Abstract
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.
Highlights
Cell Cycle Analysis—To analyze the effects of pemphigus vulgaris (PV) serum on cell cycle progression on keratinocytes, we have incubated synchronized HaCat cells with control serum (30%) and PV serum (30%) for 24 h (Fig. 1, a and b)
The major finding of the present study is that kinase machinery alterations occurring in PV acantholysis crucially involve cyclin-dependent kinase 2 (Cdk2)
We revealed that silencing of Cdk2 was able to reduce disruption of cell-cell contacts in keratinocytes exposed to PV serum
Summary
Morphological changes occurring in PV acantholytic cells have been reproduced both in cultured keratinocytes and in vivo in neonatal mice [4, 5] It is not yet clear whether PV IgG can experimentally mimic the disease on its own, since the action of serum factors other than IgG in PV blistering has been recently emphasized [6, 7]. It was believed that disruption of cell-cell contacts resulted from the simple interaction of PV autoantibodies with Dsgs by steric hindrance [8, 9] Today this model appears untenable, since (a) autoimmunity in PV is not just restricted to adhesion molecules [10], (b) serum factors other than IgG could play a role in PV pathogenesis [11], and (c) a number of signal transduction cascades seem to be crucially involved in acantholysis [12, 13]. The goal of the present study was to investigate the pathogenic molecular events involved in PV
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