Xenoestrogens disrupt type 2 collagen expression in chondrocytes in vitro.

Abstract

Purpose: In recent years chemicals with endocrine properties, called Endocrine Disrupting Compounds (EDC), have become a topic of scientific and public discussion. In the field of osteoarthritic pathologies, EDC compounds that mimic estrogenic agonists or antagonists are of major concern, because of the central role of estrogens in skeletal developmental processes (ref). EDC are acting on their target genes upon binding the aryl hydrocarbon receptor (AhR). During the ten past years, interactions between estrogen receptor (ER) and some xenobiotic-activated AhR have been well demonstrated. Very few studies have been reported on EDC effects on bone or cartilage. Our laboratory recently studied the effects of low doses and cocktail xenoestrogens on rat cartilage development in vivo, by using a rodent gestational/lactational model (T.A. Auxietre, et al, 2014). We observed transient modifications of caudal vertebral body associated with a decrease in growth plate cartilage thickness with greater impact on the hypertrophic chondrocyte zone. We inferred from these data that the tested compounds could interfere with the dynamic of chondrogenic differentiation and/or maturation processes, possibly by impacting type 2 collagen (Col2), one of the the major components of cartilage that assures cartilage function. The present work was aimed to study the in vitro effects of two xenoestrogens (genistein, G; bisphenol A, BPA) and one anti-androgen (vinclozolin, V) and its metabolite M2 on Col2 expression in two different models of cultured chondrocytes: during the process of chondrogenic induction and in mature chondrocytes. Methods: A murine stem cell line inducible towards chondrogenesis (C1) was used for dynamic studies of differentiation using chondrogenic and non-chondrogenic markers. In parallel, post-natal murine chondrocytes were used for steady-state investigations in differentiated chondrocytes in primary culture (P0) or after dedifferentiation by passages (P3) or FGF2 treatment. Biological markers were studied at the mRNA (qPCR) and protein (western blot) levels. COL2A protein expression was evaluated by western blot and immunocytochemistry by using an antibody which only recognizes the specific COL2A N-terminal propeptide. Fulvestrant and SB 203580 were used respectively as specific inhibitors of ER or p38MEK pathways. Implication of Tia1-splicing was shown after cell transfection with specific anti-Tia1 siRNAs. Results: Vinclozolin was practically inactive. M2, alone or combined with G or BPA, modified the dynamic of Col2A immature isoform of COL2 during chondrogenic induction. These compounds extended the basal expression of COL2A and delayed its replacement by the mature isoform COL2B in C1 cells. This effect was dose dependent with maximum at 10-6M. In post natal chondrocytes, COL2A expression increased with cell dedifferentiation or FGF2 treatment. EDC were inactive on differentiated chondrocytes while COL2A doubled upon M2, G and BPA addition in dedifferentiated cells. Estrogen receptor (ER) and the p38-MEK pathway were involved. As these effects were only partly transcriptional, we investigated and found an effect of FGF2 on Tia-1 splice protein expression. EDC showed no effects on other chondrogenic markers (SOX9, Aggrecan, Col10α1) nor non-chondrogenic markers. Conclusions: These data showed that xenosestrogens as well as the vinclozolin metabolite M2 modified the rate of early chondrogenic differentiation by transiently maintaining COL2A expression. Our hypothesis is that COL2A induced persistence, might provoke matrix weakening in the long term and/or sequester growth factors, eventually modifying the course of cartilage degenerative diseases such as osteoarthritis. EDC also modified COL2A expression in dedifferentiated chondrocytes such as found in aging or osteoarthritic tissues. These data are in accordance with the strong Col2A expression reported in OA cartilage but its role remains to be studied.