RESUMEN
OBJECTIVE: Fibronectin is a matrix protein that is fragmented during cartilage degradation in osteoarthritis (OA). Treatment of chondrocytes with fibronectin fragments (FN-f) has been used to model OA in vitro, but the system has not been fully characterized. This study sought to define the transcriptional response of chondrocytes to FN-f, and directly compare it to responses traditionally observed in OA. DESIGN: Normal human femoral chondrocytes isolated from tissue donors were treated with either FN-f or PBS (control) for 3, 6, or 18 h. RNA-seq libraries were compared between time-matched FN-f and control samples in order to identify changes in gene expression over time. Differentially expressed genes were compared to a published OA gene set and used for pathway, transcription factor motif, and kinome analysis. RESULTS: FN-f treatment resulted in 3,914 differentially expressed genes over the time course. Genes that are up- or downregulated in OA were significantly up- (P < 0.00001) or downregulated (P < 0.0004) in response to FN-f. Early response genes were involved in proinflammatory pathways, whereas many late response genes were involved in ferroptosis. The promoters of upregulated genes were enriched for NF-κB, AP-1, and IRF motifs. Highly upregulated kinases included CAMK1G, IRAK2, and the uncharacterized kinase DYRK3, while growth factor receptors TGFBR2 and FGFR2 were downregulated. CONCLUSIONS: FN-f treatment of normal human articular chondrocytes recapitulated many key aspects of the OA chondrocyte phenotype. This in vitro model is promising for future OA studies, especially considering its compatibility with genomics and genome-editing techniques.
Asunto(s)
Cartílago Articular/citología , Condrocitos/efectos de los fármacos , Fibronectinas/farmacología , Expresión Génica/efectos de los fármacos , Osteoartritis/genética , Proteína Quinasa Tipo 1 Dependiente de Calcio Calmodulina/efectos de los fármacos , Proteína Quinasa Tipo 1 Dependiente de Calcio Calmodulina/genética , Condrocitos/metabolismo , Fémur , Expresión Génica/genética , Humanos , Técnicas In Vitro , Factores Reguladores del Interferón/efectos de los fármacos , Factores Reguladores del Interferón/genética , Quinasas Asociadas a Receptores de Interleucina-1/efectos de los fármacos , Quinasas Asociadas a Receptores de Interleucina-1/genética , FN-kappa B/efectos de los fármacos , FN-kappa B/genética , Osteoartritis/metabolismo , Fragmentos de Péptidos/farmacología , Fenotipo , Regiones Promotoras Genéticas , Proteínas Serina-Treonina Quinasas/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/genética , Proteínas Tirosina Quinasas/efectos de los fármacos , Proteínas Tirosina Quinasas/genética , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/efectos de los fármacos , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/genética , Receptor Tipo II de Factor de Crecimiento Transformador beta/efectos de los fármacos , Receptor Tipo II de Factor de Crecimiento Transformador beta/genética , Factor de Transcripción AP-1/efectos de los fármacos , Factor de Transcripción AP-1/genéticaRESUMEN
OBJECTIVE: To determine the contribution of the gut microbiota to the development of injury-induced osteoarthritis (OA). DESIGN: OA was induced using the destabilized medial meniscus (DMM) model in 20 germ-free (GF) C57BL/6J male mice housed in a gnotobiotic facility and 23 strain-matched specific pathogen free (SPF) mice in 2 age groups -13.5 weeks avg age at DMM (17 SPF and 15 GF) and 43 weeks avg age at DMM (6 SPF and 5 GF). OA severity was measured using scores for articular cartilage structure (ACS), loss of safranin O (SafO) staining, osteophyte size, and synovial hyperplasia. Microbiome analysis by 16S rRNA amplicon sequencing was performed on stool samples and LPS and LPS binding protein (LBP) were measured in plasma. RESULTS: Compared to the SPF DMM mice, the maximum (MAX) ACS score per joint was 28% lower (p = 0.036) in GF DMM mice while the SafO sum score of all sections evaluated per joint was decreased by 31% (p = 0.009). The differences between SPF and GF mice in these scores were greater when only the younger mice were included in the analysis. The younger GF DMM mice also had significant reductions in osteophyte size (36%, P = 0.0119) and LBP (27%, P = 0.007) but not synovial scores or LPS. Differences in relative abundance of a number of Operational Taxonomic Units (OTUs) were noted between SPF mice with high vs low maximum ACS scores. CONCLUSIONS: These results suggest factors related to the gut microbiota promote the development of OA after joint injury.
Asunto(s)
Microbioma Gastrointestinal , Osteoartritis/etiología , Lesiones de Menisco Tibial/complicaciones , Proteínas de Fase Aguda , Animales , Proteínas Portadoras/sangre , Cartílago Articular/patología , Modelos Animales de Enfermedad , Microbioma Gastrointestinal/genética , Vida Libre de Gérmenes , Interleucina-6/sangre , Lipopolisacáridos/sangre , Masculino , Glicoproteínas de Membrana/sangre , Meniscos Tibiales/patología , Ratones , Ratones Endogámicos C57BL , Osteoartritis/microbiología , ARN Ribosómico 16S/genéticaRESUMEN
OBJECTIVES: The infrapatellar fat pad (IPFP), which is located underneath the patella, close to cartilage surfaces, functions in distributing mechanical load and has been shown to produce cytokines. This study aims to assess the involvement of the IPFP in the progression of post-traumatic osteoarthritis (OA) through investigating the crosstalk between the IPFP and injured cartilage in vitro. METHODS: A single blunt impact (36 MPa) on healthy bovine articular cartilage explants was used to generate traumatized cartilage. Conditioned media from IPFP and traumatized cartilage (FP-CM and TC-CM) were prepared separately. After culturing in FP-CM, the posttraumatic cartilage explants were analyzed for expression of cartilage degeneration associated genes and secretion of the interleukin (IL)-6, into the culture medium. The effect of traumatized cartilage on IPFP was studied by treating IPFP-derived adipocytes and IPFP adipose-derived stromal cells (ADSC) with TC-CM followed by analysis of cytokine expression. RESULTS: FP-CM aggravated glycosaminoglycan (GAG) release in traumatized cartilage, but did not significantly affect healthy cartilage. FP-CM raised gene expression of cyclooxygenase-2, inducible nitric oxide synthase, and IL-6 in traumatized cartilage explants, and lowered expression of tissue inhibitor of metalloproteinases-1, 2, 3, compared to non-conditioned medium. Of particular significance is that medium IL-6 levels increased substantially in both FP-CM and FP-CM treated traumatized cartilage cultures. Extrinsic IL-6 treatment of traumatized cartilage simulated part of the effects of FP-CM. TC-CM elevated levels of IL-6 expression in IPFP derived adipocytes and ADSCs. CONCLUSIONS: IPFP aggravates post-traumatized cartilage degeneration, and IL-6 is a candidate tissue degeneration mediator.
Asunto(s)
Tejido Adiposo/patología , Cartílago Articular/lesiones , Interleucina-6/fisiología , Adipocitos/metabolismo , Animales , Cartílago Articular/metabolismo , Cartílago Articular/patología , Bovinos , Ensayo de Inmunoadsorción Enzimática , Técnicas In Vitro , Osteoartritis/etiología , Rótula/patología , Células del Estroma/metabolismoRESUMEN
OBJECTIVE: We previously found in our embryonic studies that proper regulation of the chemokine CCL12 through its sole receptor CCR2, is critical for joint and growth plate development. In the present study, we examined the role of CCR2 in injury-induced-osteoarthritis (OA). METHOD: We used a murine model of injury-induced-OA (destabilization of medial meniscus, DMM), and systemically blocked CCR2 using a specific antagonist (RS504393) at different times during disease progression. We examined joint degeneration by assessing cartilage (cartilage loss, chondrocyte hypertrophy, MMP-13 expression) and bone lesions (bone sclerosis, osteophytes formation) with or without the CCR2 antagonist. We also performed pain behavioral studies by assessing the weight distribution between the normal and arthritic hind paws using the IITS incapacitance meter. RESULTS: Testing early vs delayed administration of the CCR2 antagonist demonstrated differential effects on joint damage. We found that OA changes in articular cartilage and bone were ameliorated by pharmacological CCR2 blockade, if given early in OA development: specifically, pharmacological targeting of CCR2 during the first 4 weeks (wks) following injury, reduced OA cartilage and bone damage, with less effectiveness with later treatments. Importantly, our pain-related behavioral studies showed that blockade of CCR2 signaling during early, 1-4 wks post-surgery or moderate, 4-8 wks post-surgery, OA was sufficient to decrease pain measures, with sustained improvement at later stages, after treatment was stopped. CONCLUSIONS: Our data highlight the potential efficacy of antagonizing CCR2 at early stages to slow the progression of post-injury OA and, in addition, improve pain symptoms.
Asunto(s)
Benzoxazinas/farmacología , Huesos/efectos de los fármacos , Cartílago Articular/efectos de los fármacos , Condrocitos/efectos de los fármacos , Meniscos Tibiales/efectos de los fármacos , Osteoartritis/patología , Receptores CCR2/antagonistas & inhibidores , Compuestos de Espiro/farmacología , Animales , Huesos/patología , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Hipertrofia , Metaloproteinasa 13 de la Matriz/efectos de los fármacos , Metaloproteinasa 13 de la Matriz/metabolismo , Meniscos Tibiales/cirugía , Ratones , Osteoartritis/metabolismo , Osteofito , Receptores CCR2/fisiología , Esclerosis , Lesiones de Menisco TibialRESUMEN
OBJECTIVE: We determined if the epidermal growth factor receptor ligand HB-EGF is produced in cartilage and if it regulates chondrocyte anabolic or catabolic activity. METHODS: HB-EGF expression was measured by quantitative PCR using RNA isolated from mouse knee joint tissues and from normal and osteoarthritis (OA) human chondrocytes. Immunohistochemistry was performed on normal and OA human cartilage and meniscus sections. Cultured chondrocytes were treated with fibronectin fragments (FN-f) as a catabolic stimulus and osteogenic protein 1 (OP-1) as an anabolic stimulus. Effects of HB-EGF on cell signaling were analyzed by immunoblotting of selected signaling proteins. MMP-13 was measured in conditioned media, proteoglycan synthesis was measured by sulfate incorporation, and matrix gene expression by quantitative PCR. RESULTS: HB-EGF expression was increased in 12-month old mice at 8 weeks after surgery to induce OA and increased amounts of HB-EGF were noted in human articular cartilage from OA knees. FN-f stimulated chondrocyte HB-EGF expression and HB-EGF stimulated chondrocyte MMP-13 production. However, HB-EGF was not required for FN-f stimulation of MMP-13 production. HB-EGF activated the ERK and p38 MAP kinases and stimulated phosphorylation of Smad1 at an inhibitory serine site which was associated with inhibition of OP-1 mediated proteoglycan synthesis and reduced aggrecan (ACAN) but not COL2A1 expression. CONCLUSION: HB-EGF is a new factor identified in OA cartilage that promotes chondrocyte catabolic activity while inhibiting anabolic activity suggesting it could contribute to the catabolic-anabolic imbalance seen in OA cartilage.
Asunto(s)
Condrocitos/metabolismo , Factor de Crecimiento Similar a EGF de Unión a Heparina/biosíntesis , Factor de Crecimiento Similar a EGF de Unión a Heparina/fisiología , Osteoartritis/metabolismo , Agrecanos/análisis , Animales , Proteína Morfogenética Ósea 7/farmacología , Cartílago/metabolismo , Cartílago Articular/metabolismo , Condrocitos/efectos de los fármacos , Colágeno Tipo II/análisis , Fibronectinas/farmacología , Factor de Crecimiento Similar a EGF de Unión a Heparina/análisis , Humanos , Immunoblotting , Inmunohistoquímica , Técnicas In Vitro , Articulación de la Rodilla/metabolismo , Metaloproteinasa 13 de la Matriz/análisis , Ratones , Osteoartritis de la Rodilla/metabolismo , Fosforilación , Proteoglicanos/biosíntesis , Reacción en Cadena en Tiempo Real de la Polimerasa , Transfección , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
OBJECTIVE: We investigated the roles of CXC chemokine ligand 12a (CXCL12a), also known as stromal cell-derived factor-1α (SDF-1α), in endochondral bone growth, which can give us important clues to understand the role of CXCL12a in osteoarthritis (OA). METHODS: Primary chondrocytes and tibial explants from embryonic 15.5 day-old mice were cultured with recombinant mouse CXCL12a. To assess the role of CXCL12a in chondrogenic differentiation, we conducted mesenchymal cell micromass culture. RESULTS: In tibia organ cultures, CXCL12a increased total bone length in a dose-dependent manner through proportional effects on cartilage and bone. In accordance with increased length, CXCL12a increased the protein level of proliferation markers, such as cyclin D1 and proliferating cell nuclear antigen (PCNA), in primary chondrocytes as well as in tibia organ culture. In addition, CXCL12a increased the expression of Runx2, Col10 and MMP13 in primary chondrocytes and tibia organ culture system, implying a role of CXCL12a in chondrocyte maturation. Micromass cultures of limb-bud mesenchymal progenitor cells (MPCs) revealed that CXCL12a has a limited effect on early chondrogenesis, but significantly promoted maturation of chondrocytes. CXCL12a induced the phosphorylation of p38 and Erk1/2 MAP kinases and IκB. The increased expression of cyclin D1 by CXCL12a was significantly attenuated by inhibitors of MEK1 and NF-κB. On the other hand, p38 and Erk1/2 MAP kinase and NF-κB signaling were associated with CXCL12a-induced expression of Runx2 and MMP13, the marker of chondrocyte maturation. CONCLUSION: CXCL12a promoted the proliferation and maturation of chondrocytes, which strongly suggest that CXCL12a may have a negative effect on articular cartilage and contribute to OA progression.
Asunto(s)
Quimiocina CXCL12/farmacología , Condrocitos/efectos de los fármacos , Osteogénesis/efectos de los fármacos , Animales , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Condrocitos/citología , Condrogénesis/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/fisiología , Células Madre Mesenquimatosas/efectos de los fármacos , Ratones , Técnicas de Cultivo de Órganos , Osteogénesis/fisiología , Proteínas Recombinantes/farmacología , Tibia/efectos de los fármacos , Tibia/crecimiento & desarrolloRESUMEN
The rate of endochondral bone growth determines final height in humans and is tightly controlled. Glycogen synthase kinase-3 (GSK-3) is a negative regulator of several signaling pathways that govern bone growth, such as insulin/IGF and Wnt/ß-catenin. The two GSK-3 proteins, GSK-3α and GSK-3ß, display both overlapping and distinct roles in different tissues. Here we show that pharmacological inhibition of GSK-3 signaling in a mouse tibia organ culture system results in enhanced bone growth, accompanied by increased proliferation of growth plate chondrocytes and faster turnover of hypertrophic cartilage to bone. GSK-3 inhibition rescues some, but not all, effects of phosphatidylinositide 3-kinase inhibition in this system, in agreement with the antagonistic role of these two kinases in response to signals such as IGF. However, cartilage-specific deletion of the Gsk3b gene in mice has minimal effects on skeletal growth or development. Molecular analyses demonstrated that compensatory up-regulation of GSK-3α protein levels in cartilage is the likely cause for this lack of effect. To our knowledge, this is the first tissue in which such a compensatory mechanism is described. Thus, our study provides important new insights into both skeletal development and the biology of GSK-3 proteins.