Endosteal Bone Volume Regulation and Osteoporosis

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DESCRIPTION (provided by applicant): Our long term goal in this grant (AR31062-25A2) is to identify the control molecules and their signaling pathways that contribute to the regulation of endosteal bone volume since age-related loss of endosteal bone is a major cause of senile osteoporosis. To this end, our studies during the past grant period have led to the identification of a novel molecule, claudin-18 (Cldn-18), as an important player in the regulation of endosteal bone volume. Cldn-18 is a member of a large family of transmembrane proteins that have been identified as important components of tight junction strands. Although several Cldns, including Cldn-18, have recently been shown to be expressed in bone cells, nothing is known on the role of any Cldns in bone. In preliminary studies, we have found that mice with disruption of Cldn-18 function exhibit a severe deficit in trabecular bone volume (>50%) that is not caused by changes in body weight or bone size. Instead, this reduction in trabecular bone volume in Cldn-18 knockout mice is caused by increased bone resorption as reflected by increased osteoclast (OC) number that is not compensated by a corresponding increase in osteoblast number or bone formation changes. Our studies on the mechanism by which Cldn-18 regulates OC functions have revealed exciting preliminary data to suggest that Cldn-18 effects on RANKL signaling are mediated by a novel mechanism that is independent of its known tight junction function. Based on our preliminary data and what is known on the actions of Cldns in other tissues, we propose the following hypotheses in this study: 1) Cldn-18 acts on committed cells of OC lineage to inhibit differentiation. 2) Cldn-18 effects on RANKL signaling are mediated in part via Cldn-18 interaction with a PDZ domain containing protein, Zona Occluden-2 (ZO-2), to modulate NFATc1 regulation of RANKL target genes. 3) Regulation of PDZ domain-mediated Cldn-18/ZO-2 interaction is mediated in part by RANKL-induced changes in Cldn-18 phosphorylation. To test hypothesis 1, we will isolate OC precursors from Cldn-18 knockout and wild type mice and evaluate the effects of RANKL on proliferation, activity and apoptosis of OCs. We will overexpress Cldn-18 in OC precursors from Cldn-18 knockout mice to determine if Cldn-18 overexpression impairs OC development. We will measure expression levels of marker genes and transcription factors at different stages of OC development to determine the stage of the cell and target genes influenced by Cldn-18. To evaluate the specificity of Cldn-18 action on OCs, we will determine if Cldn-18 influences the differentiation of progenitors into mature granulocytes and macrophages. To test hypothesis 2, we will test for interaction between Cldn-18 and PDZ domain containing ZO-2 in OCs as suggested by our preliminary data. To determine the role for ZO-2 in regulating OC functions, we will evaluate the consequence of overexpression or of blockade of ZO-2 on RANKL-induced OC differentiation. Because ZO-2 is known to be translocated into the nucleus to regulate transcription, we will also determine if Cldn-18 regulates nuclear transport of ZO-2 to modulate NFATc1-mediated transcriptional regulation of genes that are critical for OC differentiation. To test hypothesis 3, we will determine the role of PDZ binding motif in Cldn-18 in binding to ZO-2 and evaluate the role of RANKL-induced phosphorylation changes in Cldn-18 in regulating cellular localization of ZO-2 and Cldn-18 biological effects on OC differentiation. Our successful establishment of interaction between RANKL and Cldn-18/ZO-2 signaling pathways will provide a novel mechanism for RANKL regulation of OC differentiation and provide clues to understanding the actions of other Cldn family members in bone as well as in other tissues. PUBLIC HEALTH RELEVANCE: Developing strategies to diagnose and treat osteoporosis, a major public health threat, would require a thorough understanding of the molecular pathways and the genes involved in the bone resorption process since increased bone resorption is a major contributor to the pathogenesis of osteoporosis. Successful completion of the proposed in vivo animal studies and in vitro mechanistic studies using transgenic approaches on a novel tight junction protein, claudin-18, should lead to elucidation of the role for this gene in regulating bone resorption and thereby endosteal bone volume as suggested by our preliminary data. Because claudin-18 sequence is conserved between mice and humans, future confirmation of a role for claudin-18 in humans will eventually lead to a better understanding of why some people have high bone turnover and treatment options to correct excess bone resorption in those patients.