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Epidermal differentiation is a complex process that requires the regulated and sequential expression of various genes. Most fused-type S100 proteins are expressed in the granular layer and it is hypothesized that these proteins may be associated with cornification and barrier formation. We previously identified a member of the fused-type S100 proteins, Trichohyalin-like 1 (TCHHL1) protein. TCHHL1 is distributed in the basal layer of the normal epidermis. Furthermore, the expression is markedly increased in cancerous/non-cancerous skin samples with the hyperproliferation of keratinocytes. We herein examined the role of TCHHL1 in normal human keratinocytes (NHKs) and squamous cell carcinoma (SCC). The knockdown of TCHHL1 by transfection with TCHHL1 siRNA significantly inhibited proliferation and induced the early apoptosis of NHKs. In TCHHL1-knockdown NHKs, the level of extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation was markedly decreased. In addition, the slight inhibition of v-akt murine thymoma viral oncogene homolog (AKT) phosphorylation and upregulation of forkhead box-containing protein O1(FOXO1), B-cell lymphoma2 (BCL2) and Bcl2-like protein 11 (BCL2L11) was observed. Skin-equivalent models built by TCHHL1-knockdown NHKs showed a markedly hypoplastic epidermis. These findings highlight that TCHHL1 plays an important role in homeostasis of the normal epidermis. TCHHL1 was expressed in the growing cells of cutaneous SCC; therefore, we next examined an association with the cell growth in HSC-1 cells (a human SCC line). In HSC-1 cells, the knockdown of TCHHL1 also suppressed cell proliferation and induced apoptosis. These cells showed an inhibition of phosphorylation of ERK1/2, AKT and signal transducers and activator of transcription 3, and the significant upregulation of FOXO1, BCL2, and BCL2L11. Accordingly, TCHHL1 is associated with survival of cutaneous SCC. In addition, we hypothesize that TCHHL1 may be a novel therapeutic target in cutaneous SCC.

Introduction YLB113 is a biosimilar of the reference product (RP), etanercept, under development for treatment of patients with moderate-to-severe rheumatoid arthritis (RA) and other approved indications. A phase 3 study was conducted in Europe, Japan, and India to compare the efficacy, safety, and immunogenicity of YLB113 with the RP over a treatment period of 52 weeks. Methods Overall, 528 patients with moderate-to-severe RA receiving concomitant methotrexate were randomized to receive a once-weekly, subcutaneous dose of 50 mg YLB113 or the RP. The primary endpoint was ACR20 response rate at week 24, with similarity confirmed if the 95% confidence interval (CI) for YLB113 and the RP was within the range of − 15 to 15%. Safety and immunogenicity endpoints were assessed to week 52. Results Based on the European analysis, in the full analysis set, ACR20 response at week 24 was 83.3% and 88.5% for YLB113 and the RP, respectively. Responses were within the predefined clinical equivalence margin. The sensitivity analysis in the per protocol set revealed a similar proportion of subjects exhibiting ACR20 response at week 24 between groups, with a difference of − 5.1% (95% CI − 11.07 to 0.81). The incidence of treatment-emergent adverse events was comparable between groups, and the incidence of antidrug antibody development to week 24 favored YLB113 (0.8 vs. 8.3%). Conclusions This study demonstrated biosimilarity of YLB113 to the RP regarding efficacy, safety, and immunogenicity in patients with moderate-to-severe RA. Based on the same mechanism of action, biosimilarity could be extrapolated to other therapeutic indications approved for etanercept. Trial registration EudraCT Number: 2015-002,809-12. Electronic Supplementary Material The online version of this article (10.1007/s40744-019-00186-3) contains supplementary material, which is available to authorized users.

Compiling evidence indicates an unusual role of extracellular S100A8/A9 in cancer metastasis. S100A8/A9 secreted from either cancer cells or normal cells including epithelial and inflammatory cells stimulates cancer cells through S100A8/A9 sensor receptors in an autocrine or paracrine manner, leading to cancer cell metastatic progression. We previously reported a novel S100A8/A9 receptor, neuroplastin-β (NPTNβ), which plays a critical role in atopic dermatitis when it is highly activated in keratinocytes by an excess amount of extracellular S100A8/A9 in the inflammatory skin lesion. Interestingly, our expression profiling of NPTNβ showed significantly high expression levels in lung cancer cell lines in a consistent manner. We hence aimed to determine the significance of NPTNβ as an S100A8/A9 receptor in lung cancer. Our results showed that NPTNβ has strong ability to induce cancer-related cellular events, including anchorage-independent growth, motility and invasiveness, in lung cancer cells in response to extracellular S100A8/A9, eventually leading to the expression of a cancer disseminative phenotype in lung tissue in vivo. Mechanistic investigation revealed that binding of S100A8/A9 to NPTNβ mediates activation of NFIA and NFIB and following SPDEF transcription factors through orchestrated upstream signals from TRAF2 and RAS, which is linked to anchorage-independent growth, motility and invasiveness. Overall, our results indicate the importance of the S100A8/A9-NPTNβ axis in lung cancer disseminative progression and reveal a pivotal role of its newly identified downstream signaling, TRAF2/RAS-NFIA/NFIB-SPDEF, in linking to the aggressive development of lung cancers.

Epidermal lamellar granules transport various lipids, proteins, and protein inhibitors from the trans-Golgi network to the extracellular space, and play an important role in skin barrier formation. We elucidated the 3-dimensional structure of lamellar granules and the trans-Golgi network in normal human skin by focused ion beam scanning electron microscopy. Reconstructed focused ion beam scanning electron microscopy 3-dimensional images revealed that the overall lamellar granule structure changed from vesicular to reticular within the second layer of the stratum granulosum. Furthermore, the trans-Golgi network was well developed within this layer and spread through the cytoplasm with branched, tubular structures that connected to lamellar granules. Our study reveals the unique overall 3-dimensional structure of lamellar granules and the trans-Golgi network within the cells of the epidermis, and provides the basis for an understanding of the skin barrier formation.

Within the "seed and soil" theory of organ tropic cancer metastasis is a growing compilation of evidence that S100A8/A9 functions as a soil signal that attracts cancer cells to certain organs, which prove beneficial to their growth. S100A8/A9-sensing receptors including Toll-like receptor 4 (TLR4), advanced glycation end products (RAGE), and also important receptors we recently succeeded in identifying (EMMPRIN, NPTNβ, MCAM, and ALCAM) have the potential to become promising therapeutic targets. In our study, we prepared extracellular regions of these novel molecules and fused them to human IgG2-Fc to extend half-life expectancy, and we evaluated the anti-metastatic effects of the purified decoy proteins on metastatic cancer cells. The purified proteins markedly suppressed S100A8/A9-mediated lung tropic cancer metastasis. We hence expect that our novel biologics may become a prominent medicine to prevent cancer metastasis in clinical settings through cutting the linkage between "seed and soil".

We previously identified novel S100A8/A9 receptors, extracellular matrix metalloproteinase inducer (EMMPRIN), melanoma cell adhesion molecule (MCAM), activated leukocyte cell adhesion molecule (ALCAM), and neuroplastin (NPTN) , that are critically involved in S100A8/A9-mediated cancer metastasis and inflammation when expressed at high levels. However, little is known about the presence of any cancer-specific mechanism(s) that modifies these receptors, further inducing upregulation at protein levels without any transcriptional regulation. Expression levels of glycosyltransferase-encoding genes were examined by a PCR-based profiling array followed by confirmation with quantitative real-time PCR. Cell migration and invasion were assessed using a Boyden chamber. Western blotting was used to examine the protein level, and the RNA level was examined by Northern blotting. Immunohistochemistry was used to examine the expression pattern of -1,3-galactosyl-O-glycosyl-glycoprotein -1,6-N-acetylglucosaminyltransferase 3 (GCNT3) and MCAM in melanoma tissue. We found that GCNT3 is overexpressed in highly metastatic melanomas. Silencing and functional inhibition of GCNT3 greatly suppressed migration and invasion of melanoma cells, resulting in the loss of S100A8/A9 responsiveness. Among the novel S100A8/A9 receptors, GCNT3 favorably glycosylates the MCAM receptor, extending its half-life and leading to further elevation of S100A8/A9-mediated cellular motility in melanoma cells. GCNT3 expression is positively correlated to MCAM expression in patients with high-grade melanomas. Collectively, our results showed that GCNT3 is an upstream regulator of MCAM protein and indicate the possibility of a potential molecular target in melanoma therapeutics through abrogation of the S100A8/A9MCAM axis.

Background YLB113 is an investigational biosimilar of the reference product etanercept (ETN), being developed for the treatment of patients with moderate-to-severe rheumatoid arthritis (RA) and other approved indications of the reference product ETN. Objectives The phase 3 study of YLB113 was conducted in Europe, Japan, and India across more than 100 rheumatology clinics to compare efficacy, safety, and immunogenicity of YLB113 with ETN in patients with RA. Methods A total of 528 patients with moderate-to-severe RA receiving concomitant treatment with methotrexate were randomized to receive a once-weekly dose of 50 mg of subcutaneously administered YLB113 or ETN. The primary end point was the ACR20 response rate at Week 24, with equivalence confirmed if the 95% confidence interval (CI) was within the range of –15% to 15%. Other efficacy end points, such as DAS28 with safety and immunogenicity end points, were assessed periodically up to Week 52. Results The ACR20 response rate at Week 24 was 81.2% for YLB113 and 86.8% for ETN in the full analysis set, with a treatment difference of –5.6% (95% CI: –11.6, 0.5), which was completely within the predefined equivalence margin of −15% to 15%. The result for sensitivity analysis using the per protocol set population revealed that the proportion of subjects who showed ACR20 response at Week 24 was similar between both treatment groups, at –4.6% (95% CI: –10.1, 0.8). The incidence of treatment-emergent adverse events was comparable between YLB113 and ETN (55.5% vs 65.7%), and the incidence of antidrug antibody development up to Week 24 was in favor of YLB113 (0.8% vs 8.3%). Conclusion The present comparative study demonstrated the biosimilarity of YLB113 to ETN on the triad of efficacy, safety, and immunogenicity in patients with moderate-to-severe RA, and thus can be extrapolated to other therapeutic indications approved for ETN. The therapeutic equivalence of YLB113 and ETN in terms of the primary efficacy end point at Week 24 and long-term safety comparability until Week 52 was established with lower immunogenicity. Disclosure of Interests Hisashi Yamanaka Grant/research support from: AbbVie, Eisai, Bristol-Meyers, Novartis, Behringer, Astellas, Kaken, Nippon-Shinyaku, Pfizer, UCB, Ayumi, Ono, Daiichi-Sankyo, Taisyo-Toyama, Takeda, Tanabe-Mitsubishi, Chugai, Teijin Pharma, Torii, YLbio, Speakers bureau: Bristol-Meyers, Astellas, Pfizer, Daiichi-Sankyo, Takeda, Tanabe-Mitsubishi, Chugai, Teijin Pharma, YLbio, Naoyuki Kamatani Speakers bureau: I was a speaker in meetings and paid for the speech by YLB Pharma, Teijin Pharma, Chugai, Pfizer, Fuji Yakuhin, Sanwa Kagaku, Asahi Kasei Pharma and Asteras-Amzen BioPharma., Yoshiya Tanaka Grant/research support from: Abbvie, Astellas, Bristol-Myers Squibb, Chugai, Daiichi-Sankyo, Eisai, Mitsubishi-Tanabe, MSD, Ono, Taisho-Toyama, Takeda, Speakers bureau: Abbvie, Asahi-kasei, Astellas, Bristol-Myers Squibb, Chugai, Daiichi-Sankyo, Eli Lilly, Eisai, Glaxo-Smithkline, Janssen, Mitsubishi-Tanabe, Novartis, Pfizer Japan Inc, Sanofi, Takeda, UCB, YL Biologics, Toshihiko Hibino Consultant for: I had worked in Japanese Pharmaceutical Company (Showa Yakkako Company Co. Ltd.) as unaffiliated director until 2015 but have no conflict on business including newly built company (Ayumi Pharmaceutical Company Co. Ltd.) since then., Edit Drescher: None declared, Juan Sanchez-Burson: None declared, Manfred Rettenbacher Employee of: I’m an employee of Lupin Pharmaceuticals Ltd., Girish Bhatia: None declared, Snehal Gadve Consultant for: Was working as a consultant for YLB company, Chirag Shah Employee of: I’m an employee of Lupin Pharmaceuticals Ltd., Dhananjay Bakhle Employee of: I’m an employee of Lupin Pharmaceuticals Ltd.

Background:Eligible subjects with moderate-to-severely active rheumatoid arthritis (RA) who completed a phase 3 double-blind comparative efficacy and safety study (Study No. YLB113-002; Yamanaka et al, 2019) of 50 mg etanercept biosimilar (YLB113) or etanercept reference product (RP) by subcutaneous administration with concomitant MTX treatment for 52 weeks, were enrolled in this open label extension (OLE) study (Study No. YLB113-003) to assess the long-term safety and tolerability of YLB113 through to 3 years.Objectives:This OLE study aimed to evaluate the long-term safety of YLB113 administration in subjects with RA who received RP or YLB113 in a phase 3 active comparator study. The main parameters assessed were safety and tolerability in terms of adverse events (AEs) and injection site reactions (ISRs), incidence of immunogenicity and efficacy as DAS28 improvement (disease activity score in 28 joints).Methods:Subjects received 50 mg of YLB113 subcutaneously once every 1 to 2 weeks. Safety was assessed by AEs after study drug administration, ISRs, physical examination findings, and immunogenicity. Efficacy (DAS28 score) was assessed at the time of transition to the OLE study (Week 0), and at weeks 12, 24, 48, 72, 96 and at the end of the study.Results:201 (Full analysis set [FAS]) subjects received the study drug and 184 subjects completed the study (91.5% completion rate). The average drug exposure in 94 subjects who continued to receive YLB113 and completed the study (94.0% completion rate) and 90 subjects who switched from RP to YLB113 and completed the study (89.1% completion rate) was 103 weeks providing the long-term drug exposure data of YLB113.The CTCAE Grade of TEAEs and ADRs observed were Grade 2 or less in severity (with no Grade ≥3). The overall incidence of ISRs was 10.0% (20/201 subjects) 77 events. All the ISRs reported were Grade 1 except for one Grade 2.An overview of the AEs experienced by the subjects is summarized in the Table 1.Table 1.Overview of Adverse events in OLE studyFASn (%)Number of eventsNumber of subjects201-Treatment emergent adverse events (TEAEs)188(93.5)975Adverse drug reactions (ADRs)82(40.8)221Serious adverse events (SAEs)21(10.4)27Serious adverse drug reactions (SADRs)7(3.5)7AEs leading to premature study discontinuation4(2.0)6ADRs leading to premature study discontinuation3(1.5)4SAEs leading to premature study discontinuation3(1.5)4SADRs leading to premature study discontinuation2(1.0)2n - number of subjects with at least 1 AE in the categoryThe mean DAS28 (mean ± S.D.) of 2.22 ± 0.95 at the study transition was 2.10 ± 0.91 at Week 72 and 2.06 ± 0.89 at the end of the study. It was confirmed that DAS28 slightly decreased with time after the study transition and continued until the end of the study. The average DAS28 value remained low even with long-term administration of YLB113, suggesting that the effects of the study drug was sustained (Figure 1).Figure 1.DAS28 over time with YLB113 N: Number of subjects. * The administration period at the study completion differs for each subject, because the study transition time differs for each subject.cts who tested positive for anti-drug antibodies (ADA) at least once in the OLE study were 1.0% (2/200 subjects). All ADA were transient and disappeared at study completion. Such transient ADA formation was reported in the phase 3 study as well. In the phase 3 study, ADA formation was more frequent in subjects who received RP at 24 weeks, but all subjects were negative in the OLE study after switching. Therefore, switching did not affect immunogenicity.Conclusion:The safety, efficacy and immunogenicity profile of YLB113 was maintained over the long-term through to 3 years. Switching from RP to YLB113 did not impact safety or immunogenicity.References:[1]Yamanaka H, Kamatani N, Tanaka Y, et al. A Comparative Study to Assess the Efficacy, Safety, and Immunogenicity of YLB113 and the Etanercept Reference Product for the Treatment of Patients with Rheumatoid Arthritis. Rheumatol Ther. 2020 Mar;7(1):149-163.Disclosure of Interests:Yamanaka Hisashi Speakers bureau: YL Biologics Ltd, Consultant of: YL Biologics Ltd, Yoshiya Tanaka Speakers bureau: Received speaking fees and/or honoraria from Daiichi-Sankyo, Eli Lilly, Novartis, YL Biologics, Bristol-Myers, Eisai, Chugai, Abbvie, Astellas, Pfizer, Sanofi, Asahi-kasei, GSK, Mitsubishi-Tanabe, Gilead, Janssen., Grant/research support from: Received research grants from Abbvie, Mitsubishi-Tanabe, Chugai, Asahi-Kasei, Eisai, Takeda, Daiichi-Sankyo., Toshihiko Hibino: None declared, Chirag Shah Shareholder of: Lupin LTD, Employee of: Lupin LTD, Dhananjay Bakhle Shareholder of: As part of Employee Stock Options Plan of Lupin LTD, Employee of: Lupin LTD, Dimitris Stefanidis Employee of: Sr. Director, Global Medical Affairs Lead, Immunology Biosimilars for Viatris GmbH

The dynamic interaction between tumor cells and their microenvironment induces a proinflammatory milieu that drives cancer development and progression. The S100A8/A9 complex has been implicated in chronic inflammation, tumor development, and progression. The cancer microenvironment contributes to the up-regulation of this protein complex in many invasive tumors, which is associated with the formation of pre-metastatic niches and poor prognosis. Changing adhesive preference of cancer cells is at the core of the metastatic process that governs the reciprocal interactions of cancer cells with the extracellular matrices and neighboring stromal cells. Cell adhesion molecules (CAMs) have been confirmed to have high-level expression in various highly invasive tumors. The expression and function of CAMs are profoundly influenced by the extracellular milieu. S100A8/A9 mediates its effects by binding to cell surface receptors, such as heparan sulfate, TLR4 and RAGE on immune and tumor cells. RAGE has recently been identified as an adhesion molecule and has considerably high identity and similarity to ALCAM and MCAM, which are frequently over-expressed on metastatic malignant melanoma cells. In this study, we demonstrated that ALCAM and MCAM also function as S100A8/A9 receptors as does RAGE and induce malignant melanoma progression by NF-κB activation and ROS formation. Notably, MCAM not only activated NF-κB more prominently than ALCAM and RAGE did but also mediated intracellular signaling for the formation of lung metastasis. MCAM is known to be involved in malignant melanoma development and progression through several mechanisms. Therefore, MCAM is a potential effective target in malignant melanoma treatment.

Since metastasis accounts for the majority of cancer-associated deaths, studies on the mechanisms of metastasis are needed to establish innovative strategies for cancer treatment. We previously reported that melanoma cell adhesion molecule (MCAM) functions as a critical receptor for S100A8/A9, and binding of S100A8/A9 to MCAM results in the migration of melanoma cells to lung tissue. However, the critical role of MCAM in the original melanoma skin lesion is still not clear. In this study, we aimed to determine the importance of the S100A8/A9-MCAM axis in melanoma dissemination in a skin lesion as a critical early step for metastasis. Mechanistic studies revealed the downstream signaling of MCAM that signaled the induction of metastasis. S100A8/A9-MCAM binding activates mitogen-activated protein kinase kinase kinase 8 (MAP3K8), also termed TPL2, leading to strong activation of the transcription factor ETV4 and subsequent induction of matrix metalloproteinase-25 (MMP25), and finally to induction of melanoma lung tropic metastasis. Collectively, our results demonstrate a crucial role of the S100A8/A9-MCAM signaling axis in metastatic onset of melanoma cells and indicate that strategies targeting the identified pathway may be useful for the establishment of innovative anti-cancer therapies.

The metastatic dissemination of cancer cells to remote areas of the body is the most problematic aspect in cancer patients. Among cancers, melanomas are notoriously difficult to treat due to their significantly high metastatic potential even during early stages. Hence, the establishment of advanced therapeutic approaches to regulate metastasis is required to overcome the melanoma disease. An accumulating mass of evidence has indicated a critical role of extracellular S100A8/A9 in melanoma distant metastasis. Lung S100A8/A9 is induced by melanoma cells from distant organs and it attracts these cells to its enriched lung environment since melanoma cells possess several receptors that sense the S100A8/A9 ligand. We hence aimed to develop a neutralizing antibody against S100A8/A9 that would efficiently block melanoma lung metastasis. Our protocol provided us with one prominent antibody, Ab45 that efficiently suppressed not only S100A8/A9-mediated melanoma mobility but also lung tropic melanoma metastasis in a mouse model. This prompted us to make chimeric Ab45, a chimera antibody consisting of mouse Ab45-Fab and human IgG2-Fc. Chimeric Ab45 also showed significant inhibition of the lung metastasis of melanoma. From these results, we have high hopes that the newly produced antibody will become a potential biological tool to block melanoma metastasis in future clinical settings.

Inflammasomes are multimolecular complexes that control the inflammatory response. The function of inflammasomes in the pathogenesis of psoriasis is still unclear. To clarify the relationship between inflammasomes and the pathophysiology of psoriasis, and in particular, to identify molecules interacting with caspase-1, a crucial component of inflammasomes, scale extracts obtained from patients with psoriasis were immunoprecipitated with anti-caspase-1 antibody and analyzed by liquid chromatography coupled with electrospray tandem mass spectrometry (LC-MS/MS). The expression of the inflammasome component was assessed by immunohistochemical analysis and an in vitro assay. We identified several candidates for caspase-1-interacting proteins from the psoriatic scale extracts by immunoprecipitation and LC-MS/MS. Nucleotide-binding oligomerization domain-containing protein-like receptor family CARD domain-containing protein 4 (NLRC4) was the only inflammasome component among the candidates; thus, the protein is considered to be a key factor of inflammasomes in psoriasis. No inflammasome component was found in the extracts of atopic dermatitis or normal skin by LC-MS/MS. Immunohistochemical analysis demonstrated upregulation of NLRC4 in the lesional epidermis of some psoriatic patients whereas weak expression of NLRC4 was detected in the normal and non-lesional epidermis. The mRNA expression of the NLRC4 gene increased in keratinocytes at confluency, 48 h after air exposure and after the addition of 1.5 mmol/L calcium chloride. Our findings suggest that NLRC4 may be involved in the exacerbation or modification of psoriatic lesions.

The receptor for advanced glycation end products (RAGE) is involved in the pathogenesis of many inflammatory, degenerative, and hyperproliferative diseases, including cancer. Previously, we revealed mechanisms of downstream signaling from ligand-activated RAGE, which recruits TIRAP/MyD88. Here, we showed that DNAX-activating protein 10 (DAP10), a transmembrane adaptor protein, also binds to RAGE. By artificial oligomerization of RAGE alone or RAGE-DAP10, we found that RAGE-DAP10 heterodimer formation resulted in a marked enhancement of Akt activation, whereas homomultimeric interaction of RAGE led to activation of caspase 8. Normal human epidermal keratinocytes exposed to S100A8/A9, a ligand for RAGE, at a nanomolar concentration mimicked the pro-survival response of RAGE-DAP10 interaction, although at a micromolar concentration, the cells mimicked the pro-apoptotic response of RAGE-RAGE. In transformed epithelial cell lines, A431 and HaCaT, in which endogenous DAP10 was overexpressed, and S100A8/A9, even at a micromolar concentration, led to cell growth and survival due to RAGE-DAP10 interaction. Functional blocking of DAP10 in the cell lines abrogated the Akt phosphorylation from S100A8/A9-activated RAGE, eventually leading to an increase in apoptosis. Finally, S100A8/A9, RAGE, and DAP10 were overexpressed in the psoriatic epidermis. Our findings indicate that the functional interaction between RAGE and DAP10 coordinately regulates S100A8/A9-mediated survival and/or apoptotic response of keratinocytes.

A proteomics-based search for molecules interacting with caspase-14 identified prosaposin and epidermal mesotrypsin as candidates. Prosaposin is a precursor of four sphingolipid activator proteins (saposins A–D) that are essential for lysosomal hydrolysis of sphingolipids. Thus, we hypothesized that caspase-14 and mesotrypsin participate in processing of prosaposin. Because we identified a saposin A sequence as an interactor with these proteases, we prepared a specific antibody to saposin A and focused on saposin A-related physiological reactions. We found that mesotrypsin generated saposins A–D from prosaposin, and mature caspase-14 contributed to this process by activating mesotrypsinogen to mesotrypsin. Knockdown of these proteases markedly down-regulated saposin A synthesis in skin equivalent models. Saposin A was localized in granular cells, whereas prosaposin was present in the upper layer of human epidermis. The proximity ligation assay confirmed interaction between prosaposin, caspase-14, and mesotrypsin in the granular layer. Oil Red staining showed that the lipid envelope was significantly reduced in the cornified layer of skin from saposin A-deficient mice. Ultrastructural studies revealed severely disorganized cornified layer structure in both prosaposin- and saposin A-deficient mice. Overall, our results indicate that epidermal mesotrypsin and caspase-14 work cooperatively in prosaposin processing. We propose that they thereby contribute to permeability barrier formation in vivo.

Kallikrein-related peptidases (KLKs) have critical roles in corneocyte desquamation and are regulated by lymphoepithelial Kazal-type inhibitor (LEKTI). However, it is unclear how these proteases are activated and how activated KLKs are released from LEKTI in the upper cornified layer. Recently, we reported cloning of a PRSS3 gene product, keratinocyte-specific mesotrypsin, from a cDNA library. We hypothesized that mesotrypsin is involved in the desquamation process, and the aim of the present study was to test this idea by examining the effects of mesotrypsin on representative desquamation-related enzymes pro-KLK5 and pro-KLK7. Incubation of mesotrypsin and these zymogens resulted in generation of the active forms. KLK activities were effectively inhibited by recombinant LEKTI domains D2, D2–5, D2–6, D2–7, D5, D6, D6–9, D7, D7–9, and D10–15, whereas mesotrypsin activity was not susceptible to these domains, and in fact degraded them. Immunoelectron microscopy demonstrated that mesotrypsin was localized in the cytoplasm of granular cells and intercellular spaces of the cornified layer. Proximity ligation assay showed close association between mesotrypsin and KLKs in the granular to cornified layers. Age-dependency analysis revealed that mesotrypsin was markedly downregulated in corneocyte extract from donors in their sixties, compared with younger donors. Collectively, our findings suggest that mesotrypsin contributes to the desquamation process by activating KLKs and degrading the intrinsic KLKs’ inhibitor LEKTI.

Epidermal barrier abnormality due to filaggrin deficiency is an important predisposing factor in the development of atopic dermatitis (AD). In addition, the expression of thymic stromal lymphopoietin (TSLP) in keratinocytes (KCs), induced by barrier disruption, can promote type 2 helper T-cell polarization. Protease activity, including protease-activated receptor-2 (PAR-2), is also known to be involved in epidermal barrier function in AD. However, to our knowledge, the relationship between protease activity and filaggrin deficiency from the perspective of AD has not been elucidated. Flaky tail ( Flg ft ) mice, known to have a mutation in the filaggrin gene, were used to assess the role of protease in KCs in the steady state and the mite-induced AD-like skin inflammation model. In the steady state, the expression and activity levels of endogenous proteases, kallikreins 5, 7, and 14, in the skin and TSLP were higher in Flg ft than in control mice. In addition, activation of PAR-2 by its agonist induced the production of TSLP in KCs of Flg ft mice, which was abrogated by a newly developed PAR-2 antagonist. Application of the PAR-2 antagonist improved symptoms and basophil accumulation in Flg ft mice treated with mite extracts. These results suggest that possibly through the PAR-2 activation in KCs, filaggrin deficiency induces TSLP production and basophil accumulation, which play important roles in the establishment of AD.

The calcium-binding proteins S100A8 and S100A9 can dimerize to form calprotectin, the release of which during tissue damage has been implicated in inflammation and metastasis. However, receptor(s) mediating the physiologic and pathophysiologic effects of this damage-associated “danger signal” are uncertain. In this study, searching for candidate calprotectin receptors by affinity isolation-mass spectrometry, we identified the cell surface glycoprotein EMMPRIN/BASIGIN (CD147/BSG). EMMPRIN specifically bound to S100A9 but not S100A8. Induction of cytokines and matrix metalloproteases (MMP) by S100A9 was markedly downregulated in melanoma cells by attenuation of EMMPRIN. We found that EMMPRIN signaling used the TNF receptor–associated factor TRAF2 distinct from the known S100-binding signaling pathway mediated by RAGE (AGER). S100A9 strongly promoted migration when EMMPRIN was highly expressed, independent of RAGE, whereas EMMPRIN blockade suppressed migration by S100A9. Immunohistologic analysis of melanomas revealed that EMMPRIN was expressed at both the invasive edge of lesions and the adjacent epidermis, where S100A9 was also strongly expressed. In epidermal-specific transgenic mice, tail vein–injected melanoma accumulated in skin expressing S100A9 but not S100A8. Together, our results establish EMMPRIN as a receptor for S100A9 and suggest the therapeutic use in targeting S100A9–EMMPRIN interactions. Cancer Res; 73(1); 172–83. ©2012 AACR.

We previously reported a positive feedback loop between S100A8/A9 and proinflammatory cytokines mediated by extracellular matrix metalloproteinase inducer, an S100A9 receptor. Here, we identify neuroplastin-β as an unreported S100A8 receptor. Neuroplastin-β and extracellular matrix metalloproteinase inducer form homodimers and a heterodimer, and they are co-localized on the surface of cultured normal human keratinocytes. Knockdown of both receptors suppressed cell proliferation and proinflammatory cytokine induction. Upon stimulation with S100A8, neuroplastin-β recruited GRB2 and activated extracellular signal-regulated kinase, resulting in keratinocyte proliferation. Keratinocyte proliferation in response to inflammatory stimuli was accelerated in involucrin promoter-driven S100A8 transgenic mice. Further, S100A8 and S100A9 were strongly up-regulated and co-localized in lesional skin of atopic dermatitis patients. Our results indicate that neuroplastin-β and extracellular matrix metalloproteinase inducer form a functional heterodimeric receptor for S100A8/A9 heterodimer, followed by recruitment of specific adaptor molecules GRB2 and TRAF2, and this signaling pathway is involved in activation of both keratinocyte proliferation and skin inflammation in atopic skin. Suppression of this pathway might have potential for treatment of skin diseases associated with chronic inflammation such as atopic dermatitis.

The maturation and activation mechanisms of caspases are generally well understood, except for those of caspase-14, which is activated at the onset of keratinocyte terminal differentiation. We investigated the possible involvement of epidermal proteases expressed in the late stage of differentiation, and found that the chymotrypsin-like serine protease kallikrein-related peptidase-7 (KLK7) cleaved procaspase-14 at Tyr(178), generating an intermediate form that consists of a large (20 kDa) and a small subunit (8 kDa). We prepared an antibody directed to this cleavage site (h14Y178 Ab), and confirmed that it recognized a 20-kDa band formed when procaspase-14 was incubated with chymotrypsin or KLK7. We then constructed a constitutively active form of the intermediate, revC14-Y178. The substrate specificity of revC14-Y178 was completely different from that of caspase-14, showing broad specificity for various caspase substrates except WEHD-7-amino-4-trifluoromethylcoumarin (AFC), the preferred substrate of active, mature caspase-14. K(m) values for VEID-AFC, DEVD-AFC, LEVD-AFC, and LEHD-AFC were 0.172, 0.261, 0.504, and 0.847 μM, respectively. We confirmed that the mature form of caspase-14 was generated when procaspase-14 was incubated with KLK7 or revC14-Y178. Expression of constitutively active KLK7 in cultured keratinocytes resulted in generation of both the intermediate form and the mature form of caspase-14. Immunohistochemical analysis demonstrated that the intermediate form was localized at the granular layer. Our results indicate that regulation of procaspase-14 maturation during terminal differentiation is a unique two-step process involving KLK7 and an activation intermediate of caspase-14.

Loss-of-function mutation in the profilaggrin gene is a major risk factor for atopic dermatitis (AD). Previously, we showed that a neutral cysteine protease, bleomycin hydrolase (BH), has a role in generating natural moisturizing factors, and calpain I is an upstream protease in the filaggrin degradation pathway. Here, we investigated the transcriptional regulatory mechanisms of BH and the relevance of BH to AD. First, we cloned the 5′-flanking region of BH. Deletion analyses identified a critical region for BH promoter activity within −216 bp upstream. Electrophoretic mobility shift assay revealed that MZF-1, Sp-1, and interferon regulatory factor-1/2 could bind to this region in vitro. Moreover, site-directed mutagenesis of the MZF-1 and Sp-1 motifs markedly reduced BH promoter activity. These data indicate that BH expression is up-regulated via MZF-1 and Sp-1. Interestingly, a Th1 cytokine, IFN-γ, significantly reduced the expression of BH. Analyses with site-directed mutagenesis and small interference RNA supported the suppressing effect of IFN-γ on BH expression. On the other hand, a Th2 cytokine, IL-4, did not show any direct effect on BH expression. However, it down-regulated MZF-1 and Sp-1 in cultured keratinocytes, indicating that IL-4 could work as a suppressor in BH regulation. Lastly, we examined expression of BH in skins of patients with AD. BH activity and expression were markedly decreased in AD lesional skin, suggesting a defect of the filaggrin degradation pathway in AD. Our results suggest that BH transcription would be modulated during both differentiation and inflammation.

Members of the trypsin-like and chymotrypsin-like kallikrein family are important in the desquamation process. In this study, we isolated cDNA clones encoding trypsinogen 4 (brain trypsinogen) and a previously unreported isoform of trypsinogen from a human keratinocyte cDNA library. The nucleotide sequence of the new isoform only differs from those of trypsinogen 3 (mesotrypsinogen) and trypsinogen 4 in an exon encoding the N-terminal region, indicating that this isoform is an alternative splicing variant of the mesotrypsinogen gene PRSS3. Both isoforms contained the sequence DDDDK-I, a putative cleavage site for activation by enteropeptidase. Thus, after activation, mesotrypsin would be produced. Immunohistochemical and in situ hybridization studies revealed that trypsinogens were expressed and localized in the upper epidermis, especially in the granular layer. In cultured keratinocytes, enteropeptidase mRNA was expressed at the confluent stage, and its expression was strongly upregulated after air exposure. Interestingly, it was synthesized and localized only at the granular layer, suggesting that the generation of active mesotrypsin is restricted to this layer. The enteropeptidase-cleavage product was also found at the same layer. When a skin equivalent model was cultured in the medium without air exposure, the cornified layer was not formed, and many cells expressed trypsinogens and enteropeptidase. Those cells were found to be TUNEL positive. Because mesotrypsin is resistant to naturally occurring trypsin inhibitors, confined expression of the isoforms of mesotrypsinogens and enteropeptidase may indicate that mesotrypsin is involved in keratinocyte terminal differentiation.

Background Glycolic acid (GA) is the most commonly used alpha-hydroxy acid (AHA) for dermatologic applications, and is considered as a versatile superficial peeling agent for facial rejuvenation. Its therapeutic effect includes acceleration of epidermal turnover without apparent inflammation, and its action is pH-dependent. However, little is known about the molecular mechanism of GA-induced peeling. Objective To investigate the effects of topical application of GA on cell proliferation using a skin equivalent model and to examine the molecular mechanisms of GA-induced peeling. Methods GA solution was applied on the surface of a skin equivalent model, and cell proliferation was measured by means of BrdU-incorporation and immunohistochemical methods. Release of chemical mediators such as ATP into the medium was examined. The effects of antagonists of ion channels were also analyzed. Results At 24 h after GA application, BrdU-incorporation into basal keratinocytes was significantly increased. Induction of keratinocyte proliferation was pH-dependent, and was inhibited by antagonists of TRPV1, an acid-sensitive ion channel. Furthermore, transient ATP release was detected in the culture medium after GA stimulation, and this was also suppressed by TRPV1 antagonists. Conclusion These results suggest that one of the mechanisms of GA-induced epidermal proliferation is a growth response of basal keratinocytes to the local elevation of H + -ion concentration by infiltrated GA. This response is mediated by TRPV1 activation and ATP release. Activation of P2 receptors by the released ATP may also be involved.

Filaggrin is a component of the cornified cell envelope and the precursor of free amino acids acting as a natural moisturizing factor in the stratum corneum. Deimination is critical for the degradation of filaggrin into free amino acids. In this study, we tried to identify the enzyme(s) responsible for the cleavage of deiminated filaggrin in vitro. First, we investigated citrulline aminopeptidase activity in the extract of newborn rat epidermis by double layer fluorescent zymography and detected strong activity at neutral pH. Monitoring the citrulline-releasing activity, we purified an enzyme of 280 kDa, comprised of six identical subunits of 48 kDa. The NH2 terminus of representative tryptic peptides perfectly matched the sequence of rat bleomycin hydrolase (BH). The enzyme released various amino acids except Pro from β-naphthylamide derivatives and hydrolyzed citrulline-β-naphthylamide most effectively. Thus, to break down deiminated filaggrin, another protease would be required. Among proteases tested, calpain I degraded the deiminated filaggrin effectively into many peptides of different mass on the matrix-assisted laser desorption/ionization-time of flight mass spectrum. We confirmed that various amino acids including citrulline were released by BH from those peptides. On the other hand, caspase 14 degraded deiminated filaggrin into a few peptides of limited mass. Immunohistochemical analysis of normal human skin revealed co-localization of BH and filaggrin in the granular layer. Collectively, our results suggest that BH is essential for the synthesis of natural moisturizing factors and that calpain I would play a role as an upstream protease in the degradation of filaggrin.

Squamous cell carcinoma antigen 1 (SCCA1), which belongs to serine proteinase inhibitor (serpin) superfamily, inhibits papain-like cysteine proteinase. Recently, it has been reported that SCCA1 acts not only as a proteinase inhibitor but also as an inhibitor of UV-induced apoptosis via suppression of the activity of c-Jun NH(2)-terminal kinase (JNK1). The present study determined the crystal structure of SCCA1, suggesting that the reactive center loop (RCL) of SCCA1, a recognition site of proteinase, is very flexible and located away form the main-body of SCCA1. We show that the inhibitory effect of SCCA1 on the kinase activity of JNK1 is lost when the RCL was truncated. Furthermore, we found that a mutant protein created by replacing one amino acid in RCL maintain the suppressive activity to JNK1, whereas the inhibitory effect to proteinase is obviously decreased.

Chemical peeling with salicylic acid in polyethylene glycol vehicle (SA-PEG), which specifically acts on the stratum corneum, suppresses the development of skin tumors in UVB-irradiated hairless mice. To elucidate the mechanism through which chemical peeling with SA-PEG suppresses skin tumor development, the effects of chemical peeling on photodamaged keratinocytes and cornified envelopes (CEs) were evaluated in vivo. Among UVB-irradiated hairless mice, the structural atypia and expression of p53 protein in keratinocytes induced by UVB irradiation were intensely suppressed in the SA-PEG-treated mice 28 days after the start of weekly SA-PEG treatments when compared to that in the control UVB-irradiated mice. Incomplete expression of filaggrin and loricrin in keratinocytes from the control mice was also improved in keratinocytes from the SA-PEG-treated mice. In photo-exposed human facial skin, immature CEs were replaced with mature CEs 4 weeks after treatment with SA-PEG. Restoration of photodamaged stratum corneum by treatment with SA-PEG, which may affect remodeling of the structural environment of the keratinocytes, involved the normalization of keratinocyte differentiation and suppression of skin tumor development. These results suggest that the stratum corneum plays a protective role against carcinogenesis, and provide a novel strategy for the prevention of photo-induced skin tumors.

Transforming growth factor-beta (TGF-beta) appears to promote the regression phase of the mammalian hair cycle, in vivo in mice and in organ culture of human hair follicles. To assess the relationship between TGF-beta activity and apoptosis of epithelial cells during the murine hair cycle, we identified active TGF-beta responses using phospho-Smad2/3-specific antibodies (PS2). Strong, nuclear PS2 staining was observed in the outer root sheath throughout the anagen growth phase. Some bulb matrix cells were positive for PS2 during late anagen. Extensive, but weak, staining was observed in this region at the anagen-catagen transition. We also examined expression of TGF-beta-stimulated clone-22 (TSC-22), which is associated with TGF-beta-induced apoptosis of some cell lines. Recombinant rat TSC-22 was used to generate a rabbit anti-TSC-22 antibody useful for immunohistochemistry. TSC-22 RNA accumulation and immunoreactivity were observed in follicles throughout the murine hair cycle, including the dermal papilla and lower epithelial strand of late-catagen hair follicles. Neither the expression pattern nor the presence of nuclear TSC-22 correlated with the sites of apoptosis, suggesting that TSC-22 is not an effector of apoptosis in mouse catagen hair follicles. These studies support a complex role for TGF-beta in regulating the regression phase of the cycle, with potential for indirect promotion of apoptosis during the anagen-catagen transition.

TGF-beta plays important roles in the induction of catagen during the hair cycle. We examined whether TGF-beta2 could activate a caspase in human hair follicles. Using active caspase-9 and -3 specific antibodies, we found that TGF-beta2 activated these caspases in two regions, the lower part of the hair bulb and the outer layer of the outer root sheath. In addition, we searched for a plant extract that can effectively suppress TGF-beta action. We found that an extract of Hydrangea macrophylla reduced synthesis of a TGDbeta-inducible protein. We confirmed that the extract has a potential to promote hair elongation in the organ culture system. Furthermore, it delayed in vivo progression of catagen in a mouse model. Our results suggest that the induction of catagen by TGF-beta is mediated via activation of caspases and that a suppressor of TGF-beta could be effective in preventing male pattern baldness.

Squamous cell carcinoma antigens (SCCA) 1 and 2 are highly homologous proteins of the serpin family, although they inhibit different types of proteinases. We investigated the expression of both SCCA mRNAs in tumor tissues, in various cell lines (A431, SAS, Ca9, HeLa, SKGIIIa, HSC-2, HSC-3, HSC-4 and KB) and in HSC cell lines in the presence of tumor necrosis factor-alpha (TNF-alpha). The expression of SCCA2 mRNA could be differentiated from that of SCCA1 in tumor tissues and cell lines by means of reverse transcription-polymerase chain reaction (RT-PCR). The ratio between SCCA1 and SCCA2 mRNA expression showed selective expression of SCCA2 mRNA in SCC tissues from the uterine cervix compared to SCC tissues from the esophagus or skin. In addition, a significant level of SCCA2 mRNA expression was detected in the HSC-4 cell line, but not in Ca9, HeLa, SKG-IIIa, or HSC-3 cells. In contrast, SCCA1 mRNA was detected in all samples tested. These results suggest that the level of expression of SCCA2 mRNA detected by RT-PCR can be used to evaluate the status of SCC tumors. Next, we studied the effect of TNF-alpha on SCCA1 and SCCA2 mRNA expression in HSC cell lines. SCCA1 mRNA expression was constantly increased in the three HSC cells examined with increasing time of exposure to TNF-alpha. In contrast, SCCA2 mRNA expression was specific for HSC-4 cells. The survival rate of HSC-4 cells pretreated with TNF-alpha (6.3 ng/ml) for 48 h was found to be 72%, compared with 42% and 9% for HSC-3 and HSC-2 cells, respectively, after apoptotic stimulation by TNF-alpha (10 ng/ml) and cycloheximide (10 microg/ml) for 18 h. Furthermore, selective expression of SCCA2 mRNA in HSC-4 pretreated with TNF-alpha protected these cells from TNF-alpha-mediated apoptosis. Thus, SCCA2 overexpression in squamous tumor cells contributed to their survival by protecting them against TNF-alpha-induced apoptosis.

Squamous cell carcinoma antigen belongs to the serpin family and is used for the diagnosis and management of squamous cell carcinoma. We investigated the involvement of squamous cell carcinoma antigen in psoriasis, as it is always detected in the sera of patients with psoriasis. Squamous cell carcinoma antigen localization in psoriatic epidermis varied depending on its concentration in the patient's sera. When its level was low in serum, weak and scattered staining was observed in the granular layer. With a high concentration of squamous cell carcinoma antigen, strong staining through the suprabasal to granular layer and condensed staining around the plasma membrane or intracellular space was detected in the affected epidermis. Interestingly, squamous cell carcinoma antigen was abundant in nuclei of the granular layer cells and elongated rete ridges. Immunoelectron microscopy confirmed the localization of squamous cell carcinoma antigen in the nuclei as well as in the periphery of the cell membrane. A cDNA library was constructed from psoriatic epidermis and both clones, SCCA1 and SCCA2, were obtained. Attempts to raise specific antibodies or to prepare cRNA probes for SCCA1 and SCCA2 were unsuccessful because of their nearly identical structures. A primer pair from each reactive site sequence enabled us to give a distinctive product for SCCA1 and SCCA2 by reverse transcription polymerase chain reaction. Analysis using these primers demonstrated that the SCCA2 transcript was specifically expressed in psoriatic skin tissues. Our results suggest that overexpression of squamous cell carcinoma antigens is associated with the disease activity of psoriasis.

A proteomics-based search for molecules interacting with caspase-14 identified prosaposin and epidermal mesotrypsin as candidates. Prosaposin is a precursor of four sphingolipid activator proteins (saposins A-D) that are essential for lysosomal hydrolysis of sphingolipids. Thus, we hypothesized that caspase-14 and mesotrypsin participate in processing of prosaposin. Because we identified a saposin A sequence as an interactor with these proteases, we prepared a specific antibody to saposin A and focused on saposin A-related physiological reactions. We found that mesotrypsin generated saposins A-D from prosaposin, and mature caspase-14 contributed to this process by activating mesotrypsinogen to mesotrypsin. Knockdown of these proteases markedly down-regulated saposin A synthesis in skin equivalent models. Saposin A was localized in granular cells, whereas prosaposin was present in the upper layer of human epidermis. The proximity ligation assay confirmed interaction between prosaposin, caspase-14, and mesotrypsin in the granular layer. Oil Red staining showed that the lipid envelope was significantly reduced in the cornified layer of skin from saposin A-deficient mice. Ultrastructural studies revealed severely disorganized cornified layer structure in both prosaposin- and saposin A-deficient mice. Overall, our results indicate that epidermal mesotrypsin and caspase-14 work cooperatively in prosaposin processing. We propose that they thereby contribute to permeability barrier formation in vivo.

Unlike other caspase family members, caspase-14 shows restricted expression, being found mostly in epidermis and its appendages. It has been suggested that caspase-14 is not involved in apoptosis or inflammation, but participates in keratinocyte terminal differentiation. Its activation occurs at the corneocyte formation. In previous work, we have purified active caspase-14 from human corneocyte extracts. In addition, we have clarified activation mechanism of caspase-14, where kallikrein-related peptidase 7 (KLK7) generates an intermediate form from procaspase-14 and this form finally converts procaspase-14 to active, mature caspase-14. Here we describe techniques for measurement of caspase-14 activity using synthetic substrate, purification of caspase-14 from corneocyte extract, preparation of constitutively active caspase-14 and specific antibody, quantification of total and active caspase-14 in corneocyte extracts using ELISA, as well as methods for caspase-14 activation and its visualization by immunohistochemistry.

The regression phase of the hair cycle (catagen) is an apoptosis-driven process accompanied by terminal differentiation, proteolysis, and matrix remodeling. As an inhibitor of keratinocyte proliferation and inductor of keratinocyte apoptosis, transforming growth factor beta1 (TGF-beta1) has been proposed to play an important role in catagen regulation. This is suggested, for example, by maximal expression of TGF-beta1 and its receptors during late anagen and the onset of catagen of the hair cycle. We examined the potential involvement of TGF-beta1 in catagen control. We compared the first spontaneous entry of hair follicles into catagen between TGF-beta1 null mice and age-matched wild-type littermates, and assessed the effects of TGF-beta1 injection on murine anagen hair follicles in vivo. At day 18 p.p., hair follicles in TGF-beta1 -/- mice were still in early catagen, whereas hair follicles of +/+ littermates had already entered the subsequent resting phase (telogen). TGF-beta1-/- mice displayed more Ki-67-positive cells and fewer apoptotic cells than comparable catagen follicles from +/+ mice. In contrast, injection of TGF-beta1 into the back skin of mice induced premature catagen development. In addition, the number of proliferating follicle keratinocytes was reduced and the number of TUNEL + cells was increased in the TGF-beta1-treated mice compared to controls. Double visualization of TGF-beta type II receptor (TGFRII) and TUNEL reactivity revealed colocalization of apoptotic nuclei and TGFRII in catagen follicles. These data strongly support that TGF-beta1 ranks among the elusive endogenous regulators of catagen induction in vivo, possibly via the inhibition of keratinocyte proliferation and induction of apoptosis. Thus, TGF-betaRII agonists and antagonists may provide useful therapeutic tools for human hair growth disorders based on premature or retarded catagen development (effluvium, alopecia, hirsutism).

Unlike other caspase family members, caspase-14 shows restricted expression, being found mostly in epidermis and its appendages. It has been suggested that caspase-14 is not involved in apoptosis or inflammation, but participates in keratinocyte terminal differentiation. Its activation occurs at the corneocyte formation. In previous work, we have purified active caspase-14 from human corneocyte extracts. In addition, we have clarified activation mechanism of caspase-14, where kallikrein-related peptidase 7 (KLK7) generates an intermediate form from procaspase-14 and this form finally converts procaspase-14 to active, mature caspase-14. Here we describe techniques for measurement of caspase-14 activity using synthetic substrate, purification of caspase-14 from corneocyte extract, preparation of constitutively active caspase-14 and specific antibody, quantification of total and active caspase-14 in corneocyte extracts using ELISA, as well as methods for caspase-14 activation and its visualization by immunohistochemistry.

Loss of the nucleus is a critical step in keratinocyte terminal differentiation. To elucidate the mechanisms involved, we focused on two characteristic events: nuclear translocation of N-terminal fragment of profilaggrin and caspase-14-dependent degradation of the inhibitor of caspase-activated DNase (ICAD). First, we demonstrated that epidermal mesotrypsin liberated a 55-kDa N-terminal fragment of profilaggrin (FLG-N) and FLG-N was translocated into the nucleus. Interestingly, these cells became TUNEL positive. Mutation in the mesotrypsin-susceptible Arg-rich region between FLG-N and the first filaggrin domain abolished these changes. Furthermore, caspase-14 caused limited proteolysis of ICAD, followed by accumulation of caspase-activated DNase (CAD) in TUNEL-positive nuclei. Knockdown of both proteases resulted in a significant increase of remnant nuclei in a skin equivalent model. Immunohistochemical study revealed that both caspase-14 and mesotrypsin were markedly downregulated in parakeratotic areas of lesional skin from patients with atopic dermatitis and psoriasis. Collectively, our results indicate that at least two pathways are involved in the DNA degradation process during keratinocyte terminal differentiation.

Filaggrin protein is synthesized in the stratum granulosum of the skin and contributes to the formation of the human skin barrier. Profilaggrin is cleaved by proteolytic enzymes and converted to functional filaggrin, but its processing mechanism remains not fully elucidated. Kallikrein-related peptidase 5 (KLK5) is a major serine protease found in the skin, which is secreted from lamellar granules following its expression in the stratum granulosum and activated in the extracellular space of the stratum corneum. Here, we searched for profilaggrin-processing protease(s) by partial purification of epidermal extracts and found KLK5 as a possible candidate. We used high performance liquid chromatography coupled with electrospray tandem mass spectrometry to show that KLK5 cleaves profilaggrin. Furthermore, based on a proximity ligation assay, immunohistochemistry, and immunoelectron microscopy analysis, we reveal that KLK5 and profilaggrin co-localize in the stratum granulosum in human epidermis. KLK5 knockdown in normal cultured human epidermal keratinocytes resulted in higher levels of profilaggrin, indicating that KLK5 potentially functions in profilaggrin cleavage.