Wound Healing - Core

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 desmoglein 3 (Dsg3) and desmocollin 3 (Dsc3) are required for strong keratinocyte cohesion. Recently, we have shown that Dsg3 associates with p38 mitogenactivated protein 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 Desmogleins (Dsg) together with desmocollins (Dsc) are Ca2þ-dependent, cadherin-type transmembrane adhesion molecules that are located inside and outside of desmosomes (Delva et al., 2009). Inside desmosomes, these desmosomal cadherins are linked to the keratin filament cytoskeleton via a set of adapter proteins such as plakoglobin, plakophilins, and desmoplakin. Extradesmosomal Dsg may be attached to actin filaments (Tsang et al., 2012). Four Dsg and three Dsc isoforms exist, which show a tissue-specific distribution pattern. Dsg1/Dsc1 and Dsg3/Dsc3 are mainly restricted to stratifying epithelia of mucous membranes such as those of the oral cavity and the vagina and are highly expressed in the epidermis. Both Dsg1 and Dsg3 are targets of autoantibodies causing the autoimmune skin disease pemphigus vulgaris 1

Institute of Anatomy and Cell Biology, Ludwig-Maximilians-Universita¨t Mu¨nchen, Munich, Germany; and 2Department of Dermatology and Allergology, Ludwig-Maximilians-Universita¨t Mu¨nchen, Munich, Germany

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These authors contributed equally to this work.

Correspondence: Jens Waschke, Institute of Anatomy and Cell Biology, Department I, LMU Munich, Pettenkoferstraße 11, 80336 Mu¨nchen, Germany. E-mail: [email protected] or Volker Spindler, Institute of Anatomy and Cell Biology, Department I, LMU Munich, Pettenkoferstraße 11, 80336 Mu¨nchen, Germany. E-mail: volker.spindler@ med.uni-muenchen.de Abbreviations: Dsc3, desmocollin 3; Dsg3, desmoglein 3; ERK, extracellular signal-regulated kinase; GFP, green fluorescent protein; MEK, murine epidermal keratinocytes; p38MAPK, p38 mitogen-activated protein kinase; PKC, protein kinase C; PV, pemphigus vulgaris; PV-IgG, IgG fraction of PV patient; siRNA, small interfering RNA; wt, wild-type Received 12 December 2014; revised 1 September 2015; accepted 11 September 2015; accepted manuscript published online 30 September 2015

(PV) (Stanley and Amagai, 2006). Here, loss of Dsg3 and Dsg1 function results in reduced intercellular adhesion of keratinocytes and epithelial cells leading to mucocutaneous blisters and erosions (Waschke and Spindler, 2014). A crucial signaling event elicited by pemphigus autoantibody binding is the activation of p38 mitogen-activated protein kinase (p38MAPK), the inhibition of which in turn prevents loss of cell cohesion in vitro and blocks blistering in mouse models (Berkowitz et al., 2005, 2006). Similarly, both depletion of Dsg3 and specific interference with Dsg3 binding by monoclonal autoantibodies or small peptides yielded activation of p38MAPK (Hartlieb et al., 2014; Spindler et al., 2013). Thus, Dsg3 binding suppresses p38MAPK activity, a function that is impaired under disease conditions. However, more physiologic roles of Dsg3-dependent control of p38MAPK signaling are unknown. Skin wound repair is a highly complex process and is typically classified in three stages (Gurtner et al., 2008; Sun et al., 2014): inflammation, tissue formation, and remodeling. Stage two, besides angiogenesis and extracellular matrix generation, involves migration of keratinocytes to cover the fibrin matrix initially formed at the wound bottom. It is well established that in this regard p38MAPK activation governs keratinocyte migration. After wounding, p38MAPK is activated at the wound edge (Lambert et al., 2010), a process that may also be under the control of extracellular stimuli such as hydrogen peroxide (Loo et al., 2011) or activation of toll-like receptors (Chen et al., 2013). To allow wound healing, the desmosomes of keratinocytes need to remodel (Kitajima, 2014). In healthy conditions, desmosomes are stable structures that resist depletion of extracellular Ca2þ, which is

ª 2015 The Authors. Published by Elsevier, Inc. on behalf of the Society for Investigative Dermatology.

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surprising given the Ca2þ-dependent binding of desmosomal cadherins. This condition, named “desmosomal hyperadhesion,” is rapidly reversed after wounding both in vitro and in vivo (Thomason et al., 2012; Wallis et al., 2000). This process is only partially understood but requires the activation of protein kinase C a (PKCa). Given the fact that impairment of desmosomal adhesion (i.e., by anti-Dsg3 autoantibodies from pemphigus patients) induces both protein kinase C activation with subsequent loss of hyperadhesion as well as p38MAPK activation, we investigated the hypothesis that loss of Dsg3 contributes to the switch from an adhesive to a migratory phenotype via p38MAPK signaling. RESULTS Loss of Dsg3 function induces p38MAPK activation in vivo

We have previously shown that silencing of Dsg3 results in increased p38MAPK activity in human keratinocytes (Hartlieb et al., 2014). Here, we used Dsg3-deficient mice suffering from mucocutaneous blistering and erosions (Koch et al., 1997). The skin lesions appeared to occur randomly over the entire body of adult Dsg3 knockout mice and were often accompanied by patchy hair loss (Figure 1a). Skin erosions appeared to be more frequent compared with the initial description of the model and were not restricted to areas prone to scratching such as the snout, which may be a result of different housing or mating conditions. Interestingly, the erosions were generally prone to spontaneous wound healing. Typically, after several days, the wounds were closed again and fur was regrown. We harvested biopsies of lesional and nonlesional skin of knockout mice and sampled tissue from the same regions in wild-type (wt) littermates as controls. Whereas in wt animals the levels of p38MAPK activity were low, they were increased in lesional areas of knockout animals. Interestingly, p38MAPK activation was also detectable in nonlesional skin of most Dsg3-/- mice. An example of an ear lobe lesion is shown in Figure 1b and c. To further corroborate the effect of impaired Dsg3 function on p38MAPK activity, we stained skin sections from PV patients with a phospho-p38MAPK antibody. Compared with healthy control subjects, strong p38MAPK staining throughout the epidermis was detectable in skin sections of two PV patients (Figure 1d). Especially in areas of profound IgG fraction of PV patient (PV-IgG) binding to the keratinocyte surface, phospho-p38MAPK was found at the membrane, which is in line with data demonstrating that phospho-p38MAPK can associate with Dsg3 (Spindler et al., 2013). In addition, in samples from PV patient 2 cytoplasmic staining of p38MAPK was also detectable, which may correlate with the function of p38MAPK to regulate actin and keratin filament architecture (Berkowitz et al., 2005; Mao et al., 2014; Spindler et al., 2013). Secondary antibody controls to rule out unspecific signals are shown in Supplementary Figure S1 (see online). Taken together, loss of Dsg3 function in mice or humans was associated with p38MAPK activation. P38MAPK activation correlates with Dsg3 internalization in wounded keratinocyte monolayers

Dsg3-deficient mice show p38MAPK activation in the skin and remarkably fast wound healing. We thus hypothesized that Dsg3 and p38MAPK regulate migration of keratinocytes. As observed before by another group (Lambert et al., 2010), 302

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scratched monolayers of human keratinocytes (HaCaT cell line) displayed increased phospho-p38MAPK staining intensity at the wound edge after 15 minutes that subsided until 120 minutes (Figure 2a). This was further corroborated by Western blot analysis (Figure 2b). To demonstrate that p38MAPK is activated in Dsg3-dependent manner, we overexpressed green fluorescent protein (GFP)-tagged Dsg3 or GFP alone in HaCaT cells and analyzed p38MAPK activity after scratching (Figure 2c). p38MAPK activation was prevented in cells expressing GFP-Dsg3 (arrow), whereas nontransfected cells or cells expressing GFP alone lining the wound edge displayed increased phospho-p38MAPK staining intensity after 30 minutes. This observation was supported by experiments performed in keratinocytes isolated from wt and Dsg3 knockout mice (murine epidermal keratinocytes [MEKs]) (see Supplementary Figure S2, online). As observed before (Hartlieb et al., 2014), Dsg3-deficient MEKs demonstrate prominent phospho-p38MAPK membrane staining (red arrows), which was largely reduced in cells expressing Dsg3GFP but not GFP as control (blue arrows in magnified regions). Moreover, loss of membrane Dsg3 staining was observed 15 minutes after wounding in untreated keratinocytes lining the wound edge, that is, at a time when p38MAPK activation was evident (Figure 2d). In these cells, intracellular vesicles containing Dsg3 appeared (red arrows). However, Dsg3 depletion was more pronounced at later time points, when cells at the migration front displayed drastically reduced Dsg3 staining. These findings were also supported by Triton X100-mediated fractionation. HaCaT monolayers displayed increased Dsg3 content in the noncytoskeletal, Triton X-100soluble pool starting at 15 minutes after wounding, whereas the amount in the cytoskeletal, desmosome-containing pool was reduced (Figure 2e). This indicates that the cytoskeletal anchorage of Dsg3 is rapidly reduced after scratch wounding. Loss of Dsg3 promotes keratinocyte migration via p38MAPK signaling

To determine whether Dsg3 regulates keratinocyte migration, we silenced Dsg3 in HaCaT cells using small interfering RNA (siRNA). Keratinocytes transfected with nontarget siRNA and treated with DMSO closed the scratch to roughly 50% of the initial size after 48 hours (Figure 3a and b), which was blocked by p38MAPK inhibition using the p38MAPK-specific inhibitor SB202190 at 30 mM. However, silencing of Dsg3 resulted in significantly increased migration speed compared with nontarget siRNA controls. This acceleration of gap closure was also prevented by p38MAPK inhibition. These effects seemed to be largely dependent on migration and not on cell division because mitomycin treatment did not reduce migration of HaCaT cells (see Supplementary Figure S3a, online). Western blots were run in parallel to migration assays to ensure that silencing was detectable throughout the entire monitoring period after scratching (Supplementary Figure S3b). Furthermore, Dsg3 silencing did not affect protein levels of other desmosomal proteins apart from a slight reduction of plakoglobin (Supplementary Figure S3c). We repeated these experiments in wt and Dsg3-deficient MEKs. Again, Dsg3-deficient cells migrated significantly faster compared with wt MEKs (Figure 3c and d). Similar to the situation in HaCaT cells, both wt and Dsg3-deficient MEK

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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 with earlobe of a Dsg3þ/þ 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 representatives of six Dsg3þ/þ and Dsg3-/- mice in total. (d) Phosphop38MAPK 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, membranebound p38MAPK activation; dashed line, dermo-epidermal junction. Scale bar ¼ 20 mm.

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Desmoglein 3 and Keratinocyte Migration 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 inhibition. To further corroborate these observations, we took doughnut-shaped full-thickness biopsies from Dsg3 wt and knockout animals into culture and measured the outgrowth of keratinocytes (Figure 4a). Outgrowing cells indeed were of keratinocyte origin as they were positive for desmoplakin and Dsg3. In line with cell culture data, outgrowth from Dsg3deficient punch biopsies was significantly faster than from wt controls over a time period of 96 hours (Figure 4b and c). Again, addition of SB202190 to the culture medium significantly slowed down migration both in wt and Dsg3 knockout biopsies. Vice versa, to demonstrate that the presence of Dsg3 inhibits keratinocyte migration, we overexpressed GFP-tagged Dsg3 or GFP alone in HaCaT cells. GFP-expressing cells were dispersed throughout the monolayer next to the wound margin. This distribution pattern did not change until the wound was closed (Figure 5a). In GFP-Dsg3-expressing cells, despite relatively low transfection efficiency, a reduced wound closure was detectable, indicating an effect of Dsg3 on collective migratory behavior. After a 20-hour incubation period, Dsg3-GFP-expressing cells were very rarely found at the wound margin but rather appeared to be slower compared with nonexpressing cells (Figure 5a). Indeed, by 304


tracking individual cells constantly over this time course, the trajectories of GFP-Dsg3-expressing keratinocytes were shorter compared with GFP-expressing cells (Figure 5b), yielding a significantly reduced migration velocity (Figure 5c). Taken together, these data show that the presence of Dsg3 inhibits keratinocyte migration at least in part by suppressing p38MAPK activity. Mechanisms underlying keratinocyte migration in response to loss of Dsg3

We have shown previously that p38MAPK is located and activated in a complex with Dsg3 and plakoglobin (Spindler et al., 2013, 2014). To investigate the impact of Dsg3 loss on the localization of p38MAPK, we immunoprecipitated Dsc3 in wt and Dsg3 knockout MEKs. We found that Dsc3 is associated with Dsg3 and p38MAPK. Interestingly, in cells lacking Dsg3, the association of p38MAPK and especially of phospho-p38MAPK with Dsc3 was significantly increased (Figure 6a). This indicates that Dsg3 is not necessary for the association of p38MAPK to this complex but rather has an indirect function to suppress p38MAPK activity. Because in Dsg3 knockout cells p38MAPK inhibition did not completely prevent migration and it was shown that

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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 isolated from Dsg3þ/þ and Dsg3-/mice. Bar is 150 mm. (n ¼ 10e15 from 4 independent isolation procedures; *P < 0.05 vs. Dsg3þ/þ DMSO, #P < 0.05 vs. respective DMSO condition).

p38MAPK is necessary but not sufficient to induce migration (Li et al., 2001), we investigated other signaling molecules in the context of Dsg3. Using ELISA-based measurements, we detected RhoA activity to be significantly decreased in Dsg3 knockout MEKs (Figure 6b), which is in line with previous results in which PV-IgG led to a reduction of RhoA activity (Ro¨tzer et al., 2014; Waschke et al., 2006). Nevertheless, Yersinia pseudotuberculosis cytotoxic necrotizing factor y to specifically activate RhoA at a previously determined concentration (1 mM) did not affect scratch wound closure (Figure 6c), indicating that reduced RhoA activity in response to loss of Dsg3 does not contribute to increased migration. In contrast, inhibition of Src with 10 mM pp2 fully prevented wound closure both in wt and Dsg3 knockout MEKs (Figure 6d). However, levels of active (Tyr416phosphorylated) Src were not different in knockout versus wt cells (Figure 6e), and in contrast to p38MAPK, no activation was induced by scratch wounding (data not shown). Thus, the function of Src for keratinocyte migration appears to be independent of Dsg3. Finally, we investigated the impact of extracellular signal-regulated kinase (ERK) signaling. Interestingly, the mitogen-activated protein kinase kinase inhibitor UO126 (5 mM) had no impact on migration of Dsg3 knockout keratinocytes but significantly slowed the wound closure in wt cells (Figure 6f). This indicates that migration in response to the mitogen-activated protein kinase kinase/ERK pathway is at least in part dependent on the presence of Dsg3. Altogether, these findings underscore that regulation of cell migration is complex and that several signaling pathways are involved that in part are regulated by adhesion molecules such as Dsg3.

DISCUSSION Here, we show that Dsg3 controls keratinocyte migration and wound healing. Inhibition of p38MAPK signaling reduced migration both in nontarget as well as Dsg3-depleted cells, underscoring the notion that regulation of p38MAPK activity is important in this process. This idea is supported by in vivo data showing that Dsg3 knockout animals exhibit higher levels of active p38MAPK and remarkable erosion healing capacities and is in line with increased keratinocyte outgrowth in an ex vivo skin model. We found that Dsg3 and p38MAPK are associated with Dsc3, which indicates that adhesion-dependent regulation of p38MAPK is controlled by both Dsg3 and Dsc3. In addition, together with the observation that Dsc3 is more extensively associated with p38MAPK in cells deficient for Dsg3, this helps to solve the conundrum of how cells deficient for Dsg3 can have more activated p38MAPK at the cell membrane (Hartlieb et al., 2014), which is hard to explain if Dsg3 would be the only membrane-associated protein capable of sequestering p38MAPK. These data are consistent with the idea that Dsg3 and Dsc3 together form an adhesion-dependent signaling complex in which p38MAPK activity is increased if the function of one of the two adhesion molecules is compromised. Dsg3 together with p38MAPK controls keratinocyte migration

Migration of keratinocytes from the wound edge into the wounded area is an essential step in wound healing both in vitro and in vivo (Pastar et al., 2014). Several reports demonstrate that p38MAPK fosters keratinocyte migration. It www.jidonline.org

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Desmoglein 3 and Keratinocyte Migration 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. Dsg3þ/þ 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 et al., 2014), hydrogen peroxide (Loo et al., 2011), or tolllike-receptor 4 binding (Chen et al., 2013) promote migration via p38MAPK. Apparently, scratch wounding of keratinocyte monolayers is sufficient to induce p38MAPK activation and to subsequently modulate the expression of specific genes relevant for migration (Fitsialos et al., 2007). Disruption of contact to the basement membrane, specifically to laminin 5, was shown to promote p38MAPK activation after wounding (Harper et al., 2005), indicating that loss of cell-matrix adhesion triggers p38MAPK-dependent migration. Our data from this study identified that loss of intercellular Dsg3-mediated binding also is a cue for p38MAPK activation. A scenario is conceivable in which Dsg3 senses loss of cell-cell cohesion (i.e., induced by scratch wounding) and relays this extracellular cue into intracellular activation of p38MAPK. Indeed, it was shown recently that specific interference with Dsg3 binding via small peptides targeting the adhesive Dsg3 domain triggers p38MAPK activation (Spindler et al., 2013). Vice versa, it was demonstrated that p38MAPK promotes internalization and depletion of Dsg3 (Jolly et al., 2010). Thus, initial loss of Dsg3 binding may promote further depletion of Dsg3 levels at the wound edge via p38MAPK-dependent signaling, which is supported by our data showing that loss of Dsg3 was pronounced in cells lining the scratch at later time points (i.e., 360 minutes after wounding). This is not surprising given the fact that in addition to cell matrix contacts also cell-cell contacts such as desmosomes need to remodel to 306


re-epithelialize a wound (Krawczyk and Wilgram, 1973). p38MAPK was also shown to regulate the activity of the EGFR (Bektas et al., 2013; Lambert et al., 2010), and, in turn, EGFR activation enhances keratinocyte migration (Ando and Jensen, 1993; Barrientos et al., 2008; Sperrhacke et al., 2014). Thus, p38MAPK may be a central molecule in a signaling network regulating keratinocyte migration. We also checked for additional candidates other than p38MAPK that have been implicated both in migration and desmosome function; however, none of the molecules appeared to be both regulated by Dsg3 and influencing keratinocyte migration in our experiments. Interestingly, ERK signalingdependent migration may require Dsg3, because in Dsg3 knockout cells inhibition of ERK failed to reduce scratch wound closure. Wounded keratinocytes show remarkable similarities to keratinocytes challenged with pemphigus autoantibodies

PV is a disease characterized by antibodies targeting Dsg3, which leads to keratinocyte dissociation. Although under certain conditions loss of cell cohesion can occur independent of intracellular signaling (Mao et al., 2014; Saito et al., 2012), numerous studies demonstrate the requirement of signaling events (Kitajima, 2013; Spindler and Waschke, 2014). Interestingly, the key signaling events in PV are remarkably similar to some of those observed in migrating keratinocytes at the wound margin. Binding of PV autoantibodies leads to a quick activation of p38MAPK, which modulates intermediate filament uncoupling of desmosomes

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Desmoglein 3 and Keratinocyte Migration 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-GFPexpressing 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 was suggested that primarily the nondesmosomal Dsg3 molecules present on the cell surface and not connected to the keratin filaments sense the initial binding of autoantibodies and relay this signal into the cell, which results in pronounced p38MAPK-dependent Dsg3 depletion (Mu¨ller et al., 2008; Spindler and Waschke, 2014; Vielmuth et al., 2015). Furthermore, PKC is activated after a rapid Ca2þ influx after PV-IgG application (Osada et al., 1997; Ro¨tzer et al., 2014; Seishima et al., 1995) that switches the Ca2þindependent, hyperadhesive desmosomes found in fully confluent keratinocytes to a Ca2þ-dependent phenotype (Cirillo et al., 2010; Dehner et al., 2014). This in turn favors the loss of Dsg3, probably by reorganization of the desmosomal plaque in response to desmoplakin phosphorylation and via mechanisms involving plakophilin-1 (Dehner et al., 2014; Spindler et al., 2011; Tucker et al., 2014). In addition, EGFR phosphorylation after PV-IgG binding was

observed at least by some authors (Chernyavsky et al., 2007; Pretel et al., 2009; Schulze et al., 2012), which again may be under the control of p38MAPK (Bektas et al., 2013). Thus, in PV a set of key molecules show the same activity pattern as in migrating keratinocytes (Kitajima, 2014). Taken together, the physiologic role of the Dsg3/p38MAPK complex may be to regulate this switch depending on the actual state of epidermal integrity. In this scenario, binding of pemphigus autoantibodies would shift keratinocytes toward the migratory program by triggering this switch. However, our data also indicate that interference with p38MAPK signaling as a treatment option in pemphigus may be a double-edged sword because on one hand it may efficiently prevent blister formation, but on the other hand it may prevent keratinocytes from re-epithelializing wounds after skin blistering occurred. This raises the interesting question whether treatment options targeting desmosomal adhesion in pemphigus need to be evaluated differently for prophylaxis versus www.jidonline.org

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Desmoglein 3 and Keratinocyte Migration Figure 6. Mechanisms underlying keratinocyte migration in response to loss of Dsg3. (a) Immunoprecipitation of Dsc3 in isolated keratinocytes from Dsg3þ/þ and Dsg3-/- animals. The heavy chain of the capturing antibody served as loading control. n ¼ 4, *P < 0.05 vs. immunoprecipitation Dsg3þ/þ (n ¼ 4). (b) ELISA-based measurement of RhoA activity in wildtype and Dsg3 knockout mouse keratinocytes (n ¼ 5, *P < 0.05 vs. Dsg3þ/þ). (c) Scratch-wound assay using the RhoA-specific activator cytotoxic necrotizing factor y (n ¼ 11e14 from 4 independent isolation procedures). (d) Scratchwound 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) Scratchwound assay using the mitogenactivated 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 of p38MAPK may be effective to prevent relapses but not for treatment of skin blisters. MATERIALS AND METHODS For detailed methods please refer to the Supplementary Materials (online).

Cell culture and patient skin samples The human immortalized keratinocyte cell line HaCaT was maintained in DMEM that constantly contained 1.8 mM Ca2þ. Primary MEKs were isolated from Dsg3 knockout and corresponding wt neonatal mice as previously reported (Hartlieb et al., 2014). Cells were switched to 1.8 mM Ca2þ 24 hours before subjection to Western blot analysis, migration assay, or Rho activity measurement. Animal experiments were approved by the Regierung von Oberbayern. Diagnostic punch biopsy leftover samples taken from two PV patients’ and one control patient’s lesional skin were used after 308


the clinical diagnosis was confirmed at the Department of Dermatology. Patients had given informed and written consent, and use of the samples was approved by the Ethics Committee of the Faculty of Medicine, Ludwig-Maximilians-Universita¨t, Munich, Germany (project no. 249-12).

In vitro and ex vivo wound healing cell migration assay HaCaTs were seeded in 24-well plates with or without coverslips and transfected with nontarget or Dsg3 siRNA. After grown to confluence within 4 days, cell monolayers were manually scratched with a sterile pipette tip, washed with medium, and incubated with DMEM supplemented with the indicated mediators for 24 to 48 hours. Dsg3þ/þ and Dsg3-/- keratinocytes were switched to 1.8 mM Ca2þ 12 hours before scratching. Migration was monitored over time by bright-field microscopy (Axio Vert A1; Carl Zeiss AG, Oberkochen, Germany). For detection of p38 MAPK activation or Dsg3 protein amount, cells were seeded in six-well plates and confluent monolayers were

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Desmoglein 3 and Keratinocyte Migration scratched 10 times in a defined pattern. Cell lysates were subjected to Western blot analysis. For the ex vivo assay, skin explants were acquired from 1-day-old pups after decapitation according to an established protocol (Mazzalupo et al., 2002). Medium was changed every day, and the outgrowth of keratinocytes was monitored over time.

Live cell imaging For live cell studies, HaCaTs were seeded in m-slide eight-well chambers (Ibidi, Martinsried, Germany) and transiently transfected with 1.2 mg/ml of plasmid DNA, either pEGFP-C1-Dsg3 (kindly provided by Dr. Yasushi Hanakawa, Ehime University School of Medicine, Japan) or pEGFP-N1. Live cell imaging was performed after 24 hours in DMEM without phenol red in a constant atmosphere at 37 C with 5% CO2. Cells were imaged every 30 minutes over a time course of 20 hours. Evaluation of migration velocity was performed with Image J (www.imagej.nih.gov/ij/) and the manual tracking plugin.

Immunoprecipitation For pull-down analysis, T75 flasks of MEK cells grown to confluence were scraped in radioimmune precipitation assay buffer. Six hundred micrograms of protein were incubated for 3 hours at 4 C with 2 mg of Dsc3 antibody (Santa Cruz Biotechnology, Santa Cruz, TX). This antibody/lysate mixture was then incubated overnight with 40 ml A/G beads at 4 C. Beads were supplemented with Laemmli buffer and subjected to Western blot according to standard procedures (Ro¨tzer et al., 2015).

Image processing and statistics Images were processed using Photoshop CS5 (Adobe Systems, San Jose, CA). To evaluate in vitro wound healing or ex vivo cell outgrowth, cell-free areas were measured with Image J. For each condition the area at time point 0 hours was set to 100%, and values after 12 to 96 hours were calculated as percent of the initial scratch area. Data are presented as means standard error. Statistical analysis was performed by analysis of variance followed by Bonferroni correction for comparison of more than two groups and by paired, two-tailed Student’s t-test for comparison of two groups (Prism; GraphPad Software, La Jolla, CA). Statistical significance was assumed when P 0.05.

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The authors state no conflict of interest.

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ACKNOWLEDGMENTS

Krawczyk WS, Wilgram GF. Hemidesmosome and desmosome morphogenesis during epidermal wound healing. J Ultrastruct Res 1973;45:93e101.

We thank Martina Hitzenbichler, Cathleen Plietz, Sabine Mu¨hlsimer, and Andrea Wehmeyer for excellent technical assistance. This work was supported by grants from the Deutsche Forschungsgemeinschaft (DFG) WA2474/ 5-1 and SP1300/1-3.

Lambert S, Frankart A, Poumay Y. p38 MAPK-regulated EGFR internalization takes place in keratinocyte monolayer during stress conditions. Arch Dermatol Res 2010;302:229e33.

CONFLICT OF INTEREST

SUPPLEMENTARY MATERIAL

Li W, Nadelman C, Henry G, et al. The p38-MAPK/SAPK pathway is required for human keratinocyte migration on dermal collagen. J Invest Dermatol 2001;117:1601e11.

Supplementary material is linked to the online version of the paper at www. jidonline.org, and at http://dx.doi.org/10.1038/JID.2015.380.

Loo AEK, Ho R, Halliwell B. Mechanism of hydrogen peroxide-induced keratinocyte migration in a scratch-wound model. Free Radic Biol Med 2011;51:884e92.

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