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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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Involvement of low-level caspase-3 in loss of intercellular adhesion in vitro and in vivo.(A-C) Dissociation assay; mouse keratinocyte treated with A, 20 μg/ml AK23/mIgG; B, 20 μg/ml AK23/mIgG with 1.5mg/ml PFIgG/nhIgG or C, 1mg/ml PVIgG1/nhIgG or 4mg/ml PVIgG2/nhIgG with or without the indicated inhibitors. The number of generated fragments is presented as mean±SEM relative to DMSO/pathogenic antibody treatment set as 100%; (n, as indicated, in duplicates). (D-E) Percentage of D, PV skin blisters in AK23/PF IgG-injected neonatal mice and E, hair follicle PV blisters in AK23-injected 8-week-old mice, with or without caspase-3 inhibitor III. (n, as indicated). Data are mean±SEM, *p<0.05, **p<0.01.
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pone.0119809.g004: Involvement of low-level caspase-3 in loss of intercellular adhesion in vitro and in vivo.(A-C) Dissociation assay; mouse keratinocyte treated with A, 20 μg/ml AK23/mIgG; B, 20 μg/ml AK23/mIgG with 1.5mg/ml PFIgG/nhIgG or C, 1mg/ml PVIgG1/nhIgG or 4mg/ml PVIgG2/nhIgG with or without the indicated inhibitors. The number of generated fragments is presented as mean±SEM relative to DMSO/pathogenic antibody treatment set as 100%; (n, as indicated, in duplicates). (D-E) Percentage of D, PV skin blisters in AK23/PF IgG-injected neonatal mice and E, hair follicle PV blisters in AK23-injected 8-week-old mice, with or without caspase-3 inhibitor III. (n, as indicated). Data are mean±SEM, *p<0.05, **p<0.01.

Mentions: To address whether the low-level caspase-3 activation observed prior to loss of intercellular adhesion functionally contributes to acantholysis, we used caspase inhibitors with different specificities and targets. A dissociation assay [51] revealed that AK23-mediated loss of intercellular adhesion was significantly reduced in cultured mouse keratinocytes pre-treated with a pan-caspase and two caspase-3 inhibitors (Z-VAD-FMK, Z-DEVD-FMK (II), Ac-DEVD-CMK (III); S2B and S2C Figs. for efficiency control) (Fig. 4A). The pan-caspase inhibitor, which also targets other proteases such as calpain [58], had the strongest effect. We therefore continued to use caspase-3 inhibitor III with high specificity for caspase-3 [59]. Similar results than in mouse keratinocytes treated with AK23 were obtained for human keratinocytes (S3A Fig.) as well as for AK23/PFIgG (combined Dsg3/Dsg1 antibodies), PVIgG1- and PVIgG2-incubated mouse keratinocytes in presence of the caspase-3 inhibitor III (Fig. 4B and 4C).


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)

Involvement of low-level caspase-3 in loss of intercellular adhesion in vitro and in vivo.(A-C) Dissociation assay; mouse keratinocyte treated with A, 20 μg/ml AK23/mIgG; B, 20 μg/ml AK23/mIgG with 1.5mg/ml PFIgG/nhIgG or C, 1mg/ml PVIgG1/nhIgG or 4mg/ml PVIgG2/nhIgG with or without the indicated inhibitors. The number of generated fragments is presented as mean±SEM relative to DMSO/pathogenic antibody treatment set as 100%; (n, as indicated, in duplicates). (D-E) Percentage of D, PV skin blisters in AK23/PF IgG-injected neonatal mice and E, hair follicle PV blisters in AK23-injected 8-week-old mice, with or without caspase-3 inhibitor III. (n, as indicated). Data are mean±SEM, *p<0.05, **p<0.01.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4352034&req=5

pone.0119809.g004: Involvement of low-level caspase-3 in loss of intercellular adhesion in vitro and in vivo.(A-C) Dissociation assay; mouse keratinocyte treated with A, 20 μg/ml AK23/mIgG; B, 20 μg/ml AK23/mIgG with 1.5mg/ml PFIgG/nhIgG or C, 1mg/ml PVIgG1/nhIgG or 4mg/ml PVIgG2/nhIgG with or without the indicated inhibitors. The number of generated fragments is presented as mean±SEM relative to DMSO/pathogenic antibody treatment set as 100%; (n, as indicated, in duplicates). (D-E) Percentage of D, PV skin blisters in AK23/PF IgG-injected neonatal mice and E, hair follicle PV blisters in AK23-injected 8-week-old mice, with or without caspase-3 inhibitor III. (n, as indicated). Data are mean±SEM, *p<0.05, **p<0.01.
Mentions: To address whether the low-level caspase-3 activation observed prior to loss of intercellular adhesion functionally contributes to acantholysis, we used caspase inhibitors with different specificities and targets. A dissociation assay [51] revealed that AK23-mediated loss of intercellular adhesion was significantly reduced in cultured mouse keratinocytes pre-treated with a pan-caspase and two caspase-3 inhibitors (Z-VAD-FMK, Z-DEVD-FMK (II), Ac-DEVD-CMK (III); S2B and S2C Figs. for efficiency control) (Fig. 4A). The pan-caspase inhibitor, which also targets other proteases such as calpain [58], had the strongest effect. We therefore continued to use caspase-3 inhibitor III with high specificity for caspase-3 [59]. Similar results than in mouse keratinocytes treated with AK23 were obtained for human keratinocytes (S3A Fig.) as well as for AK23/PFIgG (combined Dsg3/Dsg1 antibodies), PVIgG1- and PVIgG2-incubated mouse keratinocytes in presence of the caspase-3 inhibitor III (Fig. 4B and 4C).

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