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Role of microRNA-23b in flow-regulation of Rb phosphorylation and endothelial cell growth.

ABSTRACT: MicroRNAs (miRs) can regulate many cellular functions, but their roles in regulating responses of vascular endothelial cells (ECs) to mechanical stimuli remain unexplored. We hypothesize that the physiological responses of ECs are regulated by not only mRNA and protein signaling networks, but also expression of the corresponding miRs. EC growth arrest induced by pulsatile shear (PS) flow is an important feature for flow regulation of ECs. miR profiling showed that 21 miRs are differentially expressed (8 up- and 13 downregulated) in response to 24-h PS as compared to static condition (ST). The mRNA expression profile indicates EC growth arrest under 24-h PS. Analysis of differentially expressed miRs yielded 68 predicted mRNA targets that overlapped with results of microarray mRNA profiling. Functional analysis of miR profile indicates that the cell cycle network is highly regulated. The upregulation of miR-23b and miR-27b was found to correlate with the PS-induced EC growth arrest. Inhibition of miR-23b using antagomir-23b oligonucleotide (AM23b) reversed the PS-induced E2F1 reduction and retinoblastoma (Rb) hypophosphorylation and attenuated the PS-induced G1/G0 arrest. Antagomir AM27b regulated E2F1 expression, but did not affect Rb and growth arrest. Our findings indicate that PS suppresses EC proliferation through the regulation of miR-23b and provide insights into the role of miRs in mechanotransduction.

SUBMITTER: Wang KC

PROVIDER: S-EPMC2840325 | biostudies-literature | 2010 Feb

REPOSITORIES: biostudies-literature

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Role of microRNA-23b in flow-regulation of Rb phosphorylation and endothelial cell growth.

Wang Kuei-Chun KC Garmire Lana Xia LX Young Angela A Nguyen Phu P Trinh Andrew A Subramaniam Shankar S Wang Nanping N Shyy John Y J JY Li Yi-Shuan YS Chien Shu S

Proceedings of the National Academy of Sciences of the United States of America 20100127 7

MicroRNAs (miRs) can regulate many cellular functions, but their roles in regulating responses of vascular endothelial cells (ECs) to mechanical stimuli remain unexplored. We hypothesize that the physiological responses of ECs are regulated by not only mRNA and protein signaling networks, but also expression of the corresponding miRs. EC growth arrest induced by pulsatile shear (PS) flow is an important feature for flow regulation of ECs. miR profiling showed that 21 miRs are differentially expr ...[more]

PMID: 20133741

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Project description:The importance of microRNA (miRNA) to vascular biology is becoming increasingly evident; however, the function of a significant number of miRNA remains to be determined. In particular, the effect of growth factor regulation of miRNAs on endothelial cell morphogenesis is incomplete. Thus, we aimed to identify miRNAs regulated by pro-angiogenic vascular endothelial growth factor (VEGF) and determine the effects of VEGF-regulated miRNAs and their targets on processes important for angiogenesis. Human umbilical vein endothelial cells (HUVECs) were thus stimulated with VEGF and miRNA levels assessed using microarrays. We found that VEGF altered expression of many miRNA, and for this study focused on one of the most significantly down-regulated miRNA in HUVECs following VEGF treatment, miR-30b. Using specific miRNA mimics, we found that overexpression of miR-30b inhibited capillary morphogenesis in vitro, while depletion of endogenous miR-30b resulted in increased capillary morphogenesis indicating the potential significance of down-regulation of miR-30b as a pro-angiogenic response to VEGF stimulation. MiR-30b overexpression in HUVEC regulated transforming growth factor beta 2 (TGFβ2) production, which led to increased phosphorylation of Smad2, indicating activation of an autocrine TGFβ signaling pathway. Up-regulation of TGFβ2 by miR-30b overexpression was found to be dependent on ATF2 activation, a transcription factor known to regulate TGFβ2 expression, as miR-30b overexpressing cells exhibited increased levels of phosphorylated ATF2 and depletion of ATF2 inhibited miR-30b-induced TGFβ2 expression. However, miR-30b effects on ATF2 were indirect and found to be via targeting of the known ATF2 repressor protein JDP2 whose mRNA levels were indirectly correlated with miR-30b levels. Increased secretion of TGFβ2 from HUVEC was shown to mediate the inhibitory effects of miR-30b on capillary morphogenesis as treatment with a neutralizing antibody to TGFβ2 restored capillary morphogenesis to normal levels in miR-30b overexpressing cells. These results support that the regulation of miR-30b by VEGF in HUVEC is important for capillary morphogenesis, as increased miR-30b expression inhibits capillary morphogenesis through enhanced expression of TGFβ2.

Project description:The retinoblastoma protein RB and the transcription factor p53 are central tumor suppressors. They are often found inactivated in various tumor types. Both proteins play central roles in regulating the cell division cycle. RB forms complexes with the E2F family of transcription factors and downregulates numerous genes. Among the RB-E2F target genes, a large number code for key cell cycle regulators. Their transcriptional repression by the RB-E2F complex is released through phosphorylation of RB, leading to expression of the cell cycle regulators. The release from repression can be prevented by the cyclin-dependent kinase inhibitor p21/CDKN1A. The CDKN1A gene is transcriptionally activated by p53. Taken together, these elements constitute the p53-p21-RB signaling pathway. Following activation of p53, for example by viral infection or induction of DNA damage, p21 expression is upregulated. High levels of p21 then result in RB-E2F complex formation and downregulation of a large number of cell cycle genes. Thus, p53-dependent transcriptional repression is indirect. The reduced expression of the many regulators leads to cell cycle arrest. Examination of the p53-p21-RB targets and genes controlled by the related p53-p21-DREAM signaling pathway reveals that there is a large overlap of the two groups. Mechanistically this can be explained by replacing RB-E2F complexes with the DREAM transcriptional repressor complex at E2F sites in target promoters. In contrast to RB-E2F, DREAM can downregulate genes also through CHR transcription factor binding sites. This results in a distinct gene set controlled by p53-p21-DREAM signaling independent of RB-E2F. Furthermore, RB has non-canonical functions without binding to E2F and DNA. Such a role of RB supporting DREAM formation may be exerted by the RB-SKP2-p27-cyclin A/E-CDK2-p130-DREAM link. In the current synopsis, the mechanism of regulation by p53-p21-RB signaling is assessed and the overlap with p53-p21-DREAM signaling is examined.

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Role of microRNA-23b in flow-regulation of Rb phosphorylation and endothelial cell growth.

Publications

Role of microRNA-23b in flow-regulation of Rb phosphorylation and endothelial cell growth.

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