Desmoglein Versus Non-desmoglein Signaling in Pemphigus Acantholysis

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115

A loss of epidermal cohesion in pemphigus vulgaris (PV) results from autoantibody action on keratinocytes (KCs) activating the signaling kinases and executioner caspases that damage KCs, causing their shrinkage, detachment from neighboring cells, and rounding up (apoptolysis). In this study, we found that PV antibody binding leads to activation of epidermal growth factor receptor kinase, Src, p38 MAPK, and JNK in KCs with time pattern variations from patient to patient. Both extrinsic and intrinsic apoptotic pathways were also activated. Although Fas ligand neutralizing antibody could inhibit the former pathway, the mechanism of activation of the latter remained unknown. PV antibodies increased cytochrome c release, suggesting damage to mitochondria. The immunoblotting experiments revealed penetration of PVIgG into the subcellular mitochondrial fraction. The antimitochondrial antibodies from different PV patients recognized distinct combinations of antigens with apparent molecular sizes of 25, 30, 35, 57, 60, and 100 kDa. Antimitochondrial antibodies were pathogenic because their absorption abolished the ability of PVIgG to cause keratinocyte detachment both in vitro and in vivo. The downstream signaling of antimitochondrial antibodies involved JNK and late p38 MAPK activation, whereas the signaling of anti-desmoglein 3 (Dsg3) antibody involved JNK and biphasic p38 MAPK activation. Using KCs grown from Dsg3؊/؊ mice, we determined that Dsg3 did not serve as a surrogate antigen allowing antimitochondrial antibodies to enter KCs. The PVIgG-induced activation of epidermal growth factor receptor and Src was affected neither in Dsg3KCs nor due to absorption of antimitochondrial antibodies. These results demonstrated that apoptolysis in PV is a complex process initiated by at least three classes of autoantibodies directed against desmosomal, mitochondrial, and other keratinocyte self-antigens. These autoantibodies synergize with the proapoptotic serum and tissue factors to trigger both extrinsic and intrinsic pathways of cell death and break the epidermal cohesion, leading to blisters. Further elucidation of the primary signaling events downstream of PV autoantigens will be crucial for the development of a more successful therapy for PV patients.Pemphigus vulgaris (PV) 3 is an IgG autoantibody-mediated disease of skin and mucosa caused by a loss of epidermal cohesion and manifested by progressive blistering and non-healing erosions. The mechanism of detachment of keratinocytes (KCs) in PV, termed acantholysis, is a subject of intensive research. Elucidation of the pathophysiologic mechanism of acantholysis should facilitate development of novel pharmacologic approaches to prevent and treat blistering without systemic corticosteroids, a mainstay of treatment of PV patients. Acantholysis can be blocked both by inhibitors of signaling kinases, such as p38 MAPK (3), mammalian target of rapamycin (4), Src, and epidermal growth factor receptor (EGFR) kinase (2, 4 -7) and by other tyrosine kinases, phospholipase C, calmodulin...

Section: Discussionsupporting

confidence: 80%

Antimitochondrial Autoantibodies in Pemphigus Vulgaris

Marchenko

Chernyavsky

Arredondo

et al. 2010

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115

“…Interestingly, we found in the present study an up-regulation (calcium channel, voltage-dependent, L type, ␣1D subunit) and down-regulation (gap junction membrane channel protein ␣1) of genes involved in calcium transport. In addition, cell signaling induced by the nondesmoglein autoantibodies produced by PV patients that may target keratinocyte acetylcholine receptors modulates Src and EGF receptor kinase, which have been shown to play an important role in regulation of intracellular adhesion (50). Moreover, we have found recently that PV IgG can recognize an antigen other than desmoglein 3 on peripheral blood mononuclear cell surface (25).…”

Section: Discussionmentioning

confidence: 81%

Evidence of Key Role of Cdk2 Overexpression in Pemphigus Vulgaris

Lanza

Cirillo

Rossiello

et al. 2008

The pathogenesis of pemphigus vulgaris (PV) is still poorly understood. Autoantibodies present in PV patients can promote detrimental effects by triggering altered transduction of signals, which results in a final acantholysis. To investigate mechanisms involved in PV, cultured keratinocytes were treated with PV serum. PV sera were able to promote the cell cycle progression, inducing the accumulation of cyclin-dependent kinase 2 (Cdk2). Microarray analysis on keratinocytes detected that PV serum induced important changes in genes coding for one and the same proteins with known biological functions involved in PV disease (560 differentially expressed genes were identified). Then, we used two different approaches to investigate the role of Cdk2. First, small interfering RNA depletion of Cdk2 prevented cell-cell detachment induced by PV sera. Second, pharmacological inhibition of Cdk2 activity through roscovitine prevented blister formation and acantholysis in the mouse model of the disease. In vivo PV serum was found to alter multiple different pathways by microarray analysis (1463 differentially expressed genes were identified). Major changes in gene expression induced by roscovitine were studied through comparison of effects of PV serum alone and in association with roscovitine. The most significantly enriched pathways were cell communication, gap junction, focal adhesion, adherens junction, and tight junction. Our data indicate that major Cdk2-dependent multiple gene regulatory events are present in PV. This alteration may influence the evolution of PV and its therapy.

“…Activation of EGFR due to binding of PV IgG to KCs is followed by phosphorylation of its downstream substrates, the MAPK ERK and the transcription factor c-Jun (18). Although both Src and EGFR kinases contribute to cell shrinkage and keratin filament aggregation, the peak of Src activity precedes that of EGFR kinase (17), indicating that engagement of Src/EGFR may be a key step that relays the signal emanating from interaction of PV IgG with various self-antigens on the keratinocyte plasma membrane to the pathway affecting adhesion complexes. Activation of PKC is also one of the earliest events of PV IgG-induced acantholysis (19).…”

mentioning

confidence: 99%

“…We have previously demonstrated that PV IgG increases the level of phosphorylation of E-cadherin by 42%, ␤-catenin by 37%, ␥-catenin by 136%, and Dsg 3 by 300% (14). Acantholysis caused by pemphigus antibodies can be blocked by inhibitors of protein kinase C (PKC), Src, p38 mitogen-activated protein kinase, epidermal growth factor receptor (EGFR) kinase, other tyrosine kinases and calmodulin (15)(16)(17). Activation of EGFR due to binding of PV IgG to KCs is followed by phosphorylation of its downstream substrates, the MAPK ERK and the transcription factor c-Jun (18).…”

mentioning

confidence: 99%

“…20). Recently, it has been demonstrated that gene silencing of Dsg 1 and/or Dsg 3 affects the ability of KCs neither to maintain a polygonal shape and form intercellular junctions nor respond to PV IgG by elevation of the activities of Src and EGFR kinase (17). This suggested that Dsg-dependent signaling is a secondary, or tertiary, pathway mediating processing and utilization of internalized Dsg molecules rather than a primary downstream signaling emanating from the cell membrane of KCs due to PV IgG binding.…”

mentioning

confidence: 99%

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Differential Coupling of M1 Muscarinic and α7 Nicotinic Receptors to Inhibition of Pemphigus Acantholysis

Chernyavsky

Arredondo

Piser

et al. 2008

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The mechanisms mediating and regulating assembly and disassembly of intercellular junctions is a subject of intensive research. The IgG autoantibodies produced in patients with the immunoblistering skin disease pemphigus vulgaris (PV) can induce keratinocyte (KC) dyshesion (acantholysis) via mechanisms that involve signaling kinases targeting intercellular adhesion molecules, thus providing a useful model to study the physiologic regulation of KC cohesion. Previous studies showed that activation of Src and protein kinase C are the earliest events in the PV IgG-induced intracellular phosphorylation cascades and that cholinergic agonists are effective for treating patients with pemphigus. In this study, we sought to elucidate the molecular mechanisms allowing cholinergic agonists to inhibit PV IgG-induced acantholysis and phosphorylation of KC adhesion molecules. The extent of acantholysis in KC monolayers correlated closely with the degree of PV IgG-induced phosphorylation of p120-and ␤-catenins, with classic isoforms of protein kinase C mediating serine phosphorylation of ␤-catenin and Src-tyrosine phosphorylation of p120-catenin. The M 1 muscarinic agonist pilocarpine blocked phosphorylation of both catenins, which could be abolised by the M 1 antagonist MT7. The ␣7 nicotinic agonist AR-R17779 inhibited phosphorylation of P120-cateinin. The ␣7 antagonist methyllycaconitine abolished the effect of AR-R17779. Okadaic acid abrogated protective effects of agonists on phosphorylation of ␤-catenin, and pervanadate, on that of p120-catenin. Stimulation of KCs with pilocarpine significantly (p < 0.05) elevated both serine/threonine and tyrosine phosphatase activities in KCs. AR-R17779 both stimulated tyrosine phosphatase and decreased PV IgGinduced Src activity. Methyllycaconitine released Src activity in intact KCs and caused acantholysis. Thus, downstream signaling from M 1 abolished PV IgG-dependent catenin phosphorylation due to activation of both serine/threonine and tyrosine phosphatases, whereas ␣7 action involved both activation of tyrosine phosphatase and inhibition of Src. These findings identified novel paradigm of regulation of signaling kinases associated with cholinergic receptors and provided mechanistic explanation of therapeutic activity of cholinomimetics in PV patients.