Cullen Kimberly A, McCool John, Anwer M Sawkat, Webster Cynthia R L
Tufts Univ. School of Veterinary Medicine, 200 Westboro Rd., Grafton, MA 01536, USA.
Am J Physiol Gastrointest Liver Physiol. 2004 Aug;287(2):G334-43. doi: 10.1152/ajpgi.00517.2003. Epub 2004 Mar 25.
cAMP has previously been shown to promote cell survival in a variety of cell types, but the downstream signaling pathway(s) of this antiapoptotic effect is unclear. Thus the role of cAMP signaling through PKA and cAMP-regulated guanine nucleotide exchange factors (cAMP-GEFs) in cAMP's antiapoptotic action was investigated in the present study. cAMP's protective effect against bile acid-, Fas ligand-, and TNF-alpha-induced apoptosis in rat hepatocytes was largely unaffected by the selective PKA inhibitor, Rp-8-(4-chlorophenylthio)-cAMP (Rp-cAMP). In contrast, a novel cAMP analog, 8-(4-chlorophenylthio)-2'-O-methyl (CPT-2-Me)-cAMP, which activated cAMP-GEFs in hepatocytes without activating PKA, protected hepatocytes against apoptosis induced by bile acids, Fas ligand, and TNF-alpha. The role of cAMP-GEF and PKA on activation of Akt, a kinase implicated in cAMP survival signaling, was investigated. Inhibition of PKA with RP-cAMP had no effect on cAMP-mediated Akt phosphorylation, whereas CPT-2-Me-cAMP, which did not activate PKA, induced phosphatidylinositol 3-kinase (PI3-kinase)-dependent activation of Akt. Pretreatment of hepatocytes with the PI3-kinase inhibitor, Ly-294002, prevented CPT-2-Me-cAMP's protective effect against bile acid and Fas ligand, but not TNF-alpha-mediated apoptosis. Glucagon, CPT-cAMP, and CPT-2-Me-cAMP all activated Rap 1, a downstream effector of cAMP-GEF. These results suggest that a PKA-independent cAMP/cAMP-GEF/Rap pathway exists in hepatocytes and that activation of cAMP-GEFs promotes Akt phosphorylation and hepatocyte survival. Thus a cAMP/cAMP-GEF/Rap/PI3-kinase/Akt signaling pathway may confer protection against bile acid- and Fas-induced apoptosis in hepatocytes.
此前已有研究表明，环磷酸腺苷（cAMP）可促进多种细胞类型的细胞存活，但其抗凋亡作用的下游信号通路尚不清楚。因此，本研究探讨了通过蛋白激酶A（PKA）和环磷酸腺苷调节的鸟嘌呤核苷酸交换因子（cAMP-GEFs）的cAMP信号在cAMP抗凋亡作用中的作用。cAMP对大鼠肝细胞中胆汁酸、Fas配体和肿瘤坏死因子-α（TNF-α）诱导的细胞凋亡的保护作用在很大程度上不受选择性PKA抑制剂Rp-8-（4-氯苯硫基）-环磷酸腺苷（Rp-cAMP）的影响。相反，一种新型的cAMP类似物8-（4-氯苯硫基）-2'-O-甲基（CPT-2-Me）-cAMP，可在不激活PKA的情况下激活肝细胞中的cAMP-GEFs，保护肝细胞免受胆汁酸、Fas配体和TNF-α诱导的细胞凋亡。研究了cAMP-GEF和PKA对Akt激活的作用，Akt是一种与cAMP存活信号相关的激酶。用RP-cAMP抑制PKA对cAMP介导的Akt磷酸化没有影响，而不激活PKA的CPT-2-Me-cAMP诱导了磷脂酰肌醇3-激酶（PI3-激酶）依赖性的Akt激活。用PI3-激酶抑制剂Ly-294002预处理肝细胞可防止CPT-2-Me-cAMP对胆汁酸和Fas配体的保护作用，但不能防止TNF-α介导的细胞凋亡。胰高血糖素、CPT-环磷酸腺苷（CPT-cAMP）和CPT-2-Me-cAMP均激活了cAMP-GEF的下游效应分子Rap 1。这些结果表明，肝细胞中存在一条不依赖PKA的cAMP/cAMP-GEF/Rap信号通路，cAMP-GEFs的激活促进了Akt磷酸化和肝细胞存活。因此，cAMP/cAMP-GEF/Rap/PI3-激酶/Akt信号通路可能对肝细胞中胆汁酸和Fas诱导的细胞凋亡具有保护作用。
