Mechanisms of JAM-A Dependent Regulation of Barrier Function Open Access

Monteiro, Ana Carolina (2015)

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The intestinal epithelial barrier is a critical homeostatic component as it selectively controls the passage of nutrients across mucosal surfaces while deterring the passage of pathogens and toxins. Intestinal barrier function is regulated by epithelial tight junctions (TJs), structures that control paracellular permeability. Junctional Adhesion Molecule-A (JAM-A) is a TJ-associated protein that regulates epithelial proliferation, migration and barrier function, however mechanisms linking JAM-A to epithelial permeability are poorly understood. We first report that JAM-A associates directly with ZO-2 and indirectly with afadin, and this complex, along with PDZ-GEF1, activates the small GTPase Rap2c. Supporting a functional link, siRNA-mediated downregulation of the above regulatory proteins resulted in enhanced permeability similar to that observed after JAM-A loss. JAM-A deficient mice and cultured epithelial cells demonstrated enhanced paracellular permeability to large molecules, revealing a potential role of JAM-A in controlling perijunctional actin cytoskeleton in addition to its previously reported role in regulating claudin proteins and small-molecule permeability. Further experiments suggested that JAM-A modulates activity of RhoA and phosphorylation of non-muscle myosin, both implicated in actomyosin contraction. Moreover, previous reports suggest that JAM-A homodimerization on the same cell surface (in cis) mediates JAM-A dependent signaling. However, there is accumulating evidence suggesting that homophilic interactions between JAM-A molecules on adjacent cells (trans) are necessary for mediating cell-cell contacts. Site directed mutagenesis along with cell-transfection and in-vitro protein interaction studies revealed that JAM-A dimerizes in both cis and trans at distinct sites on the distal-most immunoglobulin-like loop. These experiments also indicate that trans-dimerization is a low affinity interaction that likely requires the avidity supplied by JAM-A cis-dimers for assembly. The role of JAM-A trans-dimerization in cell signaling was also investigated. Cells expressing trans-dimerization null mutants displayed lower Rap2 activity compared to controls, and Rap2 activity was enhanced with cell confluence. These results suggest that JAM-A trans-dimerization may act as a barrier-inducing molecular switch that is activated when cells become confluent. Together, the studies in this thesis indicate that JAM-A multimerization may be important in recruiting a large complex of scaffold proteins to cell contacts so as to regulate epithelial barrier function.

Table of Contents

Chapter 1: Intracellular Mediators of JAM-A Dependent Epithelial Barrier Function (1)

Abstract (2)

Introduction (3)

JAM-A expression affects epithelial permeability (4)

Dimerization of JAM-A is necessary for regulation of barrier (5)

Several stimuli that alter epithelial barrier also affect JAM-A expression (8)

Putative signaling effectors downstream of JAM-A that regulate barrier (13)

Possible mechanisms that may differentiate divergent JAM-A signaling modules (21)

Conclusions (25)

Chapter 2: JAM-A associates with ZO-2, Afadin and PDZ-GEF1 to activate Rap2c and regulate epithelial barrier function. (27)

Abstract (28)

Introduction (29)

Results (33)

Discussion (58)

Chapter 3: JAM-A homodimerizes across cells at a distinct motif from JAM-A cis-dimerization to regulate Rap2 activity. (65)

Abstract (66)

Introduction (67)

Results (71)

Discussion (96)

Chapter 4: Discussion (99)

Overview and Physiological Significance (100)

JAM-A expression and dimerization in signaling (103)

Future Directions (107)

Therapeutic Implications (110)

Conclusions (112)

Chapter 5: Experimental Methods (113)

References (125)

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