Induction of dystrophin Dp71 expression during neuronal differentiation: opposite roles of Sp1 and AP2α in Dp71 promoter activity

In this study, we delineated the molecular mechanisms that modulate Dp71 expression during neuronal differentiation, using the N1E‐115 cell line. We demonstrated that Dp71 expression is up‐regulated in response to cAMP‐mediated neuronal differentiation of these cells, and that this induction is controlled at promoter level. Functional deletion analysis of the Dp71 promoter revealed that a 5′‐flanking 159‐bp DNA fragment that contains Sp1 and AP2 binding sites is necessary and sufficient for basal expression of this TATA‐less promoter, as well as for its induction during neuronal differentiation. Electrophoretic mobility shift and chromatin immunoprecipitation assays revealed that Sp1 and AP2α bind to their respective DNA elements within the Dp71 basal promoter. Overall, mutagenesis assays on the Sp1 and AP2 binding sites, over‐expression of Sp1 and AP2α, as well as knock‐down experiments on Sp1 and AP2α gene expression established that Dp71 basal expression is controlled by the combined action of Sp1 and AP2α, which act as activator and repressor, respectively. Furthermore, we demonstrated that induction of Dp71 expression in differentiated cells is the result of the maintenance of positive regulation exerted by Sp1, as well as of the loss of AP2α binding, which ultimately releases the promoter from repression.     

Transforming growth factor beta regulates clusterin gene expression via modulation of transcription factor c-Fos.

Transforming growth factor-beta (TGFbeta) induces gene expression of the glycoprotein clusterin in a variety of cell types via a consensus AP-1 binding site. Here, we demonstrate, by supershift analysis, that JunB, JunD, Fra1, Fra2, and c-Fos bound to AP-1 but that prior treatment of the cells with TGFbeta reduced dramatically c-Fos binding, suggesting that c-Fos might be playing a negative regulatory role in clusterin gene expression. Transient cotransfection assays in mink lung epithelial (CCL64) cells, using a human c-Fos expressing plasmid together with a clusterin promoter/reporter construct or the artificial TGFbeta-inducible reporter construct 3TPLux, revealed that c-Fos was indeed repressive for TGFbeta-induced promoter transactivation. Further, we demonstrate that in stable c-Fos-overexpressing cell lines, TGFbeta induction of endogenous clusterin mRNA, as well as clusterin promoter transactivation are blocked. Co-transfection with c-Fos deletion constructs revealed that the C-terminal region, including the homologue box 2 motif and the extreme C-terminal serine phosphorylation sites (Ser362 and Ser374) are required for repression of clusterin and 3TPLux transactivation. TGFbeta treatment of CCL64 cells resulted in the induction of c-Fos mRNA but caused no alternation in total c-Fos protein levels. The results suggest that the c-Fos represses clusterin gene expression, maintaining a low basal level in the absence of TGFbeta, and that TGFbeta, presumably through its effects on c-Fos protein synthesis and/or stability, abrogates the repression of c-Fos, thereby resulting in gene expression.     

Transcriptional mechanisms regulating alveolar epithelial cell-specific CCL5 secretion in pulmonary tuberculosis

CCL5 (or RANTES (regulated upon activation, normal T cell expressed and secreted)) recruits T lymphocytes and monocytes. The source and regulation of CCL5 in pulmonary tuberculosis are unclear. Infection of the human alveolar epithelial cell line (A549) by Mycobacterium tuberculosis caused no CCL5 secretion and little monocyte secretion. Conditioned medium from tuberculosis-infected human monocytes (CoMTB) stimulated significant CCL5 secretion from A549 cells and from primary alveolar, but not upper airway, epithelial cells. Differential responsiveness of small airway and normal human bronchial epithelial cells to CoMTB but not to conditioned medium from unstimulated human monocytes was specific to CCL5 and not to CXCL8. CoMTB induced CCL5 mRNA accumulation in A549 cells and induced nuclear translocation of nuclear factor kappaB (NFkappaB) subunits p50, p65, and c-rel at 1 h; nuclear binding of activator protein (AP)-1 (c-Fos, FosB, and c-Jun) at 4-8 h; and binding of NF-interleukin (IL)-6 at 24 h. CCL5 promoter-reporter analysis using deletion and site-specific mutagenesis constructs demonstrated a key role for AP-1, NF-IL-6, and NFkappaB in driving CoMTB-induced promoter activity. The IL-1 receptor antagonist inhibited A549 and small airway epithelial cell CCL5 secretion, gene expression, and promoter activity. CoMTB contained IL-1beta, and recombinant IL-1beta reproduced CoMTB effects. Monocyte alveolar, but not upper airway, epithelial cell networks in pulmonary tuberculosis cause AP-1-, NF-IL-6-, and NFkappaB-dependent CCL5 secretion. IL-1beta is the critical regulator of tuberculosis-stimulated CCL5 secretion in the lung.     

Apigenin inhibition of involucrin gene expression is associated with a specific reduction in phosphorylation of protein kinase Cdelta Tyr311

Apigenin is a plant-derived flavanoid that has significant promise as a skin cancer chemopreventive agent. In the present study, we examine the mechanism whereby apigenin regulates normal human keratinocyte differentiation. Expression of involucrin (hINV), a marker of keratinocyte differentiation, is increased by differentiating agents via a protein kinase Cdelta (PKCdelta), Ras, MEKK1, MEK3 cascade that increases AP1 factor level and AP1 factor binding to DNA elements in the hINV promoter. We show that apigenin inhibits this response. Apigenin suppresses the 12-O-tetradeconylphorbol-13-acetate-dependent increase in AP1 factor expression and binding to the hINV promoter and the increase in hINV promoter activity. Apigenin also inhibits the increase in promoter activity observed following overexpression of PKCdelta, constitutively active Ras, or MEKK1. The suppression of PKCdelta activity is associated with reduced phosphorylation of PKCdelta-Y311. The physiological importance of this phosphorylation event was confirmed by showing that the PKCdelta phosphorylation-defective mutant, PKCdelta-Y311F, is less able to increase hINV promoter activity. Activation of hINV promoter activity by the green tea polyphenol, (-)-epigellocathecin-3-gallate, is also inhibited by apigenin, suggesting that the two chemopreventive agents can produce opposing actions in keratinocytes. Additional studies show that the apigenin-dependent suppression of differentiation is associated with reduced cell proliferation but that there is no evidence of apoptosis.     

Characterization of an osteoblast-specific enhancer element in the CBFA1 gene

Cbfa1 is a critical regulator of cell differentiation expressed only in the osteochondrogenic lineage. To define the molecular basis of this cell-specific expression we analyzed the murine Cbfa1 promoter. Here we show that the first 976 bp of this promoter are specifically active in osteoblastic cells. Within this region DNase I footprinting delineated a 40-bp area (CE1) protected differently by nuclear extracts from osteoblastic cells and from non-osteoblastic cells. When multimerized, CE1 conferred an osteoblast-specific activity to a heterologous promoter in DNA transfection experiments; this enhancing ability was conserved between mouse, rat, and human CE1 present in the respective Cbfa1 promoters. CE1 site-specific mutagenesis determined that it binds NF1- and AP1-like activities. Further analyses revealed that the NF1 site acts as a repressor in non-osteoblastic cells due to the binding of NF1-A, a NF1 isoform not expressed in osteoblastic cells. In contrast, the AP1 site mediates an osteoblast-specific activation caused by the preferential binding of FosB to CE1 in osteoblastic cells. In summary, this study identified an osteoblast-specific enhancer in the Cbfa1 promoter whose activity is achieved by the combination of an inhibitory and an activatory mechanism.