Intronic CArG box regulates cysteine-rich protein 2 expression in the adult but not in developing vasculature.
OBJECTIVE
An absence of cysteine-rich protein 2 (CRP2) enhances vascular smooth muscle cell (VSMC) migration and increases neointima formation after arterial injury; therefore, CRP2 plays an important role in the response to vascular injury. The goal of the present study was to elucidate the molecular mechanisms that preserve CRP2 expression in the adult vasculature and thus might serve to inhibit the response to injury.


RESULTS
We generated a series of transgenic mice harboring potential Csrp2 regulatory regions with a lacZ reporter. We determined that the 12-kb first intron was necessary for transgene activity in adult but not in developing vasculature. Within the intron we identified a 6.3-kb region that contains 2 CArG boxes. Serum response factor preferentially bound to CArG2 box in gel mobility shift and chromatin immunoprecipitation assays; additionally, serum response factor coactivator myocardin factors activated CRP2 expression via the CArG2 box. Mutational analysis revealed that CArG2 box was important in directing lacZ expression in VSMC of adult vessels.


CONCLUSIONS
Although CRP2 expression during development is independent of CArG box regulatory sites, CRP2 expression in adult VSMC requires CArG2 element within the first intron. Our results suggest that distinct mechanisms regulate CRP2 expression in VSMC that are controlled by separate embryonic and adult regulatory modules.
Chen
Chung-Huang
CH
Institute of Cellular and System Medicine, National Health Research Institutes, 35 Keyan Road, Zhunan, Miaoli County 35053, Taiwan.
Wu
Meng-Ling
ML
Lee
Yi-Chung
YC
Layne
Matthew D
MD
Yet
Shaw-Fang
SF
D000367:Age Factors; D000375:Aging; D000818:Animals; D001158:Arteries / Q000254:growth & development / Q000378:metabolism; D001665:Binding Sites; D002478:Cells, Cultured; D047369:Chromatin Immunoprecipitation; D024202:Electrophoretic Mobility Shift Assay; D018507:Gene Expression Regulation, Developmental; D017930:Genes, Reporter; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B / Q000235:genetics / Q000378:metabolism*; D007438:Introns; D060588:LIM Domain Proteins; D007763:Lac Operon; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D009124:Muscle Proteins / Q000235:genetics / Q000378:metabolism*; D009131:Muscle, Smooth, Vascular / Q000254:growth & development / Q000378:metabolism*; D009154:Mutation; D032389:Myocytes, Smooth Muscle / Q000378:metabolism*; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D012045:Regulatory Sequences, Nucleic Acid; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D026362:Serum Response Factor / Q000378:metabolism; D015534:Trans-Activators / Q000378:metabolism; D015533:Transcriptional Activation; D014162:Transfection; D000099251:Myocardin
D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B; D060588:LIM Domain Proteins; D009124:Muscle Proteins; D009687:Nuclear Proteins; D012097:Repressor Proteins; D026362:Serum Response Factor; D015534:Trans-Activators; D000099251:Myocardin; C484376:Csrp2 protein, mouse; C077048:Hnrnpab protein, mouse