Although it is known that hormones, growth factors and integrin promote hepatocyte proliferation in liver regeneration (LR) through ERK1/2 signalling pathway, reports about regulating processes of its intracellular paths in hepatocytes of LR are limited. This study aims at exploring which paths of ERK1/2 signalling pathway participate in the regulation of rat LR, especially in hepatocyte proliferation, and how they do so. In all, 14 paths and 165 genes are known to be involved in ERK1/2 signalling pathway. Of them, 161 genes are included in Rat Genome 230 2.0 Array. This array was used to detect expression changes of genes related to ERK1/2 signalling pathway in isolated hepatocytes of rat LR, showing that 60 genes were related to hepatocytes of LR. In addition, bioinformatics and systems biology methods were used to analyse the roles of 14 above paths in regenerating hepatocytes. We found that three paths, RTK→SHC→GRB2/SOS→RAS→RAF, IntegrinΒ→FAK→RAC→PAK→RAF and GΒγ→PI3KΒγ→RAC→PAK→RAF, promoted the G1 phase progression of hepatocytes by activating ERK1/2. A further four paths, Gq→PLCΒ→PKC→SRC/PYK2→GRB2/SOS→RAS→RAF, RTK→PLCγ→PKC→SRC/PYK2→GRB2/SOS→RAS→RAF, IntegrinΒ→FAK/SRC→GRB2/SOS→RAS→RAF and IntegrinΒ→FAK→RAC→PAK→RAF, advanced the cell progression of S phase and G(2)/M checkpoint by activating ERK1/2, and so did PP1/2→Mek1/2 by decreasing the negative influence on ERK1/2. At the late phase of LR, Gαs→AC→EPAC→Rap1→Raf blocked hepatocyte proliferation by decreasing the activity of ERK1/2 and so did PP1/2→Mek1/2. In summary, 60 genes and 8 paths of ERK1/2 signalling pathway regulated hepatocyte proliferation in rat LR.
Serine peptidase inhibitor, Kazal type 3 (SPINK3) is a trypsin inhibitor, and also a growth factor that has an identical structure to epidermal growth factor (EGF), which could combine with epidermal growth factor receptor (EGFR) to promote cell proliferation. To shed light on the role and regulation mechanism of SPINK3 in rat liver regeneration (LR), Rat Genome 230 2.0 assay was used to detect the expression profiles of LR genes after partial hepatectomy (PH). The results showed that Spink3 was significantly up-regulated at 2-24 h and 72-168 h after PH. In the present study, RT-PCR and immunoblotting were used to validate the assay results. Ingenuity Pathway Analysis 9.0 (IPA) software was used to build the SPINK3 signaling regulating LR and analyze the possible mechanism. And then the expression of cell proliferation-associated gene Ccna2 was examined by RT-PCR in normal rat liver cell line BRL-3A in which Spink3 was overexpressed. The results showed that Ccna2 was significantly up-regulated in BRL-3A in which Spink3 was over-expressed. SPINK3 combining with EGFR accelerated cell proliferation during rat liver regeneration via P38, PKC, JAK-STAT and AKT pathways. Thus, SPINK3 was likely to promote hepatocytes proliferation in LR through P38, PKC, JAK-STAT and AKT.
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