IL2 Inducible T-cell Kinase, a Novel Therapeutic Target in Melanoma.

PURPOSE: IL2 inducible T-cell kinase (ITK) promoter CpG sites are hypomethylated in melanomas compared with nevi. The expression of ITK in melanomas, however, has not been established and requires elucidation. EXPERIMENTAL DESIGN: An ITK-specific monoclonal antibody was used to probe sections from deidentified, formalin-fixed paraffin-embedded tumor blocks or cell line arrays and ITK was visualized by IHC. Levels of ITK protein differed among melanoma cell lines and representative lines were transduced with four different lentiviral constructs that each contained an shRNA designed to knockdown ITK mRNA levels. The effects of the selective ITK inhibitor BI 10N on cell lines and mouse models were also determined. RESULTS: ITK protein expression increased with nevus to metastatic melanoma progression. In melanoma cell lines, genetic or pharmacologic inhibition of ITK decreased proliferation and migration and increased the percentage of cells in the G0-G1 phase. Treatment of melanoma-bearing mice with BI 10N reduced growth of ITK-expressing xenografts or established autochthonous (Tyr-Cre/Pten(null)/Braf(V600E)) melanomas. CONCLUSIONS: We conclude that ITK, formerly considered an immune cell-specific protein, is aberrantly expressed in melanoma and promotes tumor development and progression. Our finding that ITK is aberrantly expressed in most metastatic melanomas suggests that inhibitors of ITK may be efficacious for melanoma treatment. The efficacy of a small-molecule ITK inhibitor in the Tyr-Cre/Pten(null)/Braf(V600E) mouse melanoma model supports this possibility.

Targeting O-glycosyltransferase (OGT) to promote healing of diabetic skin wounds.

Non-healing wounds are a significant source of morbidity. This is particularly true for diabetic patients, who tend to develop chronic skin wounds. O-GlcNAc modification of serine and threonine residues is a common regulatory post-translational modification analogous to protein phosphorylation; increased intracellular protein O-GlcNAc modification has been observed in diabetic and hyperglycemic states. Two intracellular enzymes, UDP-N-acetylglucosamine-polypeptide ß-N-acetylglucosaminyl transferase (OGT) and O-GlcNAc-selective N-acetyl-ß-D-glucosaminidase (OGA), mediate addition and removal, respectively, of N-acetylglucosamine (GlcNAc) from intracellular protein substrates. Alterations in O-GlcNAc modification of intracellular proteins is linked to diabetes, and the increased levels of protein O-GlcNAc modification observed in diabetic tissues may in part explain some of the observed underlying pathophysiology that contributes to delayed wound healing. We have previously shown that increasing protein O-GlcNAc modification by overexpression of OGT in murine keratinocytes results in elevated protein O-GlcNAc modification and a hyperadhesive phenotype. This study was undertaken to explore the hypothesis that increased O-GlcNAc modification of cellular proteins in diabetic skin could contribute to the delayed wound healing observed in patients with diabetic skin ulcers. In the present study, we show that human keratinocytes cultured under hyperglycemic conditions display increased levels of O-GlcNAc modification as well as a delay in the rate of wound closure in vitro. We further show that specific knockdown of OGT by RNA interference (RNAi) reverses this effect, thereby opening up the opportunity for OGT-targeted therapies to promote wound healing in diabetic patients.

A pathophysiologic role for epidermal growth factor receptor in pemphigus acantholysis.

The pemphigus family of autoimmune bullous disorders is characterized by autoantibody binding to desmoglein 1 and/or 3 (dsg1/dsg3). In this study we show that EGF receptor (EGFR) is activated following pemphigus vulgaris (PV) IgG treatment of primary human keratinocytes and that EGFR activation is downstream of p38 mitogen-activated protein kinase (p38). Inhibition of EGFR blocked PV IgG-triggered dsg3 endocytosis, keratin intermediate filament retraction, and loss of cell-cell adhesion in vitro. Significantly, inhibiting EGFR prevented PV IgG-induced blister formation in the passive transfer mouse model of pemphigus. These data demonstrate cross-talk between dsg3 and EGFR, that this cross-talk is regulated by p38, and that EGFR is a potential therapeutic target for pemphigus. Small-molecule inhibitors and monoclonal antibodies directed against EGFR are currently used to treat several types of solid tumors. This study provides the experimental rationale for investigating the use of EGFR inhibitors in pemphigus.

The C5a receptor on mast cells is critical for the autoimmune skin-blistering disease bullous pemphigoid.

Bullous pemphigoid (BP) is an autoimmune skin-blistering disease characterized by the presence of autoantibodies against the hemidesmosomal proteins BP230 and BP180. In the IgG passive transfer mouse model of BP, subepidermal blistering is triggered by anti-BP180 antibodies and depends on the complement system, mast cell (MC) degranulation, and neutrophil infiltration. In this study, we have identified the signaling events that connect the activation of the complement system and MC degranulation. We found that mice deficient in MCs or the C5a receptor (C5aR) injected with pathogenic anti-BP180 IgG failed to develop subepidermal blisters and exhibited a drastic reduction in p38 MAPK phosphorylation compared with WT mice. Local reconstitution with MCs from WT but not C5aR-deficient mice restored high levels of p38 MAPK phosphorylation and subepidermal blistering in MC-deficient mice. Local injection of recombinant C5a induced phosphorylation of p38 MAPK in WT but not MC-deficient mice. Cultured mouse MCs treated with recombinant C5a exhibited a significant increase in p38 MAPK phosphorylation and MC degranulation. Taken together, these data demonstrate that C5a interacts with C5aR on MCs and that this C5a-C5aR interaction triggers activation of the p38 MAPK pathway, subsequent MC degranulation, and ultimately BP blistering.

p38MAPK signaling and desmoglein-3 internalization are linked events in pemphigus acantholysis.

Pemphigus vulgaris (PV) is an autoimmune blistering disease in which antibodies against the desmosomal cadherin, DSG3 (desmoglein-3), cause acantholysis. It has become increasingly clear that loss of cell-cell adhesion in PV is a complex and active process involving multiple signaling events such as activation of p38MAPK. It has also been demonstrated that incubating keratinocytes with PV IgG causes a redistribution of DSG3 from the cell surface to endosomes, which target these proteins for degradation. This study was undertaken to determine the relationship between p38MAPK and DSG3 endocytosis in pemphigus. In this work, we confirm that PV IgG causes internalization of cell-surface DSG3 into endosomes (as early as 4 h), which are then depleted from both detergent-soluble and detergent-insoluble pools. Cell-surface DSG3 internalization and depletion from both the detergent-soluble and detergent-insoluble fractions were blocked by the p38MAPK inhibitor SB202190. These data suggest that p38MAPK is capable of regulating PV IgG-mediated DSG3 internalization and that previously isolated mechanistic observations may be linked to a common pathway by which pemphigus autoantibodies lead to acantholysis.

Biphasic activation of p38MAPK suggests that apoptosis is a downstream event in pemphigus acantholysis.

In pemphigus vulgaris and pemphigus foliaceus (PF), autoantibodies against desmoglein-3 and desmoglein-1 induce epidermal cell detachment (acantholysis) and blistering. Activation of keratinocyte intracellular signaling pathways is emerging as an important component of pemphigus IgG-mediated acantholysis. We previously reported activation of p38 mitogen-activated protein kinase (MAPK) in response to pathogenic pemphigus vulgaris and PF IgG. Inhibition of p38MAPK blocked pemphigus IgG-induced cytoskeletal reorganization in tissue culture and blistering in pemphigus mouse models. We now extend these observations by demonstrating two peaks of p38MAPK activation in pemphigus tissue culture and mouse models. Administration of the p38MAPK inhibitor SB202190 before PF IgG injection blocked both peaks of p38MAPK phosphorylation and blister formation, consistent with our previous findings; however, administration of the inhibitor 4 h after PF IgG injection blocked only the later peak of p38MAPK activation but failed to block blistering. Examination of the temporal relationship of p38MAPK phosphorylation and apoptosis showed that apoptosis occurs at or after the second peak of p38MAPK activation. The time course of p38MAPK activation and apoptotic markers, as well as the ability of inhibitors of p38MAPK to block activation of the proapoptotic proteinase caspase-3, suggest that activation of apoptosis is downstream to, and a consequence of, p38MAPK activation in pemphigus acantholysis. Furthermore, these observations suggest that the earlier peak of p38MAPK activation is part of the mechanism leading to acantholysis, whereas the later peak of p38MAPK and apoptosis may not be essential for acantholysis.

Autoantibodies in the autoimmune disease pemphigus foliaceus induce blistering via p38 mitogen-activated protein kinase-dependent signaling in the skin.

Pemphigus foliaceus (PF) is a human autoimmune blistering disease in which a humoral immune response targeting the skin results in a loss of keratinocyte cell-cell adhesion in the superficial layers of the epidermal epithelium. In PF, desmoglein-1-specific autoantibodies induce blistering. Evidence is beginning to accumulate that activation of signaling may have an important role in the ability of pathogenic pemphigus IgGs to induce blistering and that both p38 mitogen-activated protein kinase (MAPK) and heat shock protein (HSP) 27 are part of this signaling pathway. This study was undertaken to investigate the ability of PF IgGs to activate signaling as well as the contribution of this signaling pathway to blister induction in an in vivo model of PF. Phosphorylation of both p38 MAPK and HSP25, the murine HSP27 homolog, was observed in the skin of PF IgG-treated mice. Furthermore, inhibition of p38 MAPK blocked the ability of PF IgGs to induce blistering in vivo. These results indicate that PF IgG-induced blistering is dependent on activation of p38 MAPK in the target keratinocyte. Rather than influencing the immune system, limiting the autoantibody-induced intracellular signaling response that leads to target end-organ damage may be a more viable therapeutic strategy for the treatment of autoimmune diseases. Inhibition of p38 MAPK may be an effective strategy for the treatment of PF.

p38MAPK inhibition prevents disease in pemphigus vulgaris mice.

Pemphigus vulgaris (PV) is a life-threatening autoimmune blistering skin disease characterized by detachment of keratinocytes (acantholysis). It has been proposed that PV IgG might trigger signaling and that this process may lead to acantholysis. Indeed, we recently identified a rapid and dose-dependent phosphorylation of p38 mitogen-activated protein kinase (p38MAPK) and heat shock protein (HSP) 27 after binding of PV antibodies to cultured keratinocytes. In human keratinocyte cultures, inhibitors of p38MAPK prevented PV IgG-induced phosphorylation of HSP27 and, more importantly, prevented the early cytoskeletal changes associated with loss of cell-cell adhesion. This study was undertaken to (i) determine whether p38MAPK and HSP25, the murine HSP27 homolog, were similarly phosphorylated in an in vivo model of PV and (ii) investigate the potential therapeutic use of p38MAPK inhibition to block blister formation in an animal model of PV. We now report that p38MAPK inhibitors prevented PV blistering disease in vivo. Targeting the end-organ by inhibiting keratinocyte desmosome signaling may be effective for treating desmosome autoimmune blistering disorders.

Stabilization of plakoglobin and enhanced keratinocyte cell-cell adhesion by intracellular O-glycosylation.

O-Glycosylation modifies and regulates a variety of intracellular proteins. Plakoglobin, which functions in both cell-cell adhesion and signal transduction, is modified by O-glycosylation; however, the significance is unknown. To investigate the functional consequence of plakoglobin O-glycosylation, we cloned and overexpressed in keratinocytes murine O-GlcNAc transferase (mOGT). Over expression of mOGT in murine keratinocytes resulted in (i) glycosylation of plakoglobin and (ii) increased levels of plakoglobin due to post-translational stabilization of plakoglobin. Additionally, overexpression of mOGT in keratinocytes correlated with increased staining for cell-cell adhesion proteins and greater cell-cell adhesion. These observations suggest that O-glycosylation functions to regulate the post-translational stability of plakoglobin and keratinocyte cell-cell adhesion.

Desmosome signaling. Inhibition of p38MAPK prevents pemphigus vulgaris IgG-induced cytoskeleton reorganization.

In the human autoimmune blistering disease pemphigus vulgaris (PV) pathogenic antibodies bind the desmosomal cadherin desmoglein-3 (dsg3), causing epidermal cell-cell detachment (acantholysis). Pathogenic PV dsg3 autoantibodies were used to initiate desmosome signaling in human keratinocyte cell cultures. Heat shock protein 27 (HSP27) and p38MAPK were identified as proteins rapidly phosphorylated in response to PV IgG. Inhibition of p38MAPK activity prevented PV IgG-induced HSP27 phosphorylation, keratin filament retraction, and actin reorganization. These observations suggest that PV IgG binding to dsg3 activates desmosomal signal transduction cascades leading to (i) p38MAPK and HSP27 phosphorylation and (ii) cytoskeletal reorganization, supporting a mechanistic role for signaling in PV IgG-induced acantholysis. Targeting desmosome signaling via inhibition of p38MAPK and HSP27 phosphorylation may provide novel treatments for PV and other desmosome-associated blistering diseases.

Keratinocyte adherens junctions initiate nuclear signaling by translocation of plakoglobin from the membrane to the nucleus.

Because changes in cell-cell adhesion have profound effects on cellular behavior, we hypothesized a link between the adhesion and signaling functions of plakoglobin and beta-catenin. To investigate the existence of adherens-junction-mediated signaling, we used peroxovanadate to tyrosine phosphorylate plakoglobin and beta-catenin and to dissociate adherens junctions. The distribution of plakoglobin and beta-catenin was determined by immunofluorescence, western blot analysis, pulse-chase radiolabeling, and biochemical subcellular fractionation. Coimmunoprecipitation studies from nuclear fractions, gel-shift assays, and transient transfections with T cell factor (TCF)/lymphoid enhancer factor (LEF) optimized promoter reporter constructs were used to investigate the ability of plakoglobin and beta-catenin that had redistributed from the membrane to the nucleus to form functional transcriptional regulatory complexes with TCF/LEF family member transcription factors. Tyrosine phosphorylation of plakoglobin and beta-catenin resulted in their rapid translocation from the cell membrane to the nucleus. Nuclear translocation was associated with increased plakoglobin and decreased beta-catenin binding to nuclear TCF/LEF and downregulation of gene transcription from TCF/LEF reporter constructs. These results are consistent with a signaling pathway initiated by structural changes in the adherens junction in which adherens-junction-derived plakoglobin regulates nuclear transcription by antagonizing the binding of beta-catenin to TCF/LEF proteins.