Lifang Shao

Complex regulation of human inducible nitric oxide synthase gene transcription by Stat 1 and NF-κB

The human inducible nitric oxide synthase (hiNOS) gene is expressed in several disease states and is also important in the normal immune response. Previously, we described a cytokine-responsive enhancer between −5.2 and −6.1 kb in the 5′-flanking hiNOS promoter DNA, which contains multiple nuclear factor κβ (NF-κB) elements. Here, we describe the role of the IFN-Jak kinase-Stat (signal transducer and activator of transcription) 1 pathway for regulation of hiNOS gene transcription. In A549 human lung epithelial cells, a combination of cytokines tumor necrosis factor-α, interleukin-1β, and IFN-γ (TNF-α, IL-1β, and IFN-γ) function synergistically for induction of hiNOS transcription. Pharmacological inhibitors of Jak2 kinase inhibit cytokine-induced Stat 1 DNA-binding and hiNOS gene expression. Expression of a dominant-negative mutant Stat 1 inhibits cytokine-induced hiNOS reporter expression. Site-directed mutagenesis of a cis-acting DNA element at −5.8 kb in the hiNOS promoter identifies a bifunctional NF-κB/Stat 1 motif. In contrast, gel shift assays indicate that only Stat 1 binds to the DNA element at −5.2 kb in the hiNOS promoter. Interestingly, Stat 1 is repressive to basal and stimulated iNOS mRNA expression in 2fTGH human fibroblasts, which are refractory to iNOS induction. Overexpression of NF-κB activates hiNOS promoter–reporter expression in Stat 1 mutant fibroblasts, but not in the wild type, suggesting that Stat 1 inhibits NF-κB function in these cells. These results indicate that both Stat 1 and NF-κB are important in the regulation of hiNOS transcription by cytokines in a complex and cell type-specific manner.

Identification of a negative response element in the human inducible nitric-oxide synthase (hiNOS) promoter: The role of NF-κB-repressing factor (NRF) in basal repression of the hiNOS gene

Although nuclear factor (NF)-κB plays a central role in mediating cytokine-stimulated human inducible nitric-oxide synthase (hiNOS) gene transcription, very little is known about the factors involved in silencing of the hiNOS promoter. NF-κB-repressing factor (NRF) interacts with a specific negative regulatory element (NRE) to mediate transcriptional repression of certain NF-κB responsive genes. By sequence comparison with the IFN-β and IL-8 promoters, we identified an NRE in the hiNOS promoter located at −6.7 kb upstream. In A549 and HeLa human cells, constitutive NRF mRNA expression is detected by RT-PCR. Gel shift assay showed constitutive NRF binding to the hiNOS NRE. Mutation of the −6.7-kb NRE site in the hiNOS promoter resulted in loss of NRF binding and increased basal but not cytokine-stimulated hiNOS transcription in promoter transfection experiments. Interestingly, overexpression of NRF suppressed both basal and cytokine-induced hiNOS promoter activity that depended on an intact cis-acting NRE motif. By using stably transformed HeLa cells with the tetracycline on/off expression system, reduction of cellular NRF by expressing antisense NRF increased basal iNOS promoter activity and resulted in constitutive iNOS mRNA expression. These data demonstrate that the transacting NRF protein is involved in constitutive silencing of the hiNOS gene by binding to a cis-acting NRE upstream in the hiNOS promoter.

Regulation of Human Nitric Oxide Synthase 2 Expression by Wnt β-Catenin Signaling

Nitric oxide (NO.), an important mediator of inflammation, and beta-catenin, a component of the Wnt-adenomatous polyposis coli signaling pathway, contribute to the development of cancer. We have identified two T-cell factor 4 (Tcf-4)-binding elements (TBE1 and TBE2) in the promoter of human inducible NO synthase 2 (NOS2). We tested the hypothesis that beta-catenin regulates human NOS2 gene. Mutation in either of the two TBE sites decreased the basal and cytokine-induced NOS2 promoter activity in different cell lines. The promoter activity was significantly reduced when both TBE1 and TBE2 sites were mutated (P < 0.01). Nuclear extract from HCT116, HepG2, or DLD1 cells bound to NOS2 TBE1 or TBE2 oligonucleotides in electrophoretic mobility shift assays and the specific protein-DNA complexes were supershifted with anti-beta-catenin or anti-Tcf-4 antibody. Overexpression of beta-catenin and Tcf-4 significantly increased both basal and cytokine-induced NOS2 promoter activity (P < 0.01), and the induction was dependent on intact TBE sites. Overexpression of beta-catenin or Tcf-4 increased NOS2 mRNA and protein expression in HCT116 cells. Lithium chloride (LiCl), an inhibitor of glycogen synthase kinase-3beta, increased cytosolic and nuclear beta-catenin level, NOS2 expression, and NO. production in primary human and rat hepatocytes and cancer cell lines. Treatment with Wnt-3A-conditioned medium increased beta-catenin and NOS2 expression in fetal human hepatocytes. When administered in vivo, LiCl increased hepatic beta-catenin level in a dose-dependent manner with simultaneous increase in NOS2 expression. These data are consistent with the hypothesis that beta-catenin up-regulates NOS2 and suggest a novel mechanism by which the Wnt/beta-catenin signaling pathway may contribute to cancer by increasing NO. production.

Wnt/β-Catenin Signaling Regulates Cytokine-Induced Human Inducible Nitric Oxide Synthase Expression by Inhibiting Nuclear Factor-κB Activation in Cancer Cells

The human inducible nitric oxide synthase (hiNOS) gene is regulated by nuclear factor kappaB (NF-kappaB) and has recently been shown to be a target of the Wnt/beta-catenin pathway. In this study, we tested the hypothesis that Wnt/beta-catenin signaling might regulate cytokine- or tumor necrosis factor alpha (TNFalpha)-induced hiNOS expression through interaction with NF-kappaB. A cytokine mixture of TNFalpha + interleukin (IL)-1beta + IFNgamma induced a 2- to 3-fold increase in hiNOS promoter activity in HCT116 and DLD1 colon cells, but produced a 2-fold decrease in SW480 colon cancer cells. A similar differential activity was seen in liver cancer cells (HepG2, Huh7, and Hep3B). Overexpression of beta-catenin produced a dose-dependent decrease in NF-kappaB reporter activity and decreased cytokine mixture-induced hiNOS promoter activity. Gel shift for TNFalpha-induced hiNOS NF-kappaB activation showed decreased p50 binding and decreased NF-kappaB reporter activity in the beta-catenin-mutant HAbeta18 cells. Conversely, enhanced p50 binding and increased NF-kappaB reporter activity were seen in HAbeta85 cells, which lack beta-catenin signaling. Coimmunoprecipitation confirmed that beta-catenin complexed with both p65 and p50 NF-kappaB proteins. NF-kappaB-dependent Traf1 protein expression also inversely correlated with the level of beta-catenin. Furthermore, SW480 cells stably transformed with wild-type adenomatous polyposis coli showed decreased beta-catenin protein and increased TNFalpha-induced p65 NF-kappaB binding as well as iNOS and Traf1 expression. Finally, beta-catenin inversely correlated with iNOS and Fas expression in vivo in hepatocellular carcinoma tumor samples. Our in vitro and in vivo data show that beta-catenin signaling inversely correlates with cytokine-induced hiNOS and other NF-kappaB-dependent gene expression. These findings underscore the complex role of Wnt/beta-catenin, NF-kappaB, and iNOS signaling in the pathophysiology of inflammation-associated carcinogenesis.

Ethyl pyruvate ameliorates liver ischemia-reperfusion injury by decreasing hepatic necrosis and apoptosis.

Background. Hepatic ischemia-reperfusion injury (I/R) occurs in the settings of transplantation, trauma, and elective liver resections. Reactive oxygen species (ROS) have been shown to play a major role in organ I/R injury. Pyruvate, a key intermediate in cellular metabolism, is an effective scavenger of ROS. The purpose of this study was to test the hypothesis that ethyl pyruvate (EP), a soluble pyruvate derivative, is effective in preventing hepatic I/R injury. Methods. Lewis rats underwent 60 minutes of partial warm hepatic ischemia. Three doses of EP dissolved in lactated Ringer’s solution or lactated Ringer’s solution (LR) alone were given by intravenous injection. Serum and tissue samples were obtained at 1 to 24 hours postreperfusion. Results. Serum transaminases, degree of hepatic necrosis, and neutrophil infiltration were all significantly decreased in the EP-treated rats compared with control animals. The amount of hepatic lipid peroxidation was also significantly decreased in EP-treated animals. Both circulating levels and hepatic expression of inflammatory cytokines were significantly decreased in the EP-treated animals. Furthermore, EP inhibited activation of extracellular signal-regulated kinase, p38, and c-Jun N-terminal kinase mitogen-activated protein kinases, as well as nuclear factor-&kgr;B, signaling pathways involved in cytokine release. Treatment with EP also inhibited hepatic apoptosis. Conclusion. EP has a protective effect on hepatic I/R injury, mediated in part by decreasing lipid peroxidation, down-regulation of inflammatory mediators, and inhibition of apoptosis. Strategies using this additive to LR solution should be considered in clinical settings of ischemic liver injury to decrease organ damage.

Interferon-gamma induces autophagy with growth inhibition and cell death in human hepatocellular carcinoma (HCC) cells through interferon-regulatory factor-1 (IRF-1)

Interferon-gamma (IFN-γ) is a pleiotropic cytokine with immunomodulatory, anti-viral, and anti-proliferative effects. In this study, we examined the effects of IFN-γ on autophagy and cell growth in human hepatocellular carcinoma (HCC) cells. IFN-γ inhibited cell growth of Huh7 cells with non-apoptotic cell death. IFN-γ induced autophagosome formation and conversion/turnover of microtubule associated protein 1 light chain 3 (LC3) protein. Furthermore, overexpression of IRF-1 also induced autophagy in Huh7 cells. Silencing IRF-1 expression with target small hairpin RNA blocked autophagy induced by IFN-γ. Silencing of the autophagy signals Beclin-1 or Atg5 attenuated the inhibitory effect of IFN-γ on Huh7 cells with decreased cell death. Additionally, IFN-γ activated autophagy in freshly cultured human HCC cells. Together, these findings show that IFN-γ induces autophagy through IRF-1 signaling pathway and the induction of autophagy contributes to the growth-inhibitory effect of IFN-γ with cell death in human liver cancer cells.

Identification of a classic cytokine-induced enhancer upstream in the human iNOS promoter

The human inducible NOS (iNOS) promoter transcriptionally regulated by 5 flanking region extending 16 kb upstream that contains cytokine‐responsive DNA motifs. In this study, we further identified a classic inducible enhancer located between −5 and −6 kb in the hiNOS upstream promoter. This ~1 kb promoter sequence functions as a cytokine‐inducible enhancer in an orientation‐ and position‐independent manner in human lung A549 and liver AKN1 cells. This DNA enhancer also confers cytokine inducibility to the heterologous thymidine kinase (TK) promoter. Chromatin immunoprecipitation (ChIP) analysis was applied, and confirmed cytokine‐inducible in vivo DNA‐protein interactions within this enhancer region. In vivo functional binding of both NF‐κB (p65/p50) and Stat‐1 at the −5.8 kb human iNOS promoter site was significantly increased in A549 cells after cytokine stimulation, while only Stat‐1 bound at the −5.2 kb site. These results identify the −5 to −6 kb promoter region as a classic transcriptional enhancer for the human iNOS gene and provide definitive in vivo evidence of specific NF‐κB and Stat‐1 nuclear protein binding that mediates transcription of the hiNOS gene under cytokine stimulation.—Guo, Z., Shao, L., Du, Q., Park, K. S., Geller, D. A. Identification of a classic cytokine‐induced enhancer upstream in the human iNOS promoter. FASEB J. 21, 535–542 (2006)

miRNA-939 regulates human inducible nitric oxide synthase posttranscriptional gene expression in human hepatocytes

Human inducible nitric oxide synthase (hiNOS) gene expression is regulated by transcriptional and posttranscriptional mechanisms. The purpose of this study was to determine whether specific microRNA (miRNA) directly regulate hiNOS gene expression. Sequence analysis of the 496-bp hiNOS 3′-untranslated region (3′-UTR) revealed five putative miR-939 binding sites. The hiNOS 3′-UTR conferred significant posttranscriptional blockade of luciferase activity in human A549, HCT8, and HeLa cells. The hiNOS 3′-UTR also exerted basal and cytokine-stimulated posttranscriptional repression in an orientation-dependent manner. Functional studies demonstrated that transfection of miR-939 into primary human hepatocytes (HCs) significantly inhibited cytokine-induced NO synthesis in a dose-dependent manner that was abrogated by a specific miR-939 inhibitor. MiR-939 (but not other miRNAs) abolished cytokine-stimulated hiNOS protein in human HC, but had no effect on hiNOS mRNA levels. Site-directed mutagenesis of miR-939 bindings sites at +99 or +112 bp in the hiNOS 3′-UTR increased reporter gene expression. Furthermore, intact miR-939 binding sites at +99 or +112 positions were required for posttranscriptional suppression by miR-939. Cytokine stimulation directly increased miR-939 levels in human HC. Transfection of miR-939 inhibitor (antisense miR-939) enhanced cytokine-induced hiNOS protein and increased NO synthesis in vitro in human HC. Finally, cytokine or LPS injection in vivo in mice increased hepatic miR-939 levels. Taken together, these data identify that miR-939 directly regulates hiNOS gene expression by binding in the 3′-UTR to produce a translational blockade. These findings suggest dual regulation of iNOS gene expression where cytokines induce iNOS transcription and also increase miR-939, leading to translational inhibition in a check-and-balance system.

A critical role for C/EBPbeta binding to the AABS promoter response element in the human iNOS gene.

The human iNOS (hiNOS) gene is expressed in a tissue‐specific manner, but the molecular basis for this regulation has not been elucidated. Here, we show that liver cell‐specific hiNOS gene activation involves protein‐DNA binding to an A‐activator binding site (AABS) located at ‐192 nucleotides in the hiNOS promoter region. Mutation of this site in the −7.2 kb hiNOS promoter construct inhibited basal hiNOS promoter activity in primary rat hepatocytes (77%), and two human liver cell lines, AKN‐1 (63%) and HepG2 (60%), but had no significant effect on basal hiNOS activity in three non‐hepatic human cell types. Interestingly, mutation of AABS significantly abrogated cytokine‐induced promoter activity in all cell types. C/EBPβ transcription factor bound to AABS by gel shift assay. Overexpression of C/EBPβ active form (LAP) increased hiNOS basal promoter activity ∼sixfold in liver cells, but had minimal effect in non‐hepatic cells. In contrast, overexpression of the transcriptional inhibitor (LIP) strongly suppressed both basal and cytokine‐inducible promoter activity. These data show that the cis‐acting AABS DNA element mediates liver‐specific basal hiNOS promoter activity through binding of the trans‐acting C/EBPβ factor. Further, C/EBPβ binding to AABS functions as a “switchpoint” that is necessary for cytokine‐inducible hiNOS gene expression in all cell types examined.

Hepatic B7 homolog 1 expression is essential for controlling cold ischemia/reperfusion injury after mouse liver transplantation†‡

Ischemia/reperfusion (I/R) injury remains a key risk factor significantly affecting morbidity and mortality after liver transplantation (LT). B7 homolog 1 (B7‐H1), a recently identified member of the B7 family, is known to play important roles in regulating local immune responses. We hypothesized that B7‐H1 plays crucial roles during innate immune responses induced by hepatic I/R injury, and using B7‐H1 knockout (KO) liver grafts, we tested this hypothesis in the mouse LT model with 24 hours of cold storage. Cold I/R injury in wild type (WT)‐to‐WT LT enhanced constitutive B7‐H1 expression on dendritic cells and sinusoidal endothelial cells and promptly induced B7‐H1 on hepatocytes. When B7‐H1 KO liver grafts were transplanted into WT recipients, serum alanine aminotransferase (ALT) and graft necrosis levels were significantly higher than those after WT‐to‐WT LT. Augmented tissue injury in B7‐H1 KO grafts was associated with increased frequencies and absolute numbers of graft CD3+ T cells (particularly CD8+ T cells). B7‐H1 KO grafts had significantly fewer annexin V+ CD8+ T cells, and this indicated a failure to delete infiltrating CD8+ T cells. To evaluate the relative contributions of parenchymal cell and bone marrow–derived cell (BMDC) B7‐H1 expression, we generated and transplanted into WT recipients chimeric liver grafts lacking B7‐H1 on parenchymal cells or BMDCs. A selective B7‐H1 deficiency on parenchymal cells or BMDCs resulted in similar levels of ALT and liver injury, and this suggested that parenchymal cell and BMDC B7‐H1 expression was involved in liver damage control. Human livers up‐regulated B7‐H1 expression after LT. Conclusion: The study demonstrates that graft tissue expression of B7‐H1 plays a critical role in regulating inflammatory responses during LT‐induced hepatic I/R injury, and negative coregulatory signals may have an important function in hepatic innate immune responses. (HEPATOLOGY 2011;)