Regulation of tight junction barrier function by phospholipase C Public Deposited

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  • March 21, 2019
  • Klein, Ryan Reaves
    • Affiliation: Eshelman School of Pharmacy
  • The intestinal epithelium presents a formidable barrier to the absorption of orally administered drugs and macromolecules. It is comprised of a monolayer of diverse epithelial cells connected via multi-protein junctional complexes at the apical membrane. The tight junction, the most apical component of the junctional complex, is generally considered to be the major barrier regulating the passage of molecules between adjacent cells and into the systemic circulation. Over the last fifteen years, evidence has emerged supporting a role for phospholipase C (PLC) enzymes, an important class of intracellular signaling molecules, in the regulation of tight junction function; however, unequivocal evidence to support such a role for any PLC isozyme has yet to be established. Studies in this dissertation were intended to develop a clearer understanding of the molecular mechanism involved in the regulation of intestinal epithelial tight junctions via the PLC-catalyzed signal transduction cascade. Specifically, the goal was to explore whether a cause-effect relationship exists between the inhibition of PLC activity and increased paracellular permeability via specific modulation of epithelial tight junctions. The mRNA expression profile of PLC isozymes in Caco-2 cells, an in vitro model for the human intestine, was established and directly compared to the human intestine. Results demonstrated that PLCβ1, PLCβ3, PLCγ1, PLCγ2, PLCδ3, and PLCε were all expressed at the mRNA level in Caco-2 cells. Importantly, each of these isozymes was also detected in all regions of the human small intestine. Further studies were intended to implicate PLC isozymes in the regulation of human intestinal tight junctions by determining the potency of previously reported PLC inhibitors to increase paracellular permeability and inhibit PLC activity in Caco-2 cells. The potency of a series of homologous alkylphosphocholines (APCs) to inhibit PLCβ activity varied forty five fold and correlated significantly with their potency to enhance paracellular permeability, suggesting that inhibition of PLCβ activity is associated with increased paracellular permeability. Further, structurally unrelated PLC inhibitor, U73122, also increased paracellular permeability and inhibited PLCβ activity. In order to establish a cause-effect relationship between PLC inhibition and increased paracellular permeability in epithelial cells, RNA interference was used to suppress the expression of specific PLC isozymes in MDCK cells, and the effect on tight junction function and structure was evaluated. Surprisingly, depletion of PLCβ3 and PLCγ1 (the only isozymes of their respective families detected in these cells), alone or in combination, had no impact on either tight junction assembly or on the barrier function of already formed tight junctions, suggesting that a cause-effect relationship does not exist between inhibition of these PLC families and tight junction function in epithelial cells. These results further implied that putative PLC inhibitors, APCs and U73122, increase paracellular permeability via mechanisms independent of their effects on PLC enzymes. Additional studies established that APCs disrupt apical membrane order at concentrations that also increase paracellular permeability in Caco-2 cells, providing an explanation for the observed effects on tight junction function. Unexpectedly, U73122 was found to increase the activity of hPLCβ3 in a cell free system, rather than inhibit its activity, providing an alternative hypothesis to explain the observed increase in paracellular permeability following treatment of epithelial cells with this compound. This novel interaction between U73122 and PLC was due to alkylation of the protein at cysteine residues by the highly reactive maleimide moiety in U73122. In summary, studies in this dissertation have significantly enhanced the current understanding of the role PLC enzymes play in the regulation of tight junction function, and have raised questions regarding previous reports that inhibition of these enzymes leads to increased paracellular permeability in epithelial cells.
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  • In Copyright
  • Thakker, Dhiren
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  • University of North Carolina at Chapel Hill
  • Open access

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