IFN-γ and TNF-α synergize to inhibit CTGF expression in human lung endothelial cells.

Connective tissue growth factor (CTGF/CCN2) is an angiogenetic and profibrotic factor, acting downstream of TGF-β, involved in both airway- and vascular remodeling. While the T-helper 1 (Th1) cytokine interferon-gamma (IFN-γ) is well characterized as immune-modulatory and anti-fibrotic cytokine, the role of IFN-γ in lung endothelial cells (LEC) is less defined. Tumour necrosis factor alpha (TNF-α) is another mediator that drives vascular remodeling in inflammation by influencing CTGF expression. In the present study we investigated the influence of IFN-γ and TNF-α on CTGF expression in human LEC (HPMEC-ST1.6R) and the effect of CTGF knock down on human LEC. IFN-γ and TNF-α down-regulated CTGF in human LEC at the promoter-, transcriptional- and translational-level in a dose- and time-dependent manner. The inhibitory effect of IFN-γ on CTGF-expression could be almost completely compensated by the Jak inhibitor AG-490, showing the involvement of the Jak-Stat signaling pathway. Besides the inhibitory effect of IFN-γ and TNF-α alone on CTGF expression and LEC proliferation, these cytokines had an additive inhibitory effect on proliferation as well as on CTGF expression when administered together. To study the functional role of CTGF in LEC, endogenous CTGF expression was down-regulated by a lentiviral system. CTGF silencing in LEC by transduction of CTGF shRNA reduced cell proliferation, but did not influence the anti-proliferative effect of IFN-γ and TNF-α. In conclusion, our data demonstrated that CTGF was negatively regulated by IFN-γ in LEC in a Jak/Stat signaling pathway-dependent manner. In addition, an additive effect of IFN-γ and TNF-α on inhibition of CTGF expression and cell proliferation could be found. The inverse correlation between IFN-γ and CTGF expression in LEC could mean that screwing the Th2 response to a Th1 response with an additional IFN-γ production might be beneficial to avoid airway remodeling in asthma

Functional examination of CYR61 and CTGF in mesenchymal stem cells and endothelial lung cells

Cystein rich protein 61 (CYR61/CCN1) und Connective tissue growth factor (CTGF/CCN2) stellen aufgrund ihrer Multifunktionalität zwei sehr interessante Vertreter aus der derzeit sechs Mitglieder umfassenden Familie der CCN-Proteine (CCN- CYR61/CCN1, CTGF/CCN2, NOV/CCN3, WISP1-3/CCN4-6) dar. Seit der Entdeckung von CYR61 und CTGF konnten die überlappenden, aber meist nicht redundanten zellspezifischen Effekte in verschiedenen Zellsystemen nachgewiesen werden. Die Einflüsse auf zahlreiche Prozesse wie Proliferation und Migration, aber auch Angiogenese und das Überleben von Zellen lassen eine weitreichende Bedeutung im Zusammenhang mit vielen Entwicklungsprozessen vermuten, so auch der des muskuloskelettalen Systems und der Entwicklung der Lunge. In der vorliegenden Arbeit wurden für die nähere Charakterisierung von CYR61 und CTGF humane mesenchymale Stammzellen (hMSC) und die humane primäre Lungenendothelzelllinie HPMEC-ST1.6R (human pulmonary microvascular endothelial cells) gewählt. Beide Zellsysteme sind für die Untersuchung der Funktionsfähigkeit in den verschiedenen Kompartimenten bestens geeignet. So ist die Zelllinie HPMEC-ST1.6R den primären Endothelzellen, im Vergleich mit anderen in der Forschung eingesetzten Zelllinien, in Bezug auf spezifische Oberflächenmarker am nächsten. Mesenchymale Stammzellen bilden als multipotente Zellen das Rückrat des muskuloskelettalen Systems und sind an der Homöostase des menschlichen Stütz- und Bewegungsapparates maßgeblich beteiligt. Um experimentell nutzbare Konzentrationen an rekombinanten Proteinen zu erhalten, wurde ein Baculovirus-Expressionsystems gewählt. Nach der erfolgreichen Klonierung der CTGF/Fc-Tag Sequenz in einen Expressionsvektor konnte dies auch durch Produktion in SF21-Insektenzellen erreicht und erstmalig rekombinantes CTGF/Fc von hoher Reinheit gewonnen werden. Allerdings konnte eine beständige Funktionsfähigkeit der aufgereinigten Proteine mittels eines Proliferationstestes nachfolgend nur bedingt bestätigt werden. Für die weitere Versuchsplanung, einer Untersuchung der Auswirkung von rekombinantem CTGF (rCTGF) bzw. CYR61 (rCYR61) auf die Zielzellen, musste zunächst die zelleigene ctgf bzw. cyr61 Expression herunterreguliert werden, um einen endogenen Störeffekt auszuschließen. Durch den Einsatz spezifischer shRNAs konnte ctgf/CTGF sowohl in den hMSC-, wie auch den HPMEC-ST1.6R-Zielzellen deutlich herunterreguliert und nachfolgend eine markant reduzierte Proliferation beobachtet werden. Ein Effekt für die Regulation von cyr61 blieb aus. In dieser Arbeit wurden anschließend erstmals mittels Microarray-Analysen Veränderungen im Genexpressionsmuster der ctgf herunterregulierten hMSC- bzw. Lungenendothelzellen gegenüber Kontrollzellen untersucht. Des Weiteren war die Auswirkung einer Behandlung von ctgf herunterregulierten Zielzellen mit rCTGF gegenüber unbehandelten Kontrollzellen von Interesse. Für beide Zellsysteme konnten signifikante Genregulationen nach der Behandlung mit CTGF spezifischen shRNAs gegenüber den Kontrollzellen detektiert werden, mit interessanten Genclustern im Bereich der TGF-beta (transforming growth factor ß) Signalgebung, sowie der fokalen Adhäsion (z.B. VEGF). Eine Behandlung mit rCTGF hingegen zeigte gegenüber den unbehandelten Kontrollzellen in der Auswertung der Microarray-Analyse keine signifikante Veränderung im Genexpressionsmuster. In dieser Arbeit wurden, neben einer effektiven Gewinnung von rekombinantem CTGF und der Herunterregulation der endogenen ctgf Expression, wichtige Erkenntnisse zur Biologie von CTGF (und CYR61) in mesenchymalen Stammzellen hMSC und der Lungenendothelzelllinie HPMEC-ST1.6R erlangt. Die erhaltenen Microarray-Daten bieten eine fundierte Grundlage für zahlreiche fortführende Untersuchungen.Cystein rich protein 61 (CYR61/CCN1) and connective tissue growth factor (CTGF/CCN2) are two very interesting members of the CNN family (CCN- CYR61/CCN1, CTGF/CCN2, NOV/CCN3, WISP1-3/CCN4-6) consisting of six members so far. Since its discovery the overlapping, but mostly non-redundant effects of CYR61 and CTGF were shown in different cell systems. Both proteins are linked to many different processes like proliferation and migration, but also angiogenesis and survival. They seem to be involved in very fundamental biological processes, amongst other the development of the musculoskeletal system and the lung and were analyzed in this study. To distinguish the two proteins CYR61 and CTGF, primary human mesenchymal stem cells (hMSC) and a human pulmonary endothelial cell line (HPMEC-ST1.6R) were chosen. Both cell systems are suited very well for getting more information about the function in these different compartments. So the cell line HPMEC-ST1.6R is more related to primary endothelial cells in reference to the cell surface markers, compared to other cell lines used for experimental research. Mesenchymal stem cells form the backbone of the musculoskeletal system and are involved in the homeostasis of this complex system. Getting adequate concentrations of recombinant proteins for the upcoming experiments a baculovirus expression system was chosen. After successful cloning of the CTGF/Fc-Tag sequence into an expression vector, recombinant CTGF/Fc of high purity was obtained for the first time, produced in SF21 insect cells. However the stable functioning of the proteins was partly confirmed by proliferation tests. To study the effect of recombinant CTGF or CYR61 in further experiments, the endogenous ctgf or cyr61expression had to be downregulated to avoid negative effects. By using specific shRNAs ctgf/CTGF has been downregulated in hMSC as well as HPMEC-ST1.6R cells and subsequently a reduced proliferation was observed. No effect was detected for the regulation of cyr61. In this study for the first time changes in regulation of gene expression after downregulation of ctgf in hMSC and HPMEC-ST1.6R cells were studied by microarray analyses. Furthermore to discover the effect of treating ctgf downregulated cells with recombinant CTGF compared to control cells was another aim of this experimental series. For both cell systems, significant gene regulations were detected after treatment with CTGF specific shRNAs with interesting gene cluster for TGFß-signaling as well as focal adhesion (e.g. VEGF). In contrast, no significant change in gene regulation was detected by microarray analysis after treating the target cells with rCTGF compared to non-treated cells. In summary, besides the effective preparation of rCTGF and the marked downregulation of ctgf gene expression, this study provides fundamental information about CTGF and its biology in hMSC and HPMEC-ST1.6R cells, as well. Based on the numerous detected gene regulations in the microarray analyses the study provides a basis for further experiments.

IFN-gamma and TNF-alpha synergize to inhibit CTGF expression in human lung endothelial cells

Connective tissue growth factor (CTGF/CCN2) is an angiogenetic and profibrotic factor, acting downstream of TGF-b, involved in both airway- and vascular remodeling. While the T-helper 1 (Th1) cytokine interferon-gamma (IFN-c) is well characterized as immune-modulatory and anti-fibrotic cytokine, the role of IFN-c in lung endothelial cells (LEC) is less defined. Tumour necrosis factor alpha (TNF-a) is another mediator that drives vascular remodeling in inflammation by influencing CTGF expression. In the present study we investigated the influence of IFN-c and TNF-a on CTGF expression in human LEC (HPMEC-ST1.6R) and the effect of CTGF knock down on human LEC. IFN-c and TNF-a down-regulated CTGF in human LEC at the promoter-, transcriptional- and translational-level in a dose- and time-dependent manner. The inhibitory effect of IFN-c on CTGF-expression could be almost completely compensated by the Jak inhibitor AG-490, showing the involvement of the Jak-Stat signaling pathway. Besides the inhibitory effect of IFN-c and TNF-a alone on CTGF expression and LEC proliferation, these cytokines had an additive inhibitory effect on proliferation as well as on CTGF expression when administered together. To study the functional role of CTGF in LEC, endogenous CTGF expression was down-regulated by a lentiviral system. CTGF silencing in LEC by transduction of CTGF shRNA reduced cell proliferation, but did not influence the anti-proliferative effect of IFN-c and TNF-a. In conclusion, our data demonstrated that CTGF was negatively regulated by IFN-c in LEC in a Jak/Stat signaling pathway-dependent manner. In addition, an additive effect of IFN-c and TNF-a on inhibition of CTGF expression and cell proliferation could be found. The inverse correlation between IFN-c and CTGF expression in LEC could mean that screwing the Th2 response to a Th1 response with an additional IFN-c production might be beneficial to avoid airway remodeling in asthma

Additive effect of IFN-γ and TNF-α on the inhibition of CTGF expression. A.

<p>Luminometric analysis of CTGF-Luc reporter transfected HPMEC cells: LEC were incubated with medium alone (control), TNF-α, IFN-γ, or INF-γ and TNF-α. * p<0.05 vs medium alone; # p<0.05 vs INF-γ or TNF-α alone. <b>B+C.</b> LEC were incubated with TNF-α and/or IFN-γ. CTGF mRNA expression compared to β-actin was measured by real-time PCR (B). CTGF and β-actin protein expression was determined by immunoblotting (C). Significant differences (p<0.05) compared with untreated cells are marked by *, compared with INF-γ or TNF-α are marked with #. A representative of 3 independent experiments is shown (C).</p

Effect of rCTGF on proliferation of LEC.

<p><b>A.</b> Proliferation in LEC was assessed 3 days after the addition of rCTGF at the indicated concentration. <b>B.</b> Proliferation in LEC was assessed after the addition of 100 ng/ml CTGF at the indicated time points. Values are means ± SEMs for 3 replicate experiments. Significant differences (p<0.05) compared with untreated cells are marked by *. RLU/s (relative light unit/second).</p

Effect of the Jak1-inhibitor AG-490 on IFN-γ induced inhibition of CTGF expression in LEC.

<p>LEC were incubated for different time points with 500 U/ml IFN-γ (A), with different concentrations of INF-γ for 5 min (B), or with or without the Jak-inhibitor AG-490 (30 µM) and INF-γ (500 U/ml) for 5 min (C). Stat1 phosphorylation and Stat1 expression were detected by immunoblotting with anti-phospho-Stat1 and anti-Stat1 antibodies. A representative of 3 independent experiments is shown. <b>D.</b> Luminometric analysis of CTGF-Luc reporter transfected LEC: LEC were incubated with medium alone (control) or with IFN-γ without or with the Jak-inhibitor AG-490 (30 µM). * p<0.05 vs. medium alone; # p<0.05 vs IFN-γ alone. <b>E+F.</b> LEC were incubated with IFN-γ with or without the Jak-inhibitor AG-490 (30 µM). CTGF mRNA expression compared to β-actin was measured by real-time PCR (<b>E</b>). CTGF and β-actin protein expression was determined by immunoblotting (<b>F</b>). Significant differences (p<0.05) compared with untreated cells are marked by *, compared with IFN-γ are marked with #. A representative of 3 independent experiments is shown (<b>F</b>).</p

Additive effect of IFN-γ and TNF-α on the inhibition of LEC proliferation.

<p>Proliferation was assessed 5 days after the addition of TNF-α <b>(A)</b>, IFN-γ <b>(B),</b> or TNF-α + IFN-γ <b>(C)</b> at the indicated concentration. Values are means ± SEMs for 3 replicate experiments. Significant differences (p<0.05) compared with untreated cells are marked by *, compared with INF-γ or TNF-α are marked with #.</p

Effect of TNF-α on CTGF expression in LEC.

<p><b>A.</b> Luminometric analysis of CTGF-Luc reporter transfected LEC: LEC were incubated with medium alone (control) or with decreasing concentrations of TNF-α. * p<0.05 vs medium alone. <b>B+C.</b> CTGF mRNA expression: LEC were incubated with different concentrations of TNF-α for 24 h (B) or for various duration with 20 ng/ml TNF-α (C). mRNA expression of CTGF normalized for β-actin were measured by real-time PCR. Significant differences (p<0.05) compared with untreated cells are marked by *. <b>D+E.</b> CTGF protein expression: LEC were incubated with different concentrations of TNF-α for 48 h (D) or with TNF-α (20 ng/ml) for different time periods (E). CTGF and β-actin expression was detected by immunoblotting. A representative of 3 independent experiments is shown.</p

Effect of CTGF silencing on the proliferation of LEC and IFN-γ and TNF-α mediated inhibition. A.

<p>Proliferation of LEC, in which CTGF was down-regulated by CTGF shRNA transfection (right side; “CTGF shRNA”) and of control cells, which were transfected with non-specific scrambled shRNA (left side; “control shRNA”), was assessed after the addition of 100 ng/ml rCTGF with or without the addition of IFN-γ (500 U/ml) at 72 h. Values are means ± SEMs for 3 replicate cultures. Significant differences to response to rCTGF are marked by * (p<0.05), significant differences of proliferation between CTGF shRNA and non-specific scrambled shRNA transfected cells are marked by # (p<0.05), and significant differences of proliferation between CTGF-induced proliferation and CTGF-induced proliferation with the addition of IFN-γ are marked by $ (p<0.05). <b>B.</b> Proliferation of LEC, in which CTGF was down-regulated by CTGF shRNA transfection (lined column; “CTGF shRNA”) and of control cells, transfected with non-specific scrambled shRNA (white column; “control shRNA”), was assessed 5 days after the addition of TNF-α (20 ng/ml), IFN-γ (500 U/ml), or TNF-α+IFN-γ. Values are means ± SEMs for 3 replicate experiments and present as % compared with non stimulated cells (100%; black column). Significant differences between TNF-α and/or IFN-γ treated cells to control cells are marked by * (p<0.05).</p

Effect of CTGF shRNA on basal and TGF-β1 mediated induction of CTGF mRNA- and protein-expression in LEC. A.

<p>Effect on CTGF mRNA level: Four days after downregulation of CTGF by specific shRNA LEC were cultured in serum reduced medium (2% FCS) for 24 h before treatment with TGF-ß1. RNA and protein was isolated after 24 h of incubation with 10 ng/ml TGF-ß1. CTGF RNA expression, compared to β-actin as control, was detected by RT-PCR (A). <b>B.</b> Effect on CTGF protein level: CTGF and ß-actin expression on protein level was detected by western immunoblotting (B). Significant regulations of CTGF by TGF-ß1 are marked by * (p<0.05), CTGF down-regulation by CTGF specific shRNA compared to control cells are marked by # (p<0.05).</p