The Microphthalmia (Mi) gene encodes one of the oldest recognized factors critical to osteoclast development and function. Mi is also required for development of the melanocyte lineage. The recently cloned Mi gene encodes a HLH-ZIP transcription factor, and is unique in its highly restricted tissue distribution. We have defined protein and DNA interactions of MI and found it to bind and potentially activate transcription off promoters containing its recognition element. We have recently examined an unusual rat mutant which displays severe osteoporosis which resolves with aging and shown that this results from a large deletion within Mi. We also discovered 3 dimerization partners of Mi (TFEB, TFEC, and TFE3) collectively called the """"""""MiT"""""""" family. At least one of these other factors is present in the Mi-deficient osteoclasts of this rat strain, suggesting a general a general role of this family in osteoclast function and maturation. Our studies in melanocytes have also revealed that Mi is strongly activated by MAP kinase phosphorylation following cytokine stimulation. c-Kit signaling operates through activation of Mi in this fashion. We now also know that the mechanism of this activation is phospho-Mi selective recruitment of the transcriptional co-activators p300/CBP. Importantly, our current results suggest that Mi is similarly phosphorylated in response to cytokine signaling in osteoclasts, and this response (to factors such as interleukin 6 and M-CSF) could lie critically within a major pathway of osteoclast activation. The understanding of such pathways is clearly central to future goals of modulating osteoclast function in conditions such as osteoporosis. To further enhance our understanding of this factor and extend our analysis of its actions within osteoclasts, this grant proposes to: 1) systematically examine Mi'T protein temporal expression patterns in osteoclasts throughout development and aging, 2) derive viral vectors permitting up- or down-regulation of endogenous MiT factors in osteoclasts, and examine their consequences on osteoclast development and function, 3) define mechanistic relationships between Mi and osteoclast signaling pathways of importance in bone homeostasis including other genes implicated in osteopetrosis and cytokines of functional importance, and 4) identify transcriptional target genes for Mi in osteoclasts by testing """"""""rational"""""""" candidates identified through sequence analysis of known promoters as well as a highly stringent differential display which requires up-regulation by wild type Mi, down- regulation by dominant negative Mi, as well as cytokine-mediated Mi induction in the presence of cycloheximide. Through these studies we hope to gain insight into osteoclast biology through an understanding of Mi's central role.
