ORIGINAL ARTICLE

Desmoglein 3-Dependent Signaling Regulates Keratinocyte Migration and Wound Healing Vera Ro¨tzer1,3, Eva Hartlieb1,3, Julia Winkler1, Elias Walter1, Angela Schlipp1, Miklo´s Sardy2, Volker Spindler1 and Jens Waschke1

The desmosomal transmembrane adhesion molecules 3 (Dsg3) and 3 (Dsc3) are required for strong keratinocyte cohesion. Recently, we have shown that Dsg3 associates with p38 mitogen- activated kinase (p38MAPK) and suppresses its activity. Here, we further investigated the role of Dsg3-dependent control of p38MAPK function. Dsg3-deficient mice display recurrent spontaneously healing skin erosions. In lesional and perilesional biopsies, p38MAPK activation was detectable compared with control animals. This led us to speculate that Dsg3 regulates wound repair in a p38MAPK-dependent manner. Indeed, scratch-wounded keratinocyte monolayers exhibited p38MAPK activation and loss of Dsg3 in cells lining the wound edge. Human keratinocytes after silencing of Dsg3 as well as primary cells isolated from Dsg3 knockout animals exhibited accelerated migration, which was further corroborated in an ex vivo skin outgrowth assay. Importantly, migration was efficiently blocked by inhibition of p38MAPK, indicating that p38MAPK mediates the effects observed upon loss of Dsg3. In line with this, we show that levels of active p38MAPK associated with Dsc3 are increased in Dsg3-deficient cells. These data indicate that Dsg3 controls a switch from an adhesive to a migratory keratinocyte phenotype via p38MAPK inhibition. Thus, loss of Dsg3 adhesion may foster wound closure by allowing p38MAPK-dependent migration. Journal of Investigative Dermatology (2016) 136, 301-310; doi:10.1038/JID.2015.380

INTRODUCTION (PV) (Stanley and Amagai, 2006). Here, loss of Dsg3 and (Dsg) together with (Dsc) are Dsg1 function results in reduced intercellular adhesion of þ Ca2 -dependent, -type transmembrane adhesion keratinocytes and epithelial cells leading to mucocutaneous molecules that are located inside and outside of blisters and erosions (Waschke and Spindler, 2014). A crucial (Delva et al., 2009). Inside desmosomes, these desmosomal signaling event elicited by pemphigus autoantibody binding are linked to the keratin filament cytoskeleton via a is the activation of p38 mitogen-activated protein kinase set of adapter such as plakoglobin, plakophilins, and (p38MAPK), the inhibition of which in turn prevents loss of desmoplakin. Extradesmosomal Dsg may be attached to actin cell cohesion in vitro and blocks blistering in mouse models filaments (Tsang et al., 2012). Four Dsg and three Dsc iso- (Berkowitz et al., 2005, 2006). Similarly, both depletion of forms exist, which show a tissue-specific distribution pattern. Dsg3 and specific interference with Dsg3 binding by mono- Dsg1/Dsc1 and Dsg3/Dsc3 are mainly restricted to stratifying clonal autoantibodies or small peptides yielded activation of epithelia of mucous membranes such as those of the oral p38MAPK (Hartlieb et al., 2014; Spindler et al., 2013). Thus, cavity and the vagina and are highly expressed in the Dsg3 binding suppresses p38MAPK activity, a function that is epidermis. Both Dsg1 and Dsg3 are targets of autoantibodies impaired under disease conditions. However, more physio- causing the autoimmune skin disease pemphigus vulgaris logic roles of Dsg3-dependent control of p38MAPK signaling are unknown.

1Institute of Anatomy and Cell Biology, Ludwig-Maximilians-Universita¨t Skin wound repair is a highly complex process and is Mu¨nchen, Munich, Germany; and 2Department of Dermatology and typically classified in three stages (Gurtner et al., 2008; Sun Allergology, Ludwig-Maximilians-Universita¨tMu¨nchen, Munich, Germany et al., 2014): inflammation, tissue formation, and remodel- 3 These authors contributed equally to this work. ing. Stage two, besides angiogenesis and extracellular matrix Correspondence: Jens Waschke, Institute of Anatomy and Cell Biology, generation, involves migration of keratinocytes to cover the Department I, LMU Munich, Pettenkoferstraße 11, 80336 Mu¨nchen, fibrin matrix initially formed at the wound bottom. It is well Germany. E-mail: [email protected] or Volker Spindler, established that in this regard p38MAPK activation governs Institute of Anatomy and Cell Biology, Department I, LMU Munich, Pettenkoferstraße 11, 80336 Mu¨nchen, Germany. E-mail: volker.spindler@ keratinocyte migration. After wounding, p38MAPK is acti- med.uni-muenchen.de vated at the wound edge (Lambert et al., 2010), a process that Abbreviations: Dsc3, desmocollin 3; Dsg3, desmoglein 3; ERK, extracellular may also be under the control of extracellular stimuli such as signal-regulated kinase; GFP, green fluorescent protein; MEK, murine hydrogen peroxide (Loo et al., 2011) or activation of toll-like epidermal keratinocytes; p38MAPK, p38 mitogen-activated protein kinase; receptors (Chen et al., 2013). To allow wound healing, the PKC, protein kinase C; PV, pemphigus vulgaris; PV-IgG, IgG fraction of PV patient; siRNA, small interfering RNA; wt, wild-type desmosomes of keratinocytes need to remodel (Kitajima, Received 12 December 2014; revised 1 September 2015; accepted 11 2014). In healthy conditions, desmosomes are stable struc- 2þ September 2015; accepted manuscript published online 30 September 2015 tures that resist depletion of extracellular Ca , which is

ª 2015 The Authors. Published by Elsevier, Inc. on behalf of the Society for Investigative Dermatology. www.jidonline.org 301 VRo¨tzer et al. Desmoglein 3 and Keratinocyte Migration

þ surprising given the Ca2 -dependent binding of desmosomal scratched monolayers of human keratinocytes (HaCaT cell cadherins. This condition, named “desmosomal hyper- line) displayed increased phospho-p38MAPK staining in- adhesion,” is rapidly reversed after wounding both in vitro tensity at the wound edge after 15 minutes that subsided until and in vivo (Thomason et al., 2012; Wallis et al., 2000). This 120 minutes (Figure 2a). This was further corroborated by process is only partially understood but requires the activa- Western blot analysis (Figure 2b). To demonstrate that tion of protein kinase C a (PKCa). Given the fact that p38MAPK is activated in Dsg3-dependent manner, we over- impairment of desmosomal adhesion (i.e., by anti-Dsg3 au- expressed green fluorescent protein (GFP)-tagged Dsg3 or toantibodies from pemphigus patients) induces both protein GFP alone in HaCaT cells and analyzed p38MAPK activity kinase C activation with subsequent loss of hyperadhesion as after scratching (Figure 2c). p38MAPK activation was pre- well as p38MAPK activation, we investigated the hypothesis vented in cells expressing GFP-Dsg3 (arrow), whereas non- that loss of Dsg3 contributes to the switch from an adhesive transfected cells or cells expressing GFP alone lining the to a migratory phenotype via p38MAPK signaling. wound edge displayed increased phospho-p38MAPK staining intensity after 30 minutes. This observation was supported by RESULTS experiments performed in keratinocytes isolated from wt and Loss of Dsg3 function induces p38MAPK activation in vivo Dsg3 knockout mice (murine epidermal keratinocytes [MEKs]) We have previously shown that silencing of Dsg3 results in (see Supplementary Figure S2, online). As observed before increased p38MAPK activity in human keratinocytes (Hartlieb et al., 2014), Dsg3-deficient MEKs demonstrate (Hartlieb et al., 2014). Here, we used Dsg3-deficient mice prominent phospho-p38MAPK membrane staining (red ar- suffering from mucocutaneous blistering and erosions (Koch rows), which was largely reduced in cells expressing Dsg3- et al., 1997). The skin lesions appeared to occur randomly GFP but not GFP as control (blue arrows in magnified re- over the entire body of adult Dsg3 knockout mice and were gions). Moreover, loss of membrane Dsg3 staining was often accompanied by patchy hair loss (Figure 1a). Skin observed 15 minutes after wounding in untreated keratino- erosions appeared to be more frequent compared with the cytes lining the wound edge, that is, at a time when p38MAPK initial description of the model and were not restricted to activation was evident (Figure 2d). In these cells, intracellular areas prone to scratching such as the snout, which may be a vesicles containing Dsg3 appeared (red arrows). However, result of different housing or mating conditions. Interestingly, Dsg3 depletion was more pronounced at later time points, the erosions were generally prone to spontaneous wound when cells at the migration front displayed drastically reduced healing. Typically, after several days, the wounds were closed Dsg3 staining. These findings were also supported by Triton X- again and fur was regrown. We harvested biopsies of lesional 100-mediated fractionation. HaCaT monolayers displayed and nonlesional skin of knockout mice and sampled tissue increased Dsg3 content in the noncytoskeletal, Triton X-100- from the same regions in wild-type (wt) littermates as con- soluble pool starting at 15 minutes after wounding, whereas trols. Whereas in wt animals the levels of p38MAPK activity the amount in the cytoskeletal, -containing pool were low, they were increased in lesional areas of knockout was reduced (Figure 2e). This indicates that the cytoskeletal animals. Interestingly, p38MAPK activation was also detect- anchorage of Dsg3 is rapidly reduced after scratch wounding. able in nonlesional skin of most Dsg3-/- mice. An example of an ear lobe lesion is shown in Figure 1b and c. To further Loss of Dsg3 promotes keratinocyte migration via p38MAPK corroborate the effect of impaired Dsg3 function on signaling p38MAPK activity, we stained skin sections from PV patients To determine whether Dsg3 regulates keratinocyte migration, with a phospho-p38MAPK antibody. Compared with healthy we silenced Dsg3 in HaCaT cells using small interfering RNA control subjects, strong p38MAPK staining throughout the (siRNA). Keratinocytes transfected with nontarget siRNA and epidermis was detectable in skin sections of two PV patients treated with DMSO closed the scratch to roughly 50% of the (Figure 1d). Especially in areas of profound IgG fraction of PV initial size after 48 hours (Figure 3a and b), which was patient (PV-IgG) binding to the keratinocyte surface, blocked by p38MAPK inhibition using the p38MAPK-specific phospho-p38MAPK was found at the membrane, which is in inhibitor SB202190 at 30 mM. However, silencing of Dsg3 line with data demonstrating that phospho-p38MAPK can resulted in significantly increased migration speed compared associate with Dsg3 (Spindler et al., 2013). In addition, in with nontarget siRNA controls. This acceleration of gap samples from PV patient 2 cytoplasmic staining of p38MAPK closure was also prevented by p38MAPK inhibition. These was also detectable, which may correlate with the function of effects seemed to be largely dependent on migration and not p38MAPK to regulate actin and keratin filament architecture on cell division because mitomycin treatment did not reduce (Berkowitz et al., 2005; Mao et al., 2014; Spindler et al., migration of HaCaT cells (see Supplementary Figure S3a, 2013). Secondary antibody controls to rule out unspecific online). Western blots were run in parallel to migration signals are shown in Supplementary Figure S1 (see online). assays to ensure that silencing was detectable throughout the Taken together, loss of Dsg3 function in mice or humans was entire monitoring period after scratching (Supplementary associated with p38MAPK activation. Figure S3b). Furthermore, Dsg3 silencing did not affect pro- P38MAPK activation correlates with Dsg3 internalization in tein levels of other desmosomal proteins apart from a slight wounded keratinocyte monolayers reduction of plakoglobin (Supplementary Figure S3c). Dsg3-deficient mice show p38MAPK activation in the skin We repeated these experiments in wt and Dsg3-deficient and remarkably fast wound healing. We thus hypothesized MEKs. Again, Dsg3-deficient cells migrated significantly that Dsg3 and p38MAPK regulate migration of keratinocytes. faster compared with wt MEKs (Figure 3c and d). Similar to As observed before by another group (Lambert et al., 2010), the situation in HaCaT cells, both wt and Dsg3-deficient MEK

302 Journal of Investigative Dermatology (2016), Volume 136 VRo¨tzer et al. Desmoglein 3 and Keratinocyte Migration

Figure 1. p38MAPK is activated in lesional and nonlesional skin of Dsg3-/- mice and pemphigus patients. (a) Examples of rapid healing of skin erosions (arrows) and fur regrowth in three Dsg3-/- mice. (b) Comparison of lesional earlobe of a Dsg3-/- mouse þ þ with earlobe of a Dsg3 / animal. Hematoxylin and eosin staining of this lesion shows suprabasal epidermal blistering. Scale bar ¼ 50 mm. (c) Corresponding Western blot of samples obtained from the mouse in (b) in comparison with an earlobe of a wild-type littermate. Panels show þ þ representatives of six Dsg3 / and Dsg3-/- mice in total. (d) Phospho- p38MAPK staining in biopsy samples of two pemphigus vulgaris patients in comparison with healthy control subjects. Detection of human Fc was used to visualize antibody binding. *, epidermal blister; arrows, membrane- bound p38MAPK activation; dashed line, dermo-epidermal junction. Scale bar ¼ 20 mm.

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Figure 2. p38MAPK activation correlates with Dsg3 internalization in wounded keratinocyte monolayers. (a) Phospho-p38MAPK staining in keratinocytes lining the wound edge at indicated time points after the scratch was performed. Images are representatives of four independent experiments. Scale bar ¼ 20 mm. (b) Representative Western blot of p38MAPK activity in a time course after scratch wounding (n ¼ 4). (c) Phospho-p38MAPK staining in HaCaT cells expressing GFP-Dsg3 or GFP alone 30 minutes after scratching. Arrows indicate transfected cells lining the wound edge. Bar is 25 mm and insets: 1.2 magnification of indicated area (n ¼ 4). (d) Dsg3 staining of wound edge HaCaT keratinocytes at time points indicated after wounding. Boxed areas are represented in the magnified right panels. A representative of four independent experiments is shown. Bars are 20 mm. (e) Triton fractionation of HaCaT lysates into a cytoskeletal (Tx-100 insoluble) and a noncytoskeletal (Tx-100 soluble) pool. GAPDH served as loading control for the soluble pool, desmoplakin identifies the insoluble fraction (n ¼ 3).

migration speed was significantly reduced by p38MAPK in- tracking individual cells constantly over this time course, the hibition. To further corroborate these observations, we took trajectories of GFP-Dsg3-expressing keratinocytes were doughnut-shaped full-thickness biopsies from Dsg3 wt and shorter compared with GFP-expressing cells (Figure 5b), knockout animals into culture and measured the outgrowth yielding a significantly reduced migration velocity of keratinocytes (Figure 4a). Outgrowing cells indeed were of (Figure 5c). Taken together, these data show that the presence keratinocyte origin as they were positive for desmoplakin and of Dsg3 inhibits keratinocyte migration at least in part by Dsg3. In line with cell culture data, outgrowth from Dsg3- suppressing p38MAPK activity. deficient punch biopsies was significantly faster than from wt controls over a time period of 96 hours (Figure 4b and c). Mechanisms underlying keratinocyte migration in response Again, addition of SB202190 to the culture medium signifi- to loss of Dsg3 cantly slowed down migration both in wt and Dsg3 knockout We have shown previously that p38MAPK is located and biopsies. activated in a complex with Dsg3 and plakoglobin Vice versa, to demonstrate that the presence of Dsg3 in- (Spindler et al., 2013, 2014). To investigate the impact of hibits keratinocyte migration, we overexpressed GFP-tagged Dsg3 loss on the localization of p38MAPK, we immuno- Dsg3 or GFP alone in HaCaT cells. GFP-expressing cells precipitated Dsc3 in wt and Dsg3 knockout MEKs. We were dispersed throughout the monolayer next to the wound found that Dsc3 is associated with Dsg3 and p38MAPK. margin. This distribution pattern did not change until the Interestingly, in cells lacking Dsg3, the association of wound was closed (Figure 5a). In GFP-Dsg3-expressing cells, p38MAPK and especially of phospho-p38MAPK with Dsc3 despite relatively low transfection efficiency, a reduced was significantly increased (Figure 6a). This indicates that wound closure was detectable, indicating an effect of Dsg3 Dsg3 is not necessary for the association of p38MAPK to on collective migratory behavior. After a 20-hour incubation this complex but rather has an indirect function to suppress period, Dsg3-GFP-expressing cells were very rarely found at p38MAPK activity. the wound margin but rather appeared to be slower Because in Dsg3 knockout cells p38MAPK inhibition did compared with nonexpressing cells (Figure 5a). Indeed, by not completely prevent migration and it was shown that

304 Journal of Investigative Dermatology (2016), Volume 136 VRo¨tzer et al. Desmoglein 3 and Keratinocyte Migration

Figure 3. Migration is increased in Dsg3-depleted keratinocytes in a p38MAPK-dependent manner. (a) Representative bright-field images of HaCaT keratinocyte migration subjected to nontarget (siNT) or Dsg3 siRNA (siDSG3) and treated with solvent or with the p38MAPK inhibitor SB202190 (SB20). Bar is 150 mm. (b) Wound closure expressed as the remaining area uncovered by the cells. The scratch area at time point 0 hours was set to 1 (n ¼ 4e6; *P < 0.05, #P< 0.05 vs. respective DMSO condition). (c and d) Scratch-wound closure monitored over time in cells þ þ isolated from Dsg3 / and Dsg3-/- mice. Bar is 150 mm. (n ¼ 10e15 from 4 independent isolation þ þ procedures; *P < 0.05 vs. Dsg3 / DMSO, #P < 0.05 vs. respective DMSO condition).

p38MAPK is necessary but not sufficient to induce migration DISCUSSION (Li et al., 2001), we investigated other signaling molecules in Here, we show that Dsg3 controls keratinocyte migration and the context of Dsg3. Using ELISA-based measurements, we wound healing. Inhibition of p38MAPK signaling reduced detected RhoA activity to be significantly decreased in Dsg3 migration both in nontarget as well as Dsg3-depleted cells, knockout MEKs (Figure 6b), which is in line with previous underscoring the notion that regulation of p38MAPK activity results in which PV-IgG led to a reduction of RhoA activity is important in this process. This idea is supported by in vivo (Ro¨tzer et al., 2014; Waschke et al., 2006). Nevertheless, data showing that Dsg3 knockout animals exhibit higher Yersinia pseudotuberculosis cytotoxic necrotizing factor y to levels of active p38MAPK and remarkable erosion healing specifically activate RhoA at a previously determined con- capacities and is in line with increased keratinocyte centration (1 mM) did not affect scratch wound closure outgrowth in an ex vivo skin model. We found that Dsg3 and (Figure 6c), indicating that reduced RhoA activity in response p38MAPK are associated with Dsc3, which indicates that to loss of Dsg3 does not contribute to increased migration. In adhesion-dependent regulation of p38MAPK is controlled by contrast, inhibition of Src with 10 mM pp2 fully prevented both Dsg3 and Dsc3. In addition, together with the obser- wound closure both in wt and Dsg3 knockout MEKs vation that Dsc3 is more extensively associated with (Figure 6d). However, levels of active (Tyr416- p38MAPK in cells deficient for Dsg3, this helps to solve the phosphorylated) Src were not different in knockout versus conundrum of how cells deficient for Dsg3 can have more wt cells (Figure 6e), and in contrast to p38MAPK, no acti- activated p38MAPK at the cell membrane (Hartlieb et al., vation was induced by scratch wounding (data not shown). 2014), which is hard to explain if Dsg3 would be the only Thus, the function of Src for keratinocyte migration appears to membrane-associated protein capable of sequestering be independent of Dsg3. Finally, we investigated the impact p38MAPK. These data are consistent with the idea that Dsg3 of extracellular signal-regulated kinase (ERK) signaling. and Dsc3 together form an adhesion-dependent signaling Interestingly, the mitogen-activated protein kinase kinase in- complex in which p38MAPK activity is increased if the hibitor UO126 (5 mM) had no impact on migration of Dsg3 function of one of the two adhesion molecules is knockout keratinocytes but significantly slowed the wound compromised. closure in wt cells (Figure 6f). This indicates that migration in response to the mitogen-activated protein kinase kinase/ERK Dsg3 together with p38MAPK controls keratinocyte pathway is at least in part dependent on the presence of migration Dsg3. Altogether, these findings underscore that regulation of Migration of keratinocytes from the wound edge into the cell migration is complex and that several signaling pathways wounded area is an essential step in wound healing both are involved that in part are regulated by adhesion molecules in vitro and in vivo (Pastar et al., 2014). Several reports such as Dsg3. demonstrate that p38MAPK fosters keratinocyte migration. It

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Figure 4. Keratinocyte outgrowth is increased in Dsg3-knockout skin explants in a p38MAPK-dependent manner. (a) Cells migrating out of the explants express Dsg3 and desmoplakin. (b) Wound coverage of wild-type and Dsg3-deficient explants with or without SB202190 (SB20) treatment monitored over time (n ¼ 10e14 from 4 independent isolation procedures; *P < 0.05 vs. þ þ Dsg3 / DMSO, #P < 0.05 vs. respective DMSO condition). (c) Representative images taken at time points 0, 48, and 96 hours. Scale bar ¼ 150 mm.

was shown that extracellular factors such as hypoxia (Jiang re-epithelialize a wound (Krawczyk and Wilgram, 1973). et al., 2014), hydrogen peroxide (Loo et al., 2011), or toll- p38MAPK was also shown to regulate the activity of the like-receptor 4 binding (Chen et al., 2013) promote migra- EGFR (Bektas et al., 2013; Lambert et al., 2010), and, in turn, tion via p38MAPK. Apparently, scratch wounding of kerati- EGFR activation enhances keratinocyte migration (Ando and nocyte monolayers is sufficient to induce p38MAPK Jensen, 1993; Barrientos et al., 2008; Sperrhacke et al., activation and to subsequently modulate the expression of 2014). Thus, p38MAPK may be a central molecule in a specific relevant for migration (Fitsialos et al., 2007). signaling network regulating keratinocyte migration. We also Disruption of contact to the basement membrane, specif- checked for additional candidates other than p38MAPK that ically to laminin 5, was shown to promote p38MAPK acti- have been implicated both in migration and desmosome vation after wounding (Harper et al., 2005), indicating that function; however, none of the molecules appeared to be loss of cell-matrix adhesion triggers p38MAPK-dependent both regulated by Dsg3 and influencing keratinocyte migra- migration. Our data from this study identified that loss of tion in our experiments. Interestingly, ERK signaling- intercellular Dsg3-mediated binding also is a cue for dependent migration may require Dsg3, because in Dsg3 p38MAPK activation. A scenario is conceivable in which knockout cells inhibition of ERK failed to reduce scratch Dsg3 senses loss of cell-cell cohesion (i.e., induced by wound closure. scratch wounding) and relays this extracellular cue into intracellular activation of p38MAPK. Indeed, it was shown Wounded keratinocytes show remarkable similarities to recently that specific interference with Dsg3 binding via keratinocytes challenged with pemphigus autoantibodies small peptides targeting the adhesive Dsg3 domain triggers PV is a disease characterized by antibodies targeting Dsg3, p38MAPK activation (Spindler et al., 2013). Vice versa, it was which leads to keratinocyte dissociation. Although under demonstrated that p38MAPK promotes internalization and certain conditions loss of cell cohesion can occur indepen- depletion of Dsg3 (Jolly et al., 2010). Thus, initial loss of Dsg3 dent of intracellular signaling (Mao et al., 2014; Saito et al., binding may promote further depletion of Dsg3 levels at the 2012), numerous studies demonstrate the requirement of wound edge via p38MAPK-dependent signaling, which is signaling events (Kitajima, 2013; Spindler and Waschke, supported by our data showing that loss of Dsg3 was pro- 2014). Interestingly, the key signaling events in PV are nounced in cells lining the scratch at later time points remarkably similar to some of those observed in migrating (i.e., 360 minutes after wounding). This is not surprising keratinocytes at the wound margin. Binding of PV autoanti- given the fact that in addition to cell matrix contacts also bodies leads to a quick activation of p38MAPK, which cell-cell contacts such as desmosomes need to remodel to modulates intermediate filament uncoupling of desmosomes

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Figure 5. Cells overexpressing Dsg3 migrate slower. (a) Representative images of the same areas of monolayers expressing either GFP or Dsg3-GFP right after or 20 hours after scratch wounding. (b) Example trajectories of GFP or Dsg3-GFP- expressing cells individually tracked over 20 hours. (c) Analysis of trajectories for migration speed of individually tracked keratinocytes (n 36 from 6 independent transfections. *P < 0.05 vs. GFP).

and in turn enhances Dsg3 internalization and depletion. It observed at least by some authors (Chernyavsky et al., 2007; was suggested that primarily the nondesmosomal Dsg3 Pretel et al., 2009; Schulze et al., 2012), which again may be molecules present on the cell surface and not connected to under the control of p38MAPK (Bektas et al., 2013). Thus, in the keratin filaments sense the initial binding of autoanti- PV a set of key molecules show the same activity pattern as in bodies and relay this signal into the cell, which results in migrating keratinocytes (Kitajima, 2014). pronounced p38MAPK-dependent Dsg3 depletion (Mu¨ller Taken together, the physiologic role of the Dsg3/p38MAPK et al., 2008; Spindler and Waschke, 2014; Vielmuth et al., complex may be to regulate this switch depending on the þ 2015). Furthermore, PKC is activated after a rapid Ca2 actual state of epidermal integrity. In this scenario, binding of influx after PV-IgG application (Osada et al., 1997; Ro¨tzer pemphigus autoantibodies would shift keratinocytes toward þ et al., 2014; Seishima et al., 1995) that switches the Ca2 - the migratory program by triggering this switch. However, our independent, hyperadhesive desmosomes found in fully data also indicate that interference with p38MAPK signaling þ confluent keratinocytes to a Ca2 -dependent phenotype as a treatment option in pemphigus may be a double-edged (Cirillo et al., 2010; Dehner et al., 2014). This in turn favors sword because on one hand it may efficiently prevent blis- the loss of Dsg3, probably by reorganization of the desmo- ter formation, but on the other hand it may prevent kerati- somal plaque in response to desmoplakin phosphorylation nocytes from re-epithelializing wounds after skin blistering and via mechanisms involving plakophilin-1 (Dehner et al., occurred. This raises the interesting question whether treat- 2014; Spindler et al., 2011; Tucker et al., 2014). In addi- ment options targeting desmosomal adhesion in pemphigus tion, EGFR phosphorylation after PV-IgG binding was need to be evaluated differently for prophylaxis versus

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Figure 6. Mechanisms underlying keratinocyte migration in response to loss of Dsg3. (a) Immunoprecipitation of Dsc3 in isolated keratinocytes from þ þ Dsg3 / and Dsg3-/- animals. The heavy chain of the capturing antibody served as loading control. n ¼ 4, *P < 0.05 vs. immunoprecipitation þ þ Dsg3 / (n ¼ 4). (b) ELISA-based measurement of RhoA activity in wild- type and Dsg3 knockout mouse keratinocytes (n ¼ 5, *P < 0.05 vs. þ þ Dsg3 / ). (c) Scratch-wound assay using the RhoA-specific activator cytotoxic necrotizing factor y (n ¼ 11e14 from 4 independent isolation procedures). (d) Scratch- wound assay with the Src inhibitor pp2 (n ¼ 11e16 from 4 independent isolation procedures; *P < 0.05 vs. respective DMSO condition). (e) Western blot of Src activity using pTyr416 as readout (n ¼ 4). (f) Scratch- wound assay using the mitogen- activated protein kinase kinase inhibitor UO126 (n ¼ 11e15 from 4 independent isolation procedures; *P < 0.05 vs. respective DMSO condition).

treatment of skin blisters. Some strategies such as inhibition the clinical diagnosis was confirmed at the Department of Derma- of p38MAPK may be effective to prevent relapses but not for tology. Patients had given informed and written consent, and use of treatment of skin blisters. the samples was approved by the Ethics Committee of the Faculty of Medicine, Ludwig-Maximilians-Universita¨t, Munich, Germany MATERIALS AND METHODS (project no. 249-12). For detailed methods please refer to the Supplementary Materials In vitro and ex vivo wound healing cell migration assay (online). HaCaTs were seeded in 24-well plates with or without coverslips Cell culture and patient skin samples and transfected with nontarget or Dsg3 siRNA. After grown to The human immortalized keratinocyte cell line HaCaT was main- confluence within 4 days, cell monolayers were manually scratched þ tained in DMEM that constantly contained 1.8 mM Ca2 . Primary with a sterile pipette tip, washed with medium, and incubated with MEKs were isolated from Dsg3 knockout and corresponding wt DMEM supplemented with the indicated mediators for 24 to 48 þ þ neonatal mice as previously reported (Hartlieb et al., 2014). Cells hours. Dsg3 / and Dsg3-/- keratinocytes were switched to 1.8 mM þ þ were switched to 1.8 mM Ca2 24 hours before subjection to Ca2 12 hours before scratching. Migration was monitored over time Western blot analysis, migration assay, or Rho activity measurement. by bright-field microscopy (Axio Vert A1; Carl Zeiss AG, Oberko- Animal experiments were approved by the Regierung von Ober- chen, Germany). bayern. Diagnostic punch biopsy leftover samples taken from two For detection of p38 MAPK activation or Dsg3 protein amount, PV patients’ and one control patient’s lesional skin were used after cells were seeded in six-well plates and confluent monolayers were

308 Journal of Investigative Dermatology (2016), Volume 136 VRo¨tzer et al. Desmoglein 3 and Keratinocyte Migration scratched 10 times in a defined pattern. Cell lysates were subjected Berkowitz P, Hu P, Liu Z, et al. Desmosome signaling. Inhibition of p38MAPK to Western blot analysis. prevents pemphigus vulgaris IgG-induced cytoskeleton reorganization. J Biol Chem 2005;280:23778e84. For the ex vivo assay, skin explants were acquired from 1-day-old Berkowitz P, Hu P, Warren S, Liu Z, Diaz LA, Rubenstein DS. p38MAPK in- pups after decapitation according to an established protocol hibition prevents disease in pemphigus vulgaris mice. Proc Natl Acad Sci (Mazzalupo et al., 2002). Medium was changed every day, and the USA 2006;103:12855e60. outgrowth of keratinocytes was monitored over time. Chen L, Guo S, Ranzer MJ, DiPietro LA. Toll-like receptor 4 has an essential role in early skin wound healing. J Invest Dermatol 2013;133:258e67. Live cell imaging Chernyavsky AI, Arredondo J, Vetter DE, Grando SA. Central role of For live cell studies, HaCaTs were seeded in m-slide eight-well alpha9 acetylcholine receptor in coordinating keratinocyte adhesion chambers (Ibidi, Martinsried, Germany) and transiently transfected and motility at the initiation of epithelialization. Exp Cell Res 2007;313: e with 1.2 mg/ml of plasmid DNA, either pEGFP-C1-Dsg3 (kindly 3542 55. provided by Dr. Yasushi Hanakawa, Ehime University School of Cirillo N, Lanza A, Prime SS. Induction of hyper-adhesion attenuates autoimmune-induced keratinocyte cell-cell detachment and processing of Medicine, Japan) or pEGFP-N1. Live cell imaging was performed adhesion molecules via mechanisms that involve PKC. Exp Cell Res after 24 hours in DMEM without phenol red in a constant atmo- 2010;316:580e92. sphere at 37 C with 5% CO2. Cells were imaged every 30 minutes Dehner C, Ro¨tzer V, Waschke J, Spindler V. A desmoplakin point mutation with over a time course of 20 hours. Evaluation of migration velocity was enhanced keratin association ameliorates pemphigus vulgaris autoantibody- e performed with Image J (www.imagej.nih.gov/ij/) and the manual mediated loss of cell cohesion. Am J Pathol 2014;184:2528 36. tracking plugin. Delva E, Tucker DK, Kowalczyk AP. The desmosome. Cold Spring Harb Per- spect Biol 2009;1:25e43. Immunoprecipitation Fitsialos G, Chassot AA, Turchi L, et al. Transcriptional signature of epidermal For pull-down analysis, T75 flasks of MEK cells grown to confluence keratinocytes subjected to in vitro scratch wounding reveals selective roles for ERK1/2, p38, and phosphatidylinositol 3-kinase signaling pathways. were scraped in radioimmune precipitation assay buffer. Six hundred J Biol Chem 2007;282:15090e102. micrograms of protein were incubated for 3 hours at 4 C with 2 mg Gurtner GC, Werner S, Barrandon Y, Longaker MT. Wound repair and of Dsc3 antibody (Santa Cruz Biotechnology, Santa Cruz, TX). This regeneration. Nature 2008;453:314e21. antibody/lysate mixture was then incubated overnight with 40 ml Harper EG, Alvares SM, Carter WG. Wounding activates p38 map kinase and A/G beads at 4 C. Beads were supplemented with Laemmli buffer activation transcription factor 3 in leading keratinocytes. J Cell Sci 2005;118:3471e85. and subjected to Western blot according to standard procedures (Ro¨tzer et al., 2015). Hartlieb E, Ro¨tzer V, Radeva M, Spindler V, Waschke J. Desmoglein 2 com- pensates for desmoglein 3 but does not control via regulation of p38 mitogen-activated protein kinase in keratinocytes. J Biol Chem Image processing and statistics 2014;289:17043e53. Images were processed using Photoshop CS5 (Adobe Systems, San Jiang X, Guo X, Xu X, et al. Hypoxia regulates CD9-mediated keratinocyte Jose, CA). To evaluate in vitro wound healing or ex vivo cell migration via the P38/MAPK pathway. Sci Rep 2014;4:6304. outgrowth, cell-free areas were measured with Image J. For each Jolly PS, Berkowitz P, Bektas M, et al. p38MAPK Signaling and desmoglein-3 condition the area at time point 0 hours was set to 100%, and values internalization are linked events in pemphigus acantholysis. J Biol Chem e after 12 to 96 hours were calculated as percent of the initial scratch 2010;285:8936 41. area. Data are presented as means standard error. Statistical Kitajima Y. New insights into desmosome regulation and pemphigus blis- tering as a desmosome-remodeling disease. Kaohsiung J Med Sci 2013;29: analysis was performed by analysis of variance followed by Bon- 1e13. ferroni correction for comparison of more than two groups and by Kitajima Y. 150th Anniversary series: desmosomes and autoimmune disease, paired, two-tailed Student’s t-test for comparison of two groups perspective of dynamic desmosome remodeling and its impairments in (Prism; GraphPad Software, La Jolla, CA). 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