Schaafsma Dedmer, McNeill Karol D, Stelmack Gerald L, Gosens Reinoud, Baarsma Hoeke A, Dekkers Bart G J, Frohwerk Erin, Penninks Jelte-Maarten, Sharma Pawan, Ens Karen M, Nelemans S Adriaan, Zaagsma Johan, Halayko Andrew J, Meurs Herman
Dept. of Molecular Pharmacology, University of Groningen, A. Deusinglaan 1, 9713 AV Groningen, The Netherlands.
Am J Physiol Cell Physiol. 2007 Jul;293(1):C429-39. doi: 10.1152/ajpcell.00502.2006. Epub 2007 Apr 25.
We have previously demonstrated that long-term exposure of bovine tracheal smooth muscle (BTSM) strips to insulin induces a functional hypercontractile phenotype. To elucidate molecular mechanisms by which insulin might induce maturation of contractile phenotype airway smooth muscle (ASM) cells, we investigated effects of insulin stimulation in serum-free primary BTSM cell cultures on protein accumulation of specific contractile phenotypic markers and on the abundance and stability of mRNA encoding these markers. In addition, we used microscopy to assess insulin effects on ASM cell morphology, phenotype, and induction of phosphatidylinositol (PI) 3-kinase signaling. It was demonstrated that protein and mRNA levels of smooth muscle-specific contractile phenotypic markers, including sm-myosin, are significantly increased after stimulation of cultured BTSM cells with insulin (1 microM) for 8 days compared with cells treated with serum-free media, whereas mRNA stability was unaffected. In addition, insulin treatment promoted the formation of large, elongate ASM cells, characterized by dramatic accumulation of contractile phenotype marker proteins and phosphorylated p70(S6K) (downstream target of PI 3-kinase associated with ASM maturation). Insulin effects on protein accumulation and cell morphology were abrogated by combined pretreatment with the Rho kinase inhibitor Y-27632 (1 microM) or the PI 3-kinase inhibitor LY-294002 (10 microM), indicating that insulin increases the expression of contractile phenotypic markers in BTSM in a Rho kinase- and PI 3-kinase-dependent fashion. In conclusion, insulin increases transcription and protein expression of contractile phenotypic markers in ASM. This could have important implications for the use of recently approved aerosolized insulin formulations in diabetes mellitus.
我们之前已经证明，将牛气管平滑肌（BTSM）条长期暴露于胰岛素会诱导功能性高收缩表型。为了阐明胰岛素可能诱导收缩表型气道平滑肌（ASM）细胞成熟的分子机制，我们研究了在无血清原代BTSM细胞培养物中胰岛素刺激对特定收缩表型标志物蛋白质积累以及编码这些标志物的mRNA丰度和稳定性的影响。此外，我们使用显微镜评估胰岛素对ASM细胞形态、表型以及磷脂酰肌醇（PI）3激酶信号传导诱导的影响。结果表明，与用无血清培养基处理的细胞相比，用胰岛素（1微摩尔）刺激培养的BTSM细胞8天后，包括平滑肌肌球蛋白（sm-肌球蛋白）在内的平滑肌特异性收缩表型标志物的蛋白质和mRNA水平显著增加，而mRNA稳定性未受影响。此外，胰岛素处理促进了大的、细长的ASM细胞的形成，其特征是收缩表型标志物蛋白和磷酸化的p70核糖体蛋白S6激酶（PI 3激酶的下游靶点，与ASM成熟相关）大量积累。胰岛素对蛋白质积累和细胞形态的影响可通过用Rho激酶抑制剂Y-27632（1微摩尔）或PI 3激酶抑制剂LY-294002（10微摩尔）联合预处理来消除，这表明胰岛素以Rho激酶和PI 3激酶依赖性方式增加BTSM中收缩表型标志物的表达。总之，胰岛素增加了ASM中收缩表型标志物转录和蛋白质表达。这可能对最近批准的糖尿病雾化胰岛素制剂的使用具有重要意义。
