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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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Functional contribution of low-level caspase-3 to the acantholytic process in vitro and in vivo.(A-C and E-F) Representative immunoblots and graphs for indicated proteins of A-B, culture medium of mouse keratinocytes treated with A, 20 or 80 μg/ml AK23/mIgG for the indicated time (shown are 20 μg/ml, n = 3) and B, 20 μg/ml AK23 with or without caspase-3 inhibitor for 24 hours (n = 3/group in triplicate); the mean±SEM of quantified and normalized signals for shed Dsg3 is reported as relative change compared to DMSO/AK23 set as 1, *p<0.05; (C and E-F) Triton X-100-soluble protein fractions from skin of AK23/mIgG-injected 8-week-old mice with or without caspase-3 inhibitor III treatment for the indicated time (n/group, as indicated); signals were quantified, normalized to tubulin and the mean±SEM is plotted relative to DMSO/mIgG set as 1, *p<0.05. (D) Percentage±SEM and representative immunofluorescence micrographs of mouse keratinocytes with keratin retraction and cell detachment after treatment for 48h with 80 μg/ml AK23/mIgG with or without pan-caspase and caspase-3 inhibitor III, (n = 3, 1’000 cells/group evaluated). Insets are 1.5 fold magnification of selected areas; arrow points to keratin retraction; nuclei were counterstained with Hoechst 33258; bar = 10 μm.
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pone.0119809.g005: Functional contribution of low-level caspase-3 to the acantholytic process in vitro and in vivo.(A-C and E-F) Representative immunoblots and graphs for indicated proteins of A-B, culture medium of mouse keratinocytes treated with A, 20 or 80 μg/ml AK23/mIgG for the indicated time (shown are 20 μg/ml, n = 3) and B, 20 μg/ml AK23 with or without caspase-3 inhibitor for 24 hours (n = 3/group in triplicate); the mean±SEM of quantified and normalized signals for shed Dsg3 is reported as relative change compared to DMSO/AK23 set as 1, *p<0.05; (C and E-F) Triton X-100-soluble protein fractions from skin of AK23/mIgG-injected 8-week-old mice with or without caspase-3 inhibitor III treatment for the indicated time (n/group, as indicated); signals were quantified, normalized to tubulin and the mean±SEM is plotted relative to DMSO/mIgG set as 1, *p<0.05. (D) Percentage±SEM and representative immunofluorescence micrographs of mouse keratinocytes with keratin retraction and cell detachment after treatment for 48h with 80 μg/ml AK23/mIgG with or without pan-caspase and caspase-3 inhibitor III, (n = 3, 1’000 cells/group evaluated). Insets are 1.5 fold magnification of selected areas; arrow points to keratin retraction; nuclei were counterstained with Hoechst 33258; bar = 10 μm.

Mentions: We further tested the functional contribution of caspase-3 to key events in loss of intercellular adhesion. Remodeling of desmosomes is a major feature in PV and involves for instance Dsg3 shedding as observed in PVIgG-treated human keratinocytes [44]. Desmosomal cadherins can be processed by caspase-3 followed by metalloprotease cleavage, providing a 100 kD membrane anchored and a 75 kD shed N-terminal Dsg3 fragment, respectively [13,14]. We hypothesized that Dsg3 shedding in PV might involve caspase-3. To address this possibility, equal volumes of culture supernatants from AK23/mIgG-treated mouse keratinocytes were analyzed by immunobloting. Twenty or 80 μg/ml AK23 treatment resulted in enhanced shedding of a 75 kD Dsg3 extracellular fragment into the supernatant as compared to differentiating control cells, which also exhibited some Dsg3 cleavage consistent with caspase-3 activation during differentiation [19,56] (Fig. 5A shows 20 μg/ml). The caspase-3 inhibitor III significantly reduced the AK23-mediated Dsg3 shedding (Fig. 5B).


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)

Functional contribution of low-level caspase-3 to the acantholytic process in vitro and in vivo.(A-C and E-F) Representative immunoblots and graphs for indicated proteins of A-B, culture medium of mouse keratinocytes treated with A, 20 or 80 μg/ml AK23/mIgG for the indicated time (shown are 20 μg/ml, n = 3) and B, 20 μg/ml AK23 with or without caspase-3 inhibitor for 24 hours (n = 3/group in triplicate); the mean±SEM of quantified and normalized signals for shed Dsg3 is reported as relative change compared to DMSO/AK23 set as 1, *p<0.05; (C and E-F) Triton X-100-soluble protein fractions from skin of AK23/mIgG-injected 8-week-old mice with or without caspase-3 inhibitor III treatment for the indicated time (n/group, as indicated); signals were quantified, normalized to tubulin and the mean±SEM is plotted relative to DMSO/mIgG set as 1, *p<0.05. (D) Percentage±SEM and representative immunofluorescence micrographs of mouse keratinocytes with keratin retraction and cell detachment after treatment for 48h with 80 μg/ml AK23/mIgG with or without pan-caspase and caspase-3 inhibitor III, (n = 3, 1’000 cells/group evaluated). Insets are 1.5 fold magnification of selected areas; arrow points to keratin retraction; nuclei were counterstained with Hoechst 33258; bar = 10 μm.
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pone.0119809.g005: Functional contribution of low-level caspase-3 to the acantholytic process in vitro and in vivo.(A-C and E-F) Representative immunoblots and graphs for indicated proteins of A-B, culture medium of mouse keratinocytes treated with A, 20 or 80 μg/ml AK23/mIgG for the indicated time (shown are 20 μg/ml, n = 3) and B, 20 μg/ml AK23 with or without caspase-3 inhibitor for 24 hours (n = 3/group in triplicate); the mean±SEM of quantified and normalized signals for shed Dsg3 is reported as relative change compared to DMSO/AK23 set as 1, *p<0.05; (C and E-F) Triton X-100-soluble protein fractions from skin of AK23/mIgG-injected 8-week-old mice with or without caspase-3 inhibitor III treatment for the indicated time (n/group, as indicated); signals were quantified, normalized to tubulin and the mean±SEM is plotted relative to DMSO/mIgG set as 1, *p<0.05. (D) Percentage±SEM and representative immunofluorescence micrographs of mouse keratinocytes with keratin retraction and cell detachment after treatment for 48h with 80 μg/ml AK23/mIgG with or without pan-caspase and caspase-3 inhibitor III, (n = 3, 1’000 cells/group evaluated). Insets are 1.5 fold magnification of selected areas; arrow points to keratin retraction; nuclei were counterstained with Hoechst 33258; bar = 10 μm.
Mentions: We further tested the functional contribution of caspase-3 to key events in loss of intercellular adhesion. Remodeling of desmosomes is a major feature in PV and involves for instance Dsg3 shedding as observed in PVIgG-treated human keratinocytes [44]. Desmosomal cadherins can be processed by caspase-3 followed by metalloprotease cleavage, providing a 100 kD membrane anchored and a 75 kD shed N-terminal Dsg3 fragment, respectively [13,14]. We hypothesized that Dsg3 shedding in PV might involve caspase-3. To address this possibility, equal volumes of culture supernatants from AK23/mIgG-treated mouse keratinocytes were analyzed by immunobloting. Twenty or 80 μg/ml AK23 treatment resulted in enhanced shedding of a 75 kD Dsg3 extracellular fragment into the supernatant as compared to differentiating control cells, which also exhibited some Dsg3 cleavage consistent with caspase-3 activation during differentiation [19,56] (Fig. 5A shows 20 μg/ml). The caspase-3 inhibitor III significantly reduced the AK23-mediated Dsg3 shedding (Fig. 5B).

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