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Regulation of xylosyltransferase I gene expression by interleukin 1? in human primary chondrocyte cells: mechanism and impact on proteoglycan synthesis.

ABSTRACT: Xylosyltransferase I (XT-I) is an essential enzyme of proteoglycan (PG) biosynthesis pathway catalyzing the initial and rate-limiting step in glycosaminoglycan chain assembly. It plays a critical role in the regulation of PG synthesis in cartilage; however, little is known about underlying mechanism. Here, we provide evidence that, in human primary chondrocytes, IL-1? regulates XT-I gene expression into an early phase of induction and a late phase of down-regulation. Based on promoter deletions, the region up to -850 bp was defined as a major element of XT-I gene displaying both constitutive and IL-1?-regulated promoter activity. Point mutation and signaling analyses revealed that IL-1?-induced promoter activity is achieved through AP-1 response elements and mediated by SAP/JNK and p38 signaling pathways. Transactivation and chromatin immunoprecipitation assays indicated that AP-1 is a potent transactivator of XT-I promoter and that IL-1?-induced activity is mediated through increased recruitment of AP-1 to the promoter. Finally, we show that Sp3 is a repressor of XT-I promoter and bring evidence that the repressive effect of IL-1? during the late phase is mediated through Sp3 recruitment to the promoter. This suggests that modulation of Sp3 in cartilage could prevent IL-1? inhibition of PG synthesis and limit tissue degradation.

SUBMITTER: Khair M

PROVIDER: S-EPMC3548487 | biostudies-literature | 2013 Jan

REPOSITORIES: biostudies-literature

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Regulation of xylosyltransferase I gene expression by interleukin 1β in human primary chondrocyte cells: mechanism and impact on proteoglycan synthesis.

Khair Mostafa M Bourhim Mustapha M Barré Lydia L Li Dong D Netter Patrick P Magdalou Jacques J Fournel-Gigleux Sylvie S Ouzzine Mohamed M

The Journal of biological chemistry 20121205 3

Xylosyltransferase I (XT-I) is an essential enzyme of proteoglycan (PG) biosynthesis pathway catalyzing the initial and rate-limiting step in glycosaminoglycan chain assembly. It plays a critical role in the regulation of PG synthesis in cartilage; however, little is known about underlying mechanism. Here, we provide evidence that, in human primary chondrocytes, IL-1β regulates XT-I gene expression into an early phase of induction and a late phase of down-regulation. Based on promoter deletions, ...[more]

PMID: 23223231

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Project description:OBJECTIVE: To elucidate the anti-inflammatory and anabolic effects of regulated expression of IL-4 in chondrocyte-scaffolds under in vitro inflammatory conditions. METHODS: Mature articular chondrocytes from dogs (n?=?3) were conditioned through transient transfection using pcDNA3.1.cIL-4 (constitutive) or pCOX-2.cIL-4 (cytokine-responsive) plasmids. Conditioned cells were seeded in alginate microspheres and rat-tail collagen type I matrix (CaReS®) to generate two types of tissue-engineered 3-dimensional scaffolds. Inflammatory arthritis was simulated in the packed chondrocytes through exogenous addition of recombinant canine (rc) IL-1? (100 ng/ml) plus rcTNF? (50 ng/ml) in culture media for 96 hours. Harvested cells and culture media were analyzed by various assays to monitor the anti-inflammatory and regenerative (anabolic) properties of cIL-4. RESULTS: cIL-4 was expressed from COX-2 promoter exclusively on the addition of rcIL-1? and rcTNF? while its expression from CMV promoter was constitutive. The expressed cIL-4 downregulated the mRNA expression of IL-1?, TNF?, IL-6, iNOS and COX-2 in the cells and inhibited the production of NO and PGE(2) in culture media. At the same time, it up-regulated the expression of IGF-1, IL-1ra, COL2a1 and aggrecan in conditioned chondrocytes in both scaffolds along with a diminished release of total collagen and sGAG into the culture media. An increased amount of cIL-4 protein was detected both in chondrocyte cell lysate and in concentrated culture media. Neutralizing anti-cIL-4 antibody assay confirmed that the anti-inflammatory and regenerative effects seen are exclusively driven by cIL-4. There was a restricted expression of IL-4 under COX-2 promoter possibly due to negative feedback loop while it was over-expressed under CMV promoter (undesirable). Furthermore, the anti-inflammatory /anabolic outcomes from both scaffolds were reproducible and the therapeutic effects of cIL-4 were both scaffold- and promoter-independent. CONCLUSIONS: Regulated expression of therapeutic candidate gene(s) coupled with suitable scaffold(s) could potentially serve as a useful tissue-engineering tool to devise future treatment strategies for osteoarthritis.

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Dataset Information

Regulation of xylosyltransferase I gene expression by interleukin 1? in human primary chondrocyte cells: mechanism and impact on proteoglycan synthesis.

Publications

Regulation of xylosyltransferase I gene expression by interleukin 1β in human primary chondrocyte cells: mechanism and impact on proteoglycan synthesis.

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