The M(r) 78,000
glucose-regulated
protein (
GRP78) can be induced by physiological stresses such as
glucose deprivation and
hypoxia. In solid
tumors,
hypoxia can promote malignant progression and confer resistance to irradiation and
chemotherapy by altering gene expression. Here, we investigated the molecular mechanisms and signaling pathway involved in the late and prolonged induction of the
GRP78 gene by
hypoxia in a human
gastric cancer cell line, MKN28. Nuclear run-on assays and mRNA stability measurements revealed that transcriptional activation, not stabilization of
mRNA, contributed to the dramatic induction of
GRP78 gene under
hypoxia. Induction of
GRP78 by chronic
hypoxia was completely abolished by pretreatment with
PD98059 [a specific inhibitor of
mitogen-activated
protein/
extracellular signal-regulated kinase (ERK)
kinase (MEK1)] or by overexpression of a dominant-negative MEK1 mutant, demonstrating a direct involvement of ERK in the induction of transcription at the
GRP78 promoter under these conditions. Furthermore,
hypoxia increased the transcriptional activity of a 12-O-tetradecanoylphorbol-13-acetate response element-like motif on the
GRP78 promoter and increased the abundance and
DNA binding activity of
AP-1 complex composed of c-Jun and c-Fos. A selective
protein kinase C (PKC) inhibitor,
GF109203X, inhibited the induction of
GRP78 gene expression as well as the activities of both ERK and Raf-1. Among six PKC
isoforms expressed in MKN28 cells,
PKC-epsilon expression level and
kinase activity were increased by
hypoxia. Transfection of MKN28 cells with a dominant-negative
PKC-epsilon blocked the induction of
GRP78 through ERK by
hypoxia, indicating that
PKC-epsilon directly participated in
GRP78 induction under
hypoxia. Taken together, this study shows that a PKC-epsilon-Raf-1-MEK-ERK-AP1 signaling cascade acts on a 12-O-tetradecanoylphorbol-13-acetate response element-like
element to mediate
hypoxia-induced
GRP78 expression in human
gastric cancer cells. We also confirmed in vivo the overexpression of
GRP78 in surgical specimens of human primary gastric
tumors.