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Preclinical studies identify non-apoptotic low-level caspase-3 as therapeutic target in pemphigus vulgaris.

Luyet C, Schulze K, Sayar BS, Howald D, Müller EJ, Galichet A - PLoS ONE (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Molecular Dermatology, Institute of Animal Pathology, Vetsuisse Faculty, University of Bern, Bern, Switzerland; DermFocus, Vetsuisse Faculty, University of Bern, Bern, Switzerland.

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.

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Absence of hallmarks for apoptosis in AK23-treated keratinocytes and 8-week-old mice.(A) Representative immunofluorescence micrographs for F-actin in mouse and human keratinocytes treated for 48 and 24 hours, respectively, with 20, 80 and 160 μg/ml AK23/mIgG or 20 μg/ml mitomycin C (mitC); shown are 160 μg/ml AK23/mIgG. Nuclei were counterstained with Hoechst 33258; arrows point to actin rearrangement; bar = 25μm, (n = 2/group in duplicates and 1’000 cells/group evaluated). (B-E) Representative immunoblots and graphs of Triton X-100-soluble proteins probed with indicated antibodies from B, D, mouse and human keratinocytes treated for 48 hours and 24 hours, respectively, with 20, 80 and 160 μg/ml AK23/mIgG or 20 μg/ml mitomycin C; shown are 20μg/ml (mouse cells) and 80μg/ml (human cells) AK23/mIgG; (n = 5/group); C, E, skin of 8-week-old mice injected with 12.5 μg/g b.w. AK23/mIgG for B, 30 min, 2, 24 and 48 hours and E, 30 min, 2, 5, 24 and 48 hours; shown are indicated time points; (n = 4/group). Signals were quantified, normalized to tubulin and the mean±SEM is plotted relative to mIgG set as 1.
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pone.0119809.g002: Absence of hallmarks for apoptosis in AK23-treated keratinocytes and 8-week-old mice.(A) Representative immunofluorescence micrographs for F-actin in mouse and human keratinocytes treated for 48 and 24 hours, respectively, with 20, 80 and 160 μg/ml AK23/mIgG or 20 μg/ml mitomycin C (mitC); shown are 160 μg/ml AK23/mIgG. Nuclei were counterstained with Hoechst 33258; arrows point to actin rearrangement; bar = 25μm, (n = 2/group in duplicates and 1’000 cells/group evaluated). (B-E) Representative immunoblots and graphs of Triton X-100-soluble proteins probed with indicated antibodies from B, D, mouse and human keratinocytes treated for 48 hours and 24 hours, respectively, with 20, 80 and 160 μg/ml AK23/mIgG or 20 μg/ml mitomycin C; shown are 20μg/ml (mouse cells) and 80μg/ml (human cells) AK23/mIgG; (n = 5/group); C, E, skin of 8-week-old mice injected with 12.5 μg/g b.w. AK23/mIgG for B, 30 min, 2, 24 and 48 hours and E, 30 min, 2, 5, 24 and 48 hours; shown are indicated time points; (n = 4/group). Signals were quantified, normalized to tubulin and the mean±SEM is plotted relative to mIgG set as 1.

Mentions: We also addressed occurrence of apoptosis using complementary read-outs. Cytoskeletal actin is a major target for destruction during the apoptotic process [53,54]. Consistently, mitomycin C-treated mouse keratinocytes exhibited a complete collapse of the actin cytoskeleton (Fig. 2A). In contrast, subtle changes, comparable with the retraction of actin bundles from cell boarders, reported in PV during Dsg3 endocytosis [55], were observed after 48 hours in mouse keratinocytes treated with 20, 80 or 160 μg/ml AK23 (Fig. 2A shows 160 μg/ml AK23). We also investigated primary human keratinocytes treated with AK23 under conditions of loss of adhesion (Fig. 2A; S1B Fig.) as well as mouse keratinocytes incubated with PVIgG from two different patients (containing Dsg3 [23] and Dsg3/Dsg1 antibodies, respectively) (S1C Fig.). In all cases (analyzing 1’000 cells per well in duplicates), no global actin collapse comparable to mitomycin C-treated apoptotic cells was observed. Under the same conditions we also explored the steady-state levels of full-length and cleaved poly ADP-ribose polymerase (PARP), a DNA repair enzyme that is specifically inactivated through cleavage in the early course of apoptotic cell death [54]. No evidence was obtained for reduced full-length PARP (indicative for cleavage) or increased cleaved PARP in mouse and human keratinocytes treated with AK23 (Fig. 2B). As a positive control, mitomycin C treatment resulted in complete conversion of full-length PARP to cleaved/inactivated PARP. No significant decrease in full-length PARP and no cleaved PARP were further revealed in skin extracts of adult mice between 30 minutes and 48 hours after AK23 injection (Fig. 2C and upper panel shows 48 hours).


Preclinical studies identify non-apoptotic low-level caspase-3 as therapeutic target in pemphigus vulgaris.

Luyet C, Schulze K, Sayar BS, Howald D, Müller EJ, Galichet A - PLoS ONE (2015)

Absence of hallmarks for apoptosis in AK23-treated keratinocytes and 8-week-old mice.(A) Representative immunofluorescence micrographs for F-actin in mouse and human keratinocytes treated for 48 and 24 hours, respectively, with 20, 80 and 160 μg/ml AK23/mIgG or 20 μg/ml mitomycin C (mitC); shown are 160 μg/ml AK23/mIgG. Nuclei were counterstained with Hoechst 33258; arrows point to actin rearrangement; bar = 25μm, (n = 2/group in duplicates and 1’000 cells/group evaluated). (B-E) Representative immunoblots and graphs of Triton X-100-soluble proteins probed with indicated antibodies from B, D, mouse and human keratinocytes treated for 48 hours and 24 hours, respectively, with 20, 80 and 160 μg/ml AK23/mIgG or 20 μg/ml mitomycin C; shown are 20μg/ml (mouse cells) and 80μg/ml (human cells) AK23/mIgG; (n = 5/group); C, E, skin of 8-week-old mice injected with 12.5 μg/g b.w. AK23/mIgG for B, 30 min, 2, 24 and 48 hours and E, 30 min, 2, 5, 24 and 48 hours; shown are indicated time points; (n = 4/group). Signals were quantified, normalized to tubulin and the mean±SEM is plotted relative to mIgG set as 1.
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Related In: Results  -  Collection

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pone.0119809.g002: Absence of hallmarks for apoptosis in AK23-treated keratinocytes and 8-week-old mice.(A) Representative immunofluorescence micrographs for F-actin in mouse and human keratinocytes treated for 48 and 24 hours, respectively, with 20, 80 and 160 μg/ml AK23/mIgG or 20 μg/ml mitomycin C (mitC); shown are 160 μg/ml AK23/mIgG. Nuclei were counterstained with Hoechst 33258; arrows point to actin rearrangement; bar = 25μm, (n = 2/group in duplicates and 1’000 cells/group evaluated). (B-E) Representative immunoblots and graphs of Triton X-100-soluble proteins probed with indicated antibodies from B, D, mouse and human keratinocytes treated for 48 hours and 24 hours, respectively, with 20, 80 and 160 μg/ml AK23/mIgG or 20 μg/ml mitomycin C; shown are 20μg/ml (mouse cells) and 80μg/ml (human cells) AK23/mIgG; (n = 5/group); C, E, skin of 8-week-old mice injected with 12.5 μg/g b.w. AK23/mIgG for B, 30 min, 2, 24 and 48 hours and E, 30 min, 2, 5, 24 and 48 hours; shown are indicated time points; (n = 4/group). Signals were quantified, normalized to tubulin and the mean±SEM is plotted relative to mIgG set as 1.
Mentions: We also addressed occurrence of apoptosis using complementary read-outs. Cytoskeletal actin is a major target for destruction during the apoptotic process [53,54]. Consistently, mitomycin C-treated mouse keratinocytes exhibited a complete collapse of the actin cytoskeleton (Fig. 2A). In contrast, subtle changes, comparable with the retraction of actin bundles from cell boarders, reported in PV during Dsg3 endocytosis [55], were observed after 48 hours in mouse keratinocytes treated with 20, 80 or 160 μg/ml AK23 (Fig. 2A shows 160 μg/ml AK23). We also investigated primary human keratinocytes treated with AK23 under conditions of loss of adhesion (Fig. 2A; S1B Fig.) as well as mouse keratinocytes incubated with PVIgG from two different patients (containing Dsg3 [23] and Dsg3/Dsg1 antibodies, respectively) (S1C Fig.). In all cases (analyzing 1’000 cells per well in duplicates), no global actin collapse comparable to mitomycin C-treated apoptotic cells was observed. Under the same conditions we also explored the steady-state levels of full-length and cleaved poly ADP-ribose polymerase (PARP), a DNA repair enzyme that is specifically inactivated through cleavage in the early course of apoptotic cell death [54]. No evidence was obtained for reduced full-length PARP (indicative for cleavage) or increased cleaved PARP in mouse and human keratinocytes treated with AK23 (Fig. 2B). As a positive control, mitomycin C treatment resulted in complete conversion of full-length PARP to cleaved/inactivated PARP. No significant decrease in full-length PARP and no cleaved PARP were further revealed in skin extracts of adult mice between 30 minutes and 48 hours after AK23 injection (Fig. 2C and upper panel shows 48 hours).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Molecular Dermatology, Institute of Animal Pathology, Vetsuisse Faculty, University of Bern, Bern, Switzerland; DermFocus, Vetsuisse Faculty, University of Bern, Bern, Switzerland.

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.

Show MeSH
Related in: MedlinePlus