Wnt‐mediated regulation of chondrocyte maturation: Modulation by TGF‐β

Abstract

Wnt proteins are expressed during limb morphogenesis, yet their role and mechanism of action remains unclear during long bone growth. Wnt expression, effects and modulation of signaling events by BMP and transforming growth factor-beta (TGF-β) were evaluated in chick embryonic chondrocytes. Chondrocyte cell cultures underwent spontaneous maturation with increased expression of colX and this was associated with an increase in the expression of multiple Wnts, including Wnts 4, 5a, 8c, and 9a. Both parathyroid hormone related peptide (PTHrP) and TGF-β inhibited colX, but had disparate effects on Wnt expression. While TGF-β strongly inhibited all Wnts, PTHrP did not inhibit either Wnt8c or Wnt9a and had lesser effects on the expression of the other Wnts. BMP-2 induced colX expression, and also markedly increased Wnt8c expression. Overexpression of β-Catenin and/or T cell factor (TCF)-4 also induced the type X collagen promoter. Overexpression of Wnt8c induced maturation, as did overexpression of β-Catenin. The Wnt8c/β-Catenin maturational effects were enhanced by BMP-2 and inhibited by TGF-β. TGF-β also inhibited activation of the Topflash reporter by β-Catenin, suggesting a direct inhibitory effect since the Topflash reporter contains only β-Catenin binding sequences. In turn β-Catenin inhibited activation of the p3TP-Luc reporter by TGF-β, although the effect was partial. Thus, Wnt/β-Catenin signaling is a critical regulator of the rate of chondrocyte differentiation. Moreover, this pathway is modulated by members of the TGF-β family and demonstrates the highly integrated nature of signals controlling endochondral ossification. J. Cell. Biochem. KeywordsWnt; Chondrocyte; β-Catenin; TGF-βtype X collagen Longitudinal bone growth is a highly regulated process in which chondrocytes complete sequential stages of proliferation, maturation, and hypertrophy marked by a 10-fold increase in type X collagen, and expression of alkaline phosphatase [Hunziker, 1994;Johnson and Tabin, 1997]. Local and systemic factors regulate chondrocyte maturation, and act through multiple independent signaling pathways. Several factors are known to influence the rate of chondrocyte differentiation. Transforming growth factor-beta (TGF-β) and parathyroid hormone related peptide (PTHrP) slow the rate of chondrocyte maturation, while bone morphogenetic proteins (BMPs), thyroid hormone, and retinoic acid stimulate terminal differentiation

Transforming Growth Factor-β Stimulates Cyclin D1 Expression through Activation of β-Catenin Signaling in Chondrocytes

“…thermore, we demonstrated that TGF-␤ treatment inhibits activation of the TOPflash promoter by ␤-catenin in chick upper sternal chondrocytes (6). Although these findings seemingly contradict the current observation that TGF-␤ induces ␤-catenin signaling through a SMAD3-mediated mechanism, important differences between the culture models likely account for the discrepancy in the findings.…”

“…We (6) and others (34) have shown that ␤-catenin induces prehypertrophic chondrocytes to complete terminal maturation. Overexpression of ␤-catenin or addition of Wnt3a, which induces the canonical ␤-catenin signaling pathway, stimulates expression of colX and other maturational markers (6,34).…”

“…mRNA and protein expression of TGF-␤ (including TGF-␤1, -␤2, and -␤3) and TGF-␤ receptors (including types I and II) is detected in the mouse perichondrium and in proliferating and hypertrophic chondrocytes in the growth plate from 13.5 days postcoitus until after birth (27)(28)(29)(30). In proliferating chondrocytes, TGF-␤ promotes chondrocyte proliferation (2,20), and in hypertrophic chondrocytes, TGF-␤ inhibits chondrocyte differentiation (3)(4)(5)(6).…”

“…Overexpression of ␤-catenin or addition of Wnt3a, which induces the canonical ␤-catenin signaling pathway, stimulates expression of colX and other maturational markers (6,34). Fur- Ϫ/Ϫ knock-out (KO) mice.…”

“…Although these findings seemingly contradict the current observation that TGF-␤ induces ␤-catenin signaling through a SMAD3-mediated mechanism, important differences between the culture models likely account for the discrepancy in the findings. The previous work utilized embryonic chick upper sternal chondrocytes (6). These chondrocytes express colX and spontaneously complete maturation and thus are in a relatively differentiated state at the time of harvest (6).…”