SERBP1 affects homologous recombination-mediated DNA repair by regulation of CtIP translation during S phase

DNA double-strand breaks (DSBs) are the most severe type of DNA damage and are primarily repaired by non-homologous end joining (NHEJ) and homologous recombination (HR) in the G1 and S/G2 phase, respectively. Although CtBP-interacting protein (CtIP) is crucial in DNA end resection during HR following DSBs, little is known about how CtIP levels increase in an S phase-specific manner. Here, we show that Serpine mRNA binding protein 1 (SERBP1) regulates CtIP expression at the translational level in S phase. In response to camptothecin-mediated DNA DSBs, CHK1 and RPA2 phosphorylation, which are hallmarks of HR activation, was abrogated in SERBP1-depleted cells. We identified CtIP mRNA as a binding target of SERBP1 using RNA immunoprecipitation-coupled RNA sequencing, and confirmed SERBP1 binding to CtIP mRNA in S phase. SERBP1 depletion resulted in reduction of polysome-associated CtIP mRNA and concomitant loss of CtIP expression in S phase. These effects were reversed by reconstituting cells with wild-type SERBP1, but not by SERBP1 ΔRGG, an RNA binding defective mutant, suggesting regulation of CtIP translation by SERBP1 association with CtIP mRNA. These results indicate that SERBP1 affects HR-mediated DNA repair in response to DNA DSBs by regulation of CtIP translation in S phase.     

Isoform-specific regulation of the lactate transporters MCT1 and MCT4 by contractile activity

We examined the isoform-specific regulation of monocarboxylate transporter (MCT)1 and MCT4 expression by contractile activity in red and white tibialis anterior muscles. After 1 and 3 wk of chronic muscle stimulation (24 h/day), MCT1 protein expression was increased in the red muscles (+78%, P < 0.05). In the white muscles, MCT1 was increased after 1 wk (+191%) and then was decreased after 3 wk. In the red muscle, MCT1 mRNA accumulation was increased only after 3 wk (+21%; P < 0.05). In the white muscle, MCT1 mRNA was increased after 1 wk (+30%; P < 0.05) and 3 wk (+15%; P < 0.05). MCT4 protein was not altered in either the red or white muscles after 1 or 3 wk. MCT4 mRNA was transiently lowered (approximately 15%) in both muscles in the 1st wk, but MCT4 mRNA levels were back to control levels after 3 wk. In conclusion, chronic contractile activity induces the expression of MCT1 but not MCT4. This increase in MCT1 alone was sufficient to increase lactate uptake from the circulation.     

Species-specific differential cleavage and polyadenylation of plasminogen activator inhibitor type 1 hnRNA.

Plasminogen activator inhibitor type 1 (PAI-1) is the primary physiologic inhibitor of the naturally occurring plasminogen activators. In higher primates two forms of mature PAI-1 mRNA (3.2 kb and 2.2 kb) arise by alternative cleavage and polyadenylation of PAI-1 hnRNA which is regulated in a tissue-specific fashion in humans. In other mammals only the 3.2 kb mRNA has been detected. The putative downstream polyadenylation site in humans that gives rise to the 3.2 kb PAI-1 mRNA consists of three overlapping copies of the consensus polyadenylation sequence while no consensus polyadenylation sequence is found upstream at a position that could generate the shorter mRNA species. To determine whether differential cleavage and polyadenylation of PAI-1 mRNA is due to species-specific differences in trans-acting factors that process PAI-1 mRNA or to the presence of a nonconsensus polyadenylation site acquired recently during primate evolution we prepared plasmids in which the 3' nontranslated region of the human PAI-1 gene or the mouse PAI-1 cDNA was inserted downstream of the neomycin gene in the plasmid pSV2neo. We show that the 3'-nontranslated region of the human PAI-1 gene but not the mouse PAI-1 cDNA conferred alternative cleavage and polyadenylation to the neomycin gene in transfected human Hep G2 cells as well as mouse NIH3T3 and rat L6 cells.     

Natriuretic peptides reduce plasminogen activator inhibitor-1 expression in human endothelial cells

Plasma concentrations of natriuretic peptides increase in some pathological conditions, but very little is known about the effect of these vasodilator peptides on the regulation of the blood coagulation system. The fundamental role in the regulation of fibrinolysis is played by plasminogen activator inhibitor type 1 (PAI-1). Recent studies demonstrate that natriuretic peptides can modulate PAI-1 expression in bovine aortic smooth muscle cells and rat aortic endothelial cells. In this report, we tested the effect of natriuretic peptides on PAI-1 expression in the human endothelial cell line (EA.hy 926). For this purpose, we treated the cell cultures with ANP, BNP and CNP, and modulation of PAI-1 synthesis was evaluated. We compared the effect of natriuretic peptides on synthesis and release of PAI-1 in unstimulated cells, and after activation with tumour necrosis factor alpha (TNFalpha). Natriuretic peptides abolished TNFalpha - induced upregulation of PAI-1 expression at both the PAI-1 mRNA and the antigen levels. The inhibitory efficiency was higher in the case of CNP when compared to that produced by ANP and BNP, particularly when TNFalpha-stimulated cells were used. We observed an inhibition of stimulatory effect of TNFalpha on PAI-1 expression also at the level of the PAI-1 promoter in cells transfected with a PAI-1 promoter fragment (+71 to -800). The PAI-1 promoter activity was markedly inhibited by C-type natriuretic peptide, already at a very low (0.001 micro M) concentration of the peptide.     

Reactive oxygen species upregulate expression of PAI-1 in endothelial cells

Second messengers involved in the signal transduction pathway leading to induction of the plasminogen activator inhibitor (PAI-1) have not yet been well characterized. This study focuses on the mechanisms of regulation of PAI-1 expression by reactive oxygen species (ROS) in human endothelial cells. Inhibition of the tumor necrosis factor alpha (TNFalpha?-induced expression of PAI-1 by antioxidant N-acetyl-L-cysteine (NAC) indicated redox-sensitive mechanisms involved in the signaling pathway. Because TNFalpha induces PAI-1 production in endothelial cells, and NAC attenuated this response, we attempted to investigate the possible involvement of ROS in the activation of PAI-1 by TNFalpha. Upregulation of PAI-1 expression in endothelial cells by the stimulation with TNFalpha (50 ng/ml) or H2O2 (10-200 micro M), observed by measurement of the antigen and mRNA levels, was reversed in the presence of NAC (20mM). The stimulatory effect of ROS was detected also at the level of the PAI-1 promoter in endothelial cells transfected with plasmid p800 LUC containing a PAI-1 promoter fragment (+71 to -800). The PAI-1 promoter activity was increased in the presence of ROS, and was suppressed by up to 75% in the presence of antioxidants. On the basis of this study we can conclude that reactive oxygen species play an important role in a cytokine-induced activation of PAI-1 expression, and may act as a signal transduction messenger.     

HLA-DRB1 may be antagonistically regulated by the coordinately evolved promoter and 3'-UTR under stabilizing selection

HLA-DRB1 is the most polymorphic MHC (major histocompatibility complex) class II gene in human, and plays a crucial role in the development and function of the immune system. Extensive polymorphisms exist in the promoter and 3'-UTR of HLA-DRB1, especially a LTR (Long terminal repeat) element in the promoter, which may be involved in the expression regulation. However, it remains unknown how the polymorphisms in the whole promoter region and 3'-UTR to regulate the gene expression. In this study, we investigated the extensive polymorphisms in the HLA-DRB1 promoter and 3'-UTR, and how these polymorphisms affect the gene expression in both independent and jointly manners. It was observed that most of the haplotypes in the DRB1 promoter and 3'-UTR were clustered into 4 conserved lineages (H1, H2, H3 and H4), and showed high linkage disequilibrium. Compared with H1 and H2 lineage, a LTR element in the promoter of H3 and H4 lineage significantly suppressed the promoter activity, whereas the activity of the linked 3'-UTR increased, leading to no apparent difference in the final expression product between H1/H2 and H3/H4 lineage. Nevertheless, compared with the plasmid with a promoter and 3'-UTR from the same lineage, the recombinant plasmid with a promoter from H2 and a 3'-UTR from H3 showed about double fold increased luciferase activity, Conversely, the recombinant plasmid with a promoter from H3 and a 3'-UTR from H2 resulted in about 2-fold decreased luciferase activity. These results indicate that the promoter and 3'-UTR of HLA-DRB1 may antagonistically regulate the gene expression, which may be subjected to stabilizing selection. These findings may provide a novel insight into the mechanisms of the diseases associated with HLA-DRB1 genes.