RESUMO
Vitronectin is an abundant multifunctional glycoprotein found in serum, the extracellular matrix, and bone, and is involved in diverse physiological processes. Here, we developed a new bioactive dimeric peptide (VnP-8-DN1 dimer) from a human vitronectin-derived motif (IDAAFTRINCQG; residues 206-217; VnP-8) via removal of an isoleucine residue at the N-terminus of VnP-8 and spontaneous air oxidation. The VnP-8-DN1 dimer potently enhanced cell attachment activity, and this activity was mediated by binding to cellular heparan sulfate proteoglycan receptors. Moreover, the VnP-8-DN1 dimer suppressed osteoclast differentiation by blocking the early stage of osteoclastogenesis induced by macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor-κB ligand (RANKL). Furthermore, the VnP-8-DN1 dimer decreased the bone-resorbing activity of osteoclasts and increased the survival of osteoclast precursor cells by decreasing the cellular level of c-Fms and reducing RANK expression. Taken together, these results demonstrate that the VnP-8-DN1 dimer inhibits the early stages of M-CSF- and RANK-induced osteoclast differentiation by binding to c-Fms and inhibiting M-CSF signaling.
Assuntos
Reabsorção Óssea , Fator Estimulador de Colônias de Macrófagos , Reabsorção Óssea/metabolismo , Diferenciação Celular , Humanos , Fator Estimulador de Colônias de Macrófagos/metabolismo , Fator Estimulador de Colônias de Macrófagos/farmacologia , Glicoproteínas de Membrana/metabolismo , Osteoclastos/metabolismo , Osteogênese , Ligante RANK/metabolismo , Ligante RANK/farmacologia , Receptor Ativador de Fator Nuclear kappa-B/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Vitronectina/metabolismo , Vitronectina/farmacologiaRESUMO
AIM: This study investigated whether a vitronectin-derived peptide (VnP-16) prevents and/or reverses alveolar bone resorption induced by ligature-induced periodontitis in rodents and identified the underlying mechanism. MATERIALS AND METHODS: We evaluated the effects of VnP-16 on osteogenic differentiation in human periodontal ligament cells (hPDLCs), lipopolysaccharide-induced inflammatory responses in gingival fibroblasts, and immune response in T lymphocytes. Ligature-induced periodontitis was induced by ligating the bilateral mandibular first molars for 14 days in rats and for 7 days in mice (n = 10/group). VnP-16 (100 µg/10 µl) was applied topically into the gingival sulcus of rats via intra-sulcular injection, whereas the peptide (50 µg/5 µl) was administered directly into the gingiva of mice via intra-gingival injection. To evaluate the preventive and therapeutic effects of VnP-16, micro-computed tomography analysis and histological staining were then performed. RESULTS: VnP-16 promoted osteogenic differentiation of periodontal ligament cells and inhibited the production of lipopolysaccharide-induced inflammatory mediators in gingival fibroblasts. Concomitantly, VnP-16 modulated the host immune response by reducing the number of receptor activator of NF-κB ligand (RANKL)-expressing lipopolysaccharide-stimulated CD4+ and CD8+ T cells, and by suppressing RANKL and interleukin (IL)-17A production. Furthermore, local administration of VnP-16 in rats and mice significantly prevented and reversed alveolar bone loss induced by ligature-induced periodontitis. VnP-16 enhanced osteoblastogenesis and simultaneously inhibited osteoclastogenesis and suppressed RANKL and IL-17A expression in vivo. CONCLUSIONS: Our findings suggest that VnP-16 acts as a potent therapeutic agent for preventing and treating periodontitis by regulating bone re-modelling and immune and inflammatory responses.
Assuntos
Perda do Osso Alveolar , Periodontite , Perda do Osso Alveolar/tratamento farmacológico , Perda do Osso Alveolar/prevenção & controle , Animais , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/patologia , Humanos , Interleucina-17/uso terapêutico , Ligantes , Lipopolissacarídeos/farmacologia , Camundongos , NF-kappa B , Osteogênese , Periodontite/tratamento farmacológico , Periodontite/metabolismo , Periodontite/prevenção & controle , Ligante RANK/metabolismo , Ratos , Vitronectina/uso terapêutico , Microtomografia por Raio-XRESUMO
Accumulating evidence indicates that mesenchymal stem/stromal cell-derived extracellular vesicles (MSC-EVs) exhibit immunomodulatory effects by delivering therapeutic RNAs and proteins; however, the molecular mechanism underlying the EV-mediated immunomodulation is not fully understood. In this study, we found that EVs from early-passage MSCs had better immunomodulatory potency than did EVs from late-passage MSCs in T cell receptor (TCR)- or Toll-like receptor 4 (TLR4)-stimulated splenocytes and in mice with ocular Sjögren's syndrome. Moreover, MSC-EVs were more effective when produced from 3D culture of the cells than from the conventional 2D culture. Comparative molecular profiling using proteomics and microRNA sequencing revealed the enriched factors in MSC-EVs that were functionally effective in immunomodulation. Among them, manipulation of transforming growth factor ß1 (TGF-ß1), pentraxin 3 (PTX3), let-7b-5p, or miR-21-5p levels in MSCs significantly affected the immunosuppressive effects of their EVs. Furthermore, there was a strong correlation between the expression levels of TGF-ß1, PTX3, let-7b-5p, or miR-21-5p in MSC-EVs and their suppressive function. Therefore, our comparative strategy identified TGF-ß1, PTX3, let-7b-5p, or miR-21-5p as key molecules mediating the therapeutic effects of MSC-EVs in autoimmune disease. These findings would help understand the molecular mechanism underlying EV-mediated immunomodulation and provide functional biomarkers of EVs for the development of robust EV-based therapies.
Assuntos
Proteína C-Reativa/genética , Vesículas Extracelulares/transplante , Células-Tronco Mesenquimais/citologia , MicroRNAs/genética , Componente Amiloide P Sérico/genética , Síndrome de Sjogren-Larsson/terapia , Fator de Crescimento Transformador beta1/genética , Animais , Proteína C-Reativa/metabolismo , Técnicas de Cultura de Células , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Modelos Animais de Doenças , Vesículas Extracelulares/genética , Vesículas Extracelulares/metabolismo , Perfilação da Expressão Gênica , Humanos , Células-Tronco Mesenquimais/metabolismo , Camundongos , Proteômica , Inoculações Seriadas , Componente Amiloide P Sérico/metabolismo , Síndrome de Sjogren-Larsson/genética , Síndrome de Sjogren-Larsson/metabolismo , Fator de Crescimento Transformador beta1/metabolismoRESUMO
Gö6976 is a nonglycosidic indolocarbazole compound widely used as a specific inhibitor of PKCα/ß. In experiments probing for a role of PKCα in human laminin-2-integrin-mediated cell adhesion and spreading of PC12 cells, we observed unexpected enhancements of adhesion, spreading and stress fiber formation to 1 µM Gö6976 with concomitant increase in membrane translocation of PKCδ and autophosphorylation of focal adhesion kinase (FAK). Importantly, enhanced cellular behavior and membrane translocation of PKCδ induced by Gö6976 was retained in siRNA-transfected PC12 cells to knockdown PKCα expression. Gö6976 also induced laminin-dependent cell adhesion in NIH/3T3 and CV-1 fibroblasts, suggesting of a mechanism that may be common to multiple cell-types. A specific inhibitor of PKCδ, rottlerin, completely abrogated Gö6976-dependent increase in PC12 cell adhesion to laminin as well as the activation of small GTPases, Rac1 and Cdc42, that are downstream of PKCδ in adhesion receptor signaling. siRNA knockdown of Rac1 and Cdc42 expression inhibited cell spreading and lamellipodia formation in PC12 cells. Overall, these results suggest that Gö6976 may stimulate membrane recruitment of PKCδ through a mechanism that is independent of PKCα/ß signaling. In addition, the activation of Rac1 and Cdc42 by human laminin-2-integrin-dependent activation of PKCδ/FAK signaling mediates cell spreading and lamellipodia formation in PC12 cells.
Assuntos
Carbazóis/farmacologia , Membrana Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Proteína Quinase C-delta/fisiologia , Animais , Adesão Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Membrana Celular/fisiologia , Células Cultivadas , Chlorocebus aethiops , Ativação Enzimática/efeitos dos fármacos , Células HEK293 , Humanos , Camundongos , Células NIH 3T3 , Células PC12 , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C beta , Proteína Quinase C-alfa/antagonistas & inibidores , Proteína Quinase C-delta/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Ratos , Regulação para Cima/efeitos dos fármacosRESUMO
BACKGROUND: Bone remodeling is tightly regulated through bone resorption and bone formation; imbalances in bone remodeling can cause various pathological conditions such as osteoporosis. Antiresorptive agents commonly used for treating osteoporosis do not substantially reverse osteoporotic bone loss. METHODS: We evaluated the effects of the RVYFFKGKQYWE motif (residues 270-281; VnP-16) of human vitronectin on the osteogenic differentiation of human mesenchymal stem cells (hMSCs) and osteoclastogenesis of bone marrow-derived macrophages. The effects of VnP-16 were also assessed in a mouse model of estrogen deficiency-induced osteoporosis (ovariectomized female C57BL/6 mice). To assay whether VnP-16 can reverse ovariectomy-induced bone loss, synthetic peptides or vehicle were subcutaneously injected into ovariectomized mice once a week for 4 weeks (n = 10/group). To evaluate the bone restorative effects of VnP-16, in-vivo micro-computed tomography analysis and histological staining were performed. RESULTS: VnP-16 induced osteogenic differentiation of hMSCs and inhibited the RANKL-RANK-TRAF6 axis in the osteoclastogenesis signaling pathway. Furthermore, systemic administration of VnP-16 reversed ovariectomy-induced bone loss in the femoral neck, distal femur and lumbar spine by increasing osteoblast differentiation and promoting bone formation, and concomitantly decreasing osteoclastogenesis and inhibiting bone resorption. The bone restorative effect of VnP-16 was observed one week after subcutaneous administration, and although the timing of the effect differed according to bone location, it persisted for at least 3 weeks. CONCLUSION: Our findings suggest that VnP-16 is a potential therapeutic agent for treating osteoporosis that mediates its effects through dual regulation of bone remodeling.
Assuntos
Reabsorção Óssea , Osteoporose , Feminino , Camundongos , Humanos , Animais , Vitronectina/metabolismo , Vitronectina/farmacologia , Osteogênese , Osteoclastos , Microtomografia por Raio-X , Camundongos Endogâmicos C57BL , Ovariectomia/efeitos adversos , Remodelação Óssea , Reabsorção Óssea/tratamento farmacológico , Reabsorção Óssea/complicações , Reabsorção Óssea/metabolismo , Osteoporose/tratamento farmacológico , Peptídeos/farmacologia , Peptídeos/metabolismoRESUMO
Recent research indicated that extracellular vesicles (EVs) derived from mesenchymal stem/stromal cells (MSCs) are a promising alternative to MSCs for immunomodulatory therapy. However, the contents of MSC-EVs would change as their parent MSCs change, hence the therapeutic efficacy of MSC-derived EVs (MSC-EVs) would largely depend on donors, tissue sources and culture conditions of MSCs. To overcome limitations of tissue-derived MSCs, we previously used MSCs derived from human induced pluripotent stem cells (iMSCs) to produce EVs and demonstrated their therapeutic potential in a mouse model of secondary Sjo¨gren's Syndrome. Here, we further found that EVs from early-passage iMSCs had better immunomodulatory potency than EVs from late-passage iMSCs in TLR4-stimulated splenocytes and in a mouse model of primary Sjögren's syndrome. Comparative molecular profiling using proteomics and microRNA sequencing revealed distinctive molecular profiles of iMSC-EVs with or without immunomodulation capacity. Amongst them, manipulation of TGF-ß1, miR-21 and miR-125b levels in iMSC-EVs significantly affected their immunosuppressive effects. These findings would help improve our understanding of the molecular mechanism underlying iMSC-EV-mediated immunomodulation and further provide strategies to improve regulatory function of EVs for the treatment of immune-mediated diseases.
RESUMO
Laminin-2 promotes basement membrane assembly and peripheral myelinogenesis; however, a receptor-binding motif within laminin-2 and the downstream signaling pathways for motif-mediated cell adhesion have not been fully established. The human laminin-2 alpha2 chain cDNAs cloned from human keratinocytes and fibroblasts correspond to the laminin alpha2 chain variant sequence from the human brain. Individually expressed recombinant large globular (LG) 1 protein promotes cell adhesion and has heparin binding activities. Studies with synthetic peptides delineate the DLTIDDSYWYRI motif (Ln2-P3) within the LG1 as a major site for both heparin and cell binding. Cell adhesion to LG1 and Ln2-P3 is inhibited by treatment of heparitinase I and chondroitinase ABC. Syndecan-1 from PC12 cells binds to LG1 and Ln2-P3 and colocalizes with both molecules. Suppression of syndecan-1 with RNA interference inhibits cell adhesion to LG1 and Ln2-P3. The binding of syndecan-1 with LG1 and Ln2-P3 induces the recruitment of protein kinase Cdelta (PKCdelta) into the membrane and stimulates its tyrosine phosphorylation. A decrease in PKCdelta activity significantly reduces cell adhesion to LG1 and Ln2-P3. Taken together, these results indicate that the Ln2-P3 motif and LG1 domain, containing the motif, within the human laminin-2 alpha2 chain are major ligands for syndecan-1, which mediates cell adhesion through the PKCdelta signaling pathway.
Assuntos
Adesão Celular , Membrana Celular/metabolismo , Laminina/metabolismo , Proteína Quinase C-delta/metabolismo , Sindecana-1/metabolismo , Sequência de Aminoácidos , Animais , Western Blotting , Células Cultivadas , Chlorocebus aethiops , Dicroísmo Circular , Células Epidérmicas , Epiderme/metabolismo , Fibroblastos/metabolismo , Citometria de Fluxo , Imunofluorescência , Humanos , Técnicas Imunoenzimáticas , Imunoprecipitação , Queratinócitos/metabolismo , Dados de Sequência Molecular , Células PC12 , Fosforilação , Estrutura Terciária de Proteína , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Sindecana-1/genéticaRESUMO
We previously reported that the PPFEGCIWN motif (Ln2-LG3-P2-DN3), residues 2678-2686 of the human laminin α2 chain, promotes cell attachment of normal human epidermal keratinocytes (NHEKs) and dermal fibroblasts (NHDFs); however, its in vivo effects on cutaneous wound healing have not yet been examined. In this study, we sought to determine whether Ln2-LG3-P2-DN3 could promote full-thickness cutaneous wound healing by accelerating wound reepithelialization and wound closure in vivo. Ln2-LG3-P2-DN3 had significantly higher cell attachment and spreading activities than vehicle or scrambled peptide control in both NHEKs and NHDFs in vitro. The wound area was significantly smaller in rats treated with Ln2-LG3-P2-DN3 than in those treated with vehicle or scrambled peptide in the early phase of wound healing. Furthermore, Ln2-LG3-P2-DN3 significantly accelerated wound reepithelialization relative to vehicle or scrambled peptide and promoted FAK-Tyr397 phosphorylation and Rac1 activation. Collectively, our findings suggest that the PPFEGCIWN motif has potential as a therapeutic agent for cutaneous regeneration via the acceleration of wound reepithelization and wound closure.
Assuntos
Quinase 1 de Adesão Focal/metabolismo , Guanosina Trifosfato/metabolismo , Laminina/química , Peptídeos , Cicatrização/efeitos dos fármacos , Ferimentos e Lesões , Proteínas rac1 de Ligação ao GTP/metabolismo , Motivos de Aminoácidos , Animais , Masculino , Peptídeos/química , Peptídeos/farmacologia , Ratos , Ratos Sprague-Dawley , Ferimentos e Lesões/metabolismo , Ferimentos e Lesões/terapiaRESUMO
Early implant loading is very important for reducing the duration of missing teeth in human patients. The laminin-derived peptide, DLTIDDSYWYRI motif (Ln2-P3), accelerates bone healing. Therefore, to investigate the hypothesis that Ln2-P3 increases the bone response to sandblasted, large-grit, acid-etched (SLA) titanium implants, the effect of the Ln2-P3 peptide on the osseointegration of SLA titanium implants was evaluated in vitro and in vivo. Human osteoblast-like cells were cultured on untreated, scrambled peptide (SP)-treated, and Ln2-P3-treated SLA titanium discs, and the cellular responses of these cells were evaluated. The Ln2-P3 treatment augmented osteoblast attachment and spreading, alkaline phosphatase activity, and the expression of osteogenic marker genes. Furthermore, the untreated and Ln2-P3-treated SLA titanium implants were inserted into the tibiae of rabbits for 9 and 11 days. Compared with the untreated implants, the Ln2-P3-treated implants showed a significantly higher bone-to-implant contact ratio at Day 9 after implantation and an increased bone area. The Ln2-P3 treatment of the SLA titanium implant surface augmented osteoblastic activity and accelerated peri-implant bone formation at the bone-implant interface. Overall, these results indicated that compared with the SLA titanium surface alone, the Ln2-P3 peptide-treated SLA titanium surface enhances initial osseointegration, thereby facilitating earlier implant loading.
Assuntos
Substitutos Ósseos/farmacologia , Laminina/farmacologia , Osseointegração/efeitos dos fármacos , Peptídeos/farmacologia , Titânio/farmacologia , Animais , Prótese Ancorada no Osso , Linhagem Celular , Feminino , Humanos , Osteoblastos/citologia , Osteogênese/efeitos dos fármacos , Coelhos , Tíbia/efeitos dos fármacos , Tíbia/lesões , Tíbia/fisiologia , Tíbia/cirurgiaRESUMO
PURPOSE: This study aimed to investigate the in vitro and in vivo bone-forming potential of a sandblasted, large-grit, acid-etched (SLA) titanium (Ti) surface treated with a laminin-derived functional peptide, PPFEGCIWN (DN3). MATERIALS AND METHODS: Human osteoblast-like MG63 cells were cultured with SLA Ti discs untreated or treated with DN3 or a control scrambled peptide (SP). Cell adhesion, spreading, and viability on the discs were tested. Alkaline phosphatase gene expression and enzyme activity were also evaluated. Four DN3-coated SLA Ti implants and four untreated implants were placed into the tibiae of two rabbits (two implants/tibia). Ten days later, the bone-implant interfaces were subjected to histomorphometry to measure the bone response. The surface properties of the discs and implants were determined using scanning electron, widefield confocal, and confocal laser microscopy and x-ray photoelectron spectroscopy. RESULTS: The peptide-treated and untreated discs and implants were similar in terms of physical surface properties, but the peptide-treated surfaces had significantly higher nitrogen levels (P < .05). The DN3 peptide promoted cell adhesion, spreading, and alkaline phosphatase expression and enzyme activity (P < .05). Histomorphometry of the harvested implants showed rapid bone formation and affinity of the motif. CONCLUSION: This study suggests that treatment with the cell adhesion peptide DN3 promotes bone healing at the SLA Ti surface.
Assuntos
Implantes Dentários , Titânio , Animais , Linhagem Celular , Humanos , Laminina , Microscopia Eletrônica de Varredura , Osteoblastos , Osteogênese , Peptídeos , Coelhos , Propriedades de SuperfícieRESUMO
In this study, we evaluated early bone responses to a vitronectin-derived, minimal core bioactive peptide, RVYFFKGKQYWE motif (VnP-16), both in vitro and in vivo, when the peptide was treated on sandblasted, large-grit, acid-etched (SLA) titanium surfaces. Four surface types of titanium discs and of titanium screw-shaped implants were prepared: control, SLA, scrambled peptide-treated, and VnP-16-treated surfaces. Cellular responses, such as attachment, spreading, migration, and viability of human osteoblast-like HOS and MG63 cells were evaluated in vitro on the titanium discs. Using the rabbit tibia model with the split plot design, the implants were inserted into the tibiae of four New Zealand white rabbits. After two weeks of implant insertion, the rabbits were sacrificed, the undecalcified specimens were prepared for light microscopy, and the histomorphometric data were measured. Analysis of variance tests were used for the quantitative evaluations in this study. VnP-16 was non-cytotoxic and promoted attachment and spreading of the human osteoblast-like cells. The VnP-16-treated SLA implants showed no antigenic activities at the interfaces between the bones and the implants and indicated excellent bone-to-implant contact ratios, the means of which were significantly higher than those in the SP-treated implants. VnP-16 reinforces the osteogenic potential of the SLA titanium dental implant.
RESUMO
Osteoporosis affects millions of people worldwide by promoting bone resorption and impairing bone formation. Bisphosphonates, commonly used agents to treat osteoporosis, cannot reverse the substantial bone loss that has already occurred by the time of diagnosis. Moreover, their undesirable side-effects, including osteonecrosis of the jaw, have been reported. Here, we demonstrated that a new bioactive core vitronectin-derived peptide (VnP-16) promoted bone formation by accelerating osteoblast differentiation and activity through direct interaction with ß1 integrin followed by FAK activation. Concomitantly, VnP-16 inhibited bone resorption by restraining JNK-c-Fos-NFATc1-induced osteoclast differentiation and αvß3 integrin-c-Src-PYK2-mediated resorptive function. Moreover, VnP-16 decreased the bone resorbing activity of pre-existing mature osteoclasts without changing their survival rate. Furthermore, VnP-16 had a strong anabolic effect on bone regeneration by stimulating osteoblast differentiation and increasing osteoblast number, and significantly alleviated proinflammatory cytokine-induced bone resorption by restraining osteoclast differentiation and function in murine models. Moreover, VnP-16 could reverse ovariectomy-induced bone loss by both inhibiting bone resorption and promoting bone formation. Given its dual role in promoting bone formation and inhibiting bone resorption, our results suggest that VnP-16 could be an attractive therapeutic agent for treating osteoporosis.
Assuntos
Reabsorção Óssea/tratamento farmacológico , Diferenciação Celular/efeitos dos fármacos , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Ovariectomia , Peptídeos/farmacologia , Vitronectina/química , Animais , Regeneração Óssea/efeitos dos fármacos , Reabsorção Óssea/metabolismo , Linhagem Celular , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Osteoblastos/metabolismo , Peptídeos/químicaRESUMO
Localized acidification of the osteoclast-bone interface is driven by a vacuolar-type H+-ATPase (V-ATPase) in the plasma membrane in a process thought to be associated with bone resorption. The present study investigated the mechanism underlying the roles of V-ATPase-induced acidosis in osteoclastogenesis. Active proton pumping due to increased V-ATPase activity during RANKL-induced osteoclastogenesis induced intracellular and extracellular acidification of osteoclast precursors. Subsequent analysis revealed blockage of extracellular acidification and induction of intracellular acidification by bafilomycin A1, a specific inhibitor of V-ATPase, indicating that extracellular acidification is mostly induced by V-ATPase-mediated proton pumping into extracellular space. Low-pH media controlled by HEPES-buffered conditions to mimic metabolic acidosis led to synergistic activation of RANKL-stimulated signals, including mitogen-activated protein kinases and transcription factor NF-kappaB, resulting in enhanced osteoclastogenesis. Low-pH media also upregulated the expression of osteopontin secreted into extracellular space, which is required for cell migration by binding to cell surface integrin alphavbeta3. Osteoclast precursor migration was significantly inhibited by treatment of antibodies to integrin alphavbeta3, resulting in the retardation of osteoclastogenesis. Taken together, these findings indicate that V-ATPase-driven acidosis modulates osteoclastogenesis.
Assuntos
Acidose/enzimologia , Diferenciação Celular , Movimento Celular , Osteoclastos/citologia , Osteogênese , ATPases Vacuolares Próton-Translocadoras/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Concentração de Íons de Hidrogênio , Integrina alfaVbeta3/metabolismo , Macrolídeos/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Osteoclastos/efeitos dos fármacos , Osteoclastos/enzimologia , Osteogênese/efeitos dos fármacos , Osteopontina/genética , Osteopontina/metabolismo , Ligante RANK/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais/efeitos dos fármacos , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Células-Tronco/enzimologiaRESUMO
Numerous studies implicate miR-146a as pleiotropic regulator of carcinogenesis; however, its roles in carcinogenesis are not fully understood. A clue from expression analyses of miR-146a-5p in all 13 oral squamous cell carcinoma (OSCC) cell lines examined and in OSCC tissues, whole blood and whole saliva of OSCC patients in vivo revealed that miR146a-5p expression was highly upregulated. Particularly, we widened the view of its upregulation in saliva, implicating that high miR-146a-5p expression is not only correlated closely to the development of human oral cancer, but also to a possible candidate as a diagnostic marker of OSCC. Indeed, further examination showed that exogenous miR-146a-5p expression showed pleiotropic effects on cell proliferation and apoptosis which were partially based on the contextual responses of activation of JNK, downstream of TRAF6 that was targeted by miR-146a-5p in normal human keratinocytes and OSCC cell lines. TRAF6 suppression by a TRAF6-specific siRNA resulted in contradictory consequences on cellular processes in normal and OSCC cells. Notably, TRAF6 downregulation by both miR-146a-5p and TRAF6-specific siRNA deactivated JNK in SCC-9, but not in normal human keratinocytes. In support of the proliferation-promoting effect of miR-146a-5p, silencing of endogenous miR-146a-5p significantly reduced proliferation of SCC-9. Together, these results suggest that miR-146a-5p affects proliferation and apoptosis in a cellular context-dependent manner and selectively disarms the TRAF6-mediated branch of the TGF-ß signaling in OSCC cell lines by sparing Smad4 involvement.
Assuntos
Carcinoma de Células Escamosas/genética , MicroRNAs/genética , Neoplasias Bucais/genética , Fator 6 Associado a Receptor de TNF/genética , Apoptose/genética , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Proliferação de Células/genética , Regulação Neoplásica da Expressão Gênica , Variação Genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Neoplasias Bucais/patologia , RNA Interferente Pequeno , Proteína Smad4/genética , Fator de Crescimento Transformador beta/genéticaRESUMO
Electrospinning of chitin/silk fibroin (SF) blend solutions in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) was investigated to fabricate a biomimetic nanostructured scaffolds for tissue engineering. The morphology of the electrospun chitin/SF blend nanofibers was investigated with a field emission scanning electron microscope (FE-SEM). The average diameters of chitin/SF blend fibers decreased from 920 to 340nm, with the increase of chitin content in blend compositions. The miscibility of chitin/SF blend fibers was examined by solution viscosity measurement. The chitin and SF were immiscible in the as-spun nanofibrous structure. The dimensional stability of chitin/SF blend nanofibers, with or without water vapor after-treatment, was conducted by immersing in water. As-spun SF-rich blend nanofibrous matrices were lost their fibrous structure after the water immersion for 24h, and then changed into membrane-like structure. On the contrary, nanofibrous structures of water vapor-treated SF-rich blends were almost maintained. To assay the cytocompatibility and cell behavior on the chitin/SF blend nanofibrous scaffolds, cell attachment and spreading of normal human epidermal keratinocyte and fibroblasts seeded on the scaffolds were studied. Our results indicate that chitin/SF blend nanofibrous matrix, particularly the one that contained 75% chitin and 25% SF, could be a potential candidate for tissue engineering scaffolds because it has both biomimetic three-dimensional structure and an excellent cell attachment and spreading for NHEK and NHEF.
Assuntos
Quitina/química , Fibroínas/química , Seda/química , Adolescente , Biomimética , Membrana Celular/metabolismo , Criança , Pré-Escolar , Fibroblastos/metabolismo , Humanos , Queratinócitos/metabolismo , Substâncias Macromoleculares/química , Masculino , Microscopia Eletrônica de Varredura/métodos , Nanoestruturas/química , Água/metabolismoRESUMO
Finding bioactive short peptides derived from proteins is a critical step to the advancement of tissue engineering and regenerative medicine, because the former maintains the functions of the latter without immunogenicity in biological systems. Here, we discovered a bioactive core nonapeptide sequence, PPFEGCIWN (residues 2678-2686; Ln2-LG3-P2-DN3), from the human laminin α2 chain, and investigated the role of this peptide in binding to transmembrane proteins to promote intracellular events leading to cell functions. This minimum bioactive sequence had neither secondary nor tertiary structures in a computational structure prediction. Nonetheless, Ln2-LG3-P2-DN3 bound to various cell types as actively as laminin in cell adhesion assays. The in vivo healing tests using rats revealed that Ln2-LG3-P2-DN3 promoted bone formation without any recognizable antigenic activity. Ln2-LG3-P2-DN3-treated titanium (Ti) discs and Ti implant surfaces caused the enhancement of bone cell functions in vitro and induced faster osseointegration in vivo, respectively. These findings established a minimum bioactive sequence within human laminin, and its potential application value for regenerative medicine, especially for bone tissue engineering.
Assuntos
Laminina/química , Engenharia Tecidual/métodos , Fosfatase Alcalina/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Osso e Ossos/patologia , Adesão Celular , Diferenciação Celular , Movimento Celular , Sobrevivência Celular , Células Cultivadas , Humanos , Masculino , Microscopia Eletrônica de Varredura , Microscopia de Fluorescência , Dados de Sequência Molecular , Nanopartículas/química , Osseointegração , Osteoblastos/metabolismo , Osteogênese , Células PC12 , Peptídeos/química , Coelhos , Ratos , Ratos Sprague-Dawley , Regeneração , Titânio/química , Microtomografia por Raio-XRESUMO
Laminin-derived peptide coatings can enhance epithelial cell adhesion to implants, and the positive effect of these peptides on bone cell adhesion has been anticipated. The purpose of this study was to evaluate the improvement in bone cell attachment to and activity on titanium (Ti) scaffolds coated with a laminin-derived functional peptide, Ln2-P3 (the DLTIDDSYWYRI motif). Four Ti disc surfaces were prepared, and a human osteosarcoma (HOS) cell attachment test was performed to select two candidate surfaces for peptide coating. These two candidates were then coated with Ln2-P3 peptide, a scrambled peptide, or left uncoated to measure cell attachment to each surface, following which one surface was chosen to assess alkaline phosphatase (ALP) activity and osteogenic marker gene expression with quantitative real-time PCR. On the commercially pure Ti surface, the Ln2-P3 coating significantly increased cellular ALP activity and the expression levels of ALP and bone sialoprotein mRNA as compared with the scrambled peptide-coated and uncoated surfaces. In conclusion, although further in vivo studies are needed, the findings of this in vitro study indicate that the Ln2-P3-coated implant surface promotes bone cell adhesion, which has clinical implications for reducing the overall treatment time of dental implant therapy.
Assuntos
Adesão Celular/efeitos dos fármacos , Implantes Dentários , Osteogênese , Diferenciação Celular , Linhagem Celular Tumoral , Materiais Revestidos Biocompatíveis/administração & dosagem , Materiais Revestidos Biocompatíveis/química , Humanos , Laminina/química , Osteossarcoma/metabolismo , Peptídeos/administração & dosagem , Peptídeos/química , Propriedades de Superfície , Titânio/administração & dosagem , Titânio/químicaRESUMO
Considerable effort has been directed towards replacing lost teeth using tissue-engineering methods such as titanium implants. A number of studies have tried to modify bioinert titanium surfaces by coating them with functionally bioactive molecules for faster and stronger osseointegration than pure titanium surfaces. Recently, peptides have been recognized as valuable scientific tools in the field of tissue-engineering. The DLTIDDSYWYRI motif of the human laminin-2 α2 chain has been previously reported to promote the attachment of various cell types; however, the in vivo effects of the DLTIDDSYWYRI motif on new bone formation have not yet been studied. To examine whether a laminin-2-derived peptide can promote osseointegration by accelerating new bone formation in vivo, we applied titanium implants coated with the DLTIDDSYWYRI motif in a rabbit tibia model. The application of the DLTIDDSYWYRI motif-treated implant to tibia wounds enhanced collagen deposition and alkaline phosphatase expression. It significantly promoted implant osseointegration compared with treatment with scrambled peptide-treated implants by increasing the bone-to-implant contact ratio and bone area. These findings support the hypothesis that the DLTIDDSYWYRI motif acts as an effective osseointegration accelerator by enhancing new bone formation.
Assuntos
Implantes Experimentais , Laminina/química , Osseointegração/efeitos dos fármacos , Peptídeos/farmacologia , Fosfatase Alcalina/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Biomarcadores/metabolismo , Adesão Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Colágeno/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Dados de Sequência Molecular , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Osteoblastos/enzimologia , Osteoblastos/ultraestrutura , Osteogênese/efeitos dos fármacos , Osteogênese/genética , Células PC12 , Peptídeos/química , Coelhos , Ratos , Propriedades de Superfície/efeitos dos fármacos , Titânio/farmacologiaRESUMO
Laminin α2 chain plays an important role in basement membrane assembly and peripheral myelinogenesis; however, the integrin binding motif within human laminin α2 chain and the signaling pathways downstream of this ligand-receptor interaction are poorly understood. We identified a motif, RNIPPFEGCIWN (Ln2-LG3-P2), within LG3 domain of human laminin α2 chain as a major site for both α3ß1 integrin and cellular activities such as cell adhesion, spreading, and migration. Binding of α3ß1 integrin with Ln2-LG3-P2 induced the membrane recruitment of protein kinase Cδ (PKCδ) and stimulated its tyrosine phosphorylation. The cellular activities induced by Ln2-LG3-P2 and the phosphorylation of focal adhesion kinase (FAK) were inhibited by rottlerin, a PKCδ inhibitor, but not by Gö6976, a PKCα/ß inhibitor. These results indicate that RNIPPFEGCIWN motif within human laminin α2 chain is a major ligand for α3ß1 integrin, and that binding of α3ß1 integrin mediates cellular activities through membrane recruitment and tyrosine phosphorylation of PKCδ and FAK phosphorylation.