PMA stimulates MUC5B gene expression through an Sp1-based mechanism in airway epithelial cells.

We previously showed that the MUC5B gene expression was elevated by phorbol 12-myristate 13-acetate (PMA) through an epidermal growth factor receptor-independent Ras/MEKK1/JNK and P38 signaling-based transcriptional mechanism. In the current study, we elucidated the molecular basis of this transcriptional regulation using promoter-reporter gene expression and chromatin immunoprecipitation (ChIP) assays with primary human bronchial epithelial cells that are cultured at the air-liquid interface. We have observed that PMA-induced MUC5B promoter activity is blocked by the Sp1-binding inhibitor, mithramycin A, in a dose-dependent manner. Deletion analysis with the MUC5B promoter construct demonstrated that both basal and PMA-induced promoter-reporter activities reside within the -222/-78 bp region relative to the transcriptional start site. NoShift transcriptional factor assays demonstrated that PMA stimulated Sp1 binding, but not STAT1 and c-Myc binding. Immunoprecipitation studies also verified the enhanced phosphorylation of Sp1 after PMA treatment. Site-directed mutagenesis and transfection studies demonstrated the involvement of Sp1-1 (-122/-114) and the Sp1-2 (-197/-186) cis elements in the basal and PMA-induced MUC5B promoter activity. The ChIP assay with anti-RNA polymerase II reconfirmed the PMA-induced MUC5B promoter activity by showing enhanced RNA polymerase II-DNA complex containing putative MUC5B Sp1-1, Sp1-2, or Sp1-3 sites. However, the ChIP assay using anti-Sp1 antibody demonstrated that the PMA-stimulated binding is only at Sp1-2. These results suggested an Sp1-based transcriptional mechanism with Sp1-1 as the regulator of basal MUC5B promoter activity and Sp1-2 as the regulator of PMA-induced MUC5B gene expression in the human airway epithelial cells.

Rad GTPase attenuates vascular lesion formation by inhibition of vascular smooth muscle cell migration.

BACKGROUND: Rad (Ras associated with diabetes) GTPase is a prototypic member of a new subfamily of Ras-related GTPases with unique structural features, although its physiological role remains largely unknown. In the present study, we characterized the Rad function in vascular smooth muscle cells (VSMCs) and the influence of adenovirus-mediated Rad (Ad-Rad) gene delivery on vascular remodeling after experimental angioplasty. METHODS AND RESULTS: We documented for the first time that neointimal formation using balloon-injured rat carotid arteries was associated with a significant increase in Rad expression as determined by immunohistochemistry and quantitative real-time reverse-transcriptase polymerase chain reaction. The levels of Rad expression in VSMCs were highly induced by platelet-derived growth factor and tumor necrosis factor-alpha. Morphometric analyses 14 days after injury revealed significantly diminished neointimal formation in the Ad-Rad-treated carotid arteries compared with Ad-GFP or PBS controls, whereas the mutated form of Rad GTPase, which can bind GDP but not GTP, increased neointimal formation. Overexpression of Rad significantly inhibited the attachment and migration of VSMCs. In addition, Rad expression dramatically reduced the formation of focal contacts and stress fibers in VSMCs by blocking the Rho/ROK signaling pathway. CONCLUSIONS: Our data clearly identified Rad GTPase as a novel and critical mediator that inhibits vascular lesion formation. Manipulation of the Rad signaling pathway may provide new therapeutic approaches that will limit vascular pathological remodeling.

Gene-specific TCDD suppression of RARalpha- and RXR-mediated induction of tissue transglutaminase.

The malignant human keratinocyte line SCC4 provides a model system to study the mechanism by which 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) suppresses retinoid induction of the tissue transglutaminase gene (TGM2). The current work explores the nature of TCDD suppression of retinoid action to determine whether it is gene specific, whether it is retinoid receptor isoform-dependent, and whether it requires close proximity of retinoid and TCDD response elements. First, two other retinoid-inducible genes were identified in SCC4 by microarray screening whose induction was unaffected by TCDD, clearly demonstrating the gene specificity of TCDD suppression. Second, the receptor isoform dependence of retinoid responsiveness in SCC4 was tested. TGM2 was found to be inducible by an RARalpha-specific but not by an RARgamma-selective agonist. A lack of responsiveness to RARgamma agonists was found to be characteristic of SCC4, however, inasmuch as transcription driven by a retinoid response element in transfections was also stimulated only by the alpha-specific agonist in these cells. Because SCC4 lacks expression of RARbeta, the gene specificity evidently was not attributable to differential TCDD targeting of retinoid receptor isoforms. Finally, the proximal 5 kb of the TGM2 promoter was found to be retinoid responsive in stable transfections, but the induction was not suppressed by TCDD. These results indicate that the suppressive action of TCDD occurs indirectly and through a separate DNA site likely located outside the 5-kb region, not by direct interference with retinoid action or at retinoid response elements.