PTH regulation of the human cytomegalovirus immediate-early gene promoter.
ABSTRACT Secondary hyperparathyroidism and human cytomegalovirus (hCMV) seropositivity are highly prevalent in patients undergoing renal transplantation, and both are linked to the development of chronic allograft nephropathy (CAN). We investigated the hypothesis that parathyroid hormone (PTH) 1-84 regulates hCMV immediate-early gene (IEG) promoter activation in proximal renal tubular cells. PTH 1-84 enhanced hCMV IEG promoter (-548 to +92) activity in opossum kidney cells. Deletion analysis from the 5' end of the promoter localized the PTH 1-84 associated activity to the DNA sequence between -123 and -45. Mutation of an imperfect ATF/AP-1 DNA element within this region abrogated the PTH 1-84 effect and also strongly attenuated basal gene expression. Mobility shift analyses using this DNA element revealed that a member of the ATF-1 family was in the binding complex. In summary, we present evidence for a novel pathogenic role of PTH 1-84 in promoting hCMV immediate-early gene transcription.
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ABSTRACT: Prior studies have suggested a role of the five copies of the 19-bp-repeat cyclic AMP (cAMP)-response element (CRE) in major immediate-early (MIE) promoter activation, the rate-limiting step in human cytomegalovirus (HCMV) replication. We used two different HCMV genome modification strategies to test this hypothesis in acutely infected cells. We report the following: (i) the CREs do not govern basal levels of MIE promoter activity at a high or low multiplicity of infection (MOI) in human foreskin fibroblast (HFF)- or NTera2-derived neuronal cells; (ii) serum and virion components markedly increase MIE promoter-dependent transcription at a low multiplicity of infection (MOI), but this increase is not mediated by the CREs; (iii) forskolin stimulation of the cAMP signaling pathway induces a two- to threefold increase in MIE RNA levels in a CRE-specific manner at a low MOI in both HFF- and NTera2-derived neuronal cells; and (iv) the CREs do not regulate basal levels of HCMV DNA replication at a high or low MOI in HFF. Their presence does impart a forskolin-induced increase in viral DNA replication at a low MOI but only when basal levels of MIE promoter activity are experimentally diminished. In conclusion, the 19-bp-repeat CREs add to the robust MIE promoter activity that occurs in the acutely infected stimulated cells, although the CREs' greater role may be in other settings.Journal of Virology 07/2003; 77(12):6666-75. DOI:10.1128/JVI.77.12.6666-6675.2003 · 4.44 Impact Factor
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ABSTRACT: Recently we demonstrated that the ability of tumor necrosis factor alpha (TNFalpha) to stimulate the human cytomegalovirus (HCMV) IE1/2 enhancer/promoter activity in myeloid progenitor-like cells decreases when these cells differentiate into promonocytic cells. In addition, TNFalpha stimulation in the progenitor-like cell line HL-60 was shown to be mediated by nuclear factor kappaB (NF-kappaB) activation and its binding to the 18-base pair sequence motifs of the IE1/2 enhancer. We demonstrate here that the cell differentiation-dependent reduction of TNFalpha stimulation is not due to insufficient NF-kappaB activation but correlates with increased synthesis of the monocyte differentiation-associated factors CCAAT/enhancer-binding protein (C/EBP) alpha and beta. Overexpression of C/EBPalpha/beta in HL-60 cells, which normally produce only very small amounts of C/EBP, stimulated the basal activity of the promoter in the absence of NF-kappaB but suppressed the stimulatory effect of TNFalpha. A novel C/EBP-binding site was identified in the IE1/2 enhancer directly downstream of a NF-kappaB site. In order to understand the mechanisms of interaction, we used an IE1/2 promoter mutant that failed to bind C/EBP at this position and several constructs that contained exclusively NF-kappaB- and/or C/EBP-binding sites upstream of the minimal IE1/2 promoter. We could demonstrate that C/EBPalpha/beta interacts with NF-kappaB p65 and displays inhibitory activity even in the absence of direct DNA binding by forming p65-C/EBP-containing protein complexes bound to the NF-kappaB site. Moreover, C/EBP binding to the DNA adjacent to NF-kappaB supports the down-regulatory effect of C/EBPs possibly due to stabilization of a multimeric NF-kappaB-C/EBP complex. Our results show that cell differentiation factors may interfere with TNFalpha-induced human cytomegalovirus gene (re)activation.Journal of Biological Chemistry 12/2001; 276(44):40712-20. DOI:10.1074/jbc.M009815200 · 4.57 Impact Factor
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ABSTRACT: Parathyroid hormone (PTH), a powerful bone-resorbing agent, is capable of stimulating interstitial collagenase (MMP-13) mRNA production in osteoblastic cells. In this study, a PEA3 consensus binding sequence (-80; AGGAAGT) in addition to a 'TRE-like' sequence (-89; CGACTCA) in the 5' upstream regulatory region of the rat MMP-13 gene were examined. In response to PTH, there was a time-dependent increase in binding of nuclear factors to an oligonucleotide containing the PEA3 region (-95 to -71). This increase in binding was first observed at 0.5 h, peaked at 4 h (7. 6-fold) then returned to basal levels by 24 h. Mutagenesis of the PEA3 site in a chloramphenicol acetyl transferase (CAT) construct containing 5' upstream regulatory sequence of the rat MMP-13 gene significantly decreased activation by PTH. PTH-mediated binding of nuclear factors to an oligonucleotide containing the mutant PEA3 sequence was decreased as compared with the wild type. Mutation or deletion of the TRE-like sequence affected basal as well as PTH-mediated induction of corresponding CAT constructs. Treatment with KN93, a Ca(2+)/calmodulin-dependent protein kinase II specific inhibitor, greatly reduced the amount of protein binding to the PEA3 region in response to PTH which correlated to a notable decrease in the amount of MMP-13 mRNA produced in response to PTH. Antibodies against Ets-1, cyclic AMP response element (CREB)-binding protein (CBP) and CREB were capable of supershifting proteins binding to the oligonucleotide containing the PEA3 region. These data suggest a possible co-operative interaction of factors binding to the PEA3 and TRE-like sequences and provide the first indication of a role for a calcium-mediated pathway in the PTH induction of MMP-13 mRNA in osteoblastic cells.Journal of Molecular Endocrinology 09/2000; 25(1):73-84. · 3.08 Impact Factor