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Role of protein kinase C activation in synthesis of complement components C2 and factor B in interferon-gamma-stimulated human fibroblasts, glioblastoma cell line A172 and monocytes.

ABSTRACT: The synthesis of C2 and factor B, the key components of complement system, is performed by various kinds of cells and is also up-regulated by interferon-gamma (IFN-gamma). By using human fibroblasts, human glioblastoma cell line A172 and monocytes, we investigated the signal-transduction mechanism for IFN-gamma-induced synthesis of C2 and factor B. The C2 and factor B synthesis induced by IFN-gamma in all three cell types was inhibited by a protein kinase C (PKC) inhibitor, 1-(5-isoquinolinyl-sulphonyl)-2-methylpiperazine (H-7). The depletion of PKC in these cell types after treatment with phorbol 12-myristate 13-acetate (PMA) resulted in inhibition of IFN-gamma-induced C2 production. In addition, IFN-gamma treatment elicited a decrease in cytoplasmic PKC in A172 cells, indicating that PKC is activated by IFN-gamma. These results suggest that PKC is crucial for IFN-gamma-induced C2 and factor B synthesis. Northern-blot analysis showed that the effects at H-7 were at least partly mediated by modulation of C2 and factor B mRNA abundance in A172 cells. Since treatment of fibroblasts and A172 cells with IFN-gamma had no effect on intracellular Ca2+ concentration, and since neither EGTA nor nifedipine inhibited C2 or factor B synthesis induced by IFN-gamma, we concluded that intracellular Ca2+ mobilization was not involved in the effect of IFN-gamma. In addition, genistein, herbimycin A and N-(6-aminohexyl)-5-chloro-1-naphthalene-sulphonamide (W-7) had no inhibitory effect on IFN-gamma-mediated action in any of the three cell types, which suggests that IFN-gamma acts independently of tyrosine kinases and calmodulin-dependent protein kinases.

SUBMITTER: Watanabe I

PROVIDER: S-EPMC1136379 | biostudies-other | 1995 Jan

REPOSITORIES: biostudies-other

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Project description:ImportanceLaryngotracheal stenosis (LTS) is a fibroproliferative disorder of the glottis, subglottis, and trachea. In models of fibrosis from other organ systems, the CD4+ T-cell response has been shown to regulate extracellular matrix deposition. Specifically, helper T cell 2 (TH2) promotes fibrosis, whereas TH1 and associated cytokines have been shown to be antifibrotic. However, this antifibrotic effect of the TH1 response has not been demonstrated in LTS.ObjectiveTo determine whether the TH1 cytokine interferon-γ inhibits the function of LTS-derived fibroblasts in vitro.Design, setting, and participantsThis in vitro controlled study included 6 patients with iatrogenic LTS undergoing routine surgical subglottic and tracheal dilation at a single institution. Fibroblasts were isolated from biopsy specimens of laryngotracheal scar and normal-appearing trachea. The presence of fibroblasts was confirmed by an immunohistochemical analysis. Laryngotracheal stenosis-derived fibroblasts were treated with interferon-γ and compared with untreated controls (2 sets of untreated, LTS-derived fibroblasts [media did not contain interferon-γ]) and normal airway fibroblasts (fibroblasts isolated from normal trachea). Data were collected from August 2015 through June 2016.InterventionsTreatment with interferon-γ, 10 ng/mL.Main outcomes and measuresCellular proliferation, fibrosis gene expression (using quantitative reverse transcription polymerase chain reaction analysis), soluble collagen, and cellular histologic features were assessed.ResultsAmong the 6 patients (6 women; mean [SD] age, 38.3 [17.2] years), LTS-derived fibroblast proliferation was reduced in patients who received interferon-γ treatment compared with untreated controls on days 3 (mean difference, -6515 cells; 95% CI, -10 630 to -2600 cells) to 6 (mean difference, -47 521 cells; 95% CI, -81 285 to -13 757 cells). Interferon-γ treatment reduced collagen types I and III gene expression by 86% and 68%, respectively, and resulted in lower total collagen production (10.94 vs 14.89 μg/mL). In addition, interferon-γ treatment resulted in a 32% reduction in expression of transforming growth factor β in LTS-derived fibroblasts.Conclusions and relevanceInterferon-γ reduced proliferation, soluble collagen production, and collagen expression in LTS-derived fibroblasts while also reducing the expression of the profibrotic cytokine transforming growth factor β. These findings suggest that therapeutics aimed at increasing interferon-γ and the TH1 response could attenuate LTS.

Project description:We previously identified a subset of interferon-stimulated genes (ISGs) upregulated by West Nile virus (WNV) infection in wild-type mouse embryo fibroblasts (MEFs) after viral proteins had inhibited type I interferon (IFN)-mediated JAK-STAT signaling and also in WNV-infected RIG-I-/-, MDA5-/-, STAT1-/-, STAT2-/-, IFNAR-/-, IRF3-/-, IRF7-/-, and IRF3/7-/- MEFs. In this study, ISG upregulation by WNV infection in IFNAR-/- MEFs was confirmed by transcriptome sequencing (RNA-seq). ISG upregulation by WNV infection was inhibited in RIG-I/MDA5-/- MEFs. ISGs were upregulated in IRF1-/- and IRF5-/- MEFs but only minimally upregulated in IRF3/5/7-/- MEFs, suggesting redundant IRF involvement. We previously showed that a single proximal interferon-stimulated response element (ISRE) in the Oas1a and Oas1b promoters bound the ISGF3 complex after type I IFN treatment. In this study, we used wild-type and mutant promoter luciferase reporter constructs to identify critical regions in the Oas1b and Ifit1 promoters for gene activation in infected IFNAR-/- MEFs. Two ISREs were required in both promoters. Mutation of these ISREs in an Ifit1 promoter DNA probe reduced in vitro complex formation with infected nuclear extracts. An NF-κB inhibitor decreased Ifit1 promoter activity in cells and in vitro complex formation. IRF3 and p50 promoter binding was detected by chromatin immunoprecipitation (ChIP) for upregulated ISGs with two proximal ISREs. The data indicate that ISREs function cooperatively to upregulate the expression of some ISGs when type I IFN signaling is absent, with the binding complex consisting of IRF3, IRF5, and/or IRF7 and an NF-κB component(s) as well as other, as-yet-unknown factors. IMPORTANCE Type I IFN signaling in mammalian cells induces formation of the ISGF3 transcription factor complex, which binds to interferon stimulated response elements (ISREs) in the promoters of interferon-stimulated genes (ISGs) in the cell nucleus. Flavivirus proteins counteract type I IFN signaling by preventing either the formation or nuclear localization of ISGF3. A subset of ISRE-regulated ISGs was still induced in West Nile virus (WNV)-infected mouse embryo fibroblasts (MEFs), indicating that cells have an alternative mechanism for activating these ISGs. In this study, cellular components involved in this ISG upregulation mechanism were identified using gene knockout MEFs and ChIP, and critical promoter regions for gene activation were mapped using reporter assays. The data indicate a cooperative function between two ISREs and required binding of IRF3, IRF5, and/or IRF7 and an NF-κB component(s). Moreover, type I IFN signaling-independent ISG activation requires different additional promoter activation regions than type I IFN-dependent activation.

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Role of protein kinase C activation in synthesis of complement components C2 and factor B in interferon-gamma-stimulated human fibroblasts, glioblastoma cell line A172 and monocytes.

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