RESUMO
BACKGROUND: Integrin-Binding Sialoprotein (IBSP) has been implicated in tumor progression across various cancers. However, the specific role of IBSP in breast cancer remains underexplored. There is a need to investigate the mechanisms by which IBSP influences breast cancer progression and its potential as a therapeutic target. AIMS: This study aims to elucidate the role of IBSP in breast cancer, particularly its impact on tumor progression and its relationship with prognosis. We also seek to understand the underlying mechanisms, including the involvement of the BMP-SMAD signaling pathway, and to explore the potential of targeting IBSP for therapeutic interventions. METHODS AND RESULTS: Overexpression of IBSP in breast cancer cells led to increased migration and invasion, whereas IBSP interference reduced these behaviors, indicating its role in enhancing tumor progression. Differentially expressed genes were significantly enriched in the BMP-SMAD signaling pathway, a critical pathway for osteogenic differentiation. Transcription Factor Binding: Dual luciferase reporter assays demonstrated that SMAD4 specifically binds to the IBSP promoter, establishing a regulatory link between SMAD4 and IBSP expression. Silencing IBSP (si-IBSP) mitigated the effects of SMAD4-induced tumor proliferation, confirming that IBSP acts as a downstream target of SMAD4 in the BMP signaling pathway. CONCLUSION: Our study reveals that IBSP plays a significant role in breast cancer progression through the BMP-SMAD4 signaling pathway. Targeting IBSP could be a promising therapeutic strategy for breast cancer treatment. Further research into IBSP inhibitors may offer new avenues for improving treatment outcomes and managing breast cancer more effectively.
Assuntos
Neoplasias Ósseas , Neoplasias da Mama , Proliferação de Células , Sialoproteína de Ligação à Integrina , Transdução de Sinais , Proteína Smad4 , Humanos , Neoplasias da Mama/patologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/genética , Feminino , Neoplasias Ósseas/secundário , Neoplasias Ósseas/patologia , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/genética , Proteína Smad4/metabolismo , Proteína Smad4/genética , Sialoproteína de Ligação à Integrina/metabolismo , Sialoproteína de Ligação à Integrina/genética , Camundongos , Animais , Proteínas Morfogenéticas Ósseas/metabolismo , Regulação Neoplásica da Expressão Gênica , Movimento Celular , Linhagem Celular Tumoral , Prognóstico , Camundongos NusRESUMO
Osteopontin (OPN) and Bone Sialoprotein (BSP), abundantly expressed by osteoblasts and osteoclasts, appear to have important, partly overlapping functions in bone. In gene-knockout (KO, -/-) models of either protein and their double (D)KO in the same CD1/129sv genetic background, we analyzed the morphology, matrix characteristics, and biomechanical properties of femur bone in 2 and 4 month old, male and female mice. OPN-/- mice display inconsistent, perhaps localized hypermineralization, while the BSP-/- are hypomineralized throughout ages and sexes, and the low mineralization of young DKO mice recovers with age. The higher contribution of primary bone remnants in OPN-/- shafts suggests a slow turnover, while their lower percentage in BSP-/- indicates rapid remodeling, despite FTIR-based evidence in this genotype of a high maturity of the mineralized matrix. In 3-point bending assays, OPN-/- bones consistently display higher Maximal Load, Work to Max. Load and in young mice Ultimate Stress, an intrinsic characteristic of the matrix. Young male and old female BSP-/- also display high Work to Max. Load along with low Ultimate Stress. Principal Component Analysis confirms the major role of morphological traits in mechanical competence, and evidences a grouping of the WT phenotype with the OPN-/- and of BSP-/- with DKO, driven by both structural and matrix parameters, suggesting that the presence or absence of BSP has the most profound effects on skeletal properties. Single or double gene KO of OPN and BSP thus have multiple distinct effects on skeletal phenotypes, confirming their importance in bone biology and their interplay in its regulation.
Assuntos
Sialoproteína de Ligação à Integrina , Camundongos Knockout , Osteopontina , Animais , Osteopontina/genética , Osteopontina/metabolismo , Feminino , Masculino , Camundongos , Sialoproteína de Ligação à Integrina/genética , Sialoproteína de Ligação à Integrina/metabolismo , Fenômenos Biomecânicos , Osso e Ossos/metabolismo , Densidade Óssea/fisiologia , Densidade Óssea/genética , Fêmur/metabolismo , Calcificação Fisiológica/fisiologia , Calcificação Fisiológica/genéticaRESUMO
Osteosarcoma (OS) is a malignant bone tumor that commonly affects young individuals. Circular RNAs (circRNAs) are associated with OS progression. In this study, we aimed to determine the role of hsa_circ_0001480 (circ_0001480) in OS development. OS cell invasion, viability, and colony numbers were assessed via transwell, cell counting kit-8, and colony formation assays, respectively. Tumor growth in vivo was also assessed using an OS mouse model. Additionally, targeted associations among the integrin-binding sialoprotein (IBSP), microRNA (miR)-363-3p, and circ_0001480 were evaluated via RNA immunoprecipitation and dual luciferase reporter assays, whereas their expression levels in OS cells and tissues were determined via quantitative reverse transcription-polymerase chain reaction and western blotting. Loss of circ_0001480 or IBSP significantly inhibited the proliferation and invasion of OS cells, but this effect was reversed by miR-363-3p downregulation. Moreover, circ_0001480 knockdown inhibited neoplasm growth in vivo. circ_0001480 directly bound to miR-363-3p, which further modulated IBSP. Both circ_0001480 and IBSP levels were high, whereas miR-363-3p levels were low in OS cells. Furthermore, low miR-363-3p levels attenuated the suppressive effects of circ_0001480 silencing on the proliferation and invasion of OS cells; however, loss of IBSP partially reversed these effects. Overall, our findings revealed circ_0001480 an oncogenic circRNA stimulating OS progression by modulating the miR-363-3p/IBSP pathway, suggesting its potential for OS treatment.
Assuntos
Sialoproteína de Ligação à Integrina , MicroRNAs , Osteossarcoma , RNA Circular , Osteossarcoma/patologia , Osteossarcoma/genética , Osteossarcoma/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Humanos , RNA Circular/genética , RNA Circular/metabolismo , Animais , Camundongos , Sialoproteína de Ligação à Integrina/metabolismo , Sialoproteína de Ligação à Integrina/genética , Proliferação de Células , Neoplasias Ósseas/patologia , Neoplasias Ósseas/genética , Neoplasias Ósseas/metabolismo , Progressão da Doença , Linhagem Celular Tumoral , Camundongos Nus , Camundongos Endogâmicos BALB CRESUMO
Bone sialoprotein (BSP) is a multifunctional extracellular matrix (ECM) protein present in bone and cementum. Global in vivo ablation of BSP leads to bone mineralization defects, lack of acellular cementum, and periodontal breakdown. BSP harbors three main functional domains: N-terminal collagen-binding domain, hydroxyapatite-nucleating domain, and C-terminal RGD integrin-binding signaling domain. How each of these domains contributes to BSP function(s) is not understood. We hypothesized that collagen-binding and RGD domains play distinct roles in cementoblast functions. Three CRISPR/Cas9 gene-edited cell lines were derived from control wild-type (WT) OCCM.30 murine immortalized cementoblasts: 1) deletion of the N-terminus of BSP after signal peptide, including entire collagen binding domain (Ibsp∆N-Term); 2) deletion of exon 4 (majority of collagen-binding domain; Ibsp∆Ex4); and 3) deletion of C-terminus of BSP including the integrin binding RGD domain (Ibsp∆C-Term). Compared to WT, Ibsp∆Ex4 and Ibsp∆C-Term cell lines showed reduced BSP secretion, in vitro. Abnormal cell morphology was observed in all mutant cell lines, with Ibsp∆C-Term showing highly disorganized cytoskeleton. All mutant cell lines showed significantly lower cell proliferation compared to WT at all timepoints. Ibsp∆N-Term cells showed reduced cell migration by 24 h. All mutants exhibited over 50 % significant reduced mineralization at days 6 and 10. While WT cells were largely unaffected by seeding density, mutant cells failed to mineralize at lower cell density. Mutant cell lines diverged from WT and from each other by dysregulated expression in 23 genes involved in mineralization, ECM, and cell signaling. In summary, disabling BSP functional domains led to profound and distinct changes in cementoblast cell functions, especially dysregulated gene expression and reduced mineralization, in a way did not align with a straightforward narrative where each functional domain caused specific, expected differences. Instead, the study uncovered a significant level of complexity in how different mutant forms of BSP affected cell functions, in vitro.
Assuntos
Cemento Dentário , Proteínas da Matriz Extracelular , Camundongos , Animais , Sialoproteína de Ligação à Integrina/genética , Sialoproteína de Ligação à Integrina/metabolismo , Cemento Dentário/metabolismo , Colágeno , Integrinas , OligopeptídeosRESUMO
BACKGROUND: The growing use of zirconia as a ceramic material in dentistry is attributed to its biocompatibility, mechanical properties, esthetic appearance, and reduced bacterial adhesion. These favorable properties make ceramic materials a viable alternative to commonly used titanium alloys. Mimicking the physiological properties of blood flow, particularly the mechanosignaling in endothelial cells (ECs), is crucial for enhancing our understanding of their role in the response to zirconia exposure. METHODS: In this study, EC cultures were subjected to shear stress while being exposed to zirconia for up to 3 days. The conditioned medium obtained from these cultures was then used to expose osteoblasts for a duration of 7 days. To investigate the effects of zirconia on osteoblasts, we examined the expression of genes associated with osteoblast differentiation, including Runx2, Osterix, bone sialoprotein, and osteocalcin genes. Additionally, we assessed the impact of mechanosignaling-related angiocrine factors on extracellular matrix (ECM) remodeling by measuring the activities of matrix metalloproteinases 2 and 9 (MMP2 and MMP9) during the acquisition of the osteogenic phenotype, which precedes mineralization. RESULTS: Our data revealed that mechanosignaling-related angiocrine factors play a crucial role in promoting an osteoblastic phenotype in response to zirconia exposure. Specifically, exposed osteoblasts exhibited significantly higher expression levels of genes associated with osteoblast differentiation, such as Runx2, Osterix, bone sialoprotein, and osteocalcin genes. Furthermore, the activities of MMP2 and MMP9, which are involved in ECM remodeling, were modulated by mechanosignaling-related angiocrine factors. This modulation is likely an initial event preceding the mineralization phase. CONCLUSION: Based on our findings, we propose that mechanosignaling drives the release of angiocrine factors capable of modulating the osteogenic phenotype at the biointerface with zirconia. This process creates a microenvironment that promotes wound healing and osseointegration. Moreover, these results highlight the importance of considering the mechanosignaling of endothelial cells in the modulation of bone healing and osseointegration in the context of blood vessel effects. Our data provide new insights and open avenues for further investigation into the influence of mechanosignaling on bone healing and the osseointegration of dental devices.
Assuntos
Subunidade alfa 1 de Fator de Ligação ao Core , Células Endoteliais , Osteocalcina/genética , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/farmacologia , Sialoproteína de Ligação à Integrina/genética , Sialoproteína de Ligação à Integrina/metabolismo , Sialoproteína de Ligação à Integrina/farmacologia , Células Endoteliais/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Fenótipo , Diferenciação Celular , Osteoblastos/metabolismo , Titânio/farmacologia , Propriedades de SuperfícieRESUMO
One of the major components in cementum extracellular matrix is bone sialoprotein (BSP). BSP knockout (Ibsp) mice were reported to have a nonfunctional hypo-mineralized cementum, as well as detachment and disorganization of the periodontal ligament tissue. However, studies investigating the influence of Ibsp in cementoblasts are missing yet. This study investigates the influences of Bsp in three cementoblasts cell lines (OCCM.30-WT,IbspΔNterm, and IbspKAE). The mRNA expression of cementoblast and osteoclast markers (Col1a1, Alpl, Ocn, Runx2, Ctsk, Rankl and Opg) and the cell morphology were compared. Additionally, a functional monocyte adhesion assay was performed. To understand the influence of external stimuli, the effect of Ibsp was investigated under static compressive force, mimicking the compression side of orthodontic tooth movement. Cementoblasts with genotype IbspΔNterm and IbspKAE showed slight differences in cell morphology compared to OCCM.30-WT, as well as different gene expression. Under compressive force, the Ibsp cell lines presented expression pattern markers similar to the OCCM.30-WT cell line. However, Cathepsin K was strongly upregulated in IbspΔNterm cementoblasts under compressive force. This study provides insight into the role of BSP in cementoblasts and explores the influence of BSP on periodontal ligament tissues. BSP markers in cementoblasts seem to be involved in the regulation of cementum organization as an important factor for a functional periodontium. In summary, our findings provide a basis for investigations regarding molecular biology interactions of BSP in cementoblasts, and a supporting input for understanding the periodontal and cellular cementum remodeling.
Assuntos
Cemento Dentário , Camundongos , Animais , Sialoproteína de Ligação à Integrina/genética , Sialoproteína de Ligação à Integrina/metabolismo , Cemento Dentário/metabolismo , Camundongos Knockout , Linhagem Celular , Expressão GênicaRESUMO
Bone metastases during lung cancer are common. Bone sialoprotein (BSP), a non-collagenous bone matrix protein, plays important functions in bone mineralization processes and in integrin-mediated cell-matrix interactions. Importantly, BSP induces bone metastasis in lung cancer, but the underlying mechanisms remain unclear. This study therefore sought to determine the intracellular signaling pathways responsible for BSP-induced migration and invasion of lung cancer cells to bone. Analyses of the Kaplan-Meier, TCGA, GEPIA and GENT2 databases revealed that high levels of BSP expression in lung tissue samples were associated with significantly decreased overall survival (hazard ratio = 1.17; p = 0.014) and with a more advanced clinical disease stage (F-value = 2.38, p < 0.05). We also observed that BSP-induced stimulation of matrix metalloproteinase (MMP)-14 promoted lung cancer cell migration and invasion via the PI3K/AKT/AP-1 signaling pathway. Notably, BSP promoted osteoclastogenesis in RAW 264.7 cells exposed to RANKL and BSP neutralizing antibody reduced osteoclast formation in conditioned medium (CM) from lung cancer cell lines. Finally, at 8 weeks after mice were injected with A549 cells or A549 BSP shRNA cells, the findings revealed that the knockdown of BSP expression significantly reduced metastasis to bone. These findings suggest that BSP signaling promotes lung bone metastasis via its direct downstream target gene MMP14, which reveals a novel potential therapeutic target for lung cancer bone metastases.
Assuntos
Neoplasias Ósseas , Neoplasias Pulmonares , Camundongos , Animais , Sialoproteína de Ligação à Integrina/genética , Sialoproteína de Ligação à Integrina/metabolismo , Sialoglicoproteínas/genética , Sialoglicoproteínas/metabolismo , Metaloproteinase 14 da Matriz , Fosfatidilinositol 3-Quinases , Linhagem Celular Tumoral , Neoplasias Ósseas/metabolismoRESUMO
Bone sialoprotein (BSP) is an extracellular matrix (ECM) protein associated with mineralized tissues, particularly bone and cementum. BSP includes functional domains implicated in collagen binding, hydroxyapatite nucleation, and cell signaling, although its function(s) in osteoblast and osteoclast differentiation and function remain incompletely understood. Genetic ablation of BSP in Ibsp knockout (Ibsp-/-) mice results in developmental bone mineralization and remodeling defects, with alveolar bone more severely affected than the femurs and tibias of the postcranial skeleton. The role of BSP in alveolar bone healing has not been studied. We hypothesized that BSP ablation would cause defective alveolar bone healing. We employed a maxillary first molar extraction socket healing model in 42-d postnatalIbsp-/- and wild-type (WT) control mice. Tissues were collected at 0, 7, 14, 21, and 56 d postprocedure (dpp) for analysis by micro-computed tomography (microCT), histology, in situ hybridization (ISH), immunohistochemistry (IHC), and quantitative polymerase chain reaction (qPCR) array. As expected, alveolar bone healing progressed in WT mice with increasing bone volume fraction (BV/TV), bone mineral density (BMD), and tissue mineral density (TMD), transitioning from woven to mature bone from 7 to 56 dpp. Ibsp messenger RNA (mRNA) and BSP protein were strongly expressed during alveolar bone healing in parallel with other osteogenic markers. Compared to WT, Ibsp-/- mice exhibited 50% to 70% reduced BV/TV and BMD at all time points, 7% reduced TMD at 21 dpp, abnormally increased Col1a1 and Alpl mRNA expression, and persistent presence of woven bone and increased bone marrow in healing sockets. qPCR revealed substantially dysregulated gene expression in alveolar bone of Ibsp-/- versus WT mice, with significantly disrupted expression of 45% of tested genes in functional groups, including markers for osteoblasts, osteoclasts, mineralization, ECM, cell signaling, and inflammation. We conclude that BSP is a critical and nonredundant factor for alveolar bone healing, and its absence disrupts multiple major pathways involved in appropriate healing.
Assuntos
Cemento Dentário , Osteopontina , Animais , Camundongos , Sialoproteína de Ligação à Integrina/genética , Osteopontina/metabolismo , Microtomografia por Raio-X , Cemento Dentário/metabolismo , RNA Mensageiro , Sialoglicoproteínas/metabolismoRESUMO
Bone homeostasis is the equilibrium between organic and inorganic components of the extracellular matrix (ECM) and cells. Alteration of this balance has consequences on bone mass and architecture, resulting in conditions such as osteoporosis (OP). Given ECM protein mutual regulation and their effects on bone structure and mineralization, further insight into their expression is crucial to understanding bone biology under normal and pathological conditions. This study focused on Type I Collagen, which is mainly responsible for structural properties and mineralization of bone, and selected proteins implicated in matrix composition, mineral deposition, and cell-matrix interaction such as Decorin, Osteocalcin, Osteopontin, Bone Sialoprotein 2, Osteonectin and Transforming Growth Factor beta. We developed a novel multidisciplinary approach in order to assess bone matrix in healthy and OP conditions more comprehensively by exploiting the Fourier Transform Infrared Imaging (FTIRI) technique combined with histomorphometry, Sirius Red staining, immunohistochemistry, and Western Blotting. This innovatory procedure allowed for the analysis of superimposed tissue sections and revealed that the alterations in OP bone tissue architecture were associated with warped Type I Collagen structure and deposition but not with changes in the total protein amount. The detected changes in the expression and/or cooperative or antagonist role of Decorin, Osteocalcin, Osteopontin, and Bone Sialoprotein-2 indicate the deep impact of these NCPs on collagen features of OP bone. Overall, our strategy may represent a starting point for designing targeted clinical strategies aimed at bone mass preservation and sustain the FTIRI translational capability as upcoming support for traditional diagnostic methods.
Assuntos
Osteopontina , Osteoporose , Colágeno , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Decorina/metabolismo , Cabeça do Fêmur/química , Cabeça do Fêmur/metabolismo , Cabeça do Fêmur/patologia , Análise de Fourier , Humanos , Sialoproteína de Ligação à Integrina/genética , Sialoproteína de Ligação à Integrina/metabolismo , Osteocalcina/análise , Osteocalcina/genética , Osteocalcina/metabolismo , Osteonectina , Osteopontina/genética , Osteopontina/metabolismo , Osteoporose/diagnóstico por imagem , Osteoporose/patologia , Fator de Crescimento Transformador beta/metabolismoRESUMO
OBJECTIVE: The role of circadian clock in cementogenesis is unclear. This study examines the role of REV-ERBs, one of circadian clock proteins, in proliferation, migration and mineralization of cementoblasts to fill the gap in knowledge. METHODS: Expression pattern of REV-ERBα in cementoblasts was investigated in vivo and in vitro. CCK-8 assay, scratch wound healing assay, alkaline phosphatase (ALP) and alizarin red S (ARS) staining were performed to evaluate the effects of REV-ERBs activation by SR9009 on proliferation, migration and mineralization of OCCM-30, an immortalized cementoblast cell line. Furthermore, mineralization related markers including osterix (OSX), ALP, bone sialoprotein (BSP) and osteocalcin (OCN) were evaluated. RESULTS: Strong expression of REV-ERBα was found in cellular cementum around tooth apex. Rev-erbα mRNA oscillated periodically in OCCM-30 and declined after mineralization induction. REV-ERBs activation by SR9009 inhibited proliferation but promoted migration of OCCM-30 in vitro. Results of ALP and ARS staining suggested that REV-ERBs activation negatively regulated mineralization of OCCM-30. Mechanically, REV-ERBs activation attenuated the expression of OSX and its downstream targets including ALP, BSP and OCN. CONCLUSIONS: REV-ERBs are involved in cementogenesis and negatively regulate mineralization of cementoblasts via inhibiting OSX expression. Our study provides a potential target regarding periodontal and cementum regeneration.
Assuntos
Relógios Biológicos/genética , Calcificação Fisiológica/genética , Cemento Dentário/metabolismo , Membro 1 do Grupo D da Subfamília 1 de Receptores Nucleares/genética , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Transformada , Proliferação de Células/efeitos dos fármacos , Cementogênese/efeitos dos fármacos , Cementogênese/genética , Cemento Dentário/citologia , Cemento Dentário/efeitos dos fármacos , Feminino , Regulação da Expressão Gênica , Humanos , Sialoproteína de Ligação à Integrina/genética , Sialoproteína de Ligação à Integrina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Membro 1 do Grupo D da Subfamília 1 de Receptores Nucleares/metabolismo , Osteocalcina/genética , Osteocalcina/metabolismo , Pirrolidinas/farmacologia , Transdução de Sinais , Fator de Transcrição Sp7/genética , Fator de Transcrição Sp7/metabolismo , Tiofenos/farmacologiaRESUMO
BACKGROUND: Hyaluronans exist in different forms, accordingly with molecular weight and degree of crosslinking. Here, we tested the capability to induce osteogenic differentiation in hDPSCs (human dental pulp stem cells) of three hyaluronans forms: linear pharmaceutical-grade hyaluronans at high and (HHA) low molecular weight (LHA) and hybrid cooperative complexes (HCC), containing both sizes. METHODS: hDPSCs were treated with HHA, LHA, HCC for 7, 14 and 21 days. The effects of hyaluronans on osteogenic differentiation were evaluated by qRT-PCR and WB of osteogenic markers and by Alizarin Red S staining. To identify the involved pathway, CD44 was analyzed by immunofluorescence, and YAP/TAZ expression was measured by qRT-PCR. Moreover, YAP/TAZ inhibitor-1 was used, and the loss of function of YAP/TAZ was evaluated by qRT-PCR, WB and immunofluorescence. RESULTS: We showed that all hyaluronans improves osteogenesis. Among these, HCC is the main inducer of osteogenesis, along with overexpression of bone related markers and upregulating CD44. We also found that this biological process is subordinate to the activation of YAP/TAZ pathway. CONCLUSIONS: We found that HA's molecular weight can have a relevant impact on HA performance for bone regeneration, and we unveil a new molecular mechanism by which HA acts on stem cells.
Assuntos
Osso e Ossos/citologia , Diferenciação Celular , Polpa Dentária/citologia , Ácido Hialurônico/farmacologia , Transdução de Sinais , Células-Tronco/citologia , Proteínas com Motivo de Ligação a PDZ com Coativador Transcricional/metabolismo , Proteínas de Sinalização YAP/metabolismo , Biomarcadores/metabolismo , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Separação Celular , Células Cultivadas , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Receptores de Hialuronatos/metabolismo , Sialoproteína de Ligação à Integrina/genética , Sialoproteína de Ligação à Integrina/metabolismo , Osteocalcina/genética , Osteocalcina/metabolismo , Osteogênese/efeitos dos fármacos , Osteogênese/genética , Osteopontina/genética , Osteopontina/metabolismo , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismoRESUMO
Excessive inflammation leads to periodontitis, which inhibits the osteogenic differentiation of human dental pulp stem cells (hDPSCs), irreversibly injured and difficultly repaired for the important dental pulp. Hence, it is necessary to study the functional gene to enhance the osteogenic differentiation of hDPSCs. Previous found that SNHG7 expression was increased in the osteogenic differentiation of hDPSCs. However, the regulatory functions of SNHG7 on osteogenic differentiation of hDPSCs in the inflammatory microenvironment still remains unknown. In this study, hDPSCs treatment with 50 ng/mL TNF-α to mimic the inflammatory microenvironment, then cultured in osteoblast differentiation medium for 14 days. SNHG7, miR-6512-3p, BSP, DSPP, DMP-1, RUNX2 and OPN in hDPSCs were detect by RT-qPCR. We found that SNHG7 expression was reduced during the osteogenic differentiation of hDPSCs after different concentrations TNF-α treatment. SNHG7 overexpression improved the TNF-α-induced suppression of calcium deposition, ALP activity, and the expression of BSP, DSPP, DMP-1, RUNX2 and OPN. Furthermore, SNHG7 can sponge with miR-6512-3p. miR-6512-3p expression was increased during the osteogenic differentiation of hDPSCs after different concentrations TNF-α treatment while inhibited after SNHG7 overexpression. knockdown of miR-6512-3p improved the TNF-α-induced suppression of calcium deposition, ALP activity, and the expression of BSP, DSPP, DMP-1, RUNX2 and OPN. Finally, miR-6512-3p overexpression reversed the effect of SNHG7 on the osteo/dentinogenic differentiation of TNF-α-treated hDPSCs. In conclusions, SNHG7 improves the osteogenic differentiation of hDPSCs by inhibiting miR-6512-3p expression under 50 ng/mL TNF-α-induced inflammatory environment, which provided potential targets for the treatment of periodontitis.
Assuntos
MicroRNAs/genética , Osteoblastos/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , RNA Nucleolar Pequeno/genética , Células-Tronco/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologia , Diferenciação Celular , Células Cultivadas , Microambiente Celular/efeitos dos fármacos , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Meios de Cultura/química , Meios de Cultura/farmacologia , Polpa Dentária/citologia , Polpa Dentária/metabolismo , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Regulação da Expressão Gênica , Humanos , Inflamação , Sialoproteína de Ligação à Integrina/genética , Sialoproteína de Ligação à Integrina/metabolismo , MicroRNAs/metabolismo , Modelos Biológicos , Osteoblastos/citologia , Osteoblastos/metabolismo , Osteogênese/genética , Osteopontina/genética , Osteopontina/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , RNA Nucleolar Pequeno/metabolismo , Sialoglicoproteínas/genética , Sialoglicoproteínas/metabolismo , Transdução de Sinais , Células-Tronco/citologia , Células-Tronco/metabolismoRESUMO
Polyetheretherketone (PEEK) has been widely used in the fields of orthopedics and trauma, but weak osteointegration and bacterial infection affect its long-term stability and repair effects. Surface modification is an effective way to improve the osteogenic and antibacterial activity of PEEK implants. In the present study, a layer of acrylic acid (AA) polymer coating loaded with zinc ions (Zn2+) was constructed on the surface of PEEK (PEEK-AA-Zn) using a strategy of combining plasma-induced graft polymerization with a chemical immersion technique. Successful construction of the AA coating remarkably enhanced the hydrophilicity of PEEK, and effectively loaded and released Zn2+. In vitro cell culture using MC3T3-E1 preosteoblasts showed that the Zn2+ released from PEEK-AA-Zn promoted cell proliferation and elevated gene expression levels of alkaline phosphatase (ALP), osteocalcin (OCN) and bone sialoprotein (BSP). Antibacterial tests revealed that PEEK-AA-Zn efficiently inhibited the proliferation of Staphylococcus aureus (S. aureus). These results suggest that the combined method of graft polymerization and ion incorporation endows PEEK with excellent osteogenic and antibacterial activity, which provides a wide range of possibilities for developing PEEK implants with multifunctional properties for bone tissue repair.
Assuntos
Acrilatos/química , Antibacterianos/química , Benzofenonas/química , Materiais Biocompatíveis/química , Gases em Plasma/química , Polímeros/química , Zinco/química , Animais , Antibacterianos/farmacologia , Materiais Biocompatíveis/farmacologia , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Sialoproteína de Ligação à Integrina/genética , Sialoproteína de Ligação à Integrina/metabolismo , Camundongos , Osteocalcina/genética , Osteocalcina/metabolismo , Osteogênese/efeitos dos fármacos , Polimerização , Staphylococcus aureus/efeitos dos fármacos , Propriedades de Superfície , Regulação para Cima/efeitos dos fármacosRESUMO
Bone metastasis is an incurable complication of breast cancer. In advanced stages, patients with estrogen-positive tumors experience a significantly higher incidence of bone metastasis (>87%) compared to estrogen-negative patients (<56%). To understand the mechanism of this bone-tropism of ER+ tumor, and to identify liquid biopsy biomarkers for patients with high risk of bone metastasis, the secreted extracellular vesicles and cytokines from bone-tropic breast cancer cells are examined in this study. Both exosomal miR-19a and Integrin-Binding Sialoprotein (IBSP) are found to be significantly upregulated and secreted from bone-tropic ER+ breast cancer cells, increasing their levels in the circulation of patients. IBSP is found to attract osteoclast cells and create an osteoclast-enriched environment in the bone, assisting the delivery of exosomal miR-19a to osteoclast to induce osteoclastogenesis. Our findings reveal a mechanism by which ER+ breast cancer cells create a microenvironment favorable for colonization in the bone. These two secreted factors can also serve as effective biomarkers for ER+ breast cancer to predict their risks of bone metastasis. Furthermore, our screening of a natural compound library identifies chlorogenic acid as a potent inhibitor for IBSP-receptor binding to suppress bone metastasis of ER+ tumor, suggesting its preventive use for bone recurrence in ER+ patients.
Assuntos
Neoplasias Ósseas/secundário , Neoplasias da Mama/metabolismo , Exossomos/metabolismo , Sialoproteína de Ligação à Integrina/metabolismo , MicroRNAs/metabolismo , Animais , Neoplasias Ósseas/genética , Neoplasias Ósseas/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Exossomos/genética , Feminino , Humanos , Sialoproteína de Ligação à Integrina/genética , Camundongos , Camundongos Knockout , Camundongos Nus , MicroRNAs/genética , Metástase Neoplásica , Osteoclastos/metabolismo , Receptores de Estrogênio/metabolismoRESUMO
Colorectal cancer (CRC) is a frequently occurring digestive system cancer and postoperative tumor metastasis and recurrence are the main reasons for the failure of CRC treatment. The aim of this study was to identifying and validating key genes associated with metastatic recurrence of CRC. RNA expression of three datasets (GSE17538, GSE32323, and GSE29623) was used for biomarker discovery. We identified integrin-binding sialoprotein (IBSP) as a candidate biomarker which was validated in three clinical cohorts (GSE41258, GSE21510, and GSE39582) and our clinical specimens. The results suggested that IBSP expression significantly increased at mRNA and protein levels among CRC cases, which was associated with metastatic recurrence, metastasis, high risk of recurrence, and poor survival in CRC. Consistent results were obtained in CRC cells. The relative level of serum IBSP evidently increased among CRC patients relative to normal controls, and downregulated after operation. As suggested by gene set enrichment analysis (GSEA), the IBSP level was associated with cell-matrix adhesion in CRC. Functional experiments in vitro showed that IBSP promoted the growth and aggressiveness of CRC, and the potential mechanism by which IBSP promoted carcinogenesis of CRC was the abnormal activation of Fyn/ß-catenin signaling pathway. To sum up, findings in the present work indicate that IBSP can serve as the candidate biomarker for the diagnosis, treatment, and prognosis of CRC.
Assuntos
Biomarcadores Tumorais/genética , Neoplasias Colorretais/genética , Progressão da Doença , Sialoproteína de Ligação à Integrina/genética , Recidiva Local de Neoplasia/genética , Apoptose , Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/metabolismo , Proliferação de Células , Colo/metabolismo , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/mortalidade , Neoplasias Colorretais/patologia , Bases de Dados Genéticas , Regulação para Baixo , Feminino , Humanos , Sialoproteína de Ligação à Integrina/sangue , Sialoproteína de Ligação à Integrina/metabolismo , Masculino , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/metabolismo , Recidiva Local de Neoplasia/mortalidade , Proteínas Proto-Oncogênicas c-fyn/metabolismo , RNA/metabolismo , RNA Mensageiro/metabolismo , beta Catenina/metabolismoRESUMO
This study was conducted to investigate the effect of whey protein hydrolysate (WPH) on osteogenic cell differentiation and its growth-promoting effects in rats. Alkaline phosphatase (ALP) activity and calcium deposition were measured by treating MC3T3-E1 cells with WPH, and mRNA and protein levels of factors related to osteoblast differentiation were assessed. ALP activity and calcium deposition were significantly increased in the WPH group (p < 0.001). These findings were confirmed by the upregulation of ALP, bone morphogenic protein, bone sialoprotein, and collagen at the mRNA and protein levels. Furthermore, to confirm the growth-promoting effect of WPH, bone growth was analyzed by administering 3-week-old Sprague-Dawley rats with whey protein or WPH. Moreover, serum levels of calcium, ALP, and insulin-like growth factor-1 (IGF-1) were analyzed, bone analysis was performed using micro-CT, and the size of the growth plate was measured by Cresyl violet staining. When rats were administered with a high dose of WPH (600 mg per kg per day), calcium levels decreased significantly, while ALP levels (1.14-fold; p < 0.01), IGF-1 levels, tibia length, and growth plate height increased significantly compared to those in the control group. Collectively, WPH has shown to be effective in bone differentiation and bone growth.
Assuntos
Desenvolvimento Ósseo/fisiologia , Hidrolisados de Proteína/metabolismo , Hidrolisados de Proteína/farmacologia , Proteínas do Soro do Leite/metabolismo , Proteínas do Soro do Leite/farmacologia , Animais , Biomarcadores/sangue , Proteínas Morfogenéticas Ósseas/genética , Proteínas Morfogenéticas Ósseas/metabolismo , Cálcio/sangue , Linhagem Celular , Colágeno/genética , Colágeno/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Sialoproteína de Ligação à Integrina/genética , Sialoproteína de Ligação à Integrina/metabolismo , Masculino , Osteoblastos/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Regulação para Cima , Soro do LeiteRESUMO
Cirsium setidens (Dunn) Nakai, commonly known as gondre, is a perennial herb that grows predominantly in South Korea. It contains several bioactive phytochemicals with antioxidant, anticancer, antitumor and antiinflammatory properties. The present study aimed to investigate the effects of methanolic extracts of gondre on osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs). As characterized by nuclear magnetic resonance spectroscopy and matrixassisted laser deposition/ionization (timeofflight) mass spectrometry, the methanol extract of gondre was found to be enriched with pectolinarin. After 48 h, enhanced viability of hPDLSCs was observed in the presence of gondre compared with under control conditions, suggesting the biocompatibility of gondre. Notably, biocompatibility was markedly affected by gondre concentration in cultured media. Relatively high cell viability was observed in medium containing 0.05% gondre. Furthermore, mineralization was significantly higher in hPDLSCs in the presence of gondre compared with that in control cells, indicating their mineralization potential. Increased expression of various transcription markers, such as collagen 1, runtrelated transcription factor 2, bone sialoprotein and alkaline phosphatase, was also detected when hPDLSCs were stimulated with gondre compared with in the control groups, further confirming the superior osteogenic potential of gondre extract for tissue engineering applications, particularly in bone tissues.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Cirsium/química , Osteogênese/efeitos dos fármacos , Ligamento Periodontal/citologia , Extratos Vegetais/farmacologia , Células-Tronco/efeitos dos fármacos , Adolescente , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Calcificação Fisiológica/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Expressão Gênica/efeitos dos fármacos , Humanos , Sialoproteína de Ligação à Integrina/genética , Sialoproteína de Ligação à Integrina/metabolismo , Masculino , Extratos Vegetais/química , Células-Tronco/citologia , Células-Tronco/metabolismo , Adulto JovemRESUMO
The successful implementation of adipose-derived mesenchymal stem cells (ADSCs) in bone regeneration depends on efficient osteogenic differentiation. However, a literature survey and our own experience demonstrated that current differentiation methods are not effective enough. Since the differentiation of mesenchymal stem cells (MSCs) into osteoblasts and adipocytes can be regulated by cyclic adenosine monophosphate (cAMP) signaling, we investigated the effects of cAMP activator, forskolin, and inhibitor, SQ 22,536, on the early and late osteogenic differentiation of ADSCs cultured in spheroids or in a monolayer. Intracellular cAMP concentration, protein kinase A (PKA) activity, and inhibitor of DNA binding 2 (ID2) expression examination confirmed cAMP up- and downregulation. cAMP upregulation inhibited the cell cycle and protected ADSCs from osteogenic medium (OM)-induced apoptosis. Surprisingly, the upregulation of cAMP level at the early stages of osteogenic differentiation downregulated the expression of osteogenic markers RUNX2, Osterix, and IBSP, which was more significant in spheroids, and it is used for the more efficient commitment of ADSCs into preosteoblasts, according to the previously reported protocol. However, cAMP upregulation in a culture of ADSCs in spheroids resulted in significantly increased osteocalcin production and mineralization. Thus, undifferentiated and predifferentiated ADSCs respond differently to cAMP pathway stimulation in terms of osteogenesis, which might explain the ambiguous results from the literature.
Assuntos
Tecido Adiposo/citologia , Diferenciação Celular , AMP Cíclico/metabolismo , Células-Tronco Mesenquimais/metabolismo , Osteoblastos/metabolismo , Transdução de Sinais , Células Cultivadas , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Humanos , Proteína 2 Inibidora de Diferenciação/genética , Proteína 2 Inibidora de Diferenciação/metabolismo , Sialoproteína de Ligação à Integrina/genética , Sialoproteína de Ligação à Integrina/metabolismo , Células-Tronco Mesenquimais/citologia , Osteoblastos/citologia , Osteocalcina/genética , Osteocalcina/metabolismo , Fator de Transcrição Sp7/genética , Fator de Transcrição Sp7/metabolismo , Regulação para CimaRESUMO
Strategies aimed at delaying the onset of bone tissue degeneration and the resulting skeletal fragility are key to decrease the risk of bone fracture correlated to ageing. The therapeutic properties of sulfurous thermal waters (STWs), rich in hydrogen sulfide (H2S), have been claimed for centuries. However, the direct regulation of bone cells by STWs has not been investigated yet. Here we aimed at analyzing the effect of STWs on cultured human mesenchymal stromal cells (hMSCs) derived from bone tissue. Two concentrations of STWs from 2 health spa centers in Italy (here named STW-1 and STW-2) containing, respectively, high and moderate quantities of H2S, were added to the culture media. Cytotoxicity and osteogenic differentiation were evaluated. We provided first evidence that treatment of hMSCs with STWs results in a sharp increase in intracellular H2S content, coherent with the different concentrations of H2S, thereby reveling that STWs-released H2S is internalized by cells. STWs treatment significantly induced osteogenic differentiation of hMSCs. In particular, mineral apposition was increased with a similar pattern by the two STWs, while mRNA expression of osteogenic markers (BSP, OC, RUNX-2, OPN) was differently affected. Only STW-2 induced a significant, dose-dependent increase in these gene expression. These findings support the rationale for the use of STWs as a complementary treatment of bone wasting diseases.
Assuntos
Balneologia , Diferenciação Celular/efeitos dos fármacos , Sulfeto de Hidrogênio/farmacologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Águas Minerais , Osteogênese/efeitos dos fármacos , Idoso , Células Cultivadas , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Feminino , Regulação da Expressão Gênica , Humanos , Sulfeto de Hidrogênio/metabolismo , Sialoproteína de Ligação à Integrina/genética , Sialoproteína de Ligação à Integrina/metabolismo , Itália , Masculino , Células-Tronco Mesenquimais/metabolismo , Pessoa de Meia-Idade , Osteocalcina/genética , Osteocalcina/metabolismo , Osteopontina/genética , Osteopontina/metabolismoRESUMO
The Solanum glaucophyllum Desf. has been used to treat and prevent diseases in human and veterinary medicine. On the other hand, plant poisoning causes several bone diseases, among them osteoporosis, which is characterized by osteoblastic hypoplasia. Because the osteoblast is a cell derived from the differentiation of mesenchymal stem cells (MSCs) from bone marrow, the hypothesis is that the plant reduces the osteogenic differentiation of MSCs. The objective of this study was to evaluate the effects of S. glaucophyllum Desf. extract on MSCs cultured in osteogenic differentiation medium. We determined by liquid chromatography that 1 ml of plant extract contained 3.8 µl of 1,25(OH)2 D3 (calcitriol). Four groups of MSCs cultivated in osteogenic medium were evaluated as follows: (a) treated with 100 µl of extract/L containing 0.4 µg/L of calcitriol; (b) treated with 1 ml of extract/L containing 4 µg/L of calcitriol; (c) treated with 5 ml of extract/L containing 20 µg/L of calcitriol; and (d) a control group without extract. We performed alkaline phosphatase activity assay, analysis of MTT conversion to formazan, and evaluated the percentage of cells, and number and diameter of mineralization nodules. The expression of gene transcripts for osteopontin, bone sialoprotein and BMP-2 was analysed by RT-qPCR. After 21 days, there was a significant reduction in MTT conversion to formazan in treated groups, of the cellularity in the group with 5 ml of extract/L, and in the number and size of mineralization nodules in the groups treated with 1 and 5 ml of extract/L. The 5 ml extract/L concentration also reduced transcript expression of osteopontin. It is concluded that S. glaucophyllum Desf. at concentrations of 1 and 5 ml extract/L reduced mineralized matrix synthesis in MSCs cultivated in osteogenic differentiation medium, which suggests that this is one of the mechanisms by which osteoporosis occurs in intoxicated animals.