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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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Exceptional TUNEL-positive lesions and no loss of membrane integrity in AK23-treated cultured mouse keratinocytes and 8-week-old mice or human PV patients’ biopsies.(A, B, D) Representative pictures of TUNEL staining in A, mouse keratinocyte cultures treated with 20 or 80 μg/ml AK23/mIgG for 48 hours; 20 μg/ml mitomycin C (mitC) treatment of the same culture serves as positive control for apoptosis; shown are 80 μg/ml AK23/mIgG; (n = 3 in duplicates/group and 120’000 cells/group evaluated); in B, hair follicles, back skin and hard palate of 8-week-old mice injected with 12.5 μg/g b.w. AK23/mIgG for 24h and 48h; shown are 24 hours (total mice evaluated 8; n = 2/group and time point; ≥100 hair follicles/mouse evaluated); SG: sebaceous gland, HF: hair follicle, arrows point to TUNEL positive cells in stratum corneum and sebaceous gland; in D, selected biopsies from human PV patients’ skin and oral mucosa. Note that PV patient 2 exhibits fragmentation of nuclei; DNase I-treated healthy donor skin biopsies served as positive control (high number of TUNEL positive cells) and without terminal transferase as negative control; (n = 6 PV patients, n = 2 healthy donors). Insets are twofold magnifications of selected areas. Asterisks indicate lesions. Nuclei were counterstained with Hoechst 33258. Dotted line indicates basement membrane. Bar = 75m (A), 25m (B), 125m (D). (C) Graph of LIVE/DEAD stained viable keratinocytes isolated from skin of AK23/mIgG-treated 8-week-old mice evaluated by FACS. Data are mean±SEM.
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pone.0119809.g001: Exceptional TUNEL-positive lesions and no loss of membrane integrity in AK23-treated cultured mouse keratinocytes and 8-week-old mice or human PV patients’ biopsies.(A, B, D) Representative pictures of TUNEL staining in A, mouse keratinocyte cultures treated with 20 or 80 μg/ml AK23/mIgG for 48 hours; 20 μg/ml mitomycin C (mitC) treatment of the same culture serves as positive control for apoptosis; shown are 80 μg/ml AK23/mIgG; (n = 3 in duplicates/group and 120’000 cells/group evaluated); in B, hair follicles, back skin and hard palate of 8-week-old mice injected with 12.5 μg/g b.w. AK23/mIgG for 24h and 48h; shown are 24 hours (total mice evaluated 8; n = 2/group and time point; ≥100 hair follicles/mouse evaluated); SG: sebaceous gland, HF: hair follicle, arrows point to TUNEL positive cells in stratum corneum and sebaceous gland; in D, selected biopsies from human PV patients’ skin and oral mucosa. Note that PV patient 2 exhibits fragmentation of nuclei; DNase I-treated healthy donor skin biopsies served as positive control (high number of TUNEL positive cells) and without terminal transferase as negative control; (n = 6 PV patients, n = 2 healthy donors). Insets are twofold magnifications of selected areas. Asterisks indicate lesions. Nuclei were counterstained with Hoechst 33258. Dotted line indicates basement membrane. Bar = 75m (A), 25m (B), 125m (D). (C) Graph of LIVE/DEAD stained viable keratinocytes isolated from skin of AK23/mIgG-treated 8-week-old mice evaluated by FACS. Data are mean±SEM.

Mentions: Our initial objective was to perform a systematic survey of apoptotic events in PV models and PV patients’ biopsies. Firstly, 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 (evaluating roughly 120’000 cells per experiment) under our standard conditions (20 μg/ml AK23) and even when the AK23 concentration was elevated by fourfold (Fig. 1A shows 80 μg/ml). Consistent with TUNEL negativity, visual inspection did not reveal fragmented nuclei, as is expected in case of apoptosis [15]. However, on average 19% TUNEL positive cells were counted and nuclear morphology was affected when cell death was induced with high-dose mitomycin C used as positive control.


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)

Exceptional TUNEL-positive lesions and no loss of membrane integrity in AK23-treated cultured mouse keratinocytes and 8-week-old mice or human PV patients’ biopsies.(A, B, D) Representative pictures of TUNEL staining in A, mouse keratinocyte cultures treated with 20 or 80 μg/ml AK23/mIgG for 48 hours; 20 μg/ml mitomycin C (mitC) treatment of the same culture serves as positive control for apoptosis; shown are 80 μg/ml AK23/mIgG; (n = 3 in duplicates/group and 120’000 cells/group evaluated); in B, hair follicles, back skin and hard palate of 8-week-old mice injected with 12.5 μg/g b.w. AK23/mIgG for 24h and 48h; shown are 24 hours (total mice evaluated 8; n = 2/group and time point; ≥100 hair follicles/mouse evaluated); SG: sebaceous gland, HF: hair follicle, arrows point to TUNEL positive cells in stratum corneum and sebaceous gland; in D, selected biopsies from human PV patients’ skin and oral mucosa. Note that PV patient 2 exhibits fragmentation of nuclei; DNase I-treated healthy donor skin biopsies served as positive control (high number of TUNEL positive cells) and without terminal transferase as negative control; (n = 6 PV patients, n = 2 healthy donors). Insets are twofold magnifications of selected areas. Asterisks indicate lesions. Nuclei were counterstained with Hoechst 33258. Dotted line indicates basement membrane. Bar = 75m (A), 25m (B), 125m (D). (C) Graph of LIVE/DEAD stained viable keratinocytes isolated from skin of AK23/mIgG-treated 8-week-old mice evaluated by FACS. Data are mean±SEM.
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getmorefigures.php?uid=PMC4352034&req=5

pone.0119809.g001: Exceptional TUNEL-positive lesions and no loss of membrane integrity in AK23-treated cultured mouse keratinocytes and 8-week-old mice or human PV patients’ biopsies.(A, B, D) Representative pictures of TUNEL staining in A, mouse keratinocyte cultures treated with 20 or 80 μg/ml AK23/mIgG for 48 hours; 20 μg/ml mitomycin C (mitC) treatment of the same culture serves as positive control for apoptosis; shown are 80 μg/ml AK23/mIgG; (n = 3 in duplicates/group and 120’000 cells/group evaluated); in B, hair follicles, back skin and hard palate of 8-week-old mice injected with 12.5 μg/g b.w. AK23/mIgG for 24h and 48h; shown are 24 hours (total mice evaluated 8; n = 2/group and time point; ≥100 hair follicles/mouse evaluated); SG: sebaceous gland, HF: hair follicle, arrows point to TUNEL positive cells in stratum corneum and sebaceous gland; in D, selected biopsies from human PV patients’ skin and oral mucosa. Note that PV patient 2 exhibits fragmentation of nuclei; DNase I-treated healthy donor skin biopsies served as positive control (high number of TUNEL positive cells) and without terminal transferase as negative control; (n = 6 PV patients, n = 2 healthy donors). Insets are twofold magnifications of selected areas. Asterisks indicate lesions. Nuclei were counterstained with Hoechst 33258. Dotted line indicates basement membrane. Bar = 75m (A), 25m (B), 125m (D). (C) Graph of LIVE/DEAD stained viable keratinocytes isolated from skin of AK23/mIgG-treated 8-week-old mice evaluated by FACS. Data are mean±SEM.
Mentions: Our initial objective was to perform a systematic survey of apoptotic events in PV models and PV patients’ biopsies. Firstly, 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 (evaluating roughly 120’000 cells per experiment) under our standard conditions (20 μg/ml AK23) and even when the AK23 concentration was elevated by fourfold (Fig. 1A shows 80 μg/ml). Consistent with TUNEL negativity, visual inspection did not reveal fragmented nuclei, as is expected in case of apoptosis [15]. However, on average 19% TUNEL positive cells were counted and nuclear morphology was affected when cell death was induced with high-dose mitomycin C used as positive control.

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