Suppressor of fused (Sufu) mediates the effect of parathyroid hormone-like hormone (Pthlh) on chondrocyte differentiation in the growth plate

Abstract

Growth plate chondrocytes undergo a coordinated process of differentiation, regulating long bone growth. Parathyroid hormone-like hormone (Pthlh) inhibits hypertrophic differentiation in the growth plate chondrocytes and reduces Hedgehog (Hh) signaling. In mice lacking the Hh mediator Suppressor of fused (Sufu), Pthlh treatment resulted in the up-regulation of Hh activity and an increased number of hypertrophic chondrocytes. Furthermore, Pthlh increased Sufu protein levels, and in chondrocytes lacking Sufu, it was unable to process Hh-regulated Gli transcription factors. Pthlh regulates chondrocyte differentiation and Gli activity in a Sufu-dependent manner, with Sufu acting as a molecular switch in its regulation of differentiation.

FIGURE 1.

Pthlh regulates chondrocyte differentiation in a Sufu-dependent manner. A, histological and immunohistochemical analysis of embryonic day 16.5 mouse tibial explants from Col2a1-Cre;Sufu^f/f mice or littermate mice lacking the expression of Col2a1-Cre (WT Sufu^f/f) in the absence or presence of Pthlh. The hypertrophic zone (red bars) is characterized by Col10a1 staining. The proliferative zone (black bars) is represented by the length from the end of the tibia to the beginning of the hypertrophic zone. n = 6 in each group. B, relative percentage difference of the length of the hypertrophic zone of Pthlh-, H-89-, or Pthlh/H-89-treated tibial explants compared with the controls. Data are shown as means and 95% confidence intervals. C, relative percentage difference of the length of the proliferative zone of Pthlh-, H-89-, or Pthlh/H-89-treated tibial explants compared with the controls. Data are shown as means and 95% confidence intervals.

FIGURE 2.

Kif7 is not required for the inhibitory effect of Pthlh on chondrocyte differentiation. A, Col10a1 and Safranin O (Saf O) staining of embryonic day 16.5 tibial explants of Col2a1-Cre;Kif7^f/f and Col2a1-Cre;Sufu^f/f;Kif7^f/f mice with or without Pthlh treatment. B, relative percentage difference of the length of the hypertrophic zone of Pthlh-treated tibial explants compared with the controls. Data are shown as means and 95% confidence intervals. An asterisk indicates a significant difference (p < 0.05) compared to untreated samples.

FIGURE 3.

Sufu is required for Pthlh-mediated Gli processing. A, qRT-PCR analysis of the expression levels of Gli2 and Gli3 in limbs from Sufu^f/f mice, which do not express Cre recombinase (WT Sufu^f/f), or from Col2a1-Cre;Sufu^f/f mice. B, Western analysis of Gli2 in wild-type and Sufu-deficient chondrocytes with (Pth) or without (untreated (Un)) Pthlh treatment. Actin was used as a loading control. C, Western analysis of full-length Gli3 (Gli3FL) and the Gli3 truncated repressor (Gli3R) in wild-type and Sufu-deficient chondrocytes with or without Pthlh treatment. The asterisk indicates a nonspecific band. D and E, qRT-PCR analysis of Gli1 and Ptch1 expression in wild-type and Sufu-deficient growth plate chondrocytes with or without Pthlh treatment. Data are shown as means and 95% confidence intervals. *, significant difference from controls (p < 0.05).

FIGURE 4.

Pthlh phosphorylates Sufu, resulting in inhibition of its degradation, and proposed model for how Pthlh acts through Sufu to regulate chondrocyte differentiation. A, Pthlh (PTH) treatment results in elevated levels of Sufu protein in wild-type growth plate chondrocytes. The effective deletion of Sufu in knock-out mouse chondrocytes was verified by Western analysis. Actin was used as loading control. Un, untreated. B, qRT-PCR analysis of Sufu expression levels in wild-type chondrocytes with or without Pthlh (PTHLH) treatment. C, pulse chase data showing that the rate of decline in Sufu protein slows with PTHLH treatment, a representative immunoblot and graphical data showing means and 95% confidence intervals are shown. D, Ser-346-phosphorylated Sufu levels increase with Pthlh treatment. Data are shown as means and 95% confidence intervals. *, significant difference from the control (untreated; p < 0.05). F, proposed model by which Pthlh promotes Sufu-dependent Gli2 degradation and Gli3 processing, ultimately inhibiting chondrocyte hypertrophic differentiation (circled D). E, treatment with MG132 inhibits Sufu degradation and PTH will not alter its Sufu level in the presence of MG132. A representative immunoblot and mean and 95% confidence intervals are given in a graphical form. F, Pthlh stimulates chondrocyte proliferation (circled P) in a Sufu-independent manner. Gli3R, Gli3 truncated repressor.