The BH3-only protein BIM contributes to late-stage involution in the mouse mammary gland.

After cessation of lactation, involution of the mouse mammary gland proceeds in two distinct phases, a reversible and an irreversible one, which leads to the death and removal of alveolar cells. Cell death is preceded by the loss of STAT5 activity, which abrogates cell differentiation and gain of STAT3 activity. Despite early observations implicating BCL2 (B cell lymphoma 2) family proteins in this process, recent evidence suggests that STAT3-controlled cathepsin activity is most critical for cell death at the early stage of involution. Somewhat surprisingly, this cell death associates with but does not depend on the activation of pro-apoptotic effector caspases. However, transgenic overexpression of BCL2, that blocks caspase activation, delays involution while conditional deletion of BclX accelerates this process, suggesting that BCL2 family proteins are needed for the effective execution of involution. Here, we report on the transcriptional induction of multiple pro-apoptotic BCL2 family proteins of the 'BH3-only' subgroup during involution and the rate-limiting role of BIM in this process. Loss of Bim delayed epithelial cell clearance during involution after forced weaning in mice, whereas the absence of related Bmf had minor and loss of Bad or Noxa no impact on this process. Consistent with a contribution of BCL2 family proteins to the second wave of cell death during involution, loss of Bim reduced the number of apoptotic cells in this irreversible phase. Notably, the expression changes observed within the BCL2 family did not depend on STAT3 signalling, in line with its initiating role early in the process, but rather appear to result from relief of repression by STAT5. Our findings support the existence of a signalling circuitry regulating the irreversible phase of involution in mice by engaging BH3-only protein-driven mitochondrial apoptosis.

STAT-1 expression in human glioblastoma and peritumoral tissue.

BACKGROUND: Glioblastoma is a very aggressive brain tumour with poor prognosis despite radical surgery or radiotherapy. Signal transducers and activators of transcription (STAT) proteins are important elements in intracellular signalling and part of the JAK-STAT pathway. They are activated by growth factors and cytokines and translocate into the nucleus upon activation to exert their function as transcription factors. STAT-1 can be induced by interferons and has also been found to be important in sensitizing tumours to chemotherapeutic drugs. MATERIALS AND METHODS: Forty-six glioblastoma samples have been analysed for the expression of STAT-1 by immunohistochemistry. RESULTS: In our study performed by immunohistochemistry, 22 out of 46 glioblastomas (48%) were strongly positive for staining with a STAT-1 antibody, 9 (20%) showed an intermediate reactivity, 8 (17%) low immunoreactivity, and 7 (15%) were completely negative. In the tumour tissue, STAT-1 expression was mostly localized in the cytoplasm. This location of STAT-1 suggests the predominant presence of an inactive form of STAT-1. Tumour giant cells were frequently strongly stained. Part of the peritumoral brain tissue showed strongly positively reactive glial cells. Interestingly, within the infiltration area strong STAT-1 expression was found in reactive astrocytes, glia, and particularly in microglial components. CONCLUSION: The expression of STAT-1 in the majority of glioblastomas, together with its documented role in apoptosis and in the action of chemotherapeutic drugs on tumour cell lines point to a possible function of this protein in the response of glioblastomas to chemotherapy.

Expression level-dependent contribution of glucocorticoid receptor domains for functional interaction with STAT5.

The action of the glucocorticoid receptor (GR) on beta-casein gene transcription serves as a well-studied example of a case where the action of the GR is dependent on the activity of another transcription factor, STAT5. We have investigated the domain-requirement of the GR for this synergistic response in transfection experiments employing GR mutants and CV-1 or COS-7 cells. The results were influenced by the expression levels of the GR constructs. At low expression, STAT5-dependent transactivation by mutants of the GR DNA binding domain or N-terminal transactivation domain was impaired and the antiglucocorticoid RU486 exhibited a weak agonistic activity. When the N-terminal region of the GR was exchanged with the respective domain of the progesterone receptor, STAT5-dependent transactivation was reduced at low and high expression levels. Only at high expression levels did the GR exhibit the properties of a coactivator and enhanced STAT5 activity in the absence of a functional DNA binding domain and of GR binding sites in the proximal region of the beta-casein gene promoter. Furthermore, at high GR expression levels RU486 was nearly as efficient as dexamethasone in activating transcription via the STAT5 dependent beta-casein gene promoter. The results reconcile the controversial issue regarding the DNA binding-independent action of the GR together with STAT5 and provide evidence that the mode of action of the GR depends not only on the type of the particular promoter at which it acts but also on the concentration of the GR. GR DNA binding function appears to be mandatory for beta-casein gene expression in mammary epithelial cells, since the promoter function is completely dependent on the integrity of GR binding sites in the promoter.

Synergistic and antagonistic interactions of transcription factors in the regulation of milk protein gene expression. Mechanisms of cross-talk between signalling pathways.

The stage and tissue specific expression of milk protein genes in the mammary gland is controlled by modular response regions with multiple binding sites for distinct classes of transcription factors, which either co-operate or are antagonistic. In addition, the activity of some of these factors is individually control-led by diverse extracellular signals. A well studied paradigm for a synergistic co-operation is the activation of beta-casein gene transcription by prolactin and glucocorticoids mediated by the signal transducer and activator of transcription STAT5 and the glucocorticoid receptor (GR). As an example for an antagonistic interaction we can demonstrate inhibition of prolactin signalling by TNF-alpha, which is mediated by NF-kappa B. In both cases, the interactions occur at several levels: For GR and STAT5, the synergy is discussed to be promoted by protein-protein interactions. Furthermore, we can demonstrate a co-operation between GR and STAT5 in DNA binding by a mechanism, which is dependent on the integrity of the DNA binding domain of the GR and on the existence of half-palindromic GR binding sites in the hormone response region. Indirect effects of glucocorticoids by modulation of the expression of secondary genes are also important. They might account for the observed enhancement of prolactin induced tyrosine phosphorylation of STAT5 by glucocorticoids. For NF-kappa B and STAT5, one component of the antagonism is the inhibition of STAT5 tyrosine phosphorylation by activation of NF-kappa B. Another potential mechanism is the inhibition of DNA binding of STAT5 due to overlapping binding sites for STAT5 and NF-kappa B in the beta-casein gene promoter. Thus, synergistic and antagonistic interactions between GR, NF-kappa B, and STAT5 involve (a) cross-talk mechanisms influencing the activation of STAT5 and (b) promoter-dependent interactions modulating the DNA binding activity of the transcription factors.

Activation of NF-kappaB p50/p65 is regulated in the developing mammary gland and inhibits STAT5-mediated beta-casein gene expression.

The NF-kappaB family of transcription factors regulates diverse cellular functions such as immune response and cell growth and development, and has been reported to be constitutively active in a variety of mammary carcinoma cell lines. However, its role in normal mammary gland development has not been addressed. In our study, we detected developmentally regulated NF-kappaB activity in the mammary gland of mice. During pregnancy, DNA binding activity of NF-kappaB p50/p65 increased until day 16 postcoitum and decreased with the onset of lactation, most likely due to reduced p50 and p65 protein levels in the nucleus. Cotransfection experiments performed with 293 cells revealed an inhibition of the prolactin receptor/JAK2/STAT5 pathway by NF-kappaB. In HC11 cells, NF-kappaB p50/p65 activity was inversely correlated with prolactin-induced STAT5 tyrosine phosphorylation, expression of endogenous beta-casein gene, and of a transfected beta-casein gene promoter reporter construct. This indicates a negative cross talk between NF-kappaB and the prolactin receptor/JAK2/STAT5 activation pathway, which occurs at the level of STAT5 tyrosine phosphorylation. Our results provide evidence for a role of NF-kappaB in normal mammary gland development, and indicate its function as a negative regulator of beta-casein gene expression during pregnancy by interfering with STAT5 tyrosine phosphorylation.

Cholesteryl ester transfer protein gene expression is not specifically regulated by CCAAT/enhancer-binding protein in HepG2-cells.

Cholesteryl ester transfer protein (CETP) mediates the exchange of neutral lipids among plasma lipoproteins and is expressed predominantly in liver and intestine. In band shift assays employing nuclear extracts of HepG2 cells we identified C/EBPbeta as the predominant C/EBP isoform involved in binding to the C/EBP consensus sequence within the 5' upstream region of the CETP gene. This was demonstrated by supershift experiments using antibodies specific for C/EBPalpha, C/EBPbeta and C/EBPdelta and an oligonucleotide containing a single point mutation (CAAT-->CTAT) in this site. Expression of a CETP promoter-fragment/luciferase construct in transiently transfected HepG2 and CaCo-2 cells and enhancement of promoter activity by co-transfection with human C/EBPalpha in HepG2 cells could be influenced neither by the mutation in the consensus sequence nor by elimination of this site together with a second potential binding site for C/EBP. Furthermore, transfection of HepG2 with human C/EBPalpha did not influence the synthesis of CETP by these cells. Our results indicate that the expression of C/EBP in HepG2 cells is not able (1) to influence specifically the expression of a transfected CETP promoter dependent reporter through binding to C/EBP sites in the promoter region and (2) to significantly enhance expression of the endogenous CETP gene.

Retinoic acid modulates prolactin receptor expression and prolactin-induced STAT-5 activation in breast cancer cells in vitro.

Two recent papers demonstrate that prolactin plays an important role in the induction and progression of mammary tumours. Retinoids have been shown to be potent inhibitors of breast carcinogenesis. We studied expression of prolactin receptor mRNA in human breast cancer cell lines MCF-7, SKBR-3, T47D and BT-20 treated with and without retinoids using Northern blot and a quantitative polymerase chain reaction (PCR) method. In all cell lines, all-trans- and 9-cis-retinoic acid, as well as the retinoic acid receptor gamma (RAR-gamma) selective agonists CD2325 and CD437 (1 microM), were able to down-regulate prolactin receptor. After 1 h, a significant reduction was detectable and maximal effect was achieved after 24 h of treatment. Pretreatment with retinoic acid also reduced the prolactin-/prolactin receptor-dependent signal transduction and activation of transcription 5 (STAT-5) activation in T47D cells. Cycloheximide failed to abrogate the retinoic acid-induced decline in prolactin receptor mRNA levels, indicating that this effect was not dependent upon continuing protein synthesis. Similarly, no change in the stability of prolactin receptor mRNA was observed during 12 h of retinoic acid treatment. In conclusion, our results demonstrate that retinoids are able to inhibit the expression of prolactin receptor message, which encodes an important growth factor receptor in breast cancer cells. This action could be responsible for the anti-tumour effects of retinoids.

Generation of carboxy-terminally deleted forms of STAT5 during preparation of cell extracts.

Carboxy-terminally deleted forms of STAT5 have been described to be generated in vivo either by proteolytic processing or by differential splicing mechanisms. By comparing two different cell extraction procedures, we can show that in the mammary gland carboxy-terminally deleted forms are produced in vitro and are not detectable in extracts prepared by SDS lysis.

A distal enhancer region in the human beta-casein gene mediates the response to prolactin and glucocorticoid hormones.

The 5' flanking region of the human beta-casein gene was investigated for the presence of regulatory sequences mediating the action of the lactogenic hormones prolactin and dexamethasone. DNA encompassing 9389 base pairs of the flanking region was isolated and a sequence comparison performed with regulatory regions previously identified in the beta-casein gene of rodents and ruminants. The analysis revealed the presence of a distal region between -4700 and -4550 with a high percentage of identity to the bovine beta-casein enhancer region, and a proximal region between -1 and -200 similar to the proximal promoter regions found in rodents and ruminants. Reporter gene constructs under the control of the distal or the proximal region of the human beta-casein gene were tested for their responsiveness to prolactin and dexamethasone. In transfection experiments, the distal region functioned as a lactogenic hormone inducible enhancer, whereas the proximal region exhibited low activity. In electromobility shift assays, multiple binding sites for Stat5, CCAAT/enhancer-binding proteins, and Ets domain proteins were identified in the distal human enhancer. These transcription factors have already been demonstrated as important regulators of the transcription of milk protein genes in rodents. Thus, a common set of transcription factors appears to be required for the expression of the human beta-casein gene and of milk protein genes in other species.

Transcriptional activation of c-fos by oncogenic Ha-Ras in mouse mammary epithelial cells requires the combined activities of PKC-lambda, epsilon and zeta.

The implication of protein kinase C (PKC) isoforms cPKC-alpha, nPKC-epsilon, aPKC-lambda and aPKC-zeta in the transcriptional activation of a c-fos promoter-driven CAT-reporter construct by transforming Ha-Ras has been investigated. This was achieved by employing antisense constructs encoding RNA directed against isoform-specific 5' sequences of the corresponding mRNA, and expression of PKC mutants representing either kinase-defective, dominant negative, or constitutively active forms of the PKC isoforms. The data indicate that in HC11 mouse mammary epithelial cells, transforming Ha-Ras requires the activities of the three PKC isozymes: aPKC-lambda, nPKC-epsilon and aPKC-zeta, not, however, of cPKC-alpha, for the transcriptional activation of c-fos. Co-expression of oncogenic Ha-Ras with combinations of kinase-defective, dominant negative and constitutively active mutants of the various PKC isozymes are in agreement with a tentative model suggesting that, in the signaling pathway from Ha-Ras to the c-fos promoter, aPKC-lambda acts upstream whereas aPKC-zeta functions downstream of nPKC-epsilon.

Selective coupling of STAT factors to the mouse prolactin receptor.

Prolactin has been reported to induce distinct sets of signal transducers and activators of transcription (STAT) in a cell-type-specific fashion. In the mammary epithelium, although STAT1, STAT3, STAT5A, STAT5B and STAT6 are present in a latent form, only STAT5A and STAT5B are activated. This selective activation of STAT5 by prolactin was also observed in COS-7 cells cotransfected with the long form of the mouse prolactin receptor (PRL-R) and expression vectors for STAT1, STAT3, STAT5 and STAT6. Mutated PRL-Rs and chimeric erythropoietin/gp130 (EPO/gp130) receptors with a tyrosine-containing motif attached at the carboxy terminus were employed to determine the sites in the PRL-R required for the specific activation of STAT5. The experiments revealed the importance of two motifs containing Y477 and Y578 in the PRL-R. When linked to the EPO/gp130 receptor, these sequences were sufficient to specifically induce DNA binding of STAT5 and to activate transcription from the beta-casein gene promoter. By contrast, only weakly they induced DNA binding of STAT6 and STAT3 and did not induce STAT1. A synthetic nonapeptide with phosphorylated Y477 was able to disrupt STAT5 DNA binding in vitro. Our results define structural domains within the carboxy terminus of the PRL-R which recruit STAT5 for signalling and which are capable of distinguishing STAT5 from other STAT proteins. The activity of STAT5 forms with deletions of the carboxy terminus was induced more strongly than that of their full-length counterparts 2 min after activation of the PRL-R. This effect was not dependent on the presence of Y477 and Y578 in the PRL-R, indicating that facilitated activation of short STAT5 isoforms relies on mechanisms other than increased coupling to specific regions of the PRL-R.

Granulocyte-macrophage colony-stimulating factor induces a unique set of STAT factors in murine dendritic cells.

Cytokine-mediated signaling pathways were studied in mouse dendritic cells (DC) by analysis of the activation pattern of STAT factors. Electrophoretic mobility shift assays were performed to detect STAT isoform-specific complexes. Granulocyte-macrophage colony-stimulating factor (GM-CSF) simultaneously induced complexes containing STAT1, STAT3, STAT5A, STAT5B and STAT6. In non-DC, a similar broad activation pattern of STAT factors by GM-CSF or other cytokines has not been observed so far. By comparison, in peritoneal macrophages, GM-CSF induced a complex with the properties of a truncated form of STAT5. Other cytokines tested on DC either failed to induce STAT factors [interleukin (IL)-1 beta, IL-2, IL-15], or activated the same STAT factors as observed in peritoneal macrophages (IL-4, IFN-gamma). Our results implicate a specific effect of GM-CSF on STAT signaling in DC which might account for the cell type-specific effect of this cytokine on development and function.

Promoter-dependent synergy between glucocorticoid receptor and Stat5 in the activation of beta-casein gene transcription.

Steroid hormone receptors and Stat factors comprise two distinct families of inducible transcription factors. Activation of a member of each family, namely the glucocorticoid receptor by glucocorticoids and Stat5 by prolactin, is required for the efficient induction of the expression of milk protein genes in the mammary epithelium. We have studied the mode of interaction between Stat5 and the glucocorticoid receptor in the activation of beta-casein gene transcription. The functional role of potential half-palindromic glucocorticoid receptor-binding sites mapped previously in the promoter region was investigated. beta-Casein gene promoter chloramphenicol acetyltransferase constructs containing mutations and deletions in these sites were tested for their responsiveness to the synergistic effect of prolactin and dexamethasone employing COS-7 cells or HC11 mammary epithelial cells. Synergism depended on promoter regions containing intact binding sites for the glucocorticoid receptor and Stat5. The carboxyl-terminal transactivation domains of Stat5a and Stat5b were not required for this synergism. Our results suggest that in lactogenic hormone response elements glucocorticoid receptor molecules bound to nonclassical half-palindromic sites gain competence as transcriptional activators by the interaction with Stat5 molecules binding to vicinal sites.

Mechanism of interaction between the glucocorticoid receptor and Stat5: role of DNA-binding.

The functional interaction between the glucocorticoid receptor (GR) and the signal transducer and activator of transcription-5 (Stat5) was investigated by studying the synergistic activation of beta-cascin gene transcription by prolactin and glucocorticoids. The synergism was shown to be mediated by a complex hormone response region with multiple binding sites for Stat5, the glucocorticoid receptor, and CCAAT/enhancer binding proteins (C/EBP). HC11 mammary epithelial cells, which contain physiological levels of GR and Stat5, and COS-7 cells overexpressing GR and Stat5 were employed. In both cell types intact binding sites for Stat5 and the GR were a prerequisite for the synergism, whereas C/EBP sites were only required in HC11 cells. Interestingly, the GR sites employed for the synergism were nonclassical, half palindromic sites, which did not function in the absence of activated Stat5 to mediate the action of the GR on transcription. The interaction of GR and Stat5 triggered by the unusual configuration of binding sites appears to represent a novel mechanism by which these two distinct types of transcription factors cooperate. The mode of interaction provides an efficient means to restrict gene expression to conditions where both Stat5 and the GR are activated.

Comparative study on the phosphotyrosine motifs of different cytokine receptors involved in STAT5 activation.

Several cytokines and growth factors activate transcription of their target genes via the JAK/STAT signalling pathway. It has been shown that the interaction between SH2 domains of STAT factors and receptor phosphotyrosine residues plays an essential role in the specific recruitment of STATs. For STAT5, however, the importance of receptor tyrosines is still controversial. Using a chimeric receptor system in COS-7 cells, we studied the activation of STAT5 through the interleukin-6 signal transducer gp130. In contrast to previous reports, we did not detect gp130-mediated STAT5 activation. However, STAT5 activation was achieved when tyrosine motifs of other cytokine receptors were fused to the membrane-proximal part of gp130. The comparison of the relative potency of different tyrosine motifs revealed that hydrophobic amino acids, preferentially leucine, in positions +1 and +3, and an aspartate residue in position -1 or -2 with respect to the tyrosine are likely to be required for efficient STAT5 recruitment. In summary, we show here for the first time that phosphotyrosine motifs can confer the ability to activate STAT5 to a heterologous receptor.

CCAAT/enhancer-binding protein isoforms beta and delta are expressed in mammary epithelial cells and bind to multiple sites in the beta-casein gene promoter.

Lactogenic hormone-dependent expression of the rat beta-casein gene in mammary epithelial cells is controlled via a complex regulatory region in the promoter. The sequence between -176 and -82 is the minimal region to confer the response to glucocorticoid hormone and prolactin on a heterologous promoter. The response is further enhanced by the region between -282 and -176. DNase I footprinting experiments and electromobility shift assays revealed the presence of four binding sites for CCAAT/enhancer-binding protein (C/EBP) isoforms in the hormone response region between -220 and -132. In nuclear extracts from mammary epithelial cells, the prevalent C/EBP isoform binding to these sites is beta (C/EBP-beta). C/EBP-delta is also present in mammary epithelial cells, whereas C/EBP-alpha is not detectable. The C/EBP sites are located in close proximity to the previously characterized binding sites for the prolactin-inducible mammary gland factor/signal transducer and activator of transcription-5, the nuclear factor YY1, and the glucocorticoid receptor. The importance of the two proximal C/EBP binding sites at the 5' border of the minimal region was tested by mutational analysis. Mutations of each site were found to inhibit strongly both the basal and the lactogenic hormone-induced transcription of a beta-casein gene promoter chloramphenicol acetyltransferase construct. The results implicate C/EBPs as important regulators of beta-casein gene expression in the mammary epithelium.

Involvement of Ets-related proteins in hormone-independent mammary cell-specific gene expression.

Regulatory regions have been located in the 5' flanking sequence of the mouse whey acidic protein gene which contribute to its tissue- and stage-specific expression in the mammary gland. They can be functionally separated into elements which mediate the action of lactogenic hormones prolactin and glucocorticoids and elements which control mammary cell-specific transcription in the absence of hormones. By mutational analysis, we have located a site in the whey acidic protein promoter between -120 and -100 which is important for hormone-independent promoter function. In stably transfected HC11 mammary epithelial cells, the hormone-independent activity of the mutated promoter was reduced 40-fold, whereas the capability to respond to lactogenic hormones was retained. The site was specifically recognised by two nuclear factors contained in extracts of cultivated mammary epithelial cells or mammary glands. Electrophoretic mobility shift assay, DNase I footprinting and methylation interference experiments indicated a relation of both factors to the Ets family of DNA-binding proteins. One of these factors also recognised a functionally important site in the mammary cell-specific enhancer of the mouse mammary tumor virus long terminal repeat. The results suggest that factors related to the Ets family are important determinants in mammary cell-specific gene expression.

Prolactin-dependent activation of a tyrosine phosphorylated DNA binding factor in mouse mammary epithelial cells.

The mammary gland factor MGF has been described as a developmentally and environmentally regulated nuclear factor required for transcription of the milk protein gene beta-casein. In the current study the individual role of lactogenic hormones in the activation of MGF DNA binding and the functional relation of MGF to known transcription factors was investigated by electrophoretic mobility shift assays. DNA binding of MGF was rapidly induced by PRL in mammary epithelial cells. The activation was not inhibited by the protein synthesis inhibitor cycloheximide. The effect of PRL on MGF did not require costimulation of cells with the other lactogenic hormones, insulin, and glucocorticoids. Thus, MGF is the first example of a nuclear factor directly regulated by PRL. The MGF complexes formed upon initiation of lactation in the mammary gland and upon stimulation of mammary epithelial cells with PRL migrated at the same position in electrophoretic mobility shift assay, whereas the MGF complex found in mammary gland extracts of pregnant mice exhibited a faster mobility. In cell cultures, PRL-induced activation of MGF as well as up-regulation of beta-casein gene transcription was confined to confluent cultures of mammary epithelial cells and inhibited by long term incubation of cells with epidermal growth factor. MGF was found to be related to the nuclear factors that are activated by tyrosine phosphorylation when cells are stimulated with interferons or cytokines. This notion is supported by experimental evidence for phosphorylation of MGF on tyrosine and by the similar DNA recognition motifs of MGF and cytokine-activated factors.

Activation of c-fos expression by transforming Ha-ras in HC11 mouse mammary epithelial cells is PKC-dependent and mediated by the serum response element.

The mechanism by which transforming Ha-ras induces c-fos expression in HC11 mouse mammary epithelial cells was investigated with regard to controversial data concerning the role of protein kinase C (PKC) and the required promoter elements of the fos gene. HC11 cells carrying a glucocorticoid-inducible Ha-ras (val12) construct were transfected with a chloramphenicol acetyltransferase (CAT) reporter gene under the control of a human fos promoter which includes the serum response element (SRE), the adjacent c-fos AP-1 site (FAP) and the cAMP response element (CRE). Induction of the Ha-ras gene by dexamethasone lead to a transactivation of expression of the transfected fos promoter construct which was inhibited by the PKC inhibitor BM41440 and abrogated in PKC-'depleted' cells. A similar transactivation was observed when the fos promoter construct was co-transfected with a constitutively active ras expression vector. Again, this effect was depressed by the PKC inhibitor and abolished in PKC-'depleted' cells. 'PKC-depletion' was achieved by long-term exposure to 12-O-tetradecanoylphorbol-13-acetate. This procedure was shown to deplete cells of PKC alpha and to reduce significantly PKC epsilon. Long-term exposure to bryostatin 1 selectively depletes PKC alpha. Depletion of PKC alpha by bryostatin 1 does not reduce the transcriptional activation of the SRE-FAP-TK-CAT (TK: thymidine kinase) construct by Ha-ras. In order to delineate the promoter elements mediating the transcriptional activation, constructs which lack the FAP and the CRE sites but contain an intact SRE were co-transfected with the ras construct. Elimination of the FAP and CRE sequences did not affect the transcriptional activation by Ha-ras (val12). It is concluded that in HC11 cells, transforming Ha-ras activates c-fos expression in a PKC-dependent manner, presumably implying PKC epsilon, and that the SRE is sufficient to mediate transcriptional activation.