Galectin-3 and Annexin A2 regulate the stability of cell surface proteins to control epithelial barrier function. Open Access

Rankin, Carl (2014)

Permanent URL: https://etd.library.emory.edu/concern/etds/x920fx57q?locale=en
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Abstract

The gastrointestinal epithelium functions as an important barrier that separates luminal contents from underlying tissue compartments thereby maintaining mucosal homeostasis. Mucosal wounds in inflammatory disorders compromise the critical epithelial barrier. In response to injury, intestinal epithelial cells (IECs) remodel cell matrix adhesions and intercellular junctions as they proliferate and migrate to cover denuded surfaces. AnnexinA2 (AnxA2) and Desmoglein-2 (Dsg2) proteins have been reported to regulate wound closure and epithelial barrier function. In Chapter 2 I identify a mechanism by which AnxA2 controls IEC migration and wound closure. These studies revealed that AnxA2 controls IEC migration by regulating endocytosis of β1 integrin and turnover of cell-matrix adhesions. In Chapter 3 I identify the association of a glycan binding protein, Galectin-3, with an intercellular junction protein, Dsg2, in intestinal epithelial cells. Galectin-3 stabilizes Dsg2 at the cell surface thereby influencing intercellular adhesion and intestinal epithelial barrier function.

Table of Contents

Table of Contents

Chapter 1: Molecular mechanisms of intestinal epithelial barrier regulation and repair.................................................................................................................1

1.1 Organization and function of the intestinal epithelium...........................................2

1.2 Intestinal epithelial wound closure ....................................................................2

1.3 Annexin A2 ....................................................................................................3

1.4 Intestinal epithelial cell migration ......................................................................3

1.5 Intestinal epithelial intercellular adhesion and desmosomes ..................................5

1.6 Galectin-3.......................................................................................................7

Chapter 2: Annexin A2 regulates β1 integrin internalization and intestinal epithelial cell migration ...................................................................................................21

2.1 Introduction...................................................................................................22

2.2 Materials and methods.....................................................................................24

2.3 Results...........................................................................................................27

2.3.1 AnxA2 promotes epithelial cell migration and wound closure

2.3.2 Down-regulation of AnxA2 increases cell-matrix adhesion

2.3.3 Cell surface β1 integrin stability is increased in cells with down- regulated AnxA2

2.3.4 AnxA2 promotes β1 integrin localization in endosomes

2.3.5 β1 integrin internalization is delayed in IECs with down-regulated AnxA2

2.4 Discussion........................................................................................................33

Chapter 3: Galectin-3 regulates desmoglein-2 and intestinal epithelial intercellular adhesion............................................................................................................................53

3.1 Introduction......................................................................................................54

3.2 Materials and methods.....................................................................................56

3.3 Results..............................................................................................................60

3.3.1 Gal3 associates with Dsg2 in a glycosylation dependent manner

3.3.2 Intercellular adhesion is controlled by Gal3

3.3.3 Dsg2 protein stability is influenced by Gal3

3.3.4 Inhibition of Gal3 in vivo decreases intestinal epithelial Dsg2 protein

3.4 Discussion........................................................................................................63

Chapter 4: Summary and future directions..................................................................77

4.1 Summary - AnxA2 regulation of endocytosis.............................................................78

4.2 Future directions - AnxA2 regulation of endocytosis..................................................79

4.3 Summary - Gal-3 regulation of Dsg2..........................................................................81

4.4 Future directions - Gal-3 mediated stabilization of Dsg2............................................82

References.........................................................................................................................88

Figures and Tables

Figure 1: The structure of the intestinal mucosa..................................................................9

Figure 2: Intestinal epithelial wound closure.....................................................................11

Figure 3: Mechanism of epithelial cell migration..............................................................13

Figure 4: Cell-matrix adhesions.........................................................................................15

Figure 5: Endocytic recycling of integrins.........................................................................17

Figure 6: The molecular composition of an IEC desmosome............................................19

Figure 7: AnxA2 down-regulation inhibits epithelial cell migration and wound closure................................................................................................................................37

Figure 8: Down-regulation of AnxA2 increases cell-matrix adhesion..............................39

Figure 9: Cell surface β1 integrin stability is increased following loss of AnxA2............41

Figure 10: AnxA2 promotes β1 integrin localization in early endosomes........................43

Figure 11: Down-regulation of AnxA2 delays β1 integrin internalization........................45

Figure 12: Proposed model for Annexin A2 mediated endocytosis of β1 integrin...........47

Figure 13: Gal3 associates with Dsg2 in a glycosylation dependent manner....................67

Figure 14: Intercellular adhesion is controlled by Gal3.....................................................69

Figure 15: Dsg2 protein stability is influenced by Gal3....................................................71

Figure 16: Inhibition of Gal3 in vivo decreases intestinal epithelial Dsg2 protein...........73

Figure 17: A proposed model for Gal3 lattice regulation of Dsg2 protein........................75

Figure 18: Comparison of the models generated in Chapters 2 and 3...............................84

Figure 19: AnxA2 regulates occludin but not JAM-A.......................................................86

Supplemental Figure 1.......................................................................................................49

Table 1...............................................................................................................................51

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