This document discusses cell junctions and their roles in various biological processes. It defines the main types of cell junctions, including tight junctions, gap junctions, focal adhesions, adherens junctions, and desmosomes. It provides examples of how cell junction gene expression changes during epithelial-to-mesenchymal transition, stem cell differentiation, cancer, atopic dermatitis, and the effects of probiotic bacteria on intestinal barriers. PCR array tools are presented as ways to study cell junctions and related pathways at the gene expression level to better understand their functions in normal and disease states.

1. What’s Your Cell Line Saying?
Cell Junctions & Cell Biology
George J. Quellhorst, Jr., PhD
Associate Director, R&D
Biological Content Development
.
.
.
Sample & Assay Technologies

2. Topics to be Discussed
General Definition of Cell Junctions
Specific Cell Junctions
Who, What, When, Where, and Why
Examples of Gene Expression Changes
Cancer
Atopic Dermatitis
Probiotics Intestinal Barrier
Stem Cell Differentiation
-2-
Sample & Assay Technologies

3. Cell Junctions Definition
Multi-protein Complexes
Connect Neighboring Cells or Cells To ECM Extracellularly
Connect To Cytoskeleton Intracellularly
Especially Important in Epithelial Tissue
General Function
Cellular Adhesion & Cellular Communication
Transduction of Mechanical Force
Molecular Biology of the Cell. 4th edition. Alberts B, Johnson A, Lewis J, et al. New York: Garland Science; 2002.
-3-
Sample & Assay Technologies

4. Cell Junctions Types
Tight Junctions (Occluding Junctions)
Seal adjacent epithelial cells together
Prevent passage of most dissolved molecules, membrane-bound lipids and proteins
between apical and basolateral surfaces
Gap Junctions (Communicating Junctions)
Allow adjacent cell communication; pass ions & small molecules between cytoplasms
Focal Adhesions & Hemidesmosomes
(Anchoring Junctions, Actin & Intermediate Filament Attachment Sites)
Form around integrin-mediated cell–ECM contacts
Focal adhesions connect integrins to actin filaments
Hemidesmosomes connect integrins to intermediate filaments
Adherens Junctions & Desmosomes
(Anchoring Junctions, Actin & Intermediate Filament Attachment Sites)
Form around cadherin-mediated cell–cell contacts
Adherens junctions connect cadherins to actin filaments
Desmosomes connect cadherins to intermediate filaments
-4-
Sample & Assay Technologies

5. Tight Junctions
Location
Blood–Brain Barrier
Blood Vessels
Intestines
Nephrons
Skin
Normal Processes
Immune Cell Extravasation/Diapedesis
Intestinal Absorption
Diseases
Inflammatory Bowel Disease
Epithelial-to-Mesenchymal Transition (EMT)
Components
Claudins & Occludin
Actinins & Catenins
Protein Kinases & G-Proteins
-5-
http://en.wikipedia.org/
Cell Surface Receptors
Intracellular Adaptor Proteins
Cytoskeleton Regulation
Sample & Assay Technologies

6. Gap Junctions
Location
Cardiomyocytes
Keratinocytes
Astrocytes
Endothelial Cells
Smooth Muscle Cells
Normal Processes
Excitable Cell Contraction, Neural Activity
Cellular Growth & Differentiation, Embryonic Development
http://en.wikipedia.org/
Immune Responses, Tissue Homeostasis, Metabolic Transport
Diseases
Cardiovascular Disease
Neurological Disorders
Developmental Abnormalities
Components
Innexins & Connexins
Dimerize to form channels
Receptors, Protein Kinases & G-Proteins
Regulate Connexins
-6-
Sample & Assay Technologies

7. Gap Junction and Connexin Expression in the Heart
Cx40 = GJA5
Cx43 = GJA1
Cx45 = GJC1
Severs NJ, Bruce AF, Dupont E, Rothery S. (2008) Remodelling of gap junctions
and connexin expression in diseased myocardium. Cardiovasc Res. 80:9.
-7-
Sample & Assay Technologies

8. Focal Adhesions & Hemidesmosomes
Desmosome
Location
Epithelial Cells
Normal Processes
Angiogenesis
Anchorage-Dependent Cell Survival
Cell Cycle
Cell Migration
Wound Healing
Diseases
Fibrosis
Epithelial-to-Mesenchymal Transition (EMT)
Components
Integrins
http://en.wikipedia.org/
Actin Filaments & Keratin-Based Intermediate Filaments
Focal Adhesion Kinase (PTK2 or FAK) & Integrin-Linked Kinase (ILK)
PI-3-Kinase/AKT & G-Protein Signaling
Filamin, Vinculin & Talin
-8-
Sample & Assay Technologies

9. Adherens Junctions & Desmosomes
Location
Adhesion belts linking adjacent epithelial cells
Focal contacts on lower surface of cultured fibroblasts
Normal Processes
Intestinal Absorption
Keratinization
Vascular Biology
WNT-Dependent Development
Diseases
Cardiomyopathies
Fibroproliferative Disorders
Polycystic Kidney Disease
Epithelial-To-Mesenchymal Transition (EMT)
Components
http://en.wikipedia.org/
Cadherins
Actin Filaments & Intermediate (Keratin & Desmin) Filaments
Desmocollins, Desmogleins, Nectins, & Notch Proteins
Catenins, Protein Kinases, G-Proteins (Cytoskeleton Regulation)
-9-
Sample & Assay Technologies

10. Epithelial-to-Mesenchymal Transition (EMT)
Alters Cell Junctions
EMT = Process whereby cancer cells leave primary tumor into circulation
Lose epithelial traits and gain mesenchymal stem cell markers
Reverse process at metastatic site
hTERT-immortalized primary prostate cancer cells
Versus
Same cells selected for loss of contact inhibition
Morphology and expression markers consistent with EMT
Migration and invasion assays also consistent
Agilent Whole Human Genome Microarray
Genes
Fold Change
p-value
Genes
Gap Junction
Fold Change
p-value
Tight Junction
GJB3
-87
7.30E-10
CLDN7
-20
1.20E-05
GJB6
-39
5.80E-06
OCLN
-4
4.80E-03
GJB5
-36
6.00E-10
CLDN1
-3
2.50E-02
GJB2
-20
7.70E-07
CLDN4
-2
9.60E-04
GJB4
-17
3.80E-07
Ke X.S. et al. (2008) Epithelial to mesenchymal transition of a primary prostate cell line with
switches of cell adhesion modules but without malignant transformation. PLoS One. 3:e3368.
- 10 -
Sample & Assay Technologies

11. Epithelial-to-Mesenchymal Transition (EMT)
Alters Cell Junctions
Genes
Fold Change
p-value
Genes
Desmosome
Fold Change
p-value
Desmosome
DSG3
-93
7.00E-07
DSC3
-6
7.70E-07
PPL
-39
4.90E-05
PKP2
-6
4.00E-07
PKP3
-19
5.00E-09
DSG2
-2
1.90E-03
JUP
-18
2.90E-07
DSP
-9
1.50E-03
Hemidesmosome
DSC2
-8
2.70E-07
DST
-37
5.10E-08
PKP1
-7
7.20E-06
ITGB4
-11
2.50E-05
Adherens Junction
Focal Adhesion
CDH3
-120
6.50E-08
ITGB4
-11
2.50E-05
CDH1
-24
6.90E-08
ITGB6
-6
2.60E-06
CDH2
-3
1.00E-08
CAV1
-3
9.80E-04
CTNNB1
-3
8.00E-04
PTK2
-2
1.70E-06
CTNND1
-2
1.70E-03
PARVA
2
2.80E-03
4
2.50E-06
ITGA11
4
4.60E-09
PVRL1
Ke X.S. et al. (2008) Epithelial to mesenchymal transition of a primary prostate cell line with
switches of cell adhesion modules but without malignant transformation. PLoS One. 3:e3368.
- 11 -
Sample & Assay Technologies

12. Tight Junction Defects Exist in Atopic Dermatitis
Atopic Dermatitis (AD) OR Psoriasis (PS) Versus Non-Atopic (NA)
Illumina’s BeadChips
Correlation with impaired tight junction function
Trans-Epithelial Electrical Resistance (TEER)
Also by CLDN1 knockdown which also increases keratinocyte proliferation
De Benedetto A et al. (2011) Tight junction defects in patients with atopic dermatitis. J Allergy Clin Immunol. 127:773.
- 12 -
Sample & Assay Technologies

13. Pro-Biotic Bacteria Improve Healthy Intestinal Barriers
Caco-2 treatment of pro-biotic bacteria Lactobacillus plantarum
Increases intestinal barrier function (TEER)
Increases tight junction gene expression
Anderson RC et al. (2010) Lactobacillus plantarum MB452 enhances the function of the intestinal barrier by
increasing the expression levels of genes involved in tight junction formation. BMC Microbiol. 10:316.
- 13 -
Sample & Assay Technologies

14. Cell Junction Gene Expression Changes Drive
ESC Differentiation to Endothelial & Hematopoietic Cells
Expression changes from ESC to mesoderm to terminal differentiation in all junctions
Knockdown CDH1, GJA1, TJP1 favors endothelial over hematopoietic cells
Gene
E-cad
Cldn4
Cldn6
Cx31
Cx43
Cx45
ZO-1
ZO-2
ICAM
Integrin B4
Symbol
Cdh1
Cldn4
Cldn6
Gjb3
Gja1
Gjc1
Tjp1
Tjp2
Icam1
Itgb4
Junction
Adherens
Tight
Tight
Gap
Gap
Gap
Tight, Gap, Adherens
Tight, Gap
Tight
Focal Adhesion
CDH1 GJA1 TJP1 TJP2
T (brachyury)
FLT3/KDR
TAL1
Stankovich BL, Aguayo E, Barragan F, Sharma A, and Pallavicini MG. (2011) Differential adhesion molecule expression
during murine embryonic stem cell commitment to the hematopoietic and endothelial lineages. PLoS One 6:e23810.
- 14 -
Sample & Assay Technologies

15. Summary & Conclusions
Cell junctions present in many cell types
Some cell types predominately contain one junction type
– Cardiomyocytes: Gap Junctions
Some cell types contain multiple junction types
– Epithelial Cells: Tight Junctions, Focal Adhesions & Adherens Junctions
Play role in normal biological and pathophysiological processes
Differentiation
Cancer
Epithelial Layer Function
Usually studied by traditional cell biological techniques
Immunofluorescence, Electron Microscopy
Can also be studied at the gene expression level
Permits analysis of more component genes at the same time
- 15 -
Sample & Assay Technologies

16. Available RT2 Profiler PCR Arrays
Cell Junction PathwayFinder™
Tight Junctions
Gap Junctions
Focal Adhesions
Adherens Junctions
Extracellular Matrix & Cell Adhesion Molecules
Cytoskeleton Regulators
Primary Cilia
Cell Motility
Many other areas of biological research …
- 16 -
Sample & Assay Technologies

17. To help you get started …
Two Free RT2 Profiler PCR Arrays
Both from the same pathway of your choice
With the purchase of master mix and first strand kit reagents
Sales and Technical Questions: SUPPORT@SABIOSCIENCES.COM
Questions about our Webinars: QIAWEBINARS@QIAGEN.COM
- 17 -
Sample & Assay Technologies

18. What’s Your Cell Line Saying?
Cell Junctions & Cell Biology
Q&A
George J. Quellhorst, Jr., PhD
Associate Director, R&D
Biological Content Development
.
.
.
Sample & Assay Technologies