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ER stress regulates alkaline phosphatase gene expression in vascular smooth muscle cells via an ATF4-dependent mechanism.

ABSTRACT: Vascular calcification is the deposition of hydroxyapatite crystals in the blood vessel wall. Osteogenic differentiation of vascular smooth muscle cells (VSMCs) plays a key role in this process. Increased expression of alkaline phosphatase (ALP) occurs in some in vitro models of VSMC calcification and is thought to be crucial for mineralization, however, little is known about the transcriptional regulation of ALP in VSMCs. Recently, ALP upregulation was shown to coincide with endoplasmic reticulum (ER) stress-mediated vascular calcification, specifically with expression of the transcription factor ATF4. As no direct links between ALP expression and ER stress have previously been demonstrated in VSMCs, the aim of this study was to investigate whether ATF4 interacts directly with the ALP promoter.The present study shows that ALP mRNA and activity were significantly increased by ER stress treatment of human primary VSMCs in vitro and that this was ATF4-dependent. Bioinformatics analysis predicted two ATF4 binding sites in ER-stress responsive regions of the ALP promoter (-?3631 to -?2048 bp from the first intron). However, we found that ATF4 does not bind within this fragment of the ALP promoter region.

SUBMITTER: Furmanik M

PROVIDER: S-EPMC6048897 | biostudies-other | 2018 Jul

REPOSITORIES: biostudies-other

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ER stress regulates alkaline phosphatase gene expression in vascular smooth muscle cells via an ATF4-dependent mechanism.

Furmanik Malgorzata M Shanahan Catherine M CM

BMC research notes 20180716 1

<h4>Objective</h4>Vascular calcification is the deposition of hydroxyapatite crystals in the blood vessel wall. Osteogenic differentiation of vascular smooth muscle cells (VSMCs) plays a key role in this process. Increased expression of alkaline phosphatase (ALP) occurs in some in vitro models of VSMC calcification and is thought to be crucial for mineralization, however, little is known about the transcriptional regulation of ALP in VSMCs. Recently, ALP upregulation was shown to coincide with e ...[more]

PMID: 30012221

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Project description:ObjectiveWe examined the role of syndecan-1 in modulating the phenotype of vascular smooth muscle cells in the context of endogenous inflammatory factors and altered microenvironments that occur in disease or injury-induced vascular remodeling.Methods and resultsVascular smooth muscle cells (vSMCs) display a continuum of phenotypes that can be altered during vascular remodeling. While the syndecans have emerged as powerful and complex regulators of cell function, their role in controlling vSMC phenotype is unknown. Here, we isolated vSMCs from wild type (WT) and syndecan-1 knockout (S1KO) mice. Gene expression and western blotting studies indicated decreased levels of α-smooth muscle actin (α-SMA), calponin, and other vSMC-specific differentiation markers in S1KO relative to WT cells. The spread area of the S1KO cells was found to be greater than WT cells, with a corresponding increase in focal adhesion formation, Src phosphorylation, and alterations in actin cytoskeletal arrangement. In addition, S1KO led to increased S6RP phosphorylation and decreased AKT and PKC-α phosphorylation. To examine whether these changes were present in vivo, isolated aortae from aged WT and S1KO mice were stained for calponin. Consistent with our in-vitro findings, the WT mice aortae stained higher for calponin relative to S1KO. When exposed to the inflammatory cytokine TNF-α, WT vSMCs had an 80% reduction in syndecan-1 expression. Further, with TNF-α, S1KO vSMCs produced increased pro-inflammatory cytokines relative to WT. Finally, inhibition of interactions between syndecan-1 and integrins αvβ3 and αvβ5 using the inhibitory peptide synstatin appeared to have similar effects on vSMCs as knocking out syndecan-1, with decreased expression of vSMC differentiation markers and increased expression of inflammatory cytokines, receptors, and osteopontin.ConclusionsTaken together, our results support that syndecan-1 promotes vSMC differentiation and quiescence. Thus, the presence of syndecan-1 would have a protective effect against vSMC dedifferentiation and this activity is linked to interactions with integrins αvβ3 and αvβ5.

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ER stress regulates alkaline phosphatase gene expression in vascular smooth muscle cells via an ATF4-dependent mechanism.

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ER stress regulates alkaline phosphatase gene expression in vascular smooth muscle cells via an ATF4-dependent mechanism.

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