The glucocorticoid receptor's oligomerization state is revealed to not correlate with its activity; this challenges the current prevailing view that this state defines its transcriptional activity.

Progesterone regulates diverse biological effects in a broad range of tissues, mostly by interaction with the classical progesterone receptor (PR), a member of the nuclear receptor superfamily of ligand-dependent transcription factors. Particularly in the mammary gland, progesterone plays a key role in the control of cell proliferation and differentiation (31 and references within). Accumulated evidence also indicates that progestins are involved in controlling mammary gland tumorigenesis, both in women and in animal models (6,8,15,21,24,26,31,32,48). Although the mechanisms by which progestins stimulate growth of breast cancer cells have not been completely deciphered, several lines of evidence (6, 26, 33), including our own work (3,4,25,44), have shown that convergence between progestins and growth factor (GF) signaling pathways mediates proliferative effects of progestins in mammary tumor cells.In addition to their direct transcriptional effects, rapid or nongenomic biological effects of progestins have been described in several species, including fish, amphibian, and mammalian (27). Whether the recently cloned membrane PR from humans and other vertebrates (59) is involved in mediating nongenomic progestin effects in mammalians remains to be elucidated. Nongenomic effects of progestins in breast cancer cells have been unraveled by startling reports from Auricchio and coworkers (5,13,33), who demonstrated that progestin treatment of human breast cancer T47D cells activates the signal-transducing c-Src/p21 ras /MAPK pathway, which results in cell proliferation (5,13,33). Progestin activation of the c-Src/p21 ras /MAPK pathway has also been described by Edwards and coworkers (6), who further explored mechanisms involved in progestin modulation of c-Src activity.Over the last years, a unique family of proteins, the signal transducers and activators of transcription (Stats), was found to be involved in cross talks with both steroid hormones and

The present study addresses the capacity of heregulin (HRG), a ligand of type I receptor tyrosine kinases, to transactivate the progesterone receptor (PR). For this purpose, we studied, on the one hand, an experimental model of hormonal carcinogenesis in which the synthetic progestin medroxyprogesterone acetate (MPA) induced mammary adenocarcinomas in female BALB/c mice and, on the other hand, the human breast cancer cell line T47D. HRG was able to exquisitely regulate biochemical attributes of PR in a way that mimicked PR activation by progestins. Thus, HRG treatment of primary cultures of epithelial cells of the progestindependent C4HD murine mammary tumor line and of T47D cells induced a decrease of protein levels of PRA and -B isoforms and the downregulation of progesterone-binding sites. HRG also promoted a significant increase in the percentage of PR localized in the nucleus in both cell types. DNA mobility shift assay revealed that HRG was able to induce PR binding to a progesterone response element (PRE) in C4HD and T47D cells. Transient transfections of C4HD and T47D cells with a plasmid containing a PRE upstream of a chloramphenicol acetyltransferase (CAT) gene demonstrated that HRG promoted a significant increase in CAT activity. In order to assess the molecular mechanisms underlying PR transactivation by HRG, we blocked ErbB-2 expression in C4HD and T47D cells by using antisense oligodeoxynucleotides to ErbB-2 mRNA, which resulted in the abolishment of HRG's capacity to induce PR binding to a PRE, as well as CAT activity in the transient-transfection assays. Although the inhibition of HRG binding to ErbB-3 by an anti-ErbB-3 monoclonal antibody suppressed HRG-induced PR activation, the abolishment of HRG binding to ErbB-4 had no effect on HRG activation of PR. To investigate the role of mitogen-activated protein kinases (MAPKs), we used the selective MEK1/MAPK inhibitor PD98059. Blockage of MAPK activation resulted in complete abrogation of HRG's capacity to induce PR binding to a PRE, as well as CAT activity. Finally, we demonstrate here for the first time that HRG-activated MAPK can phosphorylate both human and mouse PR in vitro.Recent evidence has clearly shown the presence of cross talks between steroid hormone and growth factor (GF) signaling pathways, which were previously thought to be distinctly separate processes. Members of the steroid receptor superfamily are heavily phosphorylated proteins that, upon ligand binding, act as nuclear transcription factors (for a review, see reference 72). Particularly, progesterone receptor (PR), the focus of the present work, is phosphorylated in the absence of hormone and undergoes an increase in phosphorylation upon hormonal stimulation (19,20,67). Although the functional role of PR phosphorylation remains elusive, increased evidence indicates that it plays a role in the regulation of PR transcriptional activity (3,7,14,22,40,66). On the other hand, most GFs bind to transmembrane receptors that carry an intrinsic activity of tyrosine kinase. Tyrosine resid...

Differential splicing from the bcl-X gene generates several isoforms with opposite effects on the apoptotic response. To explore the mechanism controlling the balance between the various isoforms, we have characterized the 5 region of the mouse bcl-X gene. We identified three new promoters in addition to the two previously described (Grillot, D. A., M., G.-G., Ekhterae, D., Duan, L., Inohara, N., Ohta, S., Seldin, M. F., and Nú ñ ez, G. (1997) J. Immunol. 158, 4750 -4757). These five promoters (P1-P5) would give rise to at least five mRNAs with different 5-untranslated region, all sharing the same translation initiation site. Except for the product of the most proximal promoter (P1), the other mRNAs are generated by alternative splicing of noncoding exons to a common acceptor site located in the first translated exon. Reverse transcriptase-polymerase chain reaction, primer extension, and RNase protection assays demonstrate a tissue-specific pattern of promoter usage. P1 and P2 are active in all tissues analyzed, whereas the other three promoter show tissue-specific activities. P3 is active in spleen, liver, and kidney, P4 is active in uterus and spleen, and P5 is active in spleen, liver, brain, and thymus. We present evidence suggesting that promoter selection influences the outcome of the splice process. Transcripts from P1 generate mainly the mRNA for the long isoform Bcl-X L , whereas transcripts from P2 generate mRNAs for the isoforms Bcl-X L , Bcl-X S , and Bcl-X ␥ and transcripts from P3 yield mainly mRNAs for the isoform Bcl-X ␥ . Our results suggest a key role of promoter choice in determining alternative splicing and, thus, the balance of Bcl-X isoforms.

Cytokine-driven activation of hepatic stellate cells (HSC) in tissue injury and inflammation is a key pathogenetic event in liver fibrogenesis leading to an expanded pool of matrix producing myofibroblasts (MFB) which represent the transformed counterpart of HSC. We hypothesize that expansion of the pool of MFB might also be accomplished by modulation of apoptosis, which plays an opposite and complementary role to mitosis in the cellular homeostasis. We characterized the susceptibility of HSC in primary culture and of MFB in secondary culture to apoptosis induced by the soluble Fas ligand (sFasL) and related the effects to the expression levels of Fas (APO-1/CD95) and some major proapoptotic and contra-apoptotic protooncogenes. MFB showed a dose-dependent apoptotic reaction upon exposure to sFasL as evidenced by a strong increase of nucleosomal DNA fragments, loss of cellular DNA, positive TUNEL reaction, and annexin staining. The effect was found only if protein synthesis (cycloheximide) or RNA synthesis (actinomycin D) were arrested. HSC maintained for various times in primary culture were completely resistant to sFasL in combination with cycloheximide, but in late primary cultures (day 7 onward) an increasing susceptibility to sFasL-mediated apoptosis was developed. By semiquantitative reverse-transcriptase polymerase chain reaction (RT-PCR) analysis and alkaline phosphatase-antialkaline phosphatase staining Fas receptor was identified both in HSC and MFB at comparable expression levels. The expression of the contra-apoptotic protooncogenes bcl-2 and bcl-xl was found to be much stronger in early HSC than in late HSC and MFB as shown by ribonuclease protection assay. The expression of bcl-2 was additionally confirmed by semiquantitative RT-PCR and immunoblotting. Proapoptotic bax was found in comparable quantities at the RNA level in HSC and MFB but at the protein level MFB showed increased bax expression. It is concluded that transformation of HSC to MFB is paralleled by an increasing sensitivity to sFasL-mediated apoptosis, which might be related to a strong decrease of bcl-2 and bcl-xl expression, leading to a preponderance of proapoptotic gene expression in MFB. Modulation of apoptotic susceptibility of transforming HSC could be an important complementary pathway in the pathogenesis of fibrosis. (HEPATOLOGY 1998;28:492-502.)

The antiapoptotic effect of melatonin has been described in several systems. In this study, the antagonistic effect of the methoxyindole on dexamethasone-induced apoptosis in mouse thymocytes was examined. Melatonin decreased both DNA fragmentation, and the number of annexin V-positive cells incubated in the presence of dexamethasone. Analysis of the expression of the members of the Bcl-2 family indicated that the synthetic glucocorticoid increased Bax protein levels without affecting the levels of Bcl-2, Bcl-XL, Bcl-XS, or Bak. This effect correlated with an increase in thymocytes bax mRNA levels. Dexamethasone also increased the release of cytochrome C from mitochondria. All of these effects were reduced in the presence of melatonin, which was ineffective per se on these parameters. In addition, the involvement of cAMP on glucocorticoid/melatonin antagonism was examined. Both melatonin and dexamethasone decreased the levels of this nucleotide in mouse thymocytes, indicating that the antagonistic action between both hormones involves a cAMP-independent pathway. In summary, the present results suggest that the antiapoptotic effect of melatonin on glucocorticoid-treated thymocytes would be a consequence of an inhibition of the mitochondrial pathway, presumably through the regulation of Bax protein levels.

a b s t r a c tHerein we describe the synthesis and properties of substituted phenylaminopyrrolo[1,2-a]quinoxalinecarboxylic acid derivatives as a novel class of potent inhibitors of the human protein kinase CK2. A set of 15 compounds was designed and synthesized using convenient and straightforward synthesis protocols. The compounds were tested for inhibition of human protein kinase CK2, which is a potential drug target for many diseases including inflammatory disorders and cancer. New inhibitors with IC 50 in the microand sub-micromolar range were identified. The most promising compound, the 4-[(3-chlorophenyl) amino]pyrrolo[1,2-a]quinoxaline-3-carboxylic acid 1c inhibited human CK2 with an IC 50 of 49 nM. Our findings indicate that pyrrolo[1,2-a]quinoxalines are a promising starting scaffold for further development and optimization of human protein kinase CK2 inhibitors.

Bcl-X exists in at least five different isoforms with complex effects on programmed cell death. Glucocorticoids and progestins control bcl-X expression and influence the ratio between bcl-X L (antiapoptotic isoform) and bcl-X S (proapoptotic isoform) in different tissues. The 5-UTR region of the mouse bcl-X gene contains at least five different promoters, which exhibit a tissuespecific pattern of promoter usage. Several mRNAs with different 5-leading exons can be generated upon promoter activation. Here we explore the potential of the various bcl-X gene promoters to be regulated by glucocorticoids or progestins. We found that the region located immediately upstream of promoter 4 (P4) contains two hormone response element (HRE)-like sequences at positions ؊3040 (HRE I) and ؊3001 (HRE II) relative to the translation initiation codon. These HRElike sequences confer hormone responsiveness to a core promoter and bind glucocorticoid or progesterone receptors in vitro. Point mutations of both HREs that prevent steroid receptor binding also eliminate hormonal inducibility. In cells treated with glucocorticoids, the hormone receptor is recruited to the P4 region containing the HREs. Analysis of the products of the endogenous bcl-X in epithelial mammary cells showed that only transcripts originating from P4 increased upon hormone treatment. This observation correlates with the induction of the bcl-X L mRNA, suggesting that P4 is one of the bcl-X promoters responsible for the generation of this antiapoptotic isoform.