RI COBRE: REGULATION OF GROWTH PLATE DEVELOPMENT BYNUCLEAR/CYTOPLASMIC FACTORS

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Our long-term goal is to understand the molecular mechanisms regulating growth plate development. The last step in maturation of the growth plate is the differentiation of proliferative chondrocytes into hypertrophic chondrocytes, which subsequently undergo endochondral ossification. Our recent findings indicate that HDAC4 nuclear-cytoplasmic shuttling and degradation plays a major role in this process. Specifically, we suspect that HDAC4 functions as a negative regulator of chondrocyte hypertrophy by binding and inhibiting Runx 2/Cbfa1 expression in the nucleus. Runx 2/Cbfa1 is a transcription factor necessary for chondrocyte differentiation and hypertrophy. However, the mechanisms underlying HDAC4 shuttling and degradation are as yet undetermined. The overall hypothesis about these mechanisms includes two parts: Hypothesis 1: HDAC4 nuclear-cytoplasmic shuttling controls chondrocyte differentiation and is dependent on the Ca2+/calmodulin signaling pathway. Specific Aims 1: To determine whether activation of the Ca2+/calmodulin signaling pathway prevents nuclear entry of HDAC4 and enhances the binding of HDAC4 to the cytoplasmic binding protein 14-3-3. This may impair HDAC4-mediated inhibition of chondrocyte differentiation in the nucleus. Hypothesis 2: HDAC4 degradation is regulated by P38 MARK by increasing expression of caspases 2 and 3. Specific Aim 2: To determine whether: 2a) caspases degrade HDAC4; 2b) p38 MARK regulates expression or activity of capsases 2 and 3; 2c) Runx2 expression is dependent on p38 MARK by in situ hybridization in p38 MARK defective growth plates; 2d) the activities of p38, ERK, and JNK are involved in the process by western blot. Project 2b will be tested using active MKK6 to elevate p38 and dominant negative p38 MARK to repress p38. Significance. The growth plate, or physis, determines the future length and shape of the human mature bone. Delineating the physiological controls of the physis could prevent disabling deformities in children without the need for surgical interventions. Prevention of leg length discrepancy, scoliosis, dwarfism, hip dysplasia and abnormal chondrocyte differentiation in osteoarthritis might also be possible once molecular triggers and stops affecting the growth plate are better understood.