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AIM: YLB113 biosimilar was evaluated in an open-label extension single-arm study to assess long-term safety, efficacy, and immunogenicity in patients with rheumatoid arthritis (RA). We also report post-hoc results on the incidence of injection-site reactions (ISRs) and injection-site erythema (ISE) from a phase III study. METHOD: Participants from the phase III, double-blind, randomized, 96 week equivalence study who completed the final visit received 50 mg YLB113 subcutaneously every 2 weeks. Key safety end points were assessed through adverse events (AEs), ISRs, ISE, and anti-drug antibody (ADA) incidence. The efficacy end point was change from baseline in Disease Activity Score 28-joint count (DAS28) over time. RESULTS: Of 201 participants, 184 (91.5%) completed the study. Treatment-emergent AEs were experienced by 93.5% and severe AEs by 10.0% of participants. The discontinuation rate due to AEs was 2.0%. Overall, 20.0% of participants reported an incidence of ISRs throughout the open-label extension study. Two participants developed ADAs, and none developed neutralizing ADAs at any time after study drug administration. The overall DAS28 (mean ± SD) change was 2.22 ± 0.95 at the study transition, 2.10 ± 0.91 at week 72, and 2.06 ± 0.89 at the end of the study. In the post-hoc analysis, YLB113 showed a statistically significant lower incidence of ISRs (10 [3.8%], P < 0.0001) and ISE (5 [1.9%], P < 0.0001) compared with the reference product Enbrel®. CONCLUSION: YLB113 demonstrated long-term safety and sustained efficacy for up to 96 weeks. Patients on YLB113 experienced significantly lower ISRs and ISE in a post-hoc analysis of the phase III study when compared with reference product.
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Antirreumáticos , Artritis Reumatoide , Biosimilares Farmacéuticos , Humanos , Etanercept/efectos adversos , Antirreumáticos/efectos adversos , Biosimilares Farmacéuticos/efectos adversos , Resultado del Tratamiento , Artritis Reumatoide/diagnóstico , Artritis Reumatoide/tratamiento farmacológico , Artritis Reumatoide/inducido químicamente , Anticuerpos , Reacción en el Punto de Inyección/diagnóstico , Reacción en el Punto de Inyección/epidemiología , Reacción en el Punto de Inyección/etiología , Método Doble CiegoAsunto(s)
Cisteína Endopeptidasas/metabolismo , Dermatitis Atópica/patología , Epidermis/enzimología , Estaciones del Año , Adolescente , Estudios de Casos y Controles , Niño , Preescolar , Cisteína Endopeptidasas/análisis , Epidermis/patología , Femenino , Voluntarios Sanos , Humanos , Japón , Masculino , Índice de Severidad de la EnfermedadRESUMEN
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.
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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.
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Metastatic breast cancer is the leading cause of cancer-associated death in women. The progression of this fatal disease is associated with inflammatory responses that promote cancer cell growth and dissemination, eventually leading to a reduction of overall survival. However, the mechanism(s) of the inflammation-boosted cancer progression remains unclear. In this study, we found for the first time that an extracellular cytokine, S100A8/A9, accelerates breast cancer growth and metastasis upon binding to a cell surface receptor, melanoma cell adhesion molecule (MCAM). Our molecular analyses revealed an important role of ETS translocation variant 4 (ETV4), which is significantly activated in the region downstream of MCAM upon S100A8/A9 stimulation, in breast cancer progression in vitro as well as in vivo. The MCAM-mediated activation of ETV4 induced a mobile phenotype called epithelial-mesenchymal transition (EMT) in cells, since we found that ETV4 transcriptionally upregulates ZEB1, a strong EMT inducer, at a very high level. In contrast, downregulation of either MCAM or ETV4 repressed EMT, resulting in greatly weakened tumor growth and lung metastasis. Overall, our results revealed that ETV4 is a novel transcription factor regulated by the S100A8/A9-MCAM axis, which leads to EMT through ZEB1 and thereby to metastasis in breast cancer cells. Thus, therapeutic strategies based on our findings might improve patient outcomes.
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Neoplasias de la Mama/genética , Transición Epitelial-Mesenquimal/genética , Proteínas Proto-Oncogénicas c-ets/genética , Animales , Neoplasias de la Mama/patología , Antígeno CD146/genética , Calgranulina A/genética , Calgranulina B/genética , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Ratones , Invasividad Neoplásica/genética , Invasividad Neoplásica/patología , Transducción de Señal/genética , Ensayos Antitumor por Modelo de Xenoinjerto , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/genéticaRESUMEN
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.
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Calgranulina A/metabolismo , Calgranulina B/metabolismo , Melanoma Experimental/patología , Melanoma/patología , Neoplasias Cutáneas/patología , Animales , Antígeno CD146/metabolismo , Calgranulina A/genética , Calgranulina B/genética , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Proteínas Ligadas a GPI/metabolismo , Células HEK293 , Humanos , Queratinocitos/patología , Neoplasias Pulmonares/secundario , Quinasas Quinasa Quinasa PAM/metabolismo , Metaloproteinasas de la Matriz Asociadas a la Membrana/metabolismo , Melanoma/terapia , Melanoma Experimental/terapia , Ratones , Ratones Endogámicos BALB C , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-ets/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/genética , Piel/patología , Neoplasias Cutáneas/terapia , Ensayos Antitumor por Modelo de Xenoinjerto , Melanoma Cutáneo MalignoRESUMEN
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-ß (NPTNß), 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 NPTNß showed significantly high expression levels in lung cancer cell lines in a consistent manner. We hence aimed to determine the significance of NPTNß as an S100A8/A9 receptor in lung cancer. Our results showed that NPTNß 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 NPTNß 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-NPTNß 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.
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Calgranulina A/metabolismo , Calgranulina B/metabolismo , Neoplasias Pulmonares/patología , Glicoproteínas de Membrana/metabolismo , Regulación hacia Arriba , Células A549 , Animales , Línea Celular Tumoral , Movimiento Celular , Progresión de la Enfermedad , Femenino , Regulación Neoplásica de la Expresión Génica , Células HEK293 , Humanos , Neoplasias Pulmonares/metabolismo , Ratones , Factores de Transcripción NFI/metabolismo , Isoformas de Proteínas/metabolismo , Proteínas Proto-Oncogénicas c-ets/metabolismo , Transducción de SeñalRESUMEN
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, NPTNß, 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".
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Calgranulina A/metabolismo , Calgranulina B/metabolismo , Melanoma Experimental/prevención & control , Melanoma Experimental/secundario , Proteínas Recombinantes de Fusión/farmacología , Animales , Basigina/química , Basigina/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Moléculas de Adhesión Celular/química , Moléculas de Adhesión Celular/farmacología , Humanos , Fragmentos Fc de Inmunoglobulinas/química , Fragmentos Fc de Inmunoglobulinas/farmacología , Inmunoglobulina G/inmunología , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundario , Melanoma Experimental/metabolismo , Melanoma Experimental/patología , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Dominios Proteicos , Receptor para Productos Finales de Glicación Avanzada/química , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
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.
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Anticuerpos Monoclonales/administración & dosificación , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/secundario , Melanoma/tratamiento farmacológico , Animales , Anticuerpos Monoclonales/farmacología , Anticuerpos Neutralizantes/administración & dosificación , Anticuerpos Neutralizantes/farmacología , Calgranulina A/inmunología , Calgranulina B/inmunología , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Neoplasias Pulmonares/metabolismo , Melanoma/metabolismo , Ratones , Resultado del Tratamiento , Microambiente Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
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.
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Diferenciación Celular , Epidermis/fisiología , Queratinocitos/fisiología , Red trans-Golgi/ultraestructura , Adulto , Anciano , Epidermis/diagnóstico por imagen , Femenino , Humanos , Imagenología Tridimensional , Queratinocitos/ultraestructura , Masculino , Microscopía Electrónica de Rastreo , Persona de Mediana EdadRESUMEN
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.
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Proteínas Adaptadoras de Señalización CARD/metabolismo , Proteínas de Unión al Calcio/metabolismo , Epidermis/patología , Inflamasomas/metabolismo , Psoriasis/inmunología , Adulto , Anciano , Biopsia , Proteínas Adaptadoras de Señalización CARD/inmunología , Proteínas de Unión al Calcio/inmunología , Caspasa 1/inmunología , Caspasa 1/metabolismo , Células Cultivadas , Dermatitis Atópica/inmunología , Dermatitis Atópica/patología , Progresión de la Enfermedad , Células Epidérmicas , Epidermis/inmunología , Femenino , Humanos , Inflamasomas/inmunología , Queratinocitos/inmunología , Queratinocitos/patología , Masculino , Persona de Mediana Edad , Psoriasis/patología , ARN Mensajero/metabolismo , Regulación hacia ArribaRESUMEN
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) ß, 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 ß-1,3-galactosyl-O-glycosyl-glycoprotein ß-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/A9-MCAM axis.
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Biomarcadores de Tumor/metabolismo , Calgranulina A/metabolismo , Calgranulina B/metabolismo , Movimiento Celular , Melanoma/patología , N-Acetilglucosaminiltransferasas/metabolismo , Apoptosis , Biomarcadores de Tumor/genética , Antígeno CD146/química , Antígeno CD146/metabolismo , Calgranulina A/genética , Calgranulina B/genética , Proliferación Celular , Regulación Neoplásica de la Expresión Génica , Humanos , Melanoma/genética , Melanoma/metabolismo , N-Acetilglucosaminiltransferasas/genética , Pronóstico , Células Tumorales CultivadasRESUMEN
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-ß as an unreported S100A8 receptor. Neuroplastin-ß 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-ß 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-ß 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.
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Basigina/metabolismo , Calgranulina A/biosíntesis , Dermatitis Atópica/metabolismo , Glicoproteínas de Membrana/biosíntesis , Regulación hacia Arriba , Animales , Basigina/genética , Calgranulina A/genética , Proliferación Celular , Células Cultivadas , Dermatitis Atópica/genética , Dermatitis Atópica/patología , Ensayo de Inmunoadsorción Enzimática , Humanos , Inmunohistoquímica , Queratinocitos/metabolismo , Queratinocitos/patología , Glicoproteínas de Membrana/genética , Ratones , Ratones Transgénicos , Transducción de SeñalRESUMEN
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.
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Calgranulina A/metabolismo , Calgranulina B/metabolismo , Neoplasias Pulmonares/secundario , Melanoma/patología , FN-kappa B/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Animales , Antígenos CD/genética , Antígenos CD/metabolismo , Apoptosis , Western Blotting , Antígeno CD146/genética , Antígeno CD146/metabolismo , Calgranulina A/genética , Calgranulina B/genética , Adhesión Celular , Moléculas de Adhesión Celular Neuronal/genética , Moléculas de Adhesión Celular Neuronal/metabolismo , Movimiento Celular , Proliferación Celular , Proteínas Fetales/genética , Proteínas Fetales/metabolismo , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Masculino , Melanoma/genética , Melanoma/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Desnudos , FN-kappa B/genética , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptor para Productos Finales de Glicación Avanzada/genética , Receptor para Productos Finales de Glicación Avanzada/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Tumorales Cultivadas , Microambiente Tumoral , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
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.
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Queratinocitos/metabolismo , Receptores Inmunológicos/metabolismo , Transducción de Señal , Calgranulina A/metabolismo , Calgranulina B/metabolismo , Células Cultivadas , Humanos , Células Asesinas Naturales/inmunología , Psoriasis/metabolismo , Interferencia de ARN , Receptor para Productos Finales de Glicación AvanzadaRESUMEN
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.
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Caspasas/metabolismo , Saposinas/metabolismo , Piel/metabolismo , Tripsina/metabolismo , Animales , Caspasas/genética , Células Cultivadas , Técnicas de Silenciamiento del Gen , Humanos , Queratinocitos/metabolismo , Ratones , Ratones Noqueados , Modelos Biológicos , Permeabilidad , Procesamiento Proteico-Postraduccional , ARN Interferente Pequeño/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saposinas/deficiencia , Saposinas/genética , Piel/ultraestructura , Tripsina/genéticaRESUMEN
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.
Asunto(s)
Caspasa 14/aislamiento & purificación , Biología Molecular/métodos , Proteínas Recombinantes/aislamiento & purificación , Apoptosis/genética , Caspasa 14/química , Caspasa 14/genética , Diferenciación Celular/genética , Línea Celular , Activación Enzimática , Humanos , Queratinocitos/enzimología , Proteínas Recombinantes/química , Proteínas Recombinantes/genéticaRESUMEN
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.
Asunto(s)
Calicreínas/metabolismo , Queratinocitos/citología , Proteínas Inhibidoras de Proteinasas Secretoras/metabolismo , Tripsina/metabolismo , Anticuerpos/química , Sitios de Unión , ADN Complementario/metabolismo , Epidermis/metabolismo , Biblioteca de Genes , Humanos , Estructura Terciaria de Proteína , Proteínas Recombinantes/metabolismo , Inhibidor de Serinpeptidasas Tipo Kazal-5RESUMEN
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.
Asunto(s)
Proteínas de Filamentos Intermediarios/metabolismo , Calicreínas/fisiología , Proteolisis , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Células Cultivadas , Proteínas Filagrina , Técnicas de Silenciamiento del Gen , Humanos , Proteínas de Filamentos Intermediarios/química , Proteínas de Filamentos Intermediarios/genética , Calicreínas/química , Queratinocitos/enzimología , Ratones , Ratones Endogámicos C57BL , Procesamiento Proteico-Postraduccional , Estructura Terciaria de Proteína , Transporte de Proteínas , ARN Interferente Pequeño/genética , Piel/citología , Piel/enzimologíaRESUMEN
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.