Pemphigus Vulgaris: Pathomechanisms of a Desmosomal Disease and Protection by Plakophilin-1 Open Access

Tucker, Dana Kirsten (2013)

Permanent URL: https://etd.library.emory.edu/concern/etds/bg257f82k?locale=en
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Abstract

Desmosomes are intercellular junctions that provide strong adhesion between epithelial cells by anchoring keratin intermediate filaments to cell-cell contact sites. Pemphigus vulgaris (PV) is a life-threatening epidermal blistering disease caused by the presence of autoantibodies against the desmosomal cadherin desmoglein 3. PV is characterized by loss of adhesion or acantholysis between the basal and suprabasal layers in the epidermis. Clinically, patients suffer from severe mucosal erosions and epidermal blistering. The mechanism by which PV IgG disrupts desmosomal adhesion is not fully understood. In my dissertation we hypothesize that PV IgG binding to desmoglein 3 promotes desmosome disassembly and perturbs desmosome assembly, thereby causing the loss of cell-cell adhesion. To test this hypothesis, primary human keratinocytes and patient IgG were used to define the morphological, biochemical and functional changes in desmosome adhesion triggered by PV IgG. Our results indicate that desmosome disassembly and the subsequent loss of cell-cell adhesion occurs sequentially in specific phases triggered by desmoglein 3 internalization and degradation. Furthermore, we found that increasing desmoglein 3 biosynthesis counteracts the increase in desmoglein 3 endocytosis and turnover caused by PV IgG. Based on these data we predicted that reinforcing desmosome adhesion by promoting assembly or slowing disassembly of desmosomes will counteract the effects of PV IgG and prevent the loss of adhesion.

We tested this prediction by exogenously expressing the desmosomal component, plakophilin-1, in basal keratinocytes to determine if this would result in desmosomes that are refractory to PV IgG. Plakophilin-1 is a differentially expressed armadillo family protein that stabilizes and increases keratinocyte adhesion by promoting desmosome formation in the suprabasal layers of the epidermis. We found that enhanced expression of plakophilin-1 blunted the effects of PV IgG and prevented the loss of cell-cell adhesion. Furthermore, plakophilin-1 expression transformed desmosome adhesion from a calcium-dependent to a calcium-independent and hyper-adhesive state. Collectively, these results demonstrate that PV IgG binding to Dsg3 triggers a series of keratinocyte responses that result in the loss of desmosomal cell-cell adhesion and that manipulating the adhesive states of desmosomes can block the pathogenic effects of PV IgG on keratinocyte adhesion.

Table of Contents

Table of Contents Page number

Chapter I. General Introduction………………….……...……....………….1-33

Significance and overview………………….…………….………………............................2

Section 1.0 Function and organization of the epidermis…………………........……...3

Section 1.1 Desmosome identification, morphology and molecular

architecture…………......……………………………………………....................................4

Section 1.2 Desmosomal cadherins - Desmogleins and Desmocollins..............…8

Section 1.3 Armadillo Proteins……………………………………………................….....12-22

1.3a Plakoglobin………......…………………………….…………...................................13

1.3b Plakophilins………......………………………………...............................…….......15

1.3c Plakophilin-1……………………………………………….....................................….17

1.3d Plakophilin-2……………………………………………......................................……19

1.3e Plakophilin-3……………………………………………....................................….….20

Section 1.4 Plakin family member - Desmoplakin...............…….……………….……..21

Section 1.5 Regulation of desmosome adhesion…....…..………........………….……..23

1.5a Calcium-dependent alterations of desmosomes……...............……...........24

Section 1.6 Figures……..…………………………….……………….....…........................28-34

Figure 1. Organization of the epidermis…………….......………....................….....28

Figure 2. Desmosome ultrastructure and molecular architecture..............…...29

Figure 3. Protein domains of the major desmosomal components...................31

Figure 4. Differentiation-specific expression patterns of

desmosomal proteins in the epidermis………....... ………..........................….....33

Figure 5. Sub-confluent keratinoctyes possess calcium-dependent

desmosomes….…….....………………….…...…………….....................................…...34

Chapter II. Introduction to Pemphigus Vulgaris…................…………….....35-49

Section 2.0 Introduction to Pemphigus………..………..............…………………….….…36

Section 2.1 Autoantibody profile and the location of blisters in pemphigus.......36

Section 2.2 Epidemiology ………………………………………………….......................….…38

Section 2.3 Diagnosis and Treatment …………………………………......……................39

Section 2.4 Pathomechanisms of PV………………………………………......................40-46

2.4a Steric hindrance model …………………...……………….......……........................41

2.4b Intracellular signaling model……………...……………….......…….......................43

2.4c Pemphigus is a disease of desmosome instability……....…......…................45

Section 2.5 Dissertation hypotheses………….....……………………………….................46

Section 2.6 Figures………………………………………………………….............................48-49

Figure 6. Autoantibody-induced loss of Desmoglein 1 and/or 3

(Dsg1, 3) adhesion and sites of blister formation in

pemphigus foliaceus, mucosal pemphigus vulgaris (PV)

and mucocutaneous pemphigus vulgaris….........…….….…...............................48

Figure 7. Histologic biopsies stained with hematoxylin

and eosin.………........................................................……….......................49

Chapter III. Desmosome Disassembly in Response to Pemphigus

Vulgaris IgG Occurs in Distinct Phases and can be

Reversed by Expression of Exogenous Dsg3…...……..........…................50-92

Section 3.0 Abstract……………………….………………………………...……........................51

Section 3.1 Introduction ………......……………………………………...………....................51

Section 3.2 Results…………………………….……………………………..............................54-61

3.2a Time Course of Desmosome Disassembly in

Response to PV IgG….………………………...……………............................................54

3.2b Non junctional desmoglein 3 is internalized and degraded

before desmosomes are disrupted by PV IgG………….……................................…56

3.2c PV IgG cause rearrangement of cell surface desmoglein 3

into linear arrays and subsequent internalization of

desmoglein 3 from cell-cell junctions….…………………..................................…....58

3.2d Expression of exogenous desmoglein 3 prevents

desmosome disassembly and loss of adhesion….....…..................................……61

Section 3.3 Discussion……………………………………………..........................……......…..62

Section 3.4 Materials and Methods……………………………….......…….……..................66

Section 3.5 Figures…………………………………………….....………….…..........................71-94

Figure 8. Time course of desmosome disassembly in

response to PV IgG.....…..…………...…………….…….............................................71

Figure 9. Desmosomes are disrupted by PV IgG but β-catenin

is minimally affected and cells remain in close

apposition...............................................................................................72

Figure 10. Non-junctional pools of desmoglein 3 are rapidly

internalized after exposure to PV IgG………...………...........................................73

Figure 11. The PV IgG-desmoglein 3 complex reorganizes

into linear arrays that exhibit retrograde movement

before entering vesicular compartments….........……..…....................................75

Figure 12. Linear arrays contain desmosomal but not adherens

junction components….....…………………...…………............................................…77

Figure 13. PV IgG-desmoglein 3 in linear arrays colocalize

with actin and align with keratin filaments………........…....................................79

Figure 14. Actin depolymerization increases PV IgG-induced

desmoglein 3 internalization………………………...............................................…...80

Figure 15. Expression of exogenous Dsg3.GFP prevents

desmoplakin mislocalization and loss of cell

adhesion in PV IgG treated keratinocytes............……..…..................................82

Supplemental Figure 1. PV IgG colocalize with desmoglein 3……..........................84

Supplemental Figure 2. PV IgG decreases steady state

desmoglein 3 protein levels…......................…...............................................85

Supplemental Figure 3. Tracer amounts of the desmoglein 3

monoclonal antibody AK23 do not cause

alterations in desmoglein 3 localization…........................................................86

Supplemental Figure 4. PV IgG and actin depolymerization act

synergistically to decrease steady

state desmosomal protein levels.…….............................................................87

Supplemental Movies 1-5…………………………......…….......…….............................88-89

Section 3.6 Current Perspectives………………………………….......…………....................90

Figure 16. Model: Pemphigus vulgaris is a disease of

desmosome instability…………………...……...………….............................................93

Chapter IV. Plakophilin-1 protects keratinocytes from pemphigus

vulgaris IgG by forming calcium-independent

desmosomes......................................................................................95-126

Section 4.0 Abstract……………..……..………….……………………………...........................96

Section 4.1 Introduction.....….….......……………..…………………………….....................96

Section 4.2 Results ………………..………………………...........……………..................…..99-105

4.2a PKP-1 promotes desmosome formation….……...........…………….....................99

4.2b PKP-1 prevents PV IgG-induced desmosome disruption

and loss of cell-cell adhesion………...............…...........................…………………...100

4.2c PKP-1 clusters desmoglein 3 with desmoplakin……............……...................102

4.2d PKP-1 expression induces the formation of

calcium-independent and hyper-adhesive desmosomes...................................103

Section 4.3 Discussion……………………...........…...…………………………......................104

Section 4.4 Materials and Methods……......…….....….......….…………………..............107

Section 4.5 Figures……………….........….....………….…………...........…............….....112-126

Figure 17. PKP-1 promotes desmosome formation……..........……........................112

Figure 18. PKP-1 protects desmosomal components from

disruption by PV IgG……………..…….....….......………...........................................114

Figure 19. PKP-1 does not prevent anti-desmoglein 3

antibodies from binding to the cell surface….....….....................................…....116

Figure 20. PKP-1 protects desmosome ultrastructure and

keratinocyte adhesion strength from

disruption by PV IgG……....................….………….............................................118

Figure 21. PKP-1 clusters the cytoplasmic tail of

desmoglein 3 with desmoplakin (DP)…....….....…....….......................................120

Figure 22. Desmoglein 3 cytoplasmic sequences mediate

co-localization with desmoplakin (DP) and

differential sensitivity to detergent pre-extraction..........................................122

Figure 23. PKP-1 induces the formation of calcium-

independent, hyper-adhesive desmosomes...........………..................................124

Figure 24. Model of interactions proposed to occur between

PKP-1 and the IL-2R-Dsg3 chimeras….....................……..................................126

Chapter V. Future Directions and Concluding Remarks…………….............127-137

Section 5.0 Future Directions…………………..........…………………………......................128

Section 5.1 Concluding Remarks……………..........………...................……………………..135

Figure 25. Pemphigus vulgaris (PV) disease model and

protection by Plakophilin-1 (PKP-1)………….................…..................................137

References…………........……………..…………....………..........…......................……...138-173

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