Department of Endocrinology, First Hospital of Harbin Medical University, Harbin, Hei Long Jiang Province 150001, PR China.
Biochem Biophys Res Commun. 2010 Jul 30;398(3):389-94. doi: 10.1016/j.bbrc.2010.06.078. Epub 2010 Jul 1.
Pancreatic ductal epithelial cells (PDECs) were induced to differentiate into insulin-producing cells by hepatocyte growth factor (HGF) in our previous study, but the mechanism through which this induction occurs is still unknown. HGF is a ligand that activates a tyrosine kinase encoded by the c-Met proto-oncogene. This activation is followed by indirect activation of multiple downstream signal transduction pathways (including MAPKs and the PI3K/AKT signaling pathways) that initiate various biological effects. Therefore, we speculated that the differentiation of PDECs is through either the MAPK signaling pathway or the PI3K/AKT signaling pathway. To test this hypothesis, isolated PDECs from adult rats were stimulated by adding HGF to their medium for 28days. Then, the expression levels of several protein kinases, including MAPKs (ERK1/2, p38, and JNK) and AKT, were determined by Western blotting to determine if specific protein kinases are activated in these pathways. Subsequently, re-isolated from adult rats and cultured PDECs were pre-treated with specific inhibitors of proteins shown to be activated in these signaling pathways; these cells were then induced to differentiate by the addition of HGF. The expression levels of protein kinases were determined by Western blotting, and the differentiation rate of insulin-positive cells was determined by flow cytometry. The change of PDEC differentiation rates were compared between the groups in which cells with or without inhibitors pretreatment to determine the specific signaling pathway(s) that may be involved in HGF-induced differentiation of PDECs. After isolating PDECs and stimulating them with HGF for 28days, the expression levels of phosphorylated ERK1/2 as well as total and phosphorylated AKT of cultured cells were significantly increased compared to the normal control group (p<0.05), suggesting that the signaling pathways involving ERK1/2 and Akt (MEK-ERK and PI3K-AKT) are activated during HGF-induced PDEC differentiation. MEK1/2 or PI3K inhibitors were separately added to the culture medium of PDECs pre-treated with HGF. These results show that compared to the HGF-treated group, the differentiation rate of insulin-positive cells was significantly decreased in the HGF/LY294002 (PI3K inhibitor) group (13.47+/-1.57% vs. 33.47+/-1.34%, p<0.05); however, the differentiation rate of insulin-positive cells was not significantly different in the HGF/PD98059 (MEK1/2 inhibitor) group. These data suggest that HGF induces PDECs to differentiate into insulin-producing cells through the PI3K/AKT signaling pathway.
在我们之前的研究中，肝细胞生长因子 (HGF) 可诱导胰腺导管上皮细胞 (PDECs) 分化为胰岛素分泌细胞，但这种诱导发生的机制尚不清楚。HGF 是一种配体，可激活原癌基因 c-Met 编码的酪氨酸激酶。这种激活随后会间接激活多个下游信号转导途径（包括 MAPK 和 PI3K/AKT 信号通路），从而引发各种生物学效应。因此，我们推测 PDECs 的分化是通过 MAPK 信号通路或 PI3K/AKT 信号通路实现的。为了验证这一假设，我们从成年大鼠中分离 PDECs，并用 HGF 刺激它们的培养基 28 天。然后，通过 Western blot 测定几种蛋白激酶（包括 MAPKs（ERK1/2、p38 和 JNK）和 AKT）的表达水平，以确定这些途径中是否有特定的蛋白激酶被激活。随后，从成年大鼠中重新分离并培养 PDECs，并用已证明在这些信号通路中被激活的蛋白的特异性抑制剂预处理这些细胞，然后再加入 HGF 诱导它们分化。通过 Western blot 测定蛋白激酶的表达水平，并通过流式细胞术测定胰岛素阳性细胞的分化率。将有无抑制剂预处理的细胞组之间的 PDEC 分化率变化进行比较，以确定可能参与 HGF 诱导的 PDECs 分化的特定信号通路。从成年大鼠中分离 PDECs 并刺激它们用 HGF 刺激 28 天后，与正常对照组相比，培养细胞中磷酸化 ERK1/2 以及总 AKT 和磷酸化 AKT 的表达水平均显著增加（p<0.05），提示在 HGF 诱导的 PDEC 分化过程中涉及 ERK1/2 和 Akt（MEK-ERK 和 PI3K-AKT）的信号通路被激活。分别将 MEK1/2 或 PI3K 抑制剂添加到用 HGF 预处理的 PDEC 的培养基中。这些结果表明，与 HGF 处理组相比，在 HGF/LY294002（PI3K 抑制剂）组中，胰岛素阳性细胞的分化率显著降低（13.47+/-1.57%对 33.47+/-1.34%，p<0.05）；然而，在 HGF/PD98059（MEK1/2 抑制剂）组中，胰岛素阳性细胞的分化率没有显著差异。这些数据表明，HGF 通过 PI3K/AKT 信号通路诱导 PDECs 分化为胰岛素分泌细胞。
