The transcription factor SOX9 is important in maintaining the chondrocyte phenotype. To identify novel genes regulated by SOX9 we investigated changes in gene expression by microarray analysis following retroviral transduction with SOX9 of a human chondrocytic cell line (SW1353). From the results the expression of a group of genes (SRPX, S100A1, APOD, RGC32, CRTL1, MYBPH, CRLF1 and SPINT1) was evaluated further in human articular chondrocytes (HACs). First, the same genes were investigated in primary cultures of HACs following SOX9 transduction, and four were found to be similarly regulated (SRPX, APOD, CRTL1 and S100A1). Second, during dedifferentiation of HACs by passage in monolayer cell culture, during which the expression of SOX9 progressively decreased, four of the genes (S100A1, RGC32, CRTL1 and SPINT1) also decreased in their expression. Third, in samples of osteoarthritic (OA) cartilage, which had decreased SOX9 expression compared with age-matched controls, there was decreased expression of SRPX, APOD, RGC32, CRTL1 and SPINT1. The results showed that a group of genes identified as being upregulated by SOX9 in the initial SW1353 screen were also regulated in expression in healthy and OA cartilage. Other genes initially identified were differently expressed in isolated OA chondrocytes and their expression was unrelated to changes in SOX9. The results thus identified some genes whose expression appeared to be linked to SOX9 expression in isolated chondrocytes and were also altered during cartilage degeneration in osteoarthritis.
Articular chondrocytes exist in an environment lacking in oxygen and nutrients due to the avascular nature of cartilage. The main source of metabolic energy is glucose, which is taken up by glucose transporters (GLUTs). In diseased joints, oxygen tensions and glucose availability alter as a result of inflammation and changes in vascularisation. Accordingly, in this study we examined the effects of hypoxia and the hypoxia mimetic cobalt chloride (CoCl 2 ) on glucose transport in equine chondrocytes and compared expression of the hypoxia responsive GLUT1 gene in normal and diseased cartilage. Monolayers of equine chondrocytes were exposed to 20% O 2 , 1% O 2 , CoCl 2 (75 mM), or a combination of 1% O 2 and CoCl 2 . Glucose uptake was measured using 2-deoxy-D-[2,6-3 H] glucose. GLUT1 protein and mRNA expression were determined by FACS analysis and qPCR, respectively. GLUT1 mRNA expression in normal and diseased cartilage was analyzed using explants derived from normal, OA, and OCD cartilage. Chondrocytes under hypoxic conditions exhibited a significantly increased glucose uptake as well as upregulated GLUT1 protein expression. GLUT1 mRNA expression significantly increased in combined hypoxia-CoCl 2 treatment. Analysis of clinical samples indicated a significant reduction in GLUT1 mRNA in OA samples. In OCD samples GLUT1 expression also decreased but did not reach statistical significance. The increase in glucose uptake and GLUT1 expression under hypoxic conditions confirms that hypoxia alters the metabolic requirements of chondrocytes. The altered GLUT1 mRNA expression in diseased cartilage with significance in OA suggests that reduced GLUT1 may contribute to the failure of OA cartilage repair. ß
The plasma serine protease activated protein C (APC) is synthesized by human chondrocytes at sites of pathological cartilage fibrillation. APC levels are increased in osteoarthritis (OA) synovial fluid, and in vitro APC has been shown to synergize with interleukin-1b (IL-1) to promote degradation from ovine cartilage. A model of equine cartilage degradation was established and used to explore corticosteroid activities. Intraarticular corticosteroids are a commonly prescribed treatment for joint disease, however their role in disease modification remains unclear. APC synergized with IL-1 or tumor necrosis factor-a (TNFa), promoting significant collagen degradation from equine cartilage explants within 4 days, but did not augment glycoaminoglycan (GAG) release. APC activated pro-matrix metalloproteinases (MMP)-2 but not pro-MMP-9, as assessed by gelatin zymography. APC did not directly activate pro-MMP-13. Dexamethasone, triamcinolone, and methylprednisolone acetate (MPA) were evaluated at concentrations between 10 À 5 M and 10 À10 M. High concentrations significantly increased GAG release from IL-1þAPC-treated explants. With the exception of MPA at 10 À10 M, all concentrations of corticosteroids caused significant decreases in IL-1þAPC-driven hydroxyproline loss. Treatment with corticosteroids suppressed expression of MMP-1, -3, and -13 mRNA. The collagenolysis associated with IL-1þAPC synergy, and the inhibition of this effect by corticosteroids may involve gelatinase activation and downregulation of MMP expression, respectively. ß
Our objective was to characterize the variation in gene expression for key genes associated with chondrogenic phenotype of osteochondrosis (OC)-affected and normal chondrocytes, and to identify whether OC chondrocytes can redifferentiate and regain a phenotype similar to normal chondrocytes if appropriate chondrogenic signals are given. Equine articular cartilage removed at surgery to treat clinically significant OC lesions was collected (n ¼ 10), and the gene expression evaluated and compared to aged-matched normal samples (n ¼ 10). Cartilage was harvested from normal (n ¼ 4) and OC (n ¼ 3) joints from horses at necropsy. Chondrogenic pellet cultures were established following monolayer proliferation. After 14 days in culture, the pellets were assessed by histochemical and pellet weight analysis, assay of glycosaminoglycan (GAG) content, and gene expression. Chondrocytes from OC cartilage expressed significantly more Coll-I, -II, -III, and -X than chondrocytes from normal cartilage (all p < 0.0001). Furthermore, OC chondrocytes expressed significantly more MMP-13, ADAMTS-4 (both p < 0.0001), and TIMP-1 (p < 0.001) and significantly less TIMP-2 and TIMP-3. Pellets created from OC chondrocytes contained significantly less GAG (p ¼ 0.0069) and expressed significantly less Sox9 and significantly more superficial zone protein (SZP) ( p ¼ 0.0105) than pellets created from normal cartilage. The results suggest that chondrocytes from OC cartilage at the time of surgical treatment have perturbations in phenotype compared to cells from normal cartilage. Despite these differences, following monolayer expansion and pellet culture under chondrogenic conditions, chondrocytes derived from OC cartilage retain some ability to undergo chondrogenic differentiation and synthesize an appropriate cartilage-like matrix. However, this chondrogenic differentiation potential is inferior to that seen in aged-matched normal chondrocytes. ß
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