Pemphigus Vulgaris: Pathomechanisms of a Desmosomal Disease and Protection by Plakophilin-1 Open Access
Tucker, Dana Kirsten (2013)
Published
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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