During human melanoma progression AP-1 binding pairs are altered with loss of c-Jun in vitro

We demonstrated previously that c-Jun, JunB and c-Fos RNA were dysregulated in metastatic melanoma cells compared with normal human melanocytes. The purpose of this study was to evaluate the distribution in composition of AP-1 dimers in human melanoma pathogenesis. We investigated AP-1 dimer pairing in radial growth phase-like (RGP) (w3211) and vertical growth phase-like (VGP) (w1205) human melanoma cells and metastatic cell lines (cloned from patients, c83-2c, c81-46A, A375, respectively) compared with melanocytes using electrophoretic mobility shift assay (EMSA), Western blot and transfection analyses. There are progressive variations in AP-1 composition in different melanoma cell lines compared with normal melanocytes, in which c-Jun, JunD and FosB were involved in AP-1 complexes. In w3211, c-Jun, JunD and Fra-1 were involved in AP-1 binding, while in w1205, overall AP-1 binding activity was decreased significantly and supershift binding was detected only with JunD antibodies. In metastatic c81-46A and A375 cells, only JunD was involved in AP-1 binding activity, but in a third (c83-2c) c-Jun, JunD and Fra-1 were present. Western blot evaluation detected c-Jun in melanocytes and w3211, but this component was decreased significantly or was not detectable in w1205, c81-46A and A375 cells. In contrast, JunD protein was elevated in c81-46A and c83-2c cells compared with melanocytes and RGP and VGP cell lines. Normal melanocytes and c83-2c cells (which have c-Jun involved in AP-1 binding), transfected with c-Jun antisense and treated with cisplatin, showed higher viability compared with untransfected cells, while in c81-46A cells (in which only JunD is detectable) no change in cell viability was observed following treatment with cisplatin and c-jun antisense transfection. A dominant-negative c-Jun mutant (TAM67) significantly increased the soft agar colony formation of w3211 and c83-2c cells. These results suggest that components of AP-1, especially c-Jun, may offer a new target for the prevention or treatment of human melanoma progression.     

Epidermal growth factor stimulate cell proliferation and inhibits prolactin secretion of the human decidual cells in culture

Epidermal growth factor (EGF) has been found to be mitogenic in a variety of tissues. We investigated the biological effect of EGF on early pregnant human decidua and the non-pregnant decidualized human endometrium in the primary cell culture. EGF had a stimulatory action on cell proliferation in the early pregnant decidual cells and an inhibitory effect on prolactin (PRL) secretion from the decidual cells. The addition of progesterone into culture medium suppressed cell proliferation of decidual cells, whereas it enhanced PRL secretion from decidua. The analysis of the specific receptor for EGF in the early pregnant decidua and non-pregnant decidualized endometrium revealed that both tissues had a single component EGF receptor with a dissociation constant of nM order. These results suggest that EGF may play a role in the growth and function of endometrial stromal cells.     

Localization of transcription factor AP-1 family proteins in ameloblast nuclei of the rat incisor.

We examined by immunocytochemistry the localization of the AP-1 family proteins c-Jun, JunB, JunD, c-Fos, FosB, Fra-1, and Fra-2 in rat incisor ameloblasts. Most of the antibodies against AP-1 family proteins, except for c-Fos-specific antibody, labeled ameloblast nuclei. The labeling intensity of the c-Jun, JunD, and Fra-2 antibodies was stronger than that of JunB, FosB, and Fra-1. Antibody reactivities of c-Jun, JunD, and Fra-2 were greatly enhanced during or after the transition zone. Furthermore, c-Jun antibodies labeled maturation ameloblasts in a cyclic pattern, which was correlated with ameloblast modulation. Disruption of ameloblast modulation by colchicine injection resulted in greatly decreased reactivity of the c-Jun antibody in the ameloblast nuclei of the maturation zone. Phospho-specific antibodies to c-Jun labeled ameloblast nuclei only weakly throughout the secretion, transition, and maturation zones. These results suggest that the stage-specific localization of AP-1 in ameloblasts is closely related to tooth enamel formation.     

Pleiotropic action of AP-1 in v-Src-transformed cells

The activation of AP-1 is a hallmark of cell transformation by tyrosine kinases. In this study, we characterize the role of AP-1 proteins in the transformation of chicken embryo fibroblasts (CEF) by v-Src. In normal CEF, the expression of a dominant negative mutant of c-Jun (TAM67) induced senescence. In contrast, three distinct phenotypes were observed when TAM67 was expressed in v-Src-transformed CEF. While senescent cells were also present, the inhibition of AP-1 caused apoptosis in a fraction of the v-Src-transformed cells. In addition, cells containing lipid-rich vesicles accumulated, suggesting that a subpopulation of the v-Src-transformed cells underwent differentiation in response to the inhibition of AP-1. JunD and Fra-2 were the main components of this factor, while c-Jun accounted for a minor fraction of AP-1 in v-Src-transformed CEF. The downregulation of c-Jun expression by short hairpin RNA (shRNA) induced senescence in normal and v-Src-transformed cells. In contrast, a high incidence of apoptosis was caused by the downregulation of JunD, suggesting that it is required for the survival of v-Src-transformed CEF. Levels of the p53 tumor suppressor were elevated under conditions of JunD inhibition. Repression of p53 by shRNA enhanced the survival and anchorage-independent proliferation of v-Src-transformed CEF with JunD/AP-1 inhibition. The inhibition of Fra-2 had no visible phenotype in normal CEF but caused the appearance of lipid-rich vesicles in v-Src-transformed CEF. Therefore, AP-1 facilitated transformation by acting as a survival factor, by inhibiting premature entry into senescence, and by blocking the differentiation of v-Src-transformed CEF.     

Helicobacter pylori and mitogen-activated protein kinases mediate activator protein-1 (AP-1) subcomponent protein expression and DNA-binding activity in gastric epithelial cells

Emerging evidence has suggested a critical role for activator protein-1 (AP)-1 in regulating various cellular functions. The goal of this study was to investigate the effects of Helicobacter pylori and mitogen-activated protein kinases (MAPK) on AP-1 subcomponents expression and AP-1 DNA-binding activity in gastric epithelial cells. We found that H. pylori infection resulted in a time- and dose-dependent increase in the expression of the proteins c-Jun, JunB, JunD, Fra-1, and c-Fos, which make up the major AP-1 DNA-binding proteins in AGS and MKN45 cells, while the expression levels of Fra-2 and FosB remained unchanged. Helicobacter pylori infection and MAPK inhibition altered AP-1 subcomponent protein expression and AP-1 DNA-binding activity, but did not change the overall subcomponent composition. Different clinical isolates of H. pylori showed various abilities to induce AP-1 DNA binding. Mutation of cagA, cagPAI, or vacA, and the nonphosphorylateable CagA mutant (cagA(EPISA)) resulted in less H. pylori-induced AP-1 DNA-binding activity, while mutation of the H. pylori flagella had no effect. extracellular signal-related kinase (ERK), p38, and c-Jun N-terminal kinase (JNK) each selectively regulated AP-1 subcomponent expression and DNA-binding activity. These results provide more insight into how H. pylori and MAPK modulate AP-1 subcomponents in gastric epithelial cells to alter the expression of downstream target genes and affect cellular functions.