PAR2 regulates regeneration, transdifferentiation, and death.
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ABSTRACT:
SUBMITTER: Piran R
PROVIDER: S-EPMC5260873 | biostudies-literature | 2016 Nov
REPOSITORIES: biostudies-literature
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PAR2 regulates regeneration, transdifferentiation, and death.
Piran Ron R Lee Seung-Hee SH Kuss Pia P Hao Ergeng E Newlin Robbin R Millán José Luis JL Levine Fred F
Cell death & disease 20161103 11
Understanding the mechanisms by which cells sense and respond to injury is central to developing therapies to enhance tissue regeneration. Previously, we showed that pancreatic injury consisting of acinar cell damage+β-cell ablation led to islet cell transdifferentiation. Here, we report that the molecular mechanism for this requires activating protease-activated receptor-2 (PAR2), a G-protein-coupled receptor. PAR2 modulation was sufficient to induce islet cell transdifferentiation in the absen ...[more]
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Project description:ObjectiveProtease-activated receptor-2 (PAR2) also known as F2RL1 is a G protein-coupled receptor that intimately correlates with cancer occurrence. DNA methylation turns out a vital mechanism regulating gene expression, while PAR2 promoter methylation is proven to be involved in cancer development. Hence, this study attempted to clarify the molecular mechanism by which PAR2 mediates lung adenocarcinoma (LUAD) progression, via identifying the effect of PAR2 promoter methylation on LUAD cell progression.MethodsAssociations of PAR2 promoter methylation with PAR2 gene expression and prognosis of LUAD patients were analyzed via bioinformatics analysis. PAR2 promoter methylation and gene expression at the cellular level were measured using methylation-specific PCR, qRT-PCR, and Western blot assays. DNA methyltransferase inhibitor 5-AzadC was used to treat cells to assess PAR2 gene expression alteration. Cell biological behaviors upon PAR2 overexpression were characterized via MTT, wound healing assay, and Transwell assay.ResultsBioinformatics analysis revealed that PAR2 promoter methylation was negatively related to PAR2 gene expression, while PAR2 promoter hypermethylation and low gene expression indicated favorable LUAD prognosis. Besides, it turned out that PAR2 presented upregulated expression and hypomethylated promoter in LUAD cells. Moreover, PAR2 gene expression was elevated in cells treated with 5-AzadC, and the proliferative, migratory, and invasive capabilities of cells with 5-AzadC or high PAR2 gene expression were all enhanced.ConclusionIn sum, PAR2 promoter hypomethylation potentiates LUAD cell progression, in turn affecting the prognosis of LUAD patients.
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Project description:Recently, we showed that pancreatitis in the context of profound β-cell deficiency was sufficient to induce islet cell transdifferentiation. In some circumstances, this effect was sufficient to result in recovery from severe diabetes. More recently, we showed that the molecular mechanism by which pancreatitis induced β-cell neogenesis by transdifferentiation was activation of an atypical GPCR called Protease-Activated Receptor 2 (PAR2). However, the ability of PAR2 to induce transdifferentiation occurred only in the setting of profound β-cell deficiency, implying the existence of a repressive factor from those cells. Here we show that the repressor from β-cells is insulin. Treatment of primary islets with a PAR2 agonist (2fLI) in combination with inhibitors of insulin secretion and signaling was sufficient to induce insulin and PAX4 gene expression. Moreover, in primary human islets, this treatment also led to the induction of bihormonal islet cells coexpressing glucagon and insulin, a hallmark of islet cell transdifferentiation. Mechanistically, insulin inhibited the positive effect of a PAR2 agonist on insulin gene expression and also led to an increase in PAX4, which plays an important role in islet cell transdifferentiation. The studies presented here demonstrate that insulin represses transdifferentiation of α- to β-cells induced by activation of PAR2. This provides a mechanistic explanation for the observation that α- to β-cell transdifferentiation occurs only in the setting of severe β-cell ablation. The mechanistic understanding of islet cell transdifferentiation and the ability to modulate that process using available pharmacological reagents represents an important step along the path towards harnessing this novel mechanism of β-cell neogenesis as a therapy for diabetes.
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Project description:There is great interest in therapeutically harnessing endogenous regenerative mechanisms to increase the number of β cells in people with diabetes. By performing whole-genome expression profiling of zebrafish islets, we identified 11 secreted proteins that are upregulated during β-cell regeneration. We then tested the proteins' ability to potentiate β-cell regeneration in zebrafish at supraphysiological levels. One protein, insulin-like growth factor (Igf) binding-protein 1 (Igfbp1), potently promoted β-cell regeneration by potentiating α- to β-cell transdifferentiation. Using various inhibitors and activators of the Igf pathway, we show that Igfbp1 exerts its regenerative effect, at least partly, by inhibiting Igf signaling. Igfbp1's effect on transdifferentiation appears conserved across species: Treating mouse and human islets with recombinant IGFBP1 in vitro increased the number of cells co-expressing insulin and glucagon threefold. Moreover, a prospective human study showed that having high IGFBP1 levels reduces the risk of developing type-2 diabetes by more than 85%. Thus, we identify IGFBP1 as an endogenous promoter of β-cell regeneration and highlight its clinical importance in diabetes.
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Project description:Type IV collagen is a major component of basement membranes and it provides structural and functional support to various cell types. Type IV collagen exists in a highly complex suprastructure form and recent studies implicate that protomer (the trimeric building unit of type IV collagen) assembly is mediated by the NC1 domain present in the C-terminus of each collagen alpha-chain polypeptide. Here we show that type IV collagen contributes to the maintenance of the epithelial phenotype of proximal tubular epithelial cells, whereas type I collagen promotes epithelial-to-mesenchymal transdifferentiation (EMT). In addition, the recombinant human alpha1NC1 domain inhibits assembly of type IV collagen NC1 hexamers and potentially disrupts the deposition of type IV collagen, facilitating EMT in vitro. Inhibition of type IV collagen assembly by the alpha1NC1 domain up-regulates the production of transforming growth factor-beta1 in proximal tubular epithelial cells, an inducer of EMT. These results strongly suggest that basement membrane architecture is pivotal for the maintenance of epithelial phenotype and that changes in basement membrane architecture potentially lead to up-regulation of transforming growth factor-beta1, which contributes to EMT during renal fibrosis.
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Project description:Protease-activated receptors PAR1 and PAR2 play an important role in the control of epithelial cell proliferation and migration. However, the survival of normal and tumor intestinal stem/progenitor cells promoted by proinflammatory mediators may be critical in oncogenesis. The glycogen synthase kinase-3β (GSK3β) pathway is overactivated in colon cancer cells and promotes their survival and drug resistance. We thus aimed to determine PAR1 and PAR2 effects on normal and tumor intestinal stem/progenitor cells and whether they involved GSK3β. First, PAR1 and PAR2 were identified in colon stem/progenitor cells by immunofluorescence. In three-dimensional cultures of murine crypt units or single tumor Caco-2 cells, PAR2 activation decreased numbers and size of normal or cancerous spheroids, and PAR2-deficient spheroids showed increased proliferation, indicating that PAR2 represses proliferation. PAR2-stimulated normal cells were more resistant to stress (serum starvation or spheroid passaging), suggesting prosurvival effects of PAR2 Accordingly, active caspase-3 was strongly increased in PAR2-deficient normal spheroids. PAR2 but not PAR1 triggered GSK3β activation through serine-9 dephosphorylation in normal and tumor cells. The PAR2-triggered GSK3β activation implicates an arrestin/PP2A/GSK3β complex that is dependent on the Rho kinase activity. Loss of PAR2 was associated with high levels of GSK3β nonactive form, strengthening the role of PAR2 in GSK3β activation. GSK3 pharmacological inhibition impaired the survival of PAR2-stimulated spheroids and serum-starved cells. Altogether our data identify PAR2/GSK3β as a novel pathway that plays a critical role in the regulation of stem/progenitor cell survival and proliferation in normal colon crypts and colon cancer.
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Project description:BackgroundDifferent factors may lead to hepatitis. Among which are liver inflammation and poisoning. We chose two hepatitis models, typical for these two underlying causes. Thus, we aimed to characterize the role of protease-activated receptor 2 (Par2) in liver regeneration and inflammation to reconcile Par2 conflicting role in many damage models, which sometimes aggravates the induced damage and sometimes alleviates it.MethodsWT and knockout (Par2KO) mice were injected with concanavalin A (ConA) to induce immune-mediated hepatitis or with carbon tetrachloride (CCl4) to elicit direct hepatic damage. To distinguish the immune component from the liver regenerative response, we conducted bone marrow (BM) replacements of WT and Par2KO mice and repeated the damage models.ResultsConA injection caused limited damage in Par2KO mice livers, while in the WT mice severe damage followed by leukocyte infiltration was evident. Reciprocal BM replacement of WT and Par2KO showed that WT BM-reconstituted Par2KO mice displayed marked liver damage, while in Par2KO BM-reconstituted WT mice, the tissue was generally protected. In the CCl4 direct damage model, hepatocytes regenerated in WT mice, whereas Par2KO mice failed to recover. Reciprocal BM replacement did not show significant differences in hepatic regeneration. In Par2KO mice, hepatitis was more apparent, while WT recovered regardless of the BM origin.ConclusionsWe conclude that Par2 activation in the immune system aggravates hepatitis and that Par2 activation in the damaged tissue promotes liver regeneration. When we incorporate this finding and revisit the literature reports, we reconciled the conflicts surrounding Par2's role in injury, recovery, and inflammation.
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Project description:UnlabelledSkin tissue expansion is a clinical procedure for skin regeneration to reconstruct cutaneous defects that can be accompanied by severe complications. The transplantation of mesenchymal stem cells (MSCs) has been proven effective in promoting skin expansion and helping to ameliorate complications; however, systematic understanding of its mechanism remains unclear. MSCs from luciferase-Tg Lewis rats were intravenously transplanted into a rat tissue expansion model to identify homing and transdifferentiation. To clarify underlying mechanisms, a systematic approach was used to identify the differentially expressed genes between mechanically stretched human MSCs and controls. The biological significance of these changes was analyzed through bioinformatic methods. We further investigated genes and pathways of interest to disclose their potential role in mechanical stretching-induced skin regeneration. Cross sections of skin samples from the expanded group showed significantly more luciferase(+) and stromal cell-derived factor 1α (SDF-1α)(+), luciferase(+)keratin 14(+), and luciferase(+)CD31(+) cells than the control group, indicating MSC transdifferentiation into epidermal basal cells and endothelial cells after SDF-1α-mediated homing. Microarray analysis suggested upregulation of genes related to hypoxia, vascularization, and cell proliferation in the stretched human MSCs. Further investigation showed that the homing of MSCs was blocked by short interfering RNA targeted against matrix metalloproteinase 2, and that mechanical stretching-induced vascular endothelial growth factor A upregulation was related to the Janus kinase/signal transducer and activator of transcription (Jak-STAT) and Wnt signaling pathways. This study determines that mechanical stretching might promote skin regeneration by upregulating MSC expression of genes related to hypoxia, vascularization, and cell proliferation; enhancing transplanted MSC homing to the expanded skin; and transdifferentiation into epidermal basal cells and endothelial cells.SignificanceSkin tissue expansion is a clinical procedure for skin regeneration to cover cutaneous defects that can be accompanied by severe complications. The transplantation of mesenchymal stem cells (MSCs) has been proven effective in promoting skin expansion and ameliorating complications. This study, which sought to provide a systematic understanding of the mechanism, determined that mechanical stretching could upregulate MSC expression of genes related to hypoxia, vascularization, and cell proliferation; enhance transplanted MSC homing to the expanded skin tissue; and promote their transdifferentiation into epidermal basal cells and endothelial cells.
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