Enzymatic catalysis in living cells enables the in-situ detection of cellular metabolites in single cells, which could contribute to early diagnosis of diseases. In this study, enzyme is packaged in amorphous metal-organic frameworks (MOFs) via a one-pot co-precipitation process under ambient conditions, exhibiting 5–20 times higher apparent activity than when the enzyme is encapsulated in corresponding crystalline MOFs. Molecular simulation and cryo-electron tomography (Cryo-ET) combined with other techniques demonstrate that the mesopores generated in this disordered and fuzzy structure endow the packaged enzyme with high enzyme activity. The highly active glucose oxidase delivered by the amorphous MOF nanoparticles allows the noninvasive and facile measurement of glucose in single living cells, which can be used to distinguish between cancerous and normal cells. For packaging enzymes into metal–organic frameworks (MOFs), crystalline MOFs are usually used. Here, the authors encapsulated enzymes in amorphous MOFs a via one-pot co-precipitation process under ambient condition, which led to higher enzymatic activity than in a corresponding crystalline MOF composite. Enzymes, as the catalysts in cells, in principle, can accelerate specific reactions to covert intracellular metabolites to detectable products. This biocatalysis in cells could provide a new method for the precise detection of metabolites in living cells. However, the delivery of enzymes and the retention of enzyme activity in cells remain challenging.Detection of intracellular metabolites is important for biomedical applications, for example, the diagnosis of cancers as well as many other diseases3. To date, there are two approaches to incorporate enzymes in MOFs. The most frequently used strategy is the adsorption of enzymes in as-synthesized crystalline MOFs7, which involves the pre-synthesis of mesoporous MOFs followed by the loading of enzyme molecules in the well-designed mesopores in MOFs that have sizes slightly larger than protein molecules. The other approach proposed by our group and others is a one-step process, in which the enzyme molecules, metal ions, and organic ligands are mixed in solution to readily form the enzyme-crystalline MOF composites13. However, the possibility of hosting molecules by amorphous MOFs (aMOFs) with disordered and fuzzy structures has not been explored.Enzymes usually lose their three-dimensional structure under harsh conditions such as high temperatures, polar organic solvents, pH extremes, and protease interactions, resulting in the serious deactivation of enzymes. Previous efforts have been made to increase enzyme stability for the wide applications in biocatalysis, biosensing, and biomedicine. Recently, as a promising candidate, crystalline metal-organic frameworks (MOFs) have been utilized to protect the encapsulated enzymes inside MOFs under harsh conditionsHere, we report the in situ packaging of enzymes in aMOFs at ambient conditions. The enzyme-aMOF composites are prepared by a one-step process, simply by mixing enzyme, metal ions, and organic ligands in an aqueous solution. The extended X-ray absorption fine structure (EXAFS) analysis and molecular dynamic (MD) simulations suggest that the formation of amorphous structures is mainly caused by coordination defects between metal ions and organic ligands. The mesopores in aMOFs as observed by cryo-electron tomography (Cryo-ET) allow the encapsulated glucose oxidase (GOx) to display 20 times higher activity than that in crystalline MOFs. The aMOFs provide a reasonable protecting effect for the encapsulated enzyme, displaying higher stability compared with the native counterpart. This high activity and stability of enzyme-aMOF composites allows its application in intracellular biosensing. The delivery of GOx by aMOFs enables noninvasive measurement of glucose in a single living cell, which identifies cancer cells from normal cells. This capability shows great promise in cancer diagnosis and understanding tumor metabolism.11 of the in situ incorporation of enzyme in a crystalline zeolitic imidazolate framework-8 (ZIF-8). After mixing 2-MeIM (40 mM), zinc acetate (10 mM), and GOx (0.25 mg mL−1) in aqueous solution at room temperature under stirring for 30 min, the product was collected via centrifugation and washed with deionized water. Scanning electron microscopy (SEM)   compared to the previous reportEM) Fig.  and tranEM) Fig.  showed tEM) Fig.  confirmeg 2-MeIM 0 mM, zin15, ball milling17, or heating19. Here, we highly suspected that, we have prepared aMOFs and their composites with protein in aqueous solution under ambient conditions by just modulating the concentration of organic ligands.Thus, we examined the crystallinity of enzyme-MOF nanocomposites by X-ray diffraction analysis. Different from pure ZIF-8 and enzyme-ZIF-8 composites, XRD patterns of nanocomposites with/without enzyme Fig.  implied 2+ and 2-MeIM. The X-ray photon spectroscopy  and PDF, G(r) of both ZIF-8 and aZIF. The experimental results agreed well with the main peaks obtained from simulation  is ca 1.19 nm22. Similarly, a linear electron density scan and its fast Fourier transformation were performed along the red dashed line in Supplementary Fig. 2 adsorption analysis  Fig.  with messis Fig.  experimesis Fig. . Please d-glucose (2-NBDG). 2-NBDG was incubated with GOx-ZIF-8 or GOx-aZIF nanocomposites under the same conditions. The fluorescence intensity of GOx-aZIF nanocomposites augmented quickly and reached a plateau after 6 min  amino]-Km, of the encapsulated GOx was 2.0 mM . In contrast, for GOx encapsulated in ZIF-8, Km was increased to 12 mM, suggesting a severe limitation for substrate transportation  and catalase (CAT) were encapsulated in aZIFs by the same procedure, producing similar nanospheres with diameters ranging from 80 to 100 nm , which can be identified by the elevated fluorescence of a •OH-sensitive fluorescent dye . Considering the acid degradation pathway for free GOx , while the peak FI for the GOx-aZIF was four times higher of that for GOx-ZIF-8. At low dosage (4.5 μg mL−1 of GOx-aZIF and GOx-ZIF-8), after delivery of enzyme in cells, the majority of GOx-aZIF and GOx-ZIF-8 was possibly deactivated before they can catalyze enough glucose, resulting in slight difference between GOx-aZIF and GOx-ZIF-8 , the difference of enzyme activity between GOx-aZIF and GOx-ZIF-8 was evident and clearly observed , because both GOx-aZIF and GOx-ZIF-8 were excessive, most of the glucose was consumed very quickly at a similar rate  from normal liver cells (L02)  to cancer cells (MGC803 < MCF-7 < 4T1). Subsequently, the DCF fluorescence intensity of different cell types was noninvasively detected after delivery of the GOx-aZIF in cells  Fig. , becausells Fig. . GOx-aZIlls Fig. ; thus, tlls Fig. . The linlls Fig. .In conclusion, we propose that aMOFs can package enzymes with highly retained activity. We studied the chemical structure of aZIF and proved that mesopores (from 1 to 10 nm) were generated in the aZIF during the formation of amorphous structure and incorporation of protein molecules. These mesopores facilitated the substrate transportation and thus greatly improved the activity of encapsulated enzyme, which was demonstrated by different types of enzymes. The delivery of GOx by aZIF allowed the dynamic detection of glucose in a single living cell, which could be used for discriminating different cell types and for distinguishing between normal and cancer cells. This also sheds light on the application in detecting other cellular metabolic processes in a mild and efficient way and promotes the development of new drug delivery systems for medical therapy.g, 5 min, ambient temperature), washed for three times with deionized water and lyophilized. Due to the excess amount of 2-MeIM, the yield was calculated based on the conversion of zinc. The product was weighed to be 1.8 ± 0.1 mg with a yield of ca. 41% according to ICP-OES results.In a typical experiment, 1 mL of zinc acetate solution (20 mM) was added into 1 mL of 2-MeIM (80 mM), followed by stirring for 30 min in a 5 mL glass bottle. The synthesized aZIF was then centrifugated  and horseradish peroxidase (0.05 mg mL−1). The increase of absorbance at 415 nm was measured by using a ultraviolet/visible (UV/Vis) spectrophotometer. The MM constant, Km, was obtained by the non-linear fitting of initial reaction rate with substrate concentration according to the MM equation.The activity of GOx in aqueous solution was measured by using glucose as the substrate in phosphate-buffered saline (PBS) (pH 7.4). In a typical measurement, 50 μL of GOx-aZIF nanocomposites or free GOx with the same amount of protein was added in PBS containing glucose (100 mM), ABTS , 0.28 mg mLp-nitrophenyl butyrate (p-NPB) as the substrate. First, p-NPB was dissolved in acetone and then diluted with PBS  containing 1.25% (w/v) Triton X-100, with a final concentration of 0.5 mM. The reaction was initiated by adding 50 µL of enzyme solution  to 950 µL of the substrate solution. The absorbance at 348 nm was recorded using a UV/Vis spectrophotometer.The hydrolytic activity of CALB-ZIF-8 was determined using 2 were maintained in Dulbecco’s modified Eagle’s medium medium containing 10% fetal bovine serum (FBS). Mouse breast cancer cell 4T1 and human normal hepatocyte L02 were maintained in RPMI-1640 medium containing 10% FBS. All cells were incubated at 37 °C in an atmosphere of 5% CO2. MCF-7 and 4T1 were from the American Type Culture Collection. MGC803 was from the China Infrastructure of Cell Line Resource. L02 was from the Cell bank of Type Culture Collection of the Chinese Academy of Sciences.The human gastric cancer cell MGC803, human breast cancer cell MCF-7, and hepatic cancer cell HepG2O2  and produce highly fluorescent DCF. Cells were primarily allowed to adhere for 24 h in 96-well plates and be washed for three times with glucose-free PBS prior to the detection. Subsequently, the DCFH-DA dye (at a work concentration of 10 µM) and the GOx-aZIF or GOx-ZIF-8 at certain concentrations were simultaneously added into the cells for dynamic detection . The fluorescent images of the cells were recorded during 4-h incubation period (37 °C and 5% CO2) via the “Operetta CLS” High Content System (PerkinElmer). Alex Fluor 488 channel (LED power) was selected to acquire the fluorescent signal from DCF, which was excited at 460–490 nm and recorded at 500–550 nm emission wavelength via standard filter sets. Ten percent power was set for the excitation, and 10 ms exposure time was controlled to avoid saturated pixels. Particularly, the instrument was equipped with a 16-bit sCMOS camera, which operated in a fast acquisition mode for exposure time ≤10 ms. The pinhole size was 55 µm.The intracellular glucose was dynamically monitored by using DCFH-DA dye, which could be de-esterified intracellularly to form DCFH. DCFH could react with HFurther information on research design is available in the Supplementary InformationDescription of Additional Supplementary FilesSupplementary Movie 1Supplementary Movie 2Supplementary Movie 3Supplementary Movie 4Supplementary Movie 5Reporting SummarySource Data


Our data suggest that D2R-dependent cross-talk modulates Wnt3a expression via an evolutionarily-conserved TCF/LEF site within the WNT3A promoter. Moreover, D2R signaling also modulates cell proliferation and modifies the pathology in a renal ischemia/reperfusion-injury disease model, via its effects on Wnt/β-catenin signaling. Together, our results suggest that D2R is a transcriptional modulator of Wnt/β-catenin signal transduction with broad implications for health and development of new therapeutics.The Wnt/β-catenin pathway is one of the most conserved signaling pathways across species with essential roles in development, cell proliferation, and disease. Wnt signaling occurs at the protein level and via β-catenin-mediated transcription of target genes. However, little is known about the underlying mechanisms regulating the expression of the key Wnt ligand Wnt3a or the modulation of its activity. Here, we provide evidence that there is significant cross-talk between the dopamine D Renal proximal tubule cells express components of the DA biosynthetic machinery as well as both D1-like  and D2-like  DA receptors7. These cells therefore endogenously synthesize DA from circulating precursors, and utilize the newly synthesized DA locally, independent of the nervous system, to signal in an autocrine/paracrine manner9. This renal DA signaling regulates sodium transport to maintain fluid and electrolyte homeostasis and normal blood pressure10. Knockdown of any of the five DA receptor subtypes consequently results in increased blood pressure in mice11. Moreover, kidney-specific downregulation of the DA D2 receptor (D2R) increases oxidative stress and causes renal tubulointerstitial fibrosis and glomerulosclerosis14. Nevertheless, the precise mechanisms of D2R signaling and their relationships to renal physiology and disease remain poorly understood.Dopamine (DA) signaling, although traditionally studied in the central nervous system, is increasingly implicated in regulating the function of peripheral organs, including the pancreas and kidney2-like DA receptors, including D2R, are class A G protein-coupled receptors (GPCRs) that signal via two downstream intracellular pathways: (i) G protein-dependent, arrestin-independent, and (ii) G protein-independent, arrestin-dependent pathways17. In the G protein-dependent pathway, D2R activation results in the recruitment of Gαi/o and ultimately leads to decreased levels of cyclic AMP (cAMP) synthesis18. In the G protein-independent, arrestin-dependent pathway, recruitment of arrestin 3 (β-arrestin-2) causes D2R internalization and inactivation of additional extracellular D2R signaling19. This β-arrestin-2-dependent pathway also leads to the recruitment of protein phosphatase 2 A (PP2A) and serine/threonine kinase AKT to the β-arrestin-2/D2R complex20. PP2A dephosphorylates AKT, leading to inactivation of AKT21. AKT regulates another key signaling kinase, glycogen synthase kinase β (GSK3β), through phosphorylation. In its non-phosphorylated state, GSK3β is constitutively active, whereas AKT-induced phosphorylation inactivates GSK3β20. This D2R and β-arrestin-2-dependent AKT regulation of GSK3β has been increasingly implicated in neuronal physiology and pathophysiology, as well as in the mechanisms of both antipsychotic drugs and lithium action24. Notably, while D2R signals via G-protein-dependent and -independent pathways in response to stimulation, evidence also suggests that there may be an additional component of constitutive activity in both pathways27.D29. The Wnt/β-catenin pathway is nearly ubiquitous across species and plays numerous critical roles in development and in both cell physiology and pathology including cancer30. This signaling pathway is comprised of Frizzled (FZD) Wnt receptors and the LRP5/6 co-receptor in association with Dishevelled, a scaffolding protein that activates a β-catenin destruction complex that includes Axin, Adenomatous polyposis coli (APC), and GSK3β32. In the absence of a stimulus, β-catenin, a transcriptional co-activator of Wnt target genes, is phosphorylated by active GSK3β and directed to the proteosomal pathway for degradation31. However, following activation of FZD/LRP5/6 by ligands, including Wnt3a, β-catenin translocates into the nucleus and binds to T-cell factor (TCF)/lymphoid enhancer-binding factor (LEF) proteins to stimulate Wnt target gene transcription34. Significantly, the ability of β-catenin to evade proteosomal destruction and initiate gene transcription is mediated by inactivation of GSK3β activity. GSK3β activity may be inhibited through phosphorylation of serine 9 or 21 through the actions of several kinases including AKT, protein kinase A (PKA), and integrin-linked kinase (ILK)36. Additionally, GSK3β activity is inhibited in response to treatment with growth factors including insulin/insulin-like growth factor I (IGF-I), nerve growth factor (NGF), or through inhibitory auto-phosphorylation42. Here, given AKT’s prominent role in D2R′s G protein-independent, arrestin-dependent pathway, we focus on D2R-mediated GSK3β inactivation via AKT phosphorylation43 and the resulting effects on β-catenin-driven changes in gene expression.In addition to AKT’s roles in β-arrestin-2-dependent D2R signaling, this kinase may also contribute to the regulation of signaling within the Wnt/β-catenin pathwayin vitro cellular and in vivo animal models in both human and mouse renal proximal tubule cells to elucidate D2R′s role in modulating the Wnt/β-catenin signaling pathway, given the importance of both D2R and Wnt signaling pathways in this cell type to kidney function including blood pressure regulation12. Using these models, we demonstrate a new paradigm by which stimulation of a GPCR, D2R, modulates Wnt/β-catenin signaling, Wnt3a expression, and cell proliferation in healthy and disease states, via its effects on gene transcription.Since AKT plays important roles in both D2R and Wnt/β-catenin signaling pathways, we asked whether there is an AKT-dependent crosstalk between these two pathways where effects on AKT activity in one pathway may modulate downstream effects in the other. Moreover, we examined whether stimulation or inactivation of D2R signaling modulates β-catenin-mediated effects on transcription of Wnt pathway target genes. We established both 14, as well as key proteins in the β-arrestin-2-dependent pathway including GSK3β, AKT, and PP2A46. However, to date, the extent of endogenous renal expression of β-arrestin-2 and its conservation across species remain unclear. We found that β-arrestin-2 was endogenously expressed in mouse renal cortex, as well as in both mouse and human renal proximal tubule cells  D2R activation leads to dephosphorylation of active, phosphorylated AKT (P-AKT) and, (2) in the setting of decreased P-AKT, repressive phosphorylation of GSK3β is also reduced, thereby increasing GSK3β kinase activity 54. We used the D2R agonist bromocriptine since bromocriptine is a highly potent and effective modulator of GSIS in INS-1E cells56. Consistent with our data in the kidney, we found that overnight treatment of INS-1E cells with bromocriptine significantly inhibited TCF/LEF transcription  increased D2R expression 10-fold  transcription is modulated by cis-regulation through β-catenin signaling64. The presence of a TCF/LEF autoregulatory element within the HyWnt promoter allows β-catenin to further increase HyWnt expression and thus potentiate Wnt signaling64. Based on D2R′s effects on β-catenin phosphorylation and TCF/LEF transcription, we asked whether similar cis-regulatory elements are used to regulate mammalian Wnt3a expression. To examine this possibility, we searched for potential TCF/LEF binding sites in the promoter region of the mammalian WNT3A gene. We constructed a phylogenetic tree of human WNT3A and discovered marked conservation across several species: human, mouse, rat, chicken, and zebrafish  of human, rat, mouse, and chicken WNT3A but only two TCF/LEF binding sites in zebrafish  model of acute kidney injury. We chose this model system given the critical role played by cell proliferation in the associated pathologyels Fig. , consistlls Fig. . By contlls Fig. . Increaslls Fig. . Confocalls Fig. . These d63. Yet, despite decades of study, many fundamental questions still remain concerning the mechanisms by which these two pathways are regulated and whether they intersect in specific cellular states or cell types. For example, though the sequence of signaling within the Wnt/β-catenin pathway has been thoroughly mapped out, the mechanisms regulating the expression of key Wnt ligands such as Wnt3a remain poorly understood. Similarly, downstream of the DA receptors, the precise physiological relevance of signaling via the G protein-dependent versus -independent pathways is largely unknown. Last but not least, despite sharing several common signaling components15, it has remained unclear whether DA and Wnt pathways modulate one another and, if so, under what contexts.Both DA and Wnt signaling pathways are nearly ubiquitous across organ systems and species. These signaling systems are integral drivers of physiological processes across development and have been implicated in a diverse array of diseases ranging from cancer to schizophrenia69. These organs have the capacity to synthesize DA, which then signals locally in an autocrine and/or paracrine manner through DA receptors, including D2R70. In the pancreatic β-cell, D2R signaling is implicated as a negative modulator of insulin secretion52. In the kidney, renal DA regulates systemic blood pressure, in part, through D2-like receptor signaling12, via its effects on fluid and electrolyte balance as well as regulation of the secretion/metabolism of hormones such as aldosterone and angiotensin II70. Moreover, genetic hypertension is associated with alterations in DA production and D2-like receptor function6. Likewise, Wnt/β-catenin pathway signaling is essential in both kidney and pancreatic development and is implicated in the pathophysiology associated in both organs72.While DA has been studied primarily in the central nervous system, it also plays important functions in diverse peripheral organs including the kidney, pancreas, and gut73. The β-arrestin-2 pathway is an important route for further downstream signaling involving AKT and GSK-3β kinases, critical signaling molecules implicated in numerous cellular functions including metabolism and proliferation74. Though the original β-arrestin-2 studies were conducted largely in rodent brain and in vitro, we show that these β-arrestin-2-dependent D2R signaling pathways are present in both human and rodent renal cells, suggesting that D2R′s capacity to modulate AKT phosphorylation and activity is a more general principle that is conserved across tissues and species. Our data also establish a model where D2R signaling via the β-arrestin-2-dependent pathway modulates TCF/LEF promoter activity through its ability to modify β-catenin’s degradation and capacity to translocate into the nucleus. This represents an important new mechanism by which GPCRs like D2R may utilize G protein-independent, β-arrestin-2-dependent signaling to modulate transcription via interactions with the Wnt/β-catenin pathway.Here, we present evidence that signaling through D2R modulates the Wnt pathway. We provide a putative mechanism for this crosstalk: D2R-dependent β-arrestin-2 signaling leads to diminished phosphorylation of β-catenin. This in turn promotes β-catenin’s translocation to the nucleus and increased TCF/LEF-mediated transcription within the Wnt/β-catenin pathway, including Wnt3a itself. In addition to G protein-dependent signaling, D2R also signals through β-arrestin-2, a protein long known to facilitate receptor internalization75, ultimately leading to changes in β-cell mass through its actions on TCF/LEF-based transcription76. Additionally, Min and colleagues reported that the effects of D2R on neuronal Wnt signaling could also be agonist-independent and based, in part, on direct physical interactions between D2R and β-catenin25. This raises the possibility that at least some of this signaling is constitutive. Indeed, our data in renal cells using D2R blockade by sulpiride or siRNA-mediated D2R knockdown indicate some degree of constitutive  regulation of Wnt signaling which is consistent with this earlier work25. Moreover, in addition to β-arrestin-2-dependent GPCR signaling, there is increasing evidence that inhibitory heterotrimeric Gαi/o proteins are also involved in Wnt-induced responses79. Therefore, we propose that D2R stimulation may also trigger recruitment and activation of sufficient Gαi/o to displace these proteins from FZD receptors, leading to a reduced G protein-dependent Wnt-stimulated response. Such a model may provide an additional explanation for our findings demonstrating that D2R stimulation by agonists including DA, quinpirole and bromocriptine reduces Wnt-dependent signaling in β-cells.Analogous to the kidney, we found that D2R activity also modulates Wnt3a-dependent β-catenin transcriptional activity in INS-1E cells, a rat pancreatic β-cell line, suggesting that D2R′s ability to modulate Wnt signaling is conserved across both species and tissues. There is precedent for GPCR modulation of Wnt/β-catenin signaling in β-cells. Earlier work demonstrated that agonists of glucagon-like peptide-1 receptor, a GPCR, could enhance β-cell Wnt signalingDRD2 SNPs that negatively regulate endogenous D2R expression and function82. Several SNPs within the human DRD2 gene have been associated with clinically relevant conditions including elevated blood pressure and essential hypertension13. Moreover, a recent study in an Asian Indian population with type 2 diabetes found that a SNP that decreased D2R expression also conferred susceptibility to chronic diabetic nephropathy83. Similarly, the combination of two highly prevalent DRD2 SNPs, rs6276 and rs6277, decreased D2R expression as a consequence of diminished D2R mRNA stability as well as via decreased receptor availability and affinity82. We found that the combined effects of these two DRD2 SNPs also diminished levels of phosphorylated β-catenin, presumably freeing more β-catenin to successfully evade degradation and thus to stimulate transcription of Wnt/β-catenin pathway target genes. Given the Wnt/β-catenin pathway’s roles in mediating cell proliferation, our results suggest that D2R signaling may utilize such an intersection with Wnt signaling to modulate β-catenin and Wnt-dependent cell proliferation. Consistent with this, recent studies suggest that D2-like receptor activation inhibits cellular proliferation while, conversely, its inhibition increases cellular proliferation87. Furthermore, in pancreatic β-cells, pharmacological D2R inhibition not only increases cell proliferation, but also decreases cell de-differentiation, collectively leading to increased β-cell mass85. Given D2R′s ability to modify β-catenin-mediated TCF/LEF transcription, we propose that the interconnections between D2R and Wnt signaling may be critical in modulating cell proliferation under physiological circumstances, at least in the cell types we assayed . Moreover, D2R′s inhibitory effects on proliferation may be a regulatory mechanism by which cells avoid uncontrolled expansion.We further dissected D2R-mediated effects on TCF/LEF transcriptional activity in human renal proximal tubule cells, taking advantage of 2 in kidney), is also implicated in these processes. Indeed, Wnt3a has been shown to mediate cell proliferation89. We used several complementary approaches to demonstrate significant increases in Wnt3a expression in response to reduced D2R expression including D2R knockdown in mouse renal proximal tubule cells in vitro and renal cortex in vivo, as well as human renal proximal tubule cells where D2R expression is reduced by the presence of SNPs. Together, our results suggest an important relationship between D2R signaling and Wnt3a expression. Furthermore, these data describe a possible new mechanism where alterations in D2R expression may trigger a corresponding change in Wnt3a levels which then alters β-catenin-mediated TCF/LEF transcription in processes including cell proliferation.Given the interconnections between D2R and Wnt/β-catenin signaling pathways, we investigated whether Wnt3a, one of the primary ligands for the Wnt receptor FZD , and thus alter levels of gene transcription93. Such a mechanism may have direct implications for β-catenin-mediated transcription since CREB-BPs are also constituents of the β-catenin/TCF transcriptional complex94. Therefore, for a Gαi/o-coupled receptor like D2R, G protein and cAMP-dependent signaling may synergize with D2R′s effects on β-catenin phosphorylation to ultimately determine the efficacy of β-catenin/TCF/LEF-mediated transcriptional regulation.At present, much of our understanding of D2R and GPCR signaling more generally derives from its acute effects on downstream G proteins. While G protein-dependent signaling is often rapid, occurring over seconds and minutes, here we show that the G protein-independent, β-arrestin-2-dependent arm of D2R signaling may also play important roles in cellular physiology and pathophysiology on a longer time scale via its effects at the transcriptional level. This suggests a paradigm where D2R utilizes multiple highly conserved downstream signaling pathways to selectively induce both acute changes in signaling and concurrent longer-term changes via its effects on transcriptional regulation. Such GPCR-modulated effects on transcription may be quite relevant physiologically to regulate cell proliferation or differentiation during development as well as in the context of disease states including diabetes or hypertension. There is precedent for GPCR regulation of transcription through the receptors’ effects on intracellular cAMP levels via G protein-dependent signaling95, our findings may shed important new light on the underlying mechanisms responsible for these profound and previously unanticipated effects on cellular physiology. These studies therefore open the door to a broader understanding of intracellular signaling and its implications in health and disease.We show that D2R modulates signaling through the Wnt/β-catenin pathway, in part, through its effects on Wnt3a expression. Our work presents a novel mechanism by which a GPCR, D2R, works through the β-arrestin-2-dependent pathway to modulate transcription. Furthermore, given the ability of D2R-blocking drugs, such as antipsychotic medications to induce metabolic syndrome consisting of disturbances such as diabetes and hypertensionThe drugs used in the present study were purchased from Sigma-Aldrich , unless indicated otherwise: plasmocin ; EGF, dexamethasone, triiodothyronine, insulin-transferrin-selenium ; penicillin/streptomycin ; fetal bovine serum (Invitrogen); amphotericin , and G418 , (−)-quinpirole hydrochloride, (S)-(−)sulpiride, LiCl, bromocriptine mesylate, L-glutamine, HEPES, sodium pyruvate, and 2-mercaptoethanol.et al.96; cells were kindly supplied by Dr. Ulrich Hopfer . These cells were differentiated into mature mouse renal proximal tubule cells (mRPTCs) and cultured to 60–70% confluence. mRPTCs were maintained in DMEM-F12 (Invitrogen) supplemented with fetal bovine serum (10%), penicillin/streptomycin (1X) and amphotericin (2.5 μg/ml). The mRPTCs were subsequently transfected with either vehicle , non-silencing AllStars Negative Control siRNA  or mouse D2R siRNA  and studied 72 hr post-transfection. For other experiments, mRPTCs were cultured to 90–95% confluence, serum-starved for 2 hr and treated for an additional 24 hr in serum-free medium with vehicle (PBS) or drug (1 μM quinpirole or 1 μM sulpiride).Undifferentiated mouse cells were cultured from progenitor kidney cells isolated from mouse embryo kidneys following the procedure described by Woost Human renal proximal tubule cells (hRPTCs) were isolated from human kidney specimens obtained solely from male patients who had unilateral nephrectomy due to renal carcinoma or trauma. All samples were completely de-identified. Informed consent was obtained from all subjects for collection of these specimens. All procedures strictly followed the guidelines of a University of Virginia institutional review board-approved protocol, as well as the Declaration of Helsinki.97. Immortalized hRPTCs were maintained at 37°C, 5% CO2 and cultured to 90–95% confluence in non-polarizing conditions in DMEM-F12 (Invitrogen) containing : plasmocin (2.5 μg/ml), EGF (10 ng/ml), dexamethasone (36 ng/ml), triiodothyronine (2 ng/ml), insulin-transferrin-selenium , penicillin/streptomycin (1X), fetal bovine serum (2%), and G418 (0.4%). The hRPTCs were also genotyped for the presence of rs6276, rs6277 DRD2 SNPs. Four cell lines from subjects bearing rs6276 and rs6277 DRD2 SNP alleles as well as four control subjects without these SNPs (D2R-WT) were studied. These two SNPs occur within and adjacent to the DRD2 gene. Because of their close proximity, they are likely to be in linkage disequilibrium which limits the possibility of identifying cell lines bearing only one of the SNPs. hRPTCs were transfected with either vehicle , non-silencing AllStars Negative Control siRNA , human D2R siRNA , or human Wnt3a siRNA  and studied 72 hr post-transfection.For isolation of the hRPTCs, only the healthy renal pole  distal from the affected part of the kidney was used; cells were subsequently immortalized, as previously describedTo test the effects of Wnt/β-catenin pathway activation on D2R mRNA expression, hRPTCs were incubated in serum-free medium in the presence or absence of either recombinant Wnt3a protein  , LiCl  or vehicle controls. mRNA was extracted from the respectively treated cells and cDNA was subsequently prepared as described below. Changes in D2R expression were measured by quantitative real-time PCR.2. The cells were cultured as described earlier51 with RPMI 1640 media , supplemented with 5% (v/v) heat inactivated fetal bovine serum, 2 mM L-glutamine, 10 mM HEPES, 1 mM sodium pyruvate, 100 units/mL penicillin, 100 μg/mL streptomycin, and 50 μM 2-mercaptoethanol. The cells were subcultured weekly in a ratio of 1:5 to 1:10 with medium renewal every 3 to 4 days.INS-1E cells  were maintained in a humidified 37 °C incubator with 5% COMouse fibroblast-derived L-Wnt3A cells  that stably express and secrete Wnt3a and the control untransfected parental L-Cell line  were purchased from ATCC . Control L-cells were cultured in Dulbecco’s Modified Eagle’s Medium (Thermo Fisher Scientific) supplemented with 10% (v/v) fetal bovine serum. L-Wnt3A cells were supplemented with the same media as control L-cells along with the addition of 0.4 mg/ml G-418. Both cell lines were subcultured in a ratio of 1:10 with medium renewal every 2 to 3 days.DRD2 under the control of cytomegalovirus promoter , using the Fugene HD transfection reagent  as per the manufacturer’s guidelines. Briefly, 4 μg of plasmid DNA were diluted to 400 μl with serum-free medium and mixed with 16 μl of Fugene HD transfection. The transfection complex was incubated for 15 min at room temperature and added dropwise onto the cells in 10 cm tissue culture plates. Three days following transfection, the hRPTCs were harvested for protein extraction and immunoblotting, total RNA preparation, and RT-qPCR, or processed for immunofluorescence staining. Transfection efficiency was 40–45%. Transfections in INS-1E cells were performed using Lipofectamine 2000 transfection reagent (Invitrogen) overnight according to manufacturer instructions.hRPTCs were transfected with an empty vector or plasmid harboring wildtype human DRD2 cDNA from plasmid RC202476 (Origene Technologies Inc.) was excised as a KpnI and FseI fragment and inserted in between the same restriction sites within the pACS plasmid98. The control AAV (CAAV) vector, which does not encode any protein, contains an EGFP cDNA in reverse orientation under the control of the CMV promoter. Recombinant AAV vector genomes were subsequently packaged into capsids from the AAV-9 serotype, via the triple transfection method in HEK 293 cells, as described previously100.To construct D2R AAV, a 1.7-kb human Drd2-specific siRNA  or non-silencing siRNA . After 48 hr, the mRPTCs were trypsinized and seeded for transient reverse transfection with a TCF/LEF luciferase reporter provided by the Cignal Reporter Assay TCF/LEF luciferase reporter system (SABiosciences-Qiagen). Following transfection, the mRPTCs underwent drug treatment with either quinpirole (1 μM) or vehicle for 24 hr; acute sulpiride treatment (1 μM) was for 6 hr. Acute recombinant Wnt3a treatment  was for 2 hr. INS-1E cells were similarly reverse-transfected using the Cignal Reporter Assay TCF/LEF luciferase reporter system, followed by treatment with either RPMI 1640 or Wnt3a-conditioned medium, in the presence or absence of the D2R agonist bromocriptine (10 µM) for 18 hr.mRPTCs were treated with 2 First Strand kit, following the manufacturer’s protocol (SABiosciences-Qiagen).Kidney samples or cell lysates were homogenized, and total RNA was extracted with Trizol  and further purified using the RNeasy RNA Extraction Mini kit (Qiagen). RNA samples were converted into first strand cDNA using an RT2 qPCR Primer Assay format, (SABiosciences-Qiagen) following manufacturer’s instructions. Primers used were: MKi-67 ; Wnt3a ; D2R ; β-arrestin-2 ; Gapdh ; all primers were commercially available from Qiagen. Data were analyzed using the ΔΔCt method and expressed as fold-change relative to control101. In the case of quantitation of β-arrestin-2 expression in control mouse and human renal samples, we specifically measured mRNA levels in arbitrary units in order to directly compare β-arrestin-2 levels of expression relative to the Gapdh control . cDNA was prepared using an RT2 First Strand kit as per the manufacturer’s protocol (SABiosciences-Qiagen). Quantitative gene expression was analyzed by real-time PCR, performed on an ABI Prism 7900 HT . The assay used SYBR Green Real-Time PCR detection method (Qiagen) and all gene-specific primer assays were commercially available in the RTFlash-frozen tissue homogenates and cell lysates were subjected to immunoblotting. The samples were homogenized or lysed in RIPA buffer, and equal amounts of protein (20 μg) were electrophoresed under reducing conditions on 4–20% gradient polyacrylamide gels and transferred onto nitrocellulose membranes. The primary antibodies used were: anti-phospho-AKT ; anti-AKT ; anti-phospho-GSK3β ; anti-GSK3β ; anti-phospho-β-catenin ; anti-β-catenin ; anti-D2R ; anti-Wnt3a ; anti-Gapdh ; anti-tubulin . Results were normalized to Gapdh or tubulin and expressed as percentage- or fold-change, relative to the average signal intensity obtained from the respective controls.4. Briefly, mouse kidneys were collected and fixed in 3.7% paraformaldehyde for 1-hr at 4°C. After washing in phosphate-buffered saline , the tissues were equilibrated with 30% sucrose in phosphate-buffered saline overnight. Paraffin-embedded tissue sections were immunostained for expression of Wnt3a using a rabbit monoclonal anti-Wnt3a antibody . We stained for Ki-67, a marker of cell proliferation, using a rabbit polyclonal anti-Ki-67 antibody . We also stained for β-catenin using a goat polyclonal anti-β-catenin antibody . DAPI was used to visualize the nuclei of individual cells. Secondary antibodies included donkey anti-rabbit Alexa Fluor 555  and a donkey anti-goat Alexa Fluor 488 . The cells were counted in at least 3 microscopy fields from each section at 10x magnification (n ≥ 3 per group). Confocal images of immunostained tissue were acquired at 20x magnification using a spinning disk confocal microscope .The procedures for immunohistochemistry were described earlier4 cells were seeded into a 96-well Isoplate (PerkinElmer Life Sciences) and grown in complete media. Bromodeoxyuridine  was added to the medium 24 hr prior to the end of the assay. The cells were subsequently fixed, the DNA was denatured, and the amount of incorporated BrdUrd was determined using an Eu-labeled monoclonal anti-BrdU antibody. Following formation of highly fluorescent chelates, fluorescent signal was detected by time-resolved fluorometry using a Victor3 multilabel reader (PerkinElmer Life Sciences). The same number of cells was seeded on a separate 96-well Isoplate and grown overnight before fixation with 4% paraformaldehyde and nuclear staining with DAPI. DAPI fluorescence was detected also using a Victor3 multilabel reader. Final results are reported as Eu fluorescence normalized to DAPI signal.Cell proliferation was assessed using the Delfia Cell Proliferation kit . Approximately 1 × 10All animals used in the study were C57BL/6 male mice  which were purchased from The Jackson Laboratory . All animals were housed and handled in accordance with all appropriate NIH guidelines. Our experimental protocol was approved by the institutional review board and ethics committee of George Washington University (IACUC protocol#A353). For all experiments, we abided by all relevant and appropriate animal care guidelines and regulations including the ARRIVE guidelines. All efforts were made to ameliorate animal suffering.102. The siRNA was loaded into the osmotic minipump with a polyethylene tube connected to the osmotic minipump. The tube was inserted underneath the kidney capsule for continuous siRNA delivery. A detailed description of the protocols used was provided previously4.Mice were anesthetized with an intraperitoneal injection of pentobarbital sodium (50 mg/kg) and placed in a supine position. The mice were treated with NS siRNA or D2R siRNA by subcapsular infusion (3 μg/day) using osmotic minipump  for 28 days in the left kidney, leaving the contralateral kidney intact4. The distal ureteral portions closest to the bladder and the renal artery supplying the target kidney were clamped off using a microvenous clip. The ureter was punctured using a tuberculin syringe fitted with a 33-gauge needle and urine was gently aspirated out. A tuberculin syringe was placed into the ureter containing ~100 μl of the respective AAV vector  and the solution was retrogradely infused into the ureter. The needle was withdrawn and a microvenous clip was placed proximal to the injection site on the ureter to prevent leakage and to attain maximum exposure to the infusion. Fifteen minutes later, the arterial and ureteral clips were sequentially removed, and the ureter was inspected for any evidence of leakage. The abdominal contents were replaced in reverse order and the incision site was closed.For retrograde ureteral infusion of AAV vectors, 14 days following the initiation of siRNA treatment, the mice were anesthetized and operated on to expose the kidneys4. In control animals, while the kidneys were exposed, the right and left renal vessels were not clamped. Upon clamp removal, a subset of the previously clamped mice were subjected to bilateral retrograde ureteral infusion of D2R AAV , as described above; the control group in this case consisted of clamped mice injected with control AAV (CAAV). The abdominal incision was closed, and the mice were allowed to recover. Fourteen days later, the mice were euthanized with an overdose of pentobarbital sodium (100 mg/kg). The kidneys, and other selected organs were harvested.The mice were anesthetized as above. Following exposure of the kidneys, both right and left renal vessels (artery and vein) were clamped with arterial clamps for 45 min to stop blood flow to both kidneysWNT3A gene orthologs in 5 species that have an annotated WNT3A gene in the UCSC genome browser: Human (hg19), Mouse (mm10), Rat (rn6), Chicken , and Zebrafish (danRer11). The 5-kb promoter region upstream of WNT3A TSS for each of the five species was searched for the eight possible WRE binding site sequences, 4 on each strand, in all available promoter isoform variants using a custom-made Python script. The same analysis was then extended to promoter regions 5-kb upstream of the DRD2 TSS, comparing the same five species for conserved binding sites: Human, Mouse, Rat, Chicken, and Zebrafish. Specifically, for the DRD2 ortholog in the danRer11 Zebrafish assembly, two different genes are listed in the UCSC genome browser: DRD2a and DRD2b. We found a conserved binding site corresponding to DRD2b which is reported in this study.On the basis of the following sequence recognized by β-catenin and several TCF/LEF transcription factors : 5′-(A/T)(A/T)CAAAG-3′ , we looked for conserved β-catenin-TCF/LEF binding sites upstream of WNT3A promoter activity was measured using the Secrete-Pair Dual Luminescence Assay System . The plasmid pWT/Wnt3a/GLuc contains a 1,721-base region from the human WNT3A promoter [−1635 to +86 of transcription start site (TSS)] driving the expression of naturally-secreted Gaussia luciferease (GLuc), and a secondary reporter with a CMV promoter driving the expression of secreted Alkaline Phosphatase (SEAP), serving as an internal control. The plasmid pMut/wnt3a/GLuc carries a mutation in which the conserved TCF/LEF binding site 5′-TTCAAAG-3′ at the position −1587 bases upstream of the TSS was mutated to 5′-CCGCGGT-3′. hRPTCs were co-transfected with NS siRNA (20 nM) or D2R siRNA (20 nM), and pWT/wnt3a/GLuc or pMut/wnt3a/GLuc. Two days later, hRPTCs were treated with fresh medium for 16 hr after which the cell culture supernatants were collected, and the luciferase activity measured via the Secrete-Pair Dual Luminescence Assay Kit . Luciferase activity results were expressed as relative light units (RLUs) normalized to SEAP activity (RLUs/SEAP).We used the Wnt3a protein coding regions (exon sequences) of human, mouse, rat, chicken, and zebrafish species to construct a phylogenetic tree using the MEGA-7 software package. The evolutionary distances in the units of base substitutions per site were calculated using the maximum composite likelihood method. There was a total of 1,052 positions after eliminating positions containing gaps and missing data. The phylogenetic tree was then constructed using the Neighbor-Joining method with the sum of branch lengths = 0.645.All results are expressed as mean ± SEM. Comparisons between experimental groups were performed using Student’s t-tests for two groups or ANOVA followed by post-hoc Holm-Sidak analyses for more than two groups. All statistical analyses were performed using the GraphPad Prism software package .Supplementary InformationDataset 1


Endothelial-to-mesenchymal transition (EndMT) plays significant roles in atherosclerosis, but the regulatory mechanisms involving lncRNAs remain to be elucidated. Here we sort to identify the role of metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) in ox-LDL-induced EndMT.−/− mice with high-fat diet, and the levels of lncRNA MALAT1 in mouse arterial tissue were detected by RT-qPCR. Cell model was established by treating human umbilical vein endothelial cells (HUVECs) with ox-LDL, and the levels of EndMT markers, such as CD31, vWF, α-SMA and Vimentin and lncRNA MALAT1 levels were detected and their correlations were analyzed. The role of MALAT1 in EndMT and its dependence on Wnt/β-catenin signaling pathway was further detected by knocking down or overexpressing MALAT1.The atherosclerosis model was established by feeding ApoEApoE−/− mice. HUVECs treated with ox-LDL showed a significant decrease in expression of CD31 and vWF, a significant increase in expression of α-SMA and vimentin, and upregulated MALAT1. An increased MALAT1 level facilitated the nuclear translocation of β-catenin induced by ox-LDL. Inhibition of MALAT1 expression reversed nuclear translocation of β-catenin and EndMT. Moreover, overexpression of MALAT1 enhanced the effects of ox-LDL on HUVEC EndMT and Wnt/β-catenin signaling activation.MALAT1 was upregulated in high-fat food fed Our study revealed that the pathological EndMT required the activation of the MALAT1-dependent Wnt/β-catenin signaling pathway, which may be important for the onset of atherosclerosis.Not applicable. Atherosclerosis is a multistep cardiovascular disease promoted by several kinds of risk factors and is one of the most common causes of mortality in the world . To dateMetastasis associated lung adenocarcinoma transcript 1 (MALAT1), which is also known as NEAT2 (noncoding nuclear-enriched abundant transcript 2) and was first identified in 2003 in non-small cell lung cancer , 15, is Wnt/β-catenin pathway, the canonical Wnt signaling, is reported to be involved in multiple aspects of the development and progression of atherosclerosis , includiIn this study, we intended to elucidate whether MALAT1 participates in the regulation of ox-LDL-induced EndMT through the Wnt/β-catenin signaling pathway. We found that MALAT1 was dysregulated in arterial tissues from ApoE−/− mice with high-fat food-induced atherosclerosis and human umbilical vein endothelial cell line (HUVECs) stimulated with ox-LDL. Overexpression of MALAT1 had similar effect as ox-LDL on EndMT of HUVECs and nuclear translocation of β-catenin. MALAT1 knockdown rescued EndMT of HUVECs via partially reversing β-catenin nuclear accumulation triggered by ox-LDL. Collectively, these data highlight the importance of MALAT1 in the development and progression of atherosclerosis.Male C57BL/6 mice (8-week old) and ApoE−/− (B6/JNju-Apoeem1Cd82/Nju) mice were obtained from Nanjing Biomedical Research Institute of Nanjing University . C57BL/6 mice were fed with standard chow diet and were used as control group. ApoE−/− mice were fed with high-fat diet (HFD) containing 21% fat, 1.25% cholesterol for 16 weeks and were used as atherosclerosis model group. All animals were kept in rooms with constant room temperature (25 °C) and with 12 h light-dark cycle (light on from 8:00 AM to 8:00 PM). The animal experiment was performed in accordance with the Animal Ethics Committee of Hebei Medical University.The thoracic aorta was harvested from mice following 16 weeks of HFD (at 24 weeks of age). After humane euthanasia, the thorax was entered and the heart swiftly exposed. The left ventricle was punctured using a 20-gauge needle, and 1x PBS (20 ml) at 4 °C was infused at 2.5 ml/min. For H&E staining, the thoracic aorta was then fixed in situ by perfusing the heart with 20 ml of 4% paraformaldehyde in PBS. The thoracic aorta was then isolated and placed in 4% paraformaldehyde for 24 h. For real-time quantitative PCR, the thoracic aorta was harvested and preserved in liquid nitrogen. The blood was harvested from mice by removing the eyeball under anesthesia following 16 weeks of HFD (at 24 weeks of age). Serum was isolated from blood by centrifugation for 15 min at 3000 rpm/min at 4 °C and stored at − 80 °C until each assay was performed.The serum total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C) levels were quantified by Hitachi 7080 automatic biochemical analyzer .After gradient dehydration and transparency, the thoracic aorta was embedded with paraffin and subsequently sectioned at 4 μm thickness. Then, the sections were stained with Hematoxylin and Eosin Staining Kit (H&E) , and then photo-graphed with Leica DM6000B fully automatic biological microscope .HUVECs were from Cell Bank of the Chinese Academy of Sciences  and were cultured in Dulbecco’s modified Eagle medium  containing 10% FBS , 2 mM glutamine, and 1% penicillin in a humidified atmosphere with 5% CO2 at 37 °C. HUVECs were treated with ox-LDL  at different concentrations  for 48 h.For MALAT1 overexpression, a pcDNA3.1 expression vector containing full-length human MALAT1 (pcDNA-MALAT1) was constructed by Shanghai Integrated Biotech Solutions . For MALAT1 knockdown, three MALAT1-targeting siRNAs  and a scrambled siRNA  (Scramble) were designed and synthesized at GenePharma . Lipofectamine™ 3000 Transfection Reagent  was used. Real-time quantitative PCR was used to validate the efficiency of MALAT1 knockdown and overexpression. For knockdown of MALAT1, HUVECs were transfected with MALAT1 siRNA or negative control siRNA for 24 h before treatment with ox-LDL (40 μg/ml) for 48 h. For overexpression of MALAT1, HUVECs were transfected with MALAT1 pcDNA or MALAT1 expressing vector for 48 h.For observing cell morphology, cellular F-actin was stained with rhodamine phalloidin , and nuclei were stained with DAPI Fluoromount-G® . Then the cells were imaged using a confocal laser scanning microscopy .-ΔΔCt method [Total RNA was extracted from fresh frozen thoracic aorta or cells collected at indicated time using Eastep® Super Total RNA Extraction Kit . The cDNA was prepared using the PrimeScript™ RT Reagent Kit with gDNA Eraser . Real-time quantitative PCR was performed in triplicates using TB Green™ Premix Ex Taq™  on applied biosystems 7300 Real-Time PCR system . The primers were purchased from Sangon Biotech CO., Ltd. . The sequences were shown in Table t method .Table 1TThe total protein was extracted from cells with ice-cold lysis buffer supplemented with protease inhibitor cocktail  and PMSF. The nuclear protein was extracted with Nuclear and Cytoplasmic Protein Extraction Kit . The protein concentration was determined with Detergent Compatible Bradford Protein Assay Kit . Proteins were separated on SDS-PAGE gels and transferred onto nitrocellulose membrane . After blocking with Odyssey® blocking buffer (PBS) , the membranes were incubated overnight with the primary antibodies of CD31 , vWF , α-SMA , vimentin , Histone H3 , β-catenin  and GAPDH  . After that, IRDye 800CW Goat anti-Rabbit IgG (H + L) Secondary Antibody , IRDye® 680LT Goat anti-Rabbit IgG (H + L) Secondary Antibody   were added and incubated for 1 h at room temperature. Then the band intensities were scanned by Odyssey  and normalized to GAPDH or Histone H3.After ox-LDL treatment or transfection, immunostaining of HUVECs was performed. Cells were fixed with 4% polyoxymethylene for 10 min at room temperature, 10 min at 4 °C and methanol for 5 min, then blocked with 1% BSA for 1 h at room temperature and incubated with primary antibodies of CD31 , vWF , α-SMA , vimentin , and β-catenin   overnight at 4 °C. After washing, Goat Anti-Rabbit IgG H&L (DyLight® 488)  was added and incubated for 1 h at room temperature, followed by nuclei staining with DAPI Fluoromount-G® . Then the cells were imaged using a confocal laser scanning microscopy .P < 0.05 was accepted as statistically significant.All graphs were created using Graphpad Prism software. All data were analyzed using SPSS version 19.0 software (SPSS lnc) and presented as mean ± SD (standard deviation). The two-tailed Student’s t-test was used to evaluate differences between two groups. Results from multiple groups were compared using one-way ANOVA test. Correlation analyses between variables were performed using the Pearson rank correlation test. Serum TC, TG and LDL-C levels in mice after 16-week HFD diet were measured. As shown in Fig. P < 0.001), and significantly negative correlation between CD31 mRNA and MALAT1, or vWF mRNA and MALAT1 . These results indicate that MALAT1 may contribute to ox-LDL-induced EndMT of HUVECs, and 40 μg/ml ox-LDL was used in following studies.Next, we investigated the expression and role of MALAT1 in HUVECs. HUVECs were treated with ox-LDL at different concentrations  for 48 h. After incubation, the HUVECs underwent an EndMT transition with the increase of ox-LDL, as confirmed by its morphological change to a spindle-shape  were used to knock down MALAT1. The knockdown efficiency of MALAT1-siRNA3 was optimal and thus MALAT1-siRNA3 was used for further experiments (Fig. To further determine the effect of MALAT1 on EndMT of HUVECs, MALAT1 was overexpressed in HUVECs. The qRT-PCR assay was applied to test the level of MALAT1 and the result showed that MALAT1 expression in pcDNA-MALAT1 transfected HUVECs was increased to 2.4 folds compared to control group (Fig. Ox-LDL can upregulate the expression of β-catenin in cell nuclei, a hallmark of Wnt/β-catenin pathway activation . TherefoTo further analyze the effect of MALAT1 on the activation of Wnt/β-catenin signaling, the expression and location of β-catenin was examined through western blot Fig. a and immThere is increasing evidence that EndMT plays important roles in the occurrence and development of atherosclerosis  and promExtensive evidence shows that MALAT1 plays critical roles in coronary atherosclerotic heart disease . For insLDL-C, especially ox-LDL, plays a central role in the development of atherosclerosis, and subclinical atherosclerosis remains common in individuals with LDL-C < 70 mg/dl . Ox-LDL Wnt/β-catenin is one of the signaling pathways involved in EndMT . When WnThis study demonstrates that MALAT1 is dysregulated in arterial tissues from atherosclerotic mice and HUVECs treated with ox-LDL, and uncovers the important role of MALAT1 in promoting ox-LDL induced HUVEC EndMT in vitro, which is dependent on Wnt/β-catenin signaling pathway. Thus, these findings might connect the ox-LDL-induced EndMT to its notorious role in atherosclerosis and highlight the importance of MALAT1 in the development of atherosclerosis.


The nephron is composed of distinct segments that perform unique physiological functions. Little is known about how multipotent nephron progenitor cells differentiate into different nephron segments. It is well known that β-catenin signaling regulates the maintenance and commitment of mesenchymal nephron progenitors during kidney development. However, it is not fully understood how it regulates nephron segmentation after nephron progenitors undergo mesenchymal-to-epithelial transition. To address this, we performed β-catenin loss-of-function and gain-of-function studies in epithelial nephron progenitors in the mouse kidney. Consistent with a previous report, the formation of the renal corpuscle was defective in the absence of β-catenin. Interestingly, we found that epithelial nephron progenitors lacking β-catenin were able to form presumptive proximal tubules but that they failed to further develop into differentiated proximal tubules, suggesting that β-catenin signaling plays a critical role in proximal tubule development. We also found that epithelial nephron progenitors lacking β-catenin failed to form the distal tubules. Expression of a stable form of β-catenin in epithelial nephron progenitors blocked the proper formation of all nephron segments, suggesting tight regulation of β-catenin signaling during nephron segmentation. This work shows that β-catenin regulates the formation of multiple nephron segments along the proximo-distal axis of the mammalian nephron. Podocytes and the Bowman’s capsule in the renal corpuscle are followed by the proximal tubule (PT), loop of Henle (LOH), and distal tubule (DT)2. Each of these segments performs unique physiological functions, indicating that different cell types are present in different nephron segments4. All nephron tubule cells are derived from the multipotent mesenchymal nephron progenitors (MNPs) which are located at the cortex of the developing kidney6. MNP undergo mesenchymal-to-epithelial transition (MET), forming the renal vesicle (RV). Epithelial nephron progenitors in the RV undergo complex morphogenesis to form the S-shaped body (SSB) which eventually develops into a nephron2  and gain-of-function (GOF) mutations of β-catenin have been powerful tools for investigating canonical Wnt signaling in vivo15. A potential caveat is that the resulting phenotypes may be, at least in part, due to defective cell adhesion since β-catenin is known to form a complex with cadherin at the plasma membrane16.β-catenin plays an essential role in mediating canonical Wnt signaling21. We have previously shown that MNPs lacking β-catenin fail to form RV17. However, due to this early developmental arrest in nephrogenesis, this study provided little information about if or how Wnt/β-catenin signaling regulates epithelial nephron progenitors (e.g. RV and its derivatives) to develop into the mature nephron. Several studies have shown that Wnt/β-catenin signaling is important at later stages of nephron development. When Ctnnb1, the gene encoding β-catenin, was deleted using Pax8Cre which targets both the developing nephron and collecting duct (CD) in the mouse kidney, formation of the Bowman’s capsule was defective22. In addition, based on the pharmacological manipulation of Wnt/β-catenin signaling in mouse kidney explants, it was proposed that Wnt/β-catenin signaling promotes the formation of distal segments of the nephron and represses the formation of proximal segments23. Taken together, these studies suggest that Wnt/β-catenin signaling may continuously regulate mammalian nephron development even after β-catenin-triggered epithelialization of MNPs.It is now well established that Wnt/β-catenin signaling is required for the maintenance and early differentiation of MNPsIn order to investigate how β-catenin signaling regulates mammalian nephron development in epithelial nephron progenitors, we have performed genetic analyses of β-catenin by specifically targeting the developing nephron in the mouse kidney. Here, we report that epithelial nephron progenitor cells lacking β-catenin can form presumptive PT cells but cannot form differentiated PT cells. We also find that β-catenin is required for the formation of DT. In summary, our data suggest that β-catenin signaling is essential for the development and maturation of multiple nephron segments in the mammalian kidney.4, the specificity of Cre is important. Wnt4GFPcre (Wnt4tm3(EGFP/cre)Amc or Wnt4tm2(EGFP/cre)Svo)25 and Pax8Cre (Pax8tm1(cre)Mbu)26 are widely used to target nephron tubules. However, the expression of these Cre lines is not exclusive to the developing nephron tubules; Wnt4GFPcre also targets the medullary stroma27 and a subset of MNPs29 while Pax8Cre targets the collecting duct in addition to the nephron lineage30. Removal of β-catenin from these non-nephron tubule cells may indirectly affect nephron segmentation. Therefore, we chose to use Osr2Cre (Osr2tm2(cre)Jian) which was previously shown to target developing nephrons in the mouse kidney31. To examine when Osr2Cre is activated and which nephron segments it targets, we performed lineage analysis employing Cre-mediated activation of a Rosa reporter 26Sortm3(CAG-EYFP)Hze)32. We found that Osr2Cre labeled mature RV and the comma-shaped body, but not nascent RV, with the Rosa reporter  and its control  kidney. We used Wt1, Lotus tetragonolobus lectin (LTL), and Slc12a1 to mark podocytes, PT, and LOH, respectively. We found that all of those nephron segments were present in the mutant kidney  was indeed deleted by Osr2Cre. Our results suggest that β-catenin is dispensable for the initial specification of podocytes, PT, and LOH during nephron development. We also found that Slc12a3+ DT cells were present in the mutant kidney 33, was considerably reduced in the mutant kidney. This result is consistent with a previous report, which showed that removal of β-catenin in renal epithelial cells by Pax8Cre causes defects in the gross architecture of glomeruli22.We observed a defective formation of the renal corpuscle in the β-catenin LOF mutant kidney by cle Fig. . Howevercle Fig. . In addiOsr2Cre is caused by a defect in the early specification of visceral epithelial cells (presumptive podocyte progenitors) or parietal epithelial cells (presumptive Bowman’s capsule progenitors), we examined the SSB stage. We found that, in both control and mutant SSBs, endothelial cells migrated into the vascular cleft which was formed by the proximal and medial segments of the SSB, suggesting that β-catenin is dispensable for the visceral epithelial cells to recruit endothelial cells  using Wnt4GFPcre (Wnt4tm3(EGFP/cre)Amc)24. We have previously shown that Wnt4GFPcre can target the nephron lineage including DT11 but since Wnt4GFPcre also targets the medullary stroma27, Wnt4GFPcre may not be suitable for studying the role of β-catenin in LOH because removal of β-catenin in the medullary stroma may affect the development of the adjacent LOH39. However, this is less of a concern for studying the DT because the DT cells are located close to the cortical region of the developing mouse kidney, away from the medullary stroma.Since Osr2Cre 40. Since the third exon encodes the N-terminus of β-catenin protein, Cre-mediated recombination results in the production of an N-terminally truncated β-catenin which is resistant to degradation. Increased abundance of β-catenin leads to the activation of canonical Wnt signaling in a ligand-independent manner40.Our β-catenin LOF studies suggest that β-catenin regulates the development of multiple nephron segments in epithelial nephron progenitors. To further explore the mechanism of β-catenin-mediated nephron development, we performed a β-catenin GOF study using Wnt4GFPcre-mediated activation of a stable form of β-catenin blocked the formation of all nephron segments . We found no evidence that constitutive activation of Wnt/β-catenin signaling in the developing nephrons promoted the formation of any specific nephron segment. Instead, we found that expression of a stable form of β-catenin in the developing nephron inhibited the formation of all nephron segments.We found that nts Fig. . No gloms Figure . Slc12a1s Figure . Given tWnt4GFPcre. In the control kidney, the proximal and medial segments of the SSB were marked by Wt1 and Jag1, respectively, and the expression domains of Wt1 and Jag1 did not overlap, showing clear separation  stage42. The distal part of the RV (the part closer to the branching tip of the collecting duct) shows differential gene expression compared to the rest of the RV  segments are established2 Figure . The proafb Fig. . The diset al. have suggested that a gradient of β-catenin activity regulates segmentation in the nascent nephron23. They manipulated Wnt/β-catenin signaling pharmacologically in the embryonic kidney explant which is suitable for studying early nephrogenesis rather than the formation of mature nephron segments. They mainly focused on how β-catenin regulates the formation of different segments in the SSB. It is unclear how the altered segmentation of the SSB influences the segmentation of the fully formed nephron. Unlike the pharmacological approach which targets every cell in the kidney explant in vitro, our genetic approach allowed us to specifically target epithelial nephron progenitors and their descendants in vivo. Although Osr2Cre is active in the mature RV  partially depends on another nephron segment (e.g. DT).We found that the β-catenin LOF mutant kidney developed structures resembling LOH as judged by the expression of ney Fig. . Therefonin Fig. , indicatnts Fig. . Taking ion Fig. , it appecre Fig. , a very e Figure . This maSix2Cre17, Wnt4Cre, or Osr2Cre blocks nephrogenesis. Although apparent phenotypes may look similar in these β-catenin GOF mutant kidneys, they show different points of developmental arrest in nephrogenesis. We have previously shown that, when Six2Cre is used, the arrest occurs at the pretubular aggregate stage17. In contrast, when Wnt4Cre or Osr2Cre is used, the developmental arrest occurs at the epithelial nephron progenitors  recapitulates the β-catenin LOF mutant phenotypes. Alternatively, manipulation of genes encoding other adhesion factors would help resolve this issue. Nonetheless, considering the critical roles of Wnt/β-catenin signaling on cell fate determination in various biological contextsGt(ROSA)26Sortm3(CAG-EYFP)Hze (Rosa26Ai3)32, Wnt4tm3(EGFP/cre)Amc (Wnt4GFPcre)24, Osr2tm2(cre)Jian (Osr2Cre or Osr2IresCre)31, Ctnnb1tm2Kem (Ctnnb1c)51, and Ctnnb1tm1Mmt (Ctnnb1ex3)40 mice were described previously.All animal procedures were approved by the Institutional Animal Care and Use Committee at Cincinnati Children’s Hospital Medical Center in accordance to their guidelines for the care and use of laboratory animals (IACUC2017–0037). Embryonic kidneys at E18.5 were fixed with 4% paraformaldehyde in PBS at room temperature for 10 min, incubated in 10% sucrose in PBS at 4 °C overnight, and imbedded in OCT. Cryosections of 10 or 12 μm were incubated in PBS containing 0.1% Triton x-100, 5% heat-inactivated sheep serum, and the following antibodies: Jag1 , Wt1 , GFP , Slc12a3 , LTL , Slc12a1 , β-catenin , β-catenin , Pecam1 , Mafb , Akap12 , Cdh6 , Hnf4a , Pax2 , Slc5a2  Fluorophore-conjugated secondary antibodies (Thermo Fisher Scientific or Jackson ImmunoResearch Laboratories) were used at a 1:500 dilution. The Jag1 antibody developed by Spyros Artavanis-Tsakonas was obtained from the Developmental Studies Hybridoma Bank, created by the NICHD of the NIH and maintained at The University of Iowa, Department of Biology, Iowa City, IA 52242. Nuclei were stained with Hoechst 33342 before mounting. Images were acquired on a Nikon TiE microscope with Andor Zyla 4.2 camera and Lumencor Spectra X light source housed at the Confocal Imaging Core at CCHMC.Supplementary figures and legends


PME-1 gene and found that human PME-1 promoter contains two lymphoid enhancer binding factor-1/T-cell factor (LEF1/TCF) cis-elements in which β-catenin serves as a co-activator. β-catenin acted on these two cis-elements and promoted PME-1 expression. GSK-3β phosphorylated β-catenin and suppressed its function in promoting PME-1 expression. Inhibition and activation of GSK-3β by PI3K-AKT pathway promoted and suppressed, respectively, PME-1 expression in primary cultured neurons, SH-SY5Y cells and in the mouse brain. These findings suggest that GSK-3β phosphorylates β-catenin and suppresses its function on PME-1 expression, resulting in an increase of PP2Ac methylation.Protein phosphatase 2A (PP2A) is the major tau phosphatase. Its activity toward tau is regulated by the methylation of PP2A catalytic subunit (PP2Ac) at Leu309. Protein phosphatase methylesterase-1 (PME-1) demethylates PP2Ac and suppresses its activity. We previously found that glycogen synthase kinase-3β (GSK-3β) suppresses PME-1 expression. However, the underlying molecular mechanism is unknown. In the present study, we analyzed the promoter of  Neuronal microtubule associated protein (MAP) tau is abnormally hyperphosphorylated and aggregated into neurofibrillary tangles (NFTs) in brains of individuals with Alzheimer’s disease (AD) and related neurodegenerative diseases , 2. The in vivo in rodent brain [Protein phosphatase 2A (PP2A) is the major tau phosphatase in human brain . It dephnt brain –13. PP2Ant brain , 15. Metnt brain –21.Methylation level of PP2Ac is determined by protein phosphatase methylesterase-1 (PME-1)  and leucWe previously reported that PI3K-GSK-3β regulates PP2Ac methylation . To confThen, we overexpressed GSK-3β in SH-SY5Y cells, and then measured the expression of PME-1 by Western blots and by quantitative real-time PCR (qPCR). We found that GSK-3β overexpression suppressed the expression of PME-1, as measured both at the mRNA level  and the To further determine the role of GSK-3β on PME-1 expression, we knocked down GSK-3β by using siRNA and measured the PME-1 expression in SH-SY5Y cells. We found that knockdown of GSK-3β increased both the mRNA  and protPME-1 gene by Genomatix’s MatInspector software [LEF1/TCF like elements located on -349 bp – -333 bp and +19 bp – +35 bp  for 24 hr to activate PI3K-AKT and analyzed the phosphorylation of GSK-3β and β-catenin by Western blots. We found that FBS induced the increase in phosphorylation of GSK-3β at Ser9 , 6B and Insulin activates PI3K signaling and suppresses GSK-3β activity , 35. To Then, we treated primary cultured neurons with 100 nM insulin for 18 hrs and analyzed PME-1 expression by Western blots . We founFurthermore, we activated GSK-3β by inhibiting PI3K/AKT signaling with LY294002 in cultured neurons and analyzed PME-1 mRNA by qPCR. We found that LY294002 suppressed the expression of PME-1 in dose-dependent manner , confirmLEF1/TCF cis-elements. We overexpressed β-catenin in SH-SY5Y cells and treated the cells with the ARA014418 for 4.5 hr. We performed ChIP assay and found that ARA014418 treatment increased the levels of co-immunoprecipitated cis-elements of LEF1/TCF-1 and LEF1/TCF-2 by β-catenin  [2-terminal domain of β- catenin are phosphorylated by GSK-3β and CK1α. Activation of Wnt signaling leads to the recruitment of Axin to the membrane and functional inactivation of the β-catenin destruction complex. This results in cytoplasmic and nuclear accumulation of β-catenin. In the nucleus, β-catenin associates with TCFs to activate transcription of Wnt signaling target genes. In the present study, ChIP assay showed that β-catenin bound to the two LEF/TCF cis-elements of PME-1 promoter and enhanced the PME-1 expression determined by using luciferase reporter.Transcription factors lymphoid enhancer-binding factors/T-cell factors (LEFs/TCFs) are known to bind to the consensus sequence CCTTTGWW, cis-element of LEF/TCF, and regulate the transcription of target genes . In the ctively) , 38. Thectively) . Withoutctively) , 39, 40.Wnt/β-catenin signaling participates in diverse biological processes including neurogenesis , axonal PP2A is also known to regulate Wnt signaling by directly dephosphorylating β-catenin at Ser 37 and Thr41 to generate active β-catenin with enhances transcriptional activity . PR55α, In summary, we found here that β-catenin promotes PME-1 transcription. GSK-3β suppresses PME-1 expression by phosphorylation of β-catenin, leading to increase of methylation of PP2Ac. Thus, we speculate that activation of GSK-3β resulting from insulin resistant in AD brain one hand causes hyperphosphorylation of tau, other hand may increase PP2A methylation through PME-1, resulting in the attenuation of hyperphosphorylation of tau.Renilla reniformis luciferase) and dual luciferase assay kit were bought from Promega . Luciferase driven by different length of or mutated promoter of human PME-1 in pGL4.10 were constructed by using human blood genomic DNA, purified by Spin Column Blood Genomic DNA Purification Kit  as template and confirmed by sequencing. Monoclonal anti-PME-1, polyclonal anti-GAPDH, monoclonal anti-demethylated-PP2Ac (DM-PP2Ac), siGSK-3β, siGSK-3α and siβ-catenin were bought from Santa Cruz . Polyclonal anti-β-catenin, anti-Non-pS-β-catenin (dephosphorylated at S33/S37/T41), anti-GSK-3β and anti-pS9-GSK-3β were purchased from Cell Signaling Technology . Monoclonal anti-HA was from Sigma . Horseradish peroxidase-conjugated anti-mouse and anti-rabbit IgG were obtained from Jackson ImmunoResearch Laboratories . ECL (enhanced chemiluminescence) kit was from Thermo Fisher Scientific .pCI/β-catenin and its mutants tagged with HA at N-terminus were constructed by PCR amplification from pcDNA3/β-catenin and confirmed by DNA sequence analysis. pGL4-1.0, pRL-TK (thymidine kinase promoter driven The hemizygous human tau transgenic (B6.Cg-Mapttm1(EGFP)Klt Tg(MAPT)8cPdav/J, Tg/hTau) mice with murine tau knockout background  obtained2. All transfections were performed with Lipofectamine 3000  according to the manufacturer’s instructions.HEK-293T and human neuroblastoma SH-SY5Y cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) or DMEM/F12 supplemented with 10% fetal bovine serum (FBS)  at 37 °C, 5% CO6 cells/ml. The cells were plated onto poly-D-lysine (100 μg/ml) coated 12-well plates and were maintained in a humidified atmosphere at 37 °C, 5% CO2. The medium was changed to neurobasal supplemented with 2% B27 (Invitrogen) containing 100 U/ml penicillin and 100 μg/ml streptomycin 4 h later. On the second day of culture, the cells were treated with 10 μM Ara-C for 24 h to inhibit non-neuronal cells. On the seventh day, the cells were treated with various concentrations of ARA014418 or LY294002 at for 4.5 h for measurement of PME-1 mRNA or treated with 100 nM insulin for 18 hr for measurement of PME-1 protein.Rat cerebral cortical neurons were isolated and cultured according to a previously described method . Briefly-ΔΔCT). The primers used for PME-1 are as follows: human PME-1 forward: 5′GTCGTCCTAAAACCTTCAAGTCTCT3′ and reverse: 5′CTCACTTCCTTCCTCATCTTCTTCT3′; mouse and rat PME-1 forward: 5′GAAGAAGATGAGGAAGGAAGTGAGT3′ and reverse: 5′AGAGCAGCAGTTTAGGAATAGGACA3′; human GAPDH forward: 5′GGTGGTCTCCTCTGACTTCAACA3′ and reverse: 5′GTTGCTGTAGCCAAATTCGTTGT3′; mouse GAPDH forward: 5′AGGTCGGTGTGAACGGATTTG3′ and reverse: 5’TGTAGACCATGTAGTTGAGGTCA3′; rat GAPDH forward 5′TGCACCACCAACTGCTTAGC3′ and reverse: 5'GGCATGGACTGTGGTCATGA3′.Total RNA was isolated from primary cultured cortical neurons or from cultured cells using the HP Total RNA Kit . One microgram of total RNA was used for first-strand cDNA synthesis with Oligo-(dT)18 by using the HiScript II Q RT SuperMix for qPCR . The target mRNA was amplified by using LightCycler® Multiplex DNA Masters  in a LightCycler® 96 system  under the condition: 95 °C 10 min, at 95 °C for 30 sec and at 60 °C for 1 min for 40 cycles. Relative levels of target mRNAs were calculated by the comparative CT (threshold cycle) method . The luciferase activity was measured by the dual luciferase assay  according to manufacturer’s manuals. The LEF1/TCF-1 and LEF1/TCF-2 cis-elements. LEF1/ TCF-1 region primer forward: 5′GTTTATCCAGCGGGGGTGCGGGTCT3′ and reverse: 5′GAAACAACGAAAAGGCCAAGGGTCTG3′; LEF1/TCF-2 region primer forward: 5′ACGCTTTGCTACAATACCTCTCCG3′ and reverse: 5′CTGGGAACCCGACGACTTTCACCGG3′.SH-SY5Y cells were transfected with pCI/β-catenin tagged with HA at the N terminus and treated with or without 20 μM ARAO14418 for 4.5 hr. Chromatin immunoprecipitation (ChIP) assay was performed using the Simple ChIP® Enzymatic Chromatin IP Kit , following the instructions of the manufacturer. The cells were fixed with 1% formaldehyde in PBS for 10 min at room temperature, and then glycine was added to a final concentration of 125 mM and incubated for 5 min at room temperature. The cells were collected in cold PBS with Protease Inhibitor Cocktail, then lysed with corresponding buffer. The nuclei were then collected and the chromatin was enzymatically digested into fragments ranging from 150 to 900 bp. ChIP was performed using protein G magnetic beads and either 2 mg of β-catenin antibody , HA antibody (Sigma), or the nonspecific IgG antibody . Protein-DNA cross-links were reversed, and the DNA was purified. The specific primers  and rabbit anti-β-catenin antibody (1:400) overnight at 4 °C. After incubation with secondary antibodies  plus Hoechst 33342 at room temperature, the cells were washed with PBS, mounted with SlowFade® Gold Antifade Mountant , and visualized with a TCS-SP5 dual photon laser-scanning confocal microscope . We used the Image J software to quantify the fluorescence intensity of the nucleus and whole cell. The ratio of fluorescence level in the nucleus and the cytoplasm  was calculated and presented as average of the cells from 4–5 fields at random.Mice were deeply anesthetized with 1.25% Avertin  and placed in a stereotaxic apparatus . After craniotomy, ARA014418 (2 μl of 5 mM dissolved in DMSO) was unilaterally injected into lateral ventricle of 6-month-old Tg/hTau mice using a 10 μl Hamilton syringe custom made with a 30-gauge/0.5 inch/ hypodermic needle . The coordinates were as follows: 0.3 mm posterior, 1.0 mm lateral to bregma, and 2.5 mm ventral to the dura surface. ARA014418 was injected at a rate of 0.66 μl/min, and the needle was kept in position for 3 min additionally before slow withdrawal to prevent leakage of the liquid infused. DMSO was injected into Tg/hTau mice of the same age by using the same approach as a vehicle control. The mice were allowed to completely recover on a soft heating pad before they were returned to their home cages. 48 hr after injection, mice were sacrificed by cervical dislocation. The cortex of mouse was homogenized as described above for Western blots and mRNA assay.t test.The GraphPad Prism 5.0 software package was used for Statistical Analysis. The data are presented as mean ± SD. For multiple-group analysis, data points were compared by one-way ANOVA with Bonferroni post-hoc test. For two-group comparison, data points were compared by the unpaired two tailed Student’s


Pseudomonas aeruginosa, strain PA01, was used as a model bacterium. Utilising a simple, cost effective fabrication process, P. aeruginosa was heat-inactivated and formulated into dissolving MNs, fabricated from aqueous blends of 20% w/w poly. The resultant MNs were of sufficient mechanical strength to consistently penetrate a validated skin model Parafilm M®, inserting to a depth of between 254 and 381 µm. MNs were successfully inserted into murine skin and partially dissolved. Analysis of MN dissolution kinetics in murine ears via optical coherence tomography showed almost complete MN dissolution 5 min post-insertion. Mice were vaccinated using these optimised MNs by application of one MN to the dorsal surface of each ear (5 min). Mice were subsequently challenged intranasally (24 h) with a live culture of P. aeruginosa (2 × 106 colony forming units). Bacterial load in the lungs of mice vaccinated with P. aeruginosa MNs was significantly (p = 0.0059) lower than those of their unvaccinated counterparts. This proof of concept work demonstrates the potential of dissolving MNs for intradermal vaccination with heat-inactivated bacteria. MNs may be a cost effective, potentially viable delivery system, which could easily be implemented in developing countries, allowing a rapid and simplified approach to vaccinating against a specific pathogen.This work describes the formulation and evaluation of dissolving microneedle patches (MNs) for intradermal delivery of heat-inactivated bacteria.  The hypodermic needle also results in the creation of biohazardous sharps waste, which pose the risk of infection to both patients and healthcare workers. An estimated 33,800 HIV infections, 1.7 million hepatitis B infections and 315,000 hepatitis C infections annually are caused from unsafe injection practices .The skin represents an attractive route for vaccination, having the advantage of increased patient compliance and increased vaccine effectiveness. The skin is densely populated with professional antigen presenting cells (APCs). Dendritic cells (DCs) present in the dermis and Langerhans cells (LCs) in the epidermis play a pivotal role in induction of immune responses, whereby they capture foreign antigens and present them in draining lymph nodes . The skiSC, and subsequently delivering their payload into viable skin  have been proposed as a novel approach to increase skin permeability and may offer an ideal platform for i.d delivery . MNs areble skin . MNs aresubjects . MNs aresubjects . Furthersubjects .Pseudomonas aeruginosa strain PA01, was chosen as a model bacterium. Utilising a simple, cost effective fabrication process, P. aeruginosa was heat-inactivated and formulated into dissolving MNs. As MNs offer the possibility for self-application, the expected benefit of MN-mediated vaccination with heat-inactivated bacteria would lie in the simplicity and cost effectiveness from the practical perspective. Whole heat-inactivated vaccines are stable, safe for administration to immunocompromised patients and can be easily freeze dried and incorporated into MNs, thus allowing ease of transport and distribution, and a cost-effective approach to vaccinating against a specific pathogen. This approach could potentially allow for mass immunisation within developing countries, helping increase vaccine coverage. To the best of our knowledge, this is the first study that uses dissolving MN arrays for delivery of a heat-inactivated bacterium, intended for vaccination against a bacteria for which no currently licenced vaccine exists.Dissolving MNs have received substantial interest for delivery of currently licenced vaccines. For example, Matsuo et al. demonstrated vaccine efficacy against tetanus and diphtheria, malaria and influenza using dissolving MNs . Moreove22.1Gantrez® S-97 (PMVE/MA), a copolymer of methyl-vinyl-ether and maleic acid , was provided by Ashland . Parafilm M®, 127 µm thick, was obtained from BRAND GMBH . Dulbecco’s phosphate buffered saline (PBS) (endotoxin tested), Luria-Bertani broth and cetrimide agar were obtained from Sigma-Aldrich . All other chemicals were of analytical reagent grade. Rompun™ 100 mg/ml, was purchased from Bayer Healthcare LLC (Bergkamen Germany). Ketalar™ 10 mg/ml ketamine hydrochloric solution was purchased from Pfizer Ltd .2.2P. aeruginosa (strain PAO1), was grown on cetrimide agar at 37 °C overnight. Post incubation a colony was selected and cultured overnight in Luria-Bertani (LB) broth (5 ml) at 37 °C in a rotatory incubator shaker (180 rpm). Post incubation, the culture (5 ml) was transferred into an Erlenmeyer flask containing a further 995 ml of LB broth and incubated as above. After incubation, the culture was transferred to 50 ml Falcon tubes and the bacteria was collected by centrifugation (4000 rpm for 20 min), and the supernatant was subsequently removed. The resultant P. aeruginosa pellets were washed twice by addition of phosphate buffer (PB), followed by centrifugation as described above. Post washing, the pellets were re-suspended in PB buffer and heat-killed by submersion in a water-bath (65 °C) for 1 h. The suspension was inoculated onto LB agar to ensure bacteria were not viable. The remaining suspension was placed in a freezer (−80 °C) for 1 h. Once frozen, each Falcon tube lid was replaced with a KimWipe  and the tubes were transferred to a side arm of a manifold-type freeze dryer (MiVac freeze dryer) under a vacuum for 48 h before being removed and stored until further use.2.3P. aeruginosa diluted in phosphate buffered saline (PBS), pH 7.4, to give a final concentration of 20% Gantrez®S-97. One hundred milligrams of this suspension, containing 200 µg of heat-inactivated P. aeruginosa was poured onto silicon micromoulds. The micromoulds contained 361 (19 × 19) pyramidal shaped needles, perpendicular to the base, 500 µm in height, 300 µm wide and with 50 µm interspacing. The arrays had areas of 0.6 cm2. A pressure of 3 bar was subsequently used to force the MN formulation into the needles of the MN mould. The MNs were dried under controlled temperature conditions (19 °C) for 24 h, before being carefully removed from the MN moulds and stored until further use. A schematic representation showing the method of production of dissolving MNs is shown in Fig. 1.MNs were prepared from aqueous blends of Gantrez® S-97. A stock of Gantrez® S-97 (40%) was diluted with the appropriate volume of a suspension of 2.4When imaging MNs using scanning electron microscopy (SEM), MNs were mounted onto an aluminium stub and observed under a TM3030 microscope . Micrographs of MN structures were obtained and the surface morphology and height/width of individual needles were examined/measured.2.5−1 until the required force of 32 N/array was applied. This has been shown to be the average maximum force a human exerts when applying MNs , a blend of a hydrocarbon wax and a polyolefin, was used as a skin simulant . Inserted MNs were immediately viewed using an EX1301 VivoSight® OCT Microscope with a hand-held probe, the contacting surface of which had an area of 1 cm2. The swept-source Fourier domain OCT system has a laser centre wave-length of 1305 ± 15 nm, facilitating real-time high resolutions imaging of the upper skin layers . The resulting 2D images were analysed using ImageJ®  software. Obtained image files had a scale of 1.0 pixel = 4.2 µm, allowing accurate measurements of MN insertion depth. Five replicates were performed per MN formulation and for each MN inserted, 20 measurements of individual MN insertion depths were taken by random selection of needles, from the 361 penetrating MNs in each array. MN insertion data was presented as (±SD) of 20 replicate measurements. The procedure was repeated at pre-determined time points .The 2.7In vivo studies were performed on C57/BL6 mice, 6–8 weeks old. Mice were randomly divided into 2 groups, those that received blank MNs (bMNs), or P. aeruginosa MNs (p.a MNs), n = 5/group. Briefly, mice were anaesthetised intraperitoneally (i.p) and immunised by manual application of one MN to the dorsal surface of each ear for 5 min, resulting in a corresponding dose of 400 µg of heat killed P. aeruginosa. At 7 days post MN application, both vaccinated and un-vaccinated mice were challenged with 2 × 106 colony forming units (CFU) of P. aeruginosa (PA01), delivered intranasally (i.n). Each mouse received a 20 µl volume of bacterial suspension, diluted in endotoxin-free PBS and administered with a micropipette. Mice were carefully monitored over the course of infection and weights were assessed. Subsequently, 24 h post-challenge, mice were sacrificed and the lungs and spleens were harvested for analysis of CFU counts.2.7.1P. aeruginosa was grown on cetrimide agar at 37 °C overnight. Preparation of bacterial inoculum for challenge study was performed by transferring a colony of P. aeruginosa into LB broth. The culture was grown overnight at 37° C under gentle agitation. Following overnight incubation, the bacteria were harvested by centrifuged for 25 min at 22° C 4000 rpm and the supernatant was carefully removed. The pellet was subsequently washed in PBS. Finally, optical density (OD) measurements were used to dilute the bacterial suspension to the desired concentration (106 CFU) using PBS and this was confirmed by plating serial dilutions of the preparations for enumeration of CFU post-overnight incubation.2.7.2. The agar plates were incubated overnight at 37 °C and the number of CFUs counted for determination of bacterial load.Lung and spleen tissue was homogenized under aseptic conditions in 1 ml of PBS in a sterile bijou using a mechanical homogeniser. Serial dilutions of the homogenized organs (100 µl) were plated onto cetrimide agar  for analysis of bacterial load2.8t-test. In all cases, p < 0.05 was considered acceptable for rejection of the null hypothesis. Statistical analysis was carried out using GraphPad Prism version 5.0 .Data was compared using an unpaired, two-tailed student’s 33.1P. aeruginosa (p.a MNs). The resulting MNs were approximately 500 µm in height, with a width at base of approximately 300 µm, and the fabrication process resulted in MNs with sharp needle tips, essential for insertion into murine skin and successful vaccine delivery. A complete array of needles was formed and MN baseplates were of sufficient strength to be easily removed from the moulds without damage being caused to the array. The addition of P. aeruginosa did not appear to affect MN structure, or the morphology of the individual needles.3.2P. aeruginosa MNs (p.a MNs) by 4.7% (p = 0.805), indicating that the loading of P. aeruginosa within the MNs did not affect their rigidity or compression characteristics. None of the MNs, or the baseplates of the MNs fractured post-application of 32 N, nor did the MNs appear to be compressed  reduced in height by 4.4% C-i and t by 4.7% C-ii, res by 4.7% C-iii , mice underwent bacterial challenge by i.n inoculation with a live culture of e models  and as sP. aeruginosa within the vaccinated group (three out of five spleens were found to be sterile), compared to the unvaccinated group, which received bMNs (one out of five spleens was found to be sterile). These results collectively demonstrate generation of a protective immune response in vivo following application of our dissolving polymeric MNs, resulting in enhanced bacterial clearance from the lungs and decreased likelihood of systemic dissemination of P. aeruginosa.At 24 h post challenge, the number of CFUs in the spleens was overall very low in both groups D, since 4P. aeruginosa (strain PA01) was used as a model bacteria, heat-inactivated and formulated into dissolving MNs. This study was conceived as a proof of concept study for investigating the feasibility of taking a highly pathogenic bacterium, incorporating it into dissolving MNs and successfully delivering the heat-inactivated bacterium in vivo, inducing protective immune responses with a view, in time of working towards more relevant bacteria of the developing world.The emergence of multidrug-resistant bacteria for which no vaccines exist, has resulted in high mortality rates with limited or no therapeutic interventions available . Thus, tMNs were prepared using Gantrez®, previously shown to have been successfully used in the formulation of dissolving MNs for delivery of model protein antigens, importantly, without deterioration of the antigens upon release from the MNs . MNs ladBacterial load post challenge is related to survival status and, therefore, the enumeration of bacteria in vaccinated mice was investigated. MN i.d vaccination resulted in reduced bacterial load in the lungs and reduced bacterial dissemination to the spleens. These results are in line with those of Yang et al. who demonstrated reduced bacterial loads, inflammatory cytokine expression (IL-6) and inflammatory cell infiltration, following intramuscular (i.m) vaccination with POH and adjuvant, in a challenge pneumonia model . Yanyan via dissolving MNs is advantageous for i.d vaccination. Firstly, heat-inactivated vaccines are safe for administration to immunocompromised patients (The use of lyophilised heat-inactivated vaccines delivered patients . Adminispatients . Also, tpatients . At prespatients . Moreovepatients . The Glopatients . Additiopatients  and may patients .via dissolving MNs results in polymer deposition in the skin. Although repeated application of dissolving MNs will not be required for vaccine delivery, regulatory bodies will undoubtedly require assurance of polymer safety. Accordingly, an understanding of the long-term effects of polymer deposition will require further investigation to ensure they do not represent a danger. While dissolving MNs are typically fabricated from biocompatible polymers, they have not been used for i.d delivery. With the MNs utilised in this particular study, each MN deposits approximately 5.4 mg of polymer per array, which is the polymer content in the needles only. As highlighted such polymer deposition may result in effects such as erythema or hepatic/lymphatic accumulation and requires further investigation (The mechanism of vaccine delivery tigation . From a P. aeruginosa. Such experiments should evaluate multiple cellular and humoral facets of the immune response and the cross-protective efficacy among different isolates of P. aeruginosa. Moreover, studies should also take into account the effects of polymer deposition and subsequent impact on immune response. In addition, prime-boost regimes and an investigation into long-term protection are warranted.As this was a proof of concept investigation, studies were not carried out to elucidate the immunological mechanism by which p.a MNs confer protective immunity. This research was designed to develop and evaluate a pre-clinical MN delivery platform for i.d vaccination with heat-inactivated bacteria. Consequently, we describe a simple, cost effective approach to vaccination that could be applicable to a range of pathogens. Future studies are warranted to elucidate the mechanism of immune responses elicited post MN-mediated vaccination against 5P. aeruginosa for i.d vaccination. Notably, MNs were fabricated using pressure only, highlighting the simplicity and cost-effectiveness in the strategy employed. In addition, in vivo murine challenge studies confirmed delivery and the generation of an immune response, resulting in reduced bacterial load in the lungs and spleens of vaccinated mice. Together, these results suggest that dissolving MN-mediated i.d vaccination is a potentially viable delivery system. This technology could simplify vaccine administration and distribution in developing countries, thus improving vaccine coverage. Heat-inactivation of bacteria and its incorporation into MNs offers a novel, simplified approach to vaccination against a specific pathogen. This is the first reported study utilising a dissolving MN platform for successful delivery of a lyophilised heat-inactivated bacterium for vaccination against a pathogen for which no currently licensed vaccine exists.The work presented here reports successful formulation and characterisation of dissolving MNs laden with heat-inactivated This was an initial, but important study highlighting the potential of MNs for delivery of heat-inactivated vaccines. We anticipate that this dissolving MN system, due to its simplicity and ease of use, could potentially have a substantial impact on the prevention of infectious disease. Dissolving MNs offer the prospect of mass vaccination and the incorporation of heat-inactivated bacteria provides a simplified approach, which could easily be implemented in countries of the developing world. This was a proof of concept study and prior to this technology being applied to patients; several additional studies are warranted, including studies to elucidate the mechanisms of induced immune protection and patient usability/acceptability profiles. This technology clearly holds much potential and warrants further investigation.