RESUMEN
OBJECTIVE: Synovial fibrosis contributes to osteoarthritis (OA) pathology, but the underlying mechanisms remain unknown. We have observed increased microRNA-27b-3p (miR-27b-3p) levels in synovial fluid of patients with late-stage radiographic knee OA. Here, we investigated the contribution of miR-27b-3p to synovial fibrosis in patients with severe knee OA and in a mouse model of knee OA. METHODS: We stained synovium sections obtained from patients with radiographic knee OA scored according to the Kellgren/Lawrence scale and mice that underwent destabilization of the medial meniscus (DMM) for miR-27b-3p using in situ hybridization. We examined the effects of intraarticular injection of miR-27b-3p mimic into naive mouse knee joints and intraarticular injection of a miR-27b-3p inhibitor into mouse knee joints after DMM. We performed transfection with miR-27b-3p mimic and miR-27b-3p inhibitor in human OA fibroblast-like synoviocytes (FLS) using reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) array, RNA sequencing, RT-qPCR, Western blotting, immunofluorescence, and migration assays. RESULTS: We observed increased miR-27b-3p expression in the synovium from patients with knee OA and in mice with DMM-induced arthritis. Injection of the miR-27b-3p mimic in mouse knee joints induced a synovial fibrosis-like phenotype, increased synovitis scores, and increased COL1A1 and α-smooth muscle actin (α-SMA) expression. In the mouse model of DMM-induced arthritis, injection of the miR-27b-3p inhibitor decreased α-SMA but did not change COL1A1 expression levels or synovitis scores. Transfection with the miR-27b-3p mimic in human OA FLS induced profibrotic responses, including increased migration and expression of key extracellular matrix (ECM) genes, but transfection with the miR-27b-3p inhibitor had the opposite effects. RNA sequencing identified a PPARG/ADAMTS8 signaling axis regulated by miR-27b-3p in OA FLS. Human OA FLS transfected with miR-27b-3p mimic and then treated with the PPARG agonist rosiglitazone or with ADAMTS8 small interfering RNA exhibited altered expression of select ECM genes. CONCLUSION: Our findings demonstrate that miR-27b-3p has a key role in ECM regulation associated with synovial fibrosis during OA.
Asunto(s)
MicroARNs , Osteoartritis de la Rodilla , Sinovitis , Animales , Humanos , Ratones , Proteínas ADAMTS/metabolismo , Fibrosis , MicroARNs/metabolismo , Osteoartritis de la Rodilla/genética , Osteoartritis de la Rodilla/metabolismo , PPAR gamma/metabolismo , Membrana Sinovial/metabolismo , Sinovitis/genética , Sinovitis/metabolismoRESUMEN
Human pregnancy-specific glycoproteins (PSGs) serve immunomodulatory and pro-angiogenic functions during pregnancy and are mainly expressed by syncytiotrophoblast cells. While PSG mRNA expression in extravillous trophoblasts (EVTs) was reported, the proteins were not previously detected. By immunohistochemistry and immunoblotting, we show that PSGs are expressed by invasive EVTs and co-localize with integrin ï¡5. In addition, we determined that native and recombinant PSG1, the most highly expressed member of the family, binds to ï¡5ï¢1 and induces the formation of focal adhesion structures resulting in adhesion of primary EVTs and EVT-like cell lines under 21% oxygen and 1% oxygen conditions. Furthermore, we found that PSG1 can simultaneously bind to heparan sulfate in the extracellular matrix and to ï¡5ï¢1 on the cell membrane. Wound healing assays and single-cell movement tracking showed that immobilized PSG1 enhances EVT migration. Although PSG1 did not affect EVT invasion in the in vitro assays employed, we found that the serum PSG1 concentration is lower in African-American women diagnosed with early-onset and late-onset preeclampsia, a pregnancy pathology characterized by shallow trophoblast invasion, than in their respective healthy controls only when the fetus was a male; therefore, the reduced expression of this molecule should be considered in the context of preeclampsia as a potential therapy.
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Integrina alfa5beta1/metabolismo , Glicoproteínas beta 1 Específicas del Embarazo/metabolismo , Trofoblastos/metabolismo , Adhesión Celular , Línea Celular , Membrana Celular/metabolismo , Movimiento Celular , Matriz Extracelular/metabolismo , Femenino , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Heparitina Sulfato/metabolismo , Humanos , Proteínas Inmovilizadas/química , Proteínas Inmovilizadas/metabolismo , Placenta/metabolismo , Preeclampsia/diagnóstico , Embarazo , Primer Trimestre del Embarazo , Glicoproteínas beta 1 Específicas del Embarazo/análisis , Glicoproteínas beta 1 Específicas del Embarazo/genética , Unión Proteica , Trofoblastos/citologíaRESUMEN
STUDY QUESTION: Do all 10 human pregnancy-specific beta 1-glycoproteins (PSGs) and murine PSG23 activate latent transforming growth factor-ß1 (TGF-ß1)? SUMMARY ANSWER: All human PSGs and murine PSG23 activated latent TGF-ß1. WHAT IS KNOWN ALREADY: Two of the 10 members of the PSG1 family, PSG1 and PSG9, were previously shown to activate the soluble small latent complex of TGF-ß1, a cytokine with potent immune suppressive functions. STUDY DESIGN, SIZE, DURATION: Recombinant PSGs were generated and tested for their ability to activate the small latent complex of TGF-ß1 in a cell-free ELISA-based assay and in a bioassay. In addition, we tested the ability of PSG1 and PSG4 to activate latent TGF-ß bound to the extracellular matrix (ECM) or on the membranes of the Jurkat human T-cell line. PARTICIPANTS/MATERIALS, SETTING, METHODS: Recombinant PSGs were generated by transient transfection and purified with a His-Trap column followed by gel filtration chromatography. The purified PSGs were compared to vehicle (PBS) used as control for their ability to activate the small latent complex of TGF-ß1. The concentration of active TGF-ß was measured in an ELISA using the TGF-ß receptor II as capture and a bioassay using transformed mink epithelial cells that express luciferase in response to active TGF-ß. The specificity of the signal was confirmed using a TGF-ß receptor inhibitor. We also measured the binding kinetics of some human PSGs for the latent-associated peptide (LAP) of TGF-ß using surface plasmon resonance and determined whether PSG1 and PSG4 could activate the large latent complex of TGF-ß1 bound to the ECM and latent TGF-ß1 bound to the cell membrane. All experiments were performed in triplicate wells and repeated three times. MAIN RESULTS AND THE ROLE OF CHANCE: All human PSGs activated the small latent complex of TGF-ß1 (P < 0.05 vs. control) and showed similar affinities (KD) for LAP. Despite the lack of sequence conservation with its human counterparts, the ability to activate latent TGF-ß1 was shared by a member of the murine PSG family. We found that PSG1 and PSG4 activated the latent TGF-ß stored in the ECM (P < 0.01) but did not activate latent TGF-ß1 bound to glycoprotein A repetitions predominant (GARP) on the surface of Jurkat T cells. LIMITATIONS, REASONS FOR CAUTION: The affinity of the interaction of LAP and PSGs was calculated using recombinant proteins, which may differ from the native proteins in their post-translational modifications. We also utilized a truncated form of murine PSG23 rather than the full-length protein. For the studies testing the ability of PSGs to activate membrane-bound TGF-ß1, we utilized the T-cell line Jurkat and Jurkat cells expressing GARP rather than primary T regulatory cells. All the studies were performed in vitro. WIDER IMPLICATIONS OF THE FINDINGS: Here, we show that all human PSGs activate TGF-ß1 and that this function is conserved in at least one member of the rodent PSG family. In vivo PSGs could potentially increase the availability of active TGF-ß1 from the soluble and matrix-bound latent forms of the cytokine contributing to the establishment of a tolerogenic environment during pregnancy. LARGE-SCALE DATA: None. STUDY FUNDING/COMPETING INTEREST(S): The research was supported by a grant from the Collaborative Health Initiative Research Program (CHIRP). No conflicts of interests are declared by the authors.
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Glicoproteínas beta 1 Específicas del Embarazo/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Ensayo de Inmunoadsorción Enzimática , Matriz Extracelular/metabolismo , Femenino , Heparitina Sulfato , Humanos , Masculino , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Embarazo , Glicoproteínas beta 1 Específicas del Embarazo/genética , Factor de Crecimiento Transformador beta1/genéticaRESUMEN
The balance between the production of reactive oxygen species and activation of antioxidant pathways is essential to maintain a normal redox state in all tissues. Oxidative stress caused by excessive oxidant species generation can cause damage to DNA and other macromolecules, affecting cell function and viability. Here we show that integrin-linked kinase (ILK) plays a key role in eliciting a protective response to oxidative damage in epidermal cells. Inactivation of the Ilk gene causes elevated levels of intracellular oxidant species (IOS) and DNA damage in the absence of exogenous oxidative insults. In ILK-deficient cells, excessive IOS production can be prevented through inhibition of NADPH oxidase activity, with a concomitant reduction in DNA damage. Additionally, ILK is necessary for DNA repair processes following UVB-induced damage, as ILK-deficient cells show a significantly impaired ability to remove cyclobutane pyrimidine dimers following irradiation. Thus, ILK is essential to maintain cellular redox balance and, in its absence, epidermal cells become more susceptible to oxidative damage through mechanisms that involve IOS production by NADPH oxidase activity.
RESUMEN
Dysregulated secretion and extracellular activation of TGF-ß1 stimulates myofibroblasts to accumulate disordered and stiff extracellular matrix (ECM) leading to fibrosis. Fibronectin immobilizes latent TGF-ß-binding protein-1 (LTBP-1) and thus stores TGF-ß1 in the ECM. Because the ED-A fibronectin splice variant is prominently expressed during fibrosis and supports myofibroblast activation, we investigated whether ED-A promotes LTBP-1-fibronectin interactions. Using stiffness-tuneable substrates for human dermal fibroblast cultures, we showed that high ECM stiffness promotes expression and colocalization of LTBP-1 and ED-A-containing fibronectin. When rescuing fibronectin-depleted fibroblasts with specific fibronectin splice variants, LTBP-1 bound more efficiently to ED-A-containing fibronectin than to ED-B-containing fibronectin and fibronectin lacking splice domains. Function blocking of the ED-A domain using antibodies and competitive peptides resulted in reduced LTBP-1 binding to ED-A-containing fibronectin, reduced LTBP-1 incorporation into the fibroblast ECM and reduced TGF-ß1 activation. Similar results were obtained by blocking the heparin-binding stretch FNIII12-13-14 (HepII), adjacent to the ED-A domain in fibronectin. Collectively, our results suggest that the ED-A domain enhances association of the latent TGF-ß1 by promoting weak direct binding to LTBP-1 and by enhancing heparin-mediated protein interactions through HepII in fibronectin.