Jo Youngah, King Steven R, Khan Shafiq A, Stocco Douglas M
Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, Texas 79430, USA.
Biol Reprod. 2005 Aug;73(2):244-55. doi: 10.1095/biolreprod.104.037721. Epub 2005 Apr 6.
This study investigated the roles of the protein kinase C (PKC) and protein kinase A (PKA) pathways in regulating constitutive steroidogenesis and steroidogenic acute regulatory (STAR; herein designated by its common name, StAR) protein in R2C Leydig tumor cells. Inhibition of PKC and phospholipase C resulted in significant decreases in steroid production, phosphorylation of cAMP-responsive element binding (CREB) protein, and Star gene transcription under basal conditions in R2C cells. These observations were corroborated in MA-10 and mLTC-1 Leydig tumor cell lines, in which activation of PKC by phorbol-12-myristate-13-acetate (PMA, 10 nM) increased CREB phosphorylation and total StAR (tot-StAR) protein expression. However, induction of StAR protein by PMA did not result in the expected concomitant increase in steroids because PKC failed to phosphorylate StAR, the biologically active form of the protein. However, in conjunction with PMA, minor increases in PKA activity using submaximal doses of (Bu)2cAMP (0.05-0.1 mM; a concentration range insufficient for induction of StAR), were able to stimulate dramatic increases in both phospho-StAR (P-StAR) and steroid production. Human chorionic gonadotropin stimulation also resulted in a further enhancement in P-StAR and progesterone production when added to PMA-treated MA-10 cells. Similar results for tot-StAR and P-StAR expression were observed in primary cultures of immature rat Leydig cells treated with PMA and submaximal doses of (Bu)2cAMP. In summary, the present study demonstrates that basal activities of both PKC and PKA play important roles in the constitutive steroidogenic characteristics of R2C cells. This study also demonstrates for the first time a role for PMA-induced PKC in StAR protein regulation and the requirement for submaximal doses of cAMP to produce steroids in Leydig cells.
本研究调查了蛋白激酶C（PKC）和蛋白激酶A（PKA）信号通路在调控R2C Leydig肿瘤细胞中组成性类固醇生成及类固醇生成急性调节蛋白（STAR；此处用其通用名StAR表示）方面的作用。在基础条件下，抑制PKC和磷脂酶C会导致R2C细胞中类固醇生成、环磷酸腺苷反应元件结合蛋白（CREB）的磷酸化以及StAR基因转录显著减少。在MA - 10和mLTC - 1 Leydig肿瘤细胞系中也得到了类似结果，其中佛波醇 - 12 - 肉豆蔻酸酯 - 13 - 乙酸酯（PMA，10 nM）激活PKC可增加CREB磷酸化及总StAR（tot - StAR）蛋白表达。然而，PMA诱导StAR蛋白并未导致预期的类固醇生成相应增加，因为PKC未能使该蛋白的生物活性形式StAR磷酸化。不过，与PMA联合使用时，用次最大剂量的（Bu）2cAMP（0.05 - 0.1 mM；该浓度范围不足以诱导StAR）轻微增加PKA活性，能够刺激磷酸化StAR（P - StAR）和类固醇生成显著增加。当添加到经PMA处理的MA - 10细胞中时，人绒毛膜促性腺激素刺激也会进一步增强P - StAR和孕酮生成。在用PMA和次最大剂量的（Bu）2cAMP处理的未成熟大鼠Leydig细胞原代培养物中，观察到了类似的tot - StAR和P - StAR表达结果。总之，本研究表明PKC和PKA的基础活性在R2C细胞的组成性类固醇生成特性中发挥重要作用。本研究还首次证明了PMA诱导的PKC在StAR蛋白调控中的作用，以及在Leydig细胞中产生类固醇需要次最大剂量的cAMP。
