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Purpose: To evaluate the potency and efficacy of a library of dopamine receptor D2 (D2R) antagonists in the mitigation of fibrotic activation in retinal pigment epithelial (RPE) cells. Methods: ARPE-19 cells were cultured and treated with methotrexate or 27 district D2R antagonists using a fibronectin deposition assay. The most potent compounds were then further assessed in assays measuring cellular proliferation, cellular migration, and profibrotic gene expression. Results: The previously established antifibrotic D2R antagonist loxapine exerted a robust and dose-dependent inhibition of fibronectin deposition, whereas methotrexate exerted minimal inhibition. The most potent D2R antagonist identified, fluphenazine, effectively blocked in vitro models of fibrosis at 300-1,000 nM concentrations. Conclusions: Here we found multiple FDA-approved D2R antagonists that potently block RPE cell fibrogenesis. These findings further support the potential of D2R antagonism as a potential therapeutic for retinal fibrotic disease.
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Pulmonary arterial hypertension (PAH) causes pulmonary vascular remodeling, increasing pulmonary vascular resistance (PVR) and leading to right heart failure and death. Matrix stiffening early in the disease promotes remodeling in pulmonary artery smooth muscle cells (PASMCs), contributing to PAH pathogenesis. Our research identified YAP and TAZ as key drivers of the mechanobiological feedback loop in PASMCs, suggesting targeting them could mitigate remodeling. However, YAP/TAZ are ubiquitously expressed and carry out diverse functions, necessitating a cell-specific approach. Our previous work demonstrated that targeting non-canonical IKB kinase TBK1 reduced YAP/TAZ activation in human lung fibroblasts. Here, we investigate non-canonical IKB kinases TBK1 and IKKε in pulmonary hypertension (PH) and their potential to modulate PASMC pathogenic remodeling by regulating YAP/TAZ. We show that TBK1 and IKKε are activated in PASMCs in a rat PH model. Inflammatory cytokines, elevated in PAH, activate these kinases in human PASMCs. Inhibiting TBK1/IKKε expression/activity significantly reduces PAH-associated PASMC remodeling, with longer-lasting effects on YAP/TAZ than treprostinil, an approved PAH therapy. These results show that non-canonical IKB kinases regulate YAP/TAZ in PASMCs and may offer a novel approach for reducing vascular remodeling in PAH.
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Hipertensión Pulmonar , Quinasa I-kappa B , Hipertensión Arterial Pulmonar , Remodelación Vascular , Animales , Humanos , Ratas , Hipertensión Pulmonar/metabolismo , Hipertensión Pulmonar/patología , Quinasa I-kappa B/metabolismo , Miocitos del Músculo Liso , Hipertensión Arterial Pulmonar/metabolismo , Hipertensión Arterial Pulmonar/patología , Arteria Pulmonar , Proteínas Señalizadoras YAP/metabolismo , Proteínas Coactivadoras Transcripcionales con Motivo de Unión a PDZ/metabolismoRESUMEN
Proliferative vitreoretinopathy (PVR), the most common cause of recurrent retinal detachment, is characterized by the formation and contraction of fibrotic membranes on the surface of the retina. There are no Food and Drug Administration (FDA)-approved drugs to prevent or treat PVR. Therefore, it is necessary to develop accurate in vitro models of the disease that will enable researchers to screen drug candidates and prioritize the most promising candidates for clinical studies. We provide a summary of recent in vitro PVR models, as well as avenues for model improvement. Several in vitro PVR models were identified, including various types of cell cultures. Additionally, novel techniques that have not been used to model PVR were identified, including organoids, hydrogels, and organ-on-a-chip models. Novel ideas for improving in vitro PVR models are highlighted. Researchers may consult this review to help design in vitro models of PVR, which will aid in the development of therapies to treat the disease.
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Desprendimiento de Retina , Vitreorretinopatía Proliferativa , Humanos , Vitreorretinopatía Proliferativa/tratamiento farmacológico , Vitreorretinopatía Proliferativa/metabolismo , RetinaRESUMEN
Pulmonary fibroblasts are the primary producers of extracellular matrix (ECM) in the lungs, and their pathogenic activation drives scarring and loss of lung function in idiopathic pulmonary fibrosis (IPF). This uncontrolled production of ECM is stimulated by mechanosignaling and transforming growth factor beta 1 (TGF-ß1) signaling that together promote transcriptional programs including Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ). G protein-coupled receptors (GPCRs) that couple to G α s have emerged as pharmacological targets to inactivate YAP/TAZ signaling and promote lung fibrosis resolution. Previous studies have shown a loss of expression of "antifibrotic GPCRs"-receptors that couple to G α s, in IPF patient-derived fibroblasts compared with non-IPF samples. Of the 14 G α s GPCRs we found to be expressed in lung fibroblasts, the dopamine receptor D1 (DRD1) was one of only two not repressed by TGF-ß1 signaling, with the ß2-adrenergic receptor being the most repressed. We compared the potency and efficacy of multiple D1 and ß2 receptor agonists +/- TGF-ß1 treatment in vitro for their ability to elevate cAMP, inhibit nuclear localization of YAP/TAZ, regulate expression of profibrotic and antifibrotic genes, and inhibit cellular proliferation and collagen deposition. Consistently, the activity of ß2 receptor agonists was lost, whereas D1 receptor agonists was maintained, after stimulating cultured lung fibroblasts with TGF-ß1. These data further support the therapeutic potential of the dopamine receptor D1 and highlight an orchestrated and pervasive loss of antifibrotic GPCRs mediated by TGF-ß1 signaling. SIGNIFICANCE STATEMENT: Idiopathic pulmonary fibrosis (IPF) is a deadly lung disease with limited therapies. GPCRs have emerged as a primary target for the development of novel antifibrotic drugs; however, a challenge to this approach is the dramatic changes in GPCR expression in response to profibrotic stimuli. Here, we investigate the impact of TGF-ß1 on the expression of antifibrotic GPCRs and show the D1 dopamine receptor expression is uniquely maintained in response to TGF-ß1, further implicating it as a compelling target to treat IPF.
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Fibrosis Pulmonar Idiopática , Factor de Crecimiento Transformador beta1 , Humanos , Fibroblastos/metabolismo , Fibrosis Pulmonar Idiopática/tratamiento farmacológico , Fibrosis Pulmonar Idiopática/metabolismo , Fibrosis Pulmonar Idiopática/patología , Pulmón , Receptores Dopaminérgicos/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Factor de Crecimiento Transformador beta1/metabolismoRESUMEN
Aberrant vascular remodeling contributes to the progression of many aging-associated diseases, including idiopathic pulmonary fibrosis (IPF), where heterogeneous capillary density, endothelial transcriptional alterations, and increased vascular permeability correlate with poor disease outcomes. Thus, identifying disease-driving mechanisms in the pulmonary vasculature may be a promising strategy to limit IPF progression. Here, we identified Ccn3 as an endothelial-derived factor that is upregulated in resolving but not in persistent lung fibrosis in mice, and whose function is critical for vascular homeostasis and repair. Loss and gain of function experiments were carried out to test the role of CCN3 in lung microvascular endothelial function in vitro through RNAi and the addition of recombinant human CCN3 protein, respectively. Endothelial migration, permeability, proliferation, and in vitro angiogenesis were tested in cultured human lung microvascular endothelial cells (ECs). Loss of CCN3 in lung ECs resulted in transcriptional alterations along with impaired wound-healing responses, in vitro angiogenesis, barrier integrity as well as an increased profibrotic activity through paracrine signals, whereas the addition of recombinant CCN3 augmented endothelial function. Altogether, our results demonstrate that the matricellular protein CCN3 plays an important role in lung endothelial function and could serve as a promising therapeutic target to facilitate vascular repair and promote lung fibrosis resolution.
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Fibrosis Pulmonar , Ratones , Humanos , Animales , Células Endoteliales/metabolismo , Proteína Hiperexpresada del Nefroblastoma/metabolismo , Células Cultivadas , Pulmón/metabolismoRESUMEN
Cellular senescence is emerging as a driver of idiopathic pulmonary fibrosis (IPF), a progressive and fatal disease with limited effective therapies. The senescence-associated secretory phenotype (SASP), involving the release of inflammatory cytokines and profibrotic growth factors by senescent cells, is thought to be a product of multiple cell types in IPF, including lung fibroblasts. NF-κB is a master regulator of the SASP, and its activity depends on the phosphorylation of p65/RelA. The purpose of this study was to assess the role of Pim-1 kinase as a driver of NF-κB-induced production of inflammatory cytokines from low-passage IPF fibroblast cultures displaying markers of senescence. Our results demonstrate that Pim-1 kinase phosphorylates p65/RelA, activating NF-κB activity and enhancing IL-6 production, which in turn amplifies the expression of PIM1, generating a positive feedback loop. In addition, targeting Pim-1 kinase with a small molecule inhibitor dramatically inhibited the expression of a broad array of cytokines and chemokines in IPF-derived fibroblasts. Furthermore, we provide evidence that Pim-1 overexpression in low-passage human lung fibroblasts is sufficient to drive premature senescence, in vitro. These findings highlight the therapeutic potential of targeting Pim-1 kinase to reprogram the secretome of senescent fibroblasts and halt IPF progression.
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Fibrosis Pulmonar Idiopática , Neumonía , Humanos , Proteínas Proto-Oncogénicas c-pim-1/metabolismo , Proteínas Proto-Oncogénicas c-pim-1/farmacología , FN-kappa B/metabolismo , Fibroblastos/metabolismo , Fibrosis Pulmonar Idiopática/metabolismo , Senescencia Celular , Pulmón/metabolismo , Neumonía/metabolismo , Citocinas/metabolismoRESUMEN
Retinal pigmented epithelial (RPE) cells play an important role in retinal fibrotic diseases such as proliferative vitreoretinopathy (PVR). The purpose of this study was to elucidate the involvement of dopamine receptor signaling in regulating the fibrotic activation of RPE cells. Dopamine receptor expression, the effect of dopamine on fibrotic activity, and dopamine production were measured in the human RPE cell line ARPE-19. The fibrotic activation of RPE cells was evaluated in response to treatments with selective dopamine receptor agonists and antagonists by measuring gene expression, migration, proliferation, and fibronectin deposition. DRD2 and DRD5 are the dominant dopaminergic receptors expressed in ARPE-19 cells and TGF-ß stimulation enhances the autocrine release of dopamine, which we show further exasperates fibrotic activation. Finally, treatment with D2 dopamine receptor antagonists or D5 dopamine receptor agonists inhibits profibrotic gene expression, migration, proliferation, and fibronectin deposition and thus may serve as effective mechanisms for treating retinal fibrosis including PVR.
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Fibronectinas , Vitreorretinopatía Proliferativa , Movimiento Celular , Dopamina/metabolismo , Agonistas de Dopamina/metabolismo , Agonistas de Dopamina/farmacología , Células Epiteliales/metabolismo , Transición Epitelial-Mesenquimal , Fibronectinas/metabolismo , Fibrosis , Humanos , Receptores Dopaminérgicos/metabolismo , Epitelio Pigmentado de la Retina/metabolismo , Vitreorretinopatía Proliferativa/metabolismo , Vitreorretinopatía Proliferativa/patologíaRESUMEN
Activating mutations in Gαq/11 are a major driver of uveal melanoma (UM), the most common intraocular cancer in adults. While progress has recently been made in targeting Gαq/11 for UM therapy, the crucial role for these proteins in normal physiology and their high structural similarity with many other important GTPase proteins renders this approach challenging. The aim of the current study was to validate whether a key regulator of Gq signaling, regulator of G protein signaling 2 (RGS2), can inhibit Gαq-mediated UM cell growth. We used two UM cell lines, 92.1 and Mel-202, which both contain the most common activating mutation GαqQ209L and developed stable cell lines with doxycycline-inducible RGS2 protein expression. Using cell viability assays, we showed that RGS2 could inhibit cell growth in both of these UM cell lines. We also found that this effect was independent of the canonical GTPase-activating protein activity of RGS2 but was dependent on the association between RGS2 and Gαq. Furthermore, RGS2 induction resulted in only partial reduction in cell growth as compared to siRNA-mediated Gαq knockdown, perhaps because RGS2 was only able to reduce mitogen-activated protein kinase signaling downstream of phospholipase Cß, while leaving activation of the Hippo signaling mediators yes-associated protein 1/TAZ, the other major pathway downstream of Gαq, unaffected. Taken together, our data indicate that RGS2 can inhibit UM cancer cell growth by associating with GαqQ209L as a partial effector antagonist.
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Melanoma , Proteínas RGS , Neoplasias de la Úvea , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Humanos , Melanoma/genética , Proteínas RGS/metabolismo , Transducción de Señal , Neoplasias de la Úvea/genéticaRESUMEN
Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease with few effective treatment options. We found a highly significant correlation between pregnancy-associated plasma protein (PAPP)-A expression in IPF lung tissue and disease severity as measured by various pulmonary and physical function tests. PAPP-A is a metalloproteinase that enhances local insulin-like growth factor (IGF) activity. We used primary cultures of normal adult human lung fibroblasts (NHLF) to test the hypothesis that PAPP-A plays an important role in the development of pulmonary fibrosis. Treatment of NHLF with pro-fibrotic transforming growth factor (TGF)-ß stimulated marked increases in IGF-I mRNA expression (>20-fold) and measurable IGF-I levels in 72-h conditioned medium (CM). TGF-ß treatment also increased PAPP-A levels in CM fourfold (p = 0.004) and proteolytic activity ~2-fold. There was an indirect effect of TGF-ß to stimulate signaling through the PI3K/Akt pathway, which was significantly inhibited by both IGF-I-inactivating and PAPP-A inhibitory antibodies. Induction of senescence in NHLF increased PAPP-A levels in CM 10-fold (p = 0.006) with attendant increased proteolytic activity. Thus, PAPP-A is a novel component of the senescent lung fibroblast secretome. In addition, NHLF secreted extracellular vehicles (EVs) with surface-bound active PAPP-A that were increased fivefold with senescence. Regulation of PAPP-A and IGF signaling by TGF-ß and cell senescence suggests an interactive cellular mechanism underlying the resistance to apoptosis and the progression of fibrosis in IPF. Furthermore, PAPP-A-associated EVs may be a means of pro-fibrotic, pro-senescent communication with other cells in the lung and, thus, a potential therapeutic target for IPF.
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Fibrosis Pulmonar Idiopática , Proteína Plasmática A Asociada al Embarazo/metabolismo , Adulto , Medios de Cultivo Condicionados/farmacología , Fibroblastos/metabolismo , Fibrosis , Humanos , Fibrosis Pulmonar Idiopática/metabolismo , Factor I del Crecimiento Similar a la Insulina/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteína Plasmática A Asociada al Embarazo/genética , Proteína Plasmática A Asociada al Embarazo/farmacología , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) are transcription cofactors implicated in the contractile and profibrotic activation of fibroblasts. Fibroblast contractile function is important in alveologenesis and in lung wound healing and fibrosis. As paralogs, YAP and TAZ may have independent or redundant roles in regulating transcriptional programs and contractile function. Using IMR-90 lung fibroblasts, microarray analysis, and traction microscopy, we tested whether independent YAP or TAZ knockdown alone was sufficient to limit transcriptional activation and contraction in vitro. Our results demonstrate limited effects of knockdown of either YAP or TAZ alone, with more robust transcriptional and functional effects observed with combined knockdown, consistent with cooperation or redundancy of YAP and TAZ in transforming growth factor ß1 (TGFß1)-induced fibroblast activation and contractile force generation. The transcriptional responses to combined YAP/TAZ knockdown were focused on a relatively small subset of genes with prominent overrepresentation of genes implicated in contraction and migration. To explore potential disease relevance of our findings, we tested primary human lung fibroblasts isolated from patients with idiopathic pulmonary fibrosis and confirmed that YAP and TAZ combined knockdown reduced the expression of three cytoskeletal genes, ACTA2, CNN1, and TAGLN. We then compared the contribution of these genes, along with YAP and TAZ, to contractile function. Combined knockdown targeting YAP/TAZ was more effective than targeting any of the individual cytoskeletal genes in reducing contractile function. Together, our results demonstrate that YAP and TAZ combine to regulate a multigene program that is essential to fibroblast contractile function.
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Fibroblastos/metabolismo , Proteínas Coactivadoras Transcripcionales con Motivo de Unión a PDZ/metabolismo , Proteínas Señalizadoras YAP/metabolismo , Fenómenos Biomecánicos/efectos de los fármacos , Línea Celular , Fibroblastos/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Factor de Crecimiento Transformador beta1/farmacologíaRESUMEN
Hepatic fibrosis is driven by deposition of matrix proteins following liver injury. Hepatic stellate cells (HSCs) drive fibrogenesis, producing matrix proteins, including procollagen I, which matures into collagen I following secretion. Disrupting intracellular procollagen processing and trafficking causes endoplasmic reticulum stress and stress-induced HSC apoptosis and thus is an attractive antifibrotic strategy. We designed an immunofluorescence-based small interfering RNA (siRNA) screen to identify procollagen I trafficking regulators, hypothesizing that these proteins could serve as antifibrotic targets. A targeted siRNA screen was performed using immunofluorescence to detect changes in intracellular procollagen I. Tumor necrosis factor receptor associated factor 2 and noncatalytic region of tyrosine kinase-interacting kinase (TNIK) was identified and interrogated in vitro and in vivo using the TNIK kinase inhibitor NCB-0846 or RNA interference-mediated knockdown. Our siRNA screen identified nine genes whose knockdown promoted procollagen I retention, including the serine/threonine kinase TNIK. Genetic deletion or pharmacologic inhibition of TNIK through the small molecule inhibitor NCB-0846 disrupted procollagen I trafficking and secretion without impacting procollagen I expression. To investigate the role of TNIK in liver fibrogenesis, we analyzed human and murine livers, finding elevated TNIK expression in human cirrhotic livers and increased TNIK expression and kinase activity in both fibrotic mouse livers and activated primary human HSCs. Finally, we tested whether inhibition of TNIK kinase activity could limit fibrogenesis in vivo. Mice receiving NCB-0846 displayed reduced CCl4 -induced fibrogenesis compared to CCl4 alone, although α-smooth muscle actin levels were unaltered. Conclusions: Our siRNA screen effectively identified TNIK as a key kinase involved in procollagen I trafficking in vitro and hepatic fibrogenesis in vivo.
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Procolágeno , Proteínas Serina-Treonina Quinasas , Animales , Hígado/metabolismo , Ratones , Procolágeno/genética , Inhibidores de Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , ARN Interferente Pequeño/genética , Factor 2 Asociado a Receptor de TNF/metabolismoRESUMEN
Yes-associated protein (YAP) and PDZ-binding motif (TAZ) have emerged as important regulators of pathologic fibroblast activation in fibrotic diseases. Agonism of Gαs-coupled G protein coupled receptors (GPCRs) provides an attractive approach to inhibit the nuclear localization and function of YAP and TAZ in fibroblasts that inhibits or reverses their pathological activation. Agonism of the dopamine D1 GPCR has proven effective in preclinical models of lung and liver fibrosis. However, the molecular mechanisms coupling GPCR agonism to YAP and TAZ inactivation in fibroblasts remain incompletely understood. Here, using human lung fibroblasts, we identify critical roles for the cAMP effectors EPAC1/2, the small GTPase RAP2c, and the serine/threonine kinase MAP4K7 as the essential elements in the downstream signaling cascade linking GPCR agonism to LATS1/2-mediated YAP and TAZ phosphorylation and nuclear exclusion in fibroblasts. We further show that this EPAC/RAP2c/MAP4K7 signaling cascade is essential to the effects of dopamine D1 receptor agonism on reducing fibroblast proliferation, contraction, and extracellular matrix production. Targeted modulation of this cascade in fibroblasts may prove a useful strategy to regulate YAP and TAZ signaling and fibroblast activities central to tissue repair and fibrosis.
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Fibroblastos/enzimología , Factores de Intercambio de Guanina Nucleótido/metabolismo , Receptores de Dopamina D1/metabolismo , Proteínas Coactivadoras Transcripcionales con Motivo de Unión a PDZ/metabolismo , Proteínas Señalizadoras YAP/metabolismo , Proteínas ras/metabolismo , Células Cultivadas , Agonistas de Dopamina/farmacología , Fibroblastos/efectos de los fármacos , Fibroblastos/patología , Fibrosis , Factores de Intercambio de Guanina Nucleótido/genética , Humanos , Fenantridinas/farmacología , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismo , Receptores de Dopamina D1/agonistas , Transducción de Señal , Proteínas Coactivadoras Transcripcionales con Motivo de Unión a PDZ/genética , Proteínas Señalizadoras YAP/genética , Proteínas ras/genéticaRESUMEN
Matrix resorption is essential to the clearance of the extracellular matrix (ECM) after normal wound healing. A disruption in these processes constitutes a main component of fibrotic diseases, characterized by excess deposition and diminished clearance of fibrillar ECM proteins, such as collagen type I. The mechanisms and stimuli regulating ECM resorption in the lung remain poorly understood. Recently, agonism of dopamine receptor D1 (DRD1), which is predominantly expressed on fibroblasts in the lung, has been shown to accelerate tissue repair and clearance of ECM following bleomycin injury in mice. Therefore, we investigated whether DRD1 receptor signaling promotes the degradation of collagen type I by lung fibroblasts. For cultured fibroblasts, we found that DRD1 agonism enhances extracellular cleavage, internalization and lysosomal degradation of collagen I mediated by cathepsin K, which results in reduced stiffness of cell-derived matrices, as measured by atomic force microscopy. In vivo agonism of DRD1 similarly enhanced fibrillar collagen degradation by fibroblasts, as assessed by tissue labeling with a collagen-hybridizing peptide. Together, these results implicate DRD1 agonism in fibroblast-mediated collagen clearance, suggesting an important role for this mechanism in fibrosis resolution.This article has an associated First Person interview with the first author of the paper.
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Colágeno Tipo I , Fibroblastos , Animales , Catepsina K/genética , Células Cultivadas , Colágeno , Colágeno Tipo I/genética , Matriz Extracelular , Pulmón , Ratones , Receptores de Dopamina D1/genéticaRESUMEN
Idiopathic pulmonary fibrosis (IPF) is a progressive disease thought to result from impaired lung repair following injury and is strongly associated with aging. While vascular alterations have been associated with IPF previously, the contribution of lung vasculature during injury resolution and fibrosis is not well understood. To compare the role of endothelial cells (ECs) in resolving and non-resolving models of lung fibrosis, we applied bleomycin intratracheally to young and aged mice. We found that injury in aged mice elicited capillary rarefaction, while injury in young mice resulted in increased capillary density. ECs from the lungs of injured aged mice relative to young mice demonstrated elevated pro-fibrotic and reduced vascular homeostasis gene expression. Among the latter, Nos3 (encoding the enzyme endothelial nitric oxide synthase, eNOS) was transiently upregulated in lung ECs from young but not aged mice following injury. Young mice deficient in eNOS recapitulated the non-resolving lung fibrosis observed in aged animals following injury, suggesting that eNOS directly participates in lung fibrosis resolution. Activation of the NO receptor soluble guanylate cyclase in human lung fibroblasts reduced TGFß-induced pro-fibrotic gene and protein expression. Additionally, loss of eNOS in human lung ECs reduced the suppression of TGFß-induced lung fibroblast activation in 2D and 3D co-cultures. Altogether, our results demonstrate that persistent lung fibrosis in aged mice is accompanied by capillary rarefaction, loss of EC identity, and impaired eNOS expression. Targeting vascular function may thus be critical to promote lung repair and fibrosis resolution in aging and IPF.
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Bleomicina/efectos adversos , Fibrosis/patología , Fibrosis Pulmonar Idiopática/inducido químicamente , Pulmón/patología , Animales , Humanos , RatonesRESUMEN
Idiopathic pulmonary fibrosis (IPF) results in scarring of the lungs by excessive extracellular matrix (ECM) production. Resident fibroblasts are the major cell type involved in ECM deposition. The biochemical pathways that facilitate pathological fibroblast activation leading to aberrant ECM deposition are not fully understood. Tank binding protein kinase-1 (TBK1) is a kinase that regulates multiple signaling pathways and was recently identified as a candidate regulator of fibroblast activation in a large-scale small-interfering RNA (siRNA) screen. To determine the effect of TBK1 on fibroblast activation, TBK1 was inhibited pharmacologically (MRT-68601) and genetically (siRNA) in normal and IPF human lung fibroblasts. Reducing the activity or expression of TBK1 led to reduction in α-smooth muscle actin stress fiber levels by 40-60% and deposition of ECM components collagen I and fibronectin by 50% in TGF-ß-stimulated normal and IPF fibroblasts. YAP and TAZ are homologous mechanoregulatory profibrotic transcription cofactors known to regulate fibroblast activation. TBK1 knockdown or inhibition decreased the total and nuclear protein levels of YAP/TAZ. Additionally, low cell-cell contact and increased ECM substrate stiffness augmented the phosphorylation and activation of TBK1, consistent with cues that regulate YAP/TAZ. The action of TBK1 toward YAP/TAZ activation was independent of LATS1/2 and canonical downstream TBK1 signaling mediator IRF3 but dependent on proteasomal machinery of the cell. This study identifies TBK1 as a fibrogenic activator of human pulmonary fibroblasts, suggesting TBK1 may be a novel therapeutic target in pulmonary fibrosis.
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Proteínas Adaptadoras Transductoras de Señales/genética , Fibroblastos/metabolismo , Fibrosis Pulmonar Idiopática/genética , Proteínas Serina-Treonina Quinasas/genética , Transactivadores/genética , Factores de Transcripción/genética , Actinas/genética , Actinas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Comunicación Celular , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Matriz Extracelular/química , Matriz Extracelular/metabolismo , Fibroblastos/efectos de los fármacos , Fibroblastos/patología , Fibronectinas/genética , Fibronectinas/metabolismo , Regulación de la Expresión Génica , Humanos , Fibrosis Pulmonar Idiopática/metabolismo , Fibrosis Pulmonar Idiopática/patología , Factor 3 Regulador del Interferón/genética , Factor 3 Regulador del Interferón/metabolismo , Pulmón/metabolismo , Pulmón/patología , Cultivo Primario de Células , Complejo de la Endopetidasa Proteasomal/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Proteínas Coactivadoras Transcripcionales con Motivo de Unión a PDZ , Factor de Crecimiento Transformador beta/farmacología , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Proteínas Señalizadoras YAPRESUMEN
A variety of G protein-coupled receptors (GPCRs) have been implicated in the pathogenesis of pulmonary fibrosis, largely through their promotion of profibrotic fibroblast activation. By contrast, recent work has highlighted the beneficial effects of Gαs-coupled GPCRs on reducing fibroblast activation and fibrosis. This review highlights how fibrosis-promoting and -inhibiting GPCR signaling converges on downstream signaling and transcriptional effectors, and how the diversity and dynamics of GPCR expression challenge efforts to identify effective therapies for idiopathic pulmonary fibrosis (IPF). Next-generation strategies to overcome these challenges, focusing on target selection, polypharmacology, and personalized medicine approaches, are discussed as a path towards more effective GPCR-targeted therapies for pulmonary fibrosis.
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Fibrosis Pulmonar Idiopática , Fibrosis , Humanos , Fibrosis Pulmonar Idiopática/tratamiento farmacológico , Polifarmacología , Receptores Acoplados a Proteínas G , Transducción de SeñalRESUMEN
Uterine fibroids (UFs) are benign myometrial neoplasms. The mechanical environment activates signaling through the Hippo pathway effectors Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding domain (TAZ) in other fibrotic disorders. Here, we assess the differences in YAP/TAZ responsiveness to signals in UF compared with myometrium (Myo). Matched samples of UF and Myo were collected. Atomic force microscopy (AFM) was used to determine in situ stiffness. Cells were plated sparsely on hydrogels or at confluence. Ten nanomolars of estradiol (E2) and 100 nM progesterone (P4) were used. Immunostaining for YAP/TAZ and extracellular matrix (ECM) proteins was performed. Cells were incubated with control or YAP1 (YAP)/WWTR1 (TAZ) small interfering RNA (siRNA). Real time qPCR was completed for connective tissue growth factor (CTGF). Cells were treated with verteporfin (a YAP inhibitor) or Y27632 (a ROCK inhibitor), and ECM gene expression was analyzed. Paired t test and Wilcoxon sign-rank test were used. AFM-measured tissue stiffness and YAP/TAZ nuclear localization in situ and in confluent cells were higher in UF compared with Myo (p < 0.05). Decreasing substrate stiffness reduced YAP/TAZ nuclear localization for both Myo and UF (p = 0.05). Stimulating cells with E2 or P4 increased YAP/TAZ nuclear localization, but only in Myo (p = 0.01). UFs had increased FN, COLI, and COLIII deposition. Following siRNA targeting, CTGF was found to be statistically decreased. Verteporfin treatment reduced cell survival and reduced FN deposition. Treatment with Y27632 demonstrated better cell tolerance and a reduction in ECM deposition. The mechanosensitive pathway may be linked to YAP/TAZ function and involved in transducing fibroid growth.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Estradiol/metabolismo , Leiomioma/metabolismo , Miometrio/metabolismo , Progesterona/metabolismo , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Neoplasias Uterinas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/antagonistas & inhibidores , Amidas/administración & dosificación , Módulo de Elasticidad/efectos de los fármacos , Inhibidores Enzimáticos/administración & dosificación , Estradiol/administración & dosificación , Proteínas de la Matriz Extracelular/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Miometrio/efectos de los fármacos , Progesterona/administración & dosificación , Piridinas/administración & dosificación , Transducción de Señal/efectos de los fármacos , Factores de Transcripción/antagonistas & inhibidores , Proteínas Coactivadoras Transcripcionales con Motivo de Unión a PDZ , Verteporfina/administración & dosificación , Proteínas Señalizadoras YAP , Quinasas Asociadas a rho/antagonistas & inhibidoresRESUMEN
OBJECTIVE: This study was designed to evaluate the roles of microRNAs (miRNAs) in slow transit constipation (STC). DESIGN: All human tissue samples were from the muscularis externa of the colon. Expression of 372 miRNAs was examined in a discovery cohort of four patients with STC versus three age/sex-matched controls by a quantitative PCR array. Upregulated miRNAs were examined by quantitative reverse transcription qPCR (RT-qPCR) in a validation cohort of seven patients with STC and age/sex-matched controls. The effect of a highly differentially expressed miRNA on a custom human smooth muscle cell line was examined in vitro by RT-qPCR, electrophysiology, traction force microscopy, and ex vivo by lentiviral transduction in rat muscularis externa organotypic cultures. RESULTS: The expression of 13 miRNAs was increased in STC samples. Of those miRNAs, four were predicted to target SCN5A, the gene that encodes the Na+ channel NaV1.5. The expression of SCN5A mRNA was decreased in STC samples. Let-7f significantly decreased Na+ current density in vitro in human smooth muscle cells. In rat muscularis externa organotypic cultures, overexpression of let-7f resulted in reduced frequency and amplitude of contraction. CONCLUSIONS: A small group of miRNAs is upregulated in STC, and many of these miRNAs target the SCN5A-encoded Na+ channel NaV1.5. Within this set, a novel NaV1.5 regulator, let-7f, resulted in decreased NaV1.5 expression, current density and reduced motility of GI smooth muscle. These results suggest NaV1.5 and miRNAs as novel diagnostic and potential therapeutic targets in STC.
Asunto(s)
Estreñimiento/fisiopatología , Regulación de la Expresión Génica , MicroARNs/genética , Proteínas Asociadas a Microtúbulos/genética , Contracción Muscular/genética , Adulto , Anciano , Biopsia con Aguja , Estudios de Casos y Controles , Colon/patología , Femenino , Motilidad Gastrointestinal/genética , Humanos , Inmunohistoquímica , Persona de Mediana Edad , Contracción Muscular/fisiología , Músculo Liso , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Valores de Referencia , Muestreo , Regulación hacia ArribaRESUMEN
Tissue fibrosis is characterized by uncontrolled deposition and diminished clearance of fibrous connective tissue proteins, ultimately leading to organ scarring. Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) have recently emerged as pivotal drivers of mesenchymal cell activation in human fibrosis. Therapeutic strategies inhibiting YAP and TAZ have been hindered by the critical role that these proteins play in regeneration and homeostasis in different cell types. Here, we find that the Gαs-coupled dopamine receptor D1 (DRD1) is preferentially expressed in lung and liver mesenchymal cells relative to other resident cells of these organs. Agonism of DRD1 selectively inhibits YAP/TAZ function in mesenchymal cells and shifts their phenotype from profibrotic to fibrosis resolving, reversing in vitro extracellular matrix stiffening and in vivo tissue fibrosis in mouse models. Aromatic l-amino acid decarboxylase [DOPA decarboxylase (DDC)], the enzyme responsible for the final step in biosynthesis of dopamine, is decreased in the lungs of subjects with idiopathic pulmonary fibrosis, and its expression inversely correlates with disease severity, consistent with an endogenous protective role for dopamine signaling that is lost in pulmonary fibrosis. Together, these findings establish a pharmacologically tractable and cell-selective approach to targeting YAP/TAZ via DRD1 that reverses fibrosis in mice.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/antagonistas & inhibidores , Proteínas de Ciclo Celular/antagonistas & inhibidores , Fibroblastos/patología , Cirrosis Hepática/patología , Fibrosis Pulmonar/patología , Receptores de Dopamina D1/agonistas , Receptores de Dopamina D1/metabolismo , Transactivadores/antagonistas & inhibidores , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Bleomicina , Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Dopa-Decarboxilasa/metabolismo , Matriz Extracelular/efectos de los fármacos , Matriz Extracelular/metabolismo , Femenino , Fibroblastos/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Células Estrelladas Hepáticas/efectos de los fármacos , Células Estrelladas Hepáticas/patología , Humanos , Pulmón/efectos de los fármacos , Pulmón/patología , Lesión Pulmonar/patología , Masculino , Ratones Endogámicos C57BL , Fenantridinas/farmacología , Fenotipo , Transporte de Proteínas/efectos de los fármacos , Interferencia de ARN , Transactivadores/metabolismo , Proteínas Señalizadoras YAPRESUMEN
Tissue fibrosis is a chronic disease driven by persistent fibroblast activation that has recently been linked to epigenetic modifications. Here, we screened a small library of epigenetic small-molecule modulators to identify compounds capable of inhibiting or reversing TGFß-mediated fibroblast activation. We identified pracinostat, an HDAC inhibitor, as a potent attenuator of lung fibroblast activation and confirmed its efficacy in patient-derived fibroblasts isolated from fibrotic lung tissue. Mechanistically, we found that HDAC-dependent transcriptional repression was an early and essential event in TGFß-mediated fibroblast activation. Treatment of lung fibroblasts with pracinostat broadly attenuated TGFß-mediated epigenetic repression and promoted fibroblast quiescence. We confirmed a specific role for HDAC-dependent histone deacetylation in the promoter region of the anti-fibrotic gene PPARGC1A (PGC1α) in response to TGFß stimulation. Finally, we identified HDAC7 as a key factor whose siRNA-mediated knockdown attenuates fibroblast activation without altering global histone acetylation. Together, these results provide novel mechanistic insight into the essential role HDACs play in TGFß-mediated fibroblast activation via targeted gene repression.