Identification of multiple IGF-1 mRNA transcripts and the effect of growth factors on the production of IGF-1, type II collagen, Decorin and Biglycan in articular chondrocytes

Desai-Abram, Sonal (2000). Identification of multiple IGF-1 mRNA transcripts and the effect of growth factors on the production of IGF-1, type II collagen, Decorin and Biglycan in articular chondrocytes. MPhil thesis The Open University.

DOI: https://doi.org/10.21954/ou.ro.0000fd1d

Abstract

This study is based on an investigation of the differential effects of basic Fibroblast Growth Factor (bFGF) on the production of Type II collagen and Insulin Like Growth Factor I (IGF I) mRNA in cultured chondrocytes and the modulation of IGF I and TGFβ by bFGF action. The regulation of IGF I production by chondrocytes in normal and OA cartilage was also studied.

The expression of mRNA for IGF I and matrix proteins was studied in cultured pig chondrocytes and human OA cartilage. Multiple transcripts of IGF I mRNA were identified by Northern blot analysis and RT-PCR in both species. Chondrocytes grown as high density monolayers or in suspension and as cultured expiants were employed to investigate which IGF I mRNA transcript was induced in cells cultured in different ways. Four IGF-I transcripts (7.6, 4.7, 1.3, and 1.1 kb) were found in cultured chondrocytes in a ratio of 1:18:2:2:. Aparent changes in the 7.6, 1.1 and 1.3kb transcripts were found in response to IGF-I and TGFβ growth factors. Total RNA was also extracted directly from samples of the femoral head of normal and OA human cartilage. The RNA was reverse transcribed into cDNA and amplified by the polymerase chain reaction. IGF I primers specific for Exons 3, 4, and 5 of the IGF I gene were synthesised for this study. An IGF I cDNA probe identified mainly a 1.1 and 1.3 kb transcripts in RNA from OA cartilage and primers specific PCR probe for exons 3 and 5 identified the larger transcripts of 7.6kb and 4.7kb with a low levels of 1.3 and l.lkb in OA cartilage.The stability of IGF I message was assessed in IGF I stimulated cells and the 4.7 kb transcript was found to be stable for up to 14 days.

The dose response and time course for the production of IGF I, Type II collagen, decorin and biglycan was studied in the presence of IGF I. Stimulation was maximal at
lower concentrations of IGF 1(10 and 100ng/ml) by day 4, but at higher concentrations (250, and 500ng/ml), the increase in expression was much less. Other mediators such as bFGF and TGFp were also tested with or without IGF I on these cells. bFGF inhibited the increased expression of Type II collagen and decorin induced in response to IGF I or TGFβ but the gene expression of IGF I or biglycan was not altered under the same condition. IL1 alone had an inhibitory effect for the production of biglycan and decorin in IGF I maintained cells, but when bFGF was also present, it diminished the inhibitory effect of IL1 for the production of biglycan.

The relevance of these findings are discussed in relation to the possible role of IGF I and other growth factors as mediators in the repair process of OA.

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