Richard A. Shapiro

Multiple NF-κB Enhancer Elements Regulate Cytokine Induction of the Human Inducible Nitric Oxide Synthase Gene

The human inducible nitric oxide synthase (iNOS) gene is overexpressed in a number of human inflammatory diseases. Previously, we observed that the human iNOS gene is transcriptionally regulated by cytokines and demonstrated that the cytokine-responsive regions are upstream of −3.8 kilobase pairs (kb). Therefore, the purpose of this study was to further localize the functional enhancer elements and to assess the role of the transcription factor NF-κB in both human liver (AKN-1) and human lung (A549) epithelial cell lines. The addition of NF-κB inhibitors significantly suppressed cytokine-stimulated iNOS mRNA expression and NO synthesis, indicating that NF-κB is involved in the induction of the human iNOS gene. Analysis of the first 4.7 kb of the 5′-flanking region demonstrated basal promoter activity and failed to show any cytokine-inducible activity. However, promoter constructs extending to −5.8 and −7.2 kb revealed 2–3-fold and 4–5-fold induction, respectively, in the presence of cytokines. DNA sequence analysis from −3.8 to −7.2 kb identified five putative NF-κB cis-regulatory transcription factor binding sites upstream of −4.7 kb. Site-directed mutagenesis of these sites revealed that the NF-κB motif at −5.8 kb is required for cytokine-induced promoter activity, while the sites at −5.2, −5.5, and −6.1 kb elicit a cooperative effect. Electromobility shift assays using a site-specific oligonucleotide and nuclear extracts from cells stimulated with cytokine-mixture, tumor necrosis factor-α or interleukin-1β, but not interferon-γ, exhibited inducible DNA binding activity for NF-κB. These data indicate that NF-κB activation is required for cytokine induction of the human iNOS gene and identifies four NF-κB enhancer elements upstream in the human iNOS promoter that confer inducibility to tumor necrosis factor-α and interleukin-1β.

Role of Toll-Like Receptors in Changes in Gene Expression and NF-κB Activation in Mouse Hepatocytes Stimulated with Lipopolysaccharide

ABSTRACT The liver is an important site of host-microbe interaction. Although hepatocytes have been reported to be responsive to lipopolysaccharide (LPS), the global gene expression changes by LPS and mechanism(s) by which LPS stimulates cultured hepatocytes remain uncertain. Cultures of primary mouse hepatocytes were incubated with LPS to assess its effects on the global gene expression, hepatic transcription factors, and mitogen-activated protein (MAP) kinase activation. DNA microarray analysis indicated that LPS modulates the selective expression of more than 80 genes and expressed sequence tags. We have shown previously that hepatocytes express CD14, which is required both for uptake and responsiveness to LPS. In other cells, responsiveness to microbial products requires expression of Toll-like receptors (TLR) and their associated accessory molecules. Hepatocytes expressed TLR1 through TLR9 as well as MyD88 and MD-2 transcripts, as shown by reverse transcriptase PCR analysis, indicating that hepatocytes express all known microbe recognition molecules. The MAP kinase extracellular signal-regulated kinase 1/2 was phosphorylated in response to LPS in mouse hepatocytes, and the levels of phosphorylation were lower in hepatocytes from TLR4-null mice. NF-κB activation was reduced in TLR4-mutant or -null hepatocytes compared to control hepatocytes, and this defect was partially restored by adenoviral transduction of mouse TLR4. Thus, hepatocytes respond to nanogram concentrations of LPS through a TLR4 response pathway.

Toll-Like Receptor-4 Inhibits Enterocyte Proliferation via Impaired β-Catenin Signaling in Necrotizing Enterocolitis

BACKGROUND & AIMS Necrotizing enterocolitis (NEC), the leading cause of gastrointestinal death from gastrointestinal disease in preterm infants, is characterized by exaggerated TLR4 signaling and decreased enterocyte proliferation through unknown mechanisms. Given the importance of beta-catenin in regulating proliferation of many cell types, we hypothesize that TLR4 impairs enterocyte proliferation in NEC via impaired beta-catenin signaling. METHODS Enterocyte proliferation was detected in IEC-6 cells or in ileum or colon from wild-type, TLR4-mutant, or TLR4(-/-) mice after induction of NEC or endotoxemia. beta-Catenin signaling was assessed by cell fractionation or immunoconfocal microscopy to detect its nuclear translocation. Activation and inhibition of beta-catenin were achieved via cDNA or small interfering RNA, respectively. TLR4 in the intestinal mucosa was inhibited with adenoviruses expressing dominant-negative TLR4. RESULTS TLR4 activation significantly impaired enterocyte proliferation in the ileum but not colon in newborn but not adult mice and in IEC-6 enterocytes. beta-Catenin activation reversed these effects in vitro. To determine the mechanisms involved, TLR4 activation phosphorylated the upstream inhibitory kinase GSK3beta, causing beta-catenin degradation. NEC in both mouse and humans was associated with decreased beta-catenin and increased mucosal GSK3beta expression. Strikingly, the inhibition of enterocyte beta-catenin signaling in NEC could be reversed, and enterocyte proliferation restored, through adenoviral-mediated inhibition of TLR4 signaling in the small intestinal mucosa. CONCLUSION We now report a novel pathway linking TLR4 with inhibition of beta-catenin signaling via GSK3beta activation, leading to reduced enterocyte proliferation in vitro and in vivo. These data provide additional insights into the pathogenesis of diseases of intestinal inflammation such as NEC.

Nitric-oxide production by murine mammary adenocarcinoma cells promotes tumor-cell invasiveness

The role of nitric oxide (NO) in tumor biology remains controversial and poorly understood. While a few reports indicate that the presence of NO in tumor cells or their micro‐environment is detrimental for tumor‐cell survival, and consequently their metastatic ability, a large body of data suggests that NO promotes tumor progression. The purpose of this study was to identify the source of NO in the spontaneously metastasizing C3‐L5 murine mammary‐adenocarcinoma model, the role of tumor‐derived NO in tumor‐cell invasiveness, and the mechanisms underlying the invasion‐stimulating effects of tumor‐derived NO. The source of NO was established by immunocytochemical localization of NO synthase (NOS) enzymes in C3‐L5 cells in vitro and transplanted tumors in vivo. An in vitro transwell Matrigel invasion assay was used to test the invasiveness of C3‐L5 cells in the presence or the absence of NO blocking agents or iNOS inducers (IFN‐γ and LPS). The mechanisms underlying the invasion‐stimulating effects of tumor‐derived NO were examined by measuring mRNA expression of matrix metalloproteinases (MMP)‐2 and ‐9, and tissue inhibitors of metalloproteinases (TIMP) 1, 2 and 3 in C3‐L5 cells in various experimental conditions. Results showed that C3‐L5 cells expressed high level of eNOS protein in vitro, and in vivo, both in primary and in metastatic tumors. C3‐L5 cells also expressed iNOS mRNA and protein when cultured in the presence of IFN‐γ and LPS. Constitutively produced NO promoted tumor‐cell invasiveness in vitro by down‐regulating TIMP 2 and TIMP 3. In addition, there was up‐regulation of MMP‐2, when extra NO was induced by IFN‐γ and LPS. In conclusion, NO produced by C3‐L5 cells promoted tumor‐cell invasiveness by altering the balance between MMP‐2 and its inhibitors TIMP‐2 and 3. Thus, our earlier observations of anti‐tumor and anti‐metastatic effects of NO inhibitors in vivo in this tumor model can be explained, at least in part, by reduced tumor‐cell invasiveness. Int. J. Cancer 81:889–896, 1999.

Signaling of high mobility group box 1 (HMGB1) through toll-like receptor 4 in macrophages requires CD14.

High mobility group box 1 (HMGB1) is a DNA-binding protein that possesses cytokinelike, proinflammatory properties when released extracellularly in the C23–C45 disulfide form. HMGB1 also plays a key role as a mediator of acute and chronic inflammation in models of sterile injury. Although HMGB1 interacts with multiple pattern recognition receptors (PRRs), many of its effects in injury models occur through an interaction with toll-like receptor 4 (TLR4). HMGB1 interacts directly with the TLR4/myeloid differentiation protein 2 (MD2) complex, although the nature of this interaction remains unclear. We demonstrate that optimal HMGB1-dependent TLR4 activation in vitro requires the coreceptor CD14. TLR4 and MD2 are recruited into CD14-containing lipid rafts of RAW264.7 macrophages after stimulation with HMGB1, and TLR4 interacts closely with the lipid raft protein GM1. Furthermore, we show that HMGB1 stimulates tumor necrosis factor (TNF)-α release in WT but not in TLR4−/−, CD14−/−, TIR domain-containing adapter-inducing interferon-β (TRIF)−/− or myeloid differentiation primary response protein 88 (MyD88)−/− macrophages. HMGB1 induces the release of monocyte chemotactic protein 1 (MCP-1), interferon gamma-induced protein 10 (IP-10) and macrophage inflammatory protein 1α (MIP-1α) in a TLR4- and CD14-dependent manner. Thus, efficient recognition of HMGB1 by the TLR4/MD2 complex requires CD14.

Calcium/Calmodulin-Dependent Protein Kinase (CaMK) IV Mediates Nucleocytoplasmic Shuttling and Release of HMGB1 during Lipopolysaccharide Stimulation of Macrophages

The chromatin-binding factor high-mobility group box 1 (HMGB1) functions as a proinflammatory cytokine and late mediator of mortality in murine endotoxemia. Although serine phosphorylation of HMGB1 is necessary for nucleocytoplasmic shuttling before its cellular release, the protein kinases involved have not been identified. To investigate if calcium/calmodulin-dependent protein kinase (CaMK) IV serine phosphorylates and mediates the release of HMGB1 from macrophages (Mφ) stimulated with LPS, RAW 264.7 cells or murine primary peritoneal Mφ were incubated with either STO609 (a CaMKIV kinase inhibitor), KN93 (a CaMKIV inhibitor), or we utilized cells from which CaMKIV was depleted by RNA interference (RNAi) before stimulation with LPS. We also compared the LPS response of primary Mφ isolated from CaMKIV+/+ and CaMKIV−/− mice. In both cell types LPS induced activation and nuclear translocation of CaMKIV, which preceded HMGB1 nucleocytoplasmic shuttling. However, Mφ treated with KN93, STO609, or CaMKIV RNAi before LPS showed reduced nucleocytoplasmic shuttling of HMGB1 and release of HMGB1 into the supernatant. Additionally, LPS induced serine phosphorylation of HMGB1, which correlated with an interaction between CaMKIV and HMGB1 and with CaMKIV phosphorylation of HMGB1 in vitro. In cells, both HMGB1 phosphorylation and interaction with CaMKIV were inhibited by STO609 or CaMKIV RNAi. Similarly, whereas CaMKIV+/+ Mφ showed serine phosphorylation of HMGB1 in response to LPS, this phosphorylation was attenuated in CaMKIV−/− Mφ. Collectively, our results demonstrate that CaMKIV promotes the nucleocytoplasmic shuttling of HMGB1 and suggest that the process may be mediated through CaMKIV-dependent serine phosphorylation of HMGB1.

Cellular-specific role of toll-like receptor 4 in hepatic ischemia-reperfusion injury in mice.

Ischemia‐reperfusion (I/R) injury is a process whereby an initial hypoxic insult and subsequent return of blood flow leads to the propagation of innate immune responses and organ injury. The necessity of the pattern recognition receptor, Toll‐like receptor (TLR)4, for this innate immune response has been previously shown. However, TLR4 is present on various cell types of the liver, both immune and nonimmune cells. Therefore, we sought to determine the role of TLR4 in individual cell populations, specifically, parenchymal hepatocytes (HCs), myeloid cells, including Kupffer cells, and dendritic cells (DCs) subsequent to hepatic I/R. When HC‐specific (Alb‐TLR4−/−) and myeloid‐cell–specific (Lyz‐TLR4−/−) TLR4 knockout (KO) mice were subjected to warm hepatic ischemia, there was significant protection in these mice, compared to wild type (WT). However, the protection afforded in these two strains was significantly less than global TLR4 KO (TLR4−/−) mice. DC‐specific TLR4−/− (CD11c‐TLR4−/−) mice had significantly increased hepatocellular damage, compared to WT mice. Circulating levels of high‐mobility group box 1 (HMGB1) were significantly reduced in Alb‐TLR4−/− mice, compared to WT, Lyz‐TLR4−/−, CD11c‐TLR4−/− mice and equivalent to global TLR4−/− mice, suggesting that TLR4‐mediated HMGB1 release from HCs may be a source of HMGB1 after I/R. HCs exposed to hypoxia responded by rapidly phosphorylating the mitogen‐activated protein kinases, c‐Jun‐N‐terminal kinase (JNK) and p38, in a TLR4‐dependent manner; inhibition of JNK decreased release of HMGB1 after both hypoxia in vitro and I/R in vivo. Conclusion: These results provide insight into the individual cellular response of TLR4. The parenchymal HC is an active participant in sterile inflammatory response after I/R through TLR4‐mediated activation of proinflammatory signaling and release of danger signals, such as HMGB1. (HEPATOLOGY 2013)

Endotoxin uptake in mouse liver is blocked by endotoxin pretreatment through a suppressor of cytokine signaling‐1–dependent mechanism

The liver is the main organ that clears lipopolysaccharide (LPS) and hepatocytes are a major cell‐type involved in LPS uptake. LPS tolerance, or desensitization, is important in negative regulation of responses to LPS, but little is known about its mechanisms in hepatocytes. Primary isolated C57BL/6 hepatocytes, and liver in vivo, internalized fluorescent LPS, and this was dependent on Toll‐like receptor 4 (TLR4) at the cell surface but not on TLR4‐TIR signaling through MyD88. LPS clearance from plasma was also TLR4‐dependent. Pretreatment of C57BL/6 hepatocytes with LPS prevented uptake of LPS 24 hours later and this LPS‐mediated suppression was dependent on TLR4 signaling through MyD88. Many regulators of TLR4 signaling have been identified and implicated in LPS desensitization, including suppressor of cytokine signaling 1 (SOCS1). SOCS1 mRNA and protein expression increased after LPS stimulation in hepatocytes and in whole liver. LPS uptake in hepatocytes and liver was significantly reduced following infection with adenoviral vectors overexpressing SOCS1. Similarly, inhibition of SOCS1 using small interfering (si)RNA‐mediated knockdown prevented LPS desensitization in hepatocytes. SOCS1 is known to interact with Toll/IL‐1 receptor associated protein (TIRAP) and cause TIRAP ubiquitination and degradation, which regulates TLR signaling. We have also shown previously that TIRAP regulates LPS uptake in hepatocytes. SOCS1 coimmunoprecipitated with TIRAP in wild type hepatocyte cell lysates up to 8 hours after LPS stimulation, but not at later times. In the same samples, ubiquitinated TIRAP was detected after 4 hours and up to 8 hours after LPS stimulation, but not at later times. Conclusion: These data indicate hepatocytes are desensitized by LPS in a TLR4 signaling‐dependent manner. LPS‐induced SOCS1 upregulation increases degradation of TIRAP and prevents subsequent LPS uptake. The exploitation of these mechanisms of LPS desensitization in the liver may be important in future sepsis therapies. (HEPATOLOGY 2009.)

Extracellular High Mobility Group Box-1 (HMGB1) Inhibits Enterocyte Migration via Activation of Toll-like Receptor-4 and Increased Cell-Matrix Adhesiveness

Toll-like receptor-4 (TLR4) is the receptor for bacterial lipopolysaccharide, yet it may also respond to a variety of endogenous molecules. Necrotizing enterocolitis (NEC) is the leading cause of death from gastrointestinal disease in newborn infants and is characterized by intestinal mucosal destruction and impaired enterocyte migration due to increased TLR4 signaling on enterocytes. The endogenous ligands for TLR4 that lead to impaired enterocyte migration remain unknown. High mobility group box-1 (HMGB1) is a DNA-binding protein that is released from injured cells during inflammation. We thus hypothesize that extracellular HMGB1 inhibits enterocyte migration via activation of TLR4 and sought to define the pathways involved. We now demonstrate that murine and human NEC are associated with increased intestinal HMGB1 expression, that serum HMGB1 is increased in murine NEC, and that HMGB1 inhibits enterocyte migration in vitro and in vivo in a TLR4-dependent manner. This finding was unique to enterocytes as HMGB1 enhanced migration of inflammatory cells in vitro and in vivo. In seeking to understand the mechanisms involved, TLR4-dependent HMGB1 signaling increased RhoA activation in enterocytes, increased phosphorylation of focal adhesion kinase, and increased phosphorylation of cofilin, resulting in increased stress fibers and focal adhesions. Using single cell force traction microscopy, the net effect of HMGB1 signaling was a TLR4-dependent increase in cell force adhesion, accounting for the impaired enterocyte migration. These findings demonstrate a novel pathway by which TLR4 activation by HMGB1 delays mucosal repair and suggest a novel potential therapeutic target in the amelioration of intestinal inflammatory diseases like NEC.

The hepatocyte as a microbial product-responsive cell.

Much research has focused on the responses to microbial products of immune cells such as monocytes, macrophages, and neutrophils. Although the liver is a primary response organ in various infections, relatively little is known about the antimicrobial responses of its major cell type, the hepatocyte. It is now known that the recognition of bacteria occurs via cell-surface proteins that are members of the Toll-like receptor (TLR) family. In addition, lipopolysaccharide (LPS) is bound by circulating LPS-binding protein (LBP) and presented to cell-surface CD14, which in turn interacts with TLR and transduces an intracellular signal. We investigated the CD14 and TLR2 responses of whole liver and isolated hepatocytes, and demonstrated that these cells can be induced to express the molecules necessary for responses to both Gram-positive and Gram-negative bacteria. Our findings may have clinical implications for pathological states such as sepsis.