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* This work was supported, in whole or in part, by National Institutes of Health Grants AR053505 and AR057001 from NIAMS (to A. S. P.) and Grant AI074957 from NIAID (to J. M. P.). This work was also supported by Penn Skin Disease Research Core Grant AR057217. The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S4 and Tables S1 and S2.
Pemphigus vulgaris (PV) is a potentially fatal blistering disease characterized by autoantibodies against the desmosomal adhesion protein desmoglein (Dsg) 3. Whether autoantibody steric hindrance or signaling through pathways such as p38 MAPK is primary in disease pathogenesis is controversial. PV mAbs that cause endocytosis of Dsg3 but do not dissociate keratinocytes because of compensatory adhesion by Dsg1 do not activate p38. The same mAbs plus exfoliative toxin to inactivate Dsg1 but not exfoliative toxin alone activate p38, suggesting that p38 activation is secondary to loss of adhesion. Mice with epidermal p38α deficiency blister after passive transfer of PV mAbs; however, acantholytic cells retain cell surface Dsg3 compared with wild-type mice. In cultured keratinocytes, p38 knockdown prevents loss of desmosomal Dsg3 by PV mAbs, and exogenous p38 activation causes internalization of Dsg3, desmocollin 3, and desmoplakin. p38α MAPK is therefore not required for the loss of intercellular adhesion in PV, but may function downstream to augment blistering via Dsg3 endocytosis. Treatments aimed at increasing keratinocyte adhesion could be used in conjunction with immunosuppressive agents, potentially leading to safer and more effective combination therapy regimens.
). The pathognomonic histologic finding in PV is suprabasal acantholysis, or the detachment of intact keratinocytes from each other because of loss of intercellular adhesion. A characteristic clinical finding in PV is Nikolsky's sign, in which blisters can be induced in otherwise normal-appearing skin by applying pressure or mechanical shear force, reflecting the loss of intercellular adhesion even in skin without overt blisters (
). The anatomic site of blister formation is thought to be due to the tissue-specific expression patterns of different Dsg isoforms, also known as the desmoglein compensation theory. Dsg3 is expressed by basal keratinocytes of mucosa and skin, whereas Dsg1 is expressed by basal keratinocytes in skin but not mucosa (
). Therefore, patients with Dsg3 autoantibodies demonstrate blistering in the mucosa, where compensatory adhesion by Dsg1 is not present (mucosal-dominant PV). In some patients who progress to develop Dsg1 in addition to Dsg3 autoantibodies, suprabasal blisters appear in both the mucosa and skin (mucocutaneous PV) (
Epitope mapping studies have shown that pathogenic PV autoantibodies preferentially bind the amino-terminal domain of Dsg3 that is predicted to form the transadhesive interface between cells, based on analogy to ultrastructural models for the classical cadherins (
), although it has been debated whether endocytosis is the result or the cause of the loss of intercellular adhesion. Additionally, other studies have shown that inhibition of various signaling pathways can prevent blister formation in the passive transfer mouse model, questioning the primacy of steric hindrance in disease pathogenesis (
p38 is a member of the MAPK family, best characterized for its role in regulating the cellular response to a variety of infectious and environmental stimuli, including thermal, osmotic, and oxidative stress (
). The four isoforms share structural and biochemical properties, including a dual Thr/Tyr motif activated by phosphorylation. Only the α and β isoforms are susceptible to inhibition by pyridinyl imidazoles such as SB202190 (
). To further define the role of p38 in desmosomal cell adhesion and PV, we studied the effects of pathogenic PV mAbs in normal and p38-silenced primary human keratinocytes as well as mice with a K14-Cre-mediated deletion of p38α MAPK in the epidermis.
The relationship between p38 activation, Dsg3 endocytosis, and the loss of intercellular adhesion in PV has been difficult to determine. A major problem in comparing data from different laboratories is that PV IgG isolated from patients' sera is heterogeneous, containing a mixture of pathogenic and nonpathogenic mAbs that can recognize Dsg3 as well as other desmosomal cadherins, with serum titers varying between different patients and even within the same patient over time. We have previously shown that recombinant nonpathogenic anti-Dsg3 mAbs cloned from a PV patient bind Dsg3 but do not cause its endocytosis or interfere with its incorporation into the desmosome; in contrast, recombinant pathogenic PV mAbs cause endocytosis of newly synthesized Dsg3, impairing desmosome assembly and consequently desmosomal adhesion (
). The advantage of our approach is that we use well characterized pathogenic PV mAbs with nonpathogenic PV mAbs as controls, thereby avoiding potential heterogeneous effects caused by polyclonal patient IgG. Additionally, all prior studies on the role of p38 MAPK in PV have been performed using only pyridinyl imidazole inhibitors, which can inhibit kinases other than p38 (
). In the current study, we have more specifically investigated the role of p38 MAPK in PV using p38 gene silencing and targeted deletion of p38α MAPK in mouse epidermal keratinocytes.
Our studies indicate that p38α MAPK is not required for the loss of intercellular adhesion in PV. Mice with a targeted deletion of p38α MAPK in the epidermis develop suprabasal blisters after passive transfer of low and high doses of PV mAbs (Fig. 2 and supplemental Table S2), indicating that the loss of Dsg3-mediated adhesion is not p38α-dependent. Furthermore, Fig. 1 indicates that Dsg3 endocytosis does not require p38 activation and suggests that p38 MAPK activation is secondary to the loss of intercellular adhesion. Taken together, these findings suggest that PV mAbs directly inhibit Dsg3-mediated adhesion, a model that has previously been supported by in vitro assays (
). Fig. 2C indicates that only focal activation of p38δ was observed in lesional keratinocytes of p38α-deficient mouse epidermis and that no activation was observed in perilesional keratinocytes, suggesting, similar to Fig. 1, that p38 activation is secondary to the loss of cell adhesion. Additionally, pyridinyl imidazole compounds such as SB202190 only inhibit p38α and p38β isoforms. However, definitive proof would require testing of mice with targeted deletion or inactivation of all p38 isoforms.
An unexpected finding in our study was that targeted deletion of p38α MAPK led to significant differences in the relative levels of cell surface Dsg3 observed in keratinocytes within the blister cavity (Fig. 2). For diagnostic studies on PV patients, direct immunofluorescence studies are performed on perilesional rather than lesional (i.e. blistered) epidermis because cell surface autoantibody staining is disrupted in keratinocytes within the blister cavity because of pathogenic autoantibody internalization. Similar to findings in PV patients, WT mice injected with pathogenic PV mAbs demonstrated loss of the linear cell surface IgG and Dsg3 staining pattern in lesional keratinocytes (Fig. 2D). In contrast, p38α-deficient keratinocytes within the blister cavity retained a significantly higher proportion of cell surface Dsg3 (Fig. 2E). These findings again support the model that the loss of intercellular adhesion does not depend on Dsg3 endocytosis. However, these data also indicate that Dsg3 endocytosis in the in vivo mouse model is impaired in the absence of p38α MAPK.
To further explore the relationship between p38 and Dsg3 endocytosis, we examined the effects of p38 activation, p38 inhibition, and p38 gene silencing in PHEK. If p38 activation is upstream of Dsg3 endocytosis, then exogenous activation of p38 should cause Dsg3 endocytosis. p38 MAPK can be activated by the protein synthesis inhibitor anisomycin and also by oxidative stress induced by H2O2. Both anisomycin and oxidative stress caused internalization of cell surface Dsg3, Dsc3, and desmoplakin (Fig. 3), with no significant effect on the localization of the adherens junction protein E-cadherin at the same time points. The effects of anisomycin and H2O2 on Dsg3 internalization are likely due to p38 activation, as p38 inhibition by SB202190 blocked these effects (Fig. 4A).
Inhibition of p38α and p38β isoforms by SB202190 also at least in part blocked the pathogenic PV mAb-induced internalization of Dsg3. Similar findings using pyridinyl imidazole inhibitors and PV patient IgG have been described previously (
). To more specifically determine the dependence of H2O2- and PV mAb-induced loss of desmosomal Dsg3 on p38, we silenced total p38 expression in primary human keratinocytes. Knockdown of p38 expression rescued the PV mAb-induced depletion of desmosomal Dsg3 and, to a lesser extent, the oxidative stress-induced depletion of desmosomal Dsg3 (Fig. 4B).
Collectively, our studies support both p38-independent and p38-dependent mechanisms of pathogenicity in PV, leading to a “two-hit” model of disease (depicted in Fig. 5). PV mAbs directly cause the initial pathogenic event in PV: the loss of Dsg-mediated adhesion (Nikolsky's sign), which is p38-independent. Fig. 1C suggests that p38 activation is secondary to the loss of cell adhesion and also indicates a p38-independent mechanism of Dsg3 endocytosis. In contrast, FIGURE 3, FIGURE 4 support a p38-dependent mechanism for Dsg3 endocytosis. A significant difference between these two mechanisms is that pathogenic PV mAbs cause endocytosis of Dsg3 but not Dsc3 (
), whereas exogenous activation of p38 leads to simultaneous loss of both cell surface Dsg3 and Dsc3. Pathogenic PV autoantibodies cause internalization of Dsg3 into an early endosomal compartment by a clathrin- and dynamin-independent mechanism (
). No significant colocalization of Dsg3 and early endosomal marker EEA-1 was observed in H2O2-treated keratinocytes, suggesting that these pathways may indeed differ. Future studies will aim to further elucidate the molecules and pathways regulating the turnover of cell surface Dsg3 under physiologic and pathophysiologic conditions.
The two-hit model is congruent with several observations on disease. First, it explains why p38 activation in a variety of skin diseases such as psoriasis does not lead to skin blistering (although loss of cell adhesion is observed in some skin cancers, such as acantholytic squamous cell carcinomas). It is also compatible with desmoglein compensation theory in that PV mAb-induced loss of desmoglein adhesion determines the site of pathology, with blister formation augmented by subsequent p38 activation. A prediction of the model is that p38 inhibition would ameliorate only p38-dependent pathologic processes (depicted on the right side of Fig. 5), which could be offset by increasing titers of pathogenic PV mAb (depicted on the left side of Fig. 5). In other words, clinical p38 inhibitors may work to prevent spontaneous blister formation but may not prevent skin fragility (Nikolsky positivity), particularly in severe cases with high titers of circulating pathogenic autoantibodies.
In summary, our data suggest that p38 activation and Dsg3 endocytosis are secondary to the loss of intercellular adhesion caused by pathogenic PV autoantibodies. Activation of p38 subsequently disrupts cell surface desmosomal cadherin localization, likely augmenting blister formation in PV. Targeting of p38 represents a novel treatment strategy for PV, which aims to block the target-organ pathologic response (i.e. increase keratinocyte adhesion) rather than generally suppress host immunity. Increased keratinocyte adhesion is thought to underlie the rapid therapeutic response of pemphigus patients to corticosteroids (
), although corticosteroids also cause immunosuppression and hyperglycemia, which are undesirable in patients with widespread blistering and risk of sepsis. Further study of pathways regulating keratinocyte adhesion may lead to safe and effective skin-targeted therapies for this life-threatening disorder.
We thank Andrea Stout, Elias Ayli, Tzvete Dentchev, John Seykora, and John Stanley for reagents, technical support, and scientific discussions.