HGF protects corneal epithelial cells from apoptosis by the PI-3K/Akt-1/bad- but not the ERK1/2-mediated signaling pathway
HGF protects corneal epithelial cells from apoptosis by the PI-3K/Akt-1/bad- but not the ERK1/2-mediated signaling pathway
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DOI：
10.1167/iovs.04-0372
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发表时间：
2004-10-01
影响因子：
4.4
通讯作者：
Bazan, HEP
中科院分区：
文献类型：
--
作者：
Kakazu, A;Chandrasekher, G;Bazan, HEP
目的。损伤后角膜上皮细胞再生过程中，细胞存活至关重要，而生长因子可能是细胞保护的基础。本研究的目的是探讨旁分泌性肝细胞生长因子（HGF）在角膜上皮细胞凋亡的预防中的作用，并确定在这一过程中的信号转导。用无营养耗尽培养基（ExM）或staurosporine （20-100 ng/mL）或钙离子载体A23187 （0.5 muM）诱导人和兔角膜上皮细胞（HCE和RCE）凋亡。通过琼脂糖凝胶DNA片段化和Hoechst染色鉴定凋亡细胞。采用Akt-1活性过表达（Akt-1 pUSEamp cDNA）和Akt mRNA特异性小干扰RNA （siRNA）。免疫荧光、Western免疫印迹和Akt激酶检测。Staurosporine、ExM和A23187诱导HCE和RCE细胞DNA断裂。HGF （20 ng/mL）与凋亡药物联合使用可显著减少DNA分解和hoechst阳性细胞的数量。在磷脂酰肌醇-3激酶（PI-3K）抑制剂（wortmannin和LY294002）存在的情况下，HGF不能克服细胞凋亡。然而，ERK1/2级联通路抑制剂PD98059在阻止HGF保护方面无效。HGF诱导了Akt-1的持续激活，活性Akt-1的过表达减少了细胞凋亡。HGF刺激Akt、糖原合成酶激酶（GSK-3）和Bad （Bcl-2家族的促凋亡成员）的下游靶点，这种作用被PI-3K抑制剂阻断，而不被ERK1/2抑制。通过Akt siRNA抑制Akt的表达导致Bad和GSK-3磷酸化水平降低。在诱导细胞凋亡时，HGF可阻止Bad向线粒体的易位，这是细胞凋亡的关键阶段。此外，在过表达活性Akt-1的上皮细胞中，Bad易位也被阻止。HGF调节角膜上皮细胞中的多种信号级联反应。结果表明，HGF以旁分泌方式通过PI-3K/Akt/Bad通路保护细胞免于凋亡，但不通过ERK1/2通路。GSK-3是PI-3K/Akt-1的靶标。
Purpose. Cell survival is critical during corneal epithelial regeneration after injury, and growth factors could be fundamental in cytoprotection. The goal of this study was to investigate the involvement of the paracrine hepatocyte growth factor (HGF) in the prevention of corneal epithelial cell apoptosis and to identify signal transducers in this process.Methods. Apoptosis in human and rabbit corneal epithelial (HCE and RCE) cells was induced with a nutrient-deprived exhausted medium (ExM) or by treatment with staurosporine (20-100 ng/mL) or the calcium ionophore A23187 (0.5 muM). Apoptotic cells were identified by DNA fragmentation in agarose gels and by Hoechst staining. Active Akt-1 overexpression (Akt-1 pUSEamp cDNA) and small interfering RNA (siRNA) specific for Akt mRNA were used. Immunofluorescence, Western immunoblot analysis, and Akt kinase assays were also used.Results. Staurosporine, ExM, and A23187 induced DNA fragmentation in HCE and RCE cells. HGF (20 ng/mL) in combination with the apoptotic agents greatly reduced DNA breakdown and the number of Hoechst-positive cells. In the presence of phosphatidylinositol-3 kinase (PI-3K) inhibitors (wortmannin and LY294002), HGF did not overcome apoptosis. However, PD98059, the ERK1/2 cascade pathway inhibitor, was ineffective in preventing HGF protection. HGF induced a sustained activation of Akt-1, and overexpression of active Akt-1 reduced apoptosis. HGF stimulated the downstream targets of Akt, glycogen synthase kinase (GSK-3), and Bad, a proapoptotic member of the Bcl-2 family, an effect that was blocked by PI-3K inhibitors but not by ERK1/2 inhibition. Suppressing the expression of Akt by Akt siRNA led to a decrease in the phosphorylation of Bad and GSK-3. Translocation of Bad to the mitochondria, a critical stage in apoptosis, was prevented by HGF when apoptosis was induced. Moreover, in epithelial cells overexpressing active Akt-1, Bad translocation was also prevented.Conclusions. HGF modulates multiple signaling cascades in corneal epithelial cells. The results demonstrated that HGF, in a paracrine fashion, protects cells from apoptosis through a PI-3K/Akt/Bad pathway but not through an ERK1/2 pathway. It was also demonstrated that GSK-3 is a target of PI-3K/Akt-1.