Claudin Expression in Alveolar Epithelia Influences Barrier Function: The Effect of Extracellular Matrix on Claudin Expression Öffentlichkeit
Findley, MK (2010)
Published
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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