Overexpression of Mos, Ras, Src, and Fos inhibits mouse mammary epithelial cell differentiation.

Mammary epithelial cells terminally differentiate in response to lactogenic hormones. We present evidence that oncoprotein overexpression is incompatible with this hormone-inducible differentiation and results in striking cellular morphological changes. In mammary epithelial cells in culture, lactogenic hormones (glucocorticoid and prolactin) activated a transfected beta-casein promoter and endogenous beta-casein gene expression. This response to lactogenic hormone treatment was paralleled by a decrease in cellular AP-1 DNA-binding activity. Expression of the mos, ras, or src (but not myc) oncogene blocked the activation of the beta-casein promoter induced by the lactogenic hormones and was associated with the maintenance of high levels of AP-1. Mos expression also increased c-fos and c-jun mRNA levels. Overexpression of Fos and Jun from transiently transfected constructs resulted in a functional inhibition of the glucocorticoid receptor in these mouse mammary epithelial cells. This finding clearly suggests that glucocorticoid receptor inhibition arising from oncogene expression will contribute to the block in hormonally induced mammary epithelial cell differentiation. Expression of Src resulted in the loss of the normal organization and morphological phenotype of mammary epithelial cells in the epithelial/fibroblastic line IM-2. Activation of a conditional c-fos/estrogen receptor gene encoding an estrogen-dependent Fos/estrogen receptor fusion protein also morphologically transformed mammary epithelial cells and inhibited initiation of mammary epithelial differentiation-associated expression of the beta-casein and WDNM 1 genes. In response to estrogen treatment, the cells displayed a high level of AP-1 DNA-binding activity. Our results demonstrate that high cellular AP-1 levels contribute to blocking the ability of mammary epithelial cells in culture to respond to lactogenic hormones. This and other studies indicate that the oncogene products Mos, Ras, and Src exert their effects, at least in part, by stimulating cellular Fos and probably cellular Jun activity.     

Inhibition of v-Mos kinase activity by protein kinase A.

We investigated the effect of cyclic AMP-dependent protein kinase (PKA ) on v-Mos kinase activity. Increase in PKA activity in vivo brought about either by forskolin treatment or by overexpression of PKA catalytic subunit resulted in a significant inhibition of v-Mos kinase activity. The purified PKA catalytic subunit was able to phosphorylate recombinant p37v-mos in vitro, suggesting that the mechanism of in vivo inhibition of v-Mos kinase involves direct phosphorylation by PKA. Combined tryptic phosphopeptide two-dimensional mapping analysis and in vitro mutagenesis studies indicated that Ser-56 is the major in vivo phosphorylation site on v-Mos. In vivo phosphorylation at Ser-56 correlated with slower migration of the v-Mos protein during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. However, even though Ser-56 was phosphorylated by PKA, this phosphorylation was not involved in the inhibition of v-Mos kinase. The alanine-for-serine substitution at residue 56 did not affect the ability of v-Mos to autophosphorylate in vitro or, more importantly, to activate MEK1 in transformed NIH 3T3 cells. We identified Ser-263 phosphorylation, the Ala-263 mutant of v-Mos was not inhibited by forskolin treatment. From our results, we propose that the known inhibitory role of PKA in the initiation of oocyte maturation in mice could be explained at least in part by its inhibition of Mos kinase.     

Interleukin-6 increases rat metalloproteinase-13 gene expression through stimulation of activator protein 1 transcription factor in cultured fibroblasts.

The role of IL-6 in collagen production and tissue remodeling is controversial. In Rat-1 fibroblasts, we measured the effect of IL-6 on matrix metalloproteinase-13 (MMP-13), c-jun, junB, and c-fos gene expression, binding of activator protein 1 (AP1) to DNA, amount of AP1 proteins, immunoreactive MMP-13 and TIMP-1 proteins, and Jun N-terminal kinase activity. We show that IL-6 increased MMP-13-mRNA and MMP-13 protein. These effects were exerted by acting on the AP1-binding site of the MMP-13 promoter, as shown by transfecting cells with reporter plasmids containing mutations in this element. Mobility shift assays demonstrated that IL-6 induced the DNA binding activity of AP1. This effect was accompanied by a marked increase in c-Jun, JunB, and c-Fos mRNA, as well as in c-Jun protein and its phosphorylated form. The latter is not due to increased Jun N-terminal kinase activity but to a decreased serine/threonine phosphatase activity. We conclude that IL-6 increases interstitial MMP-13 gene expression at the promoter level. This effect seems to be mediated by the induction of c-jun, junB, and c-fos gene expression, by the binding of AP1 to DNA, by increasing phosphorylated c-Jun, and by the inhibition of serine/threonine phosphatase activity. These effects of IL-6 might contribute to remodeling connective tissue.     

Identification of phorbol ester response elements in the promoter of Epstein-Barr virus putative lytic switch gene BZLF1

The product of the Epstein-Barr virus BZLF1 gene encodes a protein which is related to c-fos, it has been shown to bind specifically to a consensus AP-1 site, and its expression in latently Epstein-Barr virus-infected lymphocytes is sufficient to trigger the viral lytic cycle. We identified several elements within the BZLF1 promoter (Zp) which are responsive to the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), an inducer of the viral lytic cycle. These elements fall into two classes based on the factors which bind to these sequences and their resulting functional behavior. Four of the elements are homologous (ZI elements) and share homology to a protein-binding domain in the promoter region of the coordinately expressed BRLF1 gene. When cloned upstream of heterologous promoters, the ZI elements function as silencers which exhibit TPA-inducible enhancer activity. A distinct TPA-responsive element (ZII) is located near the TATA box and shares homology with the AP-1-binding site in the c-jun promoter. A synthetic oligonucleotide with a sequence corresponding to the ZII element effectively competes for binding of nuclear factors to the c-jun AP-1 site. Furthermore, we found that a complex of c-jun and c-fos bound to the ZII domain.     

Purification and identification of positive regulators binding to a novel element in the c-Jun promoter

A putative response element, GAGCCTC, was observed years ago in footprinting analysis of the c-jun promoter, and here we investigate its function in regulating c-jun expression and identify a protein complex that binds there. Electrophoretic mobility shift assays demonstrate a sequence-specific binding complex with this element in HEK293 cells. Additionally, unlabeled consensus AP-1 element DNA, but not a similar NF-jun element DNA, competes with complex formation. Mutations of this element decrease c-jun promoter reporter activity by nearly 5-fold in HEK293 cells. A new, two-step oligonucleotide trapping technique was developed to purify the element binding proteins. LC-nanospray-ESI-MS/MS identification and Western blotting show that the purified complex contains Ku80 and c-jun, which was further confirmed by antibody supershift, by immunoprecipitation with Southwestern blot or with UV cross-linking analysis in vitro as well as chromatin immunoprecipitation in vivo. c-Jun promoter activity and c-jun expression were decreased by Ku80 siRNA introduction. A mutant Ku80 plasmid with normal amino acid sequence but immune to the siRNA recovers c-jun promoter activity from siRNA inhibition. Similarly, Ku70 wild type transfection can also upregulate c-jun promoter activity. Thus, Ku80-c-jun activates c-jun expression by binding to this GAGCCTC element in the c-jun promoter and Ku70 may also serve a role.     

E5 proteins of human papillomavirus types 11 and 16 transactivate the c-fos promoter through the NF1 binding element.

Human papillomavirus type 11 (HPV-11) and HPV-16 contain an E5 gene that can induce c-fos gene expression in mouse fibroblasts. This study investigated the human c-fos promoter characteristics by mapping the c-fos promoter sequence with several deletion and point mutants that confer responsiveness to E5 of HPV-11 or HPV-16. The mutant studies show that NF1 binding sequences within the c-fos promoter were crucial for the induction of the c-fos gene by E5, and the gel shift assay study suggested that E5 of both HPV-11 and HPV-16 is associated, perhaps indirectly, with this NF1 element in the transactivation of the human c-fos promoter. Using an inducible system, we demonstrate that increased induction of the HPV-11 E5 gene in cells led to increased transactivation of the NF1 element. In addition, the transactivating activity of a series of HPV-11 E5 mutants on the NF1 element had a strong correlation with their respective transforming activities.     

Chronic Electroconvulsive Seizures Down–Regulate Expression of the Immediate-Early Genes c-fos and c-jun in Rat Cerebral Cortex

Acute seizures and other stimuli that increase neuronal activity cause a rapid induction of the immediate-early genes c-fos and c-jun, also referred to as nuclear proto-oncogenes, in the nervous system. In the present study, rats were administered one or more electroconvulsive seizures (ECS) and the responsiveness of c-fos and c-jun to an acute, "test" seizure was examined. Four hours after a single ECS, the induction of c-fos mRNA by a test seizure was blocked, in agreement with earlier findings, but by 18 h the levels of c-fos mRNA could be reinduced by the test seizure, suggesting that 1 day is sufficient to "reset" the responsiveness of this system. However, it was found that chronic, daily ECS treatments resulted in a time-dependent decrease in the expression of c-fos mRNA in response to a test seizure administered 18 h after the last daily ECS; this effect was maximal after 8-10 days of treatment, at which time the induction of c-fos mRNA by the test seizure was blocked dramatically. Chronic ECS also blocked the induction of c-jun in response to an acute, test seizure. The effect of chronic ECS on levels of Fos protein was also investigated. It was found that basal levels of Fos protein were reduced after chronic (10 days) ECS and were not induced by a test seizure. Because levels of Fos protein remain elevated 4 h after a single seizure this finding suggests that the mechanisms by which acute (4 h) and chronic (8-10 days) ECS block the induction of c-fos may differ.