No abstract

Androgen receptor (AR) may communicate with the general transcription machinery on the core promoter to exert its function as a transcriptional modulator. Our previous report demonstrated that the AR interacted with transcription factor IIH (TFIIH) under physiological conditions and that overexpression of Cdk-activating kinase, the kinase moiety of TFIIH, enhanced ARmediated transcription in prostate cancer cells. In an effort to further dissect the mechanisms implicated in AR transactivation, we report here that AR interacts with PITALRE, a kinase subunit of positive elongation factor b (P-TEFb). Cotransfection of the plasmid encoding the mutant PITALRE (mtPITALRE), defective in its RNA polymerase II COOH-terminal domain (CTD)-kinase activity, resulted in preferential inhibition of ARmediated transactivation. Indeed, AR transactivation in PC-3 cells was preferentially inhibited at the low concentration of 5,6-dicloro-1-␤-D-ribofuranosylbenzimidazole (DRB), a CTD kinase inhibitor. These results suggest that CTD phosphorylation may play an important role in AR-mediated transcription. Furthermore, a nuclear run-on transcription assay of the prostate-specific antigen gene, an androgen-inducible gene, showed that transcription efficiency of the distal region of the gene was enhanced upon androgen induction. Taken together, our reports suggest that AR interacts with TFIIH and P-TEFb and enhances the elongation stage of transcription.Molecular studies of eukaryotic transcription suggest that the process of transcription can be divided into the following steps: preinitiation complex assembly on the core promoter, initiation, promoter clearance, elongation, and termination (1). To initiate transcription, general transcription factors need to be recruited to the promoter either in a stepwise fashion or in a form of holoenzyme (1). The promoter clearance is defined as a point when RNA polymerase II leaves the initiation complex to start elongation of transcripts (2). Phosphorylation of the COOH-terminal domain (CTD) 1 of the largest subunit of RNA polymerase II is required to establish and maintain the elongation complex (3, 4). Activators have been demonstrated to stimulate one or more steps of the transcription cycle by direct or indirect communication with the general transcription factors (1, 5). In addition, activators may also interact with auxiliary factors, called coregulators, to enhance recruitment of the general transcription machinery on the promoter (6, 7). Direct interactions of activators with coregulators and/or general transcription factors have been suggested to be mechanisms for transcriptional activation (5-7). Nuclear run-on transcription and RNase protection analyses revealed three classes of activation domains (8 The AR is a member of the steroid receptor superfamily that is composed of a variable amino-terminal domain, a highly conserved DNA-binding domain, and a ligand-binding domain (9). Ligand-dependent transcriptional activation of steroid receptors is mediated by the COOH-terminal domain tha...

No abstract

During an acute phase response, interleukin-6 (IL-6) and glucocorticoids up-regulate expression of the three fibrinogen (FBG) genes (fga, fgb, and fgg) in liver and lung epithelium; however, little constitutive lung expression occurs. Recently, we showed that the magnitude of Stat3 binding to three IL-6 motifs on the human ␥FBG promoter correlates negatively with their functional activity in hepatocytes, although these cis-elements are critical for promoter activity. We determined the role of IL-6-receptor-gp130-Stat3 signaling in IL-6 activation of the ␥FBG promoter in liver and lung epithelial cells. Although IL-6 induced ␥FBG promoter activity ϳ30-fold in HepG2 cells, it was increased only 2-fold in lung A549 cells. Equivalent production of gp130 was demonstrated in both cell types by Western blotting; however, lower production of both IL-6-receptor and Stat3 explains, in part, reduced activity of the ␥FBG promoter in lung cells. Dexamethasone potentiated IL-6 induction of the ␥FBG promoter 2.3-fold in both HepG2 and A549 cells for a combined increase in promoter activity of 70-fold or 4.5-fold, respectively. Dexamethasone potentiation is likely due to the induction of IL-6-receptor expression as well as prolonged intensity and duration of Stat3 activation. By circumventing IL-6-receptorgp130-coupled signaling with ectopic expression of the granulocyte colony-stimulating factor receptor (GCSFR)-gp130(133) chimeric receptor, overexpression of Stat3 induced ␥FBG promoter activity 30-fold in A549 cells. Together, the data suggest tissue-specific differences in IL-6-receptor-gp130-coupled signaling, thereby limiting the extent of Stat3 activation and ␥FBG expression during lung inflammation.

Several families of transcription factors play important roles in modulating liver-specific gene expression during an acute phase response (APR). Stat3/APR factor is the main transactivator of gene expression by the interleukin (IL)-6 family of cytokines signaling through gp130. During an APR, fibrinogen (FBG) genes are coordinately upregulated by IL-6 and glucocorticoids. Except for rat ␥FBG, attempts to demonstrate direct binding of IL-6-activated Stat3 to FBG CTGGGAA promoter elements have not been successful. Herein we show the presence of three functional type II IL-6 response elements (IL-6REs) on the human ␥FBG promoter and that the magnitude of Stat3 binding to these elements correlates negatively with their functional activity in reporter gene assays. Stat3-specific binding to ␥FBG IL-6REs was confirmed by cross-competition with ␣ 2 -macroglobulin IL-6RE and specific interactions with anti-Stat3 in electrophoretic mobility shift assays. All type II IL-6REs contributed to full promoter activity; however, transactivation from Site II at ؊306 to ؊301 was strongest. In contrast to a previous report, IL-6 failed to induce activation of serum amyloid A-activating factor-1/c-Myc-associated zinc finger protein (SAF-1/MAZ), and mutation of the SAF-1RE had little effect on IL-6 induction of ␥FBG promoter activity. In the absence of a functional glucocorticoid receptor response element, dexamethasone potentiated IL-6-induced ␥FBG promoter activity 2-fold, requiring promoter-proximal Site I and Site II; the promoter-distal Site III had no effect on dexamethasone potentiation of IL-6-induced promoter activity. Notably the propensity for Stat3 binding to human ␥FBG IL-6REs was low compared with Stat3 binding to the ␣ 2 -macroglobulin IL-6RE. Together these data suggest that Stat3 transactivation via IL-6REs on FBG promoters likely involves participation of additional transcription factors and/or coactivators to achieve optimal coordinated up-regulation during an APR.