Promotive effect of neuropilin-1 and vascular endothelial growth factor on the proliferation of HaCaT cells

Abstract

Objective To explore the possible role of neuropilin-1 (NRP-1) and extracellular regulated protein kinases (ERK) in the mechanisms underlying the promotive effect of vascular endothelial growth factor (VEGF) on the proliferation of HaCaT cells.Methods HaCaT cells were cultured in vitro and transfected with a NRP-1 expression plasmid EX-O0008-M02.Reverse transcription (RT)-PCR and Western blot were performed to detect the mRNA and protein expression of NRP-1 in HaCaT cells respectively before and after the transfection.Some HaCaT cells were divided into three groups to be transfected with liposome (liposome control group),control plasmid pReceiver-M02 (plasmid control group),and objective plasmid EX-O0008-M02 (objective plasmid group),respectively,and each of the three groups was classified into several subgroups to be treated with phosphate buffer solution (PBS),U0126 (MEK1/2 inhibitor) and VEGF alone or in combination.After additional culture,methyl thiazolyl tetrazolium (MTT) assay was performed to determine the proliferative activity of HaCaT cells,Western blot to quantify the expression of total and phosphorylated ERK1/2 as well as proliferating cell nuclear antigen (PCNA) protein in HaCaT cells.Intergroup differences were assessed by one-way analysis of variance (ANOVA),and multiple comparisons and correction were done by using the least significant difference (LSD) test.Results RT-PCR and Western blot analysis confirmed that the transfection with NRP-1 effectively promoted the mRNA and protein expression of NRP-1 in HaCaT cells.A significant increase was observed in cellular proliferative activity (absorbence value at 570 nm) in the objective plasmid group compared with the liposome control group and plasmid control group (0.88 ± 0.14 vs.0.63 ± 0.07 and 0.62 ± 0.13,F =8.755,P ＜ 0.05),also in the VEGF-stimulated objective plasmid group compared with the VEGF-stimulated plasmid control group (1.14 ± 0.18 vs.0.88 ± 0.10,F =4.591,P ＜ 0.05).The U0126 pretreatment markedly suppressed the VEGF-induced proliferation of A375 cells in the objective plasmid group (0.50 ± 0.13 vs.1.14 ± 0.18,F =42.106,P ＜ 0.01).As Western blot analysis suggested,the objective plasmid significantly enhanced the VEGF-induced increase in ERK1/2 phosphorylation degree and PCNA expression intensity in HaCaT cells compared with the control plasmid,but the enhancing effect of objective plasmid was effectively inhibited by U0126.Conclusion The activation of ERK1/2 signaling pathway may play a key role in the NRP-1 protein-mediated promotive effect of VEGF on the proliferation of HaCaT cells. Key words: Neuropilin-1; Keratinocytes; Vascular endothelial growth factors; Diabetic foot