This project investigates osteoclast (OC) biology, and mechanisms of normal and pathological bone remodeling under 2 Specific Aims. SA 1 will continue our investigations of a novel osteopetrosis gene we discovered called Plekhm1. SA 2, will map the genetic lesion in the last unmapped rat osteopetrotic strain, op.
Aim 1 grows from our previous work which found the causative mutation in the osteopetrotic incisors absent (ia) rat in a novel gene, Plekhm1. We also showed that a PLEKHM1 frameshift mutation causes osteopetrosis in humans. Both ia rats and the human patient have TRAP+, non-resorbing osteoclasts with defective ruffled borders (RB) and defective secretion. Our colleagues also recently discovered a dominant point mutation (R714C) in PLEKHM1 in a patient with low bone mass and focal sclerosis which we propose is a gain-of- function mutation. The gene encodes a large (>1000 aa) protein with several domains suggesting modularized function. Plekhm1 localizes late to endosomes in a manner dependent on GTP-bound Rab7. Rab7+ vesicles normally move toward the nucleus, but in OCs they move to the RB. Plekhm1 has a RUN domain, 2 pleckstrin homology (PH) domains, and a C-1 domain. The ia rat protein has an intact RUN domain, lacks the rest of the protein, and fails to localize with Rab7 vesicles. We will investigate the localization of plekhm1 in resorbing vs. non-resorbing OCs and will screen candidate proteins for potential roles in a complex needed to translocate OC secretory vesicles to the RB and secrete their contents into the resorption lacuna to effect bone resorption. Under SA1.1, we will investigate the protein complex that includes plekhm1 and Rab7 by a combination of confocal and immunoelectron microscopy, IP and pull-down assays, microtubule disruption, protein crosslinking and high-sensitivity mass spectrophotometry. We will compare resorbing and non-resorbing OCs and wild type vs. ia ra OCs. Under SA1.2, we will study the ability of the PH domains to bind specific phospholipids and determine by enzyme inhibition assays if plekhm1 activity, like many OC functions, is regulated by PI3-kinase. We will also study the R714C mutation, located in the 2nd PH domain, to determine its impact on lipid binding specificity and strength. We will investigate the C-1 domain to determine if it is regulated by diacylglycerol and requires zinc ions for its activity. We will also study the effects of point mutations of candidate amino acids to determine if specific aa in the PH and C-1 domains impact their important biological binding activities. Under SA2, we will continue to map the last remaining unmapped osteopetrotic mutation in the rat, op. The op rat has large, TRAP+, non-functioning OCs. Its map position and sequencing results to date indicate it is not in a previously known osteopetrosis gene. We are maintaining an ongoing out-crossed breeding project to narrow the candidate region and are sequencing genes in the chromosome interval containing the mutation. We are currently at generation F11 and are testing for F12 breeders. When we identify the mutation, it will undoubtedly lead to additional investigations of the gene and its role in osteoclast biology.
Widespread bone disorders including osteoporosis, arthritis, periodontal bone disease, joint replacement loosening, and tumor metastasis to bone, all have in common bone loss that exceeds bone formation. Bone loss is carried out by cells called osteoclasts, and every new factor we discover that affects osteoclast activity is a potential target for therapeutics. This proposal aims to investigate new genes that regulate the formation and activity of osteoclasts to better understand bone biology in general, and potentially to find new ways to treat these widespread bone disorders.
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