Upstream regulatory elements in chick heme oxygenase-1 promoter: A study in primary cultures of chick embryo liver cells

Previously, chick heme oxygenase-1 (cHO-1) gene was cloned by us and two regions important for induction by sodium arsenite were identified. These two regions were found to contain consensus sequences of an AP-1 (-1580 to -1573) and a MRE/cMyc complex (-52 to -41). In the current study, the roles of these two elements in mediating the sodium arsenite or cobalt chloride dependent induction of cHO-1 were investigated further. DNA binding studies and site-directed mutagenesis studies indicated that both the AP-1 and MRE/cMyc elements are important for the sodium arsenite induction, while cobalt chloride induction involves only the AP-1 element. Electrophoretic mobility shift assays showed that nuclear proteins binding to the AP-1 element was increased by both sodium arsenite or cobalt chloride treatment, whereas the binding of proteins to the MRE/cMyc element showed a high basal expression in untreated cells and the binding activity was only slightly increased by sodium arsenite treatment. Site-directed mutagenesis studies showed that, to completely abolish sodium arsenite induction, both the AP-1 and MRE/cMyc elements must be mutated; mutation of either element alone resulted in only a partial effect. In contrast, a single mutation at AP-1 element was sufficient to reduce the cobalt chloride induction almost completely. The MRE/cMyc complex plays a major role in the basal level expression, and shares some similarities to the upstream stimulatory factor element (USF) identified in the promoter regions of mammalian HO-1 genes and other stress regulated genes. Because sodium arsenite is known to cause oxidative stress and because activation of AP-1 proteins has been shown to be a key step in the oxidative stress response pathway, we also explored the possibility that the induction of the cHO-1 gene by sodium arsenite is mediated through oxidative stress pathway(s) by activation of AP-1 proteins. We found that pretreatment with antioxidants (N-acetyl cysteine or quercetin) reduced the induction of the endogenous cHO-1 message or cHO-1 reporter construct activities induced by sodium arsenite or cobalt chloride. These antioxidants also reduced the protein binding activities to the AP-1 element in the electrophoretic mobility shift assays. In summary, induction of the cHO-1 gene by sodium arsenite or cobalt chloride is mediated by activation of the AP-1 element located at -1,573 to -1,580 of the 5 UTR.     

JunB and JunD regulate human heme oxygenase-1 gene expression in renal epithelial cells

Heme oxygenase-1 is a highly inducible gene, the product of which catalyzes breakdown of the prooxidant heme. The purpose of this study was to investigate the regulation of the human heme oxygenase-1 gene in renal epithelial cells. DNase I hyper-sensitivity studies identified three distal sites (HS-2, -3, and -4) corresponding to approximately -4.0, -7.2, and -9.2 kb, respectively, of the heme oxygenase-1 promoter in addition to one proximal region, HS-1, which we have shown previously to be an E box. In vivo dimethyl sulfate footprinting of the HS-2 region revealed six individual protected guanines. Two mutations within HS-2 combined with a third mutation of the proximal E box abolished hemin- and cadmium-driven heme oxygenase-1 promoter activation, suggesting that these three sites synergized for maximal heme oxygenase-1 induction. Jun proteins bound to the antioxidant response element in the HS-2 region in vitro and associated with the heme oxygenase-1 promoter in vivo. JunB and JunD contribute opposing effects; JunB activated whereas JunD repressed heme oxygenase-1 expression in human renal epithelial cells, results that were corroborated in junB(-)(/)(-) and junD(-)(/)(-) cells. We propose that heme oxygenase-1 induction is controlled by a dynamic interplay of regulatory proteins, and we provide new insights into the molecular control of the human heme oxygenase-1 gene.     

Characterization of loricrin regulation in vitro and in transgenic mice

We have previously shown that the promoter of a 6.5 kb mouse loricrin clone contains a functional AP-1 element and directs tissue-specific, but not differentiation-specific, expression. We now report the isolation of a 14-kb genomic clone containing an additional 7 kb of genomic sequence. The additional sequences limit expression of a reporter construct to differentiated keratinocytes in culture. The expression of the 6.5-kb and 14-kb loricrin constructs were also analyzed in transgenic mice. Significantly, loricrin was found in all layers of the epidermis of the 6.5-kb transgenics, including basal and spinous cells. The expression of the 14-kb clone was indistinguishable from that of the endogenous gene, confirming that the additional sequences contain negative regulatory elements that restrict loricrin expression to the granular layer in vivo. In addition, we show the AP-1 element localized in the loricrin proximal promoter is necessary but not sufficient for expression of the loricrin gene in vivo in transgenic mice. Finally, to gain further insight into how AP-1 family members regulate expression of the loricrin gene, we co-transfected the loricrin reporter constructs with expression plasmids for various fos and jun family members and demonstrated that c-Fos/Jun-B heterodimers could mimic the differentiation-specific induction of loricrin.     

Transcriptional regulation of heme oxygenase-1 gene expression by MAP kinases of the JNK and p38 pathways in primary cultures of rat hepatocytes

Heme oxygenase-1 (HO-1) gene expression is induced by various oxidative stress stimuli including sodium arsenite. Since mitogen-activated protein kinases (MAPKs) are involved in stress signaling we investigated the role of arsenite and MAPKs for HO-1 gene regulation in primary rat hepatocytes. The Jun N-terminal kinase (JNK) inhibitor SP600125 decreased sodium arsenite-mediated induction of HO-1 mRNA expression. HO-1 protein and luciferase activity of reporter gene constructs with -754 bp of the HO-1 promoter were induced by overexpression of kinases of the JNK pathway and MKK3. By contrast, overexpression of Raf-1 and ERK2 did not affect expression whereas overexpression of p38alpha, beta, and delta decreased and p38gamma increased HO-1 expression. Electrophoretic mobility shift assays (EMSA) revealed that a CRE/AP-1 element (-668/-654) bound c-Jun, a target of the JNK pathway. Deletion or mutation of the CRE/AP-1 obliterated the JNK- and c-Jun-dependent up-regulation of luciferase activity. EMSA also showed that an E-box (-47/-42) was bound by a putative p38 target c-Max. Mutation of the E-box strongly reduced MKK3, p38 isoform-, and c-Max-dependent effects on luciferase activity. Thus, the HO-1 CRE/AP-1 element mediates HO-1 gene induction via activation of JNK/c-Jun whereas p38 isoforms act through a different mechanism via the E-box.     

Pescadillo interacts with the cadmium response element of the human heme oxygenase-1 promoter in renal epithelial cells

Renal tubular cells elicit adaptive responses following exposure to nephrotoxins, such as cadmium. One response is the up-regulation of the 32-kDa redox-sensitive protein, heme oxygenase-1. Exposure of renal proximal tubular epithelial cells to 10 mum cadmium demonstrated induction ( approximately 20-fold) of heme oxygenase-1 mRNA and protein. Using a 4.5-kb human heme oxygenase-1 promoter construct, the importance of a previously identified cadmium response element (TGCTAGAT) in HeLa cells was verified in renal epithelial cells. Specific protein-DNA interaction with this sequence was demonstrated using nuclear extracts from cadmium-treated cells. Yeast one-hybrid screen of a human kidney cDNA library resulted in the identification of pescadillo, a unique nucleolar, developmental protein, as an interacting protein with the cadmium response element and was confirmed by chromatin immunoprecipitation in vivo and gel shift assays with purified glutathione S-transferase-pescadillo protein in vitro. The specificity of the DNA-protein interaction was verified by the absence of a binding complex when the core sequence of the cadmium response element was mutated or deleted. In addition, B23/nucleophosmin, another nucleolar protein, did not interact with the cadmium response sequence. Overexpression of pescadillo resulted in increased activity of the 4.5-kb human heme oxygenase-1 promoter construct but failed to activate this construct when the cadmium response sequence was mutated. The findings demonstrate the important and previously unrecognized role of pescadillo as a DNA-binding protein interacting specifically with the cadmium response element of the human heme oxygenase-1 gene.     

Cellular iron depletion weakens induction of heme oxygenase-1 by cadmium

Heme oxygenase-1 is an inducible cytoprotective gene, although its induction by environmental factors is not completely understood. This study aimed to ascertain if specific nutritive factors or related compounds influence heme oxygenase-1 expression. In HCT-116 cells, cadmium increased heme oxygenase-1 enzymatic activity. This effect of cadmium was weaker in cells made iron-deficient with the iron chelator, desferrioxamine, which was associated with repression of heme oxygenase-1 protein and mRNA expression. The repression by desferrioxamine of cadmium-induced heme oxygenase-1 upregulation was reversed upon iron replenishment of the cells. Additionally, it was found that thiol antioxidants inhibited the heme oxygenase-1 upregulation caused by cadmium and also by ethacrynic acid, which each decreased intracellular glutathione as did buthionine sulfoxamine. Interestingly, cadmium and ethacrynic acid increased nuclear translocation of Nrf2 and subsequent heme oxygenase-1 expression, but buthionine sulfoxamine did not. Furthermore, NADPH oxidase inhibitors (diphenyleneiodonium and apocynin, and a superoxide scavenger (Tiron) inhibited cadmium-induced upregulation of heme oxygenase-1. Diphenyleneiodonium was the most potent and inhibited NADPH-cytochrome P450 reductase as well, whereas apocynin and Tiron did not. It is concluded that adequate amounts of iron, which at the atomic level can serve as the pivotal element of heme in NADPH oxidase, must be present in cells to permit what appears to be thiol redox-sensitive, NADPH oxidase-dependent upregulation of heme oxygenase-1. Thus, these findings are significant because they suggest that cells without adequate iron would be unable to fully express the stress gene, heme oxygenase-1, when confronted with the toxic metal, cadmium.     

Hypertonic sodium chloride and mannitol induces COX-2 via different signaling pathways in mouse cortical collecting duct M-1 cells

The kidney cortical collecting duct is an important site for the maintenance of sodium balance. Previous studies have shown that, in renal medullary cells, hypertonic stress induces expression of cyclooxygenase-2 (COX-2) via NF-kappaB activation, but little is known about COX-2 expression in response to hypertonicity in the cortical collecting duct. Therefore, we examined the mechanism of hypertonic induction of COX-2 in M-1 cells derived from mouse cortical collecting duct. Induction of COX-2 protein was detected within 6 h of treatment with hypertonic sodium chloride. The treatment also increased COX-2 mRNA accumulation in a cycloheximide-independent manner, suggesting that ongoing protein synthesis is not required for COX-2 induction. Using reporter plasmids containing 0.2-, 0.3-, and 1.5-kb fragments of the COX-2 promoter, we found that hypertonic induction of COX-2 was due to an increase in promoter activity. The COX-2-inductive effect of hypertonicity was inhibited by SB203580, indicating that the effect is mediated by p38 MAPK. Since p38 MAPK can activate NF-kappaB, we made point mutations in the NF-kappaB binding site within the COX-2 promoter. The mutations did not block the induction of COX-2 promoter activity by hypertonic sodium chloride, and hypertonic sodium chloride failed to activate NF-kappaB binding site-driven reporter gene constructs. In contrast, hypertonic mannitol activated NF-kappaB, indicating that hypertonic mannitol and hypertonic sodium chloride activate COX-2 by different mechanisms. Thus, induction of COX-2 expression in M-1 cells by hypertonic sodium chloride does not involve activation of NF-kappaB. Furthermore, the signal transduction pathways that respond to hypertonic stress vary for different osmolytes in cortical collecting duct cells.     

Regulation of matrilysin expression in endothelium by fibroblast growth factor-2

Matrilysin (MMP7) is a secreted matrix metalloproteinase, which contributes to angiogenesis by breaking down basement membranes. We show that the angiogenic factor FGF-2 induces MMP7 expression in human endothelial cells. The promoter contains a Lef/Tcf consensus sequence, but using wildtype or Lef/Tcf-mutated promoter constructs, FGF-2-induced MMP7 reporter activity is independent from Lef/Tcf sites. Instead, we show that overexpression of a dominant negative Stat3 mutant reduces FGF-2-mediated MMP7 promoter activity. However, Stat3 does not bind to the MMP7 promoter, but activates MMP7 gene expression indirectly via AP-1. This is confirmed by MMP7 promoter constructs with mutated AP-1 sites which did not respond to FGF-2 and by siRNAs against Stat1 and Stat3, which repressed FGF-2-induced MMP7 protein expression. In conclusion, we show that FGF-2-induced MMP7 expression in endothelium depends on AP-1 and FGF-2 signaling to AP-1 involves a Stat1/3-dependent pathway.