Wnt/β-catenin pathway and osteogenesis were detected by real-time PCR, western blotting, immunofluorescence, HE staining and micro-CT. In both our in vivo and in vitro experiments, we detected higher gene and protein expression levels of the Wnt/β-catenin pathway-related factors β-catenin and cyclinD1 in adiponectin transgenic BMSCs and rats. Similar results were noted regarding the gene and protein expression levels of osteogenesis-related genes. In addition, more new bone formation was observed in the adiponectin-treated groups. Our results indicate that adiponectin could facilitate BMSC osteogenic differentiation and osteogenesis, and the Wnt/β-catenin pathway was involved in the osteogenic effect of adiponectin.Recent studies have demonstrated the stimulatory effects of adiponectin on bone formation, but the mechanism underlying these effects remains unclear. The Wnt/β-catenin pathway, one of the most important pathways in osteogenesis, has rarely been associated with the osteogenic effects of adiponectin in previous studies. The present study was designed to investigate the effects of adiponectin on bone mesenchymal stem cell (BMSC) osteogenic differentiation and bone formation through the Wnt/β-catenin pathway. We detected adiponectin receptor expression in BMSCs, constructed a recombinant adenovirus containing the human adiponectin gene, and then used the adenovirus to transfect BMSCs  It secretes many cytokines that take part in a series of pathophysiological process2. Leptin is the first fat cytokine to be found to join the metabolism of bone. It can inhibit bone formation through the central regulation of the hypothalamus, or increase the osteoprotegerin and inhibit the differentiation of osteoclast through peripheral pathway4. Visfatin, secreted by visceral fat cells, can also improve osteoblast differentiation and mineralization, and inhibit the production of osteoclasts5. Growing evidences suggest that adipose tissue is closely related to the regulation of bone metabolism.Adipose tissue, which has been regarded as a simple lipid storage organ in the past, has since become known as a vital endocrine system8. Two ApN receptors were initially identified by Yamauchi. AdipoR1 is predominantly expressed in muscle, and AdipoR2 is predominantly expressed in the liver9. These differences in the distributions of the two adiponectin receptors increase the precision and effectiveness of ApN. Berner et al. found that ApN and AdipoR1 are expressed in human osteoblasts and mouse MC3T3-E1 cells10. Shinoda and Lee subsequently detected the expression of ApN and its receptors in cells of osteoblastic and osteoclastic lineages12. Growing evidence suggests that a close relationship exists between ApN and bone metabolism, implying that ApN may play a direct role in bone metabolism by binding to its receptors13.Adiponectin (ApN), one of the adipocytokines, plays a critical role in glucose and lipid metabolism, anti-inflammation and insulin resistance attenuation14. Fewer osteoclasts and higher BMD were observed in ApN-deficient mice than in wild-type mice15. In contrast, Jiang et al. discovered that intermittent ApN administration can promote bone formation and mineralization in rabbits with mandibular osteodistraction16, and Wu et al. showed that ApN can suppress sympathetic tone and increase bone mass in mice17. Luo et al. found that sustained ApN release can promote peri-implant osteogenesis in ovariectomization rabbits by suppressing osteoclasts18. ApN can increase bone mass by suppressing osteoclastogenesis and bone resorption20, stimulating local angiogenesis22, or promoting osteogenic differentiation27. Several osteogenesis signalling pathways have been found to be activated in the presence of ApN; however, few studies have discussed the relevance of ApN and the Wnt/β-catenin pathway with respect to osteogenesis.The role of ApN in the promotion or inhibition of bone metabolism remains controversial. Lower bone mineral density (BMD) has been noted in elderly men with high circulating ApN levels29. Wnt protein acts on its cell-surface receptor to prevent β-catenin degradation30. Then, the up-regulated β-catenin translocates into the nucleus and stimulates the expression of various downstream genes31. The Wnt/β-catenin pathway has been demonstrated to promote BMSC osteogenic differentiation34. It is noteworthy that reduced bone formation and decreased β-catenin expression levels were observed in AdipoR1-knockdown BMSCs compared with wild-type BMSCs25. Accordingly, we deduced that ApN can act on BMSCs through the Wnt/β-catenin pathway.The Wnt/β-catenin pathway, also known as the canonical Wnt pathway, was found to be strongly linked with bone formationGiven the above evidence, we aimed to study the osteogenic effects of ApN through the Wnt/β-catenin pathway in ApN gene-modified BMSCs and rat tibia bone defect model. The following hypotheses were tested: (1) ApN receptors are expressed in BMSCs, (2) ApN can activate the Wnt/β-catenin pathway and promote gene-modified BMSC osteogenic differentiation and bone formation, and (3) ApN can activate the Wnt/β-catenin pathway and promote osteogenesis in transgenic mice.The third-passage BMSCs were a homogenous population and exhibited a spindle-shaped fibroblastic morphology. The flow cytometry results showed positive CD29+ (96.0%) and CD90+ (95.6%) expression and negative CD34− (0.44%) and CD45− 0.19%) expression  and no hApN expression in group B (Ad-EGFP) and group C (blank control) . All of these findings indicated that the recombinant adenovirus was successfully transfected into the BMSCs and that the target genes were expressed at their normal levels in vitro and in vivo.Thirty-six hours after transfection, several EGFP + cells were counterstained with DAPI and observed and counted under fluorescence microscopy Fig. . The traol) Fig. . All ofThe immunohistochemistry results are shown in Fig. To study the effects of hApN on Wnt/β-catenin pathway, we detected the mRNA and protein expression levels of the Wnt/β-catenin pathway-related factors β-catenin and cyclinD1 using real-time PCR, western blotting and immunofluorescence staining. After 12 h of osteogenic induction, the gene expression levels of both β-catenin and cyclinD1 were significantly elevated in group A (Ad-hApN-EGFP) compared with group B (Ad-EGFP) and group C (blank control) Fig. . The elein vitro.After 3 d and 7 d of osteogenic induction, we detected osteogenic gene expression levels by real-time PCR. The levels of these three genes, namely, osteocalcin (OCN), bone morphogenetic protein-2 (BMP-2) and Runt-related transcription factor-2 (RUNX2), were markedly increased in group A compared with the other two groups Fig. . After oIn the animal experiment, we used real-time PCR and immunofluorescence staining to detect Wnt/β-catenin pathway-related gene expression on the 3rd and 7th postoperative days. β-catenin and cyclinD1 mRNA expression levels were evidently increased in group A compared with the other two groups at the above time points. Moreover, β-catenin expression levels were elevated in all three groups on the 7th postoperative day. The immunofluorescence staining results for β-catenin are shown in Fig. P < 0.05). Three weeks after surgery, micro-CT was used to scan the new bone within the defects, and three-dimensional reconstruction was performed. Incomplete bone healing was observed, and blank areas without newly formed bone were noted in all three groups; however, the arrangement of trabecular bone was more dense, and fewer blank areas were noted in group A than in groups B and C  ApN exerts equivalent and balanced endocrine and autocrine/paracrine effects on bone, (2) ApN stimulates effective osteogenesis through its autocrine and paracrine effects, and (3) circulating ApN exerts direct/negative and indirect/positive effects on bone formation35, and a negative correlation between serum adiponectin level and bone mineralization has been reported in some studies36. Jurimae et al. noted lower bone mineral density and content in women with high circulating ApN level37. Luo et al. discovered that circulating ApN indirectly promoted osteoclast formation by suppressing osteoprotegerin production and stimulating RANKL38. These findings are not completely in conflict with our conclusions—which were based on Shinoda’s research—that circulating ApN and local ApN exert opposing effects on osteogenesis. However, the mechanism underlying this phenomenon is not clear yet.ApN is abundant in plasma10, indicating that ApN not only influences bone metabolism through its endocrine effects but also acts directly on bone-forming cells through its autocrine/paracrine effects26. Chen et al. found ApN can activate the APPL1-AMPK pathway to promote osteogenic differentiation in human adipose-derived stem cells23. Lee et al. discovered that ApN promoted osteogenic differentiation in a cyclooxygenase-2-dependent manner in BMSCs and C3H10T1/2 cells24. Luo et al. showed that ApN enhanced human osteoblast differentiation and proliferation through the MAPK signalling pathway26. Pu et al. found that ApN can promote human jaw bone marrow mesenchymal stem cell osteogenesis through the APPL1-mediated p38 MAPK pathway and increase ossification-related gene expression levels27. However, very few studies have investigated the association between ApN and the Wnt/β-catenin pathway, one of the most important pathways in osteogenesis, with respect to the regulation of bone formation and BMSC osteogenic differentiation.ApN and AdipoR1 were discovered to be ubiquitously expressed in bone-forming cells39. Once a bone fracture occurs or a bone defect develops, special factors, such as SDF-1, are released by injured tissues to mobilize BMSCs throughout the entire body. Then, BMSCs recruit and migrate to the injury sites40 while proliferating and differentiating into osteoblasts43. Yu et al. established a calvarial bone defect model in mice and found that an increased number of BMSCs egressed into the peripheral blood in ApN-infused mice compared with wild-type mice and that more new bone formation was observed in the former group of mice than in the latter group of mice44. They showed that ApN can facilitate BMSC recruitment and migration, but they did not explore whether it can promote BMSC osteogenic differentiation. Lin et al. eliminated the BMSC AdipoR1 gene, which resulted in decreased β-catenin expression, decreased osteogenesis-related gene expression, and reduced mineralization. In contrast, higher bone mineral density was observed in AdipoR1 transgenic mice25. Based on the above evidence and the findings of the present study, we believe that ApN can facilitate new bone formation and BMSC osteogenic differentiation via the Wnt/β-catenin pathway.BMSCs are multipotent cells that facilitate regeneration of bone and other mesenchymal tissues46. Therefore, the Wnt/β-catenin pathway not only participates in bone metabolism but also takes part in some diseases. Gene abnormalities affecting the Wnt/β-catenin pathway can induce colon cancer47, hair follicle tumours48, leukaemia49, mammary cancer50 and some other diseases. Interestingly, ApN was found to attenuate mammary tumourigenesis by stimulating Wnt inhibitory factor-1 and suppressing the Wnt/β-catenin pathway in human breast cancer cells52. The Wnt/β-catenin pathway has also been reported to be involved in tissue and organ fibrotic processes, such as myocardial fibrosis53, pulmonary fibrosis55 and hepatic fibrosis56, while ApN has been shown to have anti-fibrosis activity57. Reinke et al. showed that ApN can inhibit the phosphorylation of Lrp6, a co-receptor for Wnt protein58, thus suppressing the Wnt/β-catenin pathway in human dermal fibroblast59. Therefore, we speculate that a connection exists between the protective effects of ApN against tissue fibrosis and the negative actions of ApN on the Wnt/β-catenin pathway. However, in another study, the expression levels of β-catenin and the downstream genes of the canonical Wnt pathway were neither up-regulated nor down-regulated in the adipose tissue of ApN-transgenic mice compared with wild-type mice60. The above studies showed that ApN had three different effects on the canonical Wnt pathway, an inhibitory effect in human breast cancer cells and dermal fibroblasts, no influence in adipose tissue and promontory effect in BMSCs.More than 50 types of proteins are involved in the Wnt/β-catenin pathway. These proteins mediate the biological responses of various cells and play an important role in human embryonic development and in regulating the internal environment of the human bodyet al. found that ApN can inhibit β-catenin in AdipoR1/R2 gene knock-out fibroblasts59, indicating that ApN can regulate the canonical Wnt pathway through some other unknown receptors in addition to AdipoR1/R2. However, the internal environment of the body is complex and adaptable, and the factors affecting the canonical Wnt/β-catenin pathway and bone formation are more intricate. Therefore, there is still a need to explore the effects of ApN on the Wnt/β-catenin pathway further.It is noteworthy that Reinke 61. A variety of studies using gene therapy for bone defects have shown satisfactory results; however, the experiments were limited to animals. More safety and technical issues related to this therapy remain to be resolved, and an effective, secure and economical approach of administering it is still being sought. Nevertheless, gene therapy still has great clinical potential for the treatment of bone diseases.Bone defect is a challenging problem for clinicians. Identifying and developing efficient and safe methods for treating bone defects remain areas of focus for researchers. Gene therapy is a new technology used to treat diseases at the DNA level that has been shown to have long-term efficacyIn summary, the findings of the present study indicate that ApN could act on BMSCs and confirm the hypothesis that ApN could promote BMSC osteogenic differentiation and bone formation through the Wnt/β-catenin pathway in both ApN gene-modified BMSCs and ApN gene-transgenic mice. Our findings indicate that ApN may represent a strategy for treating bone defects and metabolic bone diseases. And in subsequent research, gene knock-out experiment is needed to further confirm the importance of Wnt/β-catenin pathway in the osteogenic effect of ApN.62. Ad-hApN-EGFP plaque forming units (PFUs) were 1 × 1010 per millilitre.Recombinant plasmids containing the human adiponectin (hApN) fragment were obtained from Tufts University School of Dental Medicine. Recombinant adenoviruses containing the hApN and enhanced green fluorescent protein (EGFP) genes were constructed and identified as described in previously published studies41. Then, the cells were cultured in α-MEM supplemented with 10% foetal bovine serum  at 37 °C. The medium was replaced every three days. After the cells reached 90% confluence, they were digested with trypsin enzymes for passaging. The third BMSC passage was incubated with a fluorescently labelled antibody  for 30 min and then washed with PBS. The expression levels of different cell surface markers, including CD29, CD90, CD34, and CD45, were detected by flow cytometry. Cells from the third passage were used in subsequent experiments.Sprague-Dawley (SD) rat primary BMSCs were isolated using the methods described by FriedensteinThird-generation BMSCs were seeded into a 24-well plate. Cell adherence was observed after 12 h. Following several washes with PBS, the cells were fixed in 4% paraformaldehyde at room temperature, washed with PBS again, and permeabilized with 0.5% Triton X-100 for 20 min. A Plink-2 plus Polymer HRP Detection System  and DAB Horseradish Peroxidase Color Development Kit (Beyotime) were used to detect AdipoR1 and AdipoR2 expression in BMSCs, according to the manufacturer’s instructions. The nuclei were then stained with haematoxylin again. The concentrations of the primary antibodies for AdipoR1 and AdipoR2 were 1:200 and 1:400, respectively.5 cells per millilitre. Half of the medium was changed after 24 hours. The cells were separated into the following three groups: group A (Ad-hApN-EGFP), which was incubated with 5 µl of recombinant adenovirus solution (1 × 1010 PFU/ml); group B (Ad-EGFP), which was incubated with 5 µl of adenovirus solution containing no adiponectin; and group C (blank control), which was incubated with an equal volume of serum-free medium. The multiplicity of infection (MOI) was 50. After 36 h of transfection, we used fluorescence microscopy to scan the cells, which were stained with DAPI, and calculated the transfection efficiency of the recombinant adenovirus. Then, the medium was replaced with an osteogenic induction solution and renewed every three days. Samples were collected and analysed at 12 h, 24 h, 3 d, 7 d and 20 d after osteogenic induction.The third BMSC passage was cultured in a 6-well plate at a density of 1 × 1010/ml) that was diluted with 0.25 ml of normal saline and injected into the bone defect of each tibia during surgery and on the 2nd post-operative day; group B (Ad-EGFP), which was treated with 0.25 ml of virus solution containing no adiponectin. The solution was injected into the bone defect at the above time points; group C (blank control), which was treated with the same dose normal saline. The dose was injected by the same method and at same time point as those indicated above. On days 3, 7, and 21 after surgery, the rats were killed with a narcotic overdose. The tibias were quickly removed under aseptic conditions and then preserved in liquid nitrogen after soft tissue removal and several washes with normal saline.All procedures were ratified by the Animal Care Committee of Sichuan University. All the experiments were carried out in accordance with the relevant guidelines and regulations.A total of 72 Sprague-Dawley rats were anaesthetized. Then, a longitudinal incision was made through the skin, subcutaneous tissue and periosteum along the medial aspect of the knee joint to expose the tibia. Along the medial aspect of each tibia, we used a dental engine  to create a critical 2-mm-in-diameter bone defect located one millimetre away from the growth plate. The bone marrow cavity was exposed, but the contralateral cortical bone structures remained intact. The defect was closed in layers after the procedures. All rats were intramuscularly injected with 400 thousand units of penicillin postoperatively and twice a day for three consecutive days. The rats were randomly allocated into following three equal groups: group A (Ad-hApN-EGFP), which received 5 µl of adenovirus solution  after 12 h, 24 h, 3 d and 7 d of osteogenic induction. Then, cDNA was synthesized using a PrimeScript RT Reagent Kit with a gDNA Eraser , according to the manufacturer’s instructions. Gene expression levels were quantified by an ABI PRISM 7300  with SYBR® Premix Ex Taq™ II (Takara). The following PCR cycling protocol was used: 95 °C for 20 s, followed by 40 cycles of 95 °C 10 s and 60 °C 30 s. The expression levels of the Wnt/β-catenin pathway-related genes β-catenin and cyclinD1 were detected after 12 h and 24 h of osteogenic induction. On the 3rd and 7th day after osteogenic induction, we detected the expression levels of RUNX-2, BMP-2 and OCN. Table After 12 hours and 24 hours of osteogenic induction, the adherent cells were washed with PBS, isolated by scraping, and centrifuged at 10000 rpm for 8 min, Then, BMSC cytoplasmic and nuclear proteins were isolated using a Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime). Protein concentrations were detected using a BCA Protein Assay Kit (Sigma-Aldrich). The proteins were subjected to SDS-PAGE on an 8% polyacrylamide gel and then transferred onto a PVDF membrane (Sigma-Aldrich) and incubated with 5% nonfat milk for 1 h before being washed thrice with TBST (10 mM Tris-HCL (pH 7.6)). The membranes were then incubated at 4 °C overnight with a rabbit anti-rat primary antibody for β-catenin before being incubated with an HRP-labelled secondary antibody. After 1 h, the protein bands were visualised via chemiluminescence using an ECL kit . Protein quantification was performed using ImageJ.The BMSCs were seeded in a 24-well plate for 12 h of osteogenic induction, fixed in 4% paraformaldehyde for 10 min, permeabilized with 0.25% Triton X-100 in 10% normal goat serum for 30 min, and then incubated with the indicated primary antibody  overnight at 4 °C. The following day, after being washed thrice with PBS, the cells were cultured with the appropriate secondary antibody (1:100), which was conjugated with TRIT for 30 min at room temperature. After being stained with DAPI, the cells were observed via fluorescence microscopy .After osteogenic induction for 14 d, cells were fixed with 4% paraformaldehyde for 2 min, then washed with PBS, stained by ALP staining kit mixture  according to the instructions,then visualized under microscopy. To quantify ALP activity, cells were disrupted by RIPA Lysis Buffer (Beyotime). After centrifugation, the supernatant was used to detect ALP activity by Alkalin Phosphatase Colorimetric Assay Kit (Leagene Biotech).The optical density was measured at 405 nm.2O, we visualized calcium deposits by inverted phase-contrast microscopy . The Alizarin Red-stained cells were cultured with 10% cetylpyridinium chloride solution for 25 min to quantify matrix mineralization. The absorbance of the supernatant was then measured at 562 nm.The BMSCs were cultured in 24-well plates to undergo osteogenic induction for 20 d before being fixed in 95% ethanol for 30 min and stained with 1% (w/v) AR-S (pH 4.2) for 5 min. After the cells had been washed 3 times with ddHOn the 3rd and 7th postoperative day, tibia samples were harvested, cracked and ground into powder. Total RNA was extracted using Trizol reagent, and then cDNA was synthesized using a PrimeScript RT Reagent Kit with gDNA Eraser, according to the manufacturer’s instructions. OCN, BMP-2 and RUNX-2 gene expression levels were quantified by an ABI PRISM 7300 with SYBR® Premix Ex Taq™ II. The primers used for the experiment are listed in Table On the 3rd and 7th postoperative day, tibia samples were fixed in 4% paraformaldehyde for 48 h. After several washes with PBS, the samples were decalcified using 10% EDTA decalcifying fluid (Sigma-Aldrich) for 60 d, dehydrated in graded alcohol, embedded in paraffin and cut into 5-μm-thick sections. After antigen retrieval and blocking with 10% normal goat serum for 20 min, the samples were cultured with rabbit anti-rat β-catenin (1:150) overnight at 4 °C. Then, the samples were incubated with the appropriate secondary antibody for 30 min~60 min at 37 °C before finally being evaluated with a fluorescence microscope.On the 7th postoperative day, we obtained 5-μm-thick paraffin sections and stained them with haematoxylin and eosin. We used light microscopy (100x) to scan the samples and collect images. The ratio of the new bone area to the entire sight area was calculated.Three weeks after surgery, tibia samples were harvested, fixed in 4% paraformaldehyde, and scanned by micro-CT  . Three-dimensional reconstruction was performed. BMD, BV/TV, Tb.N and Tb.Th were quantitatively analysed.P values less than 0.05 were considered statistically significant.All the data were expressed as the mean ± standard deviation and analysed using SPSS 19.0. Comparisons between groups were evaluated by one-way ANOVA.


O-triphenylmethyl andrographolide (RS-PP-050), an andrographolide analogue and determine its activity in the Wnt/β-catenin pathway. RS-PP-050 was found to potently inhibit the proliferation and survival of HT-29 CRC cells. It induces cell cycle arrest and promotes apoptotic cell death which was associated with the activation of PARP-1 and p53. Furthermore, RS-PP-050 exerts inhibitory effects on β-catenin transcription by suppressing T-cell factor/lymphocyte enhancer factor (TCF/LEF) activity in cells overexpressing β-catenin and by down-regulating the endogenous expression of Wnt target genes. RS-PP-050 also decreased the protein expression of the active form of β-catenin but functions independently of GSK-3β, a negative regulator of Wnt. Interestingly, RS-PP-050 extensively blocks phosphorylation at Ser675 of β-catenin which links to interference of the nuclear translocation of β-catenin and might contribute to Wnt inactivation. Collectively, our findings reveal the underlying anti-cancer mechanism of an andrographolide analogue and provide useful insight for exploiting a newly chemotherapeutic agent in Wnt/β-catenin-overexpressing CRC cells.The Wnt/β-catenin signaling pathway plays a key role in the progression of human colorectal cancers (CRCs) and is one of the leading targets of chemotherapy agents developed for CRC. The present study aimed to investigate the anti-cancer effects and molecular mechanisms of 19- Although the early response success rate of CRC to current chemotherapeutic drugs is high, the invariable development of drug resistance as well as patient intolerance of adverse drug side effects limits their usage2. Thus, searching for new effective chemical entities with greater specificity is urgently needed. Advancement in cancer treatment strategies depend upon gaining a better understanding of the intracellular signaling cascades and molecular pathways involved in tumor survival and progression.Colorectal cancer (CRC), is a malignancy that has one of the highest incidence rates in both males and females in the United States3. In the absence of Wnt ligands, the key effector of this signaling, β-catenin, is sequestered in a destruction complex composed of adenomatous polyposis coli (APC), casein kinase 1α (CK1α), Axin, and glycogen synthase kinase-3β (GSK-3β) which maintains the activity of cytosolic β-catenin at a low level4. Ultimately, phosphorylation of the N-terminal β-catenin by GSK-3β at Ser33/Ser37/Thr41 residues marks β-catenin for degradation in the proteasome. In cancer cells, the binding of Wnt ligands to the Frizzled (Fz) receptor and the low density lipoprotein receptor-related protein 5/6 (LRP5/6) co-receptor on the plasma membrane triggers the disassembly of the destruction complex. This results in the accumulation of free β-catenin able to translocate into the nucleus to form a complex with TCF/LEF family transcription factors to activate the transcription of Wnt target genes such as c-myc, cyclin D1, matrix metalloproteinase-7 (MMP-7), and survivin. These target genes are responsible for colon tumor proliferation, survival and metastasis8.The deregulation of Wnt/β-catenin signaling is considered to be a major oncogenic event in the initiation and progression of most colon cancers10. Under pathological conditions, however, loss or gain of function by genetic alterations of Wnt components including APC, Axin, and β-catenin, cause β-catenin to escape degradation imposed by the upstream negative regulators, allowing deregulated β-catenin transcriptional activity in the nucleus11. Because of these genetic mutations in the Wnt pathway components, inhibition upstream of β-catenin may produce the discernable effect to inactivate Wnt signaling. However, targeting events downstream of β-catenin towards the nuclear vicinity also offers a much more fascinating option for efficiently suppressing Wnt/β-catenin transactivation12. Recently, the association of protein kinase A (PKA) signaling has been implicated in positively influencing nuclear β-catenin levels and the transcriptional activity of Wnt signaling. Specific phosphorylation on Ser552 or Ser675 on the C-terminus of β-catenin by PKA leads to an increase in nuclear translocation of β-catenin and subsequently enhanced TCF/ LEF transcriptional activity in many cell types14. Aberrant accumulation of β-catenin in nucleus caused by Wnt gene mutations in CRC is thus a potential target to search for specific chemotherapeutic agents16.The level of β-catenin is negatively controlled by canonical GSK-3β-mediated phosphorylation or other degradation pathways17. A number of anti-cancer drugs originating from natural products are currently used in the clinic for treatment of CRC patients. Of these, andrographolide, a plant-based compound, that is a major constituent of Andrographis paniculata has shown great promise as an anti-cancer compound against a number of cancer cell types19. A recent study demonstrated that andrographolide promoted cell death through endoplasmic reticulum (ER) stress-mediated apoptosis through the induction of intracellular ROS generation and caused the activation of proapoptosis signaling, cell cycle arrest and inhibition of cellular pathways related to cancer cell survival20. However, it has not been used as a chemotherapeutic agent in clinic due to its low potency and poor oral bioavailability22. To overcome these limitations, chemical transformation to obtain analogues superior to the parent andrographolide has been an area of active research25. In our earlier study, a number of andrographolide analogues were developed, which showed interesting cytotoxicity profiles against a panel of cancer cell lines, acting through yet to be determined mechanisms of action27. The present study aimed to investigate the potent anti-cancer activity of 19-O-triphenylmethyl andrographolide (RS-PP-050), an andrographolide analogue, against CRC cells. The effects of the compound on the Wnt signaling pathway was explored. Here, we report that the anti-cancer activity of RS-PP-050 involves the inhibition of Wnt/β-catenin signaling via a GSK-3β-independent pathway. This is the first study which demonstrates the anti-cancer activity of the andrographolide analogue (RS-PP-050) which may hold great therapeutic potential for targeting Wnt/β-catenin-overactivated cancer cells.Natural products have been a major source of therapeutic agents for the pharmaceutical industry50 value of 11.43 ± 1.52 μM after 24 h of treatment, whereas those of SW480, and HCT116 cells were 15.88 ± 3.32 μM and 18.72 ± 1.83 μM, respectively  was higher than 100 µM. This indicates a lower cytotoxicity of RS-PP-050 to normal cells. IC50 values of RS-PP-050, andrographolide and the positive controls at various time points in HT-29 cells are shown in Table 50 = 48.64 ± 3.25 μM at 24 h). Based on the results, HT-29 cells were used for further mechanistic investigations with concentrations of RS-PP-050 ranging from 1–10 µM. RS-PP-050 was found to suppress the cellular proliferation of HT-29 in dose-dependent manner using a BrdU incorporation assay  to indicate the externalization of phosphatidylserine on the surface of the cell membrane. RS-PP-050 increased the percentage of apoptotic populations at both the early and late phases after 24 h of treatment in a dose-dependent manner Fig. . VinblasTo further investigate the effect on cell cycle distribution, the relative proportions of DNA content at different stages  were determined in HT-29 cells. After 12 h of exposure to 10 μM RS-PP-050, there was a significant reduction in the cell populations in the G1 phase, and a slight increase in the populations in the G2/M phase Fig. . In contA variety of proteins are involved in the processes of apoptotic cell death and cell cycle distribution. In the present study, the expression of PARP-1was determined by western blotting. PARP-1 is an ADP-ribosylating nuclear enzyme responsible for maintaining genome integrity. It is activated upon the induction of DNA strand breaks as well as DNA damage. The expression of cleaved PARP-1 was substantially increased by RS-PP-050 at 5 and 10 μM concentrations Fig. . This inAs induction of reactive oxygen species (ROS) production is one of the important characteristics of effective anti-cancer agents, we then determined whether the cytotoxic effects of RS-PP-050 involves the generation of ROS by using 2′,7′-Dichlorofluorescein diacetate (DCFDA), a dye that can be hydrolyzed by intracellular esterases and oxidized by ROS. ROS induction by RS-PP-050 in HT-29 cells was found to be both concentration- and time-dependent. Upon treatment with increasing RS-PP-050 concentrations, ROS production was increased and then decreased to baseline Fig. . We founAberrant activation of Wnt/β-catenin signaling is a critical step in the initiation and development of CRC. β-catenin is the pivotal mediator of this pathway. To study the effect of RS-PP-050 on the transduction of β-catenin, a luciferase reporter assay was employed. HEK293T cells were transiently transfected with a plasmid for β-catenin expression, and a TOPflash reporter plasmid, a construct containing wild-type TCF/LEF binding sites upstream of either a luciferase gene, or the gene for FOPflash, a negative control reporter to indicate the specificity on inhibition of Wnt signaling. In the control, β-catenin induced transcription of the transfected TOPflash reporter in HEK293T cells approximately 5-fold compared with cells transfected pcDNA3.1. After treatment, RS-PP-050 at concentrations of 2.5, and 5 µM significantly inhibited the TCF/LEF activity in the β-catenin-overexpressed cells Fig. . In contWe further assessed the suppressive effect of RS-PP-050 on Wnt/β-catenin signaling by determining the expression of endogenous Wnt target genes. By real-time PCR, it was found that RS-PP-050 decreased the mRNA levels of c-myc and cyclin D1 in HT-29 cells in a dose-dependent manner after 24 h of treatment Fig. . In addiTo further investigate the inhibitory mechanisms of RS-PP-050 on the regulation of β-catenin transcriptional activity, the changes in expression of Wnt proteins including total and active β-catenin were determined by immunoblotting. Following treatment with RS-PP-050 for 24 h, the expression of total β-catenin protein was not affected, but only the levels of the active form were decreased as compared to the control in HT-29 cells Fig. . These rAs only the accumulation of a non-phosphorylated β-catenin in the nucleus results in the transactivation of Wnt target genes, we examined whether the inhibitory effect of RS-PP-050 was related to changes in the subcellular localization of β-catenin. The intracellular distribution of β-catenin between the cytoplasm and nucleus was verified by western blotting. RS-PP-050 substantially decreased the levels of active β-catenin present in the nuclear fraction, compared to control, corresponding to the results found in the total lysates Fig. . While tBecause the stability of intracellular β-catenin is tightly regulated by GSK3β, a negative regulator of Wnt signaling, we next investigated the impact of RS-PP-050 on the function of GSK-3β. By using the luciferase reporter assay, HEK293T cells were transiently co-transfected with the same TOPflash reporter plasmid construct containing wild-type TCF/LEF binding sites upstream of a luciferase gene along with and β-catenin (wild-type) or S33Y (mutant β-catenin) plasmids. The luciferase activity in cells transfected with the constitutively active β-catenin mutant (S33Y) was much greater than in the cells transfected with wild-type β-catenin Fig. . Surpris11. Thus, we also examined whether RS-PP-050 downregulated β-catenin through these genetic alterations. Two CRC cell lines carrying the different mutations were used: SW480  and HCT116 . Treatment with RS-PP-050 reduces protein expression of both total and active forms of β-catenin in SW480 cells and particularly reduces the expression of the active form in HCT116 in a concentration-dependent manner , which subsequently abrogates the transcriptional activity of Wnt/β-catenin signaling inside the nucleus. This is the first evidence showing that the anti-cancer activity of an andrographolide analogue is on the level downstream of GSK-3β toward the nucleus of Wnt signaling pathway.In the present study, RS-PP-050 suppressed the TCF/LEF transcriptional activity of Wnt signaling in wild-type β-catenin-overexpression HEK293T reporter cells Fig. . As a re28. Genetic alterations of APC account for 85% of sporadic CRC cases29. While alterations of a tumor suppressor CK1, which phosphorylates the N-terminal of β-catenin at ser45 to prime GSK-3β function for subsequent degradation of β-catenin, is linked to the development of colon tumors30. By using CRC cell lines, SW480, and HCT116 cells which harbor a mutated form of APC, and CK1, respectively, RS-PP-050 was able to reduce β-catenin stabilization, indicating that RS-PP-050-mediated β-catenin degradation is not associated with APC or CK1. Thus, inhibition of downstream Wnt/β-catenin signaling is more likely to be a potential target for RS-PP-050 action.The stabilization of β-catenin is controlled by important components of a destruction complex including APC and CK131. The phosphorylation of β-catenin at ser675 by cAMP/PKA signaling has been reported to be associated with the nuclear translocation of β-catenin14. This phosphorylation causes β-catenin to interact with its coactivator, CREB-binding protein (CBP) and promotes β-catenin transcriptional activity. In this current study, the inhibition of Wnt signaling by RS-PP-050 is, at least in part, attributed to its targeting of the nuclear translocation of β-catenin by ser675 phosphorylation . Thus, the enhancement in lipophilicity potentially increases the penetration ability of the compound into the cells to efficiently kill cancer cells. This concept has been successfully demonstrated in several anti-cancer compounds modified from andrographolide34.Structurally, the cytotoxicity of andrographolide has been reported to be related to its electrophilic, α-β unsaturated γ-lactone unit which is capable of acting as a Michael acceptor to directly interact with nucleophilic biomolecules inside the cell such as GSH, DNA and proteinsIn conclusion, an andrographolide analogue RS-PP-050 acts as a potent anti-cancer compound against CRC cell growth by induction of cell cycle arrest and cellular apoptosis. It targets the Wnt signaling pathway in the nucleus by inhibiting β-catenin transcriptional activity and the nuclear accumulation of active β-catenin protein independently of GSK-3β phosphorylation. RS-PP-050 impairs the nuclear translocation of β-catenin with ser675 phosphorylation to inactivate Wnt activity. RS-PP-050 might be a promising chemotherapeutic agent used for targeting Wnt/β-catenin-overexpressing cells in human cancers and other diseases. These findings provide an instructive basis for exploiting a newly identified chemotherapeutic agent in Wnt/β-catenin-overexpressing CRC cells.2.Human embryonic kidney (HEK293T), Chang liver , and three CRC cell lines, HT-29, HCT116 and SW480 were obtained from the American Type Culture Collection (ATCC). HEK293T and Chang liver cells were maintained in Minimum Essential Media (MEM). HT-29 cells were maintained in Dulbecco’s modified Eagle’s medium: Nutrient Mix F-12 (DMEM/F12). HCT116 and SW480 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) . All growth media was supplemented with 10% fetal bovine serum (FBS) , 100 μg/ml penicillin, and 100 μg/ml streptomycin . All cells were cultured at 37 °C in a humidified incubator with 5% COO-triphenylmethyl andrographolide) are provided by Asst. Professor Dr. Rungnapha Saeeng  as previously described as compound no.1826. The chemical structures of the compounds can be seen in Supplementary Fig. TM select cDNA synthesis kit ; SYBR kit ; BCA protein assay kit, protease inhibitor cocktail, RIPA buffer, Super Signal West Pico Chemiluminescent Substrate , and phosphatase inhibitor cocktail ; The following antibodies were used: anti-β-catenin (clone H-102) and anti-lamin A/C ; anti-active-β-catenin (clone 8E7) ; anti-Phospho-β-catenin (Ser675), anti-Phospho-GSK-3β (Ser9) and anti-GSK-3β ; anti-β-actin and anti-α-tubulin (Sigma). The TCF/ LEF reporter plasmid (TOPflash) and the negative control reporter plasmid (FOPflash), wild-type β-catenin-FLAG and mutant β-catenin S33Y plasmids were described previously35.Andrographolide and a semi-synthetic andrographolide analogue, RS-PP-050 .Cells were plated at a density of 1 × 104 cells/well into 96-well plates for 24 h, and treated with various concentrations of RS-PP-050 for 24 h. After incubation, BrdU labeling solution was added, and the cells were fixed and incubated with an anti-BrdU peroxidase conjugated antibody using the cell proliferation ELISA BrdU kit according to the manufacturer’s instructions. The reaction was stopped with 1 M H2SO4 and the absorbance was measured at a wavelength of 450 nm (reference wavelength: 690 nm) using a Multiskan™ GO Microplate Spectrophotometer .Cells were plated at a density of 1 × 103 cells/well in the growth medium. Plates were further incubated for 15 days with changing of medium every 2–3 days until colonies were large enough to be visualized. Colonies were fixed with methanol and stained with 0.5% crystal violet for 1 h and dried at room temperature. Pictures of cells were taken and the colonies were counted using Image J software .Cells treated with RS-PP-050 at various concentrations were replated in 6-well plates at a density of 1 × 106 cells/dish and treated with different compounds for 24 h. Later, cells were harvested by trypsinization, centrifuged and washed twice with PBS. The cell pellets were re-suspended in FITC-labeled annexin V and PI for 15 min in dark place. For cell cycle experiments, cells were seeded in 10-cm dishes and treated with different compounds for 12–24 h. Later, cells were collected and fixed with cold 70% ethanol for at least 24 h followed by washing twice with PBS and staining with PI/RNAse solution for 15 minutes in the dark. The stained cells were analyzed using a BD FACS Calibur™ flow cytometer. The different stages of cell death and the percentage of DNA content in each phase were quantified using BD FACS Diva software version 6.1.1 (BD Bioscience).To study cellular apoptosis, cells were seeded in 10-cm dishes at a density of 2 × 104 cells/well into 96-well plates for 24 h. Cells were then treated with various concentrations of RS-PP-050 at the indicated time points in a 96-well plate. The medium was removed and 20 μM of DCFDA in DPBS were added to each well for 30 min at 37 °C in the dark. Following the incubation, cells were washed three times with DPBS and immediately analyzed at 485 and 535 nm using TECAN Spark 10 M microplate reader .Intracellular ROS levels were measured using 2′,7′-Dichlorofluorescein diacetate (DCFDA). Cells were plated at a density of 1 × 104 cells/well for 18 h. The cells were then transiently co-transfected with 0.1 μg pcDNA3.1 or β-catenin-FLAG or S33Y plasmid, 0.05 μg TOPflash or FOPflash reporter plasmid and 0.05 μg Renilla luciferase reporter plasmid for 24 h by using Lipofectamine 2000 reagent according to the manufacturer’s instructions. RS-PP-050 at various concentrations or 0.1% DMSO vehicle control was added. After 24 h, the cells were lysed and subjected for measurement of firefly luciferase activity using the Dual Luciferase Assay kit. The firefly luciferase activity in each treatment was normalized with Renilla luciferase activity as an internal control.HEK293T cells were seeded into 96-well plates at a density of 1 × 10Total RNA was isolated using the TRIzol reagent following the manufacturer’s instructions. The purity of the RNA was determined using a NanoDrop 2000C (Thermo Scientific). cDNAs were synthesized by using an iScript™ cDNA synthesis kit according to the manufacturer’s protocol and stored at −20 °C until used. Real time PCR of c-myc, cyclin D1, survivin, and MMP-7 was performed on equal amount of cDNA following the protocol provided with SYBR Green and analyzed with ABI PRISM7500 Sequence Detection System and analytical software . A panel of PCR primers were designed using NCBI/Primer-Blast follows: CyclinD1, FW 5′-GATCAAGTGTGACCCGGACTG-3′ and RW 5′-CCTTGGGGTCCATGTTCTGC-3′; C-myc, FW 5′-GCAGCGACTCTGAGGAGGAA-3′ and RW 5′ GGCCTCCAGCAGGCAGCACA-3′; Survivin, FW 5′-TGAGAACGAGCCAGACTTGG-3′ and RW 5′ TGTTCCTCTATGGGGTCGTCA-3′; MMP-7, FW 5′-TGGGAACAGGCTCAGGACTA-3′ and RW 5′-TGCATCTCCTTGAGTTTGGCT-3′; GAPDH, FW 5′-ATGCCCCCATGTTCGTCATG-3′ and RW 5′-GCAGGAGGCATTGCTGAT-GA-3′. Samples were analyzed in triplicate and the expression ratio was normalized with GAPDH.TM . The densitometry analysis was performed by using ImageJ software .The treated cells were harvested and lysed by using RIPA buffer supplemented with protease inhibitors and phosphatase inhibitor cocktail. Cytosolic and nuclear fractions were extracted by using a NE-PER Nuclear and Cytosolic Extraction kit (Thermo Scientific). Proteins were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto PVDF membranes. Blots were blocked with 5% nonfat dry milk and probed overnight at 4 °C with primary antibody. The membranes were then incubated with specific secondary antibodies. The detection of the bands was developed using ECL reagent and imaged with Amersham Hyperfilm2+/Mg2+ (PBS++), permeabilized in 0.2% Triton X-100 and blocked with 10% BSA. The slides were incubated for 2 h at room temperature with primary rabbit anti-phospho-Ser675-β-catenin (1:200), followed by a 1 h incubation in the dark with an anti-rabbit Alexa Flour 488 conjugated antibody (1:500) and DAPI for nuclei visualization. Stained cells were imaged with a confocal laser scanning microscope  at a magnification of 60x. The images were analyzed using the provided analysis software .Cells were seeded on glass cover slips in a separate 24-well plate. After treatment, cells were fixed in methanol at −20 °C, washed with cold PBS containing CaAll statistical analysis was performed using GraphPad Prism 5.01 . Data in each group were compared using a two-tailed unpaired student’s t test or one-way ANOVA with Tukey’s post-test. P-values < 0.05 and <0.01 were considered statistically significant.Supplementary Information


Different cancer types as well as many other diseases are caused by aberrant activation of the canonical Wnt signal transduction pathway, and it is especially implicated in the development and progression of colorectal cancer (CRC). The main effector protein of the canonical Wnt signaling cascade is β-catenin, which binds to the T- cell factor/lymphoid enhancer factor (TCF/LEF) and triggers the activation of Wnt target genes. Here, we identify the serine protease High-Temperature Requirement A1 (HTRA1) as a novel component of the canonical Wnt pathway. We show that the HTRA1 protein inhibits the Wnt/β-catenin signaling, in both paracrine and autocrine manners, and affects the expression of several Wnt target genes. Moreover, HTRA1 forms a complex with β-catenin and reduces the proliferation rates of cells. Taken together, our findings indicate that HTRA1 functions as a novel suppressor of the canonical Wnt signaling pathway. The expression levels of cytosolic β-catenin, the Wnt signaling pathway’s key effector, are tightly regulated by the ‘β-catenin destruction complex’, which is usually active in the absence of a Wnt signal. This protein complex encompasses large number of components including the tumor suppressor protein adenomatous polyposis coli (APC) and Axin, as well as the kinases glycogen synthase kinase (GSK3β) and casein kinase-1 (CK1), which phosphorylate β-catenin, marking it for ubiquitination following by proteasomal degradation7. Mutations in APC, Axin, or β-catenin disrupt this ‘degradation complex’, resulting in the accumulation of β-catenin and an aberrant activation of Wnt target genes. Initiation of the Wnt pathway is triggered by Wnt ligand binding to the Frizzled (Fz) transmembrane receptor and the co-receptor low-density lipoprotein receptor-related 5 (LRP5) or LRP69. This complex induces the association of Axin or the entire ‘destruction complex’ with LRP, which leads to a cascade of events that include Disheveled (Dvl) polymerization, which promotes the accumulation of unphosphorylated β-catenin that translocates into the nucleolus4. In the nucleus, β-catenin forms a complex with the TCF/LEF transcription factors and induces the upregulation of Wnt target genes. Indeed, aberrant expression of Wnt target genes is highly implicated in many cases of neoplastic transformation11. Wnt signaling is extremely intricate, regulated by other cascades and feedback loops and comprises numerous components, yet to be identified. We have successfully utilized a mammalian cells screening technique12 in order to isolate new Wnt signaling co  mponents. One of the genes identified was High-Temperature Requirement A1 (HTRA1). HTRA1 is a conserved PDZ serine protease, a member of the HTRA family of serine proteases, which is involved in numerous basal biological mechanisms in mammals13. It is a secreted enzyme with a widespread pattern of expression, and its levels in human tissues are modulated by different physiological activities14. HTRA1 is composed of four distinct protein domains: an Insulin-like growth factor binding domain (IGF-BD), a kazal-type motif (KM), a trypsin-like peptidase (proteolytic) domain, and a PDZ domain. The signal peptide (SP) in the N-terminus of the protein is essential for both the expression and the secretion of the HTRA1 protein. Human HTRA1 has been implicated in several severe pathologies such as the cerebral small vessel and arthritic diseases as well as in age-related macular degeneration17. This suggests that HTRA1 plays an important role in human physiology. In addition, several publications link the HTRA1 gene to tumorigenesis, since it has been found to be down-regulated in many tumors such as prostate cancer, medulloblastoma18, ovarian cancer19, melanoma20, lung carcinoma21, and mesothelioma22. In breast cancer, several studies indicate that HTRA1 expression is lower in estrogen-receptor(ER)-negative tumors and that down-regulation of HTRA1 is significantly correlated with a higher grade of breast carcinoma23. Moreover, poorly differentiated breast tumors and those with mutant p53 or with lymphatic infiltration have significantly lower levels of HTRA1 expression24. Recently it has been shown that epigenetic silencing of the HTRA1 gene was linked to numerous cancerous phenotypes, and specifically to colorectal cancer. Importantly, it was shown that the HTRA1 promoter was methylated and repressed in a mouse model of the human familial adenomatous polyposis disease25.The canonical Wnt signaling pathway, also called the Wnt/β-catenin signaling pathway, is transduced by the stabilization of β-catenin following the interaction between a specific Wnt ligand and its designated receptors. This important developmental pathway mainly controls embryonic growth but is also implicated in adult tissue maintenance. Thus, abnormal constitutive stimulation of the canonical Wnt cascade leads to irregular cell proliferation and promotes the progression of numerous types of human cancers, particularly the human colorectal cancers (CRCs)In the present study, we show that in accordance with its tumor suppressor traits, HTRA1 reduces the oncogenic Wnt signal, the β-catenin expression levels, and the proliferative abilities of cells. Additionally, we show that HTRA1 interacts with β-catenin and that its effect on the Wnt signal is mediated through β-catenin degradation by the proteasome. These findings indicate that HTRA1 may be a novel regulator involved in controlling the canonical Wnt cascade.12. HEK293T cells were tranfected by CaCl2 precipitation. Transfections of all other cells performed with jetPEI  following the manufacturer’s protocols.HEK293T, L-Wnt3a, L, COS-7, and the human CRC SW480 and HCT-116 cell lines were maintained in DMEM supplemented with 10% FCS as previously described12. MG132  and TGF-β  were used as indicated.HTRA1 constructs: the HTRA1-GFP-N3 plasmid was constructed by amplifying the genomic cDNA by PCR and subcloning it into enhanced (pEGFP)-N3 vector (Clontech), using the XhoI and BamHI restriction sites. The HTRA1-HA plasmid was constructed by amplifying the genomic cDNA by PCR and subcloning it into an enhanced pcDNA3.1/N-HA vector, using the XhoI and BamHI restriction sites. The HTRA1-HA and HTRA1-GFP expression vectors were used as templates for the mutagenesis of HTRA1; serine 328 was replaced with alanine (designated HTRA1-HA-S328A and HTRA1-GFP-S328A). GFP-Kazal, GFP-IGFBD, GFP-Protease, and GFP-PDZ were cloned by a PCR reaction using full-length HTRA1-pEGFP-N3 as a template, and the amplicons were inserted into the pEGFP-C2 vector  using the Gibson Chew Back and Anneal Assembly, according to the manufacturer’s protocol. All fragments containing the domains’ cDNAs were extracted from agarose gel using the DNA cleaning kit  according to the manufacturer’s instructions. All β-catenin and luciferase system constructs were obtained as previously described12.All these experiments were conducted as previously described12.COS-7, L-Wnt3a, SW480 or HCT-116 cells were grown and stained as described previouslyThe antibodies that were used include: mouse anti-β-catenin , mouse anti-active β-catenin , rabbit anti-GFP , rat anti-HA , mouse anti-FLAG , mouse anti-HTRA1 , rabbit anti-HTRA1 , Acris Antibodies GmbH), rabbit anti-HTRA1  rabbit anti-Golgin-97  Cell Signaling Technology, Inc.). Mouse anti-tubulin  was used as a loading control. Anti-rat horseradish peroxidase-conjugated secondary antibodies were obtained from Santa Cruz Biotechnology (1:5000). Anti-mouse and anti-rabbit secondary antibodies were obtained from Jackson Immuno Research  (1:10000). For IF, Alexa red and green  were used.HTRA1 small interfering RNA (siRNA) oligonucleotides were purchased from Thermo Scientific Dharmacon . The transfection reagent Interferine was purchased from Polyplus-France. Transfection was performed according to the manufacturer’s protocol. All experiments were performed in HEK293T cells using 100 nM siRNA oligonucleotides for 72 h. Non-targeting RNA oligonucleotides (Thermo Scientific Dharmacon) were used as a control. HEK293T cells were transfected 24 h after siRNA transfection with pTOPFLASH/ pFOPFLASH and β-gal plasmids to detect luciferase. Forty-eight hours post-transfection, the cells were harvested and subjected to either luciferase assay or Western blot analysis as described above. Alternatively, 72 hours after HTRA1 siRNA transfection, the cells were harvested; total RNA was extracted from the cells and converted into more stable cDNA. Then, cDNA from the cells was subjected to qRT- PCR assay.HEK293T cells were transfected with HTRA1GFP or GFP vectors and the media was collected 24–48 h later and used as HTRA1 cultured medium.Cell proliferation was measured using an XTT cell proliferation kit . Cells were grown in a 24-well plate. Twenty-four hours following transfection, cells were split into a 96-well plate. Following transfection (48 h later) the cells were treated with a XTT labeling mixture according to the manufacturer’s instructions. Cells were incubated at 37 °C, and the proliferation levels were measured every 4 h, 18 h, and 24 h.Total RNA was isolated from the cultured cells according to the protocol supplied with TRI Reagent (Sigma-Aldrich). The concentration and purity of the RNA samples were determined and total RNA was reverse transcribed (RT) with the iScript cDNA Synthesis Kit (BioRad) according to the manufacturer’s instructions.All real-time PCR reactions were performed using the CFX Connect Real-Time PCR Detection System (BioRad) and the amplifications were carried out using Fast Start Universal SYBR Green Master (Roche). The thermal cycling conditions were as follows: 50 °C for 2 min, followed by an initial denaturation step at 95 °C for 10 min, 40 cycles at 95 °C for 15 s, and 60 °C for 60 s. The experiments were carried out in three independent experiments in triplicates. β-actin was used as an endogenous control to normalize each sample.The primers for the amplifications of the specific cDNA sequences were as follows:HTRA1 Fw: 5′AAAGCCAAAGAGCTGAAGGACC3′HTRA1 Rv: 5′ACATCATTGGCGGAGACCAC3′Axin2 Fw: 5′GCAGCTCAGCAAAAAGGGAAAT3′Axin2 Rv: 5′TACATCGGGAGCACCGTCTCAT3′C-myc Fw: 5′CTCGGTGGTCTTCCCCTACCCT3′C-myc Rv: 5′TGTCCAACTTGACCCTCTTGGC3′Cyclin D Fw: 5′CATCTACACCGACAACTCCATC3′Cyclin D Rv: 5′TCTGGCATTTTGGAGAGGAAG3′β-actin Fw: 5′CCTGGCACCCAGCACAAT3′β-actin Rv: 5′GGGCCGGACTCGTCATACT3′HEK293T cells were grown in a 6-well plate and transfected with the indicated plasmids. Following transfection, cells were incubated with 50 μM serine protease inhibitor 4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF)  for 30 min at 37 °C and then harvested.12. Since most of the cDNAs used for this screen enclosed only the candidate’s C-terminal parts, the full-length HTRA1 cDNA was isolated and sub-cloned into GFP- or HA-tagged expression vectors. The HTRA1 expressing plasmids were first examined for their capacity to affect Wnt signaling in a specific manner by measuring the β-catenin/TCF-mediated transcription (using the pTOPFLASH/pFOPFLASH reporter assay). As shown in Fig. One of the genes isolated in our screen, aimed at identifying new Wnt signaling regulators, was HTRA127. Indeed, ectopic expressed HTRA1-GFP co-localizes with the Golgi marker Golgin-97 in different cell lines, which indicates that the protein enters the secretory pathway  that are generally associated with the secretion pathway , a kazal-type motif , a trypsin-like peptidase (proteolytic) domain, and a PDZ domainnes Fig. . Brieflynes Fig. . The expnes Fig. .Figure 5Numerous studies have linked HTRA1 to tumorigenesis since it has been found to be down-regulated in many tumors including prostate cancer, ovarian cancer, and melanoma. We examined the expression pattern of HTRA1 in HEK293T cells and in the CRC cell lines SW480 and HCT116. Immunostaining of endogenous HTRA1 in these cells revealed a slight reduction in HTRA1 levels in the SW480 cells  even reversed. We speculate that the changes in the levels of C-myc may be attributed to its promoter conformation, as was recently shown; (detailed model is presented in a recent study31). This model states that conformational changes in the promoter region may influence C-myc expression in CRC cells. It is possible that under certain conditions, the β-catenin/HTRA1 complex harbors high affinity to remote Wnt responsive elements (WRE), thus enhancing chromatin changes that specifically alter C-myc expression. There is emerging data suggesting that the Wnt and TGF-β pathways crosstalk in different ways. For example, it was proposed, that along the colonic crypt–villus axis, proliferation, migration, differentiation and compartmentalization are controlled by TGF-β- and Wnt gradients. The presence of TGF-β signaling and the absence of Wnt signaling in the villus compartment result in rapid cell cycle arrest and differentiation35. Thus, these pathways may control the switch between proliferation and differentiation. The relationship between the pathways is context dependent and is evident in different cell types, both in normal development and in disease37. Interestingly, it has been shown that the activity of HTRA1, which is impaired in cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL) patients, affects TGF-β signaling40. Importantly, using embryonic fibroblasts from HTRA1-knockout mice, it has been shown that HTRA1 antagonizes TGF-β signaling by cleaving its receptors to affect bone mass41 and has a role in angiogenesis via TGF-β signaling42. Our data indicates that TGF-β may overturn the effect of HTRA1 on Wnt target genes expression suggesting that the connection between HTRA1 and the Wnt pathway is governed by TGF-β. These findings should be further examined by using fibroblasts obtained from HTRA1 null mice.The HTRA1 protein is a member of the human HTRA family of serine proteases, which are found in a variety of species, including bacteria, plants, and mammals25. HTRA1 is a secreted protein with a widespread pattern of expression in the human body, and with modulated levels of expression according to the different activities of the tissues43. We tested the ability of secreted HTRA1 to suppress Wnt signaling by incubating cells with HTRA1-cultured media and by examining the TCF/β-catenin-mediated transcription. We show that GFP tagged-HTRA1 protein is secreted to the media and then can be internalized by recipient cells and affect the Wnt signal, although the effect is weaker than that of the overexpressed protein. HTRA1 is composed of four distinct domains: an Insulin-like growth factor binding domain (IGF-BD), a kazal-type motif (KM), a trypsin-like peptidase (proteolytic) domain, and a PDZ domain. The trypsin-like catalytic domain of the protein is responsible for its enzymatic activity and the serine at position 328 is crucial for its catalytic activity. We have used a catalytically inactive HTRA1 form (S328A)44 to examine its effect on Wnt signaling and on the transcription levels of several Wnt target genes. Importantly, we found that both the wild-type and the mutated forms of HTRA1 reduced the canonical Wnt signal levels to the same extent and similarly affected the expression of the Wnt target genes. Moreover, the effect of both proteins on different Wnt target genes in the presence of a serine protease inhibitor is also similar. These results indicate that HTRA1’s effect on Wnt signaling is independent of its catalytic activity, and that it is probably mediated through a different mechanism that remains to be fully understood. When the effect of each of HTRA1’s domains was examined separately, two domains exhibited behavior similar to that of the full-length protein, the Kazal domain, and the PDZ domain. Interestingly, both domains also physically interact with the β-catenin protein (not shown). This might indicate that the effect of the protein is mediated through one or both of the domains. HTRA proteins are known for their complex structural organization, which enables them to mediate a wide array of biological functions33. Although debatable, some studies claim that HTRA1 can act as a chaperone or that it harbors chaperone-like activity. In this work, we showed that the effect of HTRA1 on the Wnt signal is mediated through its interaction with the β-catenin protein. We showed that cells expressing high levels of endogenous HTRA1 exhibit lower levels of active β-catenin and vice versa. Additionally, we showed that HTRA1 and β-catenin physically interact. These findings may indicate that the effect of HTRA1 is mediated through a physical interaction with β-catenin, which results in reduced activity. In addition, we have shown that when β-catenin is mutated in a form that cannot be phosphorylated and degraded by the proteasome, HTRA1 has no effect on the Wnt signal and its ability to interact with β-catenin is abolished. Similarly, the proteasome inhibitor MG132 also ablated HTRA1’s capacity to inhibit Wnt signaling. These findings imply that β-catenin phosphorylation is crucial for HTRA1’s influence on the Wnt cascade. In order to better understand why HTRA1 is often implicated as a tumor suppressor, we examined its effect on cellular proliferation abilities, and as an indicator of the tumorigenic traits of cells. We found that overexpressed HTRA1  reduces the proliferation levels of the cells, whereas endogenous HTRA1 silencing increases cellular proliferation. This evidence supports HTRA1’s role as a tumor suppressor. Interestingly, the HA-tagged HTRA1 protein does not accumulate in the Golgi and is not secreted. A previous study showed that tags, and specifically an HA tag, can change the conformation and activity of a protein specifically in cases where protein function is based on conformational changes45. Indeed, HTRA proteins possess a complex structural organization, which enables them to fulfill a wide array of cellular functions33. Interestingly, the HA-tagged HTRA1 protein is strongly recruited to the Disheveled signalosomes. Dvl is a known activator of the Wnt cascade and is often found in puncta-like structures when overexpressed46. Interestingly, it was recently shown that Dvl utilizes conformational changes to regulate the Wnt signaling pathways. For example, a C-terminal motif can intrinsically bind the Dvl PDZ domain in order to form a “closed” conformation47. It is possible that the GFP tag mimics an interaction/modification that keeps HTRA in a conformation programmed for secretion, whereas the HA tag exposes its PDZ domain to be captured in the signalosomes by the DVl C-terminal motif. The assembly of those signalosomes that are important for the transduction of the Wnt pathway may lead to loss of HTRA1 activity. As the Wnt signaling pathway plays important roles in both normal development and pathogenesis, these findings may provide new molecular clues to the way this important pathway transduces its signal. Since the pathway is so complex and highly controlled, new genes, proteins, and other regulatory components are still frequently being identified. Characterizing these new components may shed new light on molecular events that lead to cancer development and progression and thus may hold therapeutic potential for its treatment.Next we demonstrated that silencing the expression of the endogenous HTRA1 resulted in increased Wnt signaling. It also led to elevation of all Wnt target genes that were tested: Axin2, C-myc, and cyclin D1. This strongly supports our assumption that HTRA1 functions as a novel repressor of the canonical Wnt pathway. Note that silencing of the HTRA1 gene has been previously linked to colorectal cancer, in a yet unexplained mechanism, and to numerous cancerous phenotypes such as increased proliferation, DNA instability, and failure in DNA damage checking


Due to the necessity of the low-power implementation of newly-developed electrocardiogram (ECG) sensors, exact ECG data reconstruction from the compressed measurements has received much attention in recent years. Our interest lies in improving the compression ratio (CR), as well as the ECG reconstruction performance of the sparse signal recovery. To this end, we propose a sparse signal reconstruction method by pruning-based tree search, which attempts to choose the globally-optimal solution by minimizing the cost function. In order to achieve low complexity for the real-time implementation, we employ a novel pruning strategy to avoid exhaustive tree search. Through the restricted isometry property (RIP)-based analysis, we show that the exact recovery condition of our approach is more relaxed than any of the existing methods. Through the simulations, we demonstrate that the proposed approach outperforms the existing sparse recovery methods for ECG reconstruction. K-sparse signal if It is well known that electrocardiogram (ECG) sensors enable effective medical diagnosis for heart problems, such as arrhythmia and myocardial infarction, in everyday life ,2,3. In Definition 1.The sensing matrix for any K-sparse vector j, such that Additional benefits of the CS-based ECG processing are 1) the computationally-efficient data compression and (2) the guarantee of exact reconstruction from far fewer measurements than conventional methods. While the computational efficiency of data compression can be easily demonstrated since it requires only simple linear matrix multiplication (see Definition 1), our main interest lies in the reduction in the number of required measurements ensuring exact sparse signal recovery from compressed ECG data. A popular method for identifying the sparest signal  the compuit (BP) , BP denouit (BP)  (or Lassuit (BP) ) and theuit (BP) , were init (OMP) , enablesselected ,22,23  subject to the constraint of sparsity level K. In addition, we demonstrate that this can rather reduce the running time complexity by shutting down the search in the early stage of the search tree.Our approach, referred to as tree pruning-based matching pursuit (TPMP), provides further improvement by exploiting the full dictionary information with aggressive pruning strategies for each path. To be specific, the proposed TPMP considerably reduces the computational burden of the brute-force tree search, yet achieves excellent recovery performance by jointly implementing two pruning criteria, that is (1) the pre-scanning and (2) the pruning-based tree search. In the pre-scanning stage, we greedily choose a small number of promising column indices of the sensing matrix. If we denote the set of column indices obtained in the pre-scanning stage as Θ, then we set criteria ,28,29,30While the preliminary version of this work was presented for an arbitrary system in , we showThe rest of this manuscript is organized as follows. In In this section, we introduce a low-power ECG reconstruction method based on the tree search where the system model is provided in K dominant elements of Ψ is the K-sparse signal The digitized signal j-th column of j-th entry of K elements of T to be given at the reconstruction stage, the information amount to be delivered increases from After that, Φ is a random matrix, since such a matrix is said to obey the RIP with high probability [Considering such property, one of the good choices for the sensing matrix bability .K indices in this work. That is, we select K column indices of K and K indices corresponding to maximally correlated columns with In order to achieve low complexity ECG reconstruction with improved recovery accuracy, two key pruning criteria of our method are the pre-scanning and the pruning-based tree search. In the pre-scanning stage, we greedily choose the columns of of Θ see . While ai-th layer spread branches in the . As shown in posed in . While [posed in  presentegnitude:{t^i+1, ⋯K). That is, if the ϵ  and thus the true path can survive as long as T at least once. Therefore, if we set c : If theLemma 2.: If Lemma 3.(Lemma 2.1 in ): Let I1Lemma 4.For Definition 2.: For thwhere (3-1) (Theorem 1) At least one support index should be found in the pre-scanning  (Theorem 2) At least one true path TPMP is guaranteed to exactly reconstruct If (3-1) holds, then at least one branch in each layer of the tree is the support element. Therefore, whenever there is at least one true path in current layer that is not removed by the pruning strategy, (3-1) enables the true path to proceed further. Along with (3-1), the additional condition that ensures the survival of the true path is necessary for exact support identification, which is given as (3-2). In our analysis, both (3-1) and (3-2) are guaranteed under the results in Theorems 1 and 2, respectively, and these theorems jointly provide the overall sufficient condition for exact recovery in Theorem 3.κ be the largest correlation in magnitude between ζ be the K-th largest correlation in magnitude between j-th column of κ and ζ, respectively.First, we obtain the sufficient condition for (3-1). Let Lemma 5.κ and ζ satisfy:Proof of Lemma 5.See Using Lemma 5, one can obtain the sufficient condition for (3-1).Theorem 1 (Sufficient condition for (3-1)).At least one support element is found in the pre-scanning stage under:Proof of Theorem 1.In order to choose at least one correct index in the pre-scanning stage, we should have ϵ as:Next, the condition (3-2) is guaranteed if the posterior indices of a true path always contain the support elements, that is As mentioned, the problem to choose the posterior index set for a given true path Lemma 6.If where Proof of Lemma 6.See The following theorem provides the sufficient condition for (3-2).Theorem 2 (Sufficient condition for (3-2)).The posterior index set of a true path for any Proof of Theorem 2.The element of posterior index set From Lemma 1, this inequality can be rewritten as The overall recovery condition of TPMP can be obtained by combining Theorems 1 and 2.Theorem 3 (Recovery condition of TPMP).TPMP exactly identifies the support of any K-sparse signal Proof of Theorem 3.The condition (Equation ) is obtaIt is worthwhile to note that TPMP provides a more relaxed recovery condition than conventional greedy algorithms, such as OMP K+1<1K+1 , SP (δ3KK<0.165) , gOMP δKδK2<LL+K  and MMP ented in .T always survives the pruning strategy whenever it is detected once since We also consider reconstructing ECG when the compressed signal Before we proceed, we provide a useful lemma in our analysis.Lemma 7.For any Proof of Lemma 7.Since From Lemma 2 and Definition 1, we then have:From Equations (16) and (17), we obtain the lower bound of ρ be the largest correlation in magnitude between η be the K-th largest correlation in magnitude between We first analyze the condition ensuring at least one support element is chosen by pre-scanning from noisy measurements. Let ρ and the upper bound of η.In the following lemmas, we provide the lower bound of Lemma 8.ρ satisfies:and η satisfies:Proof of Lemma 8.See The following theorem provides the condition ensuring that at least one support element is identified by the pre-scanning.Theorem 4 (Pre-scanning condition).At least one element in the support is found in the pre-scanning stage if the nonzero entries of the original sparse signal Proof of Theorem 4.It is clear that at least one support element  the posterior indices Lemma 9.For any and:respectively.Proof of Lemma 9.See The guaranteed condition of the posterior indices to satisfy Theorem 5.For any Proof of Theorem 5.Similar to Theorem 2, one can notice that the posterior indices of the true path Recall Equation  from Lemϵ. Recall that the pruning threshold is updated by the smallest residual in magnitude among all T has a smaller residual in magnitude than any possible pruning threshold ϵ and, thus, cannot be removed from the search tree.Next, we provide the guaranteed condition under which the residual of the support satisfies:∥rT∥2≤ϵfTheorem 6.The support has the minimum residual in magnitude among all possible Proof of Theorem 6.See If Theorems 5 and 6 jointly hold, then the condition that the true path Theorem 7 (Search tree condition).The true path where Proof of Theorem 7.Immediate from Theorems 5 and 6.  ☐By combining the results from Theorems 4 and 7, we obtain the sufficient condition of exact support identification from noisy measurements.Theorem 8 (Exact support identification of TPMP).The TPMP algorithm accurately identifies the support from the noisy measurement where Proof of Theorem 8.Immediate from Theorems 4 and 7.  ☐γ. If Equation  defined as CR Oracle estimator Linear MMSEBasis pursuit (BP) Orthogonal matching pursuit (OMP) Subspace pursuit (SP) Multipath matching pursuit (MMP)  with L=2Depth-first multipath matching pursuit (MMP-DF)  with L=2TPMP  basis matrix sults in , we set hysionet , and a rases see . Each reM) degrades the reconstruction performance , the TPMP algorithm performs the tree search and investigates multiple promising candidates. Further, the complexity overhead caused by the tree search is reduced by the pruning strategy. In our analysis, we analyzed the sufficient condition of TPMP for exactly identifying the support, which provides an improved recovery bound compared to the existing methods. In addition, our numerical results discussed in this work demonstrate that TPMP provides improved performance with competitive complexity with conventional algorithms.In this work, we proposed an effective ECG reconstruction method referred to as tree pruning-based matching pursuit (TPMP). In order to improve the accuracy of ECG recovery for large CR (or small


Protein arginine methyltransferases (PRMTs) regulate chromatin remodeling and gene expression. Here, we investigated whether inhibition of PRMTs affects colitis pathogenesis in mice and inflammatory bowel disease patients and further explored the underlying mechanisms. In this study, we found that protein arginine N-methyltransferase inhibitor 1 (AMI-1) treatments increased Tregs frequency, function, and reduced colitis incidence. Adoptive transfer of AMI-1-treated Tregs could reduce the colitis incidence. Colitis was associated with increased local PRMT5 expression, which was inhibited by AMI-1 treatment. Additionally, PRMT5 knockdown T cells produced a better response to TGFβ and promoted Tregs differentiation through decreased DNA methyltransferase 1 (DNMT1) expression. PRMT5 also enhanced H3K27me3 and DNMT1 binding to Foxp3 promoter, which restricted Tregs differentiation. Furthermore, PRMT5 knockdown led to decreased Foxp3 promoter methylation during Tregs induction. PRMT5 expression had a negative relationship with Tregs in UC patients, knockdown of PRMT5 expression increased Tregs frequency and decreased TNFα, IL-6, and IL-13 levels. Our study outlines a novel regulation of PRMT5 on Tregs development and function. Strategies to decrease PRMT5 expression might have therapeutic potential to control UC.Ulcerative colitis (UC) pathogenesis is related to imbalance of immune responses, and the equilibrium between inflammatory T cells and Foxp3 N-methyltransferase inhibitor 1 (AMI-1) specifically inhibits arginine methyltransferase activity and does not inhibit lysine methyltransferase activity (in vitro and in vivo models of mantle cell lymphoma (Protein arginine methyltransferases (PRMTs) are involved in posttranslational modification that can methylate arginine residues on histone and non-histone proteins, and this common modification has been engaged in protein trafficking, gene transcription, and tumor growth , 2. PRMTactivity . AMI-1 wactivity . Recentllymphoma . However+CD25− T (Teff) can be induced into the Th1, Th2, or Th17 cells under the inflammatory condition and are important mediators of inflammation in IBD (Ulcerative colitis (UC) is the primary form of chronic inflammatory bowel diseases (IBDs) and is characterized of continuous inflammation and limited solely to the mucosa and submucosa of the rectum and colon. The mechanism is related to a breakdown of immunological tolerance and that leads to pathological inflammation , 12. An n in IBD . Tregs an in IBD –17. Veltn in IBD . HoweverFoxp3 locus plays important role in the stability and functionality of Tregs, and demethylation of CpG islands in the region of Foxp3 locus-induced Foxp3 gene expression , and Tregs with the high immunosuppressive activity needs high level of Foxp3 expression and its stability , 20. Epipression , 22, whepression . IL-6 coignaling .in vitro and in vivo that could provide a choice to pharmacologically therapy. Colitis was associated with increased local PRMT5 expression, and AMI-1 treatment significantly decreased the PRMT5 expression. The specific knockdown of PRMT5 not only increased the frequency of CD4+Foxp3+ T cells from the Tregs inducing system but also maintained the Foxp3 expression when the nTregs were stimulated with IL-6 in vitro. The underlying mechanism is that the blockage of PRMT5 decreased DNMT1 expression levels, which affected Foxp3 gene expression. Furthermore, PRMT5 inhibition restored the defective Tregs frequency from the UC patients and enhanced protection from inflammatory colitis in the observed mice. Thus, targeting the PRMTs or specific knockdown of PRMT5 enhanced the Tregs levels and functions, making this a potential therapeutic strategy in the control of UC.Here, we investigated the role of the protein AMI-1 on Foxp3 expression and its influence on Tregs function in a colitis mouse model and in patients with UC. Our data showed that AMI-1 administration increased the frequency and function of Tregs SCID mice were purchased from the Shanghai Laboratory Animal Center, Chinese Academy of Sciences . The animals were housed in the animal care facilities of the Shanghai Jiao Tong University School of Medicine, Xin Hua Hospital, under pathogen-free conditions. This study was carried out in accordance with the recommendations of Institutional Animal Care and Use guidelines, Xin Hua Hospital Committee. The protocol was approved by the Institutional Animal Care and Use Committee of Xin Hua Hospital.Male C57BL/6 and n = 25, 9 men, 16 women, mean age 46.3 ± 5.2 years, range 24–62 years) were included, and Tregs in peripheral blood were investigated. Healthy individuals  served as controls. Active UC was defined as a Lichtiger score >3.24. Study was carried out in accordance with the recommendations of Xin Hua Hospital of guidelines. All of the patients provided written informed consent to participate in the study in accordance with the principles of the Declaration of Helsinki. The protocol was approved by the Xin Hua Hospital of ethics Committee.Patients with active UC .For intracellular cytokine staining, cells were stimulated with 50 ng/ml PMA and 500 ng/ml ionomycin (Sigma-Aldrich) in the presence of GolgiPlug (BD Biosciences) for 5 h. Then, the cells were fixed and permeabilized with Cytofix/Cytoperm buffer, and intracellular cytokines were stained with antibodies against Foxp3, IFN-γ, and IL-2 (eBioscience). For PRMT5 staining, the cells were stained with an antibody or isotype control antibody  for 30 min at room temperature according to the manufacturer’s instructions. Flow cytometric analysis was performed with a FACS Canto II instrument (BD Bioscience) and FlowJo software (TreeStar).Cells were directly lysed and subjected to 8% SDS-PAGE. Immunoblotting analysis was carried on by transferring proteins onto nitrocellulose membranes (Thermo Fisher Scientific) with a mini Trans-Blot apparatus (Bio-Rad). After 2 h of blocking, antibodies against PRMT1, PRMT5, DNMT1 (Abcam), and β-actin Ab (Sigma-Aldrich) were used to visualize the corresponding proteins.Total RNA was isolated from cell pellets using an RNeasy Mini Kit (Qiagen), and first-strand cDNA was subsequently synthesized with the Sensiscript RT Kit (Qiagen) according to the manufacturer’s instructions. Real-time PCR was performed using SYBR Green PCR Master Mix (Applied Biosystems). The data were collected and quantitatively analyzed on an ABI Prism 7900 Sequence Detection System (Applied Biosystems). The results are described as fold changes relative to mouse β-actin expression. The PCR primer pair sequences are shown in Table S1 in Supplementary Material.+CD25−CD45RBhi naïve T cells were purified with MACS columns (Miltenyi) from C57BL/6 mouse spleens. Then, they were activated under Tregs polarizing conditions  in the presence of T-cell-depleted splenocytes, which were utilized as the feeder cells. Finally, different AMI-1 concentrations were added to the culture system. After 3 days of culture, the cells were analyzed for Foxp3 expression.CD4+ T cells from mouse spleen were purified using the CD4+ T Cell Isolation Kit II (Miltenyi Biotec). Then CD4+CD25− (Teff) and CD4+CD25+ (2% of CD25 expression) (Tregs) T cells were sorted from purified CD4+ T cells by FACSAria (BD Biosciences) to a purity >98%.CD4+ naïve T cells in a 1:1 ratio with CD45.1+ Tregs were purified from PBS- or AMI-1-treated donors. Then, the cells were adoptively transferred into SCID mice. After 7 days, recipient spleens and lymph nodes were collected and detected the CD45.2+ CD4+ T-cell total numbers with flow cytometry.Congenic CD45.2SCID mice were intravenously injected with 1 × 106 sorted naïve T cells alone or in combination with 5 × 105 Tregs specifically treated with PRMT5 shRNA or control shRNA. Then, the mice were monitored for weight loss stool consistency, gross bleeding, and sacrificed 7 weeks after the experiment was initiated, as indicated. Proximal and distal colon sections were fixed in 4% buffered formalin and stained with H&E. Gross and histological inflammation including crypt architecture, degree of inflammatory cell infiltration, muscle thickening, goblet cell depletion, and crypt abscess was scored on a scale including normal colon, mild, moderate, and severe colitis. Images were acquired with a fluorescence microscope (Leica).C57BL/6 mice were treated orally with 3% DSS (MP Corporation) and were intraperitoneally injected with 200 mg AMI-1/kg or PBS alone daily for 7 days. Then the mice weight loss, fecal blood, and stool consistency were monitored. For the T-cell transfer model of colitis, Cell suspensions were added CFSE (Sigma-Aldrich) at a final concentration of 2 µM, 37°C for 8 min and then the cells are washed three times with PBS and suspended with complete RPMI media.+CD25+ Tregs were pretreated with the different concentration of AMI-1 under the stimulation of anti-CD3 and CD28  for 24 h, then washed three times with PBS. Then freshly isolated CFSE-labeled CD4+CD25− (Teff) cells from naïve C57BL/6 mice were cocultured with pretreated Treg cells at the indicated ratio, under the stimulation of splenic non-CD4+ cells treated with mitomycin C (Sigma-Aldrich), and anti-CD3 (2 µg/ml) for 3 days. The CFSE dilution in the Teff cells was analyzed with flow cytometry.CD46 T cells in 100 µl of Amaxa mix and transfected the cells via electroporation. After 6 h, 50 U of recombinant human IL-2 (Roche) was added to the medium for an additional 24 h. Then, we harvested the cells and purified the GFP+ cells for further studies. The purity of the sorted cells was >98%.RNA interference experiments were performed using electroporation (Amaxa) according to the manufacturer’s protocol. Briefly, we mixed 2 µg mice or human PRMT5 shRNA (Origene), respectively, or control shRNA with 1 × 106 cells) were fixed with 1% formaldehyde, and the chromatin was sonicated and pre-cleared by incubation with Protein A/G agarose/salmon sperm DNA. Then chromatin was immunoprecipitated with H3K27me3 (Abcam) and DNMT1 (Abcam) overnight at 4°C or mouse IgG monoclonal antibody followed by incubation with Protein A/G agarose/salmon sperm DNA for 1 h. The immunoprecipitates were denatured, and the DNA was purified. The amount of immunoprecipitated DNA was quantified by real-time PCR with the ABI PRISM 7500 Sequence Detection System (Applied Biosystems) using SYBR Green. The primers used for the Foxp3 promoter locus PCR analysis were as follows: forward: 5′ to 3′, TTC CTC CCG CTC TCT GAC TCT; reverse: 5′ to 3′, AAG CGC CAG TTG TGT ACA AAT ATC.ChIP assays were performed using a ChIP assay kit (Millipore) with modifications. Induced Tregs . Methylation analysis was quantified by sequencing of the genomic DNA after bisulfite conversion using the Methyl-Detector kit (Active Motif), PCR amplification, and cloning. The primer forward was 5′-TTT TAG ATG ATT TGT AAA GGG TAA AGA A and the reverse was 5′-CAA CCT AAC TTA TAA AAA ACT ACC ACA TTA TC.t-tests were used to analyze the differences between the groups. One-way ANOVAs were initially performed to determine whether an overall statistically significant change existed before using the two-tailed paired or unpaired Student’s t-tests. A value of P < 0.05 was considered statistically significant.Student’s in vitro. First, we treated the mouse splenocytes (SPs) with different AMI-1 concentrations and found that Foxp3 expressed by CD4+ T cells increased in a concentration-dependent manner  in vivo. First, we treated normal C57BL/6 mice for 7 days with 200 mg AMI-1/kg/day or PBS, and the data showed that, in the spleen and mesenteric lymph node (MLN), the frequency and absolute numbers of CD4+Foxp3+ T cells increased after the treatment of mice with AMI-1. Additionally, this treatment significantly enhanced the Tregs’ functions that can significantly inhibit Teff proliferation than control Tregs in the coculture system ; however, the PRMT1 mRNA expression level was not significantly changed  . When the Tregs had their PRMT5 levels specifically knocked down by shRNA  and stimulated with the inflammatory cytokine IL-6, the Foxp3 expression level and the Tregs inhibition function were maintained compared with the control shRNA treatment .As AMI-1 is a general protein arginine methyltransferase inhibitor, we detected the mRNA expression of PRMTs during the intestinal inflammation. The data show that the abundance of PRMT1 and PRMT5 expression was high in the colon tissue; however, the abundance of other PRMTs, including PRMT2, PRMT3, PRMT4, PRMT6, PRMT7, and PRMT10 expression levels, were relatively low (Figure SCID mice were adoptively transferred with 1 × 106 CD45.2+ naïve T cells isolated from the mesenteric lymph nodes of WT C57BL/6 mice. Once colitis developed, the mice were intravenously injected with 5 × 105 PRMT5 knockdowns or control CD45.1+ Tregs or with the same number of CD45.2+ naïve T cells and followed for 3 weeks. The PRMT5 knockdown Tregs were significantly more effective than the control Tregs in promoting weight gain and reducing diarrhea (Figure + naïve T cells displayed dense inflammatory infiltrates, the mice-receiving CD45.2+ naïve T cells plus control Tregs exhibited considerably less leukocyte infiltration. The transfer of PRMT5 knockdown Tregs led to further improvement with only minor colonic infiltrates (Figure + Tregs lacking PRMT5 exhibited increasing suppressive functions in vivo.To directly assess the contribution of PRMT5 to Tregs function during T cell-induced colitis, + cells when naïve T cells were PRMT5 knocked down and treated with TGF-β compared with control shRNA T cells (Figures Data show significantly augment proportions of Foxp3foxp3 gene expression. PRMT5 has been reported as a type II arginine methyltransferase and could silence gene expression by symmetrical di-methylation of H4R3, H2R3, and H3R8 (Foxp3 regulatory regions (foxp3 gene expression in naïve T cells. Data show that, in the in vitro system, for Tregs differentiation, when the naïve T cells were specifically knocked down of PRMT5, the DNMT1 expression level was also decreased; however, the Foxp3 gene expression level was increased (Figures Foxp3 promoter site or the inhibitory epigenetic modification of H3K27me3 by performing a ChIP analysis using anti-DNMT1 and H3K27me3 antibodies. Both DNMT1 and H3K27me3 were bound to the Foxp3 promoter region in naïve T cells and to a lesser extent when the T cells were in a Tregs differentiation system; however, when PRMT5 was specifically knocked down, the binding was significantly reduced (Figure Foxp3 promoter region, we assessed the effects of PRMT5 deletion on Foxp3 promoter methylation through bisulfite conversion, cloning, and sequencing studies. The PRMT5 knockdown cells exhibited reduced CpG methylation within the foxp3 promoter of the T cells following Tregs induction (Figure We further explored how PRMT5 mediated the observed and H3R8 –27. Addiand H3R8 . DNMT1 s regions , 29. We  Figures A,B. We nd Figure C. As PRMn Figure D.+ T cells (Figure + T cells from the UC patients were specifically knocked down of PRMT5, the DNMT1 and Foxp3 expression level was increased compared with the control shRNA treatment (Figures + T cells isolated from UC patients were treated with AMI-1 and their Foxp3 expression levels were measured. The AMI-1-treated Tregs exhibited promoted Foxp3 expression (Figure To further explore the role of PRMT5 in Tregs development in UC patients, we first detected their PRMT5 expression levels, and they were significantly increased in the patients’ CD4s Figure A. Consiss Figure B, and ths Figure C. Howeve Figures D,E. We f Figures  and foun Figures F. Additin Figure G. Our da+ Tregs and increased their suppressive capability. Consistent with the AMI-1-treatment increased Tregs suppressive function in vitro, AMI-1 administration also promoted Tregs function by inhibiting homeostatic T-cell proliferation in SCID mice. PRMT5 expression increased during experimental inflammation colitis and significantly decreased after the AMI-1 treatment. Thus, our study reveals significant new roles that PRMT inhibitors could regulate immune responses under inflammatory settings by PRMT5, which can modulate Tregs differentiation and function. Additionally, inhibition of PRMT5 decreased DNMT1 expression, which can induce CpG methylation, also effected H3K27me3 modification, and both of which had critical roles in the regulation of Foxp3 gene expression. We do not propose that Foxp3 is the only target of the PRMT inhibitor in T cells, but rather that the effects of the PRMT inhibitor on Foxp3 in Tregs constitutes an important, and previously uncharacterized, therapeutically relevant mechanism of action. Recently, Liu et al. has reported that deleting PRMT5 in bone marrow cells reduced pro- and pre-B and impaired T cell development, suggesting that it is required for lymphocyte development (Protein arginine methyltransferases regulate diverse cellular programs, like tumor development, RNA transportation, chromatin reconstruction, and cell cycle control. However, how the PRMTs regulate the immune system, especially in the regulation of Tregs development and function in an inflammatory disease setting has been elusive. In this study, we showed that AMI-1 administration increased the frequency of peripheral Foxp3elopment . So PRMTThe pathogenesis of UC is complex and involves defects in mucosal barrier function and the immune system. Despite many important findings in recent years, it is still largely unknown why the mucosal immune response is over-reactive in UC. Reduced peripheral Tregs numbers have been associated with both active CD and UC, and they were shown to be inversely correlated with disease activity , 33. ColOur data show that PRMT5 loss led to enhanced Foxp3 expression and increased Tregs function and that its reduction promoted Foxp3 demethylation and restored Tregs function to normal. Additional studies are required to further elucidate the importance and precise interactions of PRMT5 with Foxp3 using PRMT5 conditional knockout mice. A good knowledge of the interactions between PRMT5 and Foxp3 could offer new choices for developing specific small-molecule and specially targets toward Tregs, rather than using a widely used inhibitor such as AMI-1. Collectively, our study demonstrates a new mechanism that enhances Tregs number and functioning as a result of PRMT5 knockdown. We conclude that developing a small-molecule inhibitor or other strategies to inhibit the function of PRMT5 might be of therapeutic importance in the management of UC and potentially other autoimmune diseases.All of the patients provided written informed consent to participate in the study, which was carried out in accordance with the principles of the Declaration of Helsinki and was approved by the ethics committee of Xin Hua Hospital.YZ, LH, and LS designed and discussed the study. YZ and LH carried on most of the experiments, collected and analyzed data. WG and MY recruited study participants and provided clinical samples. WW helped with cell sorting. YZ, LH, YM, GX, BB, LL, HN, and LS contributed to the writing of the paper.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.