Regulation of Cyr61/CCN1 gene expression through RhoA GTPase and p38MAPK signaling pathways.
Cysteine-rich protein 61 (Cyr61/CCN1) is an angiogenic factor and a member of a family of growth factor-inducible immediate-early genes with functions in cell adhesion, proliferation and differentiation. We investigated the regulatory mechanisms and signaling pathways involved in Cyr61/CCN1gene activation in smooth muscle cells. Treatment of these cells with sphingosine 1-phosphate (S1P), a bioactive lysolipid, increased rapidly but transiently the expression of the Cyr61/CCN1 gene at both the mRNA and protein levels. Cyr61/CCN1 mRNA stability was not altered but the transcription rate of the Cyr61/CCN1 gene was increased fivefold in isolated nuclei from S1P-stimulated cells indicating that the level of control is primarily transcriptional. Transfection experiments showed that a 936-bp promoter fragment of the human Cyr61/CCN1 gene is functional and induces a reporter gene activity in S1P-treated cells. Using a combination of cis-element mutagenesis and expression of dominant negative inhibitors of transcription factors, we showed that both a CRE and AP-1 site and their cognate transcription factors, cAMP response element binding protein (CREB) and AP-1, were responsible for the promoter activity in S1P-stimulated cells. Furthermore, by using either pharmacological inhibitors or active forms of known signaling molecules, we showed that inducible Cyr61/CCN1 gene expression occurs through RhoA GTPase and that additional signaling through the p38 pathway is required. In particular, p38 seems to regulate Cyr61/CCN1 promoter activity through modulation of phosphorylation of CREB and the CREB kinase, MSK1. These findings demonstrate the transcriptional regulation of the Cyr61/CCN1 gene and provide clues to the signaling molecules and transcription factors involved in such regulation.
Han
Ji-Soo
JS
University of Pennsylvania, Department of Anatomy and Cell Biology, Philadelphia, PA 19104, USA.
Macarak
Edward
E
Rosenbloom
Joel
J
Chung
Kwang Chul
KC
Chaqour
Brahim
B
D000818:Animals; D001483:Base Sequence; D002417:Cattle; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism; D055515:Cysteine-Rich Protein 61; D005786:Gene Expression Regulation / Q000502:physiology*; D017874:Immediate-Early Proteins / Q000096:biosynthesis / Q000235:genetics*; D036341:Intercellular Signaling Peptides and Proteins / Q000096:biosynthesis / Q000235:genetics*; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D008969:Molecular Sequence Data; D032389:Myocytes, Smooth Muscle / Q000378:metabolism; D011401:Promoter Regions, Genetic; D015398:Signal Transduction / Q000502:physiology*; D018808:Transcription Factor AP-1 / Q000378:metabolism; D048051:p38 Mitogen-Activated Protein Kinases; D020742:rhoA GTP-Binding Protein / Q000378:metabolism*
C501848:CCN1 protein, human; D017362:Cyclic AMP Response Element-Binding Protein; D055515:Cysteine-Rich Protein 61; D017874:Immediate-Early Proteins; D036341:Intercellular Signaling Peptides and Proteins; D018808:Transcription Factor AP-1; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D020742:rhoA GTP-Binding Protein