Deficiency in perlecan/HSPG2 during bone development enhances osteogenesis and decreases quality of adult bone in mice

Abstract

Perlecan/HSPG2 (Pln) is a large heparan sulfate proteoglycan abundant in the extracellular matrix of cartilage and the lacunocanalicular space of adult bones. Although Pln function during cartilage development is critical, evidenced by deficiency disorders including Schwartz-Jampel Syndrome and dyssegmental dysplasia Silverman-Handmaker type, little is known about its function in development of bone shape and quality. The purpose of this study was to understand the contribution of Pln to bone geometric and mechanical properties. We used hypomorph mutant mice that secrete negligible amount of Pln into skeletal tissues and analyzed their adult bone properties using micro-computed tomography and three-point-bending tests. Bone shortening and widening in Pln mutants was observed and could be attributed to loss of growth plate organization and accelerated osteogenesis that was reflected by elevated cortical thickness at older ages. This effect was more pronounced in Pln mutant females, indicating a sex-specific effect of Pln deficiency on bone geometry. Additionally, mutant females, and to a lesser extent mutant males, increased their elastic modulus and bone mineral densities to counteract changes in bone shape, but at the expense of increased brittleness. In summary, Pln deficiency alters cartilage matrix patterning and, as we now show, coordinately influences bone formation and calcification.

Figure 1. Pln is not secreted in developing Pln mutant growth plates and co-localizes with the ER-resident protein BiP in a perinuclear intracellular compartment

Pln (red) extracellular deposition is severely decreased in Pln mutant (F-J) versus WT (A-E). Pln signal (red) is retained intracellularly in the proliferating zone of Pln mutants (F-G) and co-localizes with BiP signal (yellow in H) whereas signals for these two proteins did not overlap in WT (Pln in red and BiP in green in C). Columnar arrangement of chondrocytes in WT prehypertrophic and hypertophic zones (D) is lost in Pln mutants (I) as indicated by the disorganized aggrecan staining of the cartilage matrix in Pln mutant growth plates (compare green in D and I). Despite changes in matrix organization the overall secretion of aggrecan remained undisturbed in mutant vs. WT growth plates (compare green in E and J). Arrow in A and F indicate an increase in the diameter of Pln mutant developing bones relative to WT. Arrowhead in F points to reduced deposition of Pln in the basement membrane of a blood vessel present in tissue surrounding developing bone. Magnification bars: 200 μm (A, E, F, J), 50 μm (B, G), 10 μm (C, H), and 100 μm (D, I). The images shown are representative of immunolabelings performed on growth plates of newborn mice (n=5 per genotype)

Figure 2. Pln mutant hypertrophic zone is disorganized and expanded

Type X collagen (green) immunolabeled area overlaps with calcified deposits detected using the von Kossa method in mutant (C and D) but not in WT (A and B).

Figure 3. In vitro secretion of Pln by cultured bone progenitors prevents spontaneous differentiation, and inhibits BMP2-induced differentiation into calcifying osteoblast-like cells

Lack of extracellular expression of Pln in Pln mutants (green in B) is accompanied by increased in vitro differentiation of osteoprogenitors relative to WT (compare alkaline phosphatase activity in purple in D and C). This effect is enhanced in the presence of BMP2 (compare F to E) and in vitro mineralization can be clearly detected in Pln mutant cultures treated with BMP2 during 12 days of culture (J) whereas no mineral deposits are found in WT cultures subjected to the same culture conditions. Images representative of three biological replicates are shown.

Figure 4. Pln mutant display a short stature phenotype and mild skeletal deformities that are not associated with a decrease in skeletal bone mass

Postnatal growth curves (A) of WT, heterozygous, and Pln mutant littermates (n≥5 for each genotype). Pln mutants display a considerable growth deficiency when compared with WT and heterozygous controls of the same gender. Representative x-ray image areas of skeletally mature (control) and age-matched mutant hindlimbs (B) showed diminished length of long bones and vertebrae in Pln mutants vs. controls (see arrows). * in B indicates deformity of the ischium in Pln mutants. Mid-diaphyseal cross-sections at mid-shaft showed an increase in the bone area of mutant animals compared to gender-matched controls that was more pronounced in females than in males (C). Bone measurements showed significant decrease in Pln mutant bone length relative to wild type in all long bones examined regardless of gender (D). In contrast, wet bone weight was only significantly increased in Pln mutant male femora compared to wild type male femora (E). ***, **, and * indicate significant differences (p<0.001, p<0.01, and p<0.05, respectively) in Pln mutant relative to controls.

Figure 5. High bone mineral density in Pln mutant bones is associated with increased brittleness

Bending rigidity (EI), ultimate force, and PYD of Pln mutant and WT femurs were obtained from three-point-bending deformation curves. BMD and medial-lateral moment of inertia (I_ML) were obtained from μCT scans. Young’s moduli (E) were calculated using EI and I_ML. Changes in bone material properties are more pronounced in Pln mutant females than in Pln mutant males when compared to gender-matched controls. *p<0.05, n≥6; n.s. non significant.