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Induction of p38MAPK and HSP27 Phosphorylation in Pemphigus Patient Skin

2007; Elsevier BV; Volume: 128; Issue: 3 Linguagem: Inglês

10.1038/sj.jid.5701080

1523-1747

Paula Berkowitz, Luis A. Díaz, Russell P. Hall, David S. Rubenstein,

Coagulation, Bradykinin, Polyphosphates, and Angioedema

desmoglein heat shock protein 27 p38 mitogen-activated protein kinase pemphigus foliaceus pemphigus vulgaris TO THE EDITOR Pemphigus vulgaris (PV) is an autoimmune disease characterized by epidermal blistering involving mucosa or mucosa and skin (Ding et al., 1997Ding X. Aoki V. Mascaro Jr, J.M. Lopez-Swiderski A. Diaz L.A. Fairley J.A. Mucosal and mucocutaneous (generalized) pemphigus vulgaris show distinct autoantibody profiles.J Invest Dermatol. 1997; 109: 592-596Crossref PubMed Scopus (214) Google Scholar). In PV, autoantibodies directed against the desmosome cadherins desmoglein (dsg) 3 and dsg1 induce loss of cell–cell adhesion (acantholysis) resulting in clinical blisters. Passive transfer experiments using IgG, purified from PV patients (Anhalt et al., 1982Anhalt G.J. Labib R.S. Voorhees J.J. Beals T.F. Diaz L.A. Induction of pemphigus in neonatal mice by passive transfer of IgG from patients with the disease.N Engl J Med. 1982; 306: 1189-1196Crossref PubMed Scopus (556) Google Scholar) or highly specific anti-dsg3 monoclonal antibodies (Tsunoda et al., 2003Tsunoda K. Ota T. Aoki M. Yamada T. Nagai T. Nakagawa T. et al.Induction of pemphigus phenotype by a mouse monoclonal antibody against the amino-terminal adhesive interface of desmoglein 3.J Immunol. 2003; 170: 2170-2178Crossref PubMed Scopus (240) Google Scholar; Payne et al., 2005Payne A.S. Ishii K. Kacir S. Lin C. Li H. Hanakawa Y. et al.Genetic and functional characterization of human pemphigus vulgaris monoclonal autoantibodies isolated by phage display.J Clin Invest. 2005; 115: 888-899Crossref PubMed Scopus (170) Google Scholar), reproduce the clinical, histologic, and immunologic features of PV in the infused animals. Similarly, pemphigus foliaceus (PF) is an autoimmune blistering disease of the skin in which autoantibodies to dsg1 cause acantholysis in the subcorneal and granular layers of the epidermis. IgG purified from PF patient sera similarly reproduces disease in the passive transfer mouse model (Roscoe et al., 1985Roscoe J.T. Diaz L. Sampaio S.A. Castro R.M. Labib R.S. Takahashi Y. et al.Brazilian pemphigus foliaceus autoantibodies are pathogenic to BALB/c mice by passive transfer.J Invest Dermatol. 1985; 85: 538-541Crossref PubMed Scopus (165) Google Scholar; Rock et al., 1989Rock B. Martins C.R. Theofilopoulos A.N. Balderas R.S. Anhalt G.J. Labib R.S. et al.The pathogenic effect of IgG4 autoantibodies in endemic pemphigus foliaceus (fogo selvagem).N Engl J Med. 1989; 320: 1463-1469Crossref PubMed Scopus (234) Google Scholar). We and others have been studying the molecular mechanism by which anti-dsg autoantibodies cause acantholysis in pemphigus. Using keratinocyte tissue culture and murine in vivo models, we have demonstrated that anti-dsg3 PV IgG induce rapid, time- and dose-dependent phosphorylation of p38 mitogen-activated protein kinase (MAPK) and heat shock protein 27 (HSP27) (Berkowitz et al., 2005Berkowitz P. Hu P. Liu Z. Diaz L.A. Enghild J.J. Chua M.P. et al.Desmosome signaling: inhibition of p38MAPK prevents pemphigus vulgaris IgG-induced cytoskeleton reorganization.J Biol Chem. 2005; 280: 23778-23784Crossref PubMed Scopus (190) Google Scholar). Activation of keratinocyte p38MAPK and HSP27 by PV IgG may be relevant to the mechanism of acantholysis in PV since (i) both p38MAPK and HSP27 regulate components of the cytoskeleton including actin and intermediate filaments (Lavoie et al., 1993Lavoie J.N. Hickey E. Weber L.A. Landry J. Modulation of actin microfilament dynamics and fluid phase pinocytosis by phosphorylation of heat shock protein 27.J Biol Chem. 1993; 268: 24210-24214Abstract Full Text PDF PubMed Google Scholar, Lavoie et al., 1995Lavoie J.N. Lambert H. Hickey E. Weber L.A. Landry J. Modulation of cellular thermoresistance and actin filament stability accompanies phosphorylation-induced changes in the oligomeric structure of heat shock protein 27.Mol Cell Biol. 1995; 15: 505-516Crossref PubMed Scopus (557) Google Scholar; Benndorf et al., 1994Benndorf R. Hayess K. Ryazantsev S. Wieske M. Behlke J. Lutsch G. Phosphorylation and supramolecular organization of murine small heat shock protein HSP25 abolish its actin polymerization-inhibiting activity.J Biol Chem. 1994; 269: 20780-20784Abstract Full Text PDF PubMed Google Scholar; Guay et al., 1997Guay J. Lambert H. Gingras-Breton G. Lavoie J.N. Huot J. Landry J. Regulation of actin filament dynamics by p38 map kinase-mediated phosphorylation of heat shock protein 27.J Cell Sci. 1997; 110: 357-368Crossref PubMed Google Scholar; Perng et al., 1999Perng M.D. Cairns L. van den I.P. Prescott A. Hutcheson A.M. Quinlan R.A. Intermediate filament interactions can be altered by HSP27 and alphaB-crystallin.J Cell Sci. 1999; 112: 2099-2112PubMed Google Scholar; Geum et al., 2002Geum D. Son G.H. Kim K. Phosphorylation-dependent cellular localization and thermoprotective role of heat shock protein 25 in hippocampal progenitor cells.J Biol Chem. 2002; 277: 19913-19921Crossref PubMed Scopus (108) Google Scholar; Panasenko et al., 2003Panasenko O.O. Kim M.V. Marston S.B. Gusev N.B. Interaction of the small heat shock protein with molecular mass 25 kDa (hsp25) with actin.Eur J Biochem. 2003; 270: 892-901Crossref PubMed Scopus (43) Google Scholar; Evgrafov et al., 2004Evgrafov O.V. Mersiyanova I. Irobi J. Van Den Bosch L. Dierick I. Leung C.L. et al.Mutant small heat-shock protein 27 causes axonal Charcot–Marie–Tooth disease and distal hereditary motor neuropathy.Nat Genet. 2004; 36: 602-606Crossref PubMed Scopus (476) Google Scholar) and (ii) in human keratinocyte cultures, inhibitors of p38MAPK prevented PV IgG-induced phosphorylation of HSP27 and the early cytoskeletal changes associated with the loss of cell–cell adhesion (Berkowitz et al., 2005Berkowitz P. Hu P. Liu Z. Diaz L.A. Enghild J.J. Chua M.P. et al.Desmosome signaling: inhibition of p38MAPK prevents pemphigus vulgaris IgG-induced cytoskeleton reorganization.J Biol Chem. 2005; 280: 23778-23784Crossref PubMed Scopus (190) Google Scholar). These observations suggested that inhibition of this signaling pathway in epidermal epithelia could be used to prevent end-organ damage (e.g. blistering) caused by PV autoantibodies. Indeed, in the PV passive transfer mouse model we found that (i) p38MAPK and HSP25, the murine HSP27 homolog, are similarly phosphorylated in vivo, and significantly (ii) p38MAPK inhibitors block blister formation in this vivo model of PV (Berkowitz et al., 2006Berkowitz P. Hu P. Warren S. Liu Z. Diaz L.A. Rubenstein D.S. p38MAPK inhibition prevents disease in pemphigus vulgaris mice.Proc Natl Acad Sci USA. 2006; 103: 12855-12860Crossref PubMed Scopus (174) Google Scholar). These observations in tissue culture and animal models suggest a central role for p38MAPK and HSP27 in the mechanisms of acantholysis in PV. Importantly, targeting this signaling pathway with inhibitors may have a role in treating patients with pemphigus; however, we have yet to determine whether p38MAPK and HSP27 are similarly phosphorylated in lesional epidermis of pemphigus patients. In this study, we probe for phosphorylation of p38MAPK and HSP27 in skin biopsies of one PF and five PV patients and six specimens from control individuals affected by other skin diseases; this study was approved by the Institutional Review Board at UNC. A diagnosis of PV or PF was established by clinical, histological, and immunological criteria (Diaz and Giudice, 2000Diaz L.A. Giudice G.J. End of the century overview of skin blisters.Arch Dermatol. 2000; 136: 106-112Crossref PubMed Scopus (54) Google Scholar). The clinical and serological features of the six patients are shown in Table 1. Biopsies from control individuals showed no immunoreactants by direct immunofluorescence or histologic signs of pemphigus.Table 1Clinical and immunologic characteristics of patients from which the skin biopsies were obtainedCasesPhenotypeIIF titerDsg1-ELISADsg3-ELISAPFPF1:10,240PositiveNegativePV1mcPV1:40PositivePositivePV2mcPV1:640PositivePositivePV3mPV1:320NegativePositivePV4mcPV1:1,280PositivePositivePV5mcPV1:160PositivePositiveDsg, desmoglein; IIF, indirect immunofluorescence; PF, pemphigus foliaceus, PV, pemphigus vulgaris, m, mucosal, mc, mucocutaneous. Open table in a new tab Dsg, desmoglein; IIF, indirect immunofluorescence; PF, pemphigus foliaceus, PV, pemphigus vulgaris, m, mucosal, mc, mucocutaneous. Extracts were made from frozen archived perilesional skin biopsies in 8 M urea buffer (8 M urea, 4% CHAPS, 10 μM pepstatin, 100 μM leupeptin, 10 μM E-64, 1 mM PMSF, 1 mM Na3VO4, 50 mM NaF, 0.5 μM okadaic acid). Protein concentration was determined by Bradford method as described previously (Berkowitz et al., 2005Berkowitz P. Hu P. Liu Z. Diaz L.A. Enghild J.J. Chua M.P. et al.Desmosome signaling: inhibition of p38MAPK prevents pemphigus vulgaris IgG-induced cytoskeleton reorganization.J Biol Chem. 2005; 280: 23778-23784Crossref PubMed Scopus (190) Google Scholar). Tissue extracts were separated by 10% SDS-PAGE and immunoblotted using monoclonal antibodies against p38MAPK (Santa Cruz Biotechnology Inc., Santa Cruz, CA), phospho-p38MAPK (Cell Signaling Technology, Beverly, MA), HSP27 (ABR, Golden, CO), and phospho-HSP27 (Cell Signaling Technology). Western blots were developed by ECL reaction and the signal intensity quantified by scanning chemiluminescence on a GeneGnome scanner (Syngene Bio Imaging, Frederick,MD) using GeneSnap software. Compared to controls, increased phosphorylation of p38MAPK was observed in 1 PF and 3 of the 5 PV skin biopsies; whereas, increased HSP27 phosphorylation was observed in all PF and PV patient skin biopsies (Figure 1). In our earlier work with in vivo mouse models, we have noted degradation in the phospho-p38MAPK signal with extended sample storage time even at -70°C, this explains the discrepancy between the phospho-p38MAPK and phospho-HSP27 signals observed in 2 of the 5 PV skin biopsies (PV4 and PV5). Increased phosphorylation of p38MAPK and HSP27 was observed in both mucosal and mucocutaneous variants of PV. The observed activation of intracellular signaling events mediated by p38MAPK and HSP27 in patient skin is analogous to the increased phosphorylation of these two proteins that we have previously reported in human keratinocyte tissue culture and in vivo mouse models. Importantly, a number of p38MAPK inhibitors are currently in early-phase clinical trials for inflammatory and neoplastic diseases (O'Neill, 2006O'Neill L.A. Targeting signal transduction as a strategy to treat inflammatory diseases.Nat Rev Drug Discov. 2006; 5: 549-563Crossref PubMed Scopus (236) Google Scholar). Along with the earlier in vitro and in vivo mouse model studies, the observation that p38MAPK and HSP27 are phosphorylated in biopsies from perilesional skin of human pemphigus patients provides additional evidence to support the notion that p38MAPK inhibitors may be applicable to the therapy of patients suffering from the pemphigus family of autoimmune blistering diseases. Paula Berkowitz, Luis A. Diaz, and David S. Rubenstein are co-inventors on a patent identifying p38MAPK and HSP27 as targets for the treatment of pemphigus. Neither licensing agreements nor financial benefit has accrued to the inventors. This work was supported by the National Institutes of Health Grants RO1 AI49427 (to D.S.R.), and AR30281, AR32599 (to L.A.D.).