👤 Dean W Hum

MicroRNAs (miRNAs) down-regulate their target genes. The intronic miR-140, present in the WW domain containing E3 ubiquitin protein ligase 2 (WWP2) gene, decreases the expression of genes that play detrimental roles in osteoarthritis (OA). As the expression level of miR-140 is significantly decreased in human OA chondrocytes, we investigated its regulation in those cells. Gene expression in human chondrocytes was determined by quantitative polymerase chain reaction (qPCR) and gene silencing was done in OA chondrocytes by transient transfection with specific small interfering RNAs (siRNAs). Binding sites of the miR-140 regulatory sequence (rsmiR-140) were identified by mutagenesis and chromatin immunoprecipitation (ChIP) in OA chondrocytes. The effects of translocation on OA chondrocytes were determined by immunocytochemistry and qPCR. In contrast to miR-140, the expression of WWP2 was similar in both normal and OA cells, suggesting that miR-140 has an additional level of regulation. rsmiR-140 showed activity and predicted binding sites for nuclear matrix transcription factor 4 (NMP4), myc-associated zinc (MAZ), nuclear factor of activated T-cells (NFAT), and mothers against decapentaplegic homolog 3 (SMAD3). Silencing NFAT3 (P ≤0.01) and SMAD3 (P ≤0.05) differentially regulated miR-140 independently of WWP2. Silencing NFAT5 decreased both miR-140 and WWP2 (P ≤0.003 and P ≤0.05, respectively). NFAT3 activation increased and transforming growth factor-β (TGF-β) decreased rsmiR-140 activity. Mutagenesis of rsmiR-140 and ChIP assays identified binding sites at which NFAT3 (activator) and SMAD3 (repressor) directly regulated miR-140. TGF-β interfered with NFAT3 translocation, and subsequently with miR-140 expression. This is the first study to provide evidence of a regulatory mechanism of miR-140 independent of WWP2, and new and differential roles for NFAT3 and SMAD3 in the OA process in the regulation of miR-140 transcription. Such knowledge could advance therapeutic strategies targeting OA. Show less

Protein kinase C interacting protein (PKCI-1) was identified among the potential interactors from a yeast two hybrid screen of human brain library using N terminal of RGSZ1 as a bait. The cysteine string region, unique to the RZ subfamily, contributes to the observed interaction because PKCI-1 interacted with N-terminus of RGS17 and GAIP, but not with that of RGS2 or RGS7 where cysteine string motif is absent. The interaction between RGSZ1 and PKCI-1 was confirmed by coimmunoprecipitation and immunofluorescence. PKCI-1 and RGSZ1 could be detected by coimmunoprecipitation using 14-3-3 antibody in cells transfected with PKCI-1 or RGSZ1 respectively, but when transfected with PKCI-1 and RGSZ1 together, only RGSZ1 could be detected. Phosphorylation of Galphaz by protein kinase C (PKC) reduces the ability of the RGS to effectively function as GTPase accelerating protein for Galphaz, and interferes with ability of Galphaz to interact with betagamma complex. We investigated the roles of 14-3-3 and PKCI-1 in phosphorylation of Galphaz. Phosphorylation of Galphaz by PKC was inhibited by 14-3-3 and the presence of PKCI-1 did not provide any further inhibition. PKCI-1 interacts with mu opioid receptor and suppresses receptor desensitization and PKC related mu opioid receptor phosphorylation [W. Guang, H. Wang, T. Su, I.B. Weinstein, J.B. Wang, Mol. Pharmacol. 66 (2004) 1285.]. Previous studies have also shown that mu opioid receptor co-precipitates with RGSZ1 and influence mu receptor signaling by acting as effector antagonists [J. Garzon, M. Rodriguez-Munoz, P. Sanchez-Blazquez, Neuropharmacology 48 (2005) 853., J. Garzon, M. Rodriguez-Munoz, A. Lopez-Fando, P. Sanchez-Blazquez Neuropsychopharmacology 30 (2005) 1632.]. Inhibition of cAMP by mu opioid receptor was significantly reduced by RGSZ1 and this effect was enhanced in combination with PKCI-1. Our studies thus provide a link between the previous observations mentioned above and indicate that the major function of PKCI-1 is to modulate mu opioid receptor signaling pathway along with RGSZ1, rather than directly mediating the Galphaz RGSZ1 interaction. Show less

The newly identified apolipoprotein A5 (APOA5), selectively expressed in the liver, is a crucial determinant of plasma triglyceride levels. Because elevated plasma triglyceride concentrations constitute an independent risk factor for cardiovascular diseases, it is important to understand how the expression of this gene is regulated. In the present study, we identified the retinoic acid receptor-related orphan receptor-alpha (RORalpha) as a regulator of human APOA5 gene expression. Using electromobility shift assays, we first demonstrated that RORalpha1 and RORalpha4 proteins can bind specifically to a direct repeat 1 site present at the position -272/-260 in the APOA5 gene promoter. In addition, using transient cotransfection experiments in HepG2 and HuH7 cells, we demonstrated that both RORalpha1 and RORalpha4 strongly increase APOA5 promoter transcriptional activity in a dose-dependent manner. Finally, adenoviral overexpression of hRORalpha in HepG2 cells led to enhanced hAPOA5 mRNA accumulation. We show that the homologous region in mouse apoa5 promoter is not functional. Moreover, we show that in staggerer mice, apoa5 gene is not affected by RORalpha. These findings identify RORalpha1 and RORalpha4 as transcriptional activators of human APOA5 gene expression. These data suggest an additional important physiological role for RORalpha in the regulation of genes involved in lipid homeostasis and probably in the development of atherosclerosis. Show less

Alterations in the expression of the recently discovered apolipoprotein A5 gene strongly affect plasma triglyceride levels. In this study, we investigated the contribution of APOA5 to the liver X receptor (LXR) ligand-mediated effect on plasma triglyceride levels. Following treatment with the LXR ligand T0901317, we found that APOA5 mRNA levels were decreased in hepatoma cell lines. The observation that no down-regulation of APOA5 promoter activity was obtained by LXR-retinoid X receptor (RXR) co-transfection prompted us to explore the possible involvement of the known LXR target gene SREBP-1c (sterol regulatory element-binding protein 1c). In fact, we found that co-transfection with the active form of SREBP-1c down-regulated APOA5 promoter activity in a dose-dependent manner. We then scanned the human APOA5 promoter sequence and identified two putative E-box elements that were able to bind specifically SREBP-1c in gel-shift assays and were shown to be functional by mutation analysis. Subsequent suppression of SREBP-1 mRNA through small interfering RNA interference abolished the decrease of APOA5 mRNA in response to T0901317. Finally, administration of T0901317 to hAPOA5 transgenic mice revealed a significant decrease of APOA5 mRNA in liver tissue and circulating apolipoprotein AV protein in plasma, confirming that the described down-regulation also occurs in vivo. Taken together, our results demonstrate that APOA5 gene expression is regulated by the LXR ligand T0901317 in a negative manner through SREBP-1c. These findings may provide a new mechanism responsible for the elevation of plasma triglyceride levels by LXR ligands and support the development of selective LXR agonists, not affecting SREBP-1c, as beneficial modulators of lipid metabolism. Show less