Protein kinases G as downstream mediators of the antifibrotic effects of stimulators of soluble guanylate cyclase

Hintergrund und Ziele. Die Systemische Sklerose (SSc) ist eine prototypische fibrosierende Erkrankung, gekennzeichnet durch hohe Morbidität und Mortalität. Effektive antifibrotische Therapien sind bis jetzt für die klinische Anwendung nicht vorhanden. Stimulatoren der löslichen Guanylylcyclase (lGC, englisch sGC) werden derzeit in klinischen Studien für die Behandlung von Fibrose in SSc untersucht. Ziel dieser Arbeit war die Untersuchung der Rolle von Proteinkinasen G (PKG) als nachgeschaltete Mediatoren der sGC und des cyclischen Guanosinmonophosphat (cGMP) sowie die Charakterisierung der Fehlregulierung dieses Signalweges in SSc. Methoden. Zur Bestimmung der Expression von Proteinkinasen G 1 und 2, von sGC und von p-VASP und VASP wurden Immunfluoreszenz und/oder Western Blot eingesetzt, zur Bestimmung des Spiegels von intrazellulärem cGMP wurde ein ELISA Kit verwendet. Die Spiegel der oben genannten Proteine und Mediatoren in der Haut oder in kultivierten dermalen Fibroblasten bei gesunden Spendern wurden mit denen bei SSc Patienten verglichen. Kultivierte Fibroblasten von gesunden Spender, SSc Patienten oder PKG1 und 2 – Doppel-Knockout Mäusen wurden mit TGF-β1 und/oder dem sGC-Stimulator BAY 41-2272, oder dem PKG-Inhibitor KT5823 behandelt. Bei den behandelten Fibroblasten wurde die Bildung von Stressfasern und die Menge von α-Smooth- 7 Muscle-Actin (α-SMA) Protein, von Col1a1 mRNA und Kollagen Typ 1 Protein untersucht. Bei knockout Mäusen für PKG1 und 2 oder für sGC wurde Bleomycin appliziert, und diese anschließend mit BAY 41-2272 behandelt. Um die Fibrosebildung bei diesen Mäusen zu bestimmen, wurde die Hautdicke vermessen, der Hydroxyprolingehalt bestimmt und die Myofibroblastenanzahl quantifiziert. Ergebnisse und Beobachtungen. Wir stellten eine Fehlregulierung des Signalwegs sGC-cGMP-PKG1 und 2 bei den Patienten mit SSc fest, die auf die erhöhten TGF-β1-Spiegel zurückzuführen zu sein scheint. sGC und cGMP sind bei SSc Patienten herunterreguliert. PKG1 und PKG2 sind hochreguliert, am ehesten als frustraner Kompensationsversuch. Die Fibroblastenaktivierung wird durch sGC Stimulierung und darauffolgende Hemmung der TGF-β1-induzierten ERK-Phosphorylierung gehemmt, wobei die Hemmung oder der Knockout der PKG1 und 2 den Effekt von sGC Stimulierung aufhebt. Behandlung mit sGC Stimulatoren schützt Wildtyp-Mäuse, aber nicht die PKG 1 und 2 Doppel-Knockout Mäuse vor der durch Bleomycin-induzierten Fibrose. Schlussfolgerungen. Aus unseren Daten können wir schließen, dass PKG 1 und 2 wesentliche nachgeschaltete Mediatoren der durch Hemmung der TGF-β1-induzierten ERK-Phosphorylierung hervorgerufenen antifibrotischen Effekte von sGC Stimulatoren sind. Wir zeigen außerdem eine geringere Aktivität des antifibrotischen sGC – cGMP – PKG 1 und 2 Signalwegs in SSc, die wahrscheinlich von TGF-β1 induziert wird.Background and objectives. Systemic sclerosis (SSc) is a prototypical fibrotic disease with high morbidity and mortality. Effective antifibrotic therapies are not currently available for clinical practice. Stimulators of soluble guanylate cyclase (sGC) are currently evaluated in clinical trials for the treatment of fibrosis in SSc. We aimed in this work to evaluate the role of protein kinases G (PKG) as potential downstream mediators of sGC and of cyclic guanosine monophosphate (cGMP) and to characterize the dysregulation of this signaling pathway in SSc. Methods. Immunofluorescence and/or Western Blot were used to determine the expression levels of PKG 1 and 2, of sGC and of p-VASP and VASP. An ELISA kit was used to determine the levels of intracellular cGMP. The expression levels of the molecules above in human skin or in cultured fibroblasts was compared between healthy donors and SSc patients. Cultured fibroblasts from healthy donors, SSc patients or PKG 1 and 2 – double knockout mice were stimulated with TGF-β1 and/or treated with the sGC stimulator BAY 41-2272 or the PKG inhibitor KT5823. Stress fibers formation, the levels of α-smooth muscle actin (α-SMA) protein, of Col1a1 mRNA and type 1 collagen protein were evaluated in treated cultured fibroblasts. Knockout mice for PKG1 and 2 were challenged with bleomycin and subsequently treated with BAY 41-2272. Skin thickness, hydroxyproline content and myofibroblast counts were determined to evaluate the fibrotic remodeling in these mice. Observations and results. We observed a dysregulation of the signaling pathway sGC-cGMP-PKG1 and 2 in SSc patients, for which the elevated levels of TGF-β1 could be responsible. sGC and cGMP are downregulated in SSc patients, whereas PKG1 and 2 and upregulated, most likely as a futile compensatory mechanism. Fibroblast activation is inhibited by sGC stimulation by blocking TGF-β1-induced ERK activation, whereas inhibition or knockout of PKG 1 and 2 renders sGC stimulation ineffective. 6 Treatment with sGC stimulators protects wildtype mice, but not PKG1 and 2 – double knockout mice from bleomycin-induced fibrosis. Conclusions. We can conclude from our data that PKG 1 and 2 are essential downstream mediators of the antifibrotic effects of sGC stimulators, effects mediated by blocking TGF-β1-induced activation of ERK. We show moreover a reduced activity of the sGC-cGMP-PKG signaling pathway in SSc, likely induced by TGF-β1

Engrailed 1 coordinates cytoskeletal reorganization to induce myofibroblast differentiation

Transforming growth factor-β (TGFβ) is a key mediator of fibroblast activation in fibrotic diseases, including systemic sclerosis. Here we show that Engrailed 1 (EN1) is reexpressed in multiple fibroblast subpopulations in the skin of SSc patients. We characterize EN1 as a molecular amplifier of TGFβ signaling in myofibroblast differentiation: TGFβ induces EN1 expression in a SMAD3-dependent manner, and in turn, EN1 mediates the profibrotic effects of TGFβ. RNA sequencing demonstrates that EN1 induces a profibrotic gene expression profile functionally related to cytoskeleton organization and ROCK activation. EN1 regulates gene expression by modulating the activity of SP1 and other SP transcription factors, as confirmed by ChIP-seq experiments for EN1 and SP1. Functional experiments confirm the coordinating role of EN1 on ROCK activity and the reorganization of cytoskeleton during myofibroblast differentiation, in both standard fibroblast culture systems and in vitro skin models. Consistently, mice with fibroblast-specific knockout of En1 demonstrate impaired fibroblast-to-myofibroblast transition and are partially protected from experimental skin fibrosis

PU.1 controls fibroblast polarization and tissue fibrosis

Fibroblasts are polymorphic cells with pleiotropic roles in organ morphogenesis, tissue homeostasis and immune responses. In fibrotic diseases, fibroblasts synthesize abundant amounts of extracellular matrix, which induces scarring and organ failure. By contrast, a hallmark feature of fibroblasts in arthritis is degradation of the extracellular matrix because of the release of metalloproteinases and degrading enzymes, and subsequent tissue destruction. The mechanisms that drive these functionally opposing pro-fibrotic and pro-inflammatory phenotypes of fibroblasts remain unknown. Here we identify the transcription factor PU.1 as an essential regulator of the pro-fibrotic gene expression program. The interplay between transcriptional and post-transcriptional mechanisms that normally control the expression of PU.1 expression is perturbed in various fibrotic diseases, resulting in the upregulation of PU.1, induction of fibrosis-associated gene sets and a phenotypic switch in extracellular matrix-producing pro-fibrotic fibroblasts. By contrast, pharmacological and genetic inactivation of PU.1 disrupts the fibrotic network and enables reprogramming of fibrotic fibroblasts into resting fibroblasts, leading to regression of fibrosis in several organs

Protein kinases G are essential downstream mediators of the antifibrotic effects of sGC stimulators

OBJECTIVES Stimulators of soluble guanylate cyclase (sGC) are currently investigated in clinical trials for the treatment of fibrosis in systemic sclerosis (SSc). In this study, we aim to investigate the role of protein kinases G (PKG) as downstream mediators of sGC-cyclic guanosine monophosphate (cGMP) in SSc. METHODS Mice with combined knockout of PKG1 and 2 were challenged with bleomycin and treated with the sGC stimulator BAY 41-2272. Fibroblasts were treated with BAY 41-2272 and with the PKG inhibitor KT 5823. RESULTS PKG1 and 2 are upregulated in SSc in a transforming growth factor-β1 (TGFβ1)-dependent manner, as an attempt to compensate for the decreased signalling through the sGC-cGMP-PKG pathway. Inhibition or knockout of PKG1 and 2 abrogates the inhibitory effects of sGC stimulation on fibroblast activation in a SMAD-independent, but extracellular signal-regulated kinase (ERK)-dependent manner. In vivo, sGC stimulation fails to prevent bleomycin-induced fibrosis in PKG1 and 2 knockout mice. CONCLUSIONS Our data provide evidence that PKGs are essential mediators of the antifibrotic effects of sGC stimulators through interfering with non-canonical TGFβ signalling. TGFβ1 promotes its profibrotic effects through inhibition of sGC-cGMP-PKG signalling, sGC stimulation exerts its antifibrotic effects by inhibition of TGFβ1-induced ERK phosphorylation

Vascularised human skin equivalents as a novel in vitro model of skin fibrosis and platform for testing of antifibrotic drugs

OBJECTIVES Fibrosis is a complex pathophysiological process involving interplay between multiple cell types. Experimental modelling of fibrosis is essential for the understanding of its pathogenesis and for testing of putative antifibrotic drugs. However, most current models employ either phylogenetically distant species or rely on human cells cultured in an artificial environment. Here we evaluated the potential of vascularised in vitro human skin equivalents as a novel model of skin fibrosis and a platform for the evaluation of antifibrotic drugs. METHODS Skin equivalents were assembled on a three-dimensional extracellular matrix by sequential seeding of endothelial cells, fibroblasts and keratinocytes. Fibrotic transformation on exposure to transforming growth factor-β (TGFβ) and response to treatment with nintedanib as an established antifibrotic agent were evaluated by quantitative polymerase chain reaction (qPCR), capillary Western immunoassay, immunostaining and histology. RESULTS Skin equivalents perfused at a physiological pressure formed a mature, polarised epidermis, a stratified dermis and a functional vessel system. Exposure of these models to TGFβ recapitulated key features of SSc skin with activation of TGFβ pathways, fibroblast to myofibroblast transition, increased release of collagen and excessive deposition of extracellular matrix. Treatment with the antifibrotic agent nintedanib ameliorated this fibrotic transformation. CONCLUSION Our data provide evidence that vascularised skin equivalents can replicate key features of fibrotic skin and may serve as a platform for evaluation of antifibrotic drugs in a pathophysiologically relevant human setting

Fibroblast growth factor receptor 3 activates a network of profibrotic signaling pathways to promote fibrosis in systemic sclerosis

Aberrant activation of fibroblasts with progressive deposition of extracellular matrix is a key feature of systemic sclerosis (SSc), a prototypical idiopathic fibrotic disease. Here, we demonstrate that the profibrotic cytokine transforming growth factor β selectively up-regulates fibroblast growth factor receptor 3 (FGFR3) and its ligand FGF9 to promote fibroblast activation and tissue fibrosis, leading to a prominent FGFR3 signature in the SSc skin. Transcriptome profiling, in silico analysis and functional experiments revealed that FGFR3 induces multiple profibrotic pathways including endothelin, interleukin-4, and connective tissue growth factor signaling mediated by transcription factor CREB (cAMP response element-binding protein). Inhibition of FGFR3 signaling by fibroblast-specific knockout of FGFR3 or FGF9 or pharmacological inhibition of FGFR3 blocked fibroblast activation and attenuated experimental skin fibrosis in mice. These findings characterize FGFR3 as an upstream regulator of a network of profibrotic mediators in SSc and as a potential target for the treatment of fibrosis

The tyrosine phosphatase SHP2 controls TGFβ-induced STAT3 signaling to regulate fibroblast activation and fibrosis

Uncontrolled activation of TGFβ signaling is a common denominator of fibrotic tissue remodeling. Here we characterize the tyrosine phosphatase SHP2 as a molecular checkpoint for TGFβ-induced JAK2/STAT3 signaling and as a potential target for the treatment of fibrosis. TGFβ stimulates the phosphatase activity of SHP2, although this effect is in part counterbalanced by inhibitory effects on SHP2 expression. Stimulation with TGFβ promotes recruitment of SHP2 to JAK2 in fibroblasts with subsequent dephosphorylation of JAK2 at Y570 and activation of STAT3. The effects of SHP2 on STAT3 activation translate into major regulatory effects of SHP2 on fibroblast activation and tissue fibrosis. Genetic or pharmacologic inactivation of SHP2 promotes accumulation of JAK2 phosphorylated at Y570, reduces JAK2/STAT3 signaling, inhibits TGFβ-induced fibroblast activation and ameliorates dermal and pulmonary fibrosis. Given the availability of potent SHP2 inhibitors, SHP2 might thus be a potential target for the treatment of fibrosis