Department of Pediatrics, Nagoya University Graduate School of Medicine, Nagoya, Aichi, Japan.
Mol Cell Biochem. 2013 Jul;379(1-2):37-42. doi: 10.1007/s11010-013-1624-8. Epub 2013 Mar 31.
It remains elusive whether crosstalk exists among mitochondrial Bax, caspases, and mitogen-activated protein kinases (MAPKs), and whether epidermal growth factor (EGF), which may activate MAPKs, affects ceramide-induced apoptosis through the crosstalk in renal proximal tubular cells (RPTCs). Effect of ceramide on expression of mitochondrial Bax and phosphorylated (p)-ERK, p38MAPK and JNK, that of MAPKs inhibition, and of EGF in the presence or absence of MAPKs inhibition on ceramide-induced apoptosis were examined in HK-2 cells. Apoptosis and expression of mitochondrial Bax and p-MAPKs were measured by Hoechst 33258 staining and Western blotting. C2-ceramide, but not dihydroC2-ceramide, inactive C2-ceramide, induced apoptosis at 24 h. C2-ceramide enhanced the mitochondrial Bax expression at 1 h, which was peaked at 3-6 h and decreased at 24 h, but remained increased, compared to control. An inhibitor of caspases, zVAD-fmk, ameliorated ceramide-induced apoptosis, suggesting a role of caspases for ceramide-induced apoptosis. C2-ceramide enhanced the expression of p-ERK and p-p38MAPK, but not p-JNK, at 1 h, which was increased till 24 h. An inhibitor of ERK, PD98059, or of p38MAPK, SB202190, failed to affect C2-ceramide-induced apoptosis. EGF, which enhanced the expression of p-ERK and p-p38MAPK but not p-JNK, ameliorated C2-ceramide-induced apoptosis without affecting mitochondrial Bax. Inhibition of ERK or p38MAPK failed to abolish the protective effect of EGF on C2-ceramide-induced apoptosis. Mitochondrial Bax and caspases, but not MAPKs, play a role for ceramide-induced apoptosis in RPTCs. EGF ameliorates ceramide-induced apoptosis in Bax- and MAPKs-independent pathways. The mechanism of ceramide-induced apoptosis and anti-apoptotic effect of EGF deserves further investigations.
线粒体 Bax、胱天蛋白酶和丝裂原活化蛋白激酶（MAPK）之间是否存在串扰，以及表皮生长因子（EGF）是否可能通过肾近端管状细胞（RPTC）中的串扰影响神经酰胺诱导的细胞凋亡，这仍然难以捉摸。在 HK-2 细胞中，研究了神经酰胺对线粒体 Bax 和磷酸化（p）-ERK、p38MAPK 和 JNK 表达的影响，MAPK 抑制剂的作用，以及 MAPK 抑制剂存在或不存在时 EGF 对神经酰胺诱导的细胞凋亡的影响。通过 Hoechst 33258 染色和 Western blot 检测细胞凋亡和线粒体 Bax 和 p-MAPKs 的表达。C2-神经酰胺，但不是二氢 C2-神经酰胺、无活性 C2-神经酰胺，在 24 小时诱导细胞凋亡。C2-神经酰胺在 1 小时增强线粒体 Bax 的表达，在 3-6 小时达到高峰，在 24 小时降低，但仍高于对照。胱天蛋白酶抑制剂 zVAD-fmk 改善了神经酰胺诱导的细胞凋亡，表明胱天蛋白酶在神经酰胺诱导的细胞凋亡中起作用。C2-神经酰胺在 1 小时增强 p-ERK 和 p-p38MAPK 的表达，但不增强 p-JNK 的表达，直至 24 小时。ERK 抑制剂 PD98059 或 p38MAPK 抑制剂 SB202190 均不能影响 C2-神经酰胺诱导的细胞凋亡。增强 p-ERK 和 p-p38MAPK 表达但不增强 p-JNK 表达的 EGF 改善了 C2-神经酰胺诱导的细胞凋亡，而不影响线粒体 Bax。ERK 或 p38MAPK 的抑制作用不能消除 EGF 对 C2-神经酰胺诱导的细胞凋亡的保护作用。线粒体 Bax 和胱天蛋白酶，而不是 MAPK，在 RPTC 中的神经酰胺诱导的细胞凋亡中起作用。EGF 通过 Bax 和 MAPK 不依赖的途径改善神经酰胺诱导的细胞凋亡。神经酰胺诱导的细胞凋亡的机制和 EGF 的抗凋亡作用值得进一步研究。
