Evaluation of Gremlin-1 as a therapeutic target in metabolic dysfunction-associated steatohepatitis.

Gremlin-1 has been implicated in liver fibrosis in metabolic dysfunction-associated steatohepatitis (MASH) via inhibition of bone morphogenetic protein (BMP) signalling and has thereby been identified as a potential therapeutic target. Using rat in vivo and human in vitro and ex vivo model systems of MASH fibrosis, we show that neutralisation of Gremlin-1 activity with monoclonal therapeutic antibodies does not reduce liver inflammation or liver fibrosis. Still, Gremlin-1 was upregulated in human and rat MASH fibrosis, but expression was restricted to a small subpopulation of COL3A1/THY1+ myofibroblasts. Lentiviral overexpression of Gremlin-1 in LX-2 cells and primary hepatic stellate cells led to changes in BMP-related gene expression, which did not translate to increased fibrogenesis. Furthermore, we show that Gremlin-1 binds to heparin with high affinity, which prevents Gremlin-1 from entering systemic circulation, prohibiting Gremlin-1-mediated organ crosstalk. Overall, our findings suggest a redundant role for Gremlin-1 in the pathogenesis of liver fibrosis, which is unamenable to therapeutic targeting.

IL-20 gene expression is induced by IL-1beta through mitogen-activated protein kinase and NF-kappaB-dependent mechanisms.

IL-20 is a novel member of the IL-10 cytokine family with pleiotropic effects. Current knowledge of what triggers and regulates IL-20 gene expression is sparse. The aim of this study was to investigate the regulation of IL-20 expression in cultured normal human keratinocytes. The expression of IL-20 was rapidly induced by proinflammatory stimuli, in particular IL-1beta, IL-6, and UVB irradiation. Using kinase inhibitors and small-interfering RNA, we discovered that the p38 mitogen-activated protein kinase (MAPK) as well as inhibitory kappaB kinase-NF-kappaB signaling pathways are crucial for IL-20 expression. By electrophoretic mobility shift assay two kappaB-binding sites were identified upstream from the start codon in the IL-20 gene. Supershift analysis revealed binding of the p50/p65 heterodimer. Furthermore, the p38 MAPK was shown to exert its effects on IL-20 expression through activation of the downstream kinase mitogen- and stress-activated kinase 1 (MSK1), indicating transactivation of NF-kappaB driven IL-20 messenger RNA transcription as an important mechanism of action. IL-20 is assumed to be a key cytokine in the pathogenesis of psoriasis and possibly cancer, and therefore the p38 MAPK, MSK1, and NF-kappaB may be important new molecular targets for the modulation of IL-20 expression in these diseases.

Characterization of recombinant murine factor VIIa and recombinant murine tissue factor: a human-murine species compatibility study.

Tissue factor (TF) is believed to play an important role in coagulation, inflammation, angiogenesis and wound healing as well as in tumor growth and metastasis. To facilitate in vivo studies in experimental murine models, we have produced recombinant murine factor VII (FVII) and the ectodomain of murine TF, TF(1-223). Murine FVII was activated to FVIIa with human factor Xa and upon reaction with FFR-chloromethyl ketone converted into an active site-blocked TF antagonist, FFR-FVIIa. The activity of murine FVIIa was characterized in factor X activation assays as well as in clot assays with murine and human thromboplastin in murine and human plasma. In these assays murine FVIIa exhibited a specific activity equivalent to or higher than human FVIIa. Further analysis showed that murine FVIIa binds with high affinity to both murine and human TF, whereas the association of human FVIIa to murine TF is about three orders of magnitude weaker than the association to human TF. This difference was further emphasized by the effect of murine-and human FFR-FVIIa on bleeding in an in vivo mouse model. Intra-peritoneal administration of 1 mg/kg murine FFR-FVIIa significantly prolonged the tail-bleeding time, whereas no effect on bleeding was observed with a 25-times higher dose of the human FFR-FVIIa. Together, these data confirms the notion of poor species compatibility between human FVII and murine TF and emphasizes the requirement for autologous FVIIa in studies on the role of the TF in experimental in vivo pharmacology.

Tissue factor-factor VIIa-specific up-regulation of IL-8 expression in MDA-MB-231 cells is mediated by PAR-2 and results in increased cell migration.

Tissue factor (TF), the cellular receptor for factor VIIa (FVIIa), besides initiating blood coagulation, is believed to play an important role in tissue repair, inflammation, angiogenesis, and tumor metastasis. Like TF, the chemokine interleukin-8 (IL-8) is shown to play a critical role in these processes. To elucidate the potential mechanisms by which TF contributes to tumor invasion and metastasis, we investigated the effect of FVIIa on IL-8 expression and cell migration in a breast carcinoma cell line, MDA-MB-231, a cell line that constitutively expresses abundant TF. Expression of IL-8 mRNA in MDA-MB-231 cells was markedly up-regulated by plasma concentrations of FVII or an equivalent concentration of FVIIa (10 nM). Neither thrombin nor other proteases involved in hemostasis were effective in stimulating IL-8 in these cells. Increased transcriptional activation of the IL-8 gene is responsible for increased expression of IL-8 in FVIIa-treated cells. PAR-2-specific antibodies fully attenuated TF-FVIIa-induced IL-8 expression. Additional in vitro experiments showed that TF-FVIIa promoted tumor cell migration and invasion, active site-inactivated FVIIa, and specific antibodies against TF, PAR-2, and IL-8 inhibited TF-FVIIa-induced cell migration. In summary, the studies described herein provide insight into how TF may contribute to tumor invasion.

Imidazoline NNC77-0074 stimulates Ca2+-evoked exocytosis in INS-1E cells by a phospholipase A2-dependent mechanism.

We have previously demonstrated that the novel imidazoline compound (+)-2-(2-(4,5-dihydro-1H-imidazol-2-yl)-thiopene-2-yl-ethyl)-pyridine (NNC77-0074) increases insulin secretion from pancreatic beta-cells by stimulation of Ca(2+)-dependent exocytosis. Using capacitance measurements, we now show that NNC77-0074 stimulates exocytosis in clonal INS-1E cells. NNC77-0074-stimulated exocytosis was antagonised by the cytoplasmic phospholipase A(2) (cPLA(2)) inhibitors ACA and AACOCF(3) and in cells treated with antisense oligonucleotide against cPLA(2)alpha. NNC77-0074-evoked insulin secretion was likewise inhibited by ACA, AACOCF(3), and cPLA(2)alpha antisense oligonucleotide treatment. In pancreatic islets NNC77-0074 stimulated PLA(2) activity. We propose that cPLA(2)alpha plays an important role in the regulation of NNC77-0074-evoked exocytosis in insulin secreting beta-cells.