Abstract
The majority of pemphigus vulgaris (PV) patients suffer from a live-threatening loss of intercellular adhesion between keratinocytes (acantholysis). The disease is caused by auto-antibodies that bind to desmosomal cadherins desmoglein (Dsg) 3 or Dsg3 and Dsg1 in mucous membranes and skin. A currently unresolved controversy in PV is whether apoptosis is involved in the pathogenic process. The objective of this study was to perform preclinical studies to investigate apoptotic pathway activation in PV pathogenesis with the goal to assess its potential for clinical therapy. For this purpose, we investigated mouse and human skin keratinocyte cultures treated with PV antibodies (the experimental Dsg3 monospecific antibody AK23 or PV patients IgG), PV mouse models (passive transfer of AK23 or PVIgG into adult and neonatal mice) as well as PV patients’ biopsies (n=6). A combination of TUNEL assay, analyses of membrane integrity, early apoptotic markers such as cleaved poly-ADP-ribose polymerase (PARP) and the collapse of actin cytoskeleton failed to provide evidence for apoptosis in PV pathogenesis. However, the in vitro and in vivo PV models, allowing to monitor progression of lesion formation, revealed an early, transient and low-level caspase-3 activation. Pharmacological inhibition confirmed the functional implication of caspase-3 in major events in PV such as shedding of Dsg3, keratin retraction, proliferation including c-Myc induction, p38MAPK activation and acantholysis. Together, these data identify low-level caspase-3 activation downstream of disrupted Dsg3 trans- or cis-adhesion as a major event in PV pathogenesis that is non-synonymous with apoptosis and represents, unlike apoptotic components, a promising target for clinical therapy. At a broader level, these results posit that an impairment of adhesive functions in concert with low-level, non-lethal caspase-3 activation can evoke profound cellular changes which may be of relevance for other diseases including cancer.
Highlights
Pemphigus vulgaris (PV) is a severe autoimmune blistering disease affecting the epidermis, hair follicles and mucous membranes [1,2,3]
Cultured mouse keratinocytes treated for 48 hours with the pathogenic antibody AK23 or mouse (m)IgG as negative control were screened for TUNEL positive cells concomitantly with loss of intercellular adhesion (S1A Fig.)
As reported for human HaCat keratinocytes treated with PVIgG [35], no TUNEL positive cells were observed under our standard conditions (20 μg/ml AK23) and even when the AK23 concentration was elevated by fourfold (Fig. 1A shows 80 μg/ml)
Summary
Pemphigus vulgaris (PV) is a severe autoimmune blistering disease affecting the epidermis, hair follicles and mucous membranes [1,2,3]. It characteristically manifests as loss of intercellular adhesion (acantholysis) between basal and suprabasal keratinocytes, where desmoglein 3 (Dsg3), the major antigenic target in PV, is most abundantly expressed [4,5]. Dsg and Dsg are desmosomal cadherins and adhesive components of desmosomes These robust intercellular adhesion structures confer mechanical resistance to a variety of tissues including skin. Depending on its level of activation, caspase-3 has been proposed as a “stress intensity sensor” acting as a switch between cell survival and death [20]
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