c-fos sequence necessary for basal expression and induction by epidermal growth factor, 12-O-tetradecanoyl phorbol-13-acetate and the calcium ionophore.

We have investigated the sequence requirements for induction of the human c-fos gene by epidermal growth factor (EGF), 12-O-tetradecanoyl-13-acetate (TPA), and the calcium ionophore A23187 by transfecting c-fos promoter mutants into HeLa and A431 cells. Induction by both EGF and TPA in HeLa cells required the presence of the c-fos enhancer located at -317 to -298 relative to the mRNA cap site. A23187, however, did not induce expression of the transfected gene, even though it strongly induced expression of the endogenous gene, suggesting that it has different requirements for induction than do EGF and TPA. We have also investigated the role of promoter sequences downstream of the enhancer in general expression and induction of c-fos. A sequence between -97 and -76, which includes an 8-base-pair perfect direct repeat, was needed for efficient general expression but not for induction of the gene. A factor in nuclear extracts that bound specifically to this sequence was detected by a gel mobility shift assay. A 7-base-pair sequence, located between -63 and -57 relative to the mRNA cap site and previously shown to be important for general expression of mouse c-fos, was also important for general expression of the human gene. In addition, this element was important for inducibility by EGF and TPA, since induction was significantly reduced when internal deletion mutants that retained the enhancer but lacked the -63 to -57 sequence element were analyzed in transfecting assays.     

Molecular cloning and sequence analysis of the promoter region of mouse cyclin D1 gene: implication in phorbol ester-induced tumour promotion

Cyclin D1 is a cell cycle regulatory protein, which acts as a growth factor sensor to integrate extracellular signals with the cell cycle machinery, particularly during G1 phase of the cell cycle. Previous study using promotion-sensitive JB6 mouse epidermal cells, an in vitro model of the promotion stage of multistage carcinogenesis, showed that the expression of cyclin D1 is stimulated in the presence (but not in the absence) of 12-O-tetradecanoylphorbol-13-acetate (TPA) in these cells maintained under anchorage-independent culture conditions. In the present study, to explore the molecular basis of this observation, the promoter region of mouse cyclin D1 gene was cloned and sequenced (GenBank accession number AF212040). Dot matrix comparison of mouse, human and rat promoter sequences indicated that the mouse promoter is homologous to the human and more so to the rat promoters. The mouse promoter, like human and rat promoters, lacks canonical TATA-box or TATA-like sequence, but it has one or possibly two initiator (Inr) or Inr-like sequences. Energy dot plot analysis predicted that the mouse promoter consists of three domains: (1) the 3' domain contains NF-kappaB response element, cAMP-response element (CRE), Inr or Inr-like elements, Sp1 binding site and Oct 1 (2) the middle domain contains another Sp1 binding site, E-box and E2F binding site and (3) the 5' domain contains TPA-response element (TRE) and a tandem silencer element. The cyclin D1 promoter sequence of either promotion-sensitive or resistant JB6 mouse epidermal cells was, except for a few minor differences, essentially identical to the sequence determined for a mouse genomic clone. Since TPA is capable of stimulating the expression of cyclin D1 not only through TRE but also through CRE and NF-kappaB response element in the promoter, we tentatively propose a sequence of events that possibly leads to TPA-induced, anchorage-independent synthesis of cyclins D1 and A in the promotion-sensitive JB6 mouse epidermal cells.     

Transcriptional regulation of early growth response genes in FOS-expressing PC-12 cells.

Deregulated c-fos expression in the rat pheochromocytoma cell line, PC-12, causes pronounced downregulation of nerve growth factor (NGF)-induced c-fos and c-jun activation, accompanied by a block in NGF-induced differentiation of PC-12 cells. The FOS-expressing PC-12 cells were exposed to diverse agents such as NGF, epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6), dibutyryl cyclic adenosine 3',5' monophosphate (db cAMP), and Ca-ionophore; and the expression of egr-1, c-fos, c-jun, jun-B, and jun-D was analyzed. Pronounced downregulation of c-fos, c-jun, and--to a lesser extent--jun-B was observed on treatment with NGF, bFGF, db cAMP, and Ca-ionophore, whereas EGF-induced activation of these early response genes was not inhibited in FOS-expressing PC-12 cells. Ca-ionophore- and db cAMP-induced egr-1 activation in PC-12 fos cells was completely inhibited. Both parent and PC-12 fos cells expressed similar high basal levels of jun-D, whose expression was the least regulatable by all of these agents. Transfection of fos promoter-chloramphenicol acetyltransferase (promoter-CAT) plasmid into these stable FOS-expressing PC-12 cells revealed that these effects are exerted at the fos promoter level.