Studies have demonstrated that the retinoblastoma susceptibility gene product, RB, can either positively or negatively regulate expression of several genes through cis-acting elements in a cell-type-dependent manner. The nucleotide sequence of the retinoblastoma control element (RCE) motif, GCCACC or CCACCC, and the SpI consensus binding sequence, CCGCCC, can confer equal responsiveness to RB. Here, we report that RB activates transcription of the c-jun gene through the Spl-binding site within the c-jun promoter. Preincubation of crude nuclear extracts with monoclonal antibodies to RB results in reduction of Spl complexes in a mobility shift assay, while addition of recombinant RB in mobility shift assay mixtures with CCL64 cell extracts leads to an enhancement of DNA-binding activity of Spl. These results suggest that RB is directly or indirectly involved in Spl-DNA binding activity. A mechanism by which RB regulates transactivation is indicated by our detection of a heat-labile and protease-sensitive Spl negative regulator(s) (Spl-I) that specifically inhibits Spl binding to a c-jun Spl site. This inhibition is reversed by addition of recombinant RB protein, suggesting that RB stimulates Spl-mediated transactivation by liberating Spl from Spl-I. Additional evidence for Spl-I involvement in Spl-mediated transactivation was demonstrated by cotransfection of RB, GAL4-Spl, and a GAL4-responsive template into CV-1 cells. Finally, we have identified Spl-I, a -20-kDa protein(s) that inhibits the SpI complexes from binding to DNA and that is also an RB-associated protein. These findings provide evidence for a functional link between two distinct classes of oncoproteins, RB and c-Jun, that are involved in the control of cell growth, and also define a novel mechanism for the regulation of c-jun expression.The retinoblastoma susceptibility gene product plO5Rb (RB) is generally believed to be an important regulator in the control of cellular proliferation and regulation of the cell cycle (7,10,13,28,29). The biochemical mechanisms for RB action remain unclear, although one possibility is transcriptional control of cell growth-related genes. Indeed, six cellular genes have been identified as targets of transcriptional regulation by RB (20-22, 31, 33, 50 (20,21). Moreover, Kim et al. (20) demonstrated that the TGF-31, c-fos, and c-myc promoters can be both positively and negatively regulated by RB through the same cis-acting element, dependent upon the cell type.

NADPH oxidase is the major source of superoxide production in cardiovascular tissues. We and others reported that PG (prostaglandin) F2alpha, PDGF (platelet-derived growth factor) and angiotensin II cause hypertrophy of vascular smooth muscle cells by induction of NOX1 (NADPH oxidase 1), a catalytic subunit of NADPH oxidase. We found DPI (diphenylene iodonium), an inhibitor of flavoproteins, including NADPH oxidase itself, almost completely suppressed induction of NOX1 mRNA by PGF2alpha or PDGF in a rat vascular smooth muscle cell line, A7r5. Exploration into the site of action of DPI using various inhibitors suggested the involvement of mitochondrial oxidative phosphorylation in PGF2alpha- or PDGF-induced increase in NOX1 mRNA. In a luciferase reporter assay, activation of the CRE (cAMP-response element)-dependent gene transcription by PGF2alpha was attenuated by oligomycin, an inhibitor of mitochondrial F(o)F1-ATPase. Oligomycin and other mitochondrial inhibitors also suppressed PGF2alpha-induced phosphorylation of ATF (activating transcription factor)-1, a transcription factor of the CREB (CRE-binding protein)/ATF family. Silencing of the ATF-1 gene by RNA interference significantly reduced the induction of NOX1 by PGF2alpha or PDGF, while overexpression of ATF-1 recovered NOX1 induction suppressed by oligomycin. Taken together, ATF-1 may play a pivotal role in the up-regulation of NOX1 in rat vascular smooth muscle cells.

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Monomeric carbonyl reductases (CBRs) are enzymes that catalyze the reduction of many endogenous and xenobiotic carbonyl compounds, including steroids and prostaglandins. There are two monomeric CBR genes in the human genome, cbr1 and cbr3, which exhibit high homology in their amino acid sequences. Human CBR1 (hCBR1) is known as prostaglandin 9-keto reductase and 15-hydroxy dehydrogenase, and regulates the metastasis of cancer cells through the regulation of prostaglandin metabolism. However, there is little information concerning the molecular and enzymatic characteristics of human CBR3 (hCBR3). The present study demonstrated the tissue and cellular localization, and catalytic activity of hCBR3. Semi-quantitative PCR revealed the ubiquitous but lower expression of hCBR3 compared with that of hCBR1. Bacterially expressed hCBR3 exhibited limited catalytic activity toward menadione, 4-benzoylpyridine, and 4-nitrobenzaldehyde. Similar results were obtained when the cell lysates of CBR-overexpressing HEK293 cells were examined. Additionally, neither the prostaglandin 9-keto reductase nor the 15-hydroxy dehydrogenase activities of hCBR3 were significant. Immunofluorescence staining revealed that ectopically expressed hCBR3 proteins were localized in the cytosol of HEK293 cells. These results suggested that hCBR3 and hCBR1 play distinct physiological roles. This study expands our understanding of the relationship between the two monomeric hCBRs and prostaglandin metabolism.

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