Kim I Y, Zelner D J, Lee C
Department of Urology, Northwestern University Medical School, Chicago, Illinois 60611, USA.
Exp Cell Res. 1998 May 25;241(1):151-60. doi: 10.1006/excr.1998.4034.
LNCaP is an androgen-responsive human prostate cancer cell line that has a defective gene for ALK-5, the conventional TGF-beta receptor type I. Yet, these cells respond to exogenous TGF-beta 1 under appropriate concentrations of dihydrotestosterone (DHT). Because a heteromeric complex composed of type I and type II receptor is required for TGF-beta signaling, the expression of these receptors was investigated in LNCaP cells at following concentrations of DHT-0, 0.1, and 100 nM. These concentrations were selected because they represent the zero DHT control in which LNCaP cells are not sensitive to TGF-beta 1, the proliferative dose of DHT in which these cells are sensitive to exogenous TGF-beta 1, and the growth-arrest dose of DHT in which LNCaP exhibits signs of TGF-beta signaling but are insensitive to exogenous TGF-beta 1, respectively. Results of Western blot analysis showed that LNCaP cells express an increased level of type II receptor at 0.1 nM DHT, the TGF-beta 1-sensitive dose. However, results of competitive quantitative RT-PCR demonstrated that DHT did not significantly change the level of type II receptor mRNA, suggesting that DHT modulates the level of type II receptor at the posttranscriptional level. In contrast, ALK-5 was not detected in these cells by either Western blot analysis or RT-PCR at all concentrations of DHT used in this study. Subsequently, the expression of ALK-1, -2, and -4 in LNCaP cells was examined because these proteins have been shown to bind TGF-beta 1 in vitro. ALK-1 and -2 were detected in these cells. Further analysis by competitive quantitative RT-PCR and Western blot demonstrated that DHT did not affect the level of expression of ALK-1 and -2 in LNCaP cells. These observations, taken together, demonstrate that ALK-5 is not required for TGF-beta 1 signaling and that there may be alternative mechanism(s) for TGF-beta 1 signal transduction in some systems.
LNCaP是一种雄激素反应性人前列腺癌细胞系，其具有ALK-5（传统的I型转化生长因子β受体）的缺陷基因。然而，这些细胞在适当浓度的二氢睾酮（DHT）作用下对外源转化生长因子β1有反应。由于转化生长因子β信号传导需要由I型和II型受体组成的异源复合物，因此在LNCaP细胞中以以下DHT浓度（0、0.1和100 nM）研究了这些受体的表达。选择这些浓度是因为它们分别代表LNCaP细胞对转化生长因子β1不敏感的零DHT对照、这些细胞对外源转化生长因子β1敏感的DHT增殖剂量以及LNCaP表现出转化生长因子β信号传导迹象但对外源转化生长因子β1不敏感的DHT生长抑制剂量。蛋白质印迹分析结果显示，在0.1 nM DHT（转化生长因子β1敏感剂量）时，LNCaP细胞中II型受体水平升高。然而，竞争性定量逆转录-聚合酶链反应结果表明，DHT并未显著改变II型受体mRNA水平，这表明DHT在转录后水平调节II型受体水平。相反，在本研究中使用的所有DHT浓度下，通过蛋白质印迹分析或逆转录-聚合酶链反应均未在这些细胞中检测到ALK-5。随后，检测了LNCaP细胞中ALK-1、-2和-4的表达，因为这些蛋白已被证明在体外可结合转化生长因子β1。在这些细胞中检测到了ALK-1和-2。通过竞争性定量逆转录-聚合酶链反应和蛋白质印迹的进一步分析表明，DHT不影响LNCaP细胞中ALK-1和-2的表达水平。综上所述，这些观察结果表明，转化生长因子β1信号传导不需要ALK-5，并且在某些系统中可能存在转化生长因子β1信号转导的替代机制。
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