Advertisement

TWEAK/Fn14 Activation Contributes to the Pathogenesis of Bullous Pemphigoid

Open ArchivePublished:March 25, 2017DOI:https://doi.org/10.1016/j.jid.2017.03.019
      TWEAK participates in various cellular effects by engaging its receptor of Fn14. Increased levels of soluble TWEAK are associated with systemic autoimmunity in patients with lupus erythematosus, rheumatoid arthritis, or dermatomyositis. However, the role of TWEAK in bullous pemphigoid (BP) remains unknown. In this study, we found an elevated serum level of TWEAK and a positive correlation between serum TWEAK and anti-BP180 antibodies. Immunohistochemistry showed strong TWEAK and Fn14 expression and implied an opposite relationship between the TWEAK and BP180 expression in skin samples from BP patients. In vitro TWEAK stimuli reduced BP180 expression in HaCaT cells and inhibited the adhesion of cells to the culture dish. Consistently, the transfection of Fn14 small interfering RNA preserved BP180 and protected cells from losing adherence. Moreover, such effect of TWEAK correlated with activation of the extracellular signal-regulated kinase and NF-κB pathways and downstream ADAMs. By silencing ADAM17 with small interfering RNA, we showed that ADAM17 participated in TWEAK-induced BP180 loss. Therefore, TWEAK may contribute to the pathogenesis of BP by reducing BP180 expression and cellular adherence, involving the activation of ERK and NF-κB pathways. TWEAK may serve as a biomarker or therapeutic target of BP.

      Abbreviations:

      BP (bullous pemphigoid), ERK (extracellular signal-regulated kinase), p- (phosphorylated), qRT-PCR (quantitative real-time reverse transcriptase–PCR), siRNA (small interfering RNA), TNF (tumor necrosis factor)

      Introduction

      Bullous pemphigoid (BP) is a common autoimmune disease characterized by the appearance of subepidermal blisters in skin and the presence of circulating autoantibodies against structural components (including BP180, also called collagen XVII) of hemidesmosomes and inflammatory cell infiltration in the superficial dermis, which together cause bullae formation at the dermal-epidermal junction (
      • Hurskainen T.
      • Kokkonen N.
      • Sormunen R.
      • Jackow J.
      • Löffek S.
      • Soininen R.
      • et al.
      Deletion of the major bullous pemphigoid epitope region of collagen XVII induces blistering, autoimmunization, and itching in mice.
      ,
      • Iwata H.
      • Kamio N.
      • Aoyama Y.
      • Yamamoto Y.
      • Hirako Y.
      • Owaribe K.
      • et al.
      IgG from patients with bullous pemphigoid depletes cultured keratinocytes of the 180-kDa bullous pemphigoid antigen (type XVII collagen) and weakens cell attachment.
      ,
      • Nishie W.
      Update on the pathogenesis of bullous pemphigoid: an autoantibody-mediated blistering disease targeting collagen XVII.
      ,
      • Schmidt E.
      • Zillikens D.
      Pemphigoid diseases.
      ). BP mainly affects people 70 years or older, and the morbidity and mortality of BP increase with age because of disease-specific factors (
      • Barrick B.J.
      • Lohse C.M.
      • Lehman J.S.
      Specific causes of death in patients with bullous pemphigoid as measured by death certificate data: a retrospective cohort study.
      ,
      • Di Zenzo G.
      • Della Torre R.
      • Zambruno G.
      • Borradori L.
      Bullous pemphigoid: From the clinic to the bench.
      ). In addition, the patients with BP are more prone to malignancies, pulmonary diseases, and neurologic disorders than their counterparts (
      • Cai S.C.
      • Allen J.C.
      • Lim Y.L.
      • Tan S.H.
      • Tang M.B.
      Association of bullous pemphigoid and malignant neoplasms.
      ,
      • Di Zenzo G.
      • Della Torre R.
      • Zambruno G.
      • Borradori L.
      Bullous pemphigoid: From the clinic to the bench.
      ,
      • Kokkonen N.
      • Herukka S.K.
      • Huilaja L.
      • Kokki M.
      • Koivisto A.M.
      • Hartikainen P.
      • et al.
      Increased levels of the bullous pemphigoid BP180 autoantibody are associated with more severe dementia in Alzheimer's disease.
      ). Therefore, to understand BP mechanistically is urgent for both monitoring and treating BP and associated diseases.
      TWEAK, a member of the tumor necrosis factor (TNF) superfamily, engages with its sole signaling receptor of Fn14 and is involved in multiple biological processes, including immune responses and tissue repair (
      • Chen J.
      • Wei L.
      • Xia Y.
      The roles of tumor necrosis factor-related weak inducer of apoptosis/fibroblast growth factor-inducible 14 pathway in lupus nephritis.
      ,
      • Cheng H.
      • Zhan N.
      • Ding D.
      • Liu X.
      • Zou X.
      • Li K.
      • et al.
      HPV type 16 infection switches keratinocytes from apoptotic to proliferative fate under TWEAK/Fn14 interaction.
      ,
      • Rayego-Mateos S.
      • Morgado-Pascual J.L.
      • Sanz A.B.
      • Ramos A.M.
      • Eguchi S.
      • Batlle D.
      • et al.
      TWEAK transactivation of the epidermal growth factor receptor mediates renal inflammation.
      ,
      • Xia Y.
      • Herlitz L.C.
      • Gindea S.
      • Wen J.
      • Pawar R.D.
      • Misharin A.
      • et al.
      Deficiency of fibroblast growth factor-inducible 14 (Fn14) preserves the filtration barrier and ameliorates lupus nephritis.
      ). The major pathogenic effect of TWEAK is believed to rely on the induction of proinflammatory cytokines and chemokines, such as RANTES, MCP-1, IP-10, IL-6, and IL-8 (
      • Liu Z.C.
      • Zhou Q.L.
      • Li X.Z.
      • Yang J.H.
      • Ao X.
      • Veeraragoo P.
      • et al.
      Elevation of human tumor necrosis factor-like weak inducer of apoptosis in peripheral blood mononuclear cells is correlated with disease activity and lupus nephritis in patients with systemic lupus erythematosus.
      ). The expression of TWEAK and Fn14 is up-regulated in a number of human skin diseases (
      • Cheng H.
      • Zhan N.
      • Ding D.
      • Liu X.
      • Zou X.
      • Li K.
      • et al.
      HPV type 16 infection switches keratinocytes from apoptotic to proliferative fate under TWEAK/Fn14 interaction.
      ,
      • Cheng H.
      • Xu M.
      • Liu X.
      • Zou X.
      • Zhan N.
      • Xia Y.
      TWEAK/Fn14 activation induces keratinocyte proliferation under psoriatic inflammation.
      ,
      • Doerner J.L.
      • Wen J.
      • Xia Y.
      • Paz K.B.
      • Schairer D.
      • Wu L.
      • et al.
      TWEAK/Fn14 signaling involvement in the pathogenesis of cutaneous disease in the MRL/lpr model of spontaneous lupus.
      ,
      • Zimmermann M.
      • Koreck A.
      • Meyer N.
      • Basinski T.
      • Meiler F.
      • Simone B.
      • et al.
      TNF-like weak inducer of apoptosis (TWEAK) and TNF-α cooperate in the induction of keratinocyte apoptosis.
      ). In recent years, an increasing amount of evidence has implicated the participation of TWEAK in the pathogenesis of a wide variety of autoimmune diseases that include systemic lupus erythematosus, rheumatoid arthritis, polymyositis, and dermatomyositis (
      • Burkly L.C.
      • Michaelson J.S.
      • Zheng T.S.
      TWEAK/Fn14 pathway: an immunological switch for shaping tissue responses.
      ,
      • Peng Q.L.
      • Shu X.M.
      • Tian X.L.
      • Lu X.
      • Wang G.C.
      Expression of tumor necrosis factor-like weak inducer of apoptosis and fibroblast growth factor-inducible 14 in patients with polymyositis and dermatomyositis.
      ,
      • Xu W.D.
      • Zhao Y.
      • Liu Y.
      Role of the TWEAK/Fn14 pathway in autoimmune diseases.
      ). Moreover, TWEAK/Fn14 blockade, using genetically modified mice or neutralizing antibodies, ameliorated experimental autoimmune diseases, such as cutaneous or neuropsychiatric lupus erythematosus and nephrotoxic serum nephritis (
      • Doerner J.L.
      • Wen J.
      • Xia Y.
      • Paz K.B.
      • Schairer D.
      • Wu L.
      • et al.
      TWEAK/Fn14 signaling involvement in the pathogenesis of cutaneous disease in the MRL/lpr model of spontaneous lupus.
      ,
      • Wen J.
      • Xia Y.
      • Stock A.
      • Michaelson J.S.
      • Burkly L.C.
      • Gulinello M.
      • et al.
      Neuropsychiatric disease in murine lupus is dependent on the TWEAK/Fn14 pathway.
      ,
      • Xia Y.
      • Campbell S.R.
      • Broder A.
      • Herlitz L.
      • Abadi M.
      • Wu P.
      • et al.
      Inhibition of the TWEAK/Fn14 pathway attenuates renal disease in nephrotoxic serum nephritis.
      ,
      • Xia Y.
      • Herlitz L.C.
      • Gindea S.
      • Wen J.
      • Pawar R.D.
      • Misharin A.
      • et al.
      Deficiency of fibroblast growth factor-inducible 14 (Fn14) preserves the filtration barrier and ameliorates lupus nephritis.
      ). However, to our knowledge, there are no data on the role of TWEAK/Fn14 activation in the development of BP. Because a TWEAK/Fn14-targeting pharmaceutical is undergoing clinical trials (
      • Wajant H.
      The TWEAK-Fn14 system as a potential drug target.
      ), a further understanding of TWEAK actions in BP may guide new therapeutic approaches for this disease.
      Actually, TWEAK/Fn14 activation affects the expression of junction proteins in podocytes, glomerular endothelial cells, and tubular epithelial cells (
      • Berzal S.
      • González-Guerrero C.
      • Rayego-Mateos S.
      • Ucero Á.
      • Ocaña-Salceda C.
      • Egido J.
      • et al.
      TNF-related weak inducer of apoptosis (TWEAK) regulates junctional proteins in tubular epithelial cells via canonical NF-κB pathway and ERK activation.
      ,
      • Xia Y.
      • Herlitz L.C.
      • Gindea S.
      • Wen J.
      • Pawar R.D.
      • Misharin A.
      • et al.
      Deficiency of fibroblast growth factor-inducible 14 (Fn14) preserves the filtration barrier and ameliorates lupus nephritis.
      ). The alteration of junction proteins may further damage the linkage between the extracellular and intracellular structural elements involved in cell adhesion, or even cause cell death and loss of structural integrity (
      • Xia Y.
      • Herlitz L.C.
      • Gindea S.
      • Wen J.
      • Pawar R.D.
      • Misharin A.
      • et al.
      Deficiency of fibroblast growth factor-inducible 14 (Fn14) preserves the filtration barrier and ameliorates lupus nephritis.
      ). Considering the fact that BP correlates with the abnormalities of junction proteins and the inflammatory response of keratinocytes, we speculated about the potential effect of TWEAK/Fn14 signals on the pathogenesis of this disease. The purpose of this study was to elucidate the expression of TWEAK and Fn14 in patients with BP and the role of their interaction in the development of BP lesions.

      Results

      Serum levels of TWEAK and anti-BP180 IgG correlate positively in patients with BP

      We measured the levels of TWEAK in the sera and blister fluid of patients with BP and the sera of normal control subjects. The serum TWEAK levels in patients were significantly higher than those in normal control subjects (Figure 1a). Moreover, a positive correlation was found between the serum levels of TWEAK and anti-BP180 IgG (Figure 1b). Furthermore, the downstream proteins of RANTES, MCP-1, and IP-10 were determined, showing significant increase in sera from patients compared with normal control subjects (Figure 1c).
      Figure thumbnail gr1
      Figure 1Levels of TWEAK increase in both sera and blister fluid from patients with bullous pemphigoid (BP). (a) Serum and blister fluid levels of TWEAK were measured by ELISA. (b) In patients with BP, the relationship between serum TWEAK levels and anti-BP180 IgG titers was assessed by Spearman’s rank correlation. (c) The levels of proinflammatory cytokines were determined in serum and blister fluid samples. Number of BP patients = 16, number of healthy donors = 20. P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.

      TWEAK expression is up-regulated in skin lesions of BP

      We also assessed the TWEAK expression in blister fluid and found higher levels in BP patients than that in normal sera, though not differing from sera in BP patients (Figure 1a). Consistently, histopathological analysis showed stronger staining of both TWEAK and Fn14 in lesional skin, whereas normal or nonlesional skin displayed slight staining (Figure 2a). In contrast, the expression of BP180 was strong in normal and nonlesional tissues but weak in lesional tissues (Figure 2a). The stained epidermis was quantitated for staining intensities and showed a negative linear correlation between TWEAK (or Fn14) and BP180 expression (Figure 2b and c). The perilesional tissues showed high expression of TWEAK and Fn14 and low expression of BP180, which was similar to that of lesional samples (see Supplementary Figure S1 online).
      Figure thumbnail gr2
      Figure 2TWEAK, Fn14, and BP180 are overexpressed in skin lesions of patients with bullous pemphigoid (BP). (a) Immunohistochemistry was performed for TWEAK, Fn14, and BP180 expression in paraffin sections. (b, c) Stained epidermal region areas were quantitated by ImageJ software. The relationship between the positivity/area ratios of TWEAK (or Fn14) and BP180 staining was assessed by Spearman’s rank correlation. Number of patients = 10, number of healthy donors =10. Representative images are shown. Scale bar = 50 μm.

      TWEAK stimulation reduces BP180 expression in HaCaT cells

      The effect of TWEAK on BP180 expression was examined by culturing keratinocytes in vitro. The results showed that the mRNA level of BP180 increased but its protein expression decreased with TWEAK stimulation (Figure 3a–c). By immunofluorescence, HaCaT cells expressed less BP180 upon TWEAK stimulation (Figure 3d). In addition, there was an elevated mRNA expression of RANTES, MCP-1, IP-10, IL-6, and IL-8 in cells after TWEAK stimulation (see Supplementary Figure S2 online).
      Figure thumbnail gr3
      Figure 3TWEAK reduces BP180 expression in human keratinocytes. HaCaT cells were treated with TWEAK (100 ng/ml, for 6 hours or 48 hours). (a) The mRNA levels of BP180 were analyzed by quantitative real-time reverse transcriptase–PCR. (b, c) The BP180 protein was evaluated by Western blotting, followed by quantitation of band intensities. (d) Immunofluorescence was performed to detect BP180 expression in cells. (e) Adhesive strength indexes of cells were determined by the vibration/detachment assay. Data were from three independent experiments. Representative images are shown. Scale bar = 25 μm. ∗∗P < 0.01, ∗∗∗P < 0.001. BP, bullous pemphigoid; h, hour.
      By vibration detaching assay, the adherence of HaCaT cells to culture dish decreased significantly under TWEAK stimulation (Figure 3e). The appearance of cultures showed more detached cells in TWEAK-treated dishes (see Supplementary Figure S3 online).

      TWEAK exhibits an effect on BP180 via binding to the receptor Fn14

      Some HaCaT cells were transfected with small interfering RNA (siRNA) before TWEAK stimulation. By Western blotting, the transfection of Fn14 but not control siRNA restored BP180 expression in supernatants and lysates of cell cultures (Figure 4a and b). Similarly, immunofluorescent detection showed increased BP180 expression in the Fn14 siRNA-transfected cells when compared with controls (Figure 4c and d). Furthermore, the mRNA expression levels of RANTES, MCP-1, IP-10, IL-6, and IL-8 decreased in Fn14 siRNA-transfected cells (Figure 4e).
      Figure thumbnail gr4
      Figure 4TWEAK binding to Fn14 induces BP180 loss in human keratinocytes. HaCaT cells were transfected with Fn14 or control siRNA, followed by treatment with TWEAK (100 ng/ml, for 48 hours). (a) The mRNA levels of BP180 and Fn14 were assessed in cells. (b) The Fn14 and BP180 expression in lysates or supernatants was detected by Western blotting. (c) Immunofluorescence was carried out for BP180 expression in cells. (d) Adhesive strength indexes of cells were determined by vibration/detachment assay. (e) The mRNA levels of proinflammatory cytokines were also determined. Data were from three independent experiments. Representative images are shown. Scale bar = 25 μm. P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. BP, bullous pemphigoid; h, hour; siRNA, small interfering RNA.

      NF-κB and extracellular signal-regulated kinase (ERK) activation is involved in TWEAK-induced loss of BP180 in HaCaT cells

      Previously, we and other groups showed that both NF-κB and ERK signals are linked to the TWEAK-introduced inflammatory effect on cutaneous cells and skin tissues (
      • Cheng H.
      • Xu M.
      • Liu X.
      • Zou X.
      • Zhan N.
      • Xia Y.
      TWEAK/Fn14 activation induces keratinocyte proliferation under psoriatic inflammation.
      ,
      • Doerner J.L.
      • Wen J.
      • Xia Y.
      • Paz K.B.
      • Schairer D.
      • Wu L.
      • et al.
      TWEAK/Fn14 signaling involvement in the pathogenesis of cutaneous disease in the MRL/lpr model of spontaneous lupus.
      ). To understand the intermediate signals in TWEAK regulation of BP180 expression, the NF-κB– and ERK-related molecules were determined in HaCaT cells. The results showed that TWEAK up-regulated phosphorylated (p-)IκBα and IκBα and enhanced phosphorylation of ERK in a time-dependent manner (Figure 5a and b). Consistently, in Fn14-silenced TWEAK-treated cells, both p-ERK and p-IκBα levels were significantly lower than in control cells (see Supplementary Figure S4 online).
      Figure thumbnail gr5
      Figure 5The NF-κB and ERK pathways mediate the effect of TWEAK on BP180 loss in human keratinocytes. HaCaT cells were stimulated with TWEAK (100 ng/ml). Some cells were pretreated with the NF-κB (Bay 11-7082) or ERK (U0126) inhibitor. (a, b) Western blotting was performed for the protein expression of p-IκBα, IκBα, p-ERK, and ERK. (c) The mRNA levels of BP180 were determined. (d) The proteins of BP180, p-IκBα, IκBα, p-ERK, and ERK were detected by Western blotting. (e) The mRNA levels of proinflammatory cytokines were quantitated. In c–e, cells received 48-hour TWEAK stimulation. Data were from three independent experiments. Representative image are shown. P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. BP, bullous pemphigoid; ERK, extracellular signal-regulated kinase; h, hour; p-, phosphorylated.
      Specific inhibitors of NF-κB (Bay 11-7082) and mitogen-activated protein kinase/ERK (U0126) pathways were applied to TWEAK-stimulated cell cultures. We observed that Bay 11-7082 decreased the mRNA expression of BP180 (Figure 5c). Consistently, Western blotting showed an increase in BP180 protein upon the addition of Bay 11-7082 or plus U0126 (Figure 5d). Meanwhile, the expression of p-IkBα and p-ERK decreased in Bay 11-7082– and U0126-treated cells, respectively (Figure 5d, and see Supplementary Figure S5 online). Finally, the downstream cytokines (RANTES, MCP-1, IP-10, IL-6, and IL-8) were reduced after the administration of the two inhibitors (Figure 5e).

      ADAMs participate in TWEAK-induced BP180 loss in HaCaT cells

      Because the shedding of BP180 from the cell surface is directly associated with ADAM proteins (
      • Jacków J.
      • Schlosser A.
      • Sormunen R.
      • Nyström A.
      • Sitaru C.
      • Tasanen K.
      • et al.
      Generation of a functional non-shedding collagen XVII mouse model: Relevance of collagen XVII shedding in wound healing.
      ), we investigated the effect of TWEAK on ADAM9, ADAM10, ADAM17, and MMP-9 expression in keratinocytes. By immunohistochemistry, ADAM9, ADAM10, ADAM17, and MMP-9 were weakly expressed in normal and nonlesional skin but highly expressed in lesional skin (Figure 6a, and see Supplementary Figure S6 online). Moreover, their mRNA and protein levels increased significantly in TWEAK-stimulated HaCaT cells (Figure 6b–d, and see Supplementary Figure S6 online). However, the transfection of ADAM17 siRNA enhanced the protein expression of BP180 in these cells (Figure 6e and f), although it did not affect its mRNA expression (see Supplementary Figure S7 online). Consistently, the transfection of Fn14 siRNA down-regulated the protein expression of ADAM17 (Figure 6g and h). Moreover, both mRNA and protein levels of ADAM9, ADAM10, ADAM17, and MMP-9 were inhibited by NF-κB (Bay 11-7082) or ERK (U0126) inhibitor (see Supplementary Figure S7).
      Figure thumbnail gr6
      Figure 6ADAM17 regulates TWEAK-induced BP180 loss. HaCaT cells were stimulated with TWEAK (100 ng/ml). (a) Immunohistochemistry was performed for ADAM17 expression in skin samples from patients with bullous pemphigoid (n = 10) or healthy donors (n = 10). (b) The mRNA levels of ADAM17 were determined in HaCaT cells. (c, d) Accordingly, the ADAM17 protein was detected in these cells. (e, f) Proteins of ADAM17 and BP180 were detected in the siRNA-transfected cells. (g, h) Similarly, the ADAM17 protein was assessed in Fn14 siRNA-transfected cells. In e–h, cells received 48-h TWEAK stimulation after siRNA transfection. Data were from three independent experiments. Representative image are shown. Scale bar = 50 μm. P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. BP, bullous pemphigoid; Ctrl, control; h, hour; siRNA, small interfering RNA.

      Discussion

      In this study, we showed that both TWEAK and Fn14 are highly expressed in skin lesions of BP. Serum levels of TWEAK and anti-BP180 IgG correlate positively in patients with BP. Moreover, TWEAK stimulation reduces BP180 expression via binding to Fn14 in cultured keratinocytes. The effect of TWEAK on BP180 expression in keratinocytes involves the activation of NF-κB and ERK pathways. Furthermore, some sheddases such as ADAM17 participate in the TWEAK-induced BP180 loss in these cells. Therefore, TWEAK may contribute to the pathogenesis of BP by affecting BP180 expression and cellular adherence, involving the activation of ERK and NF-κB signals.
      Previous studies reported that TWEAK plays a role in disrupting cell junctions (
      • Berzal S.
      • González-Guerrero C.
      • Rayego-Mateos S.
      • Ucero Á.
      • Ocaña-Salceda C.
      • Egido J.
      • et al.
      TNF-related weak inducer of apoptosis (TWEAK) regulates junctional proteins in tubular epithelial cells via canonical NF-κB pathway and ERK activation.
      ,
      • Wen J.
      • Doerner J.
      • Weidenheim K.
      • Xia Y.
      • Stock A.
      • Michaelson J.S.
      • et al.
      TNF-like weak inducer of apoptosis promotes blood brain barrier disruption and increases neuronal cell death in MRL/lpr mice.
      ,
      • Xia Y.
      • Herlitz L.C.
      • Gindea S.
      • Wen J.
      • Pawar R.D.
      • Misharin A.
      • et al.
      Deficiency of fibroblast growth factor-inducible 14 (Fn14) preserves the filtration barrier and ameliorates lupus nephritis.
      ). In MRL/lpr lupus-prone mice, the Fn14 knockout reduced the brain expression of VCAM-1, ICAM-1 and fibronectin, leading to attenuation of the integrity of blood-brain barrier (
      • Wen J.
      • Doerner J.
      • Weidenheim K.
      • Xia Y.
      • Stock A.
      • Michaelson J.S.
      • et al.
      TNF-like weak inducer of apoptosis promotes blood brain barrier disruption and increases neuronal cell death in MRL/lpr mice.
      ). TWEAK/Fn14 activation can reduce the expression of nephrin, podocin, and tight junction protein-1 in podocytes and glomerular endothelial cells, preserving the integrity of renal filtration barrier (
      • Xia Y.
      • Herlitz L.C.
      • Gindea S.
      • Wen J.
      • Pawar R.D.
      • Misharin A.
      • et al.
      Deficiency of fibroblast growth factor-inducible 14 (Fn14) preserves the filtration barrier and ameliorates lupus nephritis.
      ). TWEAK also induces junctional instability in tubular epithelial cells (
      • Berzal S.
      • González-Guerrero C.
      • Rayego-Mateos S.
      • Ucero Á.
      • Ocaña-Salceda C.
      • Egido J.
      • et al.
      TNF-related weak inducer of apoptosis (TWEAK) regulates junctional proteins in tubular epithelial cells via canonical NF-κB pathway and ERK activation.
      ). In this study, we confirmed that TWEAK induces the loss of BP180 in keratinocytes and further leads to weaker cell adhesion. The silence of Fn14 expression in keratinocytes abrogates TWEAK-induced BP180 loss and proinflammatory cytokine production. Furthermore, both TWEAK and Fn14 expression is up-regulated in BP lesions, whereas BP180 expression is reduced significantly. Hence, TWEAK/Fn14 activation is pivotal in the loss of BP180 expression and cell adhesion. However, TWEAK/Fn14 activation is not bound to BP180 loss in skin. In some skin inflammation such as psoriasis and human papillomavirus infection, TWEAK induces the proliferation of keratinocytes without blister formation (
      • Cheng H.
      • Zhan N.
      • Ding D.
      • Liu X.
      • Zou X.
      • Li K.
      • et al.
      HPV type 16 infection switches keratinocytes from apoptotic to proliferative fate under TWEAK/Fn14 interaction.
      ,
      • Cheng H.
      • Xu M.
      • Liu X.
      • Zou X.
      • Zhan N.
      • Xia Y.
      TWEAK/Fn14 activation induces keratinocyte proliferation under psoriatic inflammation.
      ). This indicates that TWEAK regulates cell fate depending on local inflammatory microenvironments and that the in vitro findings need further verification through in vivo experiments.
      Serum levels of TWEAK are elevated in various autoimmune disorders, such as rheumatoid arthritis (
      • Park M.C.
      • Chung S.J.
      • Park Y.B.
      • Lee S.K.
      Relationship of serum TWEAK level to cytokine level, disease activity, and response to anti-TNF treatment in patients with rheumatoid arthritis.
      ), systemic sclerosis (
      • Yanaba K.
      • Yoshizaki A.
      • Muroi E.
      • Hara T.
      • Ogawa F.
      • Usui A.
      • et al.
      Elevated circulating TWEAK levels in systemic sclerosis: association with lower frequency of pulmonary fibrosis.
      ), and systemic lupus erythematosus (
      • Chen J.
      • Wei L.
      • Xia Y.
      The roles of tumor necrosis factor-related weak inducer of apoptosis/fibroblast growth factor-inducible 14 pathway in lupus nephritis.
      ). Similarly, we confirmed the increase of TWEAK level in sera from patients with BP. Moreover, the TWEAK level correlates positively with the serum titer of anti-BP180 IgG, which reflects the disease activity of BP (
      • Lee E.H.
      • Kim Y.H.
      • Kim S.
      • Kim S.E.
      • Kim S.C.
      Usefulness of enzyme-linked immunosorbent assay using recombinant BP180 and BP230 for serodiagnosis and monitoring disease activity of bullous pemphigoid.
      ). Therefore, soluble TWEAK may be a useful marker for monitoring the disease activity of BP. Macrophages/monocytes are the main source of TWEAK in inflammatory tissues (
      • Bird T.G.
      • Lu W.Y.
      • Boulter L.
      • Gordon-Keylock S.
      • Ridgway R.A.
      • Williams M.J.
      • et al.
      Bone marrow injection stimulates hepatic ductular reactions in the absence of injury via macrophage-mediated TWEAK signaling.
      ). Moreover, the infiltration of macrophages/monocytes is prominent in the skin lesions of patients with BP (
      • Furudate S.
      • Fujimura T.
      • Kambayashi Y.
      • Kakizaki A.
      • Aiba S.
      Comparison of CD163+ CD206+ M2 macrophages in the lesional skin of bullous pemphigoid and pemphigus vulgaris: the possible pathogenesis of bullous pemphigoid.
      ). Therefore, local macrophages/monocytes ought to be the source of TWEAK in BP lesions.
      BP180 deficiency can reduce keratinocyte-basement membrane adhesion and the size of hemidesmosome plaques (
      • Qiao H.
      • Shibaki A.
      • Long H.A.
      • Wang G.
      • Li Q.
      • Nishie W.
      • et al.
      Collagen XVII participates in keratinocyte adhesion to collagen IV, and in p38MAPK-dependent migration and cell signaling.
      ). The ectodomain of BP180 can shed constitutively from the cell surface in an ADAM-independent manner (
      • Hofmann S.C.
      • Voith U.
      • Schönau V.
      • Sorokin L.
      • Bruckner-Tuderman L.
      • Franzke C.W.
      Plasmin plays a role in the in vitro generation of the linear IgA dermatosis antigen LADB97.
      ). A series of ADAMs (ADAM8, 9, 10, 15, and 17) and MMPs (MMP-2, -9, and -13) have been suggested to participate in BP180 cleavage or BP development (
      • Franzke C.W.
      • Bruckner-Tuderman L.
      • Blobel C.P.
      Shedding of collagen XVII/BP180 in skin depends on both ADAM10 and ADAM9.
      ,
      • Laval S.
      • Laklai H.
      • Fanjul M.
      • Pucelle M.
      • Laurell H.
      • Billon-Galés A.
      • et al.
      Dual roles of hemidesmosomal proteins in the pancreatic epithelium: the phosphoinositide 3-kinase decides.
      ,
      • Niimi Y.
      • Pawankar R.
      • Kawana S.
      Increased expression of matrix metalloproteinase-2, matrix metalloproteinase-9 and matrix metalloproteinase-13 in lesional skin of bullous pemphigoid.
      ,
      • Zebrowska A.
      • Wagrowska-Danilewicz M.
      • Danilewicz M.
      • Wodz K.
      • Sokolowska M.
      • Erkiert-Polguj A.
      • et al.
      Expression of selected ADAMs in bullous pemphigoid and dermatitis herpetiformis.
      ). We found that ADAM9, ADAM10, ADAM17, and MMP-9 are highly expressed in BP lesions and increase in keratinocytes upon TWEAK/Fn14 activation. ADAM17 is the major sheddase in keratinocytes, whereas other sheddases also contribute to shedding of BP180 (
      • Franzke C.W.
      • Tasanen K.
      • Schäcke H.
      • Zhou Z.
      • Tryggvason K.
      • Mauch C.
      • et al.
      Transmembrane collagen XVII, an epithelial adhesion protein, is shed from the cell surface by ADAMs.
      ,
      • Franzke C.W.
      • Bruckner-Tuderman L.
      • Blobel C.P.
      Shedding of collagen XVII/BP180 in skin depends on both ADAM10 and ADAM9.
      ). ADAM17 expression is increased in lesional BP skin (
      • Zebrowska A.
      • Wagrowska-Danilewicz M.
      • Danilewicz M.
      • Wodz K.
      • Sokolowska M.
      • Erkiert-Polguj A.
      • et al.
      Expression of selected ADAMs in bullous pemphigoid and dermatitis herpetiformis.
      ). Moreover, TWEAK/Fn14 interaction directly induces ADAM17 activation in cultured cells (
      • Rayego-Mateos S.
      • Morgado-Pascual J.L.
      • Sanz A.B.
      • Ramos A.M.
      • Eguchi S.
      • Batlle D.
      • et al.
      TWEAK transactivation of the epidermal growth factor receptor mediates renal inflammation.
      ). In this study, we found that the transfection of Fn14 siRNA reduced ADAM17 expression and that ADAM17 siRNA treatment increased the BP180 protein level in TWEAK-stimulated keratinocytes. Hence, ADAM17 and other sheddases participate in the TWEAK modulation of BP180 loss in keratinocytes.
      TWEAK increases the mRNA level but decreases the protein level of BP180 in keratinocytes. Also, ADAM17 inhibition enhances protein expression but does not affect mRNA expression of BP180. This discrepancy suggested that ADAM17 regulates BP180 expression through potential posttranscriptional pathways. ADAM17 not only exerts proteolytic activity but also regulates signaling pathways. ADAM17 and other sheddases may act on the coiled coils in the NC16A domain, leading to the hydrolysis and cleavage of BP180, which produces a 120-kDa polypeptide (
      • Nishie W.
      • Jackow J.
      • Hofmann S.C.
      • Franzke C.W.
      • Bruckner-Tuderman L.
      Coiled coils ensure the physiological ectodomain shedding of collagen XVII.
      ). ADAM17 is dispensable for Notch1 activity (
      • Groot A.J.
      • Cobzaru C.
      • Weber S.
      • Saftig P.
      • Blobel C.P.
      • Kopan R.
      • et al.
      Epidermal ADAM17 is dispensable for notch activation.
      ,
      • Murthy A.
      • Shao Y.W.
      • Narala S.R.
      • Molyneux S.D.
      • Zúñiga-Pflücker J.C.
      • Khokha R.
      Notch activation by the metalloproteinase ADAM17 regulates myeloproliferation and atopic barrier immunity by suppressing epithelial cytokine synthesis.
      ). Moreover, TWEAK via ADAM17 can release the membrane-bound ligands of EGFR, which further triggers downstream responses, including mitogen-activated protein kinase/ERK activation and proinflammatory gene overexpression (
      • Rayego-Mateos S.
      • Morgado-Pascual J.L.
      • Sanz A.B.
      • Ramos A.M.
      • Eguchi S.
      • Batlle D.
      • et al.
      TWEAK transactivation of the epidermal growth factor receptor mediates renal inflammation.
      ). Actually, shedding of BP180 is strongly responsive to epidermal growth factor stimulation (
      • Jackow J.
      • Loffek S.
      • Nystrom A.
      • Bruckner-Tuderman L.
      • Franzke C.W.
      Collagen XVII shedding suppresses re-epithelialization by directing keratinocyte migration and dampening mTOR signaling.
      ). Hence, we suppose that the downstream signaling pathways may participate in the ADAM17 regulation of BP180 loss. Our results also showed that the inhibitors of NF-κB and ERK signaling reduce ADAM17 expression in keratinocytes under TWEAK stimulation. Methylprednisolone exerts therapeutic effects through inhibiting mitogen-activated protein kinase/ERK phosphorylation (
      • Hellberg L.
      • Samavedam U.K.
      • Holdorf K.
      • Hänsel M.
      • Recke A.
      • Beckmann T.
      • et al.
      Methylprednisolone blocks autoantibody-induced tissue damage in experimental models of bullous pemphigoid and epidermolysis bullosa acquisita through inhibition of neutrophil activation.
      ). Therefore, corticosteroids may be used to interfere with the TWEAK-mediated BP development. This speculated mechanism underlying the TWEAK-ADAMs axis in BP180 loss is illustrated in Supplementary Figure S8 online and needs to be thoroughly investigated in future studies.
      TWEAK/Fn14 activation induces the production of proinflammatory cytokines (e.g., RANTES, MCP-1, IP-10, IL-6, and IL-8) in various types of cells including keratinocytes, renal resident cells, and vascular endothelial cells (
      • Cheng H.
      • Zhan N.
      • Ding D.
      • Liu X.
      • Zou X.
      • Li K.
      • et al.
      HPV type 16 infection switches keratinocytes from apoptotic to proliferative fate under TWEAK/Fn14 interaction.
      ,
      • Wen J.
      • Doerner J.
      • Weidenheim K.
      • Xia Y.
      • Stock A.
      • Michaelson J.S.
      • et al.
      TNF-like weak inducer of apoptosis promotes blood brain barrier disruption and increases neuronal cell death in MRL/lpr mice.
      ,
      • Xia Y.
      • Herlitz L.C.
      • Gindea S.
      • Wen J.
      • Pawar R.D.
      • Misharin A.
      • et al.
      Deficiency of fibroblast growth factor-inducible 14 (Fn14) preserves the filtration barrier and ameliorates lupus nephritis.
      ). In fact, the elevation of these cytokines is prominent in sera from patients with BP (
      • Nakashima H.
      • Fujimoto M.
      • Asashima N.
      • Watanabe R.
      • Kuwano Y.
      • Yazawa N.
      • et al.
      Serum chemokine profile in patients with bullous pemphigoid.
      ). In both human and experimental murine BP, cytokines have been implicated to be essential for blister formation in this disease (
      • Hammers C.M.
      • Stanley J.R.
      Mechanisms of disease: pemphigus and bullous pemphigoid.
      ,
      • Plée J.
      • Le Jan S.
      • Giustiniani J.
      • Barbe C.
      • Joly P.
      • Bedane C.
      • et al.
      Integrating longitudinal serum IL-17 and IL-23 follow-up, along with autoantibodies variation, contributes to predict bullous pemphigoid outcome.
      ,
      • Riani M.
      • Le Jan S.
      • Plée J.
      • Durlach A.
      • Le Naour R.
      • Haegeman G.
      • et al.
      Bullous pemphigoid outcome is associated with CXCL10-induced matrix metalloproteinase 9 secretion from monocytes and neutrophils but not lymphocytes.
      ). We found that these proinflammatory cytokines have high levels in both sera and blister fluid from patients with BP. Moreover, TWEAK stimulation enhances the expression of proinflammatory cytokines in keratinocytes. Our results not only provided more evidence that cytokines are important in the pathogenesis of BP but also strongly suggested that TWEAK/Fn14 activation contributes to blister formation in BP by enhancing the inflammatory responses in keratinocytes.
      The interaction between TWEAK signals and anti-BP180 antibodies remains unclear. In the murine BP model of IgG passive transfer, anti-BP180 IgG can trigger the formation of blisters in skin by inducing BP180 degradation (
      • Lin L.
      • Betsuyaku T.
      • Heimbach L.
      • Li N.
      • Rubenstein D.
      • Shapiro S.D.
      • et al.
      Neutrophil elastase cleaves the murine hemidesmosomal protein BP180/type XVII collagen and generates degradation products that modulate experimental bullous pemphigoid.
      ). Additionally, some studies supported that the BP180 autoantibodies are the major cause of cytokine release in BP (
      • Nishie W.
      Update on the pathogenesis of bullous pemphigoid: an autoantibody-mediated blistering disease targeting collagen XVII.
      ,
      • Van den Bergh F.
      • Eliason S.L.
      • Burmeister B.T.
      • Giudice G.J.
      Collagen XVII (BP180) modulates keratinocyte expression of the proinflammatory chemokine, IL-8.
      ). The activation of complements and inflammatory pathways may be responsible for the pathogenicity of anti-BP180 IgG (
      • Nishie W.
      Update on the pathogenesis of bullous pemphigoid: an autoantibody-mediated blistering disease targeting collagen XVII.
      ). Theoretically, the pathogenic anti-BP180 antibodies may enhance Fn14 expression and trigger TWEAK/Fn14 signals. However, this study showed that TWEAK activation directly induces both BP180 loss and cytokine production of keratinocytes in anti-BP180 IgG-free media. Therefore, TWEAK/Fn14 activation plays an important role in the pathogenesis of BP as an additional contributor. TWEAK signals and anti-BP180 antibodies may act together in inflammatory cascades to aggravate the dermal-epidermal separation.
      In conclusion, the serum level of TWEAK is elevated in patients with BP, and TWEAK/Fn14 signals are activated in the skin lesions. TWEAK/Fn14 activation can reduce BP180 expression and cell adhesion of keratinocytes. The role of TWEAK in the development of BP involves the activation of NF-κB and ERK pathways and the up-regulation of ADAM17 and other sheddases in keratinocytes. TWEAK may serves as a biomarker or therapeutic target for patients with BP.

      Materials and Methods

      Sample collection

      Skin tissues (lesional, nonlesional, and perilesional) and blister fluid were collected from patients with BP (n = 23), who had not received medication or physical therapy in the past 4 weeks. Nonlesional tissues were collected from locations far from BP lesions. Normal skin tissues and sera were collected from healthy donors (n = 20). There were no statistical differences in sex or age between patients and normal control subjects (P > 0.05). Participants’ demographic characteristics are detailed in Supplementary Table S1 online. This study was performed under the supervision of the Hospital Research Ethics Committee, and written informed consent was obtained from all subjects.

      Immunohistochemistry

      As described previously (
      • Zou X.Y.
      • Ding D.
      • Zhan N.
      • Liu X.M.
      • Pan C.
      • Xia Y.M.
      Glyoxalase I is differentially expressed in cutaneous neoplasms and contributes to the progression of squamous cell carcinoma.
      ), paraffin sections were deparaffinized and rehydrated, followed by addition of Dual Endogenous Enzyme Block (DAKO, Glostrup, Denmark). Rabbit anti-Fn14 (or anti-TWEAK, anti-ADAM17, or anti-BP180) IgG (2 μg/ml; Abcam, Cambridge, MA) was used as the primary antibody. Rabbit anti-ADAM9 (or anti-ADAM10 or anti-MMP-9) IgG (2 μg/ml; Cell Signaling, Danvers, MA) was also the primary antibody. Sections were incubated with polymer-horseradish peroxidase–labeled goat anti-rabbit IgG and 3,3′-diaminobenzine-chromogen substrate (i.e., DAKO) in order before counterstaining with hematoxylin and eosin solution.
      The positively stained areas were quantified by ImageJ software (National Institutes of Health, Bethesda, MD) according to a previously described method (
      • Choudhry N.
      • Li K.
      • Zhang T.
      • Wu K.Y.
      • Song Y.
      • Farrar C.A.
      • et al.
      The complement factor 5a receptor 1 has a pathogenic role in chronic inflammation and renal fibrosis in a murine model of chronic pyelonephritis.
      ). Briefly, six to eight epidermal viewing fields were examined within each skin sample. Positively stained areas were expressed as a percentage of the whole field area (% positivity/mm2). Quantitative analyses were performed in a blinded fashion by two pathologists.

      Cell culture

      Human keratinocytes (HaCaT cell line) were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (Life Technologies, Carlsbad, CA). Before stimulation assays, keratinocytes were starved in non–fetal bovine serum-supplemented medium for 24 hours. Then, TWEAK (100 ng/ml; R&D Systems, Minneapolis, MN) was administered. In some experiments, the solutions of Bay 11-7082 (20 μmol/L, Sigma-Aldrich, St Louis, MO) and U0126 (10 μmol/L, Cell Signaling) were added and pre-incubated for 2 hours before TWEAK stimulation.

      Immunofluorescence

      As reported previously (
      • Zhang Y.
      • Yang J.
      • Jiang S.
      • Fang C.
      • Xiong L.
      • Cheng H.
      • et al.
      The lupus-derived anti-double-stranded DNA IgG contributes to myofibroblast-like phenotype in mesangial cells.
      ), cultured cells growing on a glass-bottomed culture dish (MatTek, Ashland, MA) were incubated with primary antibody targeting BP180 (2 μg/ml; Abcam) after cold acetone fixation. Then, Alexa-488 conjugated anti-rabbit IgG (2 μg/ml; Jackson ImmunoResearch, West Grove, PA) was applied. After adding 4',6-diamidino-2-phenylindole solution, the dishes were covered with slides and observed under a digital confocal microscope (Leica, Wetzlar, Germany).

      ELISA

      Serum levels of TWEAK, RANTES, MCP-1, IP-10, IL-6, and IL-8 were measured using commercial immunoassay kits (R&D Systems). MESACUP BP180-ELISA kit (MBL, Nagoya, Japan) was used for the serum titers of anti-BP180 IgG. In this kit, the NC16a recombinant proteins captured BP180 IgG in sera or fluid, and the second reaction was developed using peroxidase-conjugated goat anti-human IgG antibodies.

      siRNA transfection

      The siRNA oligonucleotides of Fn14 and ADAM17 (or control siRNAs) were purchased from Life Technologies. siRNA transfection was performed according to the recommended procedures of Lipofectamine 2000 transfection reagent (Life Technologies). Briefly, subconfluent cells in six-well plates were added with 75 pmol siRNA (in 7.5 μl of transfection reagent) for 24 hours. Then, the cells were incubated in medium for 24 hours before the next experiments. The efficiency of transfection was determined by quantitative real-time reverse transcriptase–PCR and Western blotting.

      Quantitative real-time reverse transcriptase–PCR

      Total RNA was extracted from cell lysates by using the Trizol reagent (Life Technologies). A commercial cDNA kit (Takara Bio, Kyoto, Japan) was used for reverse transcription. Next, quantitative real-time reverse transcriptase–PCR was performed on the One-Step PCR System (Applied Biosystems, Waltham, MA), with SYBR Green Master Mixes (Takara Bio) used as fluorescent dye. The PCR primers were synthesized by AuGCT DNA-SYN Biotech (Beijing, China) (see Supplementary Table S2 online).

      Western blotting

      Cell protein extracts were separated on electrophoresis gels and then transferred onto polyvinylidene difluoride membrane (Millipore, Billerica, MA). Rabbit antibodies to BP180 (recognizing amino acid residues 1300–1400, with two bands at 120 kDa and 180 kDa) or ADAM17 were purchased from Abcam. Rabbit antibodies to p-IκBα, IκBα, p-ERK, ERK, ADAM9, ADAM10, and MMP-9 were purchased from Cell Signaling. These antibodies were diluted to 0.2 μg/ml at use. Horseradish peroxidase-conjugated goat anti-rabbit IgG (1 μg/ml, Abcam) was used as the secondary antibody. Signal was developed using an ECL chemiluminescence kit (Millipore). The intensities of bands were quantitated using ImageJ software.

      Vibration detachment assay

      This assay was performed according to
      • Iwata H.
      • Kamio N.
      • Aoyama Y.
      • Yamamoto Y.
      • Hirako Y.
      • Owaribe K.
      • et al.
      IgG from patients with bullous pemphigoid depletes cultured keratinocytes of the 180-kDa bullous pemphigoid antigen (type XVII collagen) and weakens cell attachment.
      . Briefly, HaCaT cells (8 × 104) were seeded in 3.5-cm–diameter dishes and cultured for 48 hours. Then, cells were stimulated with TWEAK (100 ng/ml) for 6 or 48 hours. The adhesion of cells to the bottom of plates was assayed by determining the number of adherent cells after vibration with a vortex at grade 4.5 for 30 minutes. Cells remaining on the plate bottom were treated with 0.25% trypsin. The released cells were then counted.

      Statistical analysis

      Data were expressed as means ± standard error. GraphPad Prism version 5.0 (GraphPad Software, La Jolla, CA) was used for statistical analysis. Analysis of variance was used for the comparison of more than two groups. In comparing two groups, a two-tailed Student t test was used for statistical differences. Correlations between two parameters were analyzed using Spearman’s correlation coefficient by rank test. Differences were considered statistically significant at P < 0.05.

      Conflict of Interest

      The authors state no conflict of interest.

      Acknowledgments

      This work was supported by the National Natural Science Foundation of China (Projects No. 81472876 and No. 81630081) and by the Fundamental Research Funds for the Central Universities (No. 2015qngz01).

      Supplementary Material

      References

        • Barrick B.J.
        • Lohse C.M.
        • Lehman J.S.
        Specific causes of death in patients with bullous pemphigoid as measured by death certificate data: a retrospective cohort study.
        Int J Dermatol. 2015; 54: 56-61
        • Berzal S.
        • González-Guerrero C.
        • Rayego-Mateos S.
        • Ucero Á.
        • Ocaña-Salceda C.
        • Egido J.
        • et al.
        TNF-related weak inducer of apoptosis (TWEAK) regulates junctional proteins in tubular epithelial cells via canonical NF-κB pathway and ERK activation.
        J Cell Physiol. 2015; 230: 1580-1593
        • Bird T.G.
        • Lu W.Y.
        • Boulter L.
        • Gordon-Keylock S.
        • Ridgway R.A.
        • Williams M.J.
        • et al.
        Bone marrow injection stimulates hepatic ductular reactions in the absence of injury via macrophage-mediated TWEAK signaling.
        Proc Natl Acad Sci USA. 2013; 110: 6542-6547
        • Burkly L.C.
        • Michaelson J.S.
        • Zheng T.S.
        TWEAK/Fn14 pathway: an immunological switch for shaping tissue responses.
        Immunol Rev. 2011; 244: 99-114
        • Cai S.C.
        • Allen J.C.
        • Lim Y.L.
        • Tan S.H.
        • Tang M.B.
        Association of bullous pemphigoid and malignant neoplasms.
        JAMA Dermatol. 2015; 151: 665-667
        • Chen J.
        • Wei L.
        • Xia Y.
        The roles of tumor necrosis factor-related weak inducer of apoptosis/fibroblast growth factor-inducible 14 pathway in lupus nephritis.
        Nephrology. 2017; 22: 101-106
        • Cheng H.
        • Xu M.
        • Liu X.
        • Zou X.
        • Zhan N.
        • Xia Y.
        TWEAK/Fn14 activation induces keratinocyte proliferation under psoriatic inflammation.
        Exp Dermatol. 2016; 25: 32-37
        • Cheng H.
        • Zhan N.
        • Ding D.
        • Liu X.
        • Zou X.
        • Li K.
        • et al.
        HPV type 16 infection switches keratinocytes from apoptotic to proliferative fate under TWEAK/Fn14 interaction.
        J Invest Dermatol. 2015; 135: 2427-2436
        • Choudhry N.
        • Li K.
        • Zhang T.
        • Wu K.Y.
        • Song Y.
        • Farrar C.A.
        • et al.
        The complement factor 5a receptor 1 has a pathogenic role in chronic inflammation and renal fibrosis in a murine model of chronic pyelonephritis.
        Kidney Int. 2016; 90: 540-554
        • Di Zenzo G.
        • Della Torre R.
        • Zambruno G.
        • Borradori L.
        Bullous pemphigoid: From the clinic to the bench.
        Clin Dermatol. 2012; 30: 3-16
        • Doerner J.L.
        • Wen J.
        • Xia Y.
        • Paz K.B.
        • Schairer D.
        • Wu L.
        • et al.
        TWEAK/Fn14 signaling involvement in the pathogenesis of cutaneous disease in the MRL/lpr model of spontaneous lupus.
        J Invest Dermatol. 2015; 135: 1986-1995
        • Franzke C.W.
        • Bruckner-Tuderman L.
        • Blobel C.P.
        Shedding of collagen XVII/BP180 in skin depends on both ADAM10 and ADAM9.
        J Biol Chem. 2009; 284: 23386-23396
        • Franzke C.W.
        • Tasanen K.
        • Schäcke H.
        • Zhou Z.
        • Tryggvason K.
        • Mauch C.
        • et al.
        Transmembrane collagen XVII, an epithelial adhesion protein, is shed from the cell surface by ADAMs.
        EMBO J. 2002; 21: 5026-5035
        • Furudate S.
        • Fujimura T.
        • Kambayashi Y.
        • Kakizaki A.
        • Aiba S.
        Comparison of CD163+ CD206+ M2 macrophages in the lesional skin of bullous pemphigoid and pemphigus vulgaris: the possible pathogenesis of bullous pemphigoid.
        Dermatology. 2014; 229: 369-378
        • Groot A.J.
        • Cobzaru C.
        • Weber S.
        • Saftig P.
        • Blobel C.P.
        • Kopan R.
        • et al.
        Epidermal ADAM17 is dispensable for notch activation.
        J Invest Dermatol. 2013; 133: 2286-2288
        • Hammers C.M.
        • Stanley J.R.
        Mechanisms of disease: pemphigus and bullous pemphigoid.
        Annu Rev Pathol. 2016; 11: 175-197
        • Hellberg L.
        • Samavedam U.K.
        • Holdorf K.
        • Hänsel M.
        • Recke A.
        • Beckmann T.
        • et al.
        Methylprednisolone blocks autoantibody-induced tissue damage in experimental models of bullous pemphigoid and epidermolysis bullosa acquisita through inhibition of neutrophil activation.
        J Invest Dermatol. 2013; 33: 2390-2399
        • Hofmann S.C.
        • Voith U.
        • Schönau V.
        • Sorokin L.
        • Bruckner-Tuderman L.
        • Franzke C.W.
        Plasmin plays a role in the in vitro generation of the linear IgA dermatosis antigen LADB97.
        J Invest Dermatol. 2009; 129: 1730-1739
        • Hurskainen T.
        • Kokkonen N.
        • Sormunen R.
        • Jackow J.
        • Löffek S.
        • Soininen R.
        • et al.
        Deletion of the major bullous pemphigoid epitope region of collagen XVII induces blistering, autoimmunization, and itching in mice.
        J Invest Dermatol. 2015; 135: 1303-1310
        • Iwata H.
        • Kamio N.
        • Aoyama Y.
        • Yamamoto Y.
        • Hirako Y.
        • Owaribe K.
        • et al.
        IgG from patients with bullous pemphigoid depletes cultured keratinocytes of the 180-kDa bullous pemphigoid antigen (type XVII collagen) and weakens cell attachment.
        J Invest Dermatol. 2009; 129: 919-926
        • Jackow J.
        • Loffek S.
        • Nystrom A.
        • Bruckner-Tuderman L.
        • Franzke C.W.
        Collagen XVII shedding suppresses re-epithelialization by directing keratinocyte migration and dampening mTOR signaling.
        J Invest Dermatol. 2016; 136: 1031-1041
        • Jacków J.
        • Schlosser A.
        • Sormunen R.
        • Nyström A.
        • Sitaru C.
        • Tasanen K.
        • et al.
        Generation of a functional non-shedding collagen XVII mouse model: Relevance of collagen XVII shedding in wound healing.
        J Invest Dermatol. 2016; 136: 516-525
        • Kokkonen N.
        • Herukka S.K.
        • Huilaja L.
        • Kokki M.
        • Koivisto A.M.
        • Hartikainen P.
        • et al.
        Increased levels of the bullous pemphigoid BP180 autoantibody are associated with more severe dementia in Alzheimer's disease.
        J Invest Dermatol. 2017; 137: 71-76
        • Laval S.
        • Laklai H.
        • Fanjul M.
        • Pucelle M.
        • Laurell H.
        • Billon-Galés A.
        • et al.
        Dual roles of hemidesmosomal proteins in the pancreatic epithelium: the phosphoinositide 3-kinase decides.
        Oncogene. 2014; 3: 1934-1944
        • Lee E.H.
        • Kim Y.H.
        • Kim S.
        • Kim S.E.
        • Kim S.C.
        Usefulness of enzyme-linked immunosorbent assay using recombinant BP180 and BP230 for serodiagnosis and monitoring disease activity of bullous pemphigoid.
        Ann Dermatol. 2012; 24: 45-55
        • Lin L.
        • Betsuyaku T.
        • Heimbach L.
        • Li N.
        • Rubenstein D.
        • Shapiro S.D.
        • et al.
        Neutrophil elastase cleaves the murine hemidesmosomal protein BP180/type XVII collagen and generates degradation products that modulate experimental bullous pemphigoid.
        Matrix Biol. 2012; 31: 38-44
        • Liu Z.C.
        • Zhou Q.L.
        • Li X.Z.
        • Yang J.H.
        • Ao X.
        • Veeraragoo P.
        • et al.
        Elevation of human tumor necrosis factor-like weak inducer of apoptosis in peripheral blood mononuclear cells is correlated with disease activity and lupus nephritis in patients with systemic lupus erythematosus.
        Cytokine. 2011; 53: 295-300
        • Murthy A.
        • Shao Y.W.
        • Narala S.R.
        • Molyneux S.D.
        • Zúñiga-Pflücker J.C.
        • Khokha R.
        Notch activation by the metalloproteinase ADAM17 regulates myeloproliferation and atopic barrier immunity by suppressing epithelial cytokine synthesis.
        Immunity. 2012; 36: 105-119
        • Nakashima H.
        • Fujimoto M.
        • Asashima N.
        • Watanabe R.
        • Kuwano Y.
        • Yazawa N.
        • et al.
        Serum chemokine profile in patients with bullous pemphigoid.
        Br J Dermatol. 2007; 156: 454-459
        • Niimi Y.
        • Pawankar R.
        • Kawana S.
        Increased expression of matrix metalloproteinase-2, matrix metalloproteinase-9 and matrix metalloproteinase-13 in lesional skin of bullous pemphigoid.
        Int Arch Allergy Immunol. 2006; 139: 104-113
        • Nishie W.
        Update on the pathogenesis of bullous pemphigoid: an autoantibody-mediated blistering disease targeting collagen XVII.
        J Dermatol Sci. 2014; 73: 179-186
        • Nishie W.
        • Jackow J.
        • Hofmann S.C.
        • Franzke C.W.
        • Bruckner-Tuderman L.
        Coiled coils ensure the physiological ectodomain shedding of collagen XVII.
        J Biol Chem. 2012; 287: 29940-29948
        • Park M.C.
        • Chung S.J.
        • Park Y.B.
        • Lee S.K.
        Relationship of serum TWEAK level to cytokine level, disease activity, and response to anti-TNF treatment in patients with rheumatoid arthritis.
        Scand J Rheumatol. 2008; 37: 173-178
        • Peng Q.L.
        • Shu X.M.
        • Tian X.L.
        • Lu X.
        • Wang G.C.
        Expression of tumor necrosis factor-like weak inducer of apoptosis and fibroblast growth factor-inducible 14 in patients with polymyositis and dermatomyositis.
        Arthritis Res Ther. 2014; 16: R26
        • Plée J.
        • Le Jan S.
        • Giustiniani J.
        • Barbe C.
        • Joly P.
        • Bedane C.
        • et al.
        Integrating longitudinal serum IL-17 and IL-23 follow-up, along with autoantibodies variation, contributes to predict bullous pemphigoid outcome.
        Sci Rep. 2015; 5: 18001
        • Qiao H.
        • Shibaki A.
        • Long H.A.
        • Wang G.
        • Li Q.
        • Nishie W.
        • et al.
        Collagen XVII participates in keratinocyte adhesion to collagen IV, and in p38MAPK-dependent migration and cell signaling.
        J Invest Dermatol. 2009; 129: 2288-2295
        • Rayego-Mateos S.
        • Morgado-Pascual J.L.
        • Sanz A.B.
        • Ramos A.M.
        • Eguchi S.
        • Batlle D.
        • et al.
        TWEAK transactivation of the epidermal growth factor receptor mediates renal inflammation.
        J Pathol. 2013; 231: 480-494
        • Riani M.
        • Le Jan S.
        • Plée J.
        • Durlach A.
        • Le Naour R.
        • Haegeman G.
        • et al.
        Bullous pemphigoid outcome is associated with CXCL10-induced matrix metalloproteinase 9 secretion from monocytes and neutrophils but not lymphocytes.
        J Allergy Clin Immunol. 2017; 139: 863-872.e3
        • Schmidt E.
        • Zillikens D.
        Pemphigoid diseases.
        Lancet. 2013; 381: 320-332
        • Van den Bergh F.
        • Eliason S.L.
        • Burmeister B.T.
        • Giudice G.J.
        Collagen XVII (BP180) modulates keratinocyte expression of the proinflammatory chemokine, IL-8.
        Exp Dermatol. 2012; 21: 605-611
        • Wajant H.
        The TWEAK-Fn14 system as a potential drug target.
        Br J Pharmacol. 2013; 170: 748-764
        • Wen J.
        • Doerner J.
        • Weidenheim K.
        • Xia Y.
        • Stock A.
        • Michaelson J.S.
        • et al.
        TNF-like weak inducer of apoptosis promotes blood brain barrier disruption and increases neuronal cell death in MRL/lpr mice.
        J Autoimmun. 2015; 60: 40-50
        • Wen J.
        • Xia Y.
        • Stock A.
        • Michaelson J.S.
        • Burkly L.C.
        • Gulinello M.
        • et al.
        Neuropsychiatric disease in murine lupus is dependent on the TWEAK/Fn14 pathway.
        J Autoimmun. 2013; 43: 44-54
        • Xia Y.
        • Campbell S.R.
        • Broder A.
        • Herlitz L.
        • Abadi M.
        • Wu P.
        • et al.
        Inhibition of the TWEAK/Fn14 pathway attenuates renal disease in nephrotoxic serum nephritis.
        Clin Immunol. 2012; 145: 108-121
        • Xia Y.
        • Herlitz L.C.
        • Gindea S.
        • Wen J.
        • Pawar R.D.
        • Misharin A.
        • et al.
        Deficiency of fibroblast growth factor-inducible 14 (Fn14) preserves the filtration barrier and ameliorates lupus nephritis.
        J Am Soc Nephrol. 2015; 26: 1053-1070
        • Xu W.D.
        • Zhao Y.
        • Liu Y.
        Role of the TWEAK/Fn14 pathway in autoimmune diseases.
        Immunol Res. 2016; 64: 44-50
        • Yanaba K.
        • Yoshizaki A.
        • Muroi E.
        • Hara T.
        • Ogawa F.
        • Usui A.
        • et al.
        Elevated circulating TWEAK levels in systemic sclerosis: association with lower frequency of pulmonary fibrosis.
        J Rheumatol. 2009; 36: 1657-1662
        • Zebrowska A.
        • Wagrowska-Danilewicz M.
        • Danilewicz M.
        • Wodz K.
        • Sokolowska M.
        • Erkiert-Polguj A.
        • et al.
        Expression of selected ADAMs in bullous pemphigoid and dermatitis herpetiformis.
        J Dermatol Sci. 2009; 56: 58-61
        • Zhang Y.
        • Yang J.
        • Jiang S.
        • Fang C.
        • Xiong L.
        • Cheng H.
        • et al.
        The lupus-derived anti-double-stranded DNA IgG contributes to myofibroblast-like phenotype in mesangial cells.
        J Clin Immunol. 2012; 32: 1270-1278
        • Zimmermann M.
        • Koreck A.
        • Meyer N.
        • Basinski T.
        • Meiler F.
        • Simone B.
        • et al.
        TNF-like weak inducer of apoptosis (TWEAK) and TNF-α cooperate in the induction of keratinocyte apoptosis.
        J Allergy Clin Immunol. 2011; 127: 200-207
        • Zou X.Y.
        • Ding D.
        • Zhan N.
        • Liu X.M.
        • Pan C.
        • Xia Y.M.
        Glyoxalase I is differentially expressed in cutaneous neoplasms and contributes to the progression of squamous cell carcinoma.
        J Invest Dermatol. 2015; 135: 589-598