Galectin-3 is a downstream regulator of matrix metalloproteinase-9 function during endochondral bone formation

Abstract

Endochondral bone formation is characterized by the progressive replacement of a cartilage anlagen by bone at the growth plate with a tight balance between the rates of chondrocyte proliferation, differentiation, and cell death. Deficiency of matrix metalloproteinase-9 (MMP-9) leads to an accumulation of late hypertrophic chondrocytes. We found that galectin-3, an in vitro substrate of MMP-9, accumulates in the late hypertrophic chondrocytes and their surrounding extracellular matrix in the expanded hypertrophic cartilage zone. Treatment of wild-type embryonic metatarsals in culture with full-length galectin-3, but not galectin-3 cleaved by MMP-9, mimicked the embryonic phenotype of Mmp-9 null mice, with an increased hypertrophic zone and decreased osteoclast recruitment. These results indicate that extracellular galectin-3 could be an endogenous substrate of MMP-9 that acts downstream to regulate hypertrophic chondrocyte death and osteoclast recruitment during endochondral bone formation. Thus, the disruption of growth plate homeostasis in Mmp-9 null mice links galectin-3 and MMP-9 in the regulation of the clearance of late chondrocytes through regulation of their terminal differentiation.

Figure 1.

Accumulation of late terminally differentiated hypertrophic chondrocytes in Mmp-9 null mice. Serial sections of growth plates from 2-wk-old MMP-9 null and WT mice. (A) Safranin O/Fast Green (SO/FG) staining (a and b) and in situ hybridization with antisense probes for collagen type II (Col2; c and d) and collagen type X (Col10; e and f) show an increase in the hypertrophic chondrocyte zone (hc) but not the proliferating chondrocyte zone (pc) in MMP-9 null mice compared with WT. (B) In situ hybridization with antisense probes for late hypertrophic markers, Mmp-13 (a and c) and Osteopontin (Op; b and d) show the accumulation of late hypertrophic chondrocytes in MMP-9 null mice. (e and f) Higher magnification of Mmp-13 and Op expression pattern in MMP-9 null mice. Note that the accumulation of hypertrophic chondrocytes expressing these markers (arrows) in several rows at the chondro-osseous junction. pc, proliferating chondrocytes; hc, hypertrophic chondrocytes; tb, trabecular bone. Scale bars, 250 μm.

Figure 2.

Altered organization of the chondro-osseous junction in Mmp-9 null mice. Histological analysis of the chondro-osseous junction. (a and b) Representative pictures of semithin sections stained with Toluidine blue showing the abnormal survival of the last row of hypertrophic chondrocyte (arrows) in Mmp-9 null mice compared with WT. A capillary in close contact with last transverse septa is marked by an asterisk (^*). (c–h) Longitudinal sections through the central part of growth plates. (c and d) Alcian blue staining; note the decrease in staining in Mmp-9 null mice in the last row of hypertrophic chondrocytes and trabeculae. (e and f) PECAM staining showing no major differences in growth plate vascularization between Mmp-9 null and WT mice. (g and h) TRAP staining, note the increase in TRAP-positive cells recruited at the chondro-osseous junction in Mmp-9 null mice. hc, hypertrophic chondrocytes; tb, trabecular bone. Scale bars, 125 μm. (i) Quantification of mononuclear and multinucleated TRAP-positive cells recruited at the chondro-osseous junction, showing an increase in Mmp-9 null mice (n = 6 different metatarsals; ^*p ≤ 0.005).

Figure 3.

Accumulation of uncleaved galectin-3 in the growth plate of Mmp-9 null mice. (A) Protein extracts from WT and Mmp-9 null hypertrophic chondrocyte zones were analyzed by SDS-PAGE and silver staining. Differences in protein profiles were observed as increases in the intensity of several bands in Mmp-9 null samples, as indicated by arrows. (B) Protein extracts from WT and Mmp-9 null hypertrophic chondrocyte zones were analyzed for expression of galectin-3, tissue transglutaminase (TG2), CTGF, VEGF, and α1 chain of collagen type II (Col2; α1) by Western blotting. Note the increase in intact galectin-3 dimers and VEGF in Mmp-9 null mice compared with WT samples.

Figure 4.

Galectin-3 expression is disturbed during the initial stage of endochondral bone formation in Mmp-9 null mice. (A) Serial longitudinal sections of humeri from E15.0 WT and Mmp-9 null mice were stained with Safranin O/fast green (SO/FG; a and b) and for TRAP activity (o and p, TRAP-positive cells are indicated by black arrows). Adjacent sections were hybridized with antisense RNA probes for Galectin-3 (Gal-3, fuchsia), Collagen type 2 (Col2, red), Collagen type 10 (Col10, green), Mmp-13 (yellow), Osteopontin (Op, purple), or Mt1-mmp (orange). Note the increase in Galectin-3 expression in Mmp-9 null mice as indicated by double pink arrows. Rare Galectin-3–expressing cells are found in the perichondrium of Mmp-9 null samples (white arrow). Red dotted lines indicate the limits of Mmp-13 and Op expression domains in late HC. (B) Serial longitudinal sections of femurs from E16.0 WT mice were hybridized with antisense RNA probes for Galectin-3 and Mmp-9 as indicated. Merging of both signals shows partial overlap between Galectin-3 and Mmp-9 expression. Brackets indicate the primary ossification center in wild-type sections. Scale bars, (A) 250 μm; (B) 500 μm.

Figure 5.

Galectin-3 accumulates at the chondro-osseous junction of Mmp-9 null mice. Paraffin sections of the growth plates of 2-wk-old Mmp-9 null and WT metatarsals. (a and b) In situ hybridization with antisense RNA probe for Galectin-3. (c and d) Immunostaining with monoclonal rat anti–galectin-3. (e and f) Higher magnification of (c and d) showing the increase in galectin-3 protein in hypertrophic chondrocytes (hc) of Mmp-9 null mice. Black arrows indicate galectin-3 staining in perilacunar space. (h and i) Higher magnification of (c and d) showing the increase in recruitment of galectin-3–positive cells at the chondro-osseous junction (indicated by dotted line) in Mmp-9 null mice (black arrows) and the increase in galectin-3 staining in the pericellular matrix of the last row of hypertrophic chondrocytes (gray arrowheads). (g) Quantification of hypertrophic chondrocytes exhibiting high levels of galectin-3 immunostaining. Results are expressed as percentage of total hypertrophic chondrocytes counted. (n = 6 different metatarsals; ^*p ≤ 0.005). (j) Quantification of galectin-3–positive cells recruited at the chondro-osseous junction expressed as total number of galectin-3–positive cells per section along the ossification front. (n = 6 different metatarsals; ^*p ≤ 0.005). pc, proliferating chondrocytes; hc, hypertrophic chondrocytes; tb, trabecular bone. Scale bars, (a and b, 250 μm); (c and d, 150 μm); (e, f, h, and i, 30 μm).

Figure 6.

Ectopic addition of multimeric uncleaved galectin-3 partially mimics MMP-9 deficiency. Sections from E16.5 WT metatarsals cultured for 4 d in presence of vehicle alone, full-length mouse recombinant galectin-3–or MMP-9–cleaved galectin-3 stained with (A) Safranin O or (B) for TRAP activity. (C) Quantification of the increase in growth plate length following galectin-3 treatment. Results are expressed as the percentage of increase compared with untreated WT and the results from two representative independent experiments are shown (series 1, n = 5 for vehicle alone, n = 4 for full length galectin-3 and n = 4 for MMP-9 cleaved galectin-3), ^*p ≤ 0.005); (series 2, n = 5 for vehicle alone, n = 5 for full length galectin-3 and n = 3 for MMP-9 cleaved galectin-3, ^*p ≤ 0.005).