Cultured mouse thioglycolate-elicited peritoneal macrophages exhibit a strong block to transcriptional elongation beyond the end of the c-fos gene first exon. This block is absent in freshly isolated peritoneal cells, appears slowly during culture, and does not require adherence of the cells. The extent of this block is largely responsible for the levels of c-fos mRNA in cultured macrophages, even after modulation by agents such as the tumor promoter phorbol myristate acetate and increased intracellular cyclic AMP, which also increase the activity of the c-fos promoter. When macrophages are cultured in the absence of mobilizable calcium, the block can no longer be relieved by any inducing agent. Conversely, upon calcium influxes, there is little alteration in the level of transcriptional initiation, but transcription proceeds efficiently through the entire c-fos locus. These results suggest the presence of an intragenic calcium-responsive element in the c-fos gene and illustrate its key role in the control of c-fos gene transcription.The c-fos gene appears to constitute a master switch which is central for cell proliferation and differentiation in a wide variety of cell types. Regulation of its expression is very complex and involves both transcriptional and posttranscriptional mechanisms (for reviews, see references 18, 32, 35, 42, 46, and 47). Transcriptional control itself occurs at the levels of both initiation (13, 17, 19-21, 24, 33, 45) and elongation (5,6,16,43,44).In cultured macrophages, transcription of the c-fos gene can be induced by the stimulation of several potentially different second messenger pathways which control the functional activity of the cells (8,10,11,23,36). For instance, in a number of cell types, including macrophages, transcription of c-fos is strongly induced by the calcium ionophore A23187 (7, 8, 13, 36, 40). Recently, we have shown that mobilization of calcium from intracellular sources is required for induction of c-fos gene transcription in mouse thioglycolate-elicited (TG) peritoneal macrophages by a number of different inducing agents (10). This observation was surprising, considering that different upstream promoter elements respond, for instance, to phorbol 12-myristate 13-acetate (PMA) or increased intracellular cyclic AMP (cAMP) (4,13,14,17,38,40,41) and that induction of transcription by glucocorticoids is not known to require calcium. Macrophage activation thus seems an interesting model with which to study relationships between external signals, second messengers, and the complex regulation of c-fos expression.In this report, we show that cultured mouse inflammatory peritoneal macrophages display a strong block to transcriptional elongation beyond the end of the c-fos first exon. Induction of c-fos transcription by a number of different * Corresponding author. agents is in all cases accompanied by a relief of the elongation block with or without an additional increase in promoter activity. Furthermore, our previous observation that mobilizable intracellular calcium ...

No abstract

In numerous studies on mammary epithelial cell lines multiple factors, added to the medium or contained in the serum, were required for casein gene expression. It has been shown in these systems that the mammary gland factor (MGF) is implicated in the activation of the ␤-casein gene promoter. In the present study, we determined the relationship between known agents that affect casein gene expression and MGF activity using the properties of rabbit primary mammary epithelial cells to respond to PRL alone, when cultured in chemically defined medium. We demonstrate that MGF is rapidly activated by PRL alone or by human growth hormone, a natural ligand of many PRL receptors (PRL-Rs), in the cytoplasm and accumulated in the nucleus. The MGF activation by PRL occurred in the absence of endogenous extracellular matrix, a condition where casein synthesis is known to be markedly reduced. Different inhibitors of protein-tyrosine kinases, which have been shown to reduce casein mRNA synthesis, but not of protein kinase C, decrease the MGF activity. A tyrosine phosphatase inhibitor, sodium pervanadate, induced two GAS-binding complexes related to MGF and STAT1. Our data show that MGF is a latent cytoplasmic factor rapidly activated in mammary epithelial cells, by a mechanism involving a tyrosine kinase and a tyrosine phosphatase.

In many cell types, increased intracellular calcium gives rise to a robust induction of c-fos gene expression. Here we show that in mouse Ltk ؊ fibroblasts, calcium ionophore acts in synergy with either cAMP or PMA to strongly induce the endogenous c-fos gene. Run-on analysis shows that this corresponds to a substantial increase in active polymerases on downstream gene sequences, i.e. relief of an elongation block by calcium. Correspondingly a chimeric gene, in which the human metallothionein promoter is fused to the fos gene, is strongly induced by ionophore alone, unlike a c-fos promoter/␤-globin coding unit chimeric construct. Internal deletions in the hMT-fos reporter localize the intragenic calcium regulatory element to the 5 portion of intron 1, thereby confirming and extending previous in vitro mapping data. Ionophore induced cAMP response element-binding protein phosphorylation on Ser 133 without affecting the extracellular signal-regulated kinase cascade. Surprisingly, induction involved neither CaM-Ks nor calcineurin, while the calmodulin antagonist W7 activated c-fos transcription on its own. These data suggest that a novel calcium signaling pathway mediates intragenic regulation of c-fos expression via suppression of a transcriptional pause site.