Sample & Assay Technologies

What’s Your Cell Line Saying?

Cell Junctions & Cell Biology

.George J. Quellhorst, Jr., PhD.Associate Director, R&D

.Biological Content Development

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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

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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.

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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

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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 Cell Surface Receptors� Actinins & Catenins Intracellular Adaptor Proteins� Protein Kinases & G-Proteins Cytoskeleton Regulation

http://en.wikipedia.org/

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Gap Junctions

� Location� Cardiomyocytes� Keratinocytes� Astrocytes� Endothelial Cells� Smooth Muscle Cells

� Normal Processes� Excitable Cell Contraction, Neural Activity� Cellular Growth & Differentiation, Embryonic Development� 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

http://en.wikipedia.org/

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Gap Junction and Connexin Expression in the Heart

Severs NJ, Bruce AF, Dupont E, Rothery S. (2008) Remodelling of gap junctions and connexin expression in diseased myocardium. Cardiovasc Res. 80:9.

Cx40 = GJA5Cx43 = GJA1Cx45 = GJC1

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Focal Adhesions & Hemidesmosomes

� 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� 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

http://en.wikipedia.org/

Desmosome

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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� Cadherins� Actin Filaments & Intermediate (Keratin & Desmin) Filaments� Desmocollins, Desmogleins, Nectins, & Notch Proteins� Catenins, Protein Kinases, G-Proteins (Cytoskeleton Regulation)

http://en.wikipedia.org/

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Epithelial-to-Mesenchymal Transition (EMT)Alters Cell Junctions

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.

Genes Fold Change p-value Genes Fold Change p-value

Gap Junction 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

� 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 cellsVersus

� 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

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Epithelial-to-Mesenchymal Transition (EMT)Alters Cell Junctions

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.

Genes Fold Change p-value Genes Fold Change p-value

Desmosome 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

PVRL1 4 2.50E-06 ITGA11 4 4.60E-09

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Tight Junction Defects Exist in Atopic Dermatitis

De Benedetto A et al. (2011) Tight junction defects in patients with atopic dermatitis. J Allergy Clin Immunol. 127:773.

� 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

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Pro-Biotic Bacteria Improve Healthy Intestinal Barr iers

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.

� Caco-2 treatment of pro-biotic bacteria Lactobacillus plantarum� Increases intestinal barrier function (TEER)� Increases tight junction gene expression

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Cell Junction Gene Expression Changes DriveESC Differentiation to Endothelial & Hematopoietic Cells

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.

CDH1 GJA1 TJP1 TJP2

T (brachyury)FLT3/KDRTAL1

� Expression changes from ESC to mesoderm to terminal differentiation in all junctions� Knockdown CDH1, GJA1, TJP1 favors endothelial over hematopoietic cells

Gene Symbol JunctionE-cad Cdh1 AdherensCldn4 Cldn4 TightCldn6 Cldn6 TightCx31 Gjb3 GapCx43 Gja1 GapCx45 Gjc1 GapZO-1 Tjp1 Tight, Gap, AdherensZO-2 Tjp2 Tight, GapICAM Icam1 TightIntegrin B4 Itgb4 Focal Adhesion

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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

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Available RT 2 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 …

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To help you get started …

Two Free RT2 Profiler PCR ArraysBoth from the same pathway of your choiceWith the purchase of master mix and first strand kit reagents

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Sample & Assay Technologies

What’s Your Cell Line Saying?Cell Junctions & Cell Biology

.George J. Quellhorst, Jr., PhD.Associate Director, R&D

.Biological Content Development

Q&A