Dependence of Epithelial Intercellular Junction Biogenesis on Thapsigargin-sensitive Intracellular Calcium Stores

Abstract

Perturbation of potentially regulatable endoplasmic reticulum (ER) calcium stores with the Ca-ATPase inhibitor, thapsigargin (TG), perturbs the formation of desmosomes and tight junctions during polarized epithelial cell biogenesis, despite the development of cell contact. In a Madin-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-dependent 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 associated with the Triton X-100 insoluble cytoskeleton during intercellular junction assembly, prior treatment with 100 nM TG diminished this biochemical stabilization into the detergent-insoluble fraction, particularly in the case of ZO-1. Although spectrofluorimetric measurements in fura-2 loaded Madin-Darby canine kidney cells confirmed the occurrence of TG-mediated release of calcium from internal stores, total cytosolic calcium during junction assembly remained similar to untreated cells. Therefore, the presence of cytosolic calcium alone is not sufficient for normal intercellular junction biogenesis if intracellular 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 early (possibly common) step in polarized epithelial biogenesis.

Junctional Protein Regulation by Sphingosine Kinase 2 Contributes to Blood–Brain Barrier Protection in Hypoxic Preconditioning-Induced Cerebral Ischemic Tolerance

“…Thus, we sought to examine the cortical expression of junctional proteins in triton-insoluble fractions, which yield proteins in direct or indirect association with the cytoskeleton, presumably through linkages at cell junction sites (Stuart et al, 1996;Wang et al, 2011;Wong, 1997). First, we compared baseline expression in wild types and SphK2 knockouts, and then measured expression changes in response to HPC at a time (48 hours post-HPC) coinciding with ischemic onset in our model ( Figures 3A-3D).…”

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

“…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.…”

“…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.…”

“…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).…”