This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Malnutrition remains a worldwide problem in developing countries. In developed countries, many medical conditions, ranging from anorexia nervosa to Crohn's disease, result in severe growth impairment in children. The objective of this study is to identify mechanisms by which nutrient availability, specifically leucine which is the most abundant essential amino acids, affects chondrocyte proliferation, differentiation, deposition of extracellular matrix (ECM) and their interactions. It has long been known that nutritional status effect linear growth by modulating GH/IGF-I axis. Recently signaling mechanism by which nutrients directly regulate cells via mammalian Target Of Rapamycin (mTOR) has been elucidated. We hypothesize that leucine directly regulates chondrogenesis directly by affecting chondrocyte proliferation, differentiation and ECM deposition, 3 key components of bone growth. Our hypothesis is based on our observations. First, mTOR directly regulates chondrogenesis. MTOR inhibition results in decreased expression of Indian hedgehog (Ihh), a key regulator of chondrogenesis. Decreased Ihh could be central to mTOR action. Regulation of Ihh by mTOR is a new observation. Second, leucine restriction and mTOR inhibition result in significant reduction of ECM mRNA expression likely via miRNA regulation. Lastly, we utilized an ex vivo metatarsal explant system to demonstrate the physiological relevance of observations we made in the chondrogenic ATDC5 cell line. Based on our preliminary findings, we propose the following specific aims.
Specific Aim 1 : Identify the molecular mechanisms that mediate the effect of mTOR inhibition and leucine restriction on Ihh regulation and, as a consequence, chondrocyte growth and differentiation. .
Specific Aim 2 : Determine the effect of mTOR inhibition and leucine restriction on miRNA-29 regulation resulting in altered ECM expression and functional role in chondrocyte growth and differentiation Specific Aim 3: Utilizing a fetal metatarsal explant model, we will study the effect of mTOR inhibition and leucine restriction on bone growth, growth plate dynamics and chondrogenic markers, in particular Ihh and miRNA-29. We will study the effect of restoring Ihh and miRNA-29 expression in bones subjected to mTOR inhibition and leucine restriction. Understanding the direct effect of nutrients on bone growth may lead to a better understanding of long term bone health as well as management of the impaired growth in nutrient restriction or related medical conditions.
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