Molecular responses to bone loading in a perlecan/HSPG2-defecient mouse model

Abstract

Perlecan/HSPG2 (PLN), a large Heparan Sulfate Proteoglycan found residing in the osteocytic pericellular matrix (PCM) is a major component of the transverse tethering elements present in the lacunar-canalicular system that help relay mechanical signals. Previous studies have shown that PLN deficiency alters pericellular space and attenuates bone anabolic response to loading. Here, we use a transgenic mouse model with reduced Pln expression (termed hypo) that mimics the conditions of human Schwartz-Jampel Syndrome to study PLN’s role in osteocytic mechanosensing. We hypothesize that the presence of PLN in the PCM is essential for induction of a sustained bone anabolic response to load and that its absence in the lacunar-canalicular (LCS) space of hypo animals would alter their mRNA expression profile after loading when compared to wild type (WT) controls subjected to the same loading conditions. 19-week-old WT and hypo male mice were subjected to uniaxial-tibial compressive loading for either one session in one day or seven sessions on consecutive days and euthanized 24 hours post-load. RNA was extracted from the loaded and non-loaded tibiae of both WT and Hypo animals and the steady state mRNA levels of various bone cell marker genes and matrix proteins were compared using a real-time PCR approach. Among the genes studied, only Pln, and a couple of matrix proteinases (Mmp2 and Mmp9) showed significant difference at baseline (non-loaded conditions) between hypo mutant and WT control bones. One bout of loading was sufficient to induce a significant increase in the mRNA expression level of several markers of bone anabolism: prostaglandin E2 (Ptgs2 aka as Cox-2), the alkaline phosphatase enzyme (ALP), the membrane-associated RANK ligand (RANKL), and osteoprotegerin (OPG). This anabolic response to load was only observed in WT but not in PLN-deficient bones. Interestingly, repetitive loading (over a week period) significantly increased the transcript levels of, type I collagen (COL1A1) in both WT and hypo animals. In contrast, transcripts encoding for E11/podoplastin (gp38) which is the earliest osteocyte marker observed as the osteoblast differentiates into an osteoid and PLN transcripts themselves were upregulated only in WT animals. Additionally, Mmp2 and Mmp3 transcripts were upregulated upon loading only in hypo animals, suggestive of an increased resorptive bone phenotype in mutant animals vs. WT controls. Together, these results suggest that PLN-deficient animals are less mechanoresponsive and that PLN plays an essential function in the anabolic response that load and formation of functional osteoid in which osteocytes are embedded. The significant increase in gene expression of organic matrix components (collagen type I and PLN) with the sustained loading regimen even in the hypo mutant animals puts emphasis on the importance of loading exercises to maintain good bone health.