This proposal aims to explore the mechanisms of tight junction protein disassembly/reassembly during ATP depletion/repletion. Loss of tight junction integrity is one of the critical components of renal epithelial cell injury produced by ischemia. Previously published work from the Principal Investigator's laboratory has explored the tight junction proteins in the calcium switch model. In this model, tight junction disassembly was associated with enhanced solubility of tight junction proteins (ZO-1, ZO-2, cingulin and occludin), and reassembly was dependent on PKC activity and intracellular calcium mobilization. The present proposal aims to examine similar mechanisms during chemical ATP depletion and during the recovery from ATP depletion. There are two specific aims. First, the signaling mechanisms involved in tight junction reassembly during ATP repletion after short-term (<1hour) ATP depletion will be explored. The model systems are cultured MDCK and LLCPK-1 cells, treated with antimycin A or 2-Dexoyglucose. Reproducible ATP depletion and loss of transepithelial cell electrical resistance are reproducibly observed under these conditions. Tight junction proteins that will be examined are ZO-1, ZO-2, cingulin and occludin. The preliminary data provided show that ATP depletion is associated with immunocytochemically evident rearrangements in tight junction protein organization, association of the tight junction proteins with fodrin and formation of detergent insoluble macromolecular complexes. Evidence is presented implicating PKC, PI3kinase, heterotrimeric G-protein, and tyrosine kinase activities in the process of tight junction reassembly after the short-term ATP depletion. The first specific aim is to more fully define the signaling events that mediate tight junction reassembly. The research plan is to measure PKC, PI3kinase, and G-protein activity as a function of time after ATP repletion, and to determine whether movement of the tight junction proteins from the macromolecular weight complexes to the tight junction, as well as their phosphorylation state is dependent on these cell signaling components. The dependence of the tight junction reassembly on various signaling pathways will be studied with a number of relatively specific inhibitors, and, in the case of G-proteins, by expression dominant negative G-proteins. The second specific aim will concern itself with tight junction protein degradation, biosynthesis and assembly in cells recovering from long-term ATP depletion. The preliminary data show that transepithelial electrical resistance recovers even after prolonged ATP depletion in MDCK cells, though there is significant degradation of some tight junction proteins (occludin and ZO-2). The reassembly of tight junctions after prolonged ATP depletion is at least in part dependent on new protein synthesis. The second specific aim therefore is designed to establish whether the tight junction proteins are degraded through the ubiquitin-proteasome vs. lysosomal pathway, and whether new protein synthesis is induced after ATP repletion. The fate of pre-existing vs. newly synthesized tight junction proteins within the cell, and their respective contribution to the reassembled tight junction will be studied. The Principal Investigator first plans to establish the time-course for tight junction protein degradation, determine whether the proteins associate with ubiquitin, and whether their degradation can be blocked by inhibitors of proteasome or lysosomal degradation pathways. He plans to use Northern blots to establish whether mRNA levels for each of the tight junction proteins increases after ATP repletion, and will perform in situ hybridization studies in ischemic rat kidneys to determine the location and intensity of tight junction protein mRNA expression. He will establish in which pool pre-existing and newly synthesized tight junction proteins are found as a function of time after ATP repletion. For these studies, he will use a """"""""pulse chase"""""""" approach in cells are synthetically labeled with 35S methionine prior to ATP depletion, while the time-course of ATP repletion is performed without tracer. He will then determine total vs. radiolabeled tight junction protein in immunoprecipitates from macromolecular complex, the extractable fraction, and in the cell-surface pool.
