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Transforming growth factor beta up-regulates cysteine-rich protein 2 in vascular smooth muscle cells via activating transcription factor 2.

ABSTRACT: CRP2 (cysteine-rich protein) is a vascular smooth muscle cell (VSMC)-expressed LIM-only protein. CRP2 associates with the actin cytoskeleton and interacts with transcription factors in the nucleus to mediate smooth muscle cell gene expression. Using Csrp2 (gene symbol of the mouse CRP2 gene)-deficient mice, we previously demonstrated that an absence of CRP2 enhances VSMC migration and increases neointima formation following arterial injury. Despite its importance in vascular injury, the molecular mechanisms controlling CRP2 expression in VSMC are largely unknown. Transforming growth factor beta (TGFbeta), a key factor present in the vessel wall in the early phases of arterial response to injury, plays an important role in modulating lesion formation. Because both CRP2 and TGFbeta are mediators of VSMC responses, we examined the possibility that TGFbeta might regulate CRP2 expression. TGFbeta significantly induced CRP2 mRNA and protein expression in VSMCs. Promoter analysis identified a conserved cAMP-responsive element (CRE)-like site (TAACGTCA) in the Csrp2 promoter that was critical for basal promoter activity and response to TGFbeta. Gel mobility shift assays revealed that mainly ATF2 bound to this CRE-like element, and mutation of the CRE sequences abolished binding. TGFbeta enhanced the activation of ATF2, leading to increased phospho-ATF2 levels within the DNA-protein complexes. Furthermore, ATF2-transactivated Csrp2 promoter activity and TGFbeta enhanced this activation. In addition, a phosphorylation-negative ATF2 mutant construct decreased basal and TGFbeta-mediated Csrp2 promoter activity. Our results show for the first time in VSMC that TGFbeta activates ATF2 phosphorylation and Csrp2 gene expression via a CRE promoter element.

SUBMITTER: Lin DW

PROVIDER: S-EPMC2397482 | biostudies-literature | 2008 May

REPOSITORIES: biostudies-literature

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Transforming growth factor beta up-regulates cysteine-rich protein 2 in vascular smooth muscle cells via activating transcription factor 2.

Lin Da-Wei DW Chang Il-Chi IC Tseng Alan A Wu Meng-Ling ML Chen Chung-Huang CH Patenaude Cassandra A CA Layne Matthew D MD Yet Shaw-Fang SF

The Journal of biological chemistry 20080403 22

CRP2 (cysteine-rich protein) is a vascular smooth muscle cell (VSMC)-expressed LIM-only protein. CRP2 associates with the actin cytoskeleton and interacts with transcription factors in the nucleus to mediate smooth muscle cell gene expression. Using Csrp2 (gene symbol of the mouse CRP2 gene)-deficient mice, we previously demonstrated that an absence of CRP2 enhances VSMC migration and increases neointima formation following arterial injury. Despite its importance in vascular injury, the molecula ...[more]

PMID: 18387947

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Project description:Graphical abstract Schematic diagram of the mechanism by which H2S inhibits VSMC proliferation targeting on the FOXO1. H2S persulfidates FOXO1 at Cys457, which reduces FOXO1 phosphorylation at Ser256, prevents the FOXO1 nucleus exclusion to the cytoplasm, and further protecting against ET-1-induced VSMC proliferation. Highlights • FOXO1 is involved in the inhibitory effect of H2S on vascular smooth muscle cell proliferation.• H2S inhibits vascular smooth muscle cell proliferation by maintaining FOXO1 activity.• H2S preserves FOXO1 activity by persulfidation.• H2S persulfidates FOXO1 at Cys457 and subsequently prevents FOXO1 phosphorylation at Ser256.• The results provide new ideas for therapeutic strategies for anti-vascular remodeling. Introduction The proliferation of vascular smooth muscle cells (VSMCs) is an important physiological and pathological basis for many cardiovascular diseases. Endogenous hydrogen sulfide (H2S), the third gasotransmitter, is found to preserve vascular structure by inhibiting VSMC proliferation. However, the mechanism by which H2S suppresses VSMC proliferation has not been fully clear. Objectives This study aimed to explore whether H2S persulfidates the transcription factor FOXO1 to inhibit VSMC proliferation. Methods After the proliferation of VSMC A7r5 cells was induced by endothelin-1 (ET-1), FOXO1 phosphorylation and proliferating cell nuclear antigen (PCNA) expression were detected by Western blotting, the degree of FOXO1 nuclear exclusion and PCNA fluorescent signals in the nucleus were detected by immunofluorescence, and the persulfidation of FOXO1 was measured through a biotin switch assay. Results The results showed that ET-1 stimulation increased cell proliferation, FOXO1 phosphorylation and FOXO1 nuclear exclusion to the cytoplasm in the cells. However, pretreatment with NaHS, an H2S donor, successfully abolished the ET-1-induced increases in the VSMC proliferation, FOXO1 phosphorylation, and FOXO1 nuclear exclusion to the cytoplasm. Mechanistically, H2S persulfidated the FOXO1 protein in A7r5 and 293T cells, and the thiol reductant DTT reversed this effect. Furthermore, the C457S mutation of FOXO1 abolished the H2S-induced persulfidation of FOXO1 in the cells and the subsequent inhibitory effects on FOXO1 phosphorylation at Ser256, FOXO1 nuclear exclusion to the cytoplasm and cell proliferation. Conclusion Thus, our findings demonstrated that H2S might inhibit VSMC proliferation by persulfidating FOXO1 at Cys457 and subsequently preventing FOXO1 phosphorylation at Ser256.

Project description:Mesenchymal stem cells (MSCs) hold great promise for vascular smooth muscle regeneration. However, most studies have mainly relied on extended supplementation of sophisticated biochemical regimen to drive MSC differentiation towards vascular smooth muscle cells (vSMCs). Herein we demonstrate a concomitant method that exploits the advantages of biomimetic matrix stiffness and tethered transforming growth factor ?1 (TGF-?1) to guide vSMC commitment from human MSCs. Our designed poly(ethylene glycol) hydrogels, presenting a biomimetic stiffness and tethered TGF-?1, provide an instructive environment to potently upregulate smooth muscle marker expression in vitro and in vivo. Importantly, it significantly enhances the functional contractility of vSMCs derived from MSCs within 3 days. Interestingly, compared to non-tethered one, tethered TGF-?1 enhanced the potency of vSMC commitment on hydrogels. We provide compelling evidence that combining stiffness and tethered TGF-?1 on poly(ethylene glycol) hydrogels can be a promising approach to drastically enhance maturation and function of vSMCs from stem cell differentiation in vitro and in vivo. STATEMENT OF SIGNIFICANCE: A fast, reliable and safe regeneration of vascular smooth muscle cells (vSMCs) from stem cell differentiation is promising for vascular tissue engineering and regenerative medicine applications, but remains challenging. Herein, a photo-click hydrogel platform is devised to recapitulate the stiffness of vascular tissue and appropriate presentation of transforming growth factor ?1 (TGF-?1) to guide vSMC commitment from mesenchymal stem cells (MSCs). We demonstrate that such concomitant method drastically enhanced regeneration of mature, functional vSMCs from MSCs in vitro and in vivo within only a 3-days span. This work is not only of fundamental scientific importance, revealing how physiochemical factors and the manner of their presentation direct stem cell differentiation, but also attacks the long-standing difficulty in regenerating highly functional vSMCs within a short period.

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Transforming growth factor beta up-regulates cysteine-rich protein 2 in vascular smooth muscle cells via activating transcription factor 2.

Publications

Transforming growth factor beta up-regulates cysteine-rich protein 2 in vascular smooth muscle cells via activating transcription factor 2.

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