BONE GROWTH IN DENTAL, CRANIAL AND SKELETAL TISSUES

Thomas Jefferson University, Philadelphia PA

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Abstract

DESCRIPTION (provided by applicant): The physiological importance of apoptosis in the development and function of the growth plate is of major import. Without some mechanisms for removing terminally differentiated cells from cartilage, the plate would expand uncontrollably and there would be impaired bone formation. Accordingly, apoptosis provides a physiological mechanism for the rapid and controlled removal of terminally differentiated cells from epiphyseal tissue. Based on current studies, we now propose that apoptosis is regulated at two separate levels: a powerful late acting system that is dependent on release of specific components of the extracellular matrix and an early maturation-dependent system that prepares and sensitizes chondrocytes to apoptotic stimuli. Herein, we focus on early events in the induction of the apoptotic process. Experiments are described that explore the role of metabolic factors in the initiation of apoptosis, focusing on mitochondrial function and cellular metabolism. A second area of study that impacts on apoptosis is the control of survival responses. Accordingly, we will examine regulation of a key survival pathway and determine its relationship to metabolic factors that are common to both systems. There are three Specific Aims: The first aim is to determine at what maturation stage chondrocytes exhibit a decrease in the mitochondrial transmembrane potential and exhibit evidence of apoptosome formation and caspase activation; to ascertain if a change in transmembrane potential causes an alteration in caspase-3 activity; to learn if a low transmembrane potential increases susceptibility of chondrocytes to apoptosis. The second aim is in two parts: (A) to examine ROS/NO generation and GSH levels in relationship to transmembrane potential; to determine the role of ROS, NO and GSH in the activation of caspase-3, and/or non-cytochrome c dependent caspase systems and apoptosis; (B) to learn if Uncoupling Protein (UCP) is expressed by maturing chondrocytes: to ascertain if UCP expression is related to loss of transmembrane potential, thiol depletion, ROS generation and caspase activation. The last aim of the investigation is to examine survival pathways in maturing chondrocytes; to relate changes in transmembrane potential and ROS generation to activation of PI 3 kinase; to determine if Akt is a major target of PI 3 kinase; to relate inhibition of Akt to caspase activation and chondrocyte apoptosis.