Activating transcription factor 4 regulates stearate-induced vascular calcification

Abstract

Previously, we reported that stearate, a saturated fatty acid, promotes osteoblastic differentiation and mineralization of vascular smooth muscle cells (VSMC). In this study, we examined the molecular mechanisms by which stearate promotes vascular calcification. ATF4 is a pivotal transcription factor in osteoblastogenesis and endoplasmic reticulum (ER) stress. Increased stearate by either supplementation of exogenous stearic acid or inhibition of stearoyl-CoA desaturase (SCD) by CAY10566 induced ATF4 mRNA, phosphorylated ATF4 protein, and total ATF4 protein. Induction occurred through activation of the PERK-eIF2α pathway, along with increased osteoblastic differentiation and mineralization of VSMCs. Either stearate or the SCD inhibitor but not oleate or other fatty acid treatments also increased ER stress as determined by the expression of p-eIF2α, CHOP, and the spliced form of XBP-1, which were directly correlated with ER stearate levels. ATF4 knockdown by lentiviral ATF4 shRNA blocked osteoblastic differentiation and mineralization induced by stearate and SCD inhibition. Conversely, treatment of VSMCs with an adenovirus containing ATF4 induced vascular calcification. Our results demonstrated that activation of ATF4 mediates vascular calcification induced by stearate. Previously, we reported that stearate, a saturated fatty acid, promotes osteoblastic differentiation and mineralization of vascular smooth muscle cells (VSMC). In this study, we examined the molecular mechanisms by which stearate promotes vascular calcification. ATF4 is a pivotal transcription factor in osteoblastogenesis and endoplasmic reticulum (ER) stress. Increased stearate by either supplementation of exogenous stearic acid or inhibition of stearoyl-CoA desaturase (SCD) by CAY10566 induced ATF4 mRNA, phosphorylated ATF4 protein, and total ATF4 protein. Induction occurred through activation of the PERK-eIF2α pathway, along with increased osteoblastic differentiation and mineralization of VSMCs. Either stearate or the SCD inhibitor but not oleate or other fatty acid treatments also increased ER stress as determined by the expression of p-eIF2α, CHOP, and the spliced form of XBP-1, which were directly correlated with ER stearate levels. ATF4 knockdown by lentiviral ATF4 shRNA blocked osteoblastic differentiation and mineralization induced by stearate and SCD inhibition. Conversely, treatment of VSMCs with an adenovirus containing ATF4 induced vascular calcification. Our results demonstrated that activation of ATF4 mediates vascular calcification induced by stearate. alkaline phosphatase activating transcription factor 4 C/EBP homologous protein α-subunit of eukaryotic initiation factor 2 endoplasmic reticulum farnesoid X receptor liver X receptor nephrectomy multiplicity of infection osteocalcin osteoprotegerin phosphorylated PKR-like endoplasmic reticulum kinase stearoyl-CoA desaturase sterol regulatory element binding protein-1 unfolded protein response vascular smooth muscle cell X-box binding protein Cardiovascular disease, such as vascular calcification, is the leading cause of death in patients with chronic kidney disease, accounting for over 50% of deaths (1Mizobuchi M. Towler D. Slatopolsky E. Vascular calcification: the killer of patients with chronic kidney disease.J. Am. Soc. Nephrol. 2009; 20: 1453-1464Crossref PubMed Scopus (398) Google Scholar, 2Shanahan C.M. Crouthamel M.H. Kapustin A. Giachelli C.M. Arterial calcification in chronic kidney disease: key roles for calcium and phosphate.Circ. Res. 2011; 109: 697-711Crossref PubMed Scopus (631) Google Scholar). Vascular calcification is frequently observed in advanced atherosclerotic lesions and is a highly regulated process that recapitulates osteogenesis in bone formation. Recent in vivo and in vitro studies have implicated the involvement of numerous positive and negative regulators, including serum phosphate, several lipid-derived molecules (saturated fatty acids, oxysterol, and oxidized phospholipids), and hormonal factors (tumor necrosis factor-α, bone morphogenetic protein-2, matrix gla protein, osteocalcin, fibroblast growth factors, and Klotho) in the pathogenesis of vascular calcification (3Li X. Yang H.Y. Giachelli C.M. Role of the sodium-dependent phosphate cotransporter, Pit-1, in vascular smooth muscle cell calcification.Circ. Res. 2006; 98: 905-912Crossref PubMed Scopus (359) Google Scholar–10Kuro-o M. Matsumura Y. Aizawa H. Kawaguchi H. Suga T. Utsugi T. Ohyama Y. Kurabayashi M. Kaname T. Kume E. et al.Mutation of the mouse klotho gene leads to a syndrome resembling ageing.Nature. 1997; 390: 45-51Crossref PubMed Scopus (2784) Google Scholar). Recently, we reported that bile acid nuclear receptor, farnesoid X receptor (FXR), and oxysterol nuclear receptor, liver X receptor (LXR), elicit opposite effects on vascular calcification (11Miyazaki-Anzai S. Levi M. Kratzer A. Ting T.C. Lewis L.B. Miyazaki M. FXR activation prevents the development of vascular calcification in ApoE−/− mice with chronic kidney disease.Circ. Res. 2010; 106: 1807-1817Crossref PubMed Scopus (69) Google Scholar–13Geng Y. Hsu J.J. Lu J. Ting T.C. Miyazaki M. Demer L.L. Tintut Y. The role of cellular cholesterol metabolism in vascular cell calcification.J. Biol. Chem. 2011; 286: 33701-33706Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). We found that FXR activation attenuated CKD-dependent atherosclerotic calcification in ApoE−/− mice with 5/6 nephrectomy. FXR activation by either VP16-FXR overexpression or INT-747 (an FXR-specific agonist) treatment attenuated mineralization of vascular smooth muscle cells (VSMCs) in culture. Conversely, FXR inhibition by FXR shRNA and the dominant negative (DN) form of FXR augmented vascular calcification (11Miyazaki-Anzai S. Levi M. Kratzer A. Ting T.C. Lewis L.B. Miyazaki M. FXR activation prevents the development of vascular calcification in ApoE−/− mice with chronic kidney disease.Circ. Res. 2010; 106: 1807-1817Crossref PubMed Scopus (69) Google Scholar). In contrast to FXR, LXR activation by LXR agonists and adenovirus-mediated LXR overexpression by VP16-LXRα and VP16-LXRβ promoted mineralization of VSMCs. Conversely, LXR inhibition by dominant negative forms of LXRα and LXRβ attenuated vascular calcification in VSMCs (12Ting T.C. Miyazaki-Anzai S. Masuda M. Levi M. Demer L.L. Tintut Y. Miyazaki M. Increased lipogenesis and stearate accelerate vascular calcification in calcifying vascular cells.J. Biol. Chem. 2011; 286: 23938-23949Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). The regulation of mineralization by FXR and LXR agonists was highly correlated with changes in lipid accumulation, fatty acid synthesis, and the expression of sterol regulatory element binding protein-1 (SREBP-1). The rate of lipogenesis in VSMCs through the SREBP-1c-dependent pathway was reduced by FXR activation but increased by LXR activation (11Miyazaki-Anzai S. Levi M. Kratzer A. Ting T.C. Lewis L.B. Miyazaki M. FXR activation prevents the development of vascular calcification in ApoE−/− mice with chronic kidney disease.Circ. Res. 2010; 106: 1807-1817Crossref PubMed Scopus (69) Google Scholar, 12Ting T.C. Miyazaki-Anzai S. Masuda M. Levi M. Demer L.L. Tintut Y. Miyazaki M. Increased lipogenesis and stearate accelerate vascular calcification in calcifying vascular cells.J. Biol. Chem. 2011; 286: 23938-23949Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). SREBP-1c overexpression promoted mineralization in VSMCs, whereas SREBP-1c DN inhibited alkaline phosphatase activity and mineralization induced by LXR agonists. LXR and SREBP-1c activations increased, whereas FXR activation decreased saturated and monounsaturated fatty acids derived from lipogenesis. Furthermore, we found that stearate markedly promoted mineralization of VSMCs compared with other fatty acids. Inhibition of acetyl-CoA carboxylase or acyl-CoA synthetase reduced mineralization of VSMCs, whereas inhibition of stearoyl-CoA desaturase (which converts stearate to oleate) promoted mineralization and osteoblastic differentiation (12Ting T.C. Miyazaki-Anzai S. Masuda M. Levi M. Demer L.L. Tintut Y. Miyazaki M. Increased lipogenesis and stearate accelerate vascular calcification in calcifying vascular cells.J. Biol. Chem. 2011; 286: 23938-23949Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). We also found that increased cholesterol synthesis and uptake by LXR and SREBP-1c activations contributed to vascular calcification (13Geng Y. Hsu J.J. Lu J. Ting T.C. Miyazaki M. Demer L.L. Tintut Y. The role of cellular cholesterol metabolism in vascular cell calcification.J. Biol. Chem. 2011; 286: 33701-33706Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). Therefore, we concluded that a stearate metabolite derived from lipogenesis promotes vascular calcification. However, the molecular mechanism by which stearate metabolites promote osteoblastic differentiation and mineralization of VSMCs remains elusive. The endoplasmic reticulum (ER) is a major site for the regulation of calcium and lipid homeostasis. ER stress (also known as the unfolded protein response, UPR) is an integrated signal transduction pathway involved in the localization and folding of secreted and transmembrane proteins. A number of cellular stress conditions lead to the accumulation of unfolded or misfolded proteins in the ER lumen. The UPR is initiated by activation of three molecules: PKR-like endoplasmic reticulum kinase (PERK), inositol-requiring enzyme 1 (IRE1), and activating transcription factor 6 (ATF6) (14Haze K. Yoshida H. Yanagi H. Yura T. Mori K. Mammalian transcription factor ATF6 is synthesized as a transmembrane protein and activated by proteolysis in response to endoplasmic reticulum stress.Mol. Biol. Cell. 1999; 10: 3787-3799Crossref PubMed Scopus (1538) Google Scholar, 15Ye J. Rawson R.B. Komuro R. Chen X. Dave U.P. Prywes R. Brown M.S. Goldstein J.L. ER stress induces cleavage of membrane-bound ATF6 by the same proteases that process SREBPs.Mol. Cell. 2000; 6: 1355-1364Abstract Full Text Full Text PDF PubMed Scopus (1357) Google Scholar). Activation of PERK leads to the phosphorylation of the α-subunit of eukaryotic initiation factor 2 (eIF2α), which inhibits the assembly of the 80S ribosome and inhibits protein synthesis (16Harding H.P. Novoa I. Zhang Y. Zeng H. Wek R. Schapira M. Ron D. Regulated translation initiation controls stress-induced gene expression in mammalian cells.Mol. Cell. 2000; 6: 1099-1108Abstract Full Text Full Text PDF PubMed Scopus (2404) Google Scholar, 17Kilberg M.S. Shan J. Su N. ATF4-dependent transcription mediates signaling of amino acid limitation.Trends Endocrinol. Metab. 2009; 20: 436-443Abstract Full Text Full Text PDF PubMed Scopus (411) Google Scholar). In contrast to most proteins, activating transcription factor 4 (ATF4) is not affected by the translational attenuation of eIF2α phosphorylation because ATF4 has two small upstream open reading frames in its 5′-untranslated region. These upstream open reading frames, which prevent translation of the true ATF4 under normal conditions, are bypassed only when eIF2α is phosphorylated, and thereby permit ATF4 translation (16Harding H.P. Novoa I. Zhang Y. Zeng H. Wek R. Schapira M. Ron D. Regulated translation initiation controls stress-induced gene expression in mammalian cells.Mol. Cell. 2000; 6: 1099-1108Abstract Full Text Full Text PDF PubMed Scopus (2404) Google Scholar, 18Yang X. Matsuda K. Bialek P. Jacquot S. Masuoka H.C. Schinke T. Li S. P. et is a of and an of for Full Text Full Text PDF PubMed Scopus Google Scholar). ATF4 is a pivotal transcription factor that mediates not only ER stress but also osteoblastic differentiation X. Matsuda K. Bialek P. Jacquot S. Masuoka H.C. Schinke T. Li S. P. et is a of and an of for Full Text Full Text PDF PubMed Scopus Google Scholar, of PubMed Scopus Google Scholar). The activity of ATF4 is regulated through phosphorylation by kinase 2 and X. Matsuda K. Bialek P. Jacquot S. Masuoka H.C. Schinke T. Li S. P. et is a of and an of for Full Text Full Text PDF PubMed Scopus Google S. M. J. D. H. Y. Zhang J. K. et role of activating transcription factor 4 in the of in 2009; PubMed Scopus Google Scholar). In to ER stress such as and C/EBP homologous protein of ATF4 osteocalcin, an for the of differentiation X. Matsuda K. Bialek P. Jacquot S. Masuoka H.C. Schinke T. Li S. P. et is a of and an of for Full Text Full Text PDF PubMed Scopus Google and transcription factor in differentiation X. Matsuda K. Bialek P. Jacquot S. Masuoka H.C. Schinke T. Li S. P. et is a of and an of for Full Text Full Text PDF PubMed Scopus Google Scholar, S. M. J. D. H. Y. Zhang J. K. et role of activating transcription factor 4 in the of in 2009; PubMed Scopus Google X. the accumulation of which is determined gene expression in cells.J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). ATF4 is also for including the synthesis of the most protein found in and X. Matsuda K. Bialek P. Jacquot S. Masuoka H.C. Schinke T. Li S. P. et is a of and an of for Full Text Full Text PDF PubMed Scopus Google Scholar). has reported that ER stress the pathway is involved in differentiation induced by bone morphogenetic A. K. S. K. T. K. reticulum stress response by the pathway is involved in differentiation induced by Biol. Chem. 2011; 286: Full Text Full Text PDF PubMed Scopus Google Scholar). However, the role of ATF4 in the regulation of vascular calcification and vascular osteogenesis has to cells were by the of and in containing with either phosphate or D. S. cell a in vitro of vascular J. 2011; Google Scholar, S. P. M. of in and in gene expression with Endocrinol. PubMed Scopus Google Scholar). cells were with and fatty The fatty were as (12Ting T.C. Miyazaki-Anzai S. Masuda M. Levi M. Demer L.L. Tintut Y. Miyazaki M. Increased lipogenesis and stearate accelerate vascular calcification in calcifying vascular cells.J. Biol. Chem. 2011; 286: 23938-23949Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, N. M. S. J.L. D. et by of oxidized in oxidized that and for in Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). The was the cells were with to calcium in the was as (11Miyazaki-Anzai S. Levi M. Kratzer A. Ting T.C. Lewis L.B. Miyazaki M. FXR activation prevents the development of vascular calcification in ApoE−/− mice with chronic kidney disease.Circ. Res. 2010; 106: 1807-1817Crossref PubMed Scopus (69) Google Scholar). were a the the cells were with and with a for protein was the was a protein was in with an were by an In 1 of total was with by the and of The of was as the to a gene or the are The spliced form of X-box binding protein-1 was by with as Y. H. gene expression endoplasmic reticulum stress and in mice a PubMed Scopus Google Scholar). cells were with a multiplicity of infection of adenovirus ATF4 was the cells were with the adenovirus in with 6 the cells were with for cells were with ATF4 or PERK or were by treatment with for A was from cells with cells with ATF4 shRNA PERK shRNA and were as VSMCs, VSMCs, and VSMCs, and were The were by to a and with an ATF4 and and were to an with by an phosphorylated the were with an ATF4 and a was to the The was on a of and 4 of in and to for 2 to the which from the ER were by and acids were as Miyazaki M. K. J. of and for monounsaturated fatty acids in Res. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). was a protein cells with CAY10566 were with containing 1 were with The fatty acids were by The of the in the to acid to the in the to stearate was to stearoyl-CoA desaturase (SCD) activity as Miyazaki M. K. J. of and for monounsaturated fatty acids in Res. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). phosphatase activity was phosphate as the J.J. Lu J. M.S. Y. P. Demer L.L. Tintut Y. an LXR vascular cell 2009; PubMed Scopus Google Scholar). were from two and were reported as the for was the or with a for was and treatment induced mineralization of mouse vascular smooth muscle cell an cell as an in vitro of vascular calcification D. S. cell a in vitro of vascular J. 2011; Google Scholar). to stearate and increased calcium by and compared with treatment (12Ting T.C. Miyazaki-Anzai S. Masuda M. Levi M. Demer L.L. Tintut Y. Miyazaki M. Increased lipogenesis and stearate accelerate vascular calcification in calcifying vascular cells.J. Biol. Chem. 2011; 286: 23938-23949Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). fatty acids, such as and not mineralization of cells The of stearate In stearate treatment but not other fatty acid treatments induced osteoblastic differentiation as with activity and osteocalcin gene expression induces ATF4 expression in cells were with the fatty acids stearate oleate and for 6 or and in the of ATF4 and protein were by with was as a CHOP, and ATF4 expression in cells with the fatty acids for of in The is the expression of forms of and the is the expression of spliced forms of We examined treatment of cells with stearate or other fatty acids the expression of a pivotal transcription factor not only in osteogenesis but also in ER stress. cells were with either of stearate or fatty acid, such as or for 6 treatment increased ATF4 protein and by and compared with treatment treatment also increased ATF4 protein but the was was with stearate treatment of cells with fatty acids, including and not ATF4 protein and and protein expression of CHOP, a major ATF4 were highly induced by stearate treatment but not oleate treatment of protein as a not stearate and oleate In stearate treatment a in of the spliced form of X-box binding protein-1 of ER stress The form of in cells with stearate We also examined and effects of stearate on the expression of which is an form of ATF4 in osteoblastic cells were with stearate for to ATF4 increased compared with treatment whereas total ATF4 protein increased to 6 of stearate treatment We also examined PERK-eIF2α signaling contributed to the of ATF4 eIF2α were and induced and 2 of treatment to the of ATF4 protein was induced by 6 of treatment The of stearate on total CHOP, and protein expression a stearate treatment induced CHOP, and expression by and with protein the of CHOP, and increased with 6 of stearate treatment in a The of CHOP, and correlated with in calcium of cells by stearate treatment to the expression of involved in ER stearate treatment induced several including osteoprotegerin sodium-dependent phosphate 1 and with 6 and induces gene expression of transcription factors and cells were with stearate for the and and gene expression in of stearate metabolism ATF4 expression and ER we and CAY10566 to acyl-CoA synthetase and stearoyl-CoA two major in stearate cells were with stearate and with the of on mineralization and osteoblastic differentiation treatment reduced the expression of and total ATF4 protein protein and and with SCD inhibitor CAY10566 reduced SCD activity in cells in a in ER stearate SCD inhibition by CAY10566 treatment induced mineralization and osteoblastic differentiation and of with of vascular calcification, CAY10566 induced total and protein expression ATF4 mRNA, protein, mRNA, and were also highly and induced by CAY10566 treatment ER stress and mineralization induced by CAY10566 were correlated with in as as CAY10566 induced that CAY10566 is as as other of ER such as and which also vascular calcification and ATF4 expression and not We also examined the of CAY10566 on the expression of ATF4 and other ER stress CAY10566 treatment induced 2 of were increased by ATF4 and protein were increased by and of treatment PERK were increased by 2 of CAY10566 whereas were increased by 6 of CAY10566 treatment The of CHOP, and were induced to of CAY10566 treatment with increased ATF4 CAY10566 treatment induced sodium-dependent phosphate 1 and with and of the and not inhibition induces the expression of ER stress and and ATF4 and and of in cells with CAY10566 in the of The is the expression of forms of and the is the expression of spliced forms of and and were of CAY10566 and CAY10566 was SCD inhibition induces ATF4 and expression through the PERK-eIF2α pathway, we cells with CAY10566 for PERK knockdown inhibited the of ATF4 and protein induced by stearate In contrast to inhibition of SCD by overexpression by an adenovirus blocked mineralization of cells induced by stearate treatment with containing increased the expression of by which was to the of by The of total ATF4 and protein expression by stearate treatment was blocked by SCD overexpression only stearate but also induced ATF4 expression and mineralization We that the of to stearate is for ATF4 and we cells with knockdown reduced ATF4 expression and mineralization cells were by cells with lentiviral of cells were with containing for ATF4 or a The cells were with for to of reduced ATF4 protein and expression not In this study, we also cells with a by the as and the results are in cells and the cells were with either stearate or CAY10566 for ATF4 knockdown reduced mineralization and osteoblastic differentiation by and under conditions In ATF4 knockdown attenuated and mineralization and activity that ATF4 knockdown markedly attenuated mineralization of cells induced by CAY10566 ATF4 knockdown attenuated the of and total ATF4 protein expression induced by CAY10566 or stearate treatment not in the of and protein with the that ATF4 the expression of ER stress synthetase CHOP, and and and induced by CAY10566 treatment results were in cells by a derived from a not In contrast to knockdown of ATF4 overexpression by the adenovirus expression induced mineralization and osteoblastic differentiation of infection with increased ATF4 expression by was ATF4 in cells with stearate in and were increased by and in cells with compared with cells with ATF4 is a transcription factor that mediates not only stress but also osteoblastic differentiation bone (16Harding H.P. Novoa I. Zhang Y. Zeng H. Wek R. Schapira M. Ron D. Regulated translation initiation controls stress-induced gene expression in mammalian cells.Mol. Cell. 2000; 6: 1099-1108Abstract Full Text Full Text PDF PubMed Scopus (2404) Google Scholar, 18Yang X. Matsuda K. Bialek P. Jacquot S. Masuoka H.C. Schinke T. Li S. P. et is a of and an of for Full Text Full Text PDF PubMed Scopus Google Scholar, H. M. X. Ron D. ATF4 of in a for Metab. 2006; Full Text Full Text PDF PubMed Scopus Google H. S. Y. Zhang X. J. Lu Y. M. et transcription factor 4 differentiation in 2010; PubMed Scopus Google Scholar). However, the role of ATF4 in vascular calcification and osteogenesis in the has not The results of for the to that the activation and of ATF4 are key in the pathogenesis of vascular calcification induced by stearate. are for cells in this study, of the calcifying vascular cells in studies (11Miyazaki-Anzai S. Levi M. Kratzer A. Ting T.C. Lewis L.B. Miyazaki M. FXR activation prevents the development of vascular calcification in ApoE−/− mice with chronic kidney disease.Circ. Res. 2010; 106: 1807-1817Crossref PubMed Scopus (69) Google Scholar, 12Ting T.C. Miyazaki-Anzai S. Masuda M. Levi M. Demer L.L. Tintut Y. Miyazaki M. Increased lipogenesis and stearate accelerate vascular calcification in calcifying vascular cells.J. Biol. Chem. 2011; 286: 23938-23949Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). cells have as a cell of vascular calcification. of in calcifying vascular cells were in mouse but not lentiviral are from several for the shRNA the osteoblastic and of vascular cells but not of We reported that stearate derived from lipogenesis promotes vascular calcification of calcifying vascular cells (12Ting T.C. Miyazaki-Anzai S. Masuda M. Levi M. Demer L.L. Tintut Y. Miyazaki M. Increased lipogenesis and stearate accelerate vascular calcification in calcifying vascular cells.J. Biol. Chem. 2011; 286: 23938-23949Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). is of the major saturated fatty acids in and is through two and lipogenesis. a of stearate from stearate also through fatty acid with cells (12Ting T.C. Miyazaki-Anzai S. Masuda M. Levi M. Demer L.L. Tintut Y. Miyazaki M. Increased lipogenesis and stearate accelerate vascular calcification in calcifying vascular cells.J. Biol. Chem. 2011; 286: 23938-23949Abstract Full Text Full Text PDF PubMed Scopus (32) Google stearate most induced vascular osteoblastic differentiation and calcification in mouse VSMCs. In a number of studies cell saturated fatty acids, including stearate, have to effects ER stress E. S. et endoplasmic reticulum stress through a lipid 2009; PubMed Scopus Google Y. D. fatty acids endoplasmic reticulum stress and of in liver J. Endocrinol. Metab. 2006; PubMed Scopus Google Scholar). We that the of ATF4 through an ER stress signaling pathway mediates vascular calcification and with was that stearate treatment and induced p-eIF2α, CHOP, and eIF2α increased to the of ATF4 protein expression in response to stearate and CAY10566 that the translation of ATF4 is induced by the activation of The of stearate on vascular calcification and ATF4 is compared with other fatty acids. mineralization of proteins involved in ER stress p-eIF2α, and was in cells with oleate and other fatty acids induced mineralization and ER stress in cells to a stearate, the of ATF4 expression by treatment was attenuated by the knockdown of which stearate from These that the effects of on ATF4 expression are to an in stearate. We also found that inhibition of acyl-CoA synthetase by blocked the of ATF4 protein and expression induced by stearate with a of mineralization and osteoblastic inhibition that stearoyl-CoA or its metabolite mediates the of ATF4 and ER stress. desaturase is an ER transmembrane enzyme that the of stearate in the ER by stearate to oleate M. A. gene expression is for lipid synthesis and liver A. PubMed Scopus Google Scholar, M. H. K. X. stearoyl-CoA mice from and Metab. 6: Full Text Full Text PDF PubMed Scopus Google Scholar). CAY10566 is to the activity of and as SCD activity was in cells with of CAY10566 to stearate the treatment of cells with CAY10566 and induced p-eIF2α, and protein which directly correlated with the of SCD activity in CAY10566 treatment also increased stearate in the ER of In contrast to SCD the overexpression of attenuated ATF4 protein expression and mineralization induced by stearate In PERK knockdown inhibited the of ATF4 expression by SCD inhibition and stearate and not that PERK stearate in the ER and activated the signaling were also increased in cells with stearate and other such as the ATF6 and also stearate in the These to vascular calcification, as was reported that the pathway to vascular calcification by expression A. Chen Y. stress induces vascular calcification through of the transcription factor by Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The most in was that the knockdown of ATF4 expression by shRNA osteoblastic differentiation and mineralization of cells induced by stearate and the SCD The of factors is either to the ATF4 activity in cells or to other mechanisms to osteoblastic differentiation and mineralization of We also found that ATF4 overexpression induced osteoblastic differentiation and mineralization of These stearate promotes vascular calcification. The results of a and role for ATF4 in the regulation of vascular calcification induced by stearate and other factors, such as and oxidized not in we that or its metabolite the pathway of which leads to the of ATF4 in VSMCs, which in induces osteoblastic differentiation and mineralization of VSMCs. results that of ATF4 activity in VSMCs a for vascular calcification.