Karl-Friedrich Beck

Amplification of IL-1β-Induced Matrix Metalloproteinase-9 Expression by Superoxide in Rat Glomerular Mesangial Cells Is Mediated by Increased Activities of NF-κB and Activating Protein-1 and Involves Activation of the Mitogen-Activated Protein Kinase Pathways

The modulation of cell signaling by free radicals is important for the pathogenesis of inflammatory diseases. Recently, we have shown that NO reduces IL-1β-induced matrix metalloproteinase (MMP-9) expression in glomerular mesangial cells (MC). Here we report that exogenously administrated superoxide, generated by the hypoxanthine/xanthine oxidase system (HXXO) or by the redox cycler 2,3-dimethoxy-1,4-naphtoquinone, caused a marked amplification of IL-1β-primed, steady state, MMP-9 mRNA level and an increase in gelatinolytic activity in the conditioned medium. Superoxide generators alone were ineffective. Cytokine-induced steady state mRNA levels of TIMP-1, an endogenous inhibitor of MMP-9, were affected similarly by HXXO. Transient transfection of rat mesangial cells with 0.6 kb of the 5′-flanking region of the rat MMP-9 gene proved a transcriptional regulation of MMP-9 expression by superoxide. HXXO augmented the IL-1β-triggered nuclear translocation of p65 and c-Jun and, in parallel, increased DNA binding activities of NF-κB and AP-1. Mutation of either response element completely prevented MMP-9 promoter activation by IL-1β. Moreover, specific inhibitors of the classical extracellular signal-regulated kinase (ERK) pathway and p38 mitogen-activated protein kinase (MAPK) cascade, partially reversed the HXXO-mediated effects on MMP-9 mRNA levels, thus demonstrating involvement of ERKs and p38 MAPKs in MMP-9 expression. Furthermore, IL-1β-triggered phosphorylation of all three MAPKs, including p38-MAPK, c-Jun N-terminal kinase, and ERK, was substantially enhanced by superoxide. Our data identify superoxide as a costimulatory factor amplifying cytokine-induced MMP-9 expression by interfering with the signaling cascades leading to the activation of AP-1 and NF-κB.

Arginase 1 Overexpression in Psoriasis: Limitation of Inducible Nitric Oxide Synthase Activity as a Molecular Mechanism for Keratinocyte Hyperproliferation

Abnormal proliferation of keratinocytes in the skin appears crucial to the pathogenesis of psoriasis, but the underlying mechanisms remain unknown. Nitric oxide (NO), released from keratinocytes at high concentrations, is considered a key inhibitor of cellular proliferation and inducer of differentiation in vitro. Although high-output NO synthesis is suggested by the expression of inducible NO synthase (iNOS) mRNA and protein in psoriasis lesions, the pronounced hyperproliferation of psoriatic keratinocytes may indicate that iNOS activity is too low to effectively deliver anti-proliferative NO concentrations. Here we show that arginase 1 (ARG1), which substantially participates in the regulation of iNOS activity by competing for the common substrate L-arginine, is highly overexpressed in the hyperproliferative psoriatic epidermis and is co-expressed with iNOS. Expression of L-arginine transporter molecules is found to be normal. Treatment of primary cultured keratinocytes with Th1-cytokines, as present in a psoriatic environment, leads to de novo expression of iNOS but concomitantly a significant down-regulation of ARG1. Persistent ARG1 overexpression in psoriasis lesions, therefore, may represent a disease-associated deviation from normal expression patterns. Furthermore, the culturing of activated keratinocytes in the presence of an ARG inhibitor results in a twofold increase in nitrite accumulation providing evidence for an L-arginine substrate competition in human keratinocytes. High-output NO synthesis is indeed associated with a significant decrease in cellular proliferation as shown by down-regulation of Ki67 expression in cultured keratinocytes but also in short-term organ cultures of normal human skin. In summary, our data demonstrate for the first time a link between a human inflammatory skin disease, limited iNOS activity, and ARG1 overexpression. This link may have substantial implications for the pathophysiology of psoriasis and the development of new treatment strategies.

Regulation of gene expression by nitric oxide.

Abstract. Nitric oxide (NO) modulates transcription factors that bind specific cis-regulatory DNA responsible for coordinating the spatial and temporal patterns of gene expression that are initiated by a changing microenvironment. In this way NO helps to orchestrate gene transcription and forms the basis of functional cell responses to accommodate metabolic requirements and to coordinate endogenous defense mechanisms against a variety of stress and disease conditions. There is marked overlap between the signalling pathways triggered by NO, superoxide, and hypoxia. Understanding the redox-based regulation of signal transduction and gene expression will provide insights into how cell activities are constantly coordinated and how promising new therapies may be developed.

Cloning and sequencing of the proximal promoter of the rat iNOS gene: activation of NFκB is not sufficient for transcription of the iNOS gene in rat mesangial cells

It has previously been shown that expression of the inducible form of NO synthase (EC 1.14.23) is controlled at the transcriptional level and that induction of iNOS transcription is dependent on activation of transcription factors of the NFκB family. TNF‐α and IL‐1β synergistically stimulate iNOS transcription in rat glomerular mesangial cells. We have recently reported that endothelin‐1 completely blocks cytokine‐induced iNOS expression at the transcriptional level. To further investigate the molecular mechanisms and the role of NFκB in cytokine‐elicited iNOS transcription, we cloned a 661 bp genomic rat DNA fragment, which contains 497 bp of the proximal iNOS promoter. An NFκB‐binding site identical to that described for the murine sequence was identified and used for electrophoretic mobility shift experiments. We found that binding of NFκB is strongly induced in mesangial cells by both IL‐1β and TNF‐α. While endothelin‐1 blocks cytokine‐induced iNOS expression, it has no influence on the binding pattern of NFκB. We conclude from these data that transcription of iNOS in mesangial cells requires additional signals besides activation of NFκB.

Inhibition of Cytokine-induced Matrix Metalloproteinase 9 Expression by Peroxisome Proliferator-activated Receptor α Agonists Is Indirect and Due to a NO-mediated Reduction of mRNA Stability

Rat renal mesangial cells express high levels of matrix metalloproteinase 9 (MMP-9) in response to inflammatory cytokines such as interleukin 1β (IL-1β). We tested whether ligands of the peroxisome proliferator-activated receptor (PPARα) could influence the cytokine-induced expression of MMP-9. Different PPARα agonists dose-dependently inhibited the IL-1β-triggered increase in gelatinolytic activity mainly by decreasing the MMP-9 steady-state mRNA levels. PPARα agonists on their own had no effects on MMP-9 mRNA levels and gelatinolytic activity. Surprisingly, the reduction of MMP-9 mRNA levels by PPARα activators contrasted with an amplification of cytokine-mediated MMP-9 gene promoter activity and mRNA expression. The potentiation of MMP-9 promoter activity functionally depends on an upstream peroxisome proliferator-responsive element-like binding site, which displayed an increased DNA binding of a PPARα immunopositive complex. In contrast, the IL-1β-induced DNA-binding of nuclear factor κB was significantly impaired by PPARα agonists. Most interestingly, in the presence of an inducible nitric-oxide synthase (iNOS) inhibitor, the PPARα-mediated suppression switched to a strong amplification of IL-1β-triggered MMP-9 mRNA expression. Concomitantly, activators of PPARα potentiated the cytokine-induced iNOS expression. Using actinomycin D, we found that NO, but not PPARα activators, strongly reduced the stability of MMP-9 mRNA. In contrast, the stability of MMP-9 protein was not affected by PPARα activators. In summary, our data suggest that the inhibitory effects of PPARα agonists on cytokine-induced MMP-9 expression are indirect and primarily due to a superinduction of iNOS with high levels of NO reducing the half-life of MMP-9 mRNA.

Cooperative Binding of NF-Y and Sp1 at the DNase I-hypersensitive Site, Fatty Acid Synthase Insulin-responsive Element 1, Located at −500 in the Rat Fatty Acid Synthase Promoter

In vitro DNase I footprint analysis of the rat fatty acid synthase (FAS) promoter from −568 to −468 revealed four protein binding sites: A, B, and C boxes and the FAS insulin-responsive element 1 (FIRE1). As demonstrated by gel mobility shift analysis and supershift experiments, FIRE1, located between −516 and −498, is responsible for binding NF-Y. The C box located downstream of FIRE1 was shown by in vitro footprinting to be a Sp1 binding site, and furthermore, competition with Sp1 also abolished FIRE1 binding. Since the half-life of the Sp1·NF-Y·DNA complex is significantly longer than the half-lives of the Sp1·DNA or NF-Y·DNA complexes, the two transcription factors are deemed to bind cooperatively in the FAS promoter at −500. It is unusual that NF-Y binds at this distance from the start site of transcription. NF-Y binding sites are found in the promoters of at least three other FAS genes, viz. goose, chicken, and man. A second NF-Y binding site is located in the FAS promoter at the more usual position of −103 to −87, and it too has a neighboring Sp1 site. CTF/NF-1 competes for proteins binding to the B box. The A box binds Sp1 and contains a 12/13 match of the inverted repeat sequence responsible for binding the nuclear factor EF-C/RFX-1 in the enhancer regions of hepatitis B virus and the major histocompatibility complex class II antigen promoter. The same relative positions of NF-Y and Sp1 binding sites in the promoters of FAS genes of goose, rat, chicken, and man emphasize the involvement of these transcription factors in the diet and hormonal regulation of FAS.

Glucocorticoids inhibit lipopolysaccharide-induced up-regulation of arginase in rat alveolar macrophages

As arginase by limiting nitric oxide (NO) synthesis may play a role in airway hyperresponsiveness and glucocorticoids are known to induce the expression of arginase I in hepatic cells, glucocorticoid effects on arginase in alveolar macrophages (AMΦ) were studied. Rat AMΦ were cultured in absence or presence of test substances. Thereafter, nitrite accumulation, arginase activity, and the expression pattern of inducible NO synthase, arginase I and II mRNA (RT – PCR) and proteins (immunoblotting) were determined. Lipopolyssacharides (LPS, 20 h) caused an about 2 fold increase in arginase activity, whereas interferon‐γ (IFN‐γ), like LPS a strong inducer of NO synthesis, had no effect. Dexamethasone decreased arginase activity by about 25% and prevented the LPS‐induced increase. Mifepristone (RU‐486) as partial glucocorticoid receptor agonist inhibited LPS‐induced increase by 45% and antagonized the inhibitory effect of dexamethasone. Two different inhibitors of the NF‐κB‐pathway also prevented LPS‐induced increase in arginase activity. Rat AMΦ expressed mRNA and protein of arginase I and II, but arginase I expression was stronger. Arginase I mRNA and protein was not affected by IFN‐γ, but increased by LPS and this effect was prevented by dexamethasone. Both, LPS and IFN‐γ enhanced the levels of arginase II mRNA and protein, effects also inhibited by dexamethasone. As IFN‐γ did not affect total arginase activity, arginase II may represent only a minor fraction of total arginase activity. In rat AMΦ glucocorticoids inhibit LPS‐induced up‐regulation of arginase activity, an effect which may contribute to the beneficial effects of glucocorticoids in the treatment of inflammatory airway diseases.

Nitric oxide down-regulates the expression of the catalytic NADPH oxidase subunit Nox1 in rat renal mesangial cells

Glomerular mesangial cells can produce high amounts of nitric oxide (NO) and reactive oxygen species (ROS). Here we analyzed the impact of NO on the ROS‐generating system, particularly on the NADPH oxidase Nox1. Nox1 mRNA and protein levels were markedly decreased by treatment of mesangial cells with the NO‐releasing compound DETA‐NO in a concentration‐ and time‐dependent fashion. By altering the cGMP signaling system with different inhibitors or activators, we revealed that the effect of NO on Nox1 expression is at least in part mediated by cGMP. Analysis of a reporter construct comprising the 2547 bp of the nox1 promoter region revealed that a stimulatory effect of IL‐1β on nox1 transcription is counteracted by an inhibitory effect of IL‐1β‐evoked endogenous NO formation. Moreover, pretreatment of mesangial cells with DETA‐NO attenuated platelet‐derived growth factor (PDGF)‐BB or serum stimulated production of superoxide as assessed by real‐time EPR spectroscopy and dichlorofluorescein formation. Transfection of mesangial cells with siRNAs directed against Nox1 and Nox4 revealed that inhibition of Nox1, but not Nox4 expression, is responsible for the reduced ROS formation by NO. Obviously, there exists a fine‐tuned crosstalk between NO and ROS generating systems in the course of inflammatory diseases.

Nitric oxide induces MIP-2 transcription in rat renal mesangial cells and in a rat model of glomerulonephritis

Nitric oxide is a crucial mediator of several forms of glomerulonephritis. We examined the effects of NO on the mRNA expression pattern in glomerular mesangial cells by using a low‐stringency reverse transcriptase‐polymerase chain reaction method and detected a cDNA fragment that was induced by interleukin 1β (IL‐1β) and further up‐regulated by the NO donor diethylenetriamine‐nitric oxide (DETA‐NO). Each respective cDNA fragment was found to match with the cDNAs of rat macrophage inflammatory protein 2 (MIP‐2) and GRO/cytokine‐induced neutrophil chemoattractant 2β (CINC‐2β). Further characterization of MIP‐2 regulation by Northern blot analysis confirmed an NO‐ and IL‐1β‐dependent increase in MIP‐2 mRNA levels. Moreover, inhibition of IL‐1β‐induced endogenous NO formation by the NO‐synthase (NOS) inhibitor L‐NMMA markedly attenuated MIP‐2 protein expression. We cloned 770 bp of the 5′‐flanking region of rat MIP‐2 and fused this fragment to a luciferase reporter gene. Transfection of the construct into mesangial cells resulted in a 3.5‐fold increase in luciferase activity in cells treated with DETA‐NO when compared to controls, suggesting a transcriptional mechanism for NO‐induced MIP‐2 expression. Deletion and mutational analysis identified critical nuclear factor (NF)‐κB and NF‐IL‐6 binding sites required for NO regulation of MIP‐2. In vivo, inhibition of NO synthesis in the Thy‐1.1 model of mesangioproliferative glomerulonephritis by the specific inducible‐NOS inhibitor L‐NIL resulted in a marked reduction of MIP‐2 mRNA expression. Furthermore, infiltration of neutrophils into the glomerulus was dramatically attenuated in L‐NIL‐treated rats.

Potentiation of nitric oxide synthase expression by superoxide in interleukin 1β-stimulated rat mesangial cells

Exposure of mesangial cells to superoxide, generated by the hypoxanthine/xanthine oxidase system or by the redox cycler 2,3‐dimethoxy‐1,4‐naphthoquinone caused a concentration‐dependent amplification of interleukin (IL)‐1β‐stimulated nitrite production. The effect of superoxide was accompanied by an increase in inducible nitric oxide synthase (iNOS) protein and iNOS mRNA levels. Incubation of mesangial cells with superoxide alone did not induce iNOS expression. To elucidate whether the increase of iNOS expression is due to transcriptional upregulation we fused a 4.5‐kb genomic iNOS fragment that contains the transcriptional start site of the rat iNOS gene to a luciferase reporter gene. In transient transfection studies, superoxide caused a 10‐fold augmentation of iNOS promoter activity in IL‐1β‐challenged mesangial cells. Our data identify superoxide as a co‐stimulatory factor amplifying cytokine‐induced iNOS gene expression and subsequent nitric oxide (NO) synthesis.