Abstract
Expression of alpha-actin in smooth muscle cells (SMCs) is regulated, in part, by an intronic serum response factor (SRF)-binding CArG element. We have identified a conserved nuclear factor of activated T cells (NFAT) binding site that overlaps this CArG box and tested the hypothesis that this site plays a previously unrecognized role in regulating alpha-actin expression. A reporter construct prepared using a 56-bp region of the mouse alpha-actin first intron containing SRF, NFAT, and AP-1 sites (SNAP) acted as an enhancer element in the context of a minimal thymidine kinase promoter. Basal reporter activity following expression in SMCs was robust and sensitive to the calcineurin-NFAT pathway inhibitors cyclosporin A and FK506. Mutating either the NFAT or SRF binding site essentially abolished reporter activity, suggesting that both NFAT and SRF binding are required. Basal activity in non-smooth muscle HEK293 cells was SRF-dependent but NFAT-independent and approximately 8-fold lower than that in SMCs. Activation of NFAT in HEK293 cells induced an approximately 4-fold increase in activity that was dependent on the integrity of both NFAT and SRF binding sites. NFATc3.SRF complex formation, demonstrated by co-immunoprecipitation, was facilitated by the presence of SNAP oligonucleotide. Inhibition of the calcineurin-NFAT pathway decreased alpha-actin expression in cultured SMCs, suggesting that the molecular interaction of NFAT and SRF at SNAP may be physiologically relevant. These data provide the first evidence that NFAT and SRF may interact to cooperatively regulate SMC-specific gene expression and support a role for NFAT in the phenotypic maintenance of smooth muscle.
Highlights
Namic and maintain their differentiated phenotype through the regulated expression of a repertoire of smooth muscle-specific genes [1]
We have found that a 56-bp region of the mouse smooth muscle (SM) ␣-actin first intron that contains a highly conserved serum response factor (SRF)/nuclear factor of activated T cells (NFAT)/AP-1 (SNAP) composite binding site sequence is capable of acting as an enhancer element, increasing reporter activity in the context of a minimal thymidine kinase (TK) promoter
Our results show that inhibition of the calcineurin/NFAT pathway decreases ␣-actin expression in SMCs, consistent with a role for NFAT in the regulation of ␣-actin transcription
Summary
Cell Culture—The rat A7r5 aortic smooth muscle cell line and an HEK293 human embryonic kidney-derived cell line (BD Biosciences) were grown in Dulbecco’s modified Eagle’s medium (Invitrogen) containing 10% fetal bovine serum and penicillin-streptomycin (10 units/ ml; Invitrogen). Whole-cell extracts containing 500 g of protein were incubated with 400 ng of biotinylated oligonucleotide probes in binding buffer (20 mM Tris, pH 8.0, 1 mM EDTA, 100 mM NaCl, 0.5% Nonidet P-40, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 5 g/ml aprotinin, 9 g/ml leupeptin, 10 g/ml pepstatin, and 10 mM sodium molybdate) for 30 min on ice. Streptavidin-conjugated agarose beads (Pierce) were added (25 l of a 50% slurry), and the suspensions were placed on a gyrating shaker platform at 4 °C for 1–3 h. Co-immunoprecipitation—Whole-cell lysates from HEK293 cells (500 g total protein content/sample, treated as described in the text) were incubated with 500 ng of mouse anti-NFATc3-agarose conjugate (Santa Cruz Biotechnology) in DNA-protein binding buffer for 24 h at 4 °C with constant rocking in the presence or absence of unlabeled SNAP oligonucleotide. Because of the ease and accuracy with which multiple individual replicates can be independently analyzed, this approach is especially useful where quantitation is the goal
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