RESUMEN
Adipose tissue is an essential regulator of metabolic homeostasis. In contrast with white adipose tissue, which stores excess energy in the form of triglycerides, brown adipose tissue is thermogenic, dissipating energy as heat via the unique expression of the mitochondrial uncoupling protein UCP1. A subset of UCP1+ adipocytes develops within white adipose tissue in response to physiological stimuli; however, the developmental origin of these "brite" or "beige" adipocytes is unclear. Here, we report the identification of a BMP7-ROCK signaling axis regulating beige adipocyte formation via control of the G-actin-regulated transcriptional coactivator myocardin-related transcription factor A, MRTFA. White adipose tissue from MRTFA(-/-) mice contains more multilocular adipocytes and expresses enhanced levels of brown-selective proteins, including UCP1. MRTFA(-/-) mice also show improved metabolic profiles and protection from diet-induced obesity and insulin resistance. Our study hence unravels a central pathway driving the development of physiologically functional beige adipocytes.
Asunto(s)
Transactivadores/metabolismo , Adipogénesis , Animales , Proteína Morfogenética Ósea 7/metabolismo , Dieta , Metabolismo Energético , Resistencia a la Insulina , Masculino , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Obesidad/metabolismo , Transactivadores/genética , Factor de Crecimiento Transformador beta1/metabolismoRESUMEN
Human transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), causative pathogen of the COVID-19 pandemic, exerts a massive health and socioeconomic crisis. The virus infects alveolar epithelial type 2 cells (AT2s), leading to lung injury and impaired gas exchange, but the mechanisms driving infection and pathology are unclear. We performed a quantitative phosphoproteomic survey of induced pluripotent stem cell-derived AT2s (iAT2s) infected with SARS-CoV-2 at air-liquid interface (ALI). Time course analysis revealed rapid remodeling of diverse host systems, including signaling, RNA processing, translation, metabolism, nuclear integrity, protein trafficking, and cytoskeletal-microtubule organization, leading to cell cycle arrest, genotoxic stress, and innate immunity. Comparison to analogous data from transformed cell lines revealed respiratory-specific processes hijacked by SARS-CoV-2, highlighting potential novel therapeutic avenues that were validated by a high hit rate in a targeted small molecule screen in our iAT2 ALI system.
Asunto(s)
Células Epiteliales Alveolares/metabolismo , COVID-19/metabolismo , Fosfoproteínas/metabolismo , Proteoma/metabolismo , SARS-CoV-2/metabolismo , Células Epiteliales Alveolares/patología , Células Epiteliales Alveolares/virología , Animales , Antivirales , COVID-19/genética , COVID-19/patología , Chlorocebus aethiops , Efecto Citopatogénico Viral , Citoesqueleto , Evaluación Preclínica de Medicamentos , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/patología , Células Madre Pluripotentes Inducidas/virología , Fosfoproteínas/genética , Transporte de Proteínas , Proteoma/genética , SARS-CoV-2/genética , Transducción de Señal , Células Vero , Tratamiento Farmacológico de COVID-19RESUMEN
RhoA and its effectors, the transcriptional coactivators myocardin-related transcription factor (MRTF) and serum response factor (SRF), control epithelial phenotype and are indispensable for profibrotic epithelial reprogramming during fibrogenesis. Context-dependent control of RhoA and fibrosis-associated changes in its regulators, however, remain incompletely characterized. We previously identified the guanine nucleotide exchange factor GEF-H1 as a central mediator of RhoA activation in renal tubular cells exposed to inflammatory or fibrotic stimuli. Here we found that GEF-H1 expression and phosphorylation were strongly elevated in two animal models of fibrosis. In the Unilateral Ureteral Obstruction mouse kidney fibrosis model, GEF-H1 was upregulated predominantly in the tubular compartment. GEF-H1 was also elevated and phosphorylated in a rat pulmonary artery banding (PAB) model of right ventricular fibrosis. Prolonged stimulation of LLC-PK1 tubular cells with tumor necrosis factor (TNF)-α or transforming growth factor (TGF)-ß1 increased GEF-H1 expression and activated a luciferase-coupled GEF-H1 promoter. Knockdown and overexpression studies revealed that these effects were mediated by RhoA, cytoskeleton remodeling, and MRTF, indicative of a positive feedback cycle. Indeed, silencing endogenous GEF-H1 attenuated activation of the GEF-H1 promoter. Of importance, inhibition of MRTF using CCG-1423 prevented GEF-H1 upregulation in both animal models. MRTF-dependent increase in GEF-H1 was prevented by inhibition of the transcription factor Sp1, and mutating putative Sp1 binding sites in the GEF-H1 promoter eliminated its MRTF-dependent activation. As the GEF-H1/RhoA axis is key for fibrogenesis, this novel MRTF/Sp1-dependent regulation of GEF-H1 abundance represents a potential target for reducing renal and cardiac fibrosis.NEW & NOTEWORTHY We show that expression of the RhoA regulator GEF-H1 is upregulated in tubular cells exposed to fibrogenic cytokines and in animal models of kidney and heart fibrosis. We identify a pathway wherein GEF-H1/RhoA-dependent MRTF activation through its noncanonical partner Sp1 upregulates GEF-H1. Our data reveal the existence of a positive feedback cycle that enhances Rho signaling through control of both GEF-H1 activation and expression. This feedback loop may play an important role in organ fibrosis.
Asunto(s)
Fibrosis , Factores de Intercambio de Guanina Nucleótido Rho , Factor de Transcripción Sp1 , Transactivadores , Proteína de Unión al GTP rhoA , Animales , Factor de Transcripción Sp1/metabolismo , Factor de Transcripción Sp1/genética , Proteína de Unión al GTP rhoA/metabolismo , Proteína de Unión al GTP rhoA/genética , Factores de Intercambio de Guanina Nucleótido Rho/metabolismo , Factores de Intercambio de Guanina Nucleótido Rho/genética , Transactivadores/metabolismo , Transactivadores/genética , Ratones , Ratas , Retroalimentación Fisiológica , Masculino , Ratones Endogámicos C57BL , Humanos , Transducción de Señal , Porcinos , Fosforilación , Modelos Animales de Enfermedad , Obstrucción Ureteral/metabolismo , Obstrucción Ureteral/patología , Obstrucción Ureteral/genética , Ratas Sprague-Dawley , Línea Celular , Factores de TranscripciónRESUMEN
BACKGROUND: Abdominal aortic aneurysm (AAA) is a relatively common and often fatal condition. A major histopathological hallmark of AAA is the severe degeneration of aortic media with loss of vascular smooth muscle cells (VSMCs), which are the main source of extracellular matrix (ECM) proteins. VSMCs and ECM homeostasis are essential in maintaining structural integrity of the aorta. Cysteine-rich protein 2 (CRP2) is a VSMC-expressed protein; however, the role of CRP2 in AAA formation is unclear. METHODS: To investigate the function of CRP2 in AAA formation, mice deficient in Apoe (Apoe-/-) or both CRP2 (gene name Csrp2) and Apoe (Csrp2-/-Apoe-/-) were subjected to an angiotensin II (Ang II) infusion model of AAA formation. Aortas were harvested at different time points and histological analysis was performed. Primary VSMCs were generated from Apoe-/- and Csrp2-/-Apoe-/- mouse aortas for in vitro mechanistic studies. RESULTS: Loss of CRP2 attenuated Ang II-induced AAA incidence and severity, accompanied by preserved smooth muscle α-actin expression and reduced elastin degradation, matrix metalloproteinase 2 (MMP2) activity, deposition of collagen, particularly collagen III (Col III), aortic tensile strength, and blood pressure. CRP2 deficiency decreased the baseline MMP2 and Col III expression in VSMCs and mitigated Ang II-induced increases of MMP2 and Col III via blunting Erk1/2 signaling. Rescue experiments were performed by reintroducing CRP2 into Csrp2-/-Apoe-/- VSMCs restored Ang II-induced Erk1/2 activation, MMP2 expression and activity, and Col III levels. CONCLUSIONS: Our results indicate that in response to Ang II stimulation, CRP2 deficiency maintains aortic VSMC density, ECM homeostasis, and structural integrity through Erk1/2-Col III and MMP2 axis and reduces AAA formation. Thus, targeting CRP2 provides a potential therapeutic strategy for AAA.
Asunto(s)
Angiotensina II , Aneurisma de la Aorta Abdominal , Angiotensina II/efectos adversos , Angiotensina II/metabolismo , Animales , Aneurisma de la Aorta Abdominal/inducido químicamente , Aneurisma de la Aorta Abdominal/genética , Apolipoproteínas E/metabolismo , Colágeno/efectos adversos , Colágeno/metabolismo , Cisteína , Modelos Animales de Enfermedad , Metaloproteinasa 2 de la Matriz/genética , Metaloproteinasa 2 de la Matriz/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismoRESUMEN
Extracellular matrix (ECM) proteins, collectively known as the matrisome, include collagens, glycoproteins, and proteoglycans. Alterations in the matrisome have been implicated in the neurodegenerative pathologies including Parkinson's disease (PD). In this work, we utilized our previously published PD and control proteomics data from human prefrontal cortex and focused our analysis on the matrisome. Among matrisome proteins, we observed a significant enrichment in the expression of type I collagen in PD vs. control samples. We then performed histological analysis on the same samples used for proteomics study, and examined collagen expression using picrosirius red staining. Interestingly, we observed similar trends in collagen abundance in PD vs. control as in our matrisome analysis; thus, this and other histological analyses will be useful as a complementary technique in the future to study the matrisome in PD with a larger cohort, and it may aid in choosing regions of interest for proteomic analysis. Additionally, collagen hydroxyprolination was less variable in PD compared to controls. Glycoproteomic changes in matrisome molecules were also observed in PD relative to aged individuals, especially related to type VI collagen and versican. We further examined the list of differentially expressed matrisome molecules using network topology-based analysis and found that angiogenesis indicated by alterations in decorin and several members of the collagen family was affected in PD. These findings collectively identified matrisome changes associated with PD; further studies with a larger cohort are required to validate the current results.
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Proteínas de la Matriz Extracelular , Enfermedad de Parkinson , Proteómica , Anciano , Matriz Extracelular/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Humanos , Enfermedad de Parkinson/metabolismo , Proteoglicanos/metabolismo , Proteómica/métodosRESUMEN
Aortic carboxypeptidase-like protein (ACLP) is a collagen-binding extracellular matrix protein that has important roles in wound healing and fibrosis. ACLP contains thrombospondin repeats, a collagen-binding discoidin domain, and a catalytically inactive metallocarboxypeptidase domain. Recently, mutations in the ACLP-encoding gene, AE-binding protein 1 (AEBP1), have been discovered, leading to the identification of a new variant of Ehlers-Danlos syndrome causing connective tissue disruptions in multiple organs. Currently, little is known about the mechanisms of ACLP secretion or the role of post-translational modifications in these processes. We show here that the secreted form of ACLP contains N-linked glycosylation and that inhibition of glycosylation results in its intracellular retention. Using site-directed mutagenesis, we determined that glycosylation of Asn-471 and Asn-1030 is necessary for ACLP secretion and identified a specific N-terminal proteolytic ACLP fragment. To determine the contribution of secreted ACLP to extracellular matrix mechanical properties, we generated and mechanically tested wet-spun collagen ACLP composite fibers, finding that ACLP enhances the modulus (or stiffness), toughness, and tensile strength of the fibers. Some AEBP1 mutations were null alleles, whereas others resulted in expressed proteins. We tested the hypothesis that a recently discovered 40-amino acid mutation and insertion in the ACLP discoidin domain regulates collagen binding and assembly. Interestingly, we found that this protein variant is retained intracellularly and induces endoplasmic reticulum stress identified with an XBP1-based endoplasmic reticulum stress reporter. Our findings highlight the importance of N-linked glycosylation of ACLP for its secretion and contribute to our understanding of ACLP-dependent disease pathologies.
Asunto(s)
Carboxipeptidasas/metabolismo , Síndrome de Ehlers-Danlos/metabolismo , Matriz Extracelular/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas Represoras/metabolismo , Sustitución de Aminoácidos , Animales , Carboxipeptidasas/genética , Síndrome de Ehlers-Danlos/genética , Síndrome de Ehlers-Danlos/patología , Matriz Extracelular/genética , Matriz Extracelular/patología , Glicosilación , Ratones , Mutagénesis Sitio-Dirigida , Mutación Missense , Proteínas Represoras/genéticaRESUMEN
AEBP1 encodes the aortic carboxypeptidase-like protein (ACLP) that associates with collagens in the extracellular matrix (ECM) and has several roles in development, tissue repair, and fibrosis. ACLP is expressed in bone, the vasculature, and dermal tissues and is involved in fibroblast proliferation and mesenchymal stem cell differentiation into collagen-producing cells. Aebp1-/- mice have abnormal, delayed wound repair correlating with defects in fibroblast proliferation. In this study, we describe four individuals from three unrelated families that presented with a unique constellation of clinical findings including joint laxity, redundant and hyperextensible skin, poor wound healing with abnormal scarring, osteoporosis, and other features reminiscent of Ehlers-Danlos syndrome (EDS). Analysis of skin biopsies revealed decreased dermal collagen with abnormal collagen fibrils that were ragged in appearance. Exome sequencing revealed compound heterozygous variants in AEBP1 (c.1470delC [p.Asn490_Met495delins(40)] and c.1743C>A [p.Cys581∗]) in the first individual, a homozygous variant (c.1320_1326del [p.Arg440Serfs∗3]) in the second individual, and a homozygous splice site variant (c.1630+1G>A) in two siblings from the third family. We show that ACLP enhances collagen polymerization and binds to several fibrillar collagens via its discoidin domain. These studies support the conclusion that bi-allelic pathogenic variants in AEBP1 are the cause of this autosomal-recessive EDS subtype.
Asunto(s)
Alelos , Carboxipeptidasas/genética , Colágeno/metabolismo , Tejido Conectivo/patología , Síndrome de Ehlers-Danlos/genética , Mutación/genética , Proteínas Represoras/genética , Adulto , Secuencia de Aminoácidos , Carboxipeptidasas/química , Niño , Preescolar , Femenino , Fibroblastos/metabolismo , Fibroblastos/patología , Humanos , Masculino , Dominios Proteicos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Represoras/química , Piel/patología , Piel/ultraestructura , Adulto JovenRESUMEN
BACKGROUND: Pulmonary atelectasis is frequent in clinical settings. Yet there is limited mechanistic understanding and substantial clinical and biologic controversy on its consequences. The authors hypothesize that atelectasis produces local transcriptomic changes related to immunity and alveolar-capillary barrier function conducive to lung injury and further exacerbated by systemic inflammation. METHODS: Female sheep underwent unilateral lung atelectasis using a left bronchial blocker and thoracotomy while the right lung was ventilated, with (n = 6) or without (n = 6) systemic lipopolysaccharide infusion. Computed tomography guided samples were harvested for NextGen RNA sequencing from atelectatic and aerated lung regions. The Wald test was used to detect differential gene expression as an absolute fold change greater than 1.5 and adjusted P value (Benjamini-Hochberg) less than 0.05. Functional analysis was performed by gene set enrichment analysis. RESULTS: Lipopolysaccharide-unexposed atelectatic versus aerated regions presented 2,363 differentially expressed genes. Lipopolysaccharide exposure induced 3,767 differentially expressed genes in atelectatic lungs but only 1,197 genes in aerated lungs relative to the corresponding lipopolysaccharide-unexposed tissues. Gene set enrichment for immune response in atelectasis versus aerated tissues yielded negative normalized enrichment scores without lipopolysaccharide (less than -1.23, adjusted P value less than 0.05) but positive scores with lipopolysaccharide (greater than 1.33, adjusted P value less than 0.05). Leukocyte-related processes (e.g., leukocyte migration, activation, and mediated immunity) were enhanced in lipopolysaccharide-exposed atelectasis partly through interferon-stimulated genes. Furthermore, atelectasis was associated with negatively enriched gene sets involving alveolar-capillary barrier function irrespective of lipopolysaccharide (normalized enrichment scores less than -1.35, adjusted P value less than 0.05). Yes-associated protein signaling was dysregulated with lower nuclear distribution in atelectatic versus aerated lung (lipopolysaccharide-unexposed: 10.0 ± 4.2 versus 13.4 ± 4.2 arbitrary units, lipopolysaccharide-exposed: 8.1 ± 2.0 versus 11.3 ± 2.4 arbitrary units, effect of lung aeration, P = 0.003). CONCLUSIONS: Atelectasis dysregulates the local pulmonary transcriptome with negatively enriched immune response and alveolar-capillary barrier function. Systemic lipopolysaccharide converts the transcriptomic immune response into positive enrichment but does not affect local barrier function transcriptomics. Interferon-stimulated genes and Yes-associated protein might be novel candidate targets for atelectasis-associated injury.
Asunto(s)
Inmunidad Celular/genética , Inmunidad Celular/inmunología , Atelectasia Pulmonar/genética , Atelectasia Pulmonar/inmunología , Transcriptoma/genética , Animales , Femenino , Mediciones del Volumen Pulmonar/métodos , Atelectasia Pulmonar/diagnóstico por imagen , OvinosRESUMEN
Perlecan is a major heparan sulfate (HS) proteoglycan in the arterial wall. Previous studies have linked it to atherosclerosis. Perlecan contains a core protein and three HS side chains. Its core protein has five domains (DI-DV) with disparate structures and DII is highly homologous to the ligand-binding portion of LDL receptor (LDLR). The functional significance of this domain has been unknown. Here, we show that perlecan DII interacts with LDL. Importantly, the interaction largely relies on O-linked glycans that are only present in the secreted DII. Among the five repeat units of DII, most of the glycosylation sites are from the second unit, which is highly divergent and rich in serine and threonine, but has no cysteine residues. Interestingly, most of the glycans are capped by the negatively charged sialic acids, which are critical for LDL binding. We further demonstrate an additive effect of HS and DII on LDL binding. Unlike LDLR, which directs LDL uptake through endocytosis, this study uncovers a novel feature of the perlecan LDLR-like DII in receptor-mediated lipoprotein retention, which depends on its glycosylation. Thus, perlecan glycosylation may play a role in the early LDL retention during the development of atherosclerosis.
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Aterosclerosis/metabolismo , Lipoproteínas LDL/metabolismo , Receptores de LDL/metabolismo , Animales , Células COS , Línea Celular , Chlorocebus aethiops , Glicosilación , Células HeLa , Proteoglicanos de Heparán Sulfato/metabolismo , Humanos , Inmunohistoquímica , Microscopía Confocal , Mutagénesis Sitio-Dirigida , Ácido N-Acetilneuramínico/metabolismo , RatasRESUMEN
Idiopathic pulmonary fibrosis (IPF) is a chronic and fatal lung disease characterized by the overgrowth, hardening, and scarring of lung tissue. The exact mechanisms of how IPF develops and progresses are unknown. IPF is characterized by extracellular matrix remodeling and accumulation of active TGFß, which promotes collagen expression and the differentiation of smooth muscle α-actin (SMA)-positive myofibroblasts. Aortic carboxypeptidase-like protein (ACLP) is an extracellular matrix protein secreted by fibroblasts and myofibroblasts and is expressed in fibrotic human lung tissue and in mice with bleomycin-induced fibrosis. Importantly, ACLP knockout mice are significantly protected from bleomycin-induced fibrosis. The goal of this study was to identify the mechanisms of ACLP action on fibroblast differentiation. As primary lung fibroblasts differentiated into myofibroblasts, ACLP expression preceded SMA and collagen expression. Recombinant ACLP induced SMA and collagen expression in mouse and human lung fibroblasts. Knockdown of ACLP slowed the fibroblast-to-myofibroblast transition and partially reverted differentiated myofibroblasts by reducing SMA expression. We hypothesized that ACLP stimulates myofibroblast formation partly through activating TGFß signaling. Treatment of fibroblasts with recombinant ACLP induced phosphorylation and nuclear translocation of Smad3. This phosphorylation and induction of SMA was dependent on TGFß receptor binding and kinase activity. ACLP-induced collagen expression was independent of interaction with the TGFß receptor. These findings indicate that ACLP stimulates the fibroblast-to-myofibroblast transition by promoting SMA expression via TGFß signaling and promoting collagen expression through a TGFß receptor-independent pathway.
Asunto(s)
Carboxipeptidasas/metabolismo , Fibroblastos/citología , Fibrosis Pulmonar Idiopática/metabolismo , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Proteínas Represoras/metabolismo , Transducción de Señal/fisiología , Animales , Antibióticos Antineoplásicos/toxicidad , Bleomicina/toxicidad , Carboxipeptidasas/genética , Diferenciación Celular/fisiología , Colágeno/genética , Colágeno/metabolismo , Modelos Animales de Enfermedad , Fibroblastos/metabolismo , Células HEK293 , Humanos , Fibrosis Pulmonar Idiopática/patología , Pulmón/citología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Visón , Cultivo Primario de Células , Proteínas Represoras/genética , Mucosa Respiratoria/citología , Mucosa Respiratoria/metabolismoRESUMEN
Multiple intergenic single-nucleotide polymorphisms (SNPs) near hedgehog interacting protein (HHIP) on chromosome 4q31 have been strongly associated with pulmonary function levels and moderate-to-severe chronic obstructive pulmonary disease (COPD). However, whether the effects of variants in this region are related to HHIP or another gene has not been proven. We confirmed genetic association of SNPs in the 4q31 COPD genome-wide association study (GWAS) region in a Polish cohort containing severe COPD cases and healthy smoking controls (P = 0.001 to 0.002). We found that HHIP expression at both mRNA and protein levels is reduced in COPD lung tissues. We identified a genomic region located â¼85 kb upstream of HHIP which contains a subset of associated SNPs, interacts with the HHIP promoter through a chromatin loop and functions as an HHIP enhancer. The COPD risk haplotype of two SNPs within this enhancer region (rs6537296A and rs1542725C) was associated with statistically significant reductions in HHIP promoter activity. Moreover, rs1542725 demonstrates differential binding to the transcription factor Sp3; the COPD-associated allele exhibits increased Sp3 binding, which is consistent with Sp3's usual function as a transcriptional repressor. Thus, increased Sp3 binding at a functional SNP within the chromosome 4q31 COPD GWAS locus leads to reduced HHIP expression and increased susceptibility to COPD through distal transcriptional regulation. Together, our findings reveal one mechanism through which SNPs upstream of the HHIP gene modulate the expression of HHIP and functionally implicate reduced HHIP gene expression in the pathogenesis of COPD.
Asunto(s)
Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Elementos de Facilitación Genéticos/genética , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Polimorfismo de Nucleótido Simple/genética , Enfermedad Pulmonar Obstructiva Crónica/genética , Enfermedad Pulmonar Obstructiva Crónica/patología , Adulto , Anciano , Anciano de 80 o más Años , Alelos , Western Blotting , Bronquios/citología , Bronquios/metabolismo , Estudios de Casos y Controles , Células Cultivadas , Inmunoprecipitación de Cromatina , Mapeo Cromosómico , Cromosomas Humanos Par 4/genética , Ensayo de Cambio de Movilidad Electroforética , Femenino , Fibroblastos/citología , Fibroblastos/metabolismo , Predisposición Genética a la Enfermedad , Genotipo , Haplotipos/genética , Humanos , Pulmón/citología , Pulmón/metabolismo , Masculino , Persona de Mediana Edad , Pronóstico , Regiones Promotoras Genéticas/genética , Enfermedad Pulmonar Obstructiva Crónica/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Fumar/genética , Factor de Transcripción Sp3/metabolismoRESUMEN
BACKGROUND: Vascular smooth muscle cells (VSMCs) of the arterial wall play a critical role in the development of occlusive vascular diseases. Cysteine-rich protein 2 (CRP2) is a VSMC-expressed LIM-only protein, which functionally limits VSMC migration and protects against pathological vascular remodeling. The multifunctional cytokine TGFß has been implicated to play a role in the pathogenesis of atherosclerosis through numerous downstream signaling pathways. We showed previously that TGFß upregulates CRP2 expression; however, the detailed signaling mechanisms remain unclear. RESULTS: TGFß treatment of VSMCs activated both Smad2/3 and ATF2 phosphorylation. Individually knocking down Smad2/3 or ATF2 pathways with siRNA impaired the TGFß induction of CRP2, indicating that both contribute to CRP2 expression. Inhibiting TßRI kinase activity by SB431542 or TßRI knockdown abolished Smad2/3 phosphorylation but did not alter ATF2 phosphorylation, indicating while Smad2/3 phosphorylation was TßRI-dependent ATF2 phosphorylation was independent of TßRI. Inhibiting Src kinase activity by SU6656 suppressed TGFß-induced RhoA and ATF2 activation but not Smad2 phosphorylation. Blocking ROCK activity, the major downstream target of RhoA, abolished ATF2 phosphorylation and CRP2 induction but not Smad2 phosphorylation. Furthermore, JNK inhibition with SP600125 reduced TGFß-induced ATF2 (but not Smad2) phosphorylation and CRP2 protein expression while ROCK inhibition blocked JNK activation. These results indicate that downstream of TßRII, Src family kinase-RhoA-ROCK-JNK signaling pathway mediates TßRI-independent ATF2 activation. Promoter analysis revealed that the TGFß induction of CRP2 was mediated through the CRE and SBE promoter elements that were located in close proximity. CONCLUSIONS: Our results demonstrate that two signaling pathways downstream of TGFß converge on the CRE and SBE sites of the Csrp2 promoter to cooperatively control CRP2 induction in VSMCs, which represents a previously unrecognized mechanism of VSMC gene induction by TGFß.
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Proteínas Portadoras/metabolismo , Proteínas con Dominio LIM/metabolismo , Músculo Liso Vascular/metabolismo , Transducción de Señal , Factor de Crecimiento Transformador beta/farmacología , Factor de Transcripción Activador 2/genética , Factor de Transcripción Activador 2/metabolismo , Animales , Proteínas Portadoras/genética , Células Cultivadas , Proteínas con Dominio LIM/genética , Ratones , Músculo Liso Vascular/efectos de los fármacos , Regiones Promotoras Genéticas , Proteína Smad2/genética , Proteína Smad2/metabolismo , Proteína smad3/genética , Proteína smad3/metabolismo , Quinasas Asociadas a rho/metabolismo , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
Vascular smooth muscle cells (VSMCs) of the arterial wall normally display a differentiated and contractile phenotype. In response to arterial injury, VSMCs switch to a synthetic phenotype, contributing to vascular remodeling. Cysteine-rich protein 2 (CRP2) is a cytoskeletal protein expressed in VSMCs and blunts VSMC migration in part by sequestering the scaffolding protein p130Cas at focal adhesions. CRP2 deficiency in mice increases neointima formation following arterial injury. The goal of this study was to use Csrp2 promoter-lacZ transgenic mice to analyze CRP2 expression during VSMC phenotypic modulation. In a neointima formation model after carotid artery cessation of blood flow, lacZ reporter activity and smooth muscle (SM) α-actin expression in the media were rapidly downregulated 4 days after carotid ligation. Fourteen days after ligation, there was a high level expression of both Csrp2 promoter activity and SM α-actin protein expression in neointimal cells. In atherosclerosis prone mice fed an atherogenic diet, Csrp2 promoter activity was detected within complex atherosclerotic lesions. Interestingly, Csrp2 promoter activity was also present in the fibrous caps of complicated atherosclerotic lesions, indicating that CRP2 might contribute to plaque stability. These findings support the concept that CRP2 contributes to the phenotypic modulation of VSMCs during vascular disease. Modulating transcription to increase CRP2 expression during vascular injury might attenuate vascular remodeling. In addition, increased CRP2 expression at the fibrous caps of advanced lesions might also serve to protect atherosclerotic plaques from rupture.
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Aterosclerosis/metabolismo , Proteínas Portadoras/metabolismo , Regulación de la Expresión Génica/fisiología , Proteínas con Dominio LIM/metabolismo , Músculo Liso Vascular/metabolismo , Lesiones del Sistema Vascular/metabolismo , Actinas/metabolismo , Animales , Arterias Carótidas/cirugía , Proteínas Portadoras/genética , Movimiento Celular/fisiología , Cartilla de ADN/genética , Galactósidos , Regulación de la Expresión Génica/genética , Genotipo , Técnicas Histológicas , Inmunohistoquímica , Indoles , Proteínas con Dominio LIM/genética , Ligadura , Masculino , Ratones , Ratones Transgénicos , Neointima/fisiopatología , Reacción en Cadena de la PolimerasaRESUMEN
Transforming growth factor beta (TGF-ß) is a potent growth factor that regulates the homeostasis of native cartilage and is administered as an anabolic supplement for engineered cartilage growth. The quantification of TGF-ß activity in live tissues in situ remains a significant challenge, as conventional activity assessments (e.g., Western blotting of intracellular signaling molecules or reporter cell assays) are unable to measure absolute levels of TGF-ß activity in three-dimensional tissues. In this study, we develop a quantification platform established on TGF-ß's autoinduction response, whereby active TGF-ß (aTGF-ß) signaling in cells induces their biosynthesis and secretion of new TGF-ß in its latent form (LTGF-ß). As such, cell-secreted LTGF-ß can serve as a robust, non-destructive, label-free biomarker for quantifying in situ activity of TGF-ß in live cartilage tissues. Here, we detect LTGF-ß1 secretion levels for bovine native tissue explants and engineered tissue constructs treated with varying doses of media-supplemented aTGF-ß3 using an isoform-specific ELISA. We demonstrate that: 1) LTGF-ß secretion levels increase proportionally to aTGF-ß exposure, reaching 7.4- and 6.6-fold increases in native and engineered cartilage, respectively; 2) synthesized LTGF-ß exhibits low retention in both native and engineered cartilage tissue; and 3) secreted LTGF-ß is stable in conditioned media for 2 weeks, thus enabling a reliable biological standard curve between LTGF-ß secretion and exposed TGF-ß activity. Accordingly, we perform quantifications of TGF-ß activity in bovine native cartilage, demonstrating up to 0.59 ng/mL in response to physiological dynamic loading. We further quantify the in situ TGF-ß activity in aTGF-ß-conjugated scaffolds for engineered tissue, which exhibits 1.81 ng/mL of TGF-ß activity as a result of a nominal 3 µg/mL loading dose. Overall, cell-secreted LTGF-ß can serve as a robust biomarker to quantify in situ activity of TGF-ß in live cartilage tissue and can be potentially applied for a wide range of applications, including multiple tissue types and tissue engineering platforms with different cell populations and scaffolds.
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[This corrects the article DOI: 10.3389/fphar.2023.1243505.].
RESUMEN
Pulmonary artery hypertension (PAH) patients exhibit elevated levels of inflammatory cytokines and infiltration of inflammatory cells in the lung. Concurrently, mutations of bmpr2, the gene encoding the type II receptor of bone morphogenetic proteins (BMP), are found in â¼75% of patients with familial PAH, but a possible nexus between increased inflammation and diminished BMP signaling has hitherto remained elusive. We previously showed that BMP4 triggers nuclear localization of the Myocardin-related transcription factor A (MRTF-A) in human pulmonary artery smooth muscle cells (PASMC), resulting in the induction of contractile proteins. Here we report the BMPR2-dependent repression of a set of inflammatory mediators in response to BMP4 stimulation of PASMC. Forced expression of MRTF-A precisely emulates the anti-inflammatory effect of BMP4, while MRTF-A depletion precludes BMP4-mediated cytokine inhibition. BMP4 and MRTF-A block signaling through NF-κB, the keystone of most pathways leading to inflammatory responses, at the level of chromatin recruitment and promoter activation. Moreover, MRTF-A physically interacts with RelA/p65, the NF-κB subunit endowed with a transcription activation domain. Interestingly, the MRTF-A-NF-κB interaction is mutually antagonistic: stimulation of NF-κB signaling by TNFα, as well as p65 overexpression, hinders MRTF-A activity and the expression of contractile genes. Thus, a molecular inhibitory pathway linking BMP4 signaling, activation of MRTF-A, and inhibition of NF-κB provides insights into the etiology of PAH and a potential focus of therapeutic intervention.
Asunto(s)
Proteína Morfogenética Ósea 4/metabolismo , Proteínas de Unión al ADN/metabolismo , Hipertensión Pulmonar/metabolismo , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Proteínas de Fusión Oncogénica/metabolismo , Transducción de Señal , Proteína Morfogenética Ósea 4/genética , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/genética , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/metabolismo , Células Cultivadas , Proteínas de Unión al ADN/genética , Humanos , Hipertensión Pulmonar/genética , Hipertensión Pulmonar/patología , Hipertensión Pulmonar/terapia , Músculo Liso Vascular/patología , Miocitos del Músculo Liso/patología , Proteínas de Fusión Oncogénica/genética , Transactivadores , Factor de Transcripción ReIA/genética , Factor de Transcripción ReIA/metabolismo , Factor de Necrosis Tumoral alfa/biosíntesis , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
In adult white adipose tissue, cold or ß3-adrenoceptor activation promotes the appearance of thermogenic beige adipocytes. Our comprehensive single-cell analysis revealed that these cells arise through the reprogramming of existing adipogenic trajectories, rather than from a single precursor. These trajectories predominantly arise from SM22-expressing vascular mural progenitor cells. Central in this transition is the activation of Adrb3 in mature adipocytes, leading to subsequent upregulation of Adrb1 in primed progenitors. Under thermoneutral conditions, synergistic activation of both Adrb3 and Adrb1 recapitulates the pattern of cold-induced SM22+ cell recruitment. Lipolysis-derived eicosanoids, specifically docosahexaenoic acid (DHA) and arachidonic acid (AA) prime these processes and in vitro, were sufficient to recapitulate progenitor cells priming. Collectively, our findings provide a robust model for cold-induced beige adipogenesis, emphasizing a profound relationship between mature adipocytes and mural cells during cold acclimation, and revealing the metabolic potential of this unique cellular reservoir.
RESUMEN
Background: We hypothesize that the poor survival outcomes of end-stage kidney disease (ESKD) patients undergoing hemodialysis are associated with a low filtering efficiency and selectivity. The current gold standard criteria using single or several markers show an inability to predict or disclose the treatment effect and disease progression accurately. Methods: We performed an integrated mass spectrometry-based metabolomic and proteomic workflow capable of detecting and quantifying circulating small molecules and proteins in the serum of ESKD patients. Markers linked to cardiovascular disease (CVD) were validated on human induced pluripotent stem cell (iPSC)-derived cardiomyocytes. Results: We identified dozens of elevated molecules in the serum of patients compared with healthy controls. Surprisingly, many metabolites, including lipids, remained at an elevated blood concentration despite dialysis. These molecules and their associated physical interaction networks are correlated with clinical complications in chronic kidney disease. This study confirmed two uremic toxins associated with CVD, a major risk for patients with ESKD. Conclusion: The retained molecules and metabolite-protein interaction network address a knowledge gap of candidate uremic toxins associated with clinical complications in patients undergoing dialysis, providing mechanistic insights and potential drug discovery strategies for ESKD.
RESUMEN
Introduction: Obesity is associated with increased breast cancer incidence, recurrence, and mortality. Adipocytes and adipose-derived stem cells (ASCs), two resident cell types in adipose tissue, accelerate the early stages of breast cancer progression. It remains unclear whether obesity plays a role in the subsequent escape of malignant breast cancer cells into the local circulation. Methods: We engineered models of human breast tumors with adipose stroma that exhibited different obesity-specific alterations. We used these models to assess the invasion and escape of breast cancer cells into an empty, blind-ended cavity (as a mimic of a lymphatic vessel) for up to sixteen days. Results: Lean and obese donor-derived adipose stroma hastened escape to similar extents. Moreover, a hypertrophic adipose stroma did not affect the rate of adipose-induced escape. When admixed directly into the model tumors, lean and obese donor-derived ASCs hastened escape similarly. Conclusions: This study demonstrates that the presence of adipose cells, independently of the obesity status of the adipose tissue donor, hastens the escape of human breast cancer cells in multiple models of obesity-associated breast cancer. Supplementary Information: The online version contains supplementary material available at 10.1007/s12195-022-00750-y.