Direct Inhibition of Indian Hedgehog Expression by Parathyroid Hormone (PTH)/PTH-Related Peptide and Up-Regulation by Retinoic Acid in Growth Plate Chondrocyte Cultures

Abstract

Indian hedgehog (Ihh) is highly expressed in prehypertrophic chondrocytes in vivo and has been proposed to regulate the proliferation and maturation of chondrocytes and bone collar formation in the growth plate. In high-density cultures of rabbit growth-plate chondrocytes, Ihh mRNA was also expressed at the highest level in the prehypertrophic stage. To explore endogenous factors that regulate Ihh expression in chondrocytes, we examined the effects of various growth factors on Ihh mRNA expression in this system. Retinoic acid (RA) and bone morphogenetic protein-2 enhanced Ihh mRNA expression, whereas PTH/PTH-related peptide (PTHrP) markedly suppressed Ihh expression. RA at more than 10(-8) M induced the expression of Ihh and Patched 1 (Ptc1) within 3 h, before it increased the type X collagen mRNA level at 6-24 h. Cycloheximide blocked the up-regulation of Ihh by RA, indicating the requirement of de novo protein synthesis for this stimulation. These findings suggest that RA is involved in the up-regulation of Ihh during endochondral bone formation. In contrast to RA, PTH (1-84) at 10(-7) M abolished the mRNA expression of Ihh and Ptc1 within 2-4 h, before it suppressed the expression of type X collagen at 12-24 h. The inhibition of Ihh expression by PTH (1-84) did not require de novo protein synthesis. PTH (1-34), PTHrP (1-34), and (Bu)(2)cAMP also suppressed Ihh expression. On the other hand, Ihh has been reported to induce PTHrP synthesis in the perichondrium. Consequently, the direct inhibitory action of PTH/PTHrP on Ihh appears to be a negative feedback mechanism that prevents excess PTHrP accumulation in cartilage.

mTOR signaling contributes to chondrocyte differentiation

“…Several mechanisms, including those involving Runx-2, bone morphogenetic proteins (BMPs), retinoic acid, and the Erk and p38 MAP kinases have been shown to activate Ihh transcription (Yoshida et al, 2004(Yoshida et al, , 2001Seki and Hata, 2004;. Rapamycin did not affect Runx2 expression in our studies.…”

Indian hedgehog: Its roles and regulation in endochondral bone development

“…Yoshida et al [2001] first demonstrated this in primary chicken chondrocytes showing that PTH directly down-regulated Ihh mRNA levels, independent of its effect on differentiation. Ihh regulation was mimicked by a cAMP analog and was not blocked by either cycloheximide or actinomycin D, suggesting that PTH directly inhibits Ihh gene transcription, possibly via the cAMP/PKA pathway.…”

“…Retinoic acid (RA) has also been reported to increase Ihh levels in chondrocytes [Yoshida et al, 2001]. Yoshida et al first demonstrated in primary chicken chondrocytes that RA increased Ihh transcription in a process that required de novo protein synthesis.…”

Hedgehog signaling in animal development: paradigms and principles

“…) are divergent members of the Indian subgroup (green). Fan and Tessier-Lavigne 1994;Johnson et al 1994;Chiang et al 1996;Teillet et al 1998;Murtaugh et al 1999;Zhang et al 2001 Ihh Coordination of proliferation/ differentiation in endochondral skeleton Lankse et al 1996;Vortkamp et al 1996;St-Jacques et al 1999;Enomoto-Iwato et al 2000;Karp et al 2000;Chung et al 2001;Long et al 2001;Yoshida et al 2001 Cement gland induction…”