Stimulation of proteoglycan synthesis by glucuronosyltransferase-I gene delivery: A strategy to promote cartilage repair

Abstract

Osteoarthritis is a degenerative joint disease characterized by a progressive loss of articular cartilage components, mainly proteoglycans (PGs), leading to destruction of the tissue. We investigate a therapeutic strategy based on stimulation of PG synthesis by gene transfer of the glycosaminoglycan (GAG)-synthesizing enzyme, ␤1,3-glucuronosyltransferase-I (GlcAT-I) to promote cartilage repair. We previously reported that IL-1␤ down-regulated the expression and activity of GlcAT-I in primary rat chondrocytes. Here, by using antisense oligonucleotides, we demonstrate that GlcAT-I inhibition impaired PG synthesis and deposition in articular cartilage explants, emphasizing the crucial role of this enzyme in PG anabolism. Thus, primary chondrocytes and cartilage explants were engineered by lipid-mediated gene delivery to efficiently overexpress a human GlcAT-I cDNA. Interestingly, GlcAT-I overexpression significantly enhanced GAG synthesis and deposition as evidenced by 35 S-sulfate incorporation, histology, estimation of GAG content, and fluorophore-assisted carbohydrate electrophoresis analysis. Metabolic labeling and Western blot analyses further suggested that GlcAT-I expression led to an increase in the abundance rather than in the length of GAG chains. Importantly, GlcAT-I delivery was able to overcome IL-1␤-induced PG depletion and maintain the anabolic activity of chondrocytes. Moreover, GlcAT-I also restored PG synthesis to a normal level in cartilage explants previously depleted from endogenous PGs by IL-1␤-treatment. In concert, our investigations strongly indicated that GlcAT-I was able to control and reverse articular cartilage defects in terms of PG anabolism and GAG content associated with IL-1␤. This study provides a basis for a gene therapy approach to promote cartilage repair in degenerative joint diseases.osteoarthritis ͉ gene transfer ͉ chondrocyte ͉ glycosyltransferase ͉ glycosaminoglycan

Chondroitin Sulfate N-Acetylgalactosaminyltransferase-1 Plays a Critical Role in Chondroitin Sulfate Synthesis in Cartilage

“…In contrast, we have been unsuccessful in gene delivery to the articular cartilage (data not shown), although a recent similar study demonstrated increased CS expression in an articular cartilage explant (38). Since CS biosynthesis occurs in the Golgi apparatus (22), the addition of the enzyme to the tissue is ineffective, and overexpression in the cell is essential for this enzyme-based increase in CS levels.…”

“…However, stubs of the linkage region have not been identified in aggrecan, although such stubs are present in a parttime CS proteoglycan thrombomodulin, an integral membrane glycoprotein expressed on endothelial cell surfaces (37). Recently, GlcAT-I, which transfers a GlcUA residue to the second Gal residue in the linkage region, has been shown to increase CS biosynthesis to ϳ1.5-fold (38). Thus, an immature linkage region may be present, and the enzyme complex that includes GlcAT-I and CSGalNAcT-1 may catalyze the completion of the linkage region and CS chain initiation.…”

The effect of glycosaminoglycan loss on chondrocyte viability: A study on porcine cartilage explants

“…Durrant et al showed that cartilage explants swelled and gained Ͼ20% weight in culture (25), and Racz et al revealed that hyperosmotic conditions lead to apoptotic cell death (26). During tissue culture, additional water appears to be retained in control explants EFFECT OF GAG LOSS ON CHONDROCYTE DEATH with high GAG content and a strong negative charge (27,28). Thus, the results from our first series of studies show that GAG depletion as induced by chondroitinase ABC treatment does not directly lead to chondrocyte death.…”

Restoration of the extracellular matrix in human osteoarthritic articular cartilage by overexpression of the transcription factor SOX9

“…The transfer of the gene for a heat-shock protein (Hsp70) was also shown to afford protection against cellular injuries in chondrocytes (9). Alternatively, the delivery of sequences encoding for growth and enzymatic factors can potentially stimulate cartilage anabolism in vitro and in situ, such as insulinlike growth factor 1 (IGF-1) (10), fibroblast growth factor 2 (FGF-2) (11), bone morphogenetic protein 7 (BMP-7) (12), transforming growth factor ␤ (TGF␤) (13), and glucuronosyltransferase I (14). However, application of external stimuli to damaged articular cartilage has not yet proved sufficient in fully reestablishing an original cartilage surface, and little is known about the effects on human OA cartilage.…”