Nitric oxide is a short-lived biologic mediator for diverse cell types. Synthesis of an inducible nitric oxide synthase (NOS) in murine macrophages is stimulated by lipopolysaccharide (LPS) and interferon y. In human hepatocytes, NOS activity is induced by treatment with a combination of tumor necrosis factor, interleukin 1, interferon y, and LPS. We now report the molecular cloning and expression of an inducible human hepatocyte NOS (hep-NOS) cDNA. hep-NOS has 80% amino acid sequence homology to macrophage NOS (mac-NOS Nitric oxide (NO) is a recently recognized messenger molecule mediating diverse functions including vasodilation, neurotransmission, and antimicrobial and antitumor activities. Different cells such as macrophages (1, 2), endothelial cells (3, 4), neurons (5, 6), smooth muscle cells (7,8), and cardiac myocytes (9) produce NO from L-arginine. Constitutive and inducible isoforms of NO synthase (NOS) differ in structure and regulation (reviewed in refs. 10 and 11). Constitutive NOS has been cloned from rat cerebellum (12) and bovine (13,14) and human (15, 16) endothelial cells, whereas inducible murine macrophage NOS (mac-NOS) has been cloned from RAW 264.7 cells (17)(18)(19).Rat hepatocytes make NO in vivo during chronic hepatic inflammation (20,21) and in vitro in response to conditioned Kupffer cell supernatant (22) or to lipopolysaccharide (LPS) and the cytokines tumor necrosis factor (TNF), interleukin 1 (IL-1), and interferon y (IFN-y) (23-25). Since rats treated with LPS manifest inducible NOS in numerous tissues with few macrophages (26), it is possible that more than one isoform of inducible NOS exists. Evidence for inducible human NOS activity has been shown in patients receiving IL-2 cancer therapy (27, 28) and during sepsis (29). Recently, human hepatocytes were stimulated to produce NO by the same combination of LPS and cytokines as rat hepatocytes, providing evidence that a specific human cell expresses inducible NOS (30). We now report the cloning and functional expression of a distinct form of inducible NOS from human hepatocytes. 11 MATERIALS AND METHODSIsolation of Human Hepatocytes. Human hepatocytes were isolated from histologically normal operative wedge resections (in accordance with institutional approval) by using a modification of an in situ collagenase procedure (type IV; Sigma) (30). Briefly, hepatocytes were separated from nonparenchymal cells by differential centrifugation four times at 50 x g. The hepatocytes were then further purified over a 30% Percoll gradient at a concentration of 106 hepatocytes per ml of Percoll to obtain a highly purified cell population (31). Hepatocyte purity by microscopy was >98%, and viability consistently exceeded 95% by trypan blue exclusion.Cell Culture. Hepatocytes (5 x 106) were plated onto 100-mm gelatin-coated Petri dishes (Coming) in 6 ml of culture medium. Medium consisted of Williams media E (GIBCO) with L-arginine (0.50 mM), insulin (1 uM), Hepes (15 mM), L-glutamine, penicillin, streptomycin, and 10%o (vol/vol) low endotox...

Nitric oxide (NO-) is a short-lived mediator which can be induced in a variety of cell types and produces many physiologic and metabolic changes in target cells. The inducible or high-output NO' synthase (NOS) pathway was first characterized in macrophages activated by lipopolysaccharide (LPS) and interferon v (IFN-y). Hepatocytes also express an inducible NOS following exposure to the combination of endotoxin (LPS) and tumor necrosis factor (TNF), interleukin 1 Following the discovery of the nitric oxide (NO-) pathway and its identification as endothelium-derived relaxing factor, a variety of cell types such as macrophages (1, 2), endothelial cells (3, 4), smooth muscle cells (5), and neurons (6, 7) have been shown to produce NO' from L-arginine. Constitutive and inducible isoforms of the NO' synthase (NOS) enzyme exist, and they differ in structure and regulation (8). The neuronal constitutive NOS is a 150-kDa protein whose activity is dependent upon calcium and calmodulin (7); the inducible macrophage NOS is a 130-kDa protein which is thought to function independently of calcium/calmodulin (9, 10). The constitutive NOS cDNA has been cloned from rat cerebellum and identifies an ==10-kilobase (kb) mRNA on Northern blot analysis (11), while the inducible murine macrophage NOS has been cloned from RAW264.7 cells by three groups and identifies an -4.4-kb mRNA (12-14). The physiologic importance of NON as a vasodilator, neurotransmitter, and antimicrobial/antitumor agent is rapidly becoming apparent. Previous work showed that rat hepatocyte/Kupffer cell cocultures stimulated with lipopolysaccharide (LPS) produce large amounts of nitrite (NO-) and nitrate (NO-), the stable end products of the NO-pathway (15). Further, it was demonstrated that hepatocytes also produce NO-in vivo during chronic hepatic inflammation (16,17) and in vitro in response to conditioned Kupffer cell supernatant (18) or to a mixture of LPS and the cytokines tumor necrosis factor (TNF), interleukin 1 (IL-1), and interferon y (IFN-y) (19). Human hepatocytes were also stimulated to produce NO' by the same combination of endotoxin and cytokines as rat hepatocytes (20). However, essentially nothing has been reported about the direct signals required for inducible NOS gene activation. Therefore, the present study was undertaken to characterize the molecular regulation of the inducible rat hepatocyte NOS by endotoxin and cytokines known to up-regulate hepatocyte NO' synthesis. MATERIALS AND METHODSIsolation of Hepatocytes. Hepatocytes were isolated from male rats (200-250 g, Harlan-Sprague-Dawley) by a modification of the in situ collagenase (Sigma) perfusion technique of Seglen (21). Hepatocytes were separated from nonparenchymal cells by differential centrifugation at 50 x g and then passed over a 30%o Percoll gradient to obtain a highly purified cell population. Hepatocyte purity assessed by microscopy was >98% and viability consistently exceeded 95% by trypan blue exclusion. For the in vivo studies, hepatocytes were harvested from rats 3...

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-B in both human liver (AKN-1) and human lung (A549) epithelial cell lines. The addition of NF-B inhibitors significantly suppressed cytokinestimulated iNOS mRNA expression and NO synthesis, indicating that NF-B 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-B cis-regulatory transcription factor binding sites upstream of ؊4.7 kb. Site-directed mutagenesis of these sites revealed that the NF-B 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-␣ or interleukin-1␤, but not interferon-␥, exhibited inducible DNA binding activity for NF-B. These data indicate that NF-B activation is required for cytokine induction of the human iNOS gene and identifies four NF-B enhancer elements upstream in the human iNOS promoter that confer inducibility to tumor necrosis factor-␣ and interleukin-1␤.

The expression of inducible nitric oxide synthase (NOS2) is complex and is regulated in part by gene transcription. In this investigation we studied the regulation of NOS2 in a human liver epithelial cell line (AKN-1) which expresses high levels of NOS2 mRNA and protein in response to tumor necrosis factor a, interleukin 113, and interferon y (cytokine mix, CM). Nuclear run-on analysis revealed that CM transcript"onally activated the human NOS2 gene. To delineate the cytokine-responsive regions of the human NOS2 promoter, we stimulated AKN-1 cells with CM following transfection of NOS2 luciferase constructs. Analysis of the first 3.8 kb upstream of the NOS2 gene demonstrated basal promoter activity but failed to show any cytokine-inducible activity. However, 3-to 5-fold inductions of luciferase activity were seen in constructs extending up to -5.8 and -7.0 kb, and a 10-fold increase was seen upon transfection of a -16 kb construct.' Further analysis of various NOS2 luciferase constructs ligated upstream of the thymidine kinase promoter identified three regions containing cytokine-responsive elements in the human NOS2 gene: -3.8 to -5.8, -5.8 to -7.0, and -7.0 to -16 kb. These results are in marked contrast with the murine macrophage NOS2 promoter in which only 1 kb of the proximal 5' flanking region is necessary to confer inducibility to lipopolysaccharide and interferon y. These data demonstrate that the human NOS2 gene is transcriptionally regulated by cytokines and identify multiple cytokineresponsive regions in the 5' flanking region of the human NOS2 gene.

Background and 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 β-catenin in regulating proliferation of many cell types, we hypothesize that TLR4 impairs enterocyte proliferation in NEC via impaired β-catenin signaling.