Keratinocyte growth factor stimulates growth of MIA PaCa-2 cells through extracellular signal-regulated kinase phosphorylation

Abstract

Keratinocyte growth factor (KGF), also known as fibroblast growth factor-7, is mainly synthesized by mesenchymal cells. KGF modulates proliferation, differentiation, migration and adhesion to extracellular matrices of epithelial cells that specifically express the KGF receptor (KGFR). We previously reported that KGF is expressed in cancer cells and adjacent stromal fibroblasts in human pancreatic cancer tissues. Furthermore, KGF is thought to stimulate the growth of certain pancreatic cancer cell lines. The aim of the present study was to examine whether the mitogen-activated protein kinase (MAPK) pathway contributes to exogenous KGF-induced pancreatic cancer cell growth. Recombinant human KGF (rhKGF) was administered to MIA PaCa-2 cells, which expressed KGFR and negligible levels of KGF. Cell growth rates in MIA PaCa-2 cells were significantly increased in a dose-dependent manner following the addition of rhKGF. In the MAPK pathway, phosphorylation of extracellular signal-regulated kinase (ERK) in MIA PaCa-2 cells was increased in a dose-dependent manner, and phosphorylation of p38 was slightly increased following the administration of 100 ng/ml rhKGF. In contrast, JNK was not phosphorylated following the addition of rhKGF in MIA PaCa-2 cells. U0126, a specific inhibitor of ERK activation, decreased the rhKGF-induced phosphorylation of ERK and the growth rates of MIA PaCa-2 cells. These findings indicated that phosphorylation of the ERK signaling pathway plays a significant role in exogenous KGF-induced pancreatic cancer cell growth.

Figure 1

Cell growth assay of KGF-administered MIA PaCa-2 cells. Cells were cultured for 48 h in the presence or absence of 10 or 100 ng/ml rhKGF, and then incubated with WST-8 cell counting reagent. KGF-treated MIA PaCa-2 cells showed significantly higher growth rates, which occurred in a dose-dependent manner, than the untreated cells. ^**p<0.01.

Figure 2

Effects of rhKGF on the MAPK signaling pathway in MIA PaCa-2 cells. The phosphorylation of ERK was increased in a dose-dependent manner by the addition of rhKGF (top panel, p-ERK). p38 was moderately activated by the addition of 100 ng/ml rhKGF (third panel from top, p-p38), and JNK was not activated by the addition of rhKGF (fifth panel from top, p-JNK). The second, fourth, and bottom panels show the total ERK, total p38 and total JNK, respectively.

Figure 3

Effects of inhibition of the ERK signaling pathway on KGF-induced cell growth. (A) ERK phosphorylation by rhKGF was completely inhibited by pre-treatment with U0126. (B) Cell growth of rhKGF treated MIA PaCa-2 cells was significantly decreased when ERK activation was inhibited. ^*p<0.05.