Retinoid signaling regulates CTGF expression in hypertrophic chondrocytes with differential involvement of MAP kinases.
Retinoids are important for growth plate chondrocyte maturation, but their downstream effectors remain unclear. Recently, CTGF (CCN2) was found to regulate chondrocyte function, particularly in the hypertrophic zone. The goal of the study was to determine whether CTGF is a retinoid signaling effector molecule, how it is regulated, and how it acts.


BACKGROUND
Using a combination of in vivo and in vitro approaches, we carried out a series of studies at the cellular, biochemical, and molecular level to determine whether and how retinoid signaling is related to expression and function of connective tissue growth factor (CTGF) in chondrocyte maturation and endochondral ossification.


METHODS
Limbs of chick embryos in ovo were implanted with retinoic pan-antagonist RO 41-5253-filled beads, and phenotypic changes were assessed by in situ hybridization. CTGF gene expression and roles were tested in primary cultures of immature and hypertrophic chondrocytes. Cross-talk between retinoid signaling and other pathways was tested by determining endogenous levels of active ERK1/2 and p38 MAP kinases and phenotypic modulations exerted by specific antagonists of mitogen-activated protein (MAP) kinases and BMP signaling (Noggin).


RESULTS
Interference with retinoid signaling blocked expression of CTGF and other posthypertrophic markers in long bone anlagen in vivo and hypertrophic chondrocyte cultures, whereas all-trans-retinoic acid (RA) boosted CTGF expression and even induced it in immature proliferating cultures. Exogenous recombinant CTGF stimulated chondrocyte maturation, but failed to do so in presence of retinoid antagonists. Immunoblots showed that hypertrophic chondrocytes contained sizable levels of phosphorylated ERK1/2 and p38 MAP kinases that were dose- and time-dependently increased by RA treatment. Experimental ERK1/2 inhibition led to a severe drop in baseline and RA-stimulated CTGF expression, whereas p38 inhibition increased it markedly. These responses were gene-specific, because the opposite was seen with other hypertrophic chondrocyte genes such as collagen X and RA receptor gamma (RARgamma). Tests with Noggin showed that RA induction of CTGF expression was negatively influenced by BMP signaling, whereas induction of collagen X expression was BMP-dependent.


CONCLUSIONS
Retinoids appear to have a preeminent role in controlling expression and function of CTGF in hypertrophic and posthypertrophic chondrocytes and do so with differential cooperation and intervention of MAP kinases and BMP signaling.
Shimo
Tsuyoshi
T
Department of Orthopedic Surgery, Thomas Jefferson University College of Medicine, Philadelphia, Pennsylvania, USA. shimotsu@md.okayama-u.ac.jp
Koyama
Eiki
E
Sugito
Hiroki
H
Wu
Changshan
C
Shimo
Satoko
S
Pacifici
Maurizio
M
D000818:Animals; D015152:Blotting, Northern; D002642:Chick Embryo; D019902:Chondrocytes / Q000166:cytology* / Q000378:metabolism*; D055513:Connective Tissue Growth Factor; D004305:Dose-Response Relationship, Drug; D005786:Gene Expression Regulation; D006984:Hypertrophy / Q000473:pathology; D017874:Immediate-Early Proteins / Q000096:biosynthesis*; D015151:Immunoblotting; D017403:In Situ Hybridization; D036341:Intercellular Signaling Peptides and Proteins / Q000096:biosynthesis*; D020935:MAP Kinase Signaling System; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D008954:Models, Biological; D010641:Phenotype; D011485:Protein Binding; D012176:Retinoids / Q000378:metabolism*; D015398:Signal Transduction; D013997:Time Factors; D014212:Tretinoin / Q000378:metabolism; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
D017874:Immediate-Early Proteins; D036341:Intercellular Signaling Peptides and Proteins; D012176:Retinoids; D055513:Connective Tissue Growth Factor; D014212:Tretinoin; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D048051:p38 Mitogen-Activated Protein Kinases