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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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Transient, low-level caspase-3 activation in PV antibody-treated keratinocytes and 8-week-old mice.(A, B) Caspase-3/7 activity was measured in cells treated for indicated time points with A, 80 μg/ml AK23/mIgG and B, 2 mg/ml PVIgG1/nhIgG or 8 mg/ml PVIgG2/nhIgG: (n, as indicated, in triplicates). (C) Representative immunofluorescence micrographs and graph reporting active caspase-3 in mouse keratinocytes treated as in A and B for 1h or with 20 μg/ml mitomycin C (mitC) for 48h. Note that cells with mitotic figures (AK23 mitosis, arrow) have increased active caspase-3 and were therefore excluded from the quantification; fluorescence intensity was quantified by ImageJ and plotted (right panel); (n, as indicated, duplicates and 400 cells/group evaluated). (D) Active caspase-3 was immunoprecipitated (IP) from skin lysates of 8-week-old mice injected with 12.5 μg/g b.w. AK23/mIgG for 30 min, 2 and 24 hours and quantified by western blot analyses. One representative blot is shown. Specificity of the immunoprecipitation was assessed by non-relevant rabbit IgG (rIgG), shown on the right, (n/group as indicated). The mean±SEM of quantified and normalized signals is reported as relative change compared to mIgG set as 1, *p<0.05, **p<0.01.
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pone.0119809.g003: Transient, low-level caspase-3 activation in PV antibody-treated keratinocytes and 8-week-old mice.(A, B) Caspase-3/7 activity was measured in cells treated for indicated time points with A, 80 μg/ml AK23/mIgG and B, 2 mg/ml PVIgG1/nhIgG or 8 mg/ml PVIgG2/nhIgG: (n, as indicated, in triplicates). (C) Representative immunofluorescence micrographs and graph reporting active caspase-3 in mouse keratinocytes treated as in A and B for 1h or with 20 μg/ml mitomycin C (mitC) for 48h. Note that cells with mitotic figures (AK23 mitosis, arrow) have increased active caspase-3 and were therefore excluded from the quantification; fluorescence intensity was quantified by ImageJ and plotted (right panel); (n, as indicated, duplicates and 400 cells/group evaluated). (D) Active caspase-3 was immunoprecipitated (IP) from skin lysates of 8-week-old mice injected with 12.5 μg/g b.w. AK23/mIgG for 30 min, 2 and 24 hours and quantified by western blot analyses. One representative blot is shown. Specificity of the immunoprecipitation was assessed by non-relevant rabbit IgG (rIgG), shown on the right, (n/group as indicated). The mean±SEM of quantified and normalized signals is reported as relative change compared to mIgG set as 1, *p<0.05, **p<0.01.

Mentions: As compared to robust caspase activation in mitomycin C-treated cells (S2A Fig.), mouse keratinocytes treated with 20 or 80 μg/ml AK23 exhibited an early transient, low-level caspase-3/7 activation with a peak at 1 hour measured by two different caspase-3/7 activity assays (the substrate is specific for caspase-3 and -7) (Fig. 3A shows results of the Caspase-Glo 3/7 assay for 80 μg/ml AK23). PVIgG from two patients (containing Dsg3 [23] and Dsg3 and Dsg1 antibodies, respectively) gave similar results (Fig. 3B). In support of these findings, semi-quantitative immunofluorescence microscopy revealed a subtle but significant increase in activated caspase-3 quantified on micrographs of mouse keratinocytes 1 hour after AK23 and PVIgG exposure (Fig. 3C; quantification of 1’000 cells per well in duplicates). In AK23- and PVIgG-treated cells, active caspase-3 mainly localized to the cytoplasm while the association with DNA in mitotic cells served as a positive control in agreement with a role of caspase-3 in mitotic spindle checkpoint control [57]. Furthermore, high levels of active caspase-3 were detected in the cytoplasm and nucleus of mitomycin C-treated apoptotic cells.


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)

Transient, low-level caspase-3 activation in PV antibody-treated keratinocytes and 8-week-old mice.(A, B) Caspase-3/7 activity was measured in cells treated for indicated time points with A, 80 μg/ml AK23/mIgG and B, 2 mg/ml PVIgG1/nhIgG or 8 mg/ml PVIgG2/nhIgG: (n, as indicated, in triplicates). (C) Representative immunofluorescence micrographs and graph reporting active caspase-3 in mouse keratinocytes treated as in A and B for 1h or with 20 μg/ml mitomycin C (mitC) for 48h. Note that cells with mitotic figures (AK23 mitosis, arrow) have increased active caspase-3 and were therefore excluded from the quantification; fluorescence intensity was quantified by ImageJ and plotted (right panel); (n, as indicated, duplicates and 400 cells/group evaluated). (D) Active caspase-3 was immunoprecipitated (IP) from skin lysates of 8-week-old mice injected with 12.5 μg/g b.w. AK23/mIgG for 30 min, 2 and 24 hours and quantified by western blot analyses. One representative blot is shown. Specificity of the immunoprecipitation was assessed by non-relevant rabbit IgG (rIgG), shown on the right, (n/group as indicated). The mean±SEM of quantified and normalized signals is reported as relative change compared to mIgG set as 1, *p<0.05, **p<0.01.
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pone.0119809.g003: Transient, low-level caspase-3 activation in PV antibody-treated keratinocytes and 8-week-old mice.(A, B) Caspase-3/7 activity was measured in cells treated for indicated time points with A, 80 μg/ml AK23/mIgG and B, 2 mg/ml PVIgG1/nhIgG or 8 mg/ml PVIgG2/nhIgG: (n, as indicated, in triplicates). (C) Representative immunofluorescence micrographs and graph reporting active caspase-3 in mouse keratinocytes treated as in A and B for 1h or with 20 μg/ml mitomycin C (mitC) for 48h. Note that cells with mitotic figures (AK23 mitosis, arrow) have increased active caspase-3 and were therefore excluded from the quantification; fluorescence intensity was quantified by ImageJ and plotted (right panel); (n, as indicated, duplicates and 400 cells/group evaluated). (D) Active caspase-3 was immunoprecipitated (IP) from skin lysates of 8-week-old mice injected with 12.5 μg/g b.w. AK23/mIgG for 30 min, 2 and 24 hours and quantified by western blot analyses. One representative blot is shown. Specificity of the immunoprecipitation was assessed by non-relevant rabbit IgG (rIgG), shown on the right, (n/group as indicated). The mean±SEM of quantified and normalized signals is reported as relative change compared to mIgG set as 1, *p<0.05, **p<0.01.
Mentions: As compared to robust caspase activation in mitomycin C-treated cells (S2A Fig.), mouse keratinocytes treated with 20 or 80 μg/ml AK23 exhibited an early transient, low-level caspase-3/7 activation with a peak at 1 hour measured by two different caspase-3/7 activity assays (the substrate is specific for caspase-3 and -7) (Fig. 3A shows results of the Caspase-Glo 3/7 assay for 80 μg/ml AK23). PVIgG from two patients (containing Dsg3 [23] and Dsg3 and Dsg1 antibodies, respectively) gave similar results (Fig. 3B). In support of these findings, semi-quantitative immunofluorescence microscopy revealed a subtle but significant increase in activated caspase-3 quantified on micrographs of mouse keratinocytes 1 hour after AK23 and PVIgG exposure (Fig. 3C; quantification of 1’000 cells per well in duplicates). In AK23- and PVIgG-treated cells, active caspase-3 mainly localized to the cytoplasm while the association with DNA in mitotic cells served as a positive control in agreement with a role of caspase-3 in mitotic spindle checkpoint control [57]. Furthermore, high levels of active caspase-3 were detected in the cytoplasm and nucleus of mitomycin C-treated apoptotic cells.

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