DEPENDENCE OF EPITHELIAL INTERCELLULAR JUNCTION BIOGENESIS ON THAPSIGARGIN-SENSITIVE INTRACELLULAR CALCIUM STORES

Perturbation of potentially regulatable endoplasmic reticulum (ER) cal cium stores with the Ca-ATPase inhibitor, thapsigargin (TG), perturbs the formation of desmosomes and tight junctions during polarized epith elial cell biogenesis, despite the development of cell contact. In Mad in-Darby canine kidney cell model for intercellular junction assembly, TG treatment inhibited the development of transepithelial electrical resistance (TER), a measure of tight junction assembly, in a dose-depe ndent manner. The TG-induced inhibition of tight junction assembly was paralleled by a defect in the sorting of the tight junction protein, ZO-1. An even more dramatic delay in sorting of the desmosomal protein , desmoplakin, was observed in the presence of TG. In addition, while both ZO-1 and desmoplakin-I in control cells were shown to become asso ciated with the Triton X-100 insoluble cytoskeleton during intercellul ar junction assembly, prior treatment with 100 nM TG diminished this b iochemical stabilization into the detergent-insoluble fraction, partic ularly in the case of ZO-1. Although spectrofluorimetric measurements in fura-2 loaded Madin-Darby canine kidney cells confirmed the occurre nce of TG-mediated release of calcium form internal stores, total cyto solic calcium during junction assembly remained similar to untreated c ells. Therefore, the normal intercellular junction biogenesis if intra cellular stores are perturbed by TG. The results indicate the presence of calcium-sensitive intracellular mechanisms involved in the sorting and cytoskeletal stabilization of both tight junction and desmosomes and suggest a role for calcium-dependent signaling pathways at an earl y (possible common) step in polarized epithelial biogenesis.