Claudin Expression in Alveolar Epithelia Influences Barrier Function: The Effect of Extracellular Matrix on Claudin Expression Open Access

Findley, MK (2010)

Permanent URL: https://etd.library.emory.edu/concern/etds/9880vr465?locale=en
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Abstract


The lung is the essential respiration organ in all air breathing animals. Lung injury can drastically damage alveolar epithelium, leading to respiratory failure. This respiratory failure is caused by a loss of barrier function of the alveolar epithelia and a subsequent flood of the alveolar space with proteinaceous fluid.

The model I present here hypothesizes that injury to the alveolus stimulates a change in the ECM of the alveolar epithelium. In turn, localization and expression of tight junction proteins found in the alveolar epithelium are significantly altered. These changes in the tight junction are sufficient to alter the barrier function of the alveolus, thus leading to the flooding of the alveolar space.

Following lung injury there is a dramatic alteration of the ECM. This remodeling directly impacts the expression and function of tight junction proteins, leading to either healthy or impaired lung regeneration. In this thesis we hypothesize that although a fibronectin enriched provisional matrix accumulates during acute lung injury and this matrix promotes rapid reformation of alveolar barriers, these barriers are suboptimal as compared with barriers more slowly produced by cells on native matrix.

Our results show that cells grown on a fibronectin matrix, representative of an injury matrix, show a significant change in tight junction protein expression that is correlated with changes in barrier function. We test the hypothesis that altering the expression level of a single claudin is sufficient to induce changes in barrier function. The data in this thesis supports the conclusion that despite the ability to form a rapid barrier after injury, cells on fibronectin ultimately have suboptimal barrier function as compared with cells on native matrix. These results will have an impact on understanding a number of health issues such as chronic alcohol abuse which is known to increase the risk of acute lung injury. Chronic alcohol ingestion promotes fibronectin expression in the lung and is associated with changes of tight junction protein expression. These events lead to a decrease in the barrier function of the alveolus leaving the lung more susceptible to injury and exaggerating the lung's injury response, leading to increased mortality.

Table of Contents

Table of Contents

Chapter 1. Introduction to the Pulmonary System and Lung Injury…………………......1

I. The Pulmonary System…………………………………………………………...........................2
II. Lung Injury…………………………………………………………………….................................5


Chapter 2. Extracellular Matrix………………………………………………………….....................7
I. Laminin…………………………………………………………………………..................................8
II. Collagen………………………………………………………………………..................................9
III. Fibronectin………………………………………………………………….................................10
IV. Wound Healing………………………………………………………………................................11

Chapter 3. Tight Junctions and Claudins ………………………………………………................15
I. Tight Junctions……………………………………………………………….................................16
II. Claudin Superfamily…………………………………………………………...............................17
Members
Claudin tetraspanin structure
Claudin function in paracellular selectivity
III. Claudins Role in Tight Junctions……………………………………………..........................20
Claudin-claudin interactions
Claudin-occludin interactions
Claudin-scaffold protein interactions
IV. Functional Regulation of Claudins…………………………………………...........................23
Internalization of claudins
Palmitoylation of claudins is required for tight junction formation
Phosphorylation of claudins affects paracellular permeability
Transcriptional regulation in epithelial-mesenchymal transformation

V. Implication of Claudins in Disease…………………………………………............................26
Claudins role in cancer suggests functions beyond the regulation of permeability
Claudins are affected by growth factors and cytokines


Chapter 4. Materials and Methods………………………………………………………....................30
I. Coating transwell permeable supports with matrix components……………...............31
II. Isolation and culture of rat type II alveolar epithelial cells…………………...............31
III. Immunofluorescence staining………………………………………………............................32
IV. Immunoblot………………………………………………………………......................................33
V. Barrier function measurements………………………………………………...........................34
Fluorescent Tracers
Basic TER
VI. Using adenovirus tagged-claudins to overexpress specific claudins in alveolar
epithelial cells……………………………………………………………………...................................35


Chapter 5. Extracellular Matrix Influences Epithelial Claudin Expression and Barrier
Function…………………………………………………………………………………...............................37
I. Introduction…………………………………………………………………….................................38
II. Results……………………………………………………………………….....................................41
Effects of extracellular matrix on tight junction protein expression
Effects of extracellular matrix on barrier function


Chapter 6. Targeting Claudin-5 to Manipulate Alveolar Epithelial Barrier Function…...47
I. Introduction……………………………………………………………………..................................48
II. Results.................................................................................................50

Transfected claudins localize to the cell membranes

Claudin-5 adenovirus transfection increases ion permeability

Claudin-5 increases paracellular solute flux

Chapter 7. Discussion.................................................................................54

Referecnes................................................................................................62

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