Critical role for intracellular calcium in tight junction biogenesis

Abstract

Using the Madin Darby canine kidney (MDCK) cell "calcium switch," we have previously demonstrated that, as MDCK cells establish contact and ultimately form tight junctions, there are marked global and localized changes in intracellular calcium at the sites of cell-cell contact (Nigam et al., 1992, Proc. Natl. Acad. Sci. USA, 89:6162-6166). We have now examined whether intracellular Ca++ is critical to the biogenesis of tight junctions by chelating this ion and monitoring the formation of junctions by electrical, immunocytochemical, and biochemical criteria. Intracellular Ca++ was chelated with the cell-permeant chelators, dimethyl-BAPTA-AM and BAPTA-AM. By digital imaging of fura-2 loaded cells, it was demonstrated that both agents efficiently chelated Ca++ during the "switch" in a dose-dependent manner which paralleled their respective in vitro affinities for Ca++. Chelation of Ca++ during the switch markedly attenuated the development of transepithelial electrical resistance (TER), a measure of tight junction assembly. Immunofluorescent staining of the tight junctional protein, zonula occludens-1 (ZO-1), revealed that chelation of intracellular Ca++ retarded the movement of ZO-1 from intracellular sites to the plasma membrane during the switch. During the development of tight junctions, a fraction of ZO-1 redistributed from the Triton X-100 soluble to the Triton X-100 insoluble pool; chelation of Ca++ during the induction of cell-cell contact prevented this stabilization into the Triton X-100 insoluble fraction. Taken together, these data indicate an important role for intracellular Ca++ in tight junction biogenesis and suggest a specific role for calcium in the early sorting and possible cytoskeletal association of tight junction components.

Tight Junction Proteins Form Large Complexes and Associate with the Cytoskeleton in an ATP Depletion Model for Reversible Junction Assembly

“…TER was measured at various times after treatment with metabolic inhibitors with a Millipore ERS electrical resistance system, as described previously (8,18,19). Measurements are expressed as a percent of the initial value after subtraction of background readings.…”

“…These lesions have been reproduced in cell culture models for hypoxia-reoxygenation injury using agents that deplete cellular ATP, which has allowed for the analysis of molecular mechanisms underlying ischemic injury (15, 16). Although mechanistic insights into the disruption of the actin-based cytoskeleton are beginning to emerge, little is known about the biochemical basis of the disruption of the TJ after ischemic insult or how the TJ reassembles during recovery of epithelial cells from ischemic injury.The biochemical basis of the disassembly and reassembly of the TJ has, however, been studied in MDCK cells in a model in which extracellular calcium is manipulated: the "calcium switch" (8,(17)(18)(19)(20). When the TJ disassembles in this model under low calcium conditions, transepithelial electrical resistance (TER) is lost, and TJ proteins internalize or are diffusely distributed near the cell surface.…”

“…When extracellular calcium is raised, reassembly of the TJ appears to proceed by classical signaling pathways involving a heterotrimeric G protein, regulated intracellular calcium stores (18 -21), and protein kinase C (8). TJ proteins resort to the apico-lateral surface of the plasma membrane, TER develops, and TJ proteins become more resistant to detergent-salt extractions (8,18,19).Superficially, what is known about the behavior of the TJ in MDCK monolayers after ATP depletion and repletion resembles the disassembly of the TJ in low calcium conditions and its reassembly when external calcium is raised. However, in cell culture models for hypoxia-reoxygenation, these issues have not been examined in the same biochemical detail as with low extracellular calcium and the calcium switch.…”

“…The biochemical basis of the disassembly and reassembly of the TJ has, however, been studied in MDCK cells in a model in which extracellular calcium is manipulated: the "calcium switch" (8,(17)(18)(19)(20). When the TJ disassembles in this model under low calcium conditions, transepithelial electrical resistance (TER) is lost, and TJ proteins internalize or are diffusely distributed near the cell surface.…”

“…When extracellular calcium is raised, reassembly of the TJ appears to proceed by classical signaling pathways involving a heterotrimeric G protein, regulated intracellular calcium stores (18 -21), and protein kinase C (8). TJ proteins resort to the apico-lateral surface of the plasma membrane, TER develops, and TJ proteins become more resistant to detergent-salt extractions (8,18,19).…”