Regulation of vascular endothelial growth factor expression by insulin-like growth factor-II in human keratinocytes, differential involvement of mitogen-activated protein kinases and feedback inhibition of protein kinase C.

Abstract

Vascular endothelial growth factor (VEGF) is overexpressed in hyperproliferative diseases such as psoriasis and cancer, which are characterized by an increased angiogenesis. It was reported that insulin-like growth factor (IGF)-II is highly expressed during hepatocarcinogenesis and is increased in psoriatic lesions. The increase in IGF-II is believed to be associated with the pathogenesis of these diseases by increasing angiogenesis. In order to investigate the relationship between IGF-II and angiogenesis-related VEGF, VEGF expression in the IGF-II-treated human keratinocytes was monitored and the IGF-II signalling pathways were examined with respect to VEGF expression. Northern blot analysis for the VEGF mRNA levels and an enzyme-linked immunosorbent assay for the VEGF protein were performed to determine if IGF-II (100 ng mL(-1)) can increase the VEGF expression levels with or without a pretreatment with protein inhibitors in primary normal human keratinocytes and HaCaT cells. The mRNA and protein levels of VEGF by IGF-II were increased in a time-dependent manner and reached the maximum level 2 h and 8 h after the IGF-II treatment, respectively. However, this increase was abrogated by pretreatment with an extracellular signal-regulated kinase (ERK) inhibitor but not by a p38 inhibitor. The IGF-II-mediated VEGF induction was also effectively inhibited by a pretreatment with the tyrosine kinase inhibitor and Src inhibitor. The PI3-kinase inhibitor also inhibited the expression of VEGF by IGF-II. However, the phospholipase C (PLC) and protein kinase C (PKC) inhibitors did not block the increases of VEGF mRNA level and its protein level by IGF-II, and the PKC inhibitor instead increased VEGF expression by IGF-II. These results suggest that the tyrosine kinase-Src-ERK1/2 pathway and the PI3-kinase pathway are involved in IGF-II-mediated VEGF expression, but PKC is negatively associated in the IGF-II-mediated VEGF expression.