Hayashida Kazutaka, Johnston Douglas R, Goldberger Olga, Park Pyong Woo
Departments of Medicine, Molecular and Cellular Biology, and Molecular Virology and Microbiology, Baylor College of Medicine, Houston, Texas 77030, USA.
J Biol Chem. 2006 Aug 25;281(34):24365-74. doi: 10.1074/jbc.M509320200. Epub 2006 Jun 28.
Syndecans comprise a major family of cell surface heparan sulfate proteoglycans (HSPGs). Syndecans bind and modulate a wide variety of biological molecules through their heparan sulfate (HS) moiety. Although all syndecans contain the ligand binding HS chains, they likely perform specific functions in vivo because their temporal and spatial expression patterns are different. However, how syndecan expression is regulated has yet to be clearly defined. In this study, we examined how syndecan-1 expression is regulated in epithelial cells. Our results showed that among several bioactive agents tested, only forskolin and three isoforms of TGFbeta (TGFbeta1-TGFbeta3) significantly induced syndecan-1, but not syndecan-4, expression on various epithelial cells. Steady-state syndecan-1 mRNA was not increased by TGFbeta treatment and cycloheximide did not inhibit syndecan-1 induction by TGFbeta, indicating that TGFbeta induces syndecan-1 in a post-translational manner. However, TGFbeta induction of syndecan-1 was inhibited by transient expression of a dominant-negative construct of protein kinase A (PKA) and by specific inhibitors of PKA. Further (i) syndecan-1 cytoplasmic domains were Ser-phosphorylated when cells were treated with TGFbeta and this was inhibited by a PKA inhibitor, (ii) PKA was co-immunoprecipitated from cell lysates by anti-syndecan-1 antibodies, (iii) PKA phosphorylated recombinant syndecan-1 cytoplasmic domains in vitro, and (iv) expression of a syndecan-1 construct with its invariant Ser(286) replaced with a Gly was not induced by TGFbeta. Together, these findings define a regulatory mechanism where TGFbeta signals through PKA to phosphorylate the syndecan-1 cytoplasmic domain and increases syndecan-1 expression on epithelial cells.
Syndecans是细胞表面硫酸乙酰肝素蛋白聚糖（HSPGs）的一个主要家族。Syndecans通过其硫酸乙酰肝素（HS）部分结合并调节多种生物分子。尽管所有的Syndecans都含有与配体结合的HS链，但由于它们的时空表达模式不同，它们在体内可能执行特定的功能。然而，Syndecan的表达是如何被调控的，尚未明确界定。在本研究中，我们研究了上皮细胞中Syndecan-1的表达是如何被调控的。我们的结果表明，在测试的几种生物活性剂中，只有福司可林和三种TGFβ同工型（TGFβ1-TGFβ3）能显著诱导各种上皮细胞上Syndecan-1的表达，而不能诱导Syndecan-4的表达。TGFβ处理并未增加Syndecan-1的稳态mRNA水平，环己酰亚胺也不抑制TGFβ对Syndecan-1的诱导，这表明TGFβ以翻译后方式诱导Syndecan-1。然而，蛋白激酶A（PKA）的显性负性构建体的瞬时表达和PKA的特异性抑制剂可抑制TGFβ对Syndecan-1的诱导。进一步的研究发现：（i）用TGFβ处理细胞时，Syndecan-1的细胞质结构域会发生丝氨酸磷酸化，而PKA抑制剂可抑制这种磷酸化；（ii）抗Syndecan-1抗体可从细胞裂解物中共免疫沉淀PKA；（iii）PKA在体外可磷酸化重组Syndecan-1的细胞质结构域；（iv）将Syndecan-1构建体中不变的Ser（286）替换为Gly后，其表达不受TGFβ诱导。总之，这些发现确定了一种调节机制，即TGFβ通过PKA发出信号，使Syndecan-1的细胞质结构域磷酸化，并增加上皮细胞上Syndecan-1的表达。
