María J. López-Zabalza

p38 MAPK mediates the regulation of α1(I) procollagen mRNA levels by TNF-α and TGF-β in a cell line of rat hepatic stellate cells1

The role of members of the mitogen‐activated protein kinase (MAPK) family on tumor necrosis factor α (TNF‐α)‐mediated down‐regulation of col1a1 gene was studied. TNF‐α increased extracellular‐regulated kinase and Jun‐N‐terminal kinase phosphorylation, but these effects were not related to its inhibitory effect on α1(I) procollagen (col1a1) mRNA levels. Phosphorylation of p38 MAPK was decreased in response to TNF‐α, and the specific p38 MAPK inhibitor SB203580 mimicked the effect of TNF‐α on col1a1 mRNA levels. Transforming growth factor β (TGF‐β) increased p38 MAPK phosphorylation and SB203580 prevented the induction of col1a1 mRNA levels by TGF‐β. These results suggest that p38 MAPK plays an important role in regulating the expression of col1a1 in hepatic stellate cells in response to cytokines.

In vivo blockade of pemphigus vulgaris acantholysis by inhibition of intracellular signal transduction cascades.

Background  Pemphigus vulgaris (PV) is an autoimmune disease characterized by mucocutaneous intraepithelial blisters and pathogenic autoantibodies against desmoglein 3. The mechanism of blister formation in pemphigus has not been defined; however, in vitro data suggest a role for activation of intracellular signalling cascades.

Pemphigus vulgaris autoantibodies induce apoptosis in HaCaT keratinocytes.

Pemphigus vulgaris (PV) is an autoimmune disease characterized by binding of IgG autoantibodies to epidermal keratinocyte desmosomes. IgG autoantibodies obtained from a patient with mucocutaneous PV reacted with plakoglobin (Plkg) in addition to desmoglein‐3 (Dsg3) and Dsg1. Immunofluorescence analysis confirmed that IgG autoantibodies, unlike antibodies from a healthy volunteer, caused disruption of cell–cell contacts in HaCaT keratinocytes. Moreover, apoptosis was enhanced in cells treated with autoantibodies compared to those treated with normal antibodies. The apoptotic process induced by IgG autoantibodies was characterized by caspase‐3 activation, Bcl‐2 depletion and Bax expression. The present report demonstrates that PV IgG autoantibodies promote apoptosis in HaCaT keratinocytes.

An imbalance in Akt/mTOR is involved in the apoptotic and acantholytic processes in a mouse model of pemphigus vulgaris

Abstract:  Pemphigus vulgaris (PV) is an autoimmune blistering disease characterized by the presence of IgG autoantibodies against Dsg3. Our aim was to investigate the molecular events implicated in the development and localization of apoptosis and acantholysis in PV. We used a passive transfer mouse model together with immunohistochemical (IHC) techniques and the TUNEL assay, with quantification analysis in the basal layer of the epidermis. The activated signalling molecules analysed and apoptotic cells detected showed an identical localization. Herein, we found for the first time in vivo an increased expression of activated HER receptor isoforms in the basal layer in PV lesions. Besides, we observed the almost total lack of activated Akt compared with a higher level of activated mTOR within the basal cells of the epidermis. Our observations strongly support that the restriction of acantholysis to the basal layer may be due, at least in part, to the selective and increased presence of activated HER receptor isoforms in these cells. After phosphorylation of HER receptor isoforms, intracellular signalling pathways are activated in the basal layer. In addition, the imbalance in Akt/mTOR that takes place in the basal cells may provide intracellular signals necessary for the development of apoptosis and acantholysis.

Inducible nitric oxide synthase in human lymphomononuclear cells activated by synthetic peptides derived from extracellular matrix proteins

Synthetic peptides with sequences present in extracellular matrix proteins are capable of causing the expression of the inducible form of nitric oxide synthase (iNOS), detected by immunocytochemistry, and the release of NO by human lymphomononuclear cells incubated in their presence. Active peptides are 15‐mers containing a characteristic 2‐6‐11 motif in which the amino acid residue at position 2 is Leu, Ile, Val, Gly, Ala or Lys; the residue at position 6 is always Pro; and residue 11 is Glu or Asp. The induction of iNOS in human monocytes and macrophages could be involved in the cytotoxicity against tumor cell lines also elicited by these peptides.

Immunomodulation induced by synthetic peptides derived from Staphylococcus aureus protein A

Peptides from 10 to 22 amino acids containing sequences encompassed by Staphylococcus aureus protein A were synthesized. Some of these peptides, when present in cultures of lymphomononuclear cells from healthy donors or from cancer patients (melanoma, breast carcinoma, non-Hodgkin lymphoma and renal cell carcinoma) promoted: (i) changes in the phenotype of the lymphomononuclear population, (ii) stimulation of monocytes (release of IL-1 and TNF-alpha), and (iii) an increase in cytotoxicity against K562, Daudi and HT-29 cells. Isolated monocytes responded also to those peptides with a release of IL-1 and TNF alpha and an increase of cytotoxicity against HT-29 cells. It was found that the active peptides had the following structural pattern: a length of at least 15 amino-acid residues with a proline at position 6, valine, leucine, isoleucine, glycine, alanine or lysine at position 2, and glutamic or aspartic acid at position 11. Replacement of Pro at position 6 with any other residue turned the peptide inactive. Replacement of residues at positions 2 and 11 with amino-acid residues other than those required for activity resulted in compounds with a marked decrease in the immunomodulating properties described, or lacking these properties altogether.

Activation of human lymphomononuclear cells by peptides derived from extracellular matrix proteins

A series of peptides of 15 amino acids with sequences contained in human extracellular matrix (ECM) proteins (fibronectin, laminin A, laminin B1, tenascin, undulin, alpha 1-chain of type IV and VIII collagen and alpha 2-chain of type VIII collagen) have been synthesized. The selected structures conformed to the following pattern: (i) Pro at position 6, (ii) Leu, Lys, Ile, Val, Ala or Gly at position 2, (iii) Glu or Asp at position 11. Fibronectin and the indicated peptides, when present in cultures of lymphomononuclear cells from healthy donors, promoted stimulation of monocytes manifested by a release of IL-1 alpha, IL-beta, IL-6 and TNF alpha; an increase in the percentage of cells expressing CD14, CD16, CD11b and CD14/CD16; an increase in cytotoxicity against HT-29. Cytotoxicity against K562 and Daudi cells (targets of NK and LAK cells) was also observed together with an increase in the percentage of cells expressing CD56, CD56/CD16 (corresponding to NK cells), and CD56/CD8 (corresponding to NK-like lymphocytes), indicating a stimulation of lymphocytes. Activated monocytes and lymphocytes contained a large number of granules with DNAse activity. These results suggest that at least some of the immunological properties of ECM proteins could be accounted for by motifs fulfilling a characteristic sequence pattern shared by all of them.

Specific protein nitration in nitric oxide-induced apoptosis of human monocytes

The sustained overproduction of nitric oxide (NO) observed in inflammatory conditions can contribute to cell demise by affecting apoptosis. Nitration of tyrosine residues occurs in a range of diseases involving macrophage activation. Since NO induces apoptosis in monocytes/macrophages, we tested the hypothesis that nitration of specific proteins could result in apoptotic cell death. The peroxynitrite generator SIN-1 promoted apoptosis in monocytes based on oligonucleosomal DNA fragmentation, caspase-3 and -9 activation, Bcl-2 depletion and accumulation of Bax and p53 proteins. We also found that the signaling pathway triggered by SIN-1 was initiated through tyrosine kinase and Rac activation and resulted in increased JNK and p38 activities. Among the tyrosine-nitrated proteins, Rac and Lyn were identified. Using specific inhibitors for different signaling and effector molecules involved in the apoptotic process we demonstrate that NO, via protein-nitration, could play an important role in controlling the inflammatory response by regulation of monocyte homeostasis.

Monopathogenic vs multipathogenic explanations of pemphigus pathophysiology

This viewpoint highlights major, partly controversial concepts about the pathogenesis of pemphigus. The monopathogenic theory explains intra‐epidermal blistering through the “desmoglein (Dsg) compensation” hypothesis, according to which an antibody‐dependent disabling of Dsg 1‐ and/or Dsg 3‐mediated cell–cell attachments of keratinocytes (KCs) is sufficient to disrupt epidermal integrity and cause blistering. The multipathogenic theory explains intra‐epidermal blistering through the “multiple hit” hypothesis stating that a simultaneous and synchronized inactivation of the physiological mechanisms regulating and/or mediating intercellular adhesion of KCs is necessary to disrupt epidermal integrity. The major premise for a multipathogenic theory is that a single type of autoantibody induces only reversible changes, so that affected KCs can recover due to a self‐repair. The damage, however, becomes irreversible when the salvage pathway and/or other cell functions are altered by a partnering autoantibody and/or other pathogenic factors. Future studies are needed to (i) corroborate these findings, (ii) characterize in detail patient populations with non‐Dsg‐specific autoantibodies, and (iii) determine the extent of the contribution of non‐Dsg antibodies in disease pathophysiology.

Inhibition of FAK prevents blister formation in the neonatal mouse model of pemphigus vulgaris.

Abstract:  Pemphigus vulgaris (PV) is an autoimmune blistering skin disease characterized by suprabasal acantholysis and by autoantibodies against desmoglein 3 localized on desmosomes. In addition, caspases also seem to participate in this blistering disease. Focal adhesion kinase (FAK) is a non‐receptor tyrosine kinase involved in cytoskeleton remodelling and formation and disassembly of cell adhesion structures. We have previously demonstrated that HER (human epidermal growth factor receptor related) isoforms, Src (Rous sarcoma) and mammalian target of rapamycin (mTOR), three molecules implicated in signalling processes, take part in suprabasal acantholysis and apoptosis induced by PV‐IgG in a mouse model. Our aim was to investigate whether upregulation of FAK is implicated in the development of PV lesions. Herein, using a mouse model, PV‐IgG administration showed an increased level of FAK phosphorylated on 397 and 925 tyrosine residues in the basal layer of epidermis. When mice were pretreated with a FAK inhibitor (FI), the acantholysis of the basal layer of epidermis was absent. More interestingly, we observed that phosphorylated FAK (Y397/925) decreased when HER isoforms, Src, mTOR and pan‐caspases inhibitors were employed before PV‐IgG administration. In addition, pretreatment with the FI before PV‐IgG injection prevented the changes in both Bax and Bcl‐2 expression and caspase‐9 and caspase‐3 activities induced by PV‐IgG. Finally, FI reduced the expression of phosphorylated Src and mTOR in the basal cells of epidermis. In conclusion, our data reveal a novel role of phosphorylated FAK (Y397/925) in PV development involving HER isoforms, Src and mTOR kinases.