Alterations of collagen mRNA expression during retinoic acid induced chondrocyte modulation: Absence of untranslated α1(I) mRNA in hyaline chondrocytes

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Abstract
Retinoic acid (RA) has been shown to rapidly modulate the collagen expression pattern of chondrocytes in vitro at doses of 1–10 μM. Embryonic chicken sternal chondrocytes stop synthesizing the cartilage-specific type II collagen within 2–4 days of RA treatment and turn on the synthesis of types I and III collagen and fibronectin. While suppression of type II collagen synthesis and onset of type III collagen and fibronectin synthesis have been shown to be regulated at the transcriptional level, conflicting data are available on a possible post-translational regulation of α1(I) collagen gene expression. In this study we demonstrate by comparing a commonly used α1(I) cDNA probe from the 3′ end of the α1(I) mRNA with a newly prepared α1(I) specific cDNA probe from the 5′ end (p1E1) that—in contrast to previous reports—chicken sternal chondrocytes do not contain untranslated α1(I) mRNA which may become translatable after RA treatment. By in situ hybridization we show the absence of cytoplasmic α1(I) mRNA from chondrocytes and its presence in the perichondrium of sternal cartilage. Perichondral cells might have contaminated sternal chondrocyte preparations, explaining low levels of α1(I) mRNA seen by Northern hybridization and RNase protection assays of chicken sternal cartilage mRNA even with the p1E1 probe. We show by Northern hybridization and metabolic labeling with 3H-proline followed by SDS-gel electrophoresis that retinoic acid at 3 μM suppresses type II, IX, and X collagen gene expression within 2 days both at the mRNA and protein level and induces the onset of α1(I), α2(I), and α1(III) expression within 3 days. No expression of CRABP, the cellular retinoic acid binding protein, was seen in RA-treated or control chondrocytes, indicating that CRABP protein is not involved in the RA-induced modulation of the chondrocytes.