Advertisement

Spleen Tyrosine Kinase Mediates EGFR Signaling to Regulate Keratinocyte Terminal Differentiation

      Spleen tyrosine kinase (Syk), a nonreceptor tyrosine kinase, was initially identified as a crucial regulator in proximal immunoreceptor signaling. Additional studies have revealed its pleiotropic roles, and drugs targeting Syk are under development for inflammatory diseases. Syk expression in the skin has been detected, but its functions in the skin are still unknown. Here, we found that Syk phosphorylation and expression in primary human keratinocytes decreased gradually along with terminal differentiation. Human skin specimens showed similar in vivo patterns. Syk inhibitors or knockdown of Syk increased the expression of differentiation markers under in vitro differentiation models. Furthermore, EGFR activation prominently induced Syk phosphorylation, which could be inhibited by the EGFR inhibitor gefitinib or knockdown of EGFR. The Src inhibitor also partially attenuated EGF-induced phosphorylation of Syk. However, Syk inhibition suppressed EGF-induced phosphorylation of EGFR. Immunoprecipitation and confocal microscopy further revealed the increased molecular interaction between EGFR and Syk after EGF stimulation. This study unravels the role of Syk in EGFR-mediated signaling and reveals regulatory roles of Syk in keratinocyte differentiation, suggesting the clinical potential of topical or systemic Syk inhibitors in the treatment of skin diseases with aberrant differentiation.

      Abbreviations:

      ITAM (immunoreceptor tyrosine-based activation motif), NHEK (normal human epidermal keratinocytes), PMA (phorbol 12-myristate 13-acetate), siRNA (small interfering RNA), Syk (spleen tyrosine kinase), TGase (transglutaminase)

      Introduction

      Spleen tyrosine kinase (Syk) is a nonreceptor tyrosine kinase first found in hematopoietic lineage cells. Syk contains two tandem SRC homology 2 domains, a linker region, and one carboxy-terminal tyrosine kinase domain (
      • Mocsai A.
      • Ruland J.
      • Tybulewicz V.L.
      The SYK tyrosine kinase: a crucial player in diverse biological functions.
      ). Syk-deficient mice suffer severe hemorrhage and blocked B-cell development and die perinatally (
      • Cheng A.M.
      • Rowley B.
      • Pao W.
      • et al.
      Syk tyrosine kinase required for mouse viability and B-cell development.
      ,
      • Turner M.
      • Mee P.J.
      • Costello P.S.
      • et al.
      Perinatal lethality and blocked B-cell development in mice lacking the tyrosine kinase Syk.
      ). Syk is a crucial regulator in the proximal signaling of various receptors involved in innate and adaptive immunity (
      • Jung E.M.
      • Betancourt-Calle S.
      • Mann-Blakeney R.
      • et al.
      Sustained phospholipase D activation is associated with keratinocyte differentiation.
      ,
      • Turner M.
      • Schweighoffer E.
      • Colucci F.
      • et al.
      Tyrosine kinase SYK: essential functions for immunoreceptor signalling.
      ). In the immunoreceptor tyrosine-based activation motif (ITAM)- and hemiITAM-based signaling downstream of B-cell receptors, T-cell receptors, and Fc receptors, Syk recruitment to dual-phosphorylated ITAM triggers Syk activation, leading to diverse cellular responses (
      • Mocsai A.
      • Ruland J.
      • Tybulewicz V.L.
      The SYK tyrosine kinase: a crucial player in diverse biological functions.
      ). Syk plays a dual role in the toll-like receptor–mediated tumor necrosis factor–associated factor 6 and 3 signaling pathways to finely tune innate immune responses (
      • Lin Y.C.
      • Huang D.Y.
      • Chu C.L.
      • et al.
      The tyrosine kinase Syk differentially regulates Toll-like receptor signaling downstream of the adaptor molecules TRAF6 and TRAF3.
      ).
      Epidermal keratinocytes, the principal cell component in the skin, undergo continuous terminal differentiation to construct the outermost cornified layer of the skin, a process named cornification (
      • Candi E.
      • Schmidt R.
      • Melino G.
      The cornified envelope: a model of cell death in the skin.
      ). Cornification is considered a model of cell death and is characterized by the activation of caspase 14, a nonapoptotic caspase (
      • Galluzzi L.
      • Vitale I.
      • Abrams J.M.
      • et al.
      Molecular definitions of cell death subroutines: recommendations of the Nomenclature Committee on Cell Death 2012.
      ). Epidermal keratinocytes sequentially express distinct differentiation marker proteins such as transglutaminase (TGase) types 1, 3, and 5; involucrin, keratin 1/10; loricrin; and profilaggrin, which act as either enzymes or substrates to establish the skin barrier (
      • Candi E.
      • Schmidt R.
      • Melino G.
      The cornified envelope: a model of cell death in the skin.
      ,
      • Eckert R.L.
      • Sturniolo M.T.
      • Broome A.M.
      • et al.
      Transglutaminase function in epidermis.
      ). During the terminal differentiation process, proliferating keratinocytes gradually become growth arrested and eventually dead cornified keratinocytes. During this process EGFR expression and activation are downregulated (
      • King Jr., L.E.
      • Gates R.E.
      • Stoscheck C.M.
      • et al.
      Epidermal growth factor/transforming growth factor alpha receptors and psoriasis.
      ,
      • Nanney L.B.
      • Magid M.
      • Stoscheck C.M.
      • et al.
      Comparison of epidermal growth factor binding and receptor distribution in normal human epidermis and epidermal appendages.
      ).
      Based on the well-known immunologic functions of Syk (
      • Mocsai A.
      • Ruland J.
      • Tybulewicz V.L.
      The SYK tyrosine kinase: a crucial player in diverse biological functions.
      ,
      • Yanagi S.
      • Inatome R.
      • Takano T.
      • et al.
      Syk expression and novel function in a wide variety of tissues.
      ), the application of pharmacologic inhibitors that suppress Syk function has been investigated for the treatment of lymphoma, leukemia, asthma, allergic rhinitis, lupus erythematosus, and rheumatoid arthritis (
      • Riccaboni M.
      • Bianchi I.
      • Petrillo P.
      Spleen tyrosine kinases: biology, therapeutic targets and drugs.
      ). However, compared with the wide exploration of Syk functions in immunologic diseases, its role in normal epithelial organs such as the skin is less clear. In this study, using immunohistochemistry, Syk inhibitors, and Syk knockdown in primary human epidermal keratinocytes, we found that Syk is involved in terminal differentiation of keratinocytes and participates in EGFR-mediated signaling, indicating an important role of Syk in skin biology.

      Results

      Syk activation and expression are regulated during keratinocyte terminal differentiation

      To evaluate the in vivo pattern of Syk activation and phosphorylation, we performed immunohistochemical staining to analyze the expression of Syk and phosphorylated Syk in normal and psoriatic skin tissues. As shown in Figure 1a, c, e, and g, phosphorylated Syk expression was detected in the epidermis and was relatively prominent in the lower epidermis. Notably, phosphorylated Syk was obviously expressed in the cell membranes of keratinocytes (Figure 1a, c, e, and g, inlets). Similarly, Syk expression was low in the uppermost part of the epidermis. Phosphorylated Syk was expressed more highly in keratinocytes in psoriasis samples than in normal skin (Figure 1i and j).
      Figure 1
      Figure 1Syk phosphorylation and expression are suppressed during keratinocyte differentiation. (a–j) Immunohistochemical staining with phosphorylated Syk and Syk was performed. Two specimens from normal skin (a–d, i, j) and psoriatic skin (e–h) were analyzed. Phosphorylated Syk (a, c, e, g), Syk (b, d, f, h), anti-rabbit IgG (i), or anti-mouse IgG (j) were demonstrated. Black bar = 200 μm; white bar = 100 μm. (k) One or 4 days after subculture, NHEKs were collected for analysis of mRNA and protein expression by quantitative real-time PCR and Western blot, respectively. (l) NHEKs were treated with PMA and then Syk expression was analyzed as in (k). Quantification data of Syk protein expression are shown in the lower panels. The individual control was set as 1.0. Data were means from three independent experiments. *P < 0.05 (mean ± s.e.m. n = 3).
      Next, to investigate whether the abundant expression of Syk in primary cultured normal human epidermal keratinocytes (NHEKs) can be changed during keratinocyte differentiation, we used cell confluence and phorbol 12-myristate 13-acetate (PMA) to induce keratinocyte differentiation. As shown in Figure 1k, when NHEKs were cultured for 4 days to reach confluence, Syk mRNA expression was strongly suppressed. The expression levels of Syk and phosphorylated Syk decreased gradually after 4-day culture, irrespective of growth factors in the culture medium. When PMA was applied, Syk mRNA expression in NHEKs was decreased, especially after treatment for 24 hours. Reductions in Syk and phosphorylated Syk expression by PMA were also observed, especially at a high concentration of PMA (100 nM, 24 hours) or at the late time point (30 nM, 48 hours) (Figure 1l). Compared with confluence and PMA, the suppressive effect of calcium, another differentiation-inducing agent in cultured NHEKs, on Syk expression was weak (see Supplementary Figure S1, online).

      Inhibition of Syk promotes keratinocyte terminal differentiation

      The use of pharmacologic inhibitors of Syk has been explored to treat diverse inflammatory diseases. Thus, we evaluated the potential effects of pharmacologic inhibitors of Syk in keratinocytes. We used R406, an active metabolite of fostamatinib, to treat NHEKs and detected increased mRNA expression of various differentiation markers, including involucrin, TGase 1, keratin 10, and loricrin. The induction of keratin 10 by R406 was especially prominent (Figure 2a). As shown in Figure 2b and c, R406 induced the protein expression of various differentiation markers in time- and concentration-dependent manners, and keratin 10 and loricrin were particularly strongly induced. We also evaluated the effect of another Syk inhibitor, Bay 61-3606, on keratinocyte differentiation. Similar to R406, Bay 61-3606 induced the expression of keratinocyte differentiation markers, especially keratin 10 and loricrin (Figure 2d).
      Figure 2
      Figure 2Syk inhibitors promote the expression of keratinocyte differentiation markers. (a) NHEKs were treated with Syk inhibitor, R406, for 12 and 24 hours. Then, the mRNA expression of differentiation markers were analyzed by quantitative real-time PCR. *P < 0.05 compared with DMSO-treated control group (mean ± s.e.m. n = 3) (b) NHEKs were treated with R406 of different concentrations for 30 hours. Then, protein expression of differentiation markers were determined by Western blot. (c) NHEKs were treated with R406 (1 μM) for 1, 3, and 5 days. Then cells were collected for the protein analysis by Western blot. (d) Similar to (c), NHEKs were treated with another Syk inhibitor, Bay 61-3606 (10 μM).

      Syk expression regulates keratinocyte terminal differentiation

      Based on the prominent effects of Syk inhibitors, we evaluated the role of Syk expression in keratinocyte differentiation. Syk expression in NHEKs was knocked down by small interfering RNA (siRNA), and NHEKs were cultured until cells reached confluence. This confluence-induced differentiation model mimics the natural course of keratinocyte differentiation and exhibits various differentiation markers (
      • Green H.
      The keratinocyte as differentiated cell type.
      ). As shown in Figure 3a–d, at the fourth day after subculture when the cell density was increased, the confluence-induced mRNA expression levels of involucrin, TGase 1, keratin 10, and loricrin were more increased in Syk knockdown NHEKs than in the control siRNA group. At the protein level the induction of differentiation markers was higher in NHEKs with Syk knockdown than in the control group at the fifth day after subculture (Figure 3e). We examined if Syk also regulates PMA- and calcium-induced keratinocyte differentiation. As shown in Supplementary Figure S2 (online), 48 hours after knocking down Syk in NHEKs, we treated NHEKs with PMA and calcium for 24 hours. The protein expression levels of involucrin and TGase 1 induced by PMA or calcium were relatively higher in NHEKs with Syk knockdown than in controls (Supplementary Figure S2).
      Figure 3
      Figure 3Knockdown of Syk promotes the expression of keratinocyte differentiation markers induced by cell confluence. (a–d) One and 4 days after knocking down Syk in NHEKs, cells were collected for the analysis of mRNA expression of differentiation markers including involucrin, TGase 1, keratin 10, and loricrin by quantitative real-time PCR. *P < 0.05 compared with siControl group (mean ± s.e.m. n = 3). (e) Similarly, 1, 3, and 5 days after knocking down Syk, protein expression of differentiation markers in groups of control siRNA (siCtrl) or Syk siRNA (siSyk) were determined by Western blotting.

      Role of Syk in epidermal keratinocyte growth

      After establishing the role of Syk in keratinocyte differentiation, we further evaluated its potential regulatory role in NHEK proliferation. As shown in Figure 4a, the number of NHEKs as indexed by a crystal violet assay decreased by around 20% after treatment with the Syk inhibitors R406 and Bay 61-3606 for 3 days. Similarly, the relative cell number showed a slight (around 10%) inhibition in NHEKs after Syk knockdown (Figure 4b). A cell cycle analysis demonstrated that the S-phase of NHEKs treated with R406 was mildly but significantly reduced (Figure 4c). Based on BrdU uptake, R406 significantly reduced the proliferative effect of epidermal keratinocytes by 10% to 30% depending on the cell culture medium containing growth factor or not (Figure 4d). These results suggest that Syk may also play a role in keratinocyte growth.
      Figure 4
      Figure 4Inhibition of Syk mildly reduces keratinocyte proliferation. (a) NHEKs were treated with Syk inhibitors, R406 and Bay 61-3606 (Bay), for 3 days, and then the relative cell number was determined by the crystal violet assay. (b) Ninety-six hours after knocking down Syk in NHEKs, relative cell numbers were measured by crystal violet assay. Syk expression was also shown. (c) NHEKs were treated with the Syk inhibitor, R406 (1 μM), for 3 days, and then cell cycle was analyzed by flow cytometry. (d) After starvation for 24 hours, NHEKs were treated with R406 (1 μM) or DMSO for 2 days and then cell proliferation was analyzed by BrdU incorporation assay. The group of NHEKs treated with DMSO cultured in media containing growth factors was represented as the control. *P < 0.05 compared with DMSO group (mean ± s.e.m. n = 3).

      Cross-regulation between the activation of Syk and EGFR in epidermal keratinocytes

      EGFR signaling can affect keratinocyte differentiation and growth; accordingly, the possible roles of Syk in EGFR-activated signaling were explored. First, we assessed the role of EGFR in keratinocyte differentiation. As shown in Figure 5a, EGFR expression and EGFR phosphorylation decreased along with cell differentiation caused by confluence. EGFR knockdown by siRNA especially increased the expression of the differentiation markers keratin 10 and loricrin at cell confluence (Figure 5b). Notably, EGF prominently induced Syk phosphorylation, which persisted for more than 3 hours (Figure 5c and d). The phosphorylation of Syk induced by EGF was almost abolished by EGFR knockdown or gefitinib treatment (Figure 5c and d), confirming that Syk is a downstream signaling molecule of EGFR. Src has crucial roles in EGFR-activated signaling (
      • Egloff A.M.
      • Grandis J.R.
      Targeting epidermal growth factor receptor and SRC pathways in head and neck cancer.
      ,
      • Xi S.
      • Zhang Q.
      • Dyer K.F.
      • et al.
      Src kinases mediate STAT growth pathways in squamous cell carcinoma of the head and neck.
      ) and Src family kinases are upstream signals for Syk activation in multiple ITAM-containing immunoreceptors (
      • Mocsai A.
      • Ruland J.
      • Tybulewicz V.L.
      The SYK tyrosine kinase: a crucial player in diverse biological functions.
      ); these observations were verified using the Src-family kinase inhibitor PP2. We found that EGF-induced Syk phosphorylation was partially suppressed by PP2, implying that Src mediates EGF-induced Syk activation (Figure 5d). Since Syk inhibition strongly promoted the expression of keratin 10 and loricrin, similar to the effect of EGFR knockdown, we further investigated the roles of Syk in EGFR activation. As shown in Figure 5e, R406 obviously suppressed EGF-induced phosphorylation of EGFR. Knockdown of Syk showed a similar but relatively mild effect.
      Figure 5
      Figure 5Syk is involved in EGFR-mediated signaling in keratinocytes. (a) Expression of EGFR and phosphorylated EGFR in NHEKs 1, 3, and 5 days after subculture was determined by Western blot. Lower panels were quantification data. The individual control was set as 1.0. Data were means from three independent experiments. *P < 0.05 (mean ± s.e.m. n = 3). (b) Keratinocyte differentiation markers were evaluated 1, 3, and 5 days after knocking down EGFR. (c) Twenty-four hours after knocking down EGFR, NHEKs were treated with EGF (50 ng/ml) after starvation for another 48 hours. (d) After pretreatment of gefitinib (10 μM) or PP2 (10 μM), Syk phosphorylation was determined. (e) After pretreatment of R406 (1 μM), EGFR phosphorylation was evaluated after EGF (50 ng/ml) treatment. Twenty-four hours after knocking down Syk, NHEKs were starved for another 48 hours and then treated similarly.

      Interactions between Syk and EGFR in epidermal keratinocytes

      We next investigated the subcellular localization of Syk and EGFR to examine their interactions. As shown in Figure 6a, before stimulation with EGF, EGFR was primarily detected on the plasma membrane, whereas Syk was detected in the plasma membrane, nuclei, and cytoplasm. The distribution of Src expression was quite similar to that of EGFR. Mild co-expression of EGFR and Syk was detected in the plasma membrane. After stimulation with EGF, the co-localization of EGFR and Syk was increased and moved from plasma membrane to perinuclei between 30 minutes and 1 hour and then returned back to the plasma membrane at 3 hours. These findings reveal subcellular interactions between Syk and EGFR in epidermal keratinocytes. To verify the molecular interaction between EGFR and Syk in NHEKs, we used immunoprecipitation to demonstrate that in addition to the interaction between EGFR and Src, both Syk and Src interacted with EGFR, and such interactions were particularly enhanced after EGF stimulation (Figure 6b). We also investigated the interaction between Syk and EGFR in differentiated keratinocytes. Confocal microscopy showed very subtle change in the subcellular localization of Syk or co-localization of EGFR and Syk after induction of differentiation (see Supplementary Figure S3, online). Based on immunoprecipitation in cells under PMA or calcium treatment or at confluence, we found that Syk and Src association with EGFR was weaker (Figure 6c).
      Figure 6
      Figure 6Interaction between Syk and EGFR in response to EGF stimulation. (a) NHEKs were stained with anti-EGFR (red, R), anti-Syk (green, G), and anti-Src (white, W) antibodies and with DAPI (blue) for nuclei. Individual and overlaid fluorescent images were shown after EGF stimulation. White bar = 20 μm. Arrowheads indicate the co-localization of Syk and EGFR. Data were representative of three independent experiments. Images were acquired using a Zeiss LSM780 confocal microscope. (b) After EGF (50 ng/ml) treatment, immunoprecipitation with EGFR antibody was performed to detect the interaction with Syk. (c) NHEKs treated with CaCl2 or PMA for 24 hours or without treatments (C) confluent NHEKs (Conf.) were subjected for immunoprecipitation with EGFR antibody to detect the interaction with Syk.

      Discussion

      Because of the embryonic or perinatal lethality of Syk-deficient mice (
      • Cheng A.M.
      • Rowley B.
      • Pao W.
      • et al.
      Syk tyrosine kinase required for mouse viability and B-cell development.
      ,
      • Turner M.
      • Mee P.J.
      • Costello P.S.
      • et al.
      Perinatal lethality and blocked B-cell development in mice lacking the tyrosine kinase Syk.
      ), it is difficult to observe the skin phenotype of adult Syk-deficient mice. The development of mice after epidermis-specific Syk knockout may reveal the in vivo roles of Syk in skin keratinocytes. Syk functions in the skin have rarely been studied. One previous study indicated that Syk regulates UV-induced metalloproteinase expression in skin dermal fibroblasts (
      • Papazoglou E.
      • Huang Z.Y.
      • Sunkari C.
      • et al.
      The role of Syk kinase in ultraviolet-mediated skin damage.
      ). However, many potential biologic roles of Syk in epidermal keratinocytes remain unknown. In our study we used pharmacologic inhibitors of Syk and Syk knockdown to examine the roles of Syk in epidermal keratinocytes.
      The regulatory roles of Syk in immune systems have been widely explored (
      • Mocsai A.
      • Ruland J.
      • Tybulewicz V.L.
      The SYK tyrosine kinase: a crucial player in diverse biological functions.
      ). Syk may act as a positive or negative regulator in the signaling pathways of innate and adaptive immunoreceptors. It is not surprising that Syk is abundantly expressed in epidermal keratinocytes because keratinocytes express multiple innate immunoreceptors and play critical roles in innate immunity to maintain skin homeostasis (
      • Kobayashi M.
      • Yoshiki R.
      • Sakabe J.
      • et al.
      Expression of toll-like receptor 2, NOD2 and dectin–1 and stimulatory effects of their ligands and histamine in normal human keratinocytes.
      ,
      • McInturff J.E.
      • Modlin R.L.
      • Kim J.
      The role of toll-like receptors in the pathogenesis and treatment of dermatological disease.
      ). Skin keratinocytes undergo the well-coordinated process of terminal differentiation to form the skin barrier. In fact, in addition to being a unique model of cell death, keratinocyte terminal differentiation is defensive in nature, and various components involved in innate immunity are upregulated during this process (
      • Elias P.M.
      Stratum corneum defensive functions: an integrated view.
      ,
      • Nestle F.O.
      • Di Meglio P.
      • Qin J.Z.
      • et al.
      Skin immune sentinels in health and disease.
      ). In this study we demonstrated that Syk, an immunoregulator, is involved in keratinocyte differentiation. More interestingly, Syk activation and expression were gradually downregulated along with keratinocyte differentiation, as evidenced by immunohistochemical staining and cell studies (Figure 1). We also showed that Syk inhibitors or Syk knockdown increase the expression of keratinocyte differentiation markers, suggesting a negative regulatory role of Syk in keratinocyte differentiation.
      Most previous studies of Syk functions in nonskin epithelial cells have focused on carcinogenesis. Syk expression is low or undetectable in invasive breast carcinomas, and the reintroduction of Syk into cancer cell lines inhibits tumor growth and metastasis in animal models (
      • Coopman P.J.
      • Do M.T.
      • Barth M.
      • et al.
      The Syk tyrosine kinase suppresses malignant growth of human breast cancer cells.
      ). These findings suggest that Syk modulates epithelial cell growth and acts as a tumor suppressor in breast carcinomas. Syk is downregulated in oral squamous cell carcinoma specimens and is regarded as a candidate tumor suppressor gene (
      • Ogane S.
      • Onda T.
      • Takano N.
      • et al.
      Spleen tyrosine kinase as a novel candidate tumor suppressor gene for human oral squamous cell carcinoma.
      ). In squamous cell carcinoma of the head and neck, high Syk expression in tumor specimens is associated with reduced patient survival (
      • Luangdilok S.
      • Box C.
      • Patterson L.
      • et al.
      Syk tyrosine kinase is linked to cell motility and progression in squamous cell carcinomas of the head and neck.
      ). In addition to epithelial tumors, Syk acts as a tumor suppressor to induce senescence-like growth arrest in melanomas (
      • Bailet O.
      • Fenouille N.
      • Abbe P.
      • et al.
      Spleen tyrosine kinase functions as a tumor suppressor in melanoma cells by inducing senescence-like growth arrest.
      ).
      As a critical regulator in immunoreceptor-mediated signaling, Syk phosphorylation is triggered by the activation of B-cell receptors, T-cell receptors, and Fc receptors and by several pattern recognition receptors (
      • Mocsai A.
      • Ruland J.
      • Tybulewicz V.L.
      The SYK tyrosine kinase: a crucial player in diverse biological functions.
      ). We demonstrated that EGFR activation strongly increases Syk phosphorylation, and this effect lasted for more than 3 hours. Induced and even basal phosphorylation of Syk were almost completely abolished by the EGFR inhibitor gefitinib and EGFR knockdown. This finding indicates that Syk is an important signaling molecule downstream of the EGFR pathway, in addition to its well-known roles in downstream immunoreceptor signaling. Additionally, we showed that Src kinase inhibitor, PP2, partially suppresses EGFR activation-induced Syk phosphorylation. These findings reveal that Syk is critically involved in the EGFR-mediated signaling pathway.
      The EGFR/ligand system plays key roles in essential cellular functions. Skin keratinocytes are a rich source of several EGFR ligands (
      • Pastore S.
      • Mascia F.
      • Mariani V.
      • et al.
      The epidermal growth factor receptor system in skin repair and inflammation.
      ) and autocrine action of EGFR ligands is involved in epidermal homeostasis, hair follicle development, and keratinocyte stem cell behavior (
      • Nanba D.
      • Toki F.
      • Barrandon Y.
      • et al.
      Recent advances in the epidermal growth factor receptor/ligand system biology on skin homeostasis and keratinocyte stem cell regulation.
      ). EGFR signaling in the skin is involved in epidermal keratinocyte growth regulation, re-epithelialization of epidermal wound healing, inflammatory responses, differentiation, and proliferation of keratinocyte stem cells (
      • Gazel A.
      • Blumenberg M.
      Transcriptional effects of inhibiting epidermal growth factor receptor in keratinocytes.
      ,
      • Jensen K.B.
      • Collins C.A.
      • Nascimento E.
      • et al.
      Lrig1 expression defines a distinct multipotent stem cell population in mammalian epidermis.
      ,
      • Pastore S.
      • Mascia F.
      • Mariani V.
      • et al.
      The epidermal growth factor receptor system in skin repair and inflammation.
      ). Recently, we identified a role of EGFR in psoriasis via upregulation of decoy receptor 3, an immune modulator and proinflammatory mediator, in human keratinocytes (
      • Wu N.L.
      • Huang D.Y.
      • Hsieh S.L.
      • et al.
      EGFR-driven up-regulation of decoy receptor 3 in keratinocytes contributes to the pathogenesis of psoriasis.
      ). To date, studies suggesting a functional link between Syk and EGFR are limited. Our data revealed the importance of Syk in EGFR-mediated signaling, and these results may promote further studies of the role of Syk in regulating EGFR-mediated inflammatory diseases.
      Previous studies have shown that Syk is associated with EGFR in some cell types (
      • Luangdilok S.
      • Box C.
      • Patterson L.
      • et al.
      Syk tyrosine kinase is linked to cell motility and progression in squamous cell carcinomas of the head and neck.
      ,
      • Ruschel A.
      • Ullrich A.
      Protein tyrosine kinase Syk modulates EGFR signalling in human mammary epithelial cells.
      ). High Syk expression is correlated with reduced survival in squamous cell carcinomas of the head and neck, and Syk inhibition reduces cell migration and invasion (
      • Luangdilok S.
      • Box C.
      • Patterson L.
      • et al.
      Syk tyrosine kinase is linked to cell motility and progression in squamous cell carcinomas of the head and neck.
      ). However, downregulation of endogenous Syk expression in breast epithelial cells increases the ligand-induced activity of EGFR (
      • Ruschel A.
      • Ullrich A.
      Protein tyrosine kinase Syk modulates EGFR signalling in human mammary epithelial cells.
      ). In our study Syk was associated with EGFR and mediated its signaling; it also negatively regulated keratinocyte differentiation. Furthermore, the expression of phosphorylated Syk was obviously located in the cell membrane of epidermal keratinocytes, especially in psoriasis specimens. This staining pattern is similar to that observed in our confocal imaging study, which reveals relatively strong co-localization of Syk and EGFR in the cell membrane at basal status.
      Based on the important roles of Syk in immunologic functions, Syk inhibition is a promising treatment for immunologic or inflammatory diseases (
      • Kaur M.
      • Singh M.
      • Silakari O.
      Inhibitors of switch kinase ‘spleen tyrosine kinase’ in inflammation and immune-mediated disorders: a review.
      ). In addition to the accumulating experimental studies of the therapeutic potential of Syk inhibitors, the use of fostamatinib (R788), a Syk inhibitor, to treat rheumatoid arthritis has been examined in a phase III clinical trial (
      • Scott I.C.
      • Scott D.L.
      Spleen tyrosine kinase inhibitors for rheumatoid arthritis: where are we now?.
      ). Nevertheless, because of the ubiquitous expression of Syk in various tissues and cells, it is interesting to investigate the effects of Syk inhibitors on other organs, including the skin. Based on our results, the Syk inhibitor R406, which is an active metabolite of fostamatinib, prominently induced the expression of differentiation markers, suggesting potential effects of Syk inhibitors on skin keratinocytes. In addition, our immunohistochemical study shows high expression of phosphorylated Syk in psoriatic epidermis samples. Our recent study also reveals that Syk mediates IL-17A–induced signaling to regulate chemokine C-C motif ligand 20 expression in epidermal keratinocytes (
      • Wu N.L.
      • Huang D.Y.
      • Tsou H.N.
      • et al.
      Syk mediates IL-17-induced CCL20 expression by targeting Act1-dependent K63-linked ubiquitination of TRAF6.
      ), implying potential roles of Syk inhibitors in the treatment of psoriasis. Additional studies are needed to determine the effects of diverse Syk inhibitors on the skin. Although no topical Syk inhibitor is used in clinical applications, their potential effects on the skin are of interest. Based on our results, animal models should be used to evaluate the effects of topical Syk inhibitors on the recovery of skin barrier function or skin diseases characterized by aberrant keratinocyte differentiation and inflammation, such as psoriasis.
      In conclusion, we revealed the crucial role of Syk in skin biology. Syk acts as a negative regulator in epidermal keratinocyte differentiation, and is also involved in EGFR signaling, which may contribute, at least in part, to its regulatory role in keratinocyte terminal differentiation. These findings not only extend the current understanding of Syk but also pave the way to develop pharmacologic agents to treat skin disorders via Syk targeting.

      Methods

      Immunohistochemical staining

      Paraffin-embedded skin specimens were prepared retrospectively for immunohistochemical studies. The experiments were conducted according to the Declaration of Helsinki principles and approved by the Ethics Committee of Mackay Memorial Hospital. Briefly, formalin-fixed and paraffin-embedded tissue sections were processed and subjected to staining with Dako Autostainer Link48 (Dako, Glostrup, Denmark) and the primary antibodies against phosphorylated Syk (Abgent, San Diego, CA) or Syk (Santa Cruz Biotechnology, Santa Cruz, CA).

      Cell culture

      NHEKs were obtained from normal adult human foreskin and isolated as described previously (
      • Wu N.L.
      • Lee T.A.
      • Tsai T.L.
      • et al.
      TRAIL-induced keratinocyte differentiation requires caspase activation and p63 expression.
      ). The experiments were conducted according to the Declaration of Helsinki principles and approved by the Ethics Committee of Mackay Memorial Hospital (Institutional Review Board codes 10MMHIS179 and 15MMHIS019e). Written, informed consent was obtained from each donor before experiments were performed.

      Reagents

      Antibodies against TGase 1, involucrin, and Syk and horseradish peroxidase-coupled secondary antibodies were purchased from Santa Cruz Biotechnology. Antibodies for human keratin 10 and loricrin were purchased from Covance (Emeryville, CA). Antibodies for EGFR, Src, phosphorylated EGFR, and phosphorylated Syk were purchased from Cell Signaling Technology (Beverly, MA). The antibody against β-actin was obtained from Upstate Biotechnology (Charlottesville, VA). The enhanced chemiluminescence reagent was obtained from Perkin Elmer (Wellesley, MA). Soluble recombinant human EGF was purchased from PeproTech (Rocky Hill, NJ). Gefitinib was purchased from Cayman Chemical Company (Ann Arbor, MI). PP2 was purchased from Calbiochem (San Diego, CA). R406 was purchased from Selleck Chemicals LLC (Houston, TX).

      Immunoblotting

      Protein expression was determined in cell lysates by electrophoresis and immunoblotting as previously described (
      • Wu N.L.
      • Huang D.Y.
      • Hsieh S.L.
      • et al.
      EGFR-driven up-regulation of decoy receptor 3 in keratinocytes contributes to the pathogenesis of psoriasis.
      ).

      Immunoprecipitation

      NHEKs with the indicated treatments were harvested. Immunoprecipitation experiments were performed with specific antibodies, and then an immunoblotting analysis was performed as described previously (
      • Lin Y.C.
      • Huang D.Y.
      • Chu C.L.
      • et al.
      The tyrosine kinase Syk differentially regulates Toll-like receptor signaling downstream of the adaptor molecules TRAF6 and TRAF3.
      ).

      Real-time PCR

      The primer sequence pairs used for quantitative real-time PCR were involucrin (NM_005547): 5′-TCAATACCCATCAGGAGCAAATG-3′ and 5′-GAGCTCGACA GGCACCTTCT-3′; TGase 1 (NM_000359): 5′-TCTTCAAGAACCCCCTTCCC-3′ and 5′-TCTGTAACCCAGAGCCTTCG A-3′; keratin 10 (NM_000421.3): 5′-ATAAT GCCAACATCCTGCTTCA-3′ and 5′-AGGCCGTTGATGTCAGCCT-3′; loricrin (NM_000427.2): 5′-TCATGATGCTACCCGAGGTT TG-3′ and 5′-CAGAA CTAGATGCAGCCGGAGA-3′; and the housekeeping gene cyclophilin A (NM_21130): 5′-CCCACCGTGTTCTTCGACAT-3′ and 5′-CCAGTGCTCAGAGCACGAAA-3′. The experiment and data analysis were conducted as previously described (
      • Wu N.L.
      • Lee T.A.
      • Tsai T.L.
      • et al.
      TRAIL-induced keratinocyte differentiation requires caspase activation and p63 expression.
      ).

      Small interfering RNA

      siRNA targeting mRNA degradation of human Syk and EGFR and scramble nonspecific siRNA were purchased from Dharmacon Research Inc. (Lafayette, CO). The procedure was performed as described previously (
      • Wu N.L.
      • Lee T.A.
      • Tsai T.L.
      • et al.
      TRAIL-induced keratinocyte differentiation requires caspase activation and p63 expression.
      ).

      Crystal violet assay

      To evaluate the relative cell numbers, NHEKs with the indicated treatments were cultured in media for days. After cells were rinsed with phosphate-buffered saline, they were fixed with methanol for 10 minutes at room temperature and stained with 0.1% crystal violet. The relative cell number was determined by measuring the absorbance of the dissolved dye at 540 nm after elution with 33% acetic acid.

      BrdU cell proliferation assay

      Briefly, NHEKs were starved for 48 hours and then treated with indicated drugs and cultured in media with or without growth factors for an additional 24 hours. Cells were treated with BrdU, and the cell proliferation rate was determined by colorimetric BrdU cell proliferation ELISA according to the manufacturer’s instructions (Roche, Mannheim, Germany).

      Cell cycle analysis by flow cytometry

      NHEKs with the indicated treatments were harvested and subjected to a cell cycle analysis by flow cytometry (BD Biosciences, Franklin Lakes, NJ) as described previously (
      • Su R.Y.
      • Chao Y.
      • Chen T.Y.
      • et al.
      5-Aminoimidazole-4-carboxamide riboside sensitizes TRAIL- and TNF{alpha}-induced cytotoxicity in colon cancer cells through AMP-activated protein kinase signaling.
      ).

      Immunofluorescence and confocal microscopy

      NHEKs were fixed with 4% paraformaldehyde at 37 °C for 20 minutes and then permeabilized with 0.2% Triton X-100 for 15 minutes. After blocking with 5% BSA with normal IgG (1:300) for 1 hour, immunostaining was performed with primary antibodies against Syk (Santa Cruz Biotechnology), EGFR (Santa Cruz Biotechnology), and hSrc-647nm (Abcam, Cambridge, UK) in 1% BSA overnight at 4 °C. After washing with phosphate-buffered saline, cells were incubated with secondary antibodies in 1% BSA in phosphate-buffered saline solution for 1 hour at room temperature and then mounted with DAPI Fluoromount-G (SouthernBiotech, Birmingham, AL). Images were acquired using a confocal microscope (model LSM780; Carl Zeiss MicroImaging GmbH, Jena, Germany).

      Statistical analysis

      Data were expressed as means ± s.e.m. Student’s t-tests were used to assess the statistical significance of differences. P < 0.05 was considered to be statistically significant.

      Conflict of Interest

      The authors state no conflict of interest.

      Acknowledgments

      This work was supported by research grants from the Ministry of Science and Technology, Taiwan (NSC 102-2325-B-002-033, NSC 101-2314-B-195-003-, MOST 103-2314-B-195-012-MY2), Academia Sinica (IBMS-CRC103-P01), and TMUTOP103005-5. We thank the staff of the imaging core at the First Core Labs, National Taiwan University College of Medicine, for technical assistance.

      Supplementary Material

      References

        • Bailet O.
        • Fenouille N.
        • Abbe P.
        • et al.
        Spleen tyrosine kinase functions as a tumor suppressor in melanoma cells by inducing senescence-like growth arrest.
        Cancer Res. 2009; 69: 2748-2756
        • Candi E.
        • Schmidt R.
        • Melino G.
        The cornified envelope: a model of cell death in the skin.
        Nat Rev Mol Cell Biol. 2005; 6: 328-340
        • Cheng A.M.
        • Rowley B.
        • Pao W.
        • et al.
        Syk tyrosine kinase required for mouse viability and B-cell development.
        Nature. 1995; 378: 303-306
        • Coopman P.J.
        • Do M.T.
        • Barth M.
        • et al.
        The Syk tyrosine kinase suppresses malignant growth of human breast cancer cells.
        Nature. 2000; 406: 742-747
        • Eckert R.L.
        • Sturniolo M.T.
        • Broome A.M.
        • et al.
        Transglutaminase function in epidermis.
        J Invest Dermatol. 2005; 124: 481-492
        • Egloff A.M.
        • Grandis J.R.
        Targeting epidermal growth factor receptor and SRC pathways in head and neck cancer.
        Semin Oncol. 2008; 35: 286-297
        • Elias P.M.
        Stratum corneum defensive functions: an integrated view.
        J Invest Dermatol. 2005; 125: 183-200
        • Galluzzi L.
        • Vitale I.
        • Abrams J.M.
        • et al.
        Molecular definitions of cell death subroutines: recommendations of the Nomenclature Committee on Cell Death 2012.
        Cell Death Differ. 2012; 19: 107-120
        • Gazel A.
        • Blumenberg M.
        Transcriptional effects of inhibiting epidermal growth factor receptor in keratinocytes.
        Dermatol Sinica. 2013; 31: 107-119
        • Green H.
        The keratinocyte as differentiated cell type.
        Harvey Lect. 1980; 74: 101-139
        • Jensen K.B.
        • Collins C.A.
        • Nascimento E.
        • et al.
        Lrig1 expression defines a distinct multipotent stem cell population in mammalian epidermis.
        Cell Stem Cell. 2009; 4: 427-439
        • Jung E.M.
        • Betancourt-Calle S.
        • Mann-Blakeney R.
        • et al.
        Sustained phospholipase D activation is associated with keratinocyte differentiation.
        Carcinogenesis. 1999; 20: 569-576
        • Kaur M.
        • Singh M.
        • Silakari O.
        Inhibitors of switch kinase ‘spleen tyrosine kinase’ in inflammation and immune-mediated disorders: a review.
        Eur J Med Chem. 2013; 67: 434-446
        • King Jr., L.E.
        • Gates R.E.
        • Stoscheck C.M.
        • et al.
        Epidermal growth factor/transforming growth factor alpha receptors and psoriasis.
        J Invest Dermatol. 1990; 95: 10S-12S
        • Kobayashi M.
        • Yoshiki R.
        • Sakabe J.
        • et al.
        Expression of toll-like receptor 2, NOD2 and dectin–1 and stimulatory effects of their ligands and histamine in normal human keratinocytes.
        Br J Dermatol. 2009; 160: 297-304
        • Lin Y.C.
        • Huang D.Y.
        • Chu C.L.
        • et al.
        The tyrosine kinase Syk differentially regulates Toll-like receptor signaling downstream of the adaptor molecules TRAF6 and TRAF3.
        Sci Signal. 2013; 6: ra71
        • Luangdilok S.
        • Box C.
        • Patterson L.
        • et al.
        Syk tyrosine kinase is linked to cell motility and progression in squamous cell carcinomas of the head and neck.
        Cancer Res. 2007; 67: 7907-7916
        • McInturff J.E.
        • Modlin R.L.
        • Kim J.
        The role of toll-like receptors in the pathogenesis and treatment of dermatological disease.
        J Invest Dermatol. 2005; 125: 1-8
        • Mocsai A.
        • Ruland J.
        • Tybulewicz V.L.
        The SYK tyrosine kinase: a crucial player in diverse biological functions.
        Nat Rev Immunol. 2010; 10: 387-402
        • Nanba D.
        • Toki F.
        • Barrandon Y.
        • et al.
        Recent advances in the epidermal growth factor receptor/ligand system biology on skin homeostasis and keratinocyte stem cell regulation.
        J Dermatol Sci. 2013; 72: 81-86
        • Nanney L.B.
        • Magid M.
        • Stoscheck C.M.
        • et al.
        Comparison of epidermal growth factor binding and receptor distribution in normal human epidermis and epidermal appendages.
        J Invest Dermatol. 1984; 83: 385-393
        • Nestle F.O.
        • Di Meglio P.
        • Qin J.Z.
        • et al.
        Skin immune sentinels in health and disease.
        Nat Rev Immunol. 2009; 9: 679-691
        • Ogane S.
        • Onda T.
        • Takano N.
        • et al.
        Spleen tyrosine kinase as a novel candidate tumor suppressor gene for human oral squamous cell carcinoma.
        Int J Cancer. 2009; 124: 2651-2657
        • Papazoglou E.
        • Huang Z.Y.
        • Sunkari C.
        • et al.
        The role of Syk kinase in ultraviolet-mediated skin damage.
        Br J Dermatol. 2011; 165: 69-77
        • Pastore S.
        • Mascia F.
        • Mariani V.
        • et al.
        The epidermal growth factor receptor system in skin repair and inflammation.
        J Invest Dermatol. 2008; 128: 1365-1374
        • Riccaboni M.
        • Bianchi I.
        • Petrillo P.
        Spleen tyrosine kinases: biology, therapeutic targets and drugs.
        Drug Discov Today. 2010; 15: 517-530
        • Ruschel A.
        • Ullrich A.
        Protein tyrosine kinase Syk modulates EGFR signalling in human mammary epithelial cells.
        Cell Signal. 2004; 16: 1249-1261
        • Scott I.C.
        • Scott D.L.
        Spleen tyrosine kinase inhibitors for rheumatoid arthritis: where are we now?.
        Drugs. 2014; 74: 415-422
        • Su R.Y.
        • Chao Y.
        • Chen T.Y.
        • et al.
        5-Aminoimidazole-4-carboxamide riboside sensitizes TRAIL- and TNF{alpha}-induced cytotoxicity in colon cancer cells through AMP-activated protein kinase signaling.
        Mol Cancer Therap. 2007; 6: 1562-1571
        • Turner M.
        • Mee P.J.
        • Costello P.S.
        • et al.
        Perinatal lethality and blocked B-cell development in mice lacking the tyrosine kinase Syk.
        Nature. 1995; 378: 298-302
        • Turner M.
        • Schweighoffer E.
        • Colucci F.
        • et al.
        Tyrosine kinase SYK: essential functions for immunoreceptor signalling.
        Immunol Today. 2000; 21: 148-154
        • Wu N.L.
        • Huang D.Y.
        • Hsieh S.L.
        • et al.
        EGFR-driven up-regulation of decoy receptor 3 in keratinocytes contributes to the pathogenesis of psoriasis.
        Biochim Biophys Acta. 2013; 1832: 1538-1548
        • Wu N.L.
        • Huang D.Y.
        • Tsou H.N.
        • et al.
        Syk mediates IL-17-induced CCL20 expression by targeting Act1-dependent K63-linked ubiquitination of TRAF6.
        J Invest Dermatol. 2015; 135: 490-498
        • Wu N.L.
        • Lee T.A.
        • Tsai T.L.
        • et al.
        TRAIL-induced keratinocyte differentiation requires caspase activation and p63 expression.
        J Invest Dermatol. 2011; 131: 874-883
        • Xi S.
        • Zhang Q.
        • Dyer K.F.
        • et al.
        Src kinases mediate STAT growth pathways in squamous cell carcinoma of the head and neck.
        J Biol Chem. 2003; 278: 31574-31583
        • Yanagi S.
        • Inatome R.
        • Takano T.
        • et al.
        Syk expression and novel function in a wide variety of tissues.
        Biochem Biophys Res Commun. 2001; 288: 495-498