Proteoglycan desulfation determines the efficiency of chondrocyte autophagy and the extent of FGF signaling during endochondral ossification - PubMed (original) (raw)

Proteoglycan desulfation determines the efficiency of chondrocyte autophagy and the extent of FGF signaling during endochondral ossification

Carmine Settembre et al. Genes Dev. 2008.

Abstract

Cartilage extracellular matrix (ECM) contains large amounts of proteoglycans made of a protein core decorated by highly sulfated sugar chains, the glycosaminoglycans (GAGs). GAGs desulfation, a necessary step for their degradation, is exerted by sulfatases that are activated by another enzyme, Sulfatase-Modifying Factor 1 (SUMF1), whose inactivation in humans leads to severe skeletal abnormalities. We show here that despite being expressed in both osteoblasts and chondrocytes Sumf1 does not affect osteoblast differentiation. Conversely, in chondrocytes it favors ECM production and autophagy and promotes proliferation and differentiation by limiting FGF signaling. Thus, proteoglycan desulfation is a critical regulator of chondrogenesis.

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Figures

Figure 1.

Figure 1.

Skeletal development in Sumf1−/− embryos and mice. (A–C) X-gal-stained Sumf1+/− E12.5, E14.5, and E16.5 embryos. LacZ was expressed in all cartilagineous elements starting at E14.5. (D) Femoral growth plate section showing LacZ staining in proliferating chondrocytes and osteoblasts. (E) In situ hybridization of Sumf1 in E16.5 growth plate. (F_–_H) Alcian blue/alizarin red staining of E16.5 (F), newborn (G), and P4 (H) wild-type and Sumf1−/− embryos and mice. (G,H) Femur and tibia magnification of newborn and P4 _Sumf1_−/− and wild-type mice.

Figure 2.

Figure 2.

Sumf1−/− deficiency affects chondrogenesis but not osteogenesis. (A) Hematoxylin/eosin (H/E) staining and in situ hybridization analysis of femoral sections. No differences between Sumf1−/− and wild-type growth plates are observed in E14.5 embryos, while in E16.5 (B) and newborn (C) there is a progressive shortening of both proliferative and hypertrophic area in mutant mice. Expression of α_1(II)_ and α_1(X) Collagen_ was decreased in newborn Sumf1−/− compared with wild-type mice. α_1(I) Collagen_ expression was similar in wild-type and Sumf1−/− samples at all stages analyzed. (D) Von Kossa-Van Gieson staining of femoral sections showed normal mineralization (black staining) in E16.5 and newborn Sumf1−/− mice. Magnification: A–C, 100×, D, 50×.

Figure 3.

Figure 3.

Defective ECM in _Sumf1_−/− growth plate. (A) Chondrocyte number in the growth plate proliferative zone. Values are the mean ± SD. Student’s test (*) P < 0.05. (B) Toluidin blue staining of chondrocostal cartilage of newborn Sumf1−/− and wild-type littermates. Note the presence of cytoplasmatic vacuolization in Sumf1−/− chondrocytes. (C) EM analysis of E14.5 Sumf1−/− and wild-type chondrocytes showing no evidence of lysosomal vacuolization in Sumf1−/− embryos. (D,E) Cytoplasmatic vacuoles in E16.5 (D) and newborn (E) Sumf1−/− chondrocytes filled with amorphous material (GAGs) and partially degraded collagen fiber (E, inset). (F) Sumf1−/− osteoblasts are less affected than chondrocytes by lysosomal vacuolization. (G) ECM proteoglycan produced by Sumf1−/− and wild-type primary chondrocytes labeled with 3H-glucosamine and Na35SO4. ECM amount was estimated by 3H and 35S incorporation and normalized for cells number. (H) Decreased alcian blue staining in P7 Sumf1−/− compared with wild-type growth plate.

Figure 4.

Figure 4.

Abnormal autophagy in Sumf1−/− chondrocytes. (A) Number of BrdU positive cells present in the proliferative zone of the growth plate. Values are the mean ± SD. Student’s test (*) P < 0.05. (B) EM analysis of newborn chondrochostal cartilage revealed the presence of autophagosomes in wild-type chondrocytes. (Boxed inset) Note the double membrane vesicles surrounding a portion of cytoplasm (arrows). (C) Chondrocyte from newborn Sumf1−/− showing more autophagosomes (AV) surrounded by enlarged lysosomes (L). (D) Confocal microscopy analysis of Sumf1−/−;GFP-LC3 and wt;GFP-LC3 growth plate. In GFP-LC3 chondrocytes (left) the GFP fluorescence was more diffused throughout the cytoplasm while in Sumf1−/−;GFP-LC3 it was aggregated in cytoplasmatic dots (right). (E) Western blot analysis showing a 2.5-fold increase in LC3II level in newborn Sumf1−/− chondrocytes. No difference was observed in osteoblasts. Values shown are means of triplicate experiments. (F) Abnormal autophagy in Sumf1−/− chondrocytes during serum and nutrient starvation. Wild-type and Sumf1−/− chondrocytes were starved for the indicated period of time, harvested, and subjected to LC3 immunoblotting. Sumf1−/− chondrocytes and wild type stimulated with Baf presented an increased amount of LC3II compared with wild-type chondrocytes at all time points analyzed. (G) ATP amount is decreased in wild-type chondrocytes when autophagy is inhibited with Baf. Sumf1−/− chondrocytes displayed a lower level of ATP compared with wild-type and Baf treatment did not affected significantly ATP concentration. (H) Wild-type and Sumf1−/− chondrocytes were cultured in serum and glucose-free medium for 2 d. Wild-type chondrocytes were also treated with baf and 3-methyladenine, another inhibitor of autophagy. Cell viability was monitored after 12 h, 24 h, and 48 h. Error bars represent SEM. Student’s test (*) P < 0.05;

(**)P < 0.01.

Figure 5.

Figure 5.

Sumf1 regulates FGF18 activity during endochondral ossification. (A) H/E staining of femurs showing shortening of Sumf1−/− bone length (arrowheads) and its rescue in Sumf1−/−;fgf18+/− mice. Brdu staining (B) and in situ expression of α1(II) Collagen (C) and α1(X) Collagen (D) in wild-type (left), Sumf1−/− (middle), and Sumf1−/−;fgf18+/− mice. (E) Quantification of femoral length, BrdU index, and cell number in newborn wild-type, Sumf1−/−, and Sumf1−/−;fgf18+/− mice. At least three mice were analyzed per each genotype. Error bars represent SEM. Student’s test (*) P < 0.05;

(**) P < 0.01. (F) Primary chondrocytes from wild-type and Sumf1−/− mice treated with Fgf18 (20 ng/mL) for the indicated period of time. Note the more sustained phosophorylation of ERK and 70S6k in Sumf1−/− than in wild-type chondrocytes following Fgf18 treatment.

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