Function Of Skeletal Matrix Genes

National Institute Of Dental & Craniofacial Research

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Abstract

Small Leucine Rich Proteoglycans and Skeletal Aging The SLRP family is composed of 17 members sub-divided into classes (I-V) based on their amino acid sequence and genomic organization. All members of the SLRP family (excluding asporin) have extensive post-translational glycosylation on a relatively small protein core backbone composed of repeat structures rich in leucine. For years, evidence has been mounting about the importance of SLRPs in skeletal function. We have focused on the SRLP biglycan (Bgn) and fibromodulin (Fmod) because of their high level of expression in bones and teeth. Taken together our work highlights the fact that they are not essential for bone development but, rather, appear to play a role in skeletal aging. For this reason, we have focused on roles for Col6, Bgn and Fmod in bone turnover in mature bone. Bgn and Fmod are widely expressed in the extra-cellular matrix (ECM) of mineralized tisssues and made by bone marrow stromal cells, osteoblasts, and osteocytes. We previously generated a Bgn/Fmod double knock-out (DKO) mouse model and showed these mice have a 3-fold increase in osteoclastogenesis compared with Wild type (WT) controls resulting in a markedly low bone mass (LBM) phenotype observed as early as 1 month and getting worse with age. Obstruction of osteoclast differentiation, recruitment, activity and life span as in the case of bisphosphonate treatment, leads to increase in bone mineral density (BMD), as well as many other bone parameters including trabecular number and thickness and better geometrical properties of bone. To try and rescue/repair the LBM phenotype of Bgn/Fmod DKO mice by suppressing osteoclast formation and activity, 3- and 26-week-old Bgn/Fmod DKO mice and age/gender matched WT controls were injected with the anti-osteoclast agent OPG-fc for 6 weeks after which bone parameters were evaluated using DEXA, microtomographic (CT) and serum biomarkers analyses. In the appendicular skeleton, OPG-Fc treatment improved some morphometric and geometric parameters in both the trabecular and cortical compartments of the long bone in Bgn/Fmod DKO female and male mice, especially in the repair module. The effect of the treatment was not to the same extent in the Bgn/Fmod DKO and WT controls implicating Bgn and Fmod in the regulation of the mechanism of action of OPG-Fc in bone homeostasis. In addition, our results indicate that the treatment was not able to prevent or ameliorate the formation of ectopic ossifications in tendons, which are common lesions seen in aged joints and are one of the phenotypical hallmarks of our Bgn/Fmod DKO model. When skulls were analyzed by microCT we found that OPG-Fc treatment recovered some parameters of LBM phenotype in the craniofacial skeleton albeit with some adverse effects including abnormally high bone density. We conclude from this data that SLRPs expressed and secreted by bone-forming cells interact with molecules in the ECM niche to control OPG attenuation of osteoclast development. Using OPG-Fc as treatment alleviated, yet did not restore, the severe osteopenia and mineralized tissue structural abnormalities that Bgn/Fmod DKO mice suffer from. In summary, we found that the LBM phenotype of the Bgn/Fmod DKO model could be partially rescued by OPG-Fc treatment in mature mice, but in young mice, even though effective in enhancing BMD, treatment was harmful to the skeletal structure independent of genotype. While overactive osteoclastogenesis in the Bgn/Fmod DKO model appeared to be reduced by the OPG-Fc treatment and somewhat beneficial to the mineralized tissue, the nature, quality and strength of the new bone produced is not known. Further experiments will be needed to determine if additional factors could be used to achieve full effectiveness of OPG-Fc in cases of ECM disruption caused by loss of Bgn/Fmod. Col6 regulates bone mass by inhibiting osteoclast function Bone is a highly dynamic tissue that depends on specialized cell types to regulate its formation and structural integrity. Bones are formed by the anabolic actions of osteoblasts. Osteoblasts have a close relationship with catabolic osteoclasts that resorb or digest bones. The coupled actions of bone resorption and subsequent bone formation is known as bone turnover, a process that replaces and renews bone tissue that has micro-damage from impact or aging. An imbalance of bone turnover results in either osteopetrosis, which is excess bone matrix, or osteopenia, which is insufficient bone matrix. In both cases, the bone is fragile and prone to breakage. Identifying novel factors that affect the osteoblast-osteoclast relationship will improve our understanding of bone turnover and pave the way for future bone therapies. Type VI collagen (Col6) is most often composed of three different -chains (1, 2, 3) with three other minor chains (4, 5, and 6) found at low levels. Mutations in any one of the three major chains of human type VI collagen cause Bethlem myopathy or Ullrich congenital muscular dystrophy (UCMD), diseases that present with mild to severe muscle weakness, respectively. While type VI collagen is found in many musculoskeletal tissues, its functions in bone are just beginning to be investigated. Assembly of the type VI collagen triple helix begins at the C-terminus and the triple helical monomer is flanked by two large multidomain globular regions. Dimers are assembled from head-to-tail staggered monomers. Then, dimers align side-by-side to form tetramers, which are then secreted. Outside the cell, tetramers assemble end-to-end into microfibril structures that appear as beads when visualized by the electron microscope. The importance of the ColVI2 chain in the assembly, secretion and subsequent microfibril formation was highlighted in a UCMD patient with compound heterozygous mutations in Col6a2. The mutant collagen was found to be retained intracellularly, preventing normal folding and microfibril assembly. To investigate the role of ColVI in bone and its mechanistic foundation, the skeletal phenotype of mice globally deficient in the ColVI was generated by depleting the alpha 2 chain (Col6a2-KO) in mice. Using the newly generated mouse model devoid of Col6, we showed that Col6 regulates trabecular bone mass in both the femur and spine by controlling the balance between bone formation and resporption. The reduced bone mass observed in Col62-KO mice arises from increased osteoclastogenesis with no apparent effect on bone formation. RNAseq analysis of mRNA extracted from the bones of Col62-KO mice showed deregulation of bone remodeling pathways, and in addition, pointed to a possible link to TNF alpha. We found that there is a direct interaction between Col6a2 and TNF and that Col6a2 attenuates TNF-induced osteoclastogenesis to subsequently influence bone mass. We are currently examining Col6a2 function in the periodontal ligament and surrounding alveolar bone and in the temporomandibular joint that are all tissues critical to oral health.