Hautmann M B, Adam P J, Owens G K
Department of Molecular Physiology and Biological Physics, University of Virginia Health Sciences Center, Charlottesville 22908, USA.
Arterioscler Thromb Vasc Biol. 1999 Sep;19(9):2049-58. doi: 10.1161/01.atv.19.9.2049.
Transforming growth factor-beta (TGF-beta) has been shown to stimulate smooth muscle (SM) alpha-actin expression in smooth muscle cells (SMCs) and non-SMCs. We previously demonstrated that the 2 CArG boxes A and B and a novel TGF-beta control element (TCE) located within the first 125 bp of the SM alpha-actin promoter were required for TGF-beta inducibility of SM alpha-actin in SMCs. The aims of the present study were (1) to determine whether the TCE exhibits SMC specificity or contributes to TGF-beta induction of SM alpha-actin expression in non-SMCs (ie, endothelial cells and fibroblasts) and (2) to determine whether TGF-beta can induce expression of multiple TCE-containing SMC differentiation marker genes, such as SM22alpha, h(1) calponin, and SM myosin heavy chain (SM MHC) in non-SMCs. Results of transient transfection assays demonstrated that mutation of CArG A, CArG B, or the TCE within a 125-bp promoter context completely abolished TGF-beta inducibility of SM alpha-actin in endothelial cells and fibroblasts. However, in contrast to observations in SMCs, inclusion of regions upstream from (-155) completely repressed TGF-beta responsiveness in non-SMCs. Electrophoretic mobility shift assays showed that TGF-beta enhanced binding of a serum response factor to the CArG elements and the binding of an as-yet-unidentified factor to the TCE in endothelial cells and fibroblasts, but to a much lesser extent compared with SMCs. TGF-beta also stimulated expression of the SMC differentiation marker SM22alpha in non-SMCs. However, in contrast to SMCs, TGF-beta did not induce expression of h(1) calponin and SM MHC in non-SMCs. In summary, these results suggest a conserved role for CArG A, CArG B, and the TCE in TGF-beta-induced expression of SM alpha-actin in SMCs and non-SMCs that is modified by a complex interplay of positive- and negative-acting cis elements in a cell-specific manner. Furthermore, observations that TGF-beta stimulated expression of several early but not late differentiation markers in non-SMCs indicate that TGF-beta alone is not sufficient to induce transdifferentiation of non-SMCs into SMCs.
转化生长因子-β（TGF-β）已被证明可刺激平滑肌细胞（SMC）和非SMC中的平滑肌（SM）α-肌动蛋白表达。我们之前证明，SMα-肌动蛋白启动子前125 bp内的2个CArG框A和B以及一个新的TGF-β控制元件（TCE）是TGF-β诱导SMC中SMα-肌动蛋白所必需的。本研究的目的是：（1）确定TCE是否具有SMC特异性或是否有助于TGF-β诱导非SMC（即内皮细胞和成纤维细胞）中SMα-肌动蛋白的表达；（2）确定TGF-β是否能诱导非SMC中多个含TCE的SMC分化标记基因的表达，如SM22α、h(1)钙调蛋白和SM肌球蛋白重链（SM MHC）。瞬时转染分析结果表明，在125 bp启动子背景下，CArG A、CArG B或TCE的突变完全消除了内皮细胞和成纤维细胞中TGF-β对SMα-肌动蛋白的诱导能力。然而，与在SMC中的观察结果相反，包含（-155）上游区域完全抑制了非SMC中TGF-β的反应性。电泳迁移率变动分析表明，TGF-β增强了血清反应因子与CArG元件的结合以及一种尚未鉴定的因子与内皮细胞和成纤维细胞中TCE的结合，但与SMC相比程度要小得多。TGF-β还刺激了非SMC中SMC分化标记SM22α的表达。然而，与SMC相反，TGF-β并未诱导非SMC中h(1)钙调蛋白和SM MHC的表达。总之，这些结果表明CArG A,CArG B和TCE在TGF-β诱导的SMC和非SMC中SMα-肌动蛋白表达中具有保守作用，这种作用以细胞特异性方式受到正负作用顺式元件复杂相互作用的修饰。此外，TGF-β刺激非SMC中几种早期而非晚期分化标记表达的观察结果表明仅TGF-β不足以诱导非SMC向SMC的转分化。
