Interactions of DPP-4 and integrin β1 influences endothelial-to-mesenchymal transition
2015; Elsevier BV; Volume: 88; Issue: 3 Linguagem: Inglês
10.1038/ki.2015.103
ISSN1523-1755
AutoresSen Shi, Swayam Prakash Srivastava, Megumi Kanasaki, Jianhua He, Munehiro Kitada, Takako Nagai, Kyoko Nitta, Susumu Takagi, Keizo Kanasaki, Daisuke Koya,
Tópico(s)Peptidase Inhibition and Analysis
ResumoIntegrin β1 and dipeptidyl peptidase (DPP)-4 play roles in endothelial cell biology. Vascular endothelial growth factor (VEGF)-A inhibits endothelial-to-mesenchymal transition (EndMT) through VEGF-R2, but through VEGF-R1 promotes EndMT by reducing the bioavailability of VEGF-A. Here we tested whether DPP-4-integrin β1 interactions have a role in EndMT in the renal fibrosis of diabetic nephropathy. In streptozotocin-induced fibrotic kidneys in diabetic CD-1 mice, levels of endothelial DPP-4, integrin β1, and phospho-integrin β1 were all higher and associated with plasma cystatin C elevation. The DPP-4 inhibitor linagliptin ameliorated kidney fibrosis, reduced plasma cystatin C levels, and suppressed endothelial levels of DPP-4, integrin β1, and phospho-integrin β1. In cultured endothelial cells, DPP-4 and integrin β1 physically interacted. Suppression of DPP-4 by siRNA was associated with suppression of integrin β1 and vice versa. Knockdown of either integrin β1 or DPP-4 resulted in the silencing of TGF-β2-induced TGF-β receptor heterodimer formation, smad3 phosphorylation, and EndMT. DPP-4 negatively regulated endothelial viability signaling by VEGF-R2 suppression and VEGF-R1 induction in endothelial cells. Thus, DPP-4 and integrin β1 interactions regulate key endothelial cell signal transduction in both physiological and pathological conditions including EndMT. Hence, inhibiting DPP-4 may be a therapeutic target for treating kidney fibrosis in diabetes. Integrin β1 and dipeptidyl peptidase (DPP)-4 play roles in endothelial cell biology. Vascular endothelial growth factor (VEGF)-A inhibits endothelial-to-mesenchymal transition (EndMT) through VEGF-R2, but through VEGF-R1 promotes EndMT by reducing the bioavailability of VEGF-A. Here we tested whether DPP-4-integrin β1 interactions have a role in EndMT in the renal fibrosis of diabetic nephropathy. In streptozotocin-induced fibrotic kidneys in diabetic CD-1 mice, levels of endothelial DPP-4, integrin β1, and phospho-integrin β1 were all higher and associated with plasma cystatin C elevation. The DPP-4 inhibitor linagliptin ameliorated kidney fibrosis, reduced plasma cystatin C levels, and suppressed endothelial levels of DPP-4, integrin β1, and phospho-integrin β1. In cultured endothelial cells, DPP-4 and integrin β1 physically interacted. Suppression of DPP-4 by siRNA was associated with suppression of integrin β1 and vice versa. Knockdown of either integrin β1 or DPP-4 resulted in the silencing of TGF-β2-induced TGF-β receptor heterodimer formation, smad3 phosphorylation, and EndMT. DPP-4 negatively regulated endothelial viability signaling by VEGF-R2 suppression and VEGF-R1 induction in endothelial cells. Thus, DPP-4 and integrin β1 interactions regulate key endothelial cell signal transduction in both physiological and pathological conditions including EndMT. Hence, inhibiting DPP-4 may be a therapeutic target for treating kidney fibrosis in diabetes. Kidney fibrosis is the final common consequence of any type of kidney disease, including diabetic nephropathy. Kidney fibrosis is caused by prolonged insults that are associated with the dysregulation of normal wound healing and the excess accumulation of extracellular matrix (ECM) components.1.Kanasaki K. Taduri G. Koya D. Diabetic nephropathy: the role of inflammation in fibroblast activation and kidney fibrosis.Front Endocrinol (Lausanne). 2013; 4: 7PubMed Google Scholar Kidney fibroblasts have an essential role in fibrotic processes. Among the diverse origins of matrix-producing fibroblasts, endothelial-to-mesenchymal transition (EndMT) is an important source of fibroblasts.2.Zeisberg E.M. Potenta S.E. Sugimoto H. et al.Fibroblasts in kidney fibrosis emerge via endothelial-to-mesenchymal transition.J Am Soc Nephrol. 2008; 19: 2282-2287Crossref PubMed Scopus (681) Google Scholar, 3.He J. Xu Y. Koya D. et al.Role of the endothelial-to-mesenchymal transition in renal fibrosis of chronic kidney disease.Clin Exp Nephrol. 2013; 17: 488-497Crossref PubMed Scopus (137) Google Scholar, 4.Zeisberg E.M. Tarnavski O. Zeisberg M. et al.Endothelial-to-mesenchymal transition contributes to cardiac fibrosis.Nat Med. 2007; 13: 952-961Crossref PubMed Scopus (1627) Google Scholar EndMT is a cellular adaptation process in which vascular endothelial cells acquire myofibroblast features that are characterized by an ability to express mesenchymal cell characteristics related to tissue fibrogenesis. The EndMT is relevant to cardiac valve formation during development.5.Kovacic J.C. Mercader N. Torres M. et al.Epithelial-to-mesenchymal and endothelial-to-mesenchymal transition: from cardiovascular development to disease.Circulation. 2012; 125: 1795-1808Crossref PubMed Scopus (294) Google Scholar In addition, the EndMT has emerged as an important component in a variety of organ fibrosis conditions such as cardiac, pulmonary, and kidney fibrosis.6.Piera-Velazquez S. Li Z. Jimenez S.A. Role of endothelial-mesenchymal transition (EndoMT) in the pathogenesis of fibrotic disorders.Am J Pathol. 2011; 179: 1074-1080Abstract Full Text Full Text PDF PubMed Scopus (422) Google Scholar,7.Li J. Bertram J.F. Endothelial-myofibroblast transition, a new player in diabetic renal fibrosis.Nephrology (Carlton). 2010; 15: 507-512Crossref PubMed Scopus (83) Google Scholar Transforming growth factor (TGF)-β is the most potent inducer of EndMT and is the primary cytokine associated with the pathogenesis of fibroproliferative diseases.8.Sheppard D. Transforming growth factor beta: a central modulator of pulmonary and airway inflammation and fibrosis.Proc Am Thorac Soc. 2006; 3: 413-417Crossref PubMed Scopus (247) Google Scholar,9.Vallance B.A. Gunawan M.I. Hewlett B. et al.TGF-beta1 gene transfer to the mouse colon leads to intestinal fibrosis.Am J Physiol Gastrointest Liver Physiol. 2005; 289: G116-G128Crossref PubMed Scopus (128) Google Scholar Our previous studies showed that the dipeptidyl peptidase (DPP)-4 inhibitor linagliptin inhibited kidney fibrosis in diabetic mice and such anti-fibrotic effects were associated with EndMT suppression.10.Kanasaki K. Shi S. Kanasaki M. et al.Linagliptin-mediated DPP-4 inhibition ameliorates kidney fibrosis in streptozotocin-induced diabetic mice by inhibiting endothelial-to-mesenchymal transition in a therapeutic regimen.Diabetes. 2014; 63: 2120-2131Crossref PubMed Scopus (267) Google Scholar DPP-4 is a cell surface aminopeptidase that was originally characterized as a T-cell differentiation antigen (CD26). DPP-4 is ubiquitously distributed and responsible for the cleavage of numerous substrates.11.Fleischer B. CD26: a surface protease involved in T-cell activation.Immunol Today. 1994; 15: 180-184Abstract Full Text PDF PubMed Scopus (54) Google Scholar Furthermore, DPP-4 is a well-documented drug target for the treatment of type 2 diabetes because DPP-4 cleaves pro-insulinotropic incretin hormones.12.Drucker D.J. Therapeutic potential of dipeptidyl peptidase IV inhibitors for the treatment of type 2 diabetes.Expert Opin Investig Drugs. 2003; 12: 87-100Crossref PubMed Scopus (232) Google Scholar Recently, pleiotropic beneficial effects of DPP-4 inhibitors were reported in both clinical research and preclinical experiments.13.Lee Y.K. Song S.O. Kim K.J. et al.Glycemic effectiveness of metformin-based dual-combination therapies with sulphonylurea, pioglitazone, or DPP-4-inhibitor in drug-naive korean type 2 diabetic patients.Diabetes Metab J. 2013; 37: 465-474Crossref PubMed Scopus (19) Google Scholar, 14.Abd El Motteleb D.M. Elshazly S.M. Renoprotective effect of sitagliptin against hypertensive nephropathy induced by chronic administration of L-NAME in rats: role of GLP-1 and GLP-1 receptor.Eur J Pharmacol. 2013; 720: 158-165Crossref PubMed Scopus (32) Google Scholar, 15.Dos Santos L. Salles T.A. Arruda-Junior D.F. et al.Circulating dipeptidyl peptidase IV activity correlates with cardiac dysfunction in human and experimental heart failure.Circ Heart Fail. 2013; 6: 1029-1038Crossref PubMed Scopus (94) Google Scholar, 16.Sortino M.A. Sinagra T. Canonico P.L. Linagliptin: a thorough characterization beyond its clinical efficacy.Front Endocrinol. 2013; 4: 16Crossref PubMed Scopus (25) Google Scholar DPP-4 is one of the matrix-consisting/interacting proteins and is associated with integrin β1.17.Ghersi G. Zhao Q. Salamone M. et al.The protease complex consisting of dipeptidyl peptidase IV and seprase plays a role in the migration and invasion of human endothelial cells in collagenous matrices.Cancer Res. 2006; 66: 4652-4661Crossref PubMed Scopus (114) Google Scholar The loss of DPP-4 expression at the cell surface is associated with decreased integrin β1 phosphorylation at the S785 residue, which has a key role in integrin β1 cellular adhesion to the ECM.18.Sato T. Yamochi T. Yamochi T. et al.CD26 regulates p38 mitogen-activated protein kinase-dependent phosphorylation of integrin beta1, adhesion to extracellular matrix, and tumorigenicity of T-anaplastic large cell lymphoma Karpas 299.Cancer Res. 2005; 65: 6950-6956Crossref PubMed Scopus (58) Google Scholar Integrins are cell surface receptors that have key roles in cell migration and adhesion by providing a physical interaction between the ECM, cytoskeleton, and transmitting signals.19.Hynes R.O. Integrins: bidirectional, allosteric signaling machines.Cell. 2002; 110: 673-687Abstract Full Text Full Text PDF PubMed Scopus (6875) Google Scholar Integrin β1 forms at least 12 different types of integrins by interacting with different α chains of the integrin family and possesses diverse physiological and pathological functions.20.Barczyk M. Carracedo S. Gullberg D. Integrins.Cell Tissue Res. 2010; 339: 269-280Crossref PubMed Scopus (1165) Google Scholar,21.Campbell I.D. Humphries M.J. Integrin structure, activation, and interactions.Cold Spring Harbor Perspect Biol. 2011; 3: a004994Crossref Scopus (693) Google Scholar Blocking integrin β1 signaling diminishes the fibrosis progression.22.Liu S. Kapoor M. Denton C.P. et al.Loss of beta1 integrin in mouse fibroblasts results in resistance to skin scleroderma in a mouse model.Arthritis Rheum. 2009; 60: 2817-2821Crossref PubMed Scopus (76) Google Scholar CD26 potentiates tumor cell invasion through its interaction with α5β1 integrin.23.Okamoto T. Iwata S. Yamazaki H. et al.CD9 negatively regulates CD26 expression and inhibits CD26-mediated enhancement of invasive potential of malignant mesothelioma cells.PLoS One. 2014; 9: e86671Crossref PubMed Scopus (30) Google Scholar Here, we hypothesized that the DPP-4–integrin β1 interaction is a therapeutic target for kidney fibrosis in diabetes through normalization of endothelial cell homeostasis and inhibition of EndMT. To determine the pathological significance of the interaction between DPP-4 and integrin β1, we analyzed streptozotocin (STZ)-induced diabetic male CD-1 mice, a murine model with extensive diabetes-associated kidney fibrosis.24.Sugimoto H. Grahovac G. Zeisberg M. et al.Renal fibrosis and glomerulosclerosis in a new mouse model of diabetic nephropathy and its regression by bone morphogenic protein-7 and advanced glycation end product inhibitors.Diabetes. 2007; 56: 1825-1833Crossref PubMed Scopus (187) Google Scholar STZ-induced diabetic CD-1 mice exhibited elevated blood glucose levels and lower body weight. Linagliptin treatment in STZ-induced diabetic CD-1 mice did not affect the blood glucose levels and body weight of the mice (Supplementary Figure S1A and S1B online). STZ-induced diabetic CD-1 mice also exhibited kidney fibrosis in a time-dependent manner, associated with elevated levels of plasma cystatin C and higher urine albumin excretion (Supplementary Figure S1C and S1D online). At 20 weeks after the initiation of diabetes, the diabetic mice displayed severe kidney fibrosis compared with control mice (Supplementary Figure S2A–S2E, S2H–S2L online). Linagliptin treatment in diabetic mice from 20 weeks after diabetes induction ameliorated the kidney fibrosis associated with cystatin C suppression in a time-dependent manner (Supplementary Figure S1C and S2F–S2G, S2M–S2N online). Morphometric analysis of the kidneys revealed that diabetic mice displayed significantly enlarged glomeruli, mesangial expansion, and relative areas of Masson-trichrome-positive interstitial fibrosis; however, linagliptin inhibited these pathological alterations in the diabetic kidneys of CD-1 mice (Supplementary Figure S2O–S2Q online). Immunofluorescence analysis revealed that phospho-integrin β1 levels in endothelial cells in diabetic kidneys were significantly higher than in control kidneys, and these increased levels of phospho-integrin β1 were suppressed in linagliptin-treated diabetic mouse kidneys (Figure 1a–c). DPP-4 is immunolabeled on the glomerular basement membrane, tubules, and CD31-positive endothelial cells in the kidneys of STZ-induced diabetic CD-1 mice.10.Kanasaki K. Shi S. Kanasaki M. et al.Linagliptin-mediated DPP-4 inhibition ameliorates kidney fibrosis in streptozotocin-induced diabetic mice by inhibiting endothelial-to-mesenchymal transition in a therapeutic regimen.Diabetes. 2014; 63: 2120-2131Crossref PubMed Scopus (267) Google Scholar Here, we found that the expression of integrin β1 protein, integrin β1 phosphorylation, and DPP-4 protein was induced in diabetic kidneys compared with control kidneys and that linagliptin suppressed all of these inductions (Figure 1d–i). The effects of integrin β1 were mediated through the formation of a heterodimer with α-subunits such as α3, α5, or αv on endothelial cells.20.Barczyk M. Carracedo S. Gullberg D. Integrins.Cell Tissue Res. 2010; 339: 269-280Crossref PubMed Scopus (1165) Google Scholar,21.Campbell I.D. Humphries M.J. Integrin structure, activation, and interactions.Cold Spring Harbor Perspect Biol. 2011; 3: a004994Crossref Scopus (693) Google Scholar Immunofluorescence analysis revealed that α3 and αv were immunolabeled in both non-endothelial glomerular cells and endothelial cells of the kidney, whereas integrin α5 was dominantly expressed in endothelial cells (Supplementary Figure S3A–S3J online). Western blot analysis revealed that STZ-induced diabetic mice had increased expression levels of DPP-4, TGF-β receptor (TGF-βR) 1, and TGF-βR2 compared with control kidneys (Figure 1j–m). Integrin β1 protein levels and phospho-integrin β1 levels were also increased in diabetic kidneys (Figure 1n–p). The increased expression levels of DPP-4, integrin β1, phospho-integrin β1, TGF-βR1, and TGF-βR2 were all suppressed by linagliptin treatment (Figure 1j–p). Download .jpg (.05 MB) Help with files Supplementary Figure 1 Download .jpg (.12 MB) Help with files Supplementary Figure 2-1 Download .jpg (.06 MB) Help with files Supplementary Figure 2-2 Download .jpg (.08 MB) Help with files Supplementary Figure 3 Utilizing small interfering RNA (siRNA)-mediated knockdown of integrin β1 or DPP-4, we investigated the role of the interaction between integrin β1 and DPP-4 on EndMT induction. Western blot analysis revealed that TGF-β2 induced the suppression of the endothelial-specific protein von Willebrand factor with concomitant induction of αSMA and SM22α in cultured endothelial cells, suggesting that TGF-β2 induces EndMT. siRNA-mediated integrin β1 knockdown resulted in the suppression of TGF-β2-induced EndMT (Figure 2a). Furthermore, integrin β1 deletion was also associated with DPP-4 protein level suppression (Figure 2a). TGF-β2-induced phosphorylation of smad3 was also suppressed by integrin β1 deletion (Figure 2b and c). To further confirm the role of integrin β1 in EndMT induction and DPP-4 activity, we treated endothelial cells with an integrin β1 activation antibody (9EG7) and found that integrin β1 activation induced EndMT, as shown by the suppression of the endothelial marker CD31 and the induction of mesenchymal markers (SM22α and αSMA), DPP-4, and TGF-βR1 compared with cells treated with control immunoglobulin G (Figure 2d). The activation of integrin β1-induced EndMT was inhibited by DPP-4 deletion, revealing the DPP-4-dependent induction of EndMT (Figure 2e). DPP-4 deletion by siRNA in endothelial cells abolished TGF-β2-induced EndMT, integrin β1 protein expression, and phosphorylated-integrin β1 compared with cells transfected with control siRNA (Figure 2f). To confirm the role of the mechanisms underlying the interaction between DPP-4 and integrin β1 on EndMT induction, we analyzed the levels of TGF-βRs and the protein interaction of TGF-βRs, both of which are critical for TGF-β signal transduction.25.Dennler S. Goumans M.J. ten Dijke P. Transforming growth factor beta signal transduction.J Leukoc Biol. 2002; 71: 731-740PubMed Google Scholar Western blot analysis revealed that both TGF-βR1 and TGF-βR2 were increased by TGF-β2 stimulation and were decreased when either integrin β1 or DPP-4 was suppressed via siRNA (Figure 3a and b). The Duolink In Situ proximity ligation assay was performed, and DPP-4 and integrin β1 were shown to be in close proximity on the endothelial cells (Figure 3c). The close proximity between DPP-4 and integrin β1 was increased in TGF-β2-treated cells; this TGF-β2-induced proximity of these two molecules was suppressed by linagliptin (Figure 3c). Such close proximity between DPP-4 and integrin β1 increased in the endothelial cells cultured in the medium with a high glucose concentration (30 mmol/l) compared with the cells cultured in normal glucose medium (5.5 mmol/l glucose; Supplementary Figure S4A–S4D online). The close proximity between TGF-βR1 and TGF-βR2 suggests that TGF-β2 induced TGF-βR heterodimer formation; however, the proximity between TGF-βR1 and TGF-βR2 was suppressed in either integrin β1 or DPP-4 siRNA-transfected cells compared with the cells transfected with control siRNA (Figure 3d–f). This finding suggests that the interaction between integrin β1 and DPP-4 is essential for TGF-β-induced receptor heterodimerization and subsequent intracellular signal transduction. When treated with integrin β1 activation antibody (9EG7), the proximity of DPP-4-integrin β1 or TGF-βR1-R2 was increased when compared with control immunoglobulin G treatment (Figure 3g and h). We also performed an immunoprecipitation assay and found that the interaction between DPP-4 and integrin β1 increased in TGF-β2-treated cells and was suppressed by linagliptin (Figure 4a–c). Download .jpg (.05 MB) Help with files Supplementary Figure 4Figure 4TGF-β2-induced DPP-4 and integrin β1 physical interaction was suppressed by linagliptin in the immunoprecipitation assay. (a) Western blot analysis of immunoprecipitation combined with DPP-4 antibody. (b) Western blot analysis of immunoprecipitation combined with integrin β1 antibody. (c) Western blot analysis for DPP-4 and integrin β1 without immunoprecipitation. Representative results from 3 independent analyses are shown. DPP-4, dipeptidyl peptidase-4; TGF-β, transforming growth factor-β.View Large Image Figure ViewerDownload (PPT) Regarding the β-subunits of integrins, β1 and β3 are two major integrins involved in the cell–ECM interaction.20.Barczyk M. Carracedo S. Gullberg D. Integrins.Cell Tissue Res. 2010; 339: 269-280Crossref PubMed Scopus (1165) Google Scholar,21.Campbell I.D. Humphries M.J. Integrin structure, activation, and interactions.Cold Spring Harbor Perspect Biol. 2011; 3: a004994Crossref Scopus (693) Google Scholar To investigate whether integrin β3 is also associated with DPP-4, we performed siRNA-mediated knockdown of integrin β3 and analyzed the DPP-4 protein levels in endothelial cells. In contrast with integrin β1 (Figure 2a), integrin β3 deletion in endothelial cells increased the DPP-4 protein level but did not alter the DPP-4 level under stimulation with TGF-β2 (Supplementary Figure S5A and S5B online). Download .jpg (.05 MB) Help with files Supplementary Figure 5 Both TGF-β and vascular endothelial growth factor (VEGF) signaling have important physiological and pathological roles in diabetic nephropathy.26.Fujimoto M. Maezawa Y. Yokote K. et al.Mice lacking Smad3 are protected against streptozotocin-induced diabetic glomerulopathy.Biochem Biophys Res Commun. 2003; 305: 1002-1007Crossref PubMed Scopus (167) Google Scholar, 27.Wang A. Ziyadeh F.N. Lee E.Y. et al.Interference with TGF-beta signaling by Smad3-knockout in mice limits diabetic glomerulosclerosis without affecting albuminuria.Am J Physiol Renal Physiol. 2007; 293: F1657-F1665Crossref PubMed Scopus (107) Google Scholar, 28.Chen S. Iglesias-de la Cruz M.C. Jim B. et al.Reversibility of established diabetic glomerulopathy by anti-TGF-beta antibodies in db/db mice.Biochem Biophys Res Commun. 2003; 300: 16-22Crossref PubMed Scopus (125) Google Scholar VEGF-A has biologically significant roles through the VEGF-receptor (-R) 2 in endothelial cells, and VEGF-R1 binds to VEGF-A but may not contribute to cell survival.29.Olsson A.K. Dimberg A. Kreuger J. et al.VEGF receptor signalling-in control of vascular function.Nat Rev Mol Cell Biol. 2006; 7: 359-371Crossref PubMed Scopus (2455) Google Scholar EndMT is negatively regulated by VEGF–VEGF-R2 signaling; VEGF-R1-mediated trapping of VEGF-A results in the reduction of bioavailable VEGF-A, inducing EndMT.30.Medici D. Shore E.M. Lounev V.Y. et al.Conversion of vascular endothelial cells into multipotent stem-like cells.Nat Med. 2010; 16: 1400-1406Crossref PubMed Scopus (550) Google Scholar When the endothelial cells were stimulated with TGF-β2, VEGF-R1 was induced; however, linagliptin suppressed TGF-β2-stimulated VEGF-R1 levels (Figure 5a and b). Next, we performed DPP-4 knockdown in endothelial cells using specific siRNA and found that DPP-4 knockdown resulted in the suppression of VEGF-R1 and in the induction of VEGF-R2 levels (Figure 5c–e). By contrast, DPP-4 complementary DNA-overexpressing endothelial cells displayed VEGF-R1 induction, VEGF-R2 suppression, and resistance to the VEGF-A-mediated phosphorylation of extracellular signal–regulated kinase (ERK; Figure 5f–i), suggesting that VEGF-A-stimulated endothelial cell viability signaling was compromised in cells with DPP-4 overexpression. We also performed a tube formation assay in endothelial cells and found that TGF-β2 decreased tube formation compared with the control group (Figure 5j and k). VEGF incubation with TGF-β2-pretreated cells had a small but significant effect on tube formation (Figure 5l). Linagliptin treatment further enhanced tube formation on Matrigel (Figure 5m and o). According to the MTT cell viability assay, TGF-β2-suppressed endothelial cell viability was reversed by linagliptin (Figure 5p) and DPP-4 or integrin β1 siRNA transfection (Figure 5q and r). When analyzing in vivo kidney samples, immunofluorescence and western blot analysis revealed that the VEGF-R1 levels on the endothelial cells of the diabetic kidney were significantly elevated compared with control kidneys. These elevated VEGF-R1 levels in diabetic kidneys were suppressed by linagliptin (Figure 6a–c and g), whereas the endothelial VEGF-R2 levels were remarkably lower in diabetic kidneys compared with control kidneys. Linagliptin treatment restored the VEGF-R2 levels to normal levels (Figure 6d–g).Figure 6VEGF-R1 and R2 expression in the kidney. (a–f) Immunofluorescence microscopy analysis of diabetic mice. (a–c) VEGF-R1/CD31; (d–f) VEGF-R2/CD31. Merged images are shown. The original magnification was × 300. Scale bar = 50 μm in each panel. Representative pictures from three mice are shown. The inset in each figure shows enlarged image of the indicated area. (g) Western blot analysis for VEGF-R1 and VEGF-R2 in the kidney, which was normalized with GAPDH. VEGF-R, vascular endothelial growth factor receptor.View Large Image Figure ViewerDownload (PPT) Endothelial cell damage is a major therapeutic target for diabetic complications and EndMT development, which is important when considering organ fibrosis in diabetes. In this study, we found a new profibrotic molecular mechanism associated with the interaction between DPP-4 and integrin β1. DPP-4-associated EndMT was inhibited by integrin β1 deletion. In addition, DPP-4 or integrin β1 deficiency resulted in the inhibition of TGF-β2-stimulated heterodimer formation of TGF-βRs. Finally, the interaction between DPP-4 and integrin β1 induced VEGF-R1 expression with the concomitant suppression of VEGF-R2 levels. These results indicate that the interaction between DPP-4 and integrin β1 may be a therapeutic target for kidney fibrosis in diabetes. Integrin β1 has been implicated in the diverse roles of biological processes, including cell migration, adhesion, angiogenesis, basement membrane formation, and cell cycle control.31.Mulrooney J.P. Hong T. Grabel L.B. Serine 785 phosphorylation of the beta1 cytoplasmic domain modulates beta1A-integrin-dependent functions.J Cell Sci. 2001; 114: 2525-2533PubMed Google Scholar, 32.Kanasaki K. Yu W. von Bodungen M. et al.Loss of beta1-integrin from urothelium results in overactive bladder and incontinence in mice: a mechanosensory rather than structural phenotype.FASEB J. 2013; 27: 1950-1961Crossref PubMed Scopus (33) Google Scholar, 33.Kanasaki K. Kanda Y. Palmsten K. et al.Integrin beta1-mediated matrix assembly and signaling are critical for the normal development and function of the kidney glomerulus.Dev Biol. 2008; 313: 584-593Crossref PubMed Scopus (110) Google Scholar, 34.Tanjore H. Zeisberg E.M. Gerami-Naini B. et al.Beta1 integrin expression on endothelial cells is required for angiogenesis but not for vasculogenesis.Dev Dyn. 2008; 237: 75-82Crossref PubMed Scopus (69) Google Scholar The levels of integrin β1, DPP-4, and phosphorylated-integrin β1 protein were all increased in endothelial cells in STZ-induced diabetic kidneys and were suppressed by linagliptin treatment. In a recent study, we showed that the anti-fibrotic effects of the DPP-4 inhibitor linagliptin were associated with EndMT inhibition,10.Kanasaki K. Shi S. Kanasaki M. et al.Linagliptin-mediated DPP-4 inhibition ameliorates kidney fibrosis in streptozotocin-induced diabetic mice by inhibiting endothelial-to-mesenchymal transition in a therapeutic regimen.Diabetes. 2014; 63: 2120-2131Crossref PubMed Scopus (267) Google Scholar and EndMT has emerged as an important origin of ECM-producing mesenchymal cells.2.Zeisberg E.M. Potenta S.E. Sugimoto H. et al.Fibroblasts in kidney fibrosis emerge via endothelial-to-mesenchymal transition.J Am Soc Nephrol. 2008; 19: 2282-2287Crossref PubMed Scopus (681) Google Scholar Integrin β1 deletion in skin fibroblasts has resulted in resistance in a bleomycin-induced skin scleroderma animal model.22.Liu S. Kapoor M. Denton C.P. et al.Loss of beta1 integrin in mouse fibroblasts results in resistance to skin scleroderma in a mouse model.Arthritis Rheum. 2009; 60: 2817-2821Crossref PubMed Scopus (76) Google Scholar TGF-β2-induced EndMT was suppressed by integrin β1 deletion, and this effect of integrin β1 deletion was associated with DPP-4 suppression. By contrast, the activation of integrin β1 by a specific antibody induced EndMT associated with DPP-4 induction. Integrin β1 interacted with TGF-β/smad signaling and affected EMT in kidney tubular epithelial cells and tubulointerstitial fibrosis.35.Yeh Y.C. Wei W.C. Wang Y.K. et al.Transforming growth factor-{beta}1 induces Smad3-dependent {beta}1 integrin gene expression in epithelial-to-mesenchymal transition during chronic tubulointerstitial fibrosis.Am J Pathol. 2010; 177: 1743-1754Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar,36.Xiwu C. Hongtao W. Hong-Jun L. Integrin-mediated type II TGF-β receptor tyrosine dephosphorylation controls SMAD-dependent profibrotic signaling.J Clin Invest. 2014; 124: 3295-3310Crossref PubMed Scopus (50) Google Scholar The DPP-4 inhibitor linagliptin inhibits TGF-β2-induced smad3 phosphorylation, and integrin β1 deletion was associated with the suppression of DPP-4, TGF-βR interaction, and phospho-smad3 levels. By utilizing the Duolink In Situ Proximity Ligation Assay, we found that DPP-4 and integrin β1 were in close proximity on the endothelial cells. This close proximity was a physical interaction as confirmed by an immunoprecipitation assay. This close proximity between DPP-4 and integrin β1 was increased after TGF-β2 treatment in endothelial cells; such TGF-β2-induced pro
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