CELLULAR AND MOLECULAR TOXICITY OF LEAD

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Previous studies have established that production of osteocalcin, the major non-collagenous, calcium-binding, bone matrix protein is very sensitive to inhibition by Pb2+. Lead (5 to 25 uM) significantly decreased osteocalcin mRNA and secreted protein. These Pb2 concentrations do not inhibit general protein or RNA synthesis. The effects of Pb2+ on osteocalcin production do not appear to be the result of down-regulation of the vitamin D receptor. And, Pb2+ does not perturb post-translational processes, as the osteocalcin does not accumulate inside the osteoblastic cells. The central concept of this application is that lead toxicity in general is mediated via perturbation of the signal transduction processes. The hypothesis to be tested is that lead down-regulates the expression of the osteocalcin gene by perturbing the phosphorylation state of the VDR. Specifically, lead may directly activate PKC beta resulting in phosphorylation of the VDR in the DNA binding domain, thereby reducing VD3-dependent transactivation. In particular, the experimental design is directed at determining the actions of lead on the non-genomic and the genomic functions of VD3 (Aims 1-3 and 4-5 respectively) in the osteoblastic bone cell line (ROS 1712.8). Specific Aims of this proposal are to; 1. Characterize the actions of Pb2+ on the VD3-stimulated rCa2+)i signal. The interactive effects of Pb2 on the generation and clearance of the VD3-induced [Ca2+]i signals in lead intoxicated cells will be determined using an interactive laser cytometer and Ca2+ indicator dyes. 2. Characterize the Pb2+- and VD3-dependent translocation and regulation of protein kinase C and to investigate the effects on PKC and PKA activities using Western blot analysis, fluorescence confocal microscopy and phosphorylation assays. 3. Characterize the biological consequences of lead and VD3 modulated protein kinase activities on phosphorylation of the VDR by measuring the interactive effects of Pb2+ and VD3 on 32P-incorporation into the VDR by SDS-PAGE and autoradiography. 4. Identify and characterize the negative effects of lead on VD3-dependent transcription of the rat osteocalcin gene. A series of deletion constructs of the 5' rat and human osteocalcin genes promoter regions fused to the CAT reporter gene will be used to identify the Pb2+ sensitive site(s) in the 5' flanking region of the osteocalcin gene. Gel shift assays will be used to monitor the direct and indirect actions of Pb2+ on VDR binding to specific VDREs. 5. Distinguish the effects of Pb2+ on the transcriptional activity of the VDR from the effects of Pb2+ on other transcriptional processes. The effect of lead on the transcription rate and integrity of the osteocalcin hRNA will be determined by nuclear run-on and mRNA stability experiments. In summary, this proposed research will use cellular and molecular techniques to elucidate actions of lead on VD3-dependent hormone signal transduction and gene regulatory processes in osteoblastic bone cells. The results and concepts developed from this project will further our understanding of the skeleton as a target of lead toxicity, skeletal metabolism of lead, and the physiological factors which mobilize skeletal lead. Moreover, the results and concepts defining the fundamental actions of lead on cellular and molecular processes derived from this research will enhance our understanding the effects of lead on growth, hypertension, behavior, and learning deficits.