5' CArG degeneracy in smooth muscle alpha-actin is required for injury-induced gene suppression in vivo

Abstract

CC(A/T)6GG-dependent (CArG-dependent) and serum response factor-dependent (SRF-dependent) mechanisms are required for gene expression in smooth muscle cells (SMCs). However, an unusual feature of many SMC-selective promoter CArG elements is that they contain a conserved single G or C substitution in their central A/T-rich region, which reduces binding affinity for ubiquitously expressed SRF. We hypothesized that this CArG degeneracy contributes to cell-specific expression of smooth muscle alpha-actin in vivo, since substitution of c-fos consensus CArGs for the degenerate CArGs resulted in relaxed specificity in cultured cells. Surprisingly, our present results show that these substitutions have no effect on smooth muscle-specific transgene expression during normal development and maturation in transgenic mice. However, these substitutions significantly attenuated injury-induced downregulation of the mutant transgene under conditions where SRF expression was increased but expression of myocardin, a smooth muscle-selective SRF coactivator, was decreased. Finally, chromatin immunoprecipitation analyses, together with cell culture studies, suggested that myocardin selectively enhanced SRF binding to degenerate versus consensus CArG elements. Our results indicate that reductions in myocardin expression and the degeneracy of CArG elements within smooth muscle promoters play a key role in phenotypic switching of smooth muscle cells in vivo, as well as in mediating responses of CArG-dependent smooth muscle genes and growth regulatory genes under conditions in which these 2 classes of genes are differentially expressed.

Figure 1

Schematic diagram of wild-type and SRE-substituted mutant/LacZ promoter constructs used to generate transgenic mice. To determine the importance of the reduced SRF binding affinity of the degenerate CArGs for controlling SM α-actin expression, we generated mutant LacZ promoter constructs in which a c-fos SRE consensus CArG was substituted for the CArG-A and/or CArG-B element(s) within the SM α-actin promoter previously shown to mimic expression of the endogenous gene. The resulting mutations are shown in bold.

Figure 2

SRE-substituted transgenic mice showed no loss of SMC-specific SM α-actin expression during normal development and maturation. (A) Analysis of LacZ expression in whole adult tissues of WT and SRE-AB transgenic mice indicated that the SRE substitutions had no effect on transgene expression in adult tissues. Sm. int., small intestine. (B) Analysis of LacZ expression in E16.5 whole embryos from wild-type and SRE-AB transgenic mice indicated no loss of specificity upon replacement of SM α-actin CArG-A and CArG-B with the c-fos SRE consensus CArG. (C) Histological analyses of adult tissues indicated no loss of SMC-specific SM α-actin expression in SRE-substituted transgenic mice. Tissues were paraffin-embedded, sectioned, and stained with H&E. LacZ expression was specific to SMCs in wild-type aorta and in aortas of SRE-AB transgenic mice across multiple independent founder lines. Magnification, ×40. In SRE-substituted transgenic mice, the same LacZ expression pattern was found in all SMC-containing tissues examined, including esophagus and small intestine (data not shown). (D) Histological examination of LacZ- and eosin-stained aortas of E16.5 embryos from wild-type and SRE-substituted transgenic mice indicated no loss of specificity in the mutants. LacZ expression was restricted to the SMC-containing layers of all tissues examined, including the aorta (magnification, ×40), esophagus, bronchi, and small intestine (data not shown).

Figure 3

Oligonucleotides containing the c-fos SRE consensus CArG substitution competed for SRF binding activity more effectively than did wild-type SM α-actin oligonucleotides (containing CArG-A and/or CArG-B) in EMSAs. (A) In vitro translated SRF and a 95-bp radiolabeled probe harboring the CArG-containing region of the SM α-actin promoter were used for EMSAs. Unlabeled 95-bp double-stranded oligonucleotides containing either the SM α-actin 5′ CArGs (WT) or the SRE consensus CArG substituted for CArG-A (SRE-A), CArG-B (SRE-B), or CArG-A and CArG-B (SRE-AB) in the context of the SM α-actin promoter were used as cold competitors at approximately 50-, 100-, and 200-fold excess over labeled probe. The SRE CArG in the context of the c-fos promoter (fos) was used as a cold competitor at approximately 50-fold excess over labeled probe. The SRF band was supershifted (SRF SS) by the addition of 2 μg of anti-SRF rabbit polyclonal antibody. Unprog. lysate, unprogrammed control lysate. (B) Densitometry was performed on the SRF bands (see Figure 5A) and results were plotted relative to maximal SRF binding to the radiolabeled probe in the absence of cold competitor. Results are representative of 3 independent experiments. Statistical analyses were performed using 1-way ANOVA. We found statistically significant differences between percentage of SRF binding in the presence of WT cold competitor and percentage of SRF binding in the presence of SRE-AB, SRE-A, or SRE-B cold competitor under all but 1 condition (×50 WT versus ×50 SRE-AB) across multiple experiments (data not shown).

Figure 4

Analysis of LacZ expression in mouse carotid arteries indicated that the SRE-AB transgene is differentially expressed compared with the wild-type SM α-actin transgene 7 days following wire injury. Results are representative of at least 5 animals from each of 2 WT and 2 SRE-AB independent transgenic founder lines. Small arrowheads indicate the internal elastic lamina, and large arrowheads indicate the external elastic lamina. Magnification is indicated in each panel.

Figure 5

Temporal expression analysis by real-time RT-PCR of endogenous SRF in balloon-injured carotid arteries showed increased SRF expression following injury. SRF expression was normalized to 18S rRNA expression in the injured and uninjured contralateral control vessel. Each time point represents the mean ± SE of the injured (SRF:18S) vessel normalized to the uninjured (SRF:18S) vessel (n = 4 animals per time point).

Figure 6

Myocardin can differentially regulate SRF binding to degenerate versus consensus CArGs and is decreased following vascular injury. (A) Results of ChIP assays in cultured rat aortic SMCs indicate that SRF binding is enhanced at the CArG-containing region of the SM α-actin promoter but not the c-fos promoter in response to myocardin overexpression. Quantitative PCR was used to detect CArG-containing regions of the SM α-actin and c-fos promoters in chromatin fragments immunoprecipitated with an SRF antibody. Data represent the mean ± SE of the fold increase in SRF association in cells overexpressing myocardin versus control cells in 3 independent experiments. A fold increase value of 1 indicates no change in SRF association in cells overexpressing myocardin versus control cells. *P < 0.05 compared with SRF association at the c-fos CArG region under the same conditions. (B) Temporal expression analysis by real-time RT-PCR of endogenous myocardin in balloon-injured rat carotid arteries showed decreased myocardin expression following injury. Myocardin expression was normalized to 18S rRNA expression in the injured and uninjured contralateral control vessel. Each time point represents the mean ± SE of the injured vessel (myocardin:18S) normalized to that of the uninjured (myocardin:18S) vessel (n = 4 animals per time point). *P < 0.05 compared with myocardin expression prior to injury. (C) Effect of substitution of CArG-A and CArG-B with the c-fos SRE CArG on myocardin responsiveness of SM α-actin promoter activity. SM α-actin/luciferase and SRE-AB/luciferase promoter constructs were cotransfected with myocardin into rat aortic SMCs and assayed for luciferase activity. The activity was normalized for protein content. Normalized promoter activities of SM α-actin/luciferase and SRE-AB/luciferase in the absence of myocardin were set to 1. Fold induction over basal promoter activity in response to myocardin was calculated. Values represent the mean ± SE of 3 independent experiments. *P < 0.05 compared with WT fold induction under the same conditions.