Fujita Takayuki, Otsu Kouji, Oshikawa Jin, Hori Hideaki, Kitamura Hitoshi, Ito Takaaki, Umemura Satoshi, Minamisawa Susumu, Ishikawa Yoshihiro
Cardiovascular Research Institute, Department of Pathology and Department of Medicine, Yokohama City University Graduate School of Medical Science, Fukuura, Yokohama, Japan.
J Cell Mol Med. 2006 Jan-Mar;10(1):216-24. doi: 10.1111/j.1582-4934.2006.tb00302.x.
Caveolin, a major protein component of caveolae, directly interacts with multiple signaling molecules, such as Ras and growth factor receptors, and inhibits their function. However, the role of the second messenger system in mediating this inhibition by caveolin remains poorly understood. We examined the role of Ca2+-dependent signal in caveolin- mediated growth inhibition using a rat cardiac myoblast cell line (H9C2), in which the expression of caveolin- 3, the muscle specific subtype, can be induced using the LacSwitch system. Upon induction with IPTG and serum-starvation, the expression of caveolin-3 was increased by 3.3-fold relative to that of mock-induced cells. The recombinant caveolin-3 was localized to the same subcellular fraction as endogenous caveolin-3 after sucrose gradient purification. Angiotensin II enhanced ERK phosphorylation, but this enhancement was significantly decreased in caveolin-3-induced cells in comparison to that in mock-induced cells. Similarly, when cells were stimulated with fetal calf serum, DNA synthesis, as determined by [3H]-thymidine incorporation, was significantly decreased in caveolin- 3-induced cells. When cells were treated with Ca2+ chelator (BAPTA and EGTA), however, this attenuation was blunted. Calphostin (PKC inhibitor), but not cyclosporine A treatment (calcineurin inhibitor), blunted this attenuation in caveolin-3 induced cells. Our findings suggest that caveolin exhibits growth inhibition in a Ca2+-dependent manner, most likely through PKC, in cardiac myoblasts.
小窝蛋白是小窝的主要蛋白质成分，它直接与多种信号分子相互作用，如Ras和生长因子受体，并抑制它们的功能。然而，第二信使系统在介导小窝蛋白的这种抑制作用中的作用仍知之甚少。我们使用大鼠心肌成肌细胞系（H9C2）研究了Ca2+依赖性信号在小窝蛋白介导的生长抑制中的作用，在该细胞系中，可使用LacSwitch系统诱导肌肉特异性亚型小窝蛋白-3的表达。用异丙基-β-D-硫代半乳糖苷（IPTG）诱导并血清饥饿后，与模拟诱导的细胞相比，小窝蛋白-3的表达增加了3.3倍。蔗糖梯度纯化后，重组小窝蛋白-3与内源性小窝蛋白-3定位于相同的亚细胞组分。血管紧张素II增强了细胞外信号调节激酶（ERK）的磷酸化，但与模拟诱导的细胞相比，在小窝蛋白-3诱导的细胞中这种增强作用明显减弱。同样，当用胎牛血清刺激细胞时，通过[3H]胸苷掺入法测定，小窝蛋白-3诱导的细胞中的DNA合成显著减少。然而，当用Ca2+螯合剂（乙二醇双四乙酸（BAPTA）和乙二醇双乙醚二胺四乙酸（EGTA））处理细胞时，这种减弱作用被减弱。钙泊三醇（蛋白激酶C（PKC）抑制剂）而非环孢素A处理（钙调神经磷酸酶抑制剂）减弱了小窝蛋白-3诱导细胞中的这种减弱作用。我们的研究结果表明，在心肌成肌细胞中，小窝蛋白以Ca2+依赖性方式发挥生长抑制作用，最有可能是通过PKC。
