Pancreatic β-cell protection from inflammatory stress by the endoplasmic reticulum proteins thrombospondin 1 and mesencephalic astrocyte-derived neutrotrophic factor (MANF)
2017; Elsevier BV; Volume: 292; Issue: 36 Linguagem: Inglês
10.1074/jbc.m116.769877
ISSN1083-351X
AutoresDaniel A. Cunha, Monia Cito, Fabio Arturo Grieco, Cristina Cosentino, Tatiana Danilova, Laurence Ladrière, Maria Lindahl, Andrii Domanskyi, Marco Bugliani, Piero Marchetti, Décio L. Eizirik, Miriam Cnop,
Tópico(s)Endoplasmic Reticulum Stress and Disease
ResumoCytokine-induced endoplasmic reticulum (ER) stress is one of the molecular mechanisms underlying pancreatic β-cell demise in type 1 diabetes. Thrombospondin 1 (THBS1) was recently shown to promote β-cell survival during lipotoxic stress. Here we show that ER-localized THBS1 is cytoprotective to rat, mouse, and human β-cells exposed to cytokines or thapsigargin-induced ER stress. THBS1 confers cytoprotection by maintaining expression of mesencephalic astrocyte-derived neutrotrophic factor (MANF) in β-cells and thereby prevents the BH3-only protein BIM (BCL2-interacting mediator of cell death)-dependent triggering of the mitochondrial pathway of apoptosis. Prolonged exposure of β-cells to cytokines or thapsigargin leads to THBS1 and MANF degradation and loss of this prosurvival mechanism. Approaches that sustain intracellular THBS1 and MANF expression in β-cells should be explored as a cytoprotective strategy in type 1 diabetes. Cytokine-induced endoplasmic reticulum (ER) stress is one of the molecular mechanisms underlying pancreatic β-cell demise in type 1 diabetes. Thrombospondin 1 (THBS1) was recently shown to promote β-cell survival during lipotoxic stress. Here we show that ER-localized THBS1 is cytoprotective to rat, mouse, and human β-cells exposed to cytokines or thapsigargin-induced ER stress. THBS1 confers cytoprotection by maintaining expression of mesencephalic astrocyte-derived neutrotrophic factor (MANF) in β-cells and thereby prevents the BH3-only protein BIM (BCL2-interacting mediator of cell death)-dependent triggering of the mitochondrial pathway of apoptosis. Prolonged exposure of β-cells to cytokines or thapsigargin leads to THBS1 and MANF degradation and loss of this prosurvival mechanism. Approaches that sustain intracellular THBS1 and MANF expression in β-cells should be explored as a cytoprotective strategy in type 1 diabetes. Type 1 and type 2 diabetes mellitus (T1D 3The abbreviations used are: T1D, type 1 diabetes; T2D, type 2 diabetes; ER, endoplasmic reticulum; PERK, protein kinase R-like endoplasmic reticulum kinase; MANF, mesencephalic astrocyte-derived neutrotrophic factor; BiP, binding immunoglobulin protein; DCF, 2‘,7‘-dichlorofluorescein diacetate; THBS, thrombospondin; PUMA, p53-upregulated modulator of apoptosis; DP5, death protein 5; BIM, BCL2-interacting mediator of cell death; BAD, BCL-2-associated death promoter; BAX, BCL-2-associated protein X; NOD, nonobese diabetic; THA, thapsigargin; ad, adenovirus; Luc, luciferase. 3The abbreviations used are: T1D, type 1 diabetes; T2D, type 2 diabetes; ER, endoplasmic reticulum; PERK, protein kinase R-like endoplasmic reticulum kinase; MANF, mesencephalic astrocyte-derived neutrotrophic factor; BiP, binding immunoglobulin protein; DCF, 2‘,7‘-dichlorofluorescein diacetate; THBS, thrombospondin; PUMA, p53-upregulated modulator of apoptosis; DP5, death protein 5; BIM, BCL2-interacting mediator of cell death; BAD, BCL-2-associated death promoter; BAX, BCL-2-associated protein X; NOD, nonobese diabetic; THA, thapsigargin; ad, adenovirus; Luc, luciferase. and T2D, respectively) are characterized by failure of the pancreatic β-cells. In T1D, this is caused by autoimmune aggression against β-cells that leads to progressive β-cell dysfunction and death. The pathogenesis of T2D is characterized by different degrees of β-cell failure/loss relative to variable degrees of insulin resistance (1.Cnop M. Welsh N. Jonas J.C. Jörns A. Lenzen S. Eizirik D.L. Mechanisms of pancreatic β-cell death in type 1 and type 2 diabetes: many differences, few similarities.Diabetes. 2005; 54: S97-S107Crossref PubMed Scopus (1177) Google Scholar). Apoptosis seems to be the main form of β-cell death in both forms of the disease. In T1D, invading immune cells trigger β-cell death by cell-to-cell contact and local production of proinflammatory cytokines, such as IL-1β and IFN-γ (2.Eizirik D.L. Colli M.L. Ortis F. The role of inflammation in insulitis and β-cell loss in type 1 diabetes.Nat. Rev. Endocrinol. 2009; 5: 219-226Crossref PubMed Scopus (737) Google Scholar). The recent observation that gene expression in laser-captured islets from recent-onset T1D patients (3.Lundberg M. Krogvold L. Kuric E. Dahl-Jørgensen K. Skog O. Expression of interferon-stimulated genes in insulitic pancreatic islets of patients recently diagnosed with type 1 diabetes.Diabetes. 2016; 65: 3104-3110Crossref PubMed Scopus (72) Google Scholar) is remarkably similar to gene expression in human islets exposed in vitro to IL-1β and IFN-γ (4.Eizirik D.L. Sammeth M. Bouckenooghe T. Bottu G. Sisino G. Igoillo-Esteve M. Ortis F. Santin I. Colli M.L. Barthson J. Bouwens L. Hughes L. Gregory L. Lunter G. Marselli L. et al.The human pancreatic islet transcriptome: expression of candidate genes for type 1 diabetes and the impact of pro-inflammatory cytokines.PLoS Genet. 2012; 8: e1002552Crossref PubMed Scopus (324) Google Scholar) supports the idea that these cytokines (or other cytokines that elicit similar signal transduction) play a role in the human disease. In the context of T2D, the metabolic stress of chronic exposure to elevated levels of saturated free fatty acids, such as palmitate, and glucose contribute to β-cell dysfunction and apoptosis (5.Cnop M. Fatty acids and glucolipotoxicity in the pathogenesis of type 2 diabetes.Biochem. Soc. Trans. 2008; 36: 348-352Crossref PubMed Scopus (153) Google Scholar, 6.Poitout V. Robertson R.P. Glucolipotoxicity: fuel excess and β-cell dysfunction.Endocr. Rev. 2008; 29: 351-366Crossref PubMed Scopus (820) Google Scholar). It is of high interest to identify approaches that prevent both immune-mediated and metabolic β-cell demise; such an approach would be very useful in the prevention or early treatment of T1D and T2D. This task is made difficult, however, by the fact that proinflammatory cytokines (4.Eizirik D.L. Sammeth M. Bouckenooghe T. Bottu G. Sisino G. Igoillo-Esteve M. Ortis F. Santin I. Colli M.L. Barthson J. Bouwens L. Hughes L. Gregory L. Lunter G. Marselli L. et al.The human pancreatic islet transcriptome: expression of candidate genes for type 1 diabetes and the impact of pro-inflammatory cytokines.PLoS Genet. 2012; 8: e1002552Crossref PubMed Scopus (324) Google Scholar) and palmitate (7.Cnop M. Abdulkarim B. Bottu G. Cunha D.A. Igoillo-Esteve M. Masini M. Turatsinze J.V. Griebel T. Villate O. Santin I. Bugliani M. Ladriere L. Marselli L. McCarthy M.I. Marchetti P. et al.RNA sequencing identifies dysregulation of the human pancreatic islet transcriptome by the saturated fatty acid palmitate.Diabetes. 2014; 63: 1978-1993Crossref PubMed Scopus (189) Google Scholar) induce different gene networks and lead to pancreatic β-cell apoptosis by different mechanisms (1.Cnop M. Welsh N. Jonas J.C. Jörns A. Lenzen S. Eizirik D.L. Mechanisms of pancreatic β-cell death in type 1 and type 2 diabetes: many differences, few similarities.Diabetes. 2005; 54: S97-S107Crossref PubMed Scopus (1177) Google Scholar). One cellular stress response that is, however, present in β-cells in both forms of diabetes is endoplasmic reticulum (ER) stress (8.Marchetti P. Bugliani M. Lupi R. Marselli L. Masini M. Boggi U. Filipponi F. Weir G.C. Eizirik D.L. Cnop M. The endoplasmic reticulum in pancreatic β cells of type 2 diabetes patients.Diabetologia. 2007; 50: 2486-2494Crossref PubMed Scopus (322) Google Scholar, 9.Marhfour I. Lopez X.M. Lefkaditis D. Salmon I. Allagnat F. Richardson S.J. Morgan N.G. Eizirik D.L. Expression of endoplasmic reticulum stress markers in the islets of patients with type 1 diabetes.Diabetologia. 2012; 55: 2417-2420Crossref PubMed Scopus (166) Google Scholar). Pharmacological modulation of the ER stress response might therefore hold promise for β-cell therapy (10.Engin F. Yermalovich A. Nguyen T. Ngyuen T. Hummasti S. Fu W. Eizirik D.L. Mathis D. Hotamisligil G.S. Restoration of the unfolded protein response in pancreatic β cells protects mice against type 1 diabetes.Sci. Transl. Med. 2013; 5: 211ra156Crossref PubMed Scopus (205) Google Scholar, 11.Cunha D.A. Ladrière L. Ortis F. Igoillo-Esteve M. Gurzov E.N. Lupi R. Marchetti P. Eizirik D.L. Cnop M. Glucagon-like peptide-1 agonists protect pancreatic β-cells from lipotoxic endoplasmic reticulum stress through upregulation of BiP and JunB.Diabetes. 2009; 58: 2851-2862Crossref PubMed Scopus (179) Google Scholar). The multimeric Ca2+-binding glycoprotein thrombospondin 1 (THBS1) protects cardiomyocytes against ER stress via activation of ATF6 and downstream chaperones (12.Lynch J.M. Maillet M. Vanhoutte D. Schloemer A. Sargent M.A. Blair N.S. Lynch K.A. Okada T. Aronow B.J. Osinska H. Prywes R. Lorenz J.N. Mori K. Lawler J. Robbins J. Molkentin J.D. A thrombospondin-dependent pathway for a protective ER stress response.Cell. 2012; 149: 1257-1268Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). We have shown recently that THBS1 protects human and rodent β-cells from palmitate-induced apoptosis (13.Cunha D.A. Cito M. Carlsson P.O. Vanderwinden J.M. Molkentin J.D. Bugliani M. Marchetti P. Eizirik D.L. Cnop M. Thrombospondin 1 protects pancreatic β-cells from lipotoxicity via the PERK-NRF2 pathway.Cell Death Differ. 2016; 23: 1995-2006Crossref PubMed Scopus (50) Google Scholar). Different from cardiomyocytes, however, this takes place through activation of the ER stress transducer protein kinase R-like endoplasmic reticulum kinase (PERK) and the downstream transcription factor NRF2, increasing the β-cell capacity to withstand oxidative stress induced by saturated fatty acids (13.Cunha D.A. Cito M. Carlsson P.O. Vanderwinden J.M. Molkentin J.D. Bugliani M. Marchetti P. Eizirik D.L. Cnop M. Thrombospondin 1 protects pancreatic β-cells from lipotoxicity via the PERK-NRF2 pathway.Cell Death Differ. 2016; 23: 1995-2006Crossref PubMed Scopus (50) Google Scholar). Here we tested whether THBS1 is equally beneficial to rodent and human β-cells exposed to cytokines or chemically induced ER stress. THBS1 was clearly protective, but this was mediated by a different mechanism compared with protection against lipotoxic β-cell demise (13.Cunha D.A. Cito M. Carlsson P.O. Vanderwinden J.M. Molkentin J.D. Bugliani M. Marchetti P. Eizirik D.L. Cnop M. Thrombospondin 1 protects pancreatic β-cells from lipotoxicity via the PERK-NRF2 pathway.Cell Death Differ. 2016; 23: 1995-2006Crossref PubMed Scopus (50) Google Scholar) or cardiomyopathy (12.Lynch J.M. Maillet M. Vanhoutte D. Schloemer A. Sargent M.A. Blair N.S. Lynch K.A. Okada T. Aronow B.J. Osinska H. Prywes R. Lorenz J.N. Mori K. Lawler J. Robbins J. Molkentin J.D. A thrombospondin-dependent pathway for a protective ER stress response.Cell. 2012; 149: 1257-1268Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar); namely, through induction of the mesencephalic astrocyte-derived neutrotrophic factor (MANF). This raises the intriguing possibility that the multifunctional protein THBS1 changes roles and/or partner affinities in a cell- or stress-specific manner, as suggested recently for other complex biological systems (14.Hu J.X. Thomas C.E. Brunak S. Network biology concepts in complex disease comorbidities.Nat. Rev. Genet. 2016; 17: 615-629Crossref PubMed Scopus (196) Google Scholar). Furthermore, these findings indicate that THBS1-inducing agents may represent a novel strategy for β-cell protection in both T1D and T2D. Knockdown of THBS1 in rat INS-1E cells by two independent siRNAs did not affect basal expression of cleaved caspase 9 and 3 and apoptosis (Fig. 1, A and B), but it augmented caspase cleavage and cell death following exposure to the chemical ER stressor thapsigargin (an inhibitor of the SERCA2 pump that depletes ER Ca2+) and the proinflammatory cytokines IL-1β and IFN-γ, which cause ER stress, at least in part, via inhibition of SERCA2 (15.Cardozo A.K. Ortis F. Storling J. Feng Y.M. Rasschaert J. Tonnesen M. Van Eylen F. Mandrup-Poulsen T. Herchuelz A. Eizirik D.L. Cytokines downregulate the sarcoendoplasmic reticulum pump Ca2+ ATPase 2b and deplete endoplasmic reticulum Ca2+, leading to induction of endoplasmic reticulum stress in pancreatic β-cells.Diabetes. 2005; 54: 452-461Crossref PubMed Scopus (426) Google Scholar). The cleavage of both caspase 9 and 3 indicates that THBS1 knockdown favors activation of the intrinsic pathway of apoptosis by thapsigargin and cytokines (16.Gurzov E.N. Eizirik D.L. Bcl-2 proteins in diabetes: mitochondrial pathways of β-cell death and dysfunction.Trends Cell Biol. 2011; 21: 424-431Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar). These findings were confirmed in human islets silenced for THBS1 (Fig. 1, C and D). Apoptosis was also detected by immunostaining for cleaved caspase 3 in THBS1-depleted human β-cells exposed to thapsigargin (Fig. 1E and supplemental Fig. S1) or cytokines (supplemental Fig. S1). Islets isolated from THBS1 knock-out mice were also significantly sensitized to thapsigargin and cytokines (Fig. 1F); in both cases, lack of THBS1 sensitized the cells to thapsigargin- and cytokine-induced apoptosis. In mirror experiments, THBS1 was overexpressed in INS-1E cells (Fig. 1G) and human islet cells (Fig. 1I) using an adenoviral vector. THBS1 overexpression partially reduced thapsigargin- and cytokine-induced caspase 9 and 3 cleavage (Fig. 1G) and apoptosis in rat β-cells (Fig. 1H) and human islet cells (Fig. 1J). Because the above findings indicate that THBS1 protects human, rat, and mouse β-cells from cytokine- and ER stress–induced cell death, we evaluated whether these stresses affect THBS1 expression. Exposure of human islets to two different ER stressors, brefeldin A (which blocks transfer of cargo from the ER to the Golgi) and thapsigargin, decreased THBS1 mRNA expression by nearly 80% (Fig. 2A). A similar inhibition was observed in IL-1β and IFN-γ-exposed human islets (Fig. 2A), and these findings were confirmed at the protein level for thapsigargin and cytokines (Fig. 2B). Thapsigargin, which rapidly induces severe ER stress in INS-1E cells (15.Cardozo A.K. Ortis F. Storling J. Feng Y.M. Rasschaert J. Tonnesen M. Van Eylen F. Mandrup-Poulsen T. Herchuelz A. Eizirik D.L. Cytokines downregulate the sarcoendoplasmic reticulum pump Ca2+ ATPase 2b and deplete endoplasmic reticulum Ca2+, leading to induction of endoplasmic reticulum stress in pancreatic β-cells.Diabetes. 2005; 54: 452-461Crossref PubMed Scopus (426) Google Scholar), already reduced THBS1 protein expression by 2 h, with a progressive decrease up to 24 h (Fig. 2C). Cytokines, which induce ER stress more slowly in INS-1E cells (by 6–8 h) (15.Cardozo A.K. Ortis F. Storling J. Feng Y.M. Rasschaert J. Tonnesen M. Van Eylen F. Mandrup-Poulsen T. Herchuelz A. Eizirik D.L. Cytokines downregulate the sarcoendoplasmic reticulum pump Ca2+ ATPase 2b and deplete endoplasmic reticulum Ca2+, leading to induction of endoplasmic reticulum stress in pancreatic β-cells.Diabetes. 2005; 54: 452-461Crossref PubMed Scopus (426) Google Scholar), inhibited THBS1 expression by 8 h, with a nadir at 24 h. These observations suggest that chemical or cytokine-mediated ER stress progressively decreases THBS1 expression in β-cells, sensitizing these cells to a pro-apoptotic outcome. In addition to transcriptional inhibition (Fig. 2A), proteasomal degradation contributes to THBS1 depletion, as proteasome inhibition by MG132 prevented the loss of THBS1 (Fig. 2E). THBS1 may reside in the ER and/or be secreted into the extracellular milieu (13.Cunha D.A. Cito M. Carlsson P.O. Vanderwinden J.M. Molkentin J.D. Bugliani M. Marchetti P. Eizirik D.L. Cnop M. Thrombospondin 1 protects pancreatic β-cells from lipotoxicity via the PERK-NRF2 pathway.Cell Death Differ. 2016; 23: 1995-2006Crossref PubMed Scopus (50) Google Scholar, 17.Bornstein P. Diversity of function is inherent in matricellular proteins: an appraisal of thrombospondin 1.J. Cell Biol. 1995; 130: 503-506Crossref PubMed Scopus (582) Google Scholar, 18.Veliceasa D. Ivanovic M. Hoepfner F.T. Thumbikat P. Volpert O.V. Smith N.D. Transient potential receptor channel 4 controls thrombospondin-1 secretion and angiogenesis in renal cell carcinoma.FEBS J. 2007; 274: 6365-6377Crossref PubMed Scopus (64) Google Scholar). We thus investigated whether intra- or extracellular THBS1 mediates cytoprotection against cytokines or thapsigargin (Fig. 1). Conditioned medium from INS-1E cells overexpressing THBS1 (Fig. 3A) or exogenously added recombinant THBS1 (Fig. 3B), shown previously by us to transduce signals in β-cells (13.Cunha D.A. Cito M. Carlsson P.O. Vanderwinden J.M. Molkentin J.D. Bugliani M. Marchetti P. Eizirik D.L. Cnop M. Thrombospondin 1 protects pancreatic β-cells from lipotoxicity via the PERK-NRF2 pathway.Cell Death Differ. 2016; 23: 1995-2006Crossref PubMed Scopus (50) Google Scholar), failed to protect INS-1E cells against thapsigargin- or cytokine-induced apoptosis. On the other hand, an adenovirus for THBS1 containing the ER retention sequence KDEL (13.Cunha D.A. Cito M. Carlsson P.O. Vanderwinden J.M. Molkentin J.D. Bugliani M. Marchetti P. Eizirik D.L. Cnop M. Thrombospondin 1 protects pancreatic β-cells from lipotoxicity via the PERK-NRF2 pathway.Cell Death Differ. 2016; 23: 1995-2006Crossref PubMed Scopus (50) Google Scholar) was cytoprotective to the same extent as the control THBS1-FLAG adenovirus (Fig. 3, C and D). Islet cells from THBS1−/− mice have increased susceptibility to cell death induced by ER stressors or cytokines (Fig. 1F), and we next evaluated whether ER-retained THBS1-KDEL could rescue this phenotype. The THBS1-KDEL adenovirus markedly induced THBS1 mRNA expression under both control or stressed conditions (Fig. 3E). As in human islet cells (Fig. 1J), THBS1+/+ mouse islet cells were protected against thapsigargin or cytokines (Fig. 3F). Importantly, overexpression of ER-retained THBS1 in islet cells that lack endogenous THBS1 (THBS1−/−) abrogated the marked susceptibility to thapsigargin or cytokines (Fig. 3F), indicating that the ER-retained form of THBS1 is necessary and sufficient for the cytoprotective effects. It has been shown that THBS1 protects cardiomyocytes against ER stress by activating the transcription factor ATF6 and, thereby, up-regulating protective chaperones such as binding immunoglobulin protein (BiP) (12.Lynch J.M. Maillet M. Vanhoutte D. Schloemer A. Sargent M.A. Blair N.S. Lynch K.A. Okada T. Aronow B.J. Osinska H. Prywes R. Lorenz J.N. Mori K. Lawler J. Robbins J. Molkentin J.D. A thrombospondin-dependent pathway for a protective ER stress response.Cell. 2012; 149: 1257-1268Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). This does not, however, explain β-cell protection by THBS1. THBS1 silencing did not impair ATF6 activity, as assessed using a luciferase reporter, or BiP expression (supplemental Fig. S2, A and B). In keeping with previous findings (12.Lynch J.M. Maillet M. Vanhoutte D. Schloemer A. Sargent M.A. Blair N.S. Lynch K.A. Okada T. Aronow B.J. Osinska H. Prywes R. Lorenz J.N. Mori K. Lawler J. Robbins J. Molkentin J.D. A thrombospondin-dependent pathway for a protective ER stress response.Cell. 2012; 149: 1257-1268Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 13.Cunha D.A. Cito M. Carlsson P.O. Vanderwinden J.M. Molkentin J.D. Bugliani M. Marchetti P. Eizirik D.L. Cnop M. Thrombospondin 1 protects pancreatic β-cells from lipotoxicity via the PERK-NRF2 pathway.Cell Death Differ. 2016; 23: 1995-2006Crossref PubMed Scopus (50) Google Scholar), THBS1 knockdown reduced signaling in the PERK pathway (supplemental Fig. S2A). Conversely, THBS1 overexpression did not induce expression of BiP protein or mRNA, nor did it induce the ATF6 target GRP94 (supplemental Fig. S2, C and D). Our recent findings indicate that THBS1 protects β-cells from lipotoxicity via the PERK-NRF2 pathway and consequent up-regulation of antioxidant defenses (13.Cunha D.A. Cito M. Carlsson P.O. Vanderwinden J.M. Molkentin J.D. Bugliani M. Marchetti P. Eizirik D.L. Cnop M. Thrombospondin 1 protects pancreatic β-cells from lipotoxicity via the PERK-NRF2 pathway.Cell Death Differ. 2016; 23: 1995-2006Crossref PubMed Scopus (50) Google Scholar). THBS1 loss or gain of function did not, however, alter oxidative stress (supplemental Fig. S3, A and B). Cytokines, but not thapsigargin, increased oxidation of 2′,7′-dichlorofluorescein diacetate (DCF), and this was not altered by modulation of THBS1 expression (supplemental Fig. S3, A and B). In line with these findings, neither THBS1 silencing nor overexpression modified expression of the antioxidant genes GSTm1, catalase, and SOD2 (supplemental Fig. S3, C and D). THBS1 knockdown did not affect the activity of an antioxidant response element luciferase reporter (supplemental Fig. S3E), which we found previously to be activated by palmitate via up-regulation of NRF2 (13.Cunha D.A. Cito M. Carlsson P.O. Vanderwinden J.M. Molkentin J.D. Bugliani M. Marchetti P. Eizirik D.L. Cnop M. Thrombospondin 1 protects pancreatic β-cells from lipotoxicity via the PERK-NRF2 pathway.Cell Death Differ. 2016; 23: 1995-2006Crossref PubMed Scopus (50) Google Scholar). As a whole, these observations indicate that THBS1 protects β-cells against palmitate or cytokines/ER stressors by different mechanisms. We next explored alternative mechanisms underlying the THBS1 cytoprotection and examined the expression of MANF, shown previously to be up-regulated by THBS (12.Lynch J.M. Maillet M. Vanhoutte D. Schloemer A. Sargent M.A. Blair N.S. Lynch K.A. Okada T. Aronow B.J. Osinska H. Prywes R. Lorenz J.N. Mori K. Lawler J. Robbins J. Molkentin J.D. A thrombospondin-dependent pathway for a protective ER stress response.Cell. 2012; 149: 1257-1268Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). MANF is an ER stress–regulated protein that is crucial for postnatal mouse β-cell survival (19.Lindahl M. Danilova T. Palm E. Lindholm P. Võikar V. Hakonen E. Ustinov J. Andressoo J.O. Harvey B.K. Otonkoski T. Rossi J. Saarma M. MANF is indispensable for the proliferation and survival of pancreatic β cells.Cell Rep. 2014; 7: 366-375Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). Thapsigargin and cytokines inhibited MANF protein expression, and this was aggravated by THBS1 knockdown (Fig. 4A). Palmitate, on the other hand, did not reduce MANF expression (Fig. 4A). A time course analysis in INS-1E cells exposed to cytokines showed that MANF expression already decreased at 4 h, and more so in THBS1-depleted cells (Fig. 4B). On the other hand, adenoviral THBS1 overexpression induced and preserved MANF expression for up to 8 h of cytokine exposure (Fig. 4C). MANF depletion by cytokines was not associated with a change in its subcellular localization, which remained cytoplasmic (Fig. 4D). As for THBS1, proteasome inhibition by MG132 prevented the cytokine-induced loss of MANF protein (Fig. 4E). We next examined the role of MANF directly using two independent siRNAs. Efficient MANF knockdown (Fig. 4F) did not affect basal cell survival but sensitized INS-1E cells to both thapsigargin- and cytokine-induced apoptosis (Fig. 4G). A similar sensitization was seen in human insulin-producing EndoC-βH1 cells (Fig. 4H). Apoptosis was further confirmed by immunostaining for cleaved caspase 3 in MANF-depleted thapsigargin- or cytokine-treated EndoC-βH1 cells (supplemental Fig. S4). MANF silencing also induced caspase 3 cleavage in insulin-stained primary human β-cells exposed to thapsigargin (Fig. 4I and supplemental Fig. S5) or cytokines (supplemental Fig. S5). We next examined whether cytoprotection could be achieved by exogenously added MANF protein. Recombinant MANF protein failed to protect clonal rat INS-1E cells against thapsigargin or cytokines (Fig. 5A). In clonal human EndoC-βH1 cells, however, partial protection was observed from both stresses (Fig. 5B). Comparable cytoprotection was seen in dispersed mouse islet cells (Fig. 5C). Apoptosis was further assessed by TUNEL assay, confirming that recombinant MANF decreases thapsigargin-induced apoptosis in insulin-positive mouse β-cells (Fig. 5D). Taken together, these data show significant protection conferred by exogenous MANF in mouse and human but not rat β-cells, pointing to a species difference. To evaluate whether MANF is indeed an important mediator of the protective effects of THBS1, we adenovirally overexpressed THBS1 and knocked down MANF in parallel (Fig. 6A). Inhibition of MANF abrogated the beneficial effects of THBS1 overexpression in INS-1E cells (Fig. 6B). Similarly, efficient THBS1 overexpression and MANF silencing in human islet cells (Fig. 6C) abolished the cytoprotection conferred by THBS1 (Fig. 6D). In mirror experiments, lentiviral MANF overexpression concurrent to THBS1 silencing (Fig. 6E) completely prevented sensitization to cytokine-induced apoptosis (Fig. 6F). These data show that MANF is necessary and sufficient for THBS1-mediated β-cell protection against cytokines or ER stress. We next evaluated the effector mechanisms downstream of THBS1-MANF that regulate β-cell apoptosis. Previous data indicate that the BH3-only proteins PUMA (p53-upregulated modulator of apoptosis, also known as BBC3) (20.Gurzov E.N. Germano C.M. Cunha D.A. Ortis F. Vanderwinden J.M. Marchetti P. Zhang L. Eizirik D.L. p53 up-regulated modulator of apoptosis (PUMA) activation contributes to pancreatic β-cell apoptosis induced by proinflammatory cytokines and endoplasmic reticulum stress.J. Biol. Chem. 2010; 285: 19910-19920Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar), death protein 5 (DP5) (21.Gurzov E.N. Ortis F. Cunha D.A. Gosset G. Li M. Cardozo A.K. Eizirik D.L. Signaling by IL-1β+IFN-γ and ER stress converge on DP5/Hrk activation: a novel mechanism for pancreatic β-cell apoptosis.Cell Death Differ. 2009; 16: 1539-1550Crossref PubMed Scopus (134) Google Scholar), and BCL2-interacting mediator of cell death (BIM) (22.Barthson J. Germano C.M. Moore F. Maida A. Drucker D.J. Marchetti P. Gysemans C. Mathieu C. Nuñez G. Jurisicova A. Eizirik D.L. Gurzov E.N. Cytokines tumor necrosis factor-α and interferon-γ induce pancreatic β-cell apoptosis through STAT1-mediated Bim protein activation.J. Biol. Chem. 2011; 286: 39632-39643Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar) are critical mediators of cytokine-induced apoptosis, whereas the Bcl-2 family proteins Bcl-2 and Bcl-XL antagonize these effects (16.Gurzov E.N. Eizirik D.L. Bcl-2 proteins in diabetes: mitochondrial pathways of β-cell death and dysfunction.Trends Cell Biol. 2011; 21: 424-431Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar). Knockdown or overexpression of THBS1 did not modify the expression of DP5, PUMA, BIM, Bcl-2, and Bcl-XL in INS-1E cells (supplemental Fig. S6, A–E). BH3-only proteins may be a necessary component for cytokine-induced apoptosis, even under conditions where their expression is not up-regulated (16.Gurzov E.N. Eizirik D.L. Bcl-2 proteins in diabetes: mitochondrial pathways of β-cell death and dysfunction.Trends Cell Biol. 2011; 21: 424-431Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar, 23.Marroquí L. Santin I. Dos Santos R.S. Marselli L. Marchetti P. Eizirik D.L. BACH2, a candidate risk gene for type 1 diabetes, regulates apoptosis in pancreatic β-cells via JNK1 modulation and crosstalk with the candidate gene PTPN2.Diabetes. 2014; 63: 2516-2527Crossref PubMed Scopus (71) Google Scholar). To test whether this was the case here, we silenced THBS1 in parallel with BIM, DP5, PUMA, or BCL-2-associated death promoter (BAD) (Fig. 7A). Efficient knockdown was achieved for BIM (by 52%; Fig. 7, B and D), DP5 (silencing of mRNA by 63%), PUMA (by 74%), and BAD (by 89%). BIM silencing, but not DP5, PUMA, or BAD knockdown, partially prevented cytokine-induced apoptosis in THBS1-depleted β-cells (Fig. 7A). In a similar experimental approach, BIM also mediated apoptosis induced by thapsigargin (Fig. 7, B and C). Because THBS1 cytoprotection requires MANF expression (see above), we examined whether BIM also mediates cell death induced by MANF deficiency. BIM knockdown fully abrogated sensitization to cytokine-induced apoptosis in MANF-depleted cells (Fig. 7, D and E). These observations indicate that BIM is the downstream mediator of β-cell apoptosis following inhibition of the THBS1–MANF pathway. The local release of proinflammatory mediators by infiltrating immune cells contributes to pancreatic β-cell demise in T1D. Cytokine-induced ER stress is one of the molecular mechanisms activating the mitochondrial pathway of apoptosis in β-cells (9.Marhfour I. Lopez X.M. Lefkaditis D. Salmon I. Allagnat F. Richardson S.J. Morgan N.G. Eizirik D.L. Expression of endoplasmic reticulum stress markers in the islets of patients with type 1 diabetes.Diabetologia. 2012; 55: 2417-2420Crossref PubMed Scopus (166) Google Scholar, 10.Engin F. Yermalovich A. Nguyen T. Ngyuen T. Hummasti S. Fu W. Eizirik D.L. Mathis D. Hotamisligil G.S. Restoration of the unfolded protein response in pancreatic β cells protects mice against type 1 diabetes.Sci. Transl. Med. 2013; 5: 211ra156Crossref PubMed Scopus (205) Google Scholar, 24.Tersey S.A. Nishiki Y. Templin A.T. Cabrera S.M. Stull N.D. Colvin S.C. Evans-Molina C. Rickus J.L. Maier B. Mirmira R.G. Islet β-cell endoplasmic reticulum stress precedes the onset of type 1 diabetes in the nonobese diabetic mouse model.Diabetes. 2012; 61: 818-827Crossref PubMed Scopus (253) Google Scholar). This stress response also plays a role in β-cell failure in T2D and in monogenic forms of diabetes (25.Cnop M. Foufelle F. Velloso L.A. Endoplasmic reticulum stress, obesity and diabetes.Trends Mol. Med. 2012; 18: 59-68Abstract Full Text Full Text PDF PubMed Scopus (472) Google Scholar, 26.Abdulkarim B. Nicolino M. Igoillo-Esteve M. Daures M. Romero S. Philippi A. Senée V. L
Referência(s)