin concentration than E3 ligases. In this regard, two ubiquitin-specific proteases have been reported. First, USP34 can rescue Axin1 from PARylation-dependent degradation10. However, USP34 mediates the nuclear localization of Axin1, and it positively regulates Wnt signaling pathway. Second, a recent genetic screening using CRISPR/CAS9 method revealed USP7 as a negative regulator of Wnt signaling pathway as a deubiquitinase for Axin11. USP7 interacts with Axin1 and removes ubiquitin moieties conjugated by both SIAH1/2 and RNF146. Importantly, our study demonstrates that there is not only a protective mechanism by deubiquitinating enzymes, but also an alternative protection mechanism for Axin1, which has not been known to date. For instance, even though UCHL5 is also classified as a deubiquitinating enzyme, its deubiquitinating activity is dispensable for Axin1 stabilization. Consistently, Wild-type and catalytically inactive UCHL5 comparably suppressed the activity of Wnt signaling pathway, and had almost a similar efficacy on Axin1 stabilization. Interestingly, this nonenzymatic effect of UCHL5 on Axin1 stabilization is clearly achieved by protecting Axin1 from ubiquitination. Our ubiquitination assay results showed that Axin1 was ubiquitinated less under the UCHL5-overexpressed condition. In contrast, the ubiquitination of Axin1 was enhanced under the UCHL5-depleted condition. However, UCHL5 recombinant proteins did not remove polyubiquitination of Axin1. These results suggest that UCHL5 may physically block an Axin1-targeting E3 ligase unknown to this study. Notably, we suppose that this mechanism is independent of SIAH1/2 and Tankyrase/RNF146, since the depletion of UCHL5 still led to the destabilization of Axin1 mutants (VxP mutants), which have a resistance against SIAH1/2-mediated degradation , and ubiquitination at K54 blocks DIX-dependent polymerization by interfering with the DIX-DIX interface22. We think that UCHL5-associated ubiquitination in Axin1 DIX domain may also work in the same way to disrupt Axin1 polymerization. However, the molecular mechanism of DIX ubiquitination blockage by UCHL5 remains ambiguous. Intriguingly, our results indicate that UCHL5 blocks DIX ubiquitination without interacting with the DIX domain. Therefore, this is likely to require Axin1’s dynamic conformational changes in consequence of its intra-molecular interactions. These intra-molecular interactions are regulated by either GSK3ß-mediated phosphorylation or Wnt signal activation23. Coincidentally, UCHL5 interacts with Axin1 through multiple domains  where intra-molecular interactions arise. In all likelihood, these multiple interactions allow UCHL5 to protect the DIX domain from ubiquitination at all times despite the dynamic conformational change of Axin1. Furthermore, the depletion of UCHL5 also led to the additional ubiquitination in N-terminal end of Axin1, which may suggest another functional layer of UCHL5 even though it is not related to the stability of Axin1 and not addressed in this study. Therefore, future studies based on protein structure and post-translational modifications must be conducted to understand the exact molecular mechanism.As for the functional activity of Axin, its DIX-dependent polymerization is necessaryAnother interesting part catching our attention was that the altered expression of UCHL5 (OE or KD) only showed a significant change in Wnt signal activity under the Wnt-active condition. We presume the physiological role of UCHL5 is to protect ß-catenin destruction complex from its inhibition by Wnt-activation, thereby keeping a certain threshold level of signal responsiveness against Wnt stimulation. That way, the biological environment (cell or tissue) can stably secure Wnt-off state. Hence, UCHL5 KD or its impaired function gets the environment sensitized to Wnt stimulation.Xenopus embryos and HepG2 liver cancer cell24. In another recent study using endometrial cancer cells, UCHL5 reportedly promotes cancer growth by positively regulating the Wnt signaling pathway25. Interestingly, our current study using HeLa, MCF7, SW480, and A549 showed a negative effect of UCHL5 on Wnt signaling pathway , pCS4-HA (XhoI/XbaI), and pEBG (Cla/Not1). pCS2 + ubiquitin-HA and pCS-ubiquitin-Flag were kindly provided by Dr. Eek-Hoon Jho. GSK3 and ß-catenin were PCR-amplified from cDNA of HEK293T and inserted into pCS4-3HA and pCS2 + GFP, respectively.UCHL5 was amplified from cDNA of HEK293T by PCR and inserted into pCS2 + (XhoI/XbaI), pCS4-6Flag (BamH1/ClaI) or pCS4-3HA (BamH1/ClaI) vector. Catalytically inactive UCHL5 (Mut.UCHL5) was previously described and generated by PCR-directed mutagenesis2.HeLa, MCF7, SW480, and A549 cells (ATCC) were maintained in DMEM High glucose (Hyclone) supplemented with 10% FBS (Hyclone) and 1% penicillin–streptomycin (Sigma P0781) at 37 °C with 5% COStable KD HeLa cells expressing short hairpin RNA (shRNA) for UCHL5 depletion were generated by infection of lentiviral package. Two independent shRNA sequences for UCHL5 were: 5′-TCCCGACTTGACACGATATTT-3′ and.All plasmids were transfected using Lipofectamin 2000 (Invitrogen), according to the manufacturer’s protocol.P < 0.05, ** P < 0.005.Cells were seeded in a 24 well plate. 250 ng of TOPFlash and 50 ng of Renilla were transfected the following day. 24 h after transfection, cells were lysed and subjected to luciferase reporter assay using Dual-Luciferase® Reporter Assay System (Promega), according to the manufacturer’s protocol. Duplicates were repeated three times independently, and average figures were displayed with standard deviation (SD). P-value was calculated using two-tailed test, * https://www.thermofisher.com /trizol.html; TRIzol User Guide). For cDNA synthesis, 1ug of total RNA was reacted with M-MLV reverse transcriptase (Promega) and oligo(dT) 15 primer according to the manufacturer's protocol (https://www.promega.com › Resources › Protocols).The extraction of total RNA was performed using TRIZOL reagent (Invitrogen) according to the manufacturer's protocol . PCR conditions were 95 °C (10 min) and 40 cycles of 95 °C (30 s) and 60 °C (1 min). Data are displayed as mean with SD from triplicates. P value was calculated using two-tailed test, *P < 0.05, **P < 0.005.We performed quantitative real-time PCR (qPCR) analysis as described beforePrimers used were:For western blot analysis, cells were harvested in cold PBS and lysed in RIPA buffer . Samples were cleared by centrifugation and then separated on SDS-PAGE gels, followed by transfer to PVDF or nitrocellulose membrane at 100 V for 2 h at 4 °C. Western blot images were acquired using LAS-4000 system (FujiFilm).For immunoprecipitation analysis, cells were lysed in modified RIPA buffer . 1 mg of lysates were incubated with antibodies and Protein A-Sepharose® 4B. After that, immunoprecipitates were subjected to western blot analysis.For in vitro binding assay, 3 μg of GST or GST-Axin1  was incubated with UCHL5 (R&D system) in modified RIPA buffer at 4 °C for 6 h. Glutathione-Sepharose beads (Peptron) were added to pulldown the protein complexes. After that, beads were washed four times using IP buffer and then, samples were analyzed by western blot analysis.For ubiquitination assay, cells were incubated with 20 μM MG132 (Sigma) for 5 h. After that, cells were rinsed with cold PBS twice and boiled in SDS lysis buffer  at 100 °C for 5 min. the boiled samples were diluted 10 times with modified RIPA buffer, and then the samples were chilled at 4 °C for 1 h. After that, samples were subjected to immunoprecipitation and western blot analysis sequentially.24. For in vitro ubiquitination analysis, both myc-Axin1 and Flag-Ub were transfected to HeLa cells. After 48 h, cells were lysed in IP buffer. Cell lysates were centrifuged and then, supernatants were sequentially incubated with indicated antibody and Protein G sepharose (Invitrogen) at 4 °C for 3 h. Beads were washed 3 times with in vitro DUB buffer (50 mM Tris–Cl (pH7.5), 150 mM NaCl, 1 mM EDTA and 1 mM DTT). Washed beads were incubated with indicated amount of UCHL5 recombinant proteins (R&D system) at 37 °C for 2 h in 50 μl of in vitro DUB buffer. The reaction was terminated by adding SDS sample buffer and subsequent boiling at 95 °C for 10 min. Samples were analyzed by western blot analysis.We performed in vitro ubiquitination assay as described beforeCells were plated on coverslips and cultured in 12 well plates. 48 h after transfection, cells were washed with PBS and fixed in 4% PFA for 5 min at room temperature. For immunostaining, cells were permeabilized with 0.2% Triton X-100 in PBS for 5 min, followed by blocking with 10% goat-serum for 1 h. After that, cells were incubated with primary antibodies (1:200) overnight, and secondary antibodies  for 2-5 h. The stained cells were mounted in UltraCruz® Aqueous Mounting Medium with DAPI (Santa Cruz). Images were acquired using FV3000 confocal laser scanning microscope (Olympus). The images were quantified using the default ‘Analyze particles’ plugin in ImageJ.t-test. Asterisks (*) indicate statistical significance .A minimum of three independent replicates were performed for each experiment. The error bars shown represent the standard deviation (SD) of at least three independent experiments, Statistical relevance was determined using a two-tailed Student’s Supplementary Figures.