Existence of different Fos/Jun complexes during the G0-to-G1 transition and during exponential growth in mouse fibroblasts: differential role of Fos proteins.

We have determined the different Fos/Jun complexes present in Swiss 3T3 cells either following serum stimulation of quiescent cells or during exponential growth by immunoprecipitation analyses. We have shown that while c-Fos is the major Fos protein associated with the Jun proteins (c-Jun, JunB, and JunD) soon after serum stimulation, at later times Fra-1 and Fra-2 are the predominant Fos proteins associated with the different Jun proteins. During exponential growth, the synthesis of Fra-1 and Fra-2 is maintained at a significant level, in contrast to c-Fos and FosB, which are expressed at very low or undetectable levels. Consequently, Fra-1 and Fra-2 are the main Fos proteins complexed with the Jun proteins in asynchronously growing cells. To determine whether the Fos proteins are differentially required during the G0-to-G1 transition and exponential growth for the entrance into S phase, we microinjected affinity-purified antibodies directed against c-Fos, FosB, Fra-1, and Fra-2. We have found that while the activities of c-Fos and FosB are required mostly during the G0-to-G1 transition, Fra-1 and Fra-2 are involved both in the G0-to-G1 transition and in asynchronous growth.     

Signal transductions induced by bone morphogenetic protein-2 and transforming growth factor-beta in normal human osteoblastic cells

Transforming growth factor beta (TGF-beta) activates Ras/MAPK signaling in many cell types. Because TGF-beta and BMP-2 exert similar effects, we examined if this signaling is stimulated by both factors and analyzed the relationship between this signaling and the Smads in osteoblasts. BMP-2 and TGF-beta stimulated Ras, MAPK, and AP-1 activities. The DNA binding activities of c-Fos, FosB/Delta FosB, Fra-1, Fra-2, and JunB were up-regulated whereas JunD activity was decreased. c-Fos, FosB/Delta FosB, and JunB were associated with Smad4. The stimulation of AP-1 by BMP-2 and TGF-beta was dependent on Smad signaling, and anti-Smad4 antibody interfered with AP-1 activity. Thus, BMP-2 and TGF-beta activate both Ras/MAPK/AP-1 and Smad signaling in osteoblasts with Smads modulating AP-1 activity. To determine the roles of MAPK in BMP-2 and TGF-beta function, we analyzed the effect of ERK and p38 inhibitors on the regulation of bone matrix protein expression and JunB and JunD levels by these two factors. ERK and p38 mediated TGF-beta suppression of osteocalcin and JunD as well as stimulation of JunB. p38 was essential in BMP-2 up-regulation of type I collagen, fibronectin, osteopontin, osteocalcin, and alkaline phosphatase activity whereas ERK mediated BMP-2 stimulation of fibronectin and osteopontin. Thus, ERK and p38 differentially mediate TGF-beta and BMP-2 function in osteoblasts.     

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.     

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.     

Expression of different Jun and Fos proteins during the G0-to-G1 transition in mouse fibroblasts: in vitro and in vivo associations.

We have characterized the expression of c-Jun, JunB, JunD, c-Fos, and FosB proteins following serum stimulation of quiescent Swiss 3T3 cells by immunoprecipitation analyses. The synthesis of the three Jun proteins rapidly increases following stimulation, remaining at a significant level for at least 8 h. JunB protein presents the highest expression of all. FosB, like c-Fos, is transiently induced. Pulse-chase experiments show that all of the proteins except JunD are short-lived. We have shown that c-Fos and FosB form complexes in vivo with the different Jun proteins and that JunB complexes are predominant. In vitro association and competition experiments show that the affinities between the different Fos and Jun proteins are similar. This finding, together with the in vivo observations described above, suggests that the proportion of the different Jun/Fos heterodimers is governed by the concentration of the different components. The Fos and Jun proteins are phosphoproteins, and some remain relatively highly phosphorylated in their heterodimeric form.