RESUMO
OBJECTIVE: Temporomandibular joint (TMJ) diseases predominantly afflict women, suggesting a role of estrogen in the disease etiology. Previously, we determined that decreased occlusal loading (DOL) inhibited collagen type II (Col2) expression in the mandibular condylar cartilage (MCC) of female wild-type (WT) mice whereas no change was observed in males. This decrease in chondrogenesis was abolished by estrogen receptor beta (ERß) deficiency in females. Therefore, the goal of this study was to examine the role of estradiol - ERß signaling in mediating DOL effects in male mice to further decipher sex differences. METHODS: Male 21 day-old WT and ERßKO male mice were treated with either placebo or estradiol and exposed to normal or DOL for 4 weeks. Cartilage thickness and cell proliferation, gene expression and immunohistochemistry of chondrogenic markers and estrogen receptor alpha (ERα), and analysis of bone histomorphometry via microCT were completed to ascertain the effect of estradiol on DOL effects to the TMJ. RESULTS: ERßKO male mice lack a MCC phenotype. In both genotypes, estradiol treatment increased Col2 gene expression and trabecular thickness. DOL in combination with estradiol treatment caused a significant increase in Col2 gene expression in both genotypes. CONCLUSIONS: The sex differences in DOL-induced inhibition of Col2 expression do not appear to be mediated by differences in estradiol levels between male and female mice. Greater understanding on the role of estrogen and altered loading are critical in order to decipher the sex dimorphism of TMJ disorders.
Assuntos
Cartilagem Articular/efeitos dos fármacos , Estradiol/farmacologia , Receptor beta de Estrogênio/genética , Estrogênios/farmacologia , Articulação Temporomandibular/efeitos dos fármacos , Animais , Cartilagem Articular/metabolismo , Proliferação de Células/efeitos dos fármacos , Condrogênese/efeitos dos fármacos , Condrogênese/genética , Colágeno Tipo II/efeitos dos fármacos , Colágeno Tipo II/metabolismo , Receptor alfa de Estrogênio/metabolismo , Receptor beta de Estrogênio/metabolismo , Expressão Gênica , Masculino , Côndilo Mandibular/diagnóstico por imagem , Côndilo Mandibular/efeitos dos fármacos , Camundongos , Camundongos Knockout , Fatores Sexuais , Articulação Temporomandibular/metabolismo , Articulação Temporomandibular/fisiopatologia , Suporte de Carga/fisiologia , Microtomografia por Raio-XRESUMO
OBJECTIVE: We aimed to determine the minimum mechanical impact to cause microstructural damage in the network of collagen (microcracking) within human cartilage and hypothesized that energies below 0.1 J or 1 mJ/mm3 would suffice. DESIGN: We completed 108 low-energy impact tests (0.05, 0.07, or 0.09 J; 0.75 or 1.0 m/s2) using healthy cartilage specimens from six male donors (30.2 ± 8.8 yrs old). Before and after impact we acquired, imaging the second harmonic generation (SHG), ten images from each specimen (50 µm depth, 5 µm step size), resulting in 2160 images. We quantified both the presence and morphology of microcracks. We then correlated test parameters (predictors) impact energy/energy dissipation density, nominal stress/stress rate, and strain/strain rate to microcracking and tested for significance. Where predictors significantly correlated with microstructural outcomes we fitted binary logistic regression plots with 95% confidence intervals (CIs). RESULTS: No specimens presented visible damage following impact. We found that impact energy/energy dissipation density and nominal stress/stress rate were significant (P < 0.05) predictors of microcracking while both strain and strain rate were not. In our test configuration, an impact energy density of 2.93 mJ/mm3, an energy dissipation density of 1.68 mJ/mm3, a nominal stress of 4.18 MPa, and a nominal stress rate of 689 MPa/s all corresponded to a 50% probability of microcracking in the network of collagen. CONCLUSIONS: An impact energy density of 1.0 mJ/mm3 corresponded to a â¼20% probability of microcracking. Such changes may initiate a degenerative cascade leading to post-traumatic osteoarthritis.
Assuntos
Cartilagem Articular/lesões , Colágeno/metabolismo , Estresse Mecânico , Adulto , Fenômenos Biomecânicos , Cartilagem Articular/metabolismo , Cartilagem Articular/patologia , Humanos , Modelos Logísticos , Masculino , Microscopia de Geração do Segundo Harmônico , Adulto JovemRESUMO
The role that transduced mouse bone marrow stromal cells (mBMSCs) engineered to overexpress human bone morphogenetic protein 2 (BMP-2) play in healing critical-sized skeletal defects is largely unknown. We evaluated the interaction between host osteoprogenitor cells and donor mBMSCs transduced with either a lentiviral (LV) vector-expressing red fluorescent protein (RFP) with or without BMP-2 that were implanted into a critical-sized femoral defect. Radiographs taken at the time of killing were evaluated using a five-point scaled scoring system. Frozen histologic sections were analyzed to assess both the transduced cells' role in bone repair and the local osteoprogenitor response. There was complete radiographic bridging in 94% of group I (LV-RFPch-BMP-2-cmyc) and 100% of group III (recombinant human BMP-2) specimens. Radiographs demonstrated a lack of healing in group II (LV-RFPch). Mouse BMSCs transduced with an LV-RFPch-BMP-2 vector were able to induce host cells to differentiate down an osteoblastic lineage and heal a critical-sized defect. However, the donor cells appeared to be functioning as a delivery vehicle of BMP-2 rather than actually differentiating into osteoblasts capable of participating in bone repair as evidenced by a lack of colocalization of the transduced cells to the sites of skeletal repair where the host progenitor cells were found.
Assuntos
Células da Medula Óssea/metabolismo , Proteína Morfogenética Óssea 2/metabolismo , Regeneração Óssea , Fêmur/citologia , Fêmur/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Cicatrização , Animais , Células Cultivadas , Vetores Genéticos , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Proteínas Recombinantes/metabolismo , Células Estromais/metabolismo , Tíbia/citologia , Tíbia/metabolismo , Transdução GenéticaRESUMO
OBJECTIVE: Temporomandibular joint (TMJ) diseases predominantly afflict women, suggesting a role for female hormones in the disease process. However, little is known about the role of estrogen receptor (ER) signaling in regulating mandibular condylar cartilage growth. Therefore, the goal of this study was to examine the effects of altered estrogen levels on the mandibular condylar cartilage in wild type (WT) and ER beta Knockout (KO) mice. MATERIALS AND METHODS: 21-day-old female WT (n = 37) and ER beta KO mice (n = 36) were either sham operated or ovariectomized, and treated with either placebo or estradiol. The mandibular condylar cartilage was evaluated by histomorphometry, proliferation was analyzed by double ethynyl-2'-deoxyuridine/bromodeoxyuridine (EdU/BrdU) labeling, and assays on gene and protein expression of chondrocyte maturation markers were performed. RESULTS: In WT mice, ovariectomy caused a significant increase in mandibular condylar cartilage cell numbers, a significant increase in Sox9 expression and a significant increase in proliferation compared with sham operated WT mice. In contrast, ovariectomy did not cause any of these effects in the ER beta KO mice. Estrogen replacement treatment in ovariectomized WT mice caused a significant decrease in ER alpha expression and a significant increase in Sost expression compared with ovariectomized mice treated with placebo. Estrogen replacement treatment in ovariectomized ER beta KO mice caused a significant increase in Col2 expression, no change in ER alpha expression, and a significant increase in Sost expression. CONCLUSION: Estrogen via ER beta inhibits proliferation and ER alpha expression while estrogen independent of ER beta induces Col2 and Sost expression.
Assuntos
Receptor beta de Estrogênio/genética , Estrogênios/genética , Regulação da Expressão Gênica no Desenvolvimento , Côndilo Mandibular/crescimento & desenvolvimento , RNA/genética , Transtornos da Articulação Temporomandibular/genética , Articulação Temporomandibular/crescimento & desenvolvimento , Proteínas Adaptadoras de Transdução de Sinal , Animais , Cartilagem Articular/crescimento & desenvolvimento , Cartilagem Articular/metabolismo , Modelos Animais de Doenças , Receptor beta de Estrogênio/biossíntese , Receptor beta de Estrogênio/uso terapêutico , Estrogênios/biossíntese , Estrogênios/uso terapêutico , Feminino , Glicoproteínas/biossíntese , Glicoproteínas/genética , Imuno-Histoquímica , Peptídeos e Proteínas de Sinalização Intercelular , Masculino , Côndilo Mandibular/metabolismo , Camundongos , Camundongos Knockout , Articulação Temporomandibular/efeitos dos fármacos , Articulação Temporomandibular/metabolismo , Transtornos da Articulação Temporomandibular/tratamento farmacológico , Transtornos da Articulação Temporomandibular/metabolismoRESUMO
'Ex vivo' gene therapy using viral vectors to overexpress BMP-2 is shown to heal critical-sized bone defects in experimental animals. To increase its safety, we constructed a dual-expression lentiviral vector to overexpress BMP-2 or luciferase and an HSV1-tk analog, Δtk (LV-Δtk-T2A-BMP-2/Luc). We hypothesized that administering ganciclovir (GCV) will eliminate the transduced cells at the site of implantation. The vector-induced expression of BMP-2 and luciferase in a mouse stromal cell line (W-20-17 cells) and mouse bone marrow cells (MBMCs) was reduced by 50% compared with the single-gene vector. W-20-17 cells were more sensitive to GCV compared with MBMCs (90-95% cell death at 12 days with GCV at 1 µg ml(-1) in MBMCs vs 90-95% cell death at 5 days by 0.1 µg ml(-1) of GCV in W-20-17 cells). Implantation of LV-Δtk-T2A-BMP-2 transduced MBMCs healed a 2 mm femoral defect at 4 weeks. Early GCV treatment (days 0-14) postoperatively blocked bone formation confirming a biologic response. Delayed GCV treatment starting at day 14 for 2 or 4 weeks reduced the luciferase signal from LV-Δtk-T2A-Luc-transduced MBMCs, but the signal was not completely eliminated. These data suggest that this suicide gene strategy has potential for clinical use in the future, but will need to be optimized for increased efficiency.
Assuntos
Células da Medula Óssea/metabolismo , Fraturas do Fêmur/terapia , Genes Transgênicos Suicidas , Terapia Genética/métodos , Simplexvirus/enzimologia , Células Estromais/metabolismo , Timidina Quinase/metabolismo , Animais , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/virologia , Transplante de Medula Óssea/métodos , Proteína Morfogenética Óssea 2/genética , Proteína Morfogenética Óssea 2/metabolismo , Linhagem Celular , Terapia Combinada/efeitos adversos , Fraturas do Fêmur/patologia , Ganciclovir/farmacologia , Vetores Genéticos/administração & dosagem , Vetores Genéticos/efeitos adversos , Humanos , Lentivirus/efeitos dos fármacos , Lentivirus/genética , Luciferases/metabolismo , Masculino , Camundongos , Células Estromais/efeitos dos fármacos , Células Estromais/virologia , Timidina Quinase/genética , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
INTRODUCTION: Primary leiomyosarcoma of the parotid gland is an extremely rare neoplasm, develops from smooth muscle cells. Its primary origin in the face and especially in the salivary glands is even more rare. CASE REPORT: A 15-year-old boy, with no prior history was hospitalized for swelling in the left parotid area having appeared 5 months before. The mass was painless and there was no facial paralysis. CT scan showed a tumoral process of mixed density in the left parotid gland. Thoracoabdominal CT scan was normal. Conservative parotidectomy was performed. The histological diagnosis was primary leiomyosarcoma of the parotid gland. DISCUSSION: Five per cent of salivary gland primitive tumors are of mesenchymatous origin, of which 0.3 to 1.5% are sarcoma. The diagnosis of leiomyosarcoma of the parotid gland is confirmed by histological and immunohistochemical assessment. Surgery sometimes combined to radiochemotherapy seems to be the treatment of choice.
Assuntos
Leiomiossarcoma/diagnóstico , Neoplasias Parotídeas/diagnóstico , Adolescente , Biópsia , Humanos , Leiomiossarcoma/cirurgia , Imageamento por Ressonância Magnética , Masculino , Glândula Parótida/cirurgia , Neoplasias Parotídeas/cirurgiaRESUMO
OBJECTIVE: Little is known about the natural progression of the disease process of temporomandibular joint (TMJ) osteoarthritis (OA), which affects approximately 1% of the US population. The goal of this study was to examine the early microarchitectural and molecular changes in the condylar cartilage and subchondral bone in biglycan/fibromodulin (Bgn/Fmod) double-deficient mice, which develop TMJ-OA at 6 months. METHODS: TMJs from 3-month-old (n=44) and 9-month-old (n=52) wild-type (WT n=46) and Bgn/Fmod (n=50) double-deficient mice were evaluated. Micro-CT analysis of the subchondral bone (n=24), transmission electron microscopy for condylar cartilage fibril diameters (n=26), and real-time PCR analysis for gene expression for bone and cartilage maturation markers (n=45) was performed. RESULTS: A statistically significant increase in collagen fibril diameter of the condylar cartilage and a decrease in expression of Parathyroid related protein in the mandibular condylar head were observed in the 3-month Bgn/Fmod double-deficient mice compared to WT controls. The 9-month Bgn/Fmod double-deficient mouse demonstrated an increase in bone volume and total volume in subchondral bone, and an increase in the expression of Collagen Type X and Aggrecan in the mandibular condylar head compared to the WT controls. CONCLUSION: We found that changes in the microarchitecture of the condylar cartilage preceded changes in the subchondral bone during OA in the TMJ in Bgn/Fmod double-deficient mice.
Assuntos
Proteínas da Matriz Extracelular/deficiência , Côndilo Mandibular/patologia , Osteoartrite/patologia , Proteoglicanas/deficiência , Transtornos da Articulação Temporomandibular/patologia , Agrecanas/biossíntese , Agrecanas/genética , Animais , Biglicano , Cartilagem Articular/patologia , Colágeno Tipo X/biossíntese , Colágeno Tipo X/genética , Modelos Animais de Doenças , Fibromodulina , Expressão Gênica , Côndilo Mandibular/metabolismo , Camundongos , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Osteoartrite/metabolismo , Proteína Relacionada ao Hormônio Paratireóideo/biossíntese , Proteína Relacionada ao Hormônio Paratireóideo/genética , Transtornos da Articulação Temporomandibular/metabolismo , Microtomografia por Raio-XRESUMO
Tumor necrosis factor (TNF)-alpha is known for its osteoclastogenic and resorptive activities. Induction of osteoclastogenesis by receptor activator of NF-kappaB ligand (RANKL) is accompanied by increased TNF-alpha expression. In the present study we investigated the mechanism by which RANKL induces expression of TNF-alpha in osteoclast precursors. The macrophage-like cell-line, RAW 264.7 was used as a model for osteoclast precursors. To examine if RANKL-mediated increase in TNF-alpha expression involves increased stability of its transcript, RAW264.7 cells were treated with or without RANKL, and then a transcription inhibitor was added. At different time points, TNF-alpha and L32 mRNA levels were examined. TNF-alpha mRNA stability was not altered by RANKL. We next measured directly the transcription rate of TNF-alpha by a run-on assay and found that RANKL increases TNF-alpha transcription rate by 2.9-fold in RAW264.7 cells. We further characterized this transcriptional induction of TNF-alpha by RANKL. Gel shift assays using nuclear extracts derived from RANKL-treated RAW264.7 cells show increased specific NF-kappaB binding activity on the murine TNF-alpha promoter. Gliotoxin, known for its ability to inhibit NF-kappaB activation blocked RANKL-induced TNF-alpha expression. We finally used 1,260 bp of the murine TNF-alpha promoter fused to luciferase, as well as four mutants of this promoter carrying mutations in each of the four NF-kappaB sites to stably transfect RAW 264.7 cells. Reporter activity was increased in response to RANKL in wild type promoter transfected cells, whereas treatment of the mutants' transfected cells did not elicit reporter activity. In conclusion, RANKL induces TNF-alpha expression via a transcriptional mechanism, depending on the NF-kappaB sites in the TNF promoter.
Assuntos
Proteínas de Transporte/fisiologia , Regulação da Expressão Gênica/fisiologia , Glicoproteínas de Membrana/fisiologia , Transcrição Gênica/fisiologia , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Animais , Medula Óssea , Proteínas de Transporte/farmacologia , Linhagem Celular , Macrófagos/metabolismo , Masculino , Glicoproteínas de Membrana/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , NF-kappa B/metabolismo , Osteoclastos/metabolismo , Regiões Promotoras Genéticas/efeitos dos fármacos , Ligante RANK , RNA Mensageiro/metabolismo , Receptor Ativador de Fator Nuclear kappa-B , Células-Tronco/metabolismo , Transcrição Gênica/efeitos dos fármacosRESUMO
BACKGROUND: Intracellular signaling triggered by bone morphogenetic proteins (BMPs) results in activated Smad complexes that regulate transcription of BMP-responsive genes. However, the low specificity of Smad binding to regulatory sequences implies that additional tissue-specific transcription factors are also needed. Runx2 (Cbfal) is a transcription factor required for bone formation. We have examined the role of Smads and Runx2 in BMP induction of type X collagen, which is a marker of chondrocyte hypertrophy leading to endochondral bone formation. METHODS: Pre-hypertrophic chondrocytes from the cephalic portion of the chick embryo sternum were placed in culture in the presence or absence of rhBMP-2. Cultures were transiently transfected with DNA containing the BMP-responsive type X collagen promoter upstream of the luciferase gene. The cultures were also transfected with plasmids, causing over-expression of Smads or Runx2, or both. After 24-48 hours, cell extracts were examined for levels of luciferase expression. RESULTS: In the presence of BMP-2, chondrocytes over-expressing BMP-activated Smadl or Smad5 showed significant enhancement of luciferase production compared with that seen with BMP alone. This enhancement was not observed with over-expression of Smad2, a transforming growth factor beta (TGF-beta)-activated Smad. Overexpression of Runx2 in BMP-treated cultures increased transcriptional activity to levels similar to those seen with Smads 1 or 5. When chondrocytes were simultaneously transfected with both Runx2 and Smad 1 or 5, promoter activity was further increased, indicating that BMP-stimulated Smad activity can be augmented by increasing the levels of Runx2. CONCLUSIONS: These results implicate the skeletal tissue transcription factor Runx2 in regulation of chondrocyte hypertrophy and suggest that maximal transcription of the type X collagen gene in pre-hypertrophic chondrocytes involves interaction of BMP-stimulated Smads with Runx2. CLINICAL RELEVANCE: Many skeletal abnormalities are associated with impaired regulation of chondrocyte hypertrophy in growth plates. These studies demonstrate that both BMP-activated Smads and Runx2 levels can modulate chondrocyte transition to hypertrophy.
Assuntos
Proteínas Morfogenéticas Ósseas/fisiologia , Condrócitos/fisiologia , Proteínas de Ligação a DNA/fisiologia , Proteínas de Neoplasias , Proteínas Proto-Oncogênicas , Transdução de Sinais , Transativadores/fisiologia , Fatores de Transcrição/fisiologia , Animais , Células Cultivadas , Embrião de Galinha , Condrócitos/patologia , Colágeno/fisiologia , Subunidade alfa 1 de Fator de Ligação ao Core , Subunidade alfa 2 de Fator de Ligação ao Core , Subunidades alfa de Fatores de Ligação ao Core , Proteínas de Ligação a DNA/genética , Hipertrofia , Luciferases/fisiologia , Fosfoproteínas/fisiologia , Regiões Promotoras Genéticas , Proteínas Smad , Proteína Smad5 , Transativadores/genética , Fatores de Transcrição/genética , Transcrição Gênica , TransfecçãoRESUMO
The runt related transcription factor CBFA1 (AML3/PEBP2alphaA/RUNX2) regulates expression of several bone- and cartilage-related genes and is required for bone formation in vivo. The gene regulatory mechanisms that control activation and repression of CBFA1 gene transcription during osteoblast differentiation and skeletal development are essential for proper execution of the osteogenic program. We have therefore defined functional contributions of 5' regulatory sequences conserved in rat, mouse and human CBFA1 genes to transcription. Deletion analysis reveals that 0.6 kB of the bone-related rat or mouse CBFA1 promoter (P1, MASNS protein isoform) is sufficient to confer transcriptional activation, and that there are multiple promoter domains which positively and negatively regulate transcription. Progressive deletion of promoter segments between nt -351 and -92 causes a striking 30- to 100-fold combined decrease in promoter activity. Additionally, 5' UTR sequences repress reporter gene transcription 2- to 3-fold. Our data demonstrate that CBFA1 is a principal DNA binding protein interacting with the 5' region of the CBFA1 gene in osseous cells, that there are at least three CBFA1 recognition motifs in the rat CBFA1 promoter, and that there are three tandemly repeated CBFA1 sites within the 5' UTR. We find that forced expression of CBFA1 protein downregulates CBFA1 promoter activity and that a single CBFA1 site is sufficient for transcriptional autosuppression. Thus, our data indicate that the CBFA1 gene is autoregulated in part by negative feedback on its own promoter to stringently control CBFA1 gene expression and function during bone formation.
Assuntos
Osso e Ossos/metabolismo , Proteínas de Neoplasias , Fatores de Transcrição/genética , Regiões 5' não Traduzidas , Animais , Sequência de Bases , Linhagem Celular , Clonagem Molecular , Sequência Consenso , Subunidade alfa 1 de Fator de Ligação ao Core , DNA Complementar/genética , Regulação para Baixo , Regulação da Expressão Gênica , Humanos , Camundongos , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Ratos , Transcrição Gênica , TransfecçãoRESUMO
To determine the local mechanisms involved in the effects of skeletal unloading on bone formation, we studied the temporal pattern of mRNA levels for insulin-like growth factor-I (IGF-I), IGF-I receptor type I (IGF-IR), and transforming growth factor beta receptor type II (TGF-betaRII) in relation to osteoblast phenotypic markers and osteoblast activity in hindlimb suspended rats. Skeletal unloading decreased bone volume and the mineralizing and osteoblastic surfaces at 4, 7, and 14 days in the tibial metaphysis, whereas the mineral appositional rate returned to normal at 14 days of suspension. RT-PCR analysis showed that skeletal unloading decreased type 1 collagen (Col 1) and osteocalcin (OC) mRNA levels in metaphyseal bone at days 4 and 7, and the levels returned to normal at 14 days of suspension. Unloading also decreased mRNA levels for IGF-I, IGF-IR, and TGF-betaRII at 4-7 days in the metaphyseal bone. However, IGF-I and IGF-IR levels rose above normal at 14 days of suspension. The biphasic changes in IGF-I mRNA levels were strongly correlated with Col 1 and OC mRNA levels. The associated biphasic pattern of IGF-I/IGF-IR expression, osteoblast markers, and osteoblast activity strongly suggests an important role for IGF-I signaling in the local effect of skeletal unloading on metaphyseal bone formation.
Assuntos
Fêmur/fisiologia , Fator de Crescimento Insulin-Like I/biossíntese , Osteoblastos/citologia , Tíbia/fisiologia , Animais , Antígenos de Diferenciação , Diferenciação Celular , Fator de Crescimento Insulin-Like I/genética , Masculino , Tamanho do Órgão , Proteínas Serina-Treonina Quinases , RNA Mensageiro/análise , Ratos , Ratos Wistar , Receptor IGF Tipo 1/biossíntese , Receptor IGF Tipo 1/genética , Receptor do Fator de Crescimento Transformador beta Tipo II , Receptores de Fatores de Crescimento Transformadores beta/biossíntese , Receptores de Fatores de Crescimento Transformadores beta/genética , Fator de Crescimento Transformador beta/biossíntese , Fator de Crescimento Transformador beta/genética , Suporte de CargaRESUMO
The cyclin-dependent kinase (cdk) inhibitors are key regulators of cell cycle progression. p27 and p21 are members of the Cip/Kip family of cdk inhibitors and regulate cell growth by inactivating cell cycle stage-specific CDK-cyclin complexes. Because down-regulation of osteoprogenitor proliferation is a critical step for osteoblast differentiation, we investigated expression of p27 and p21 during development of the osteoblast phenotype in rat calvarial osteoblasts and in proliferating and growth-inhibited osteosarcoma ROS 17/2.8 cells. Expression of these proteins indicates that p21, which predominates in the growth period, is related to proliferation control. p27 levels are maximal postproliferatively, suggesting a role in the transition from cell proliferation to osteoblast differentiation. We directly examined the role of p27 during differentiation of osteoprogenitor cells derived from the bone marrow (BM) of p27-/- mice. BM cells from p27 null mice exhibited increased proliferative activity compared with BM cells from wild-type mice and formed an increased number and larger size of osteoblastic colonies, which further differentiated to the mineralization stage. Although p27-/- adherent marrow cells proliferate faster, they retain competency for differentiation, which may result, in part, from observed higher p21 levels compared with wild type. Histological studies of p27-/- bones also showed an increased cellularity in the marrow cavity compared with the p27+/+. The increased proliferation in bone does not lead to tumorigenesis, in contrast to observed adenomas in the null mice. Taken together, these findings indicate that p27 plays a key role in regulating osteoblast differentiation by controlling proliferation-related events in bone cells.
Assuntos
Proteínas de Ciclo Celular , Ciclo Celular , Proteínas Associadas aos Microtúbulos/fisiologia , Osteoblastos/citologia , Proteínas Supressoras de Tumor , Animais , Neoplasias Ósseas/patologia , Calcificação Fisiológica , Cálcio/análise , Contagem de Células , Diferenciação Celular , Divisão Celular , Células Cultivadas , Inibidor de Quinase Dependente de Ciclina p21 , Inibidor de Quinase Dependente de Ciclina p27 , Ciclinas/fisiologia , DNA/análise , Camundongos , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/deficiência , Proteínas Associadas aos Microtúbulos/genética , Proteínas de Neoplasias/deficiência , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/fisiologia , Osteossarcoma/patologia , Ratos , Crânio/citologia , Crânio/embriologia , Células Estromais/citologia , Células Tumorais CultivadasRESUMO
Although calcitonin gene-related peptide (CGRP) may act as a local factor in bone, its mechanisms of action on osteoblasts are not well understood. We previously showed the presence of CGRP transcripts and peptide in human OHS-4 osteoblastic cells. The authors investigated the expression of CGRP receptor (CGRP-R) and its intracellular signalling properties in OHS-4 cells. Semi-quantitative RT-PCR analysis showed that OHS-4 cells express much more CGRP-R than calcitonin (CT)-R transcripts. After amplification of CGRP-R by RT-PCR and cloning of amplified fragments, the predicted CGRP-R sequence in OHS-4 cells was found to share 100% identity with human lung CGRP-R. Biochemical analysis showed that hCGRP did not increase intracellular cAMP levels in synchronized OHS-4 cells whatever was the cell cycle position. However, adenylate cyclase activity was functional, as human parathyroid hormone increased cAMP levels. In contrast, hCGRP induced a rapid, transient and dose-dependent increase in free cytosolic calcium levels. The data show that CGRP increases intracellular free Ca2+concentration but is not coupled to adenylate cyclase in CGRP receptor-positive OHS-4 osteosarcoma cells, suggesting that CGRP induces downstream events driven by phospholipase C in these cells.
Assuntos
Peptídeo Relacionado com Gene de Calcitonina/farmacologia , Cálcio/metabolismo , AMP Cíclico/biossíntese , Osteossarcoma/metabolismo , Receptores de Peptídeo Relacionado com o Gene de Calcitonina/metabolismo , Animais , Sequência de Bases , Calcitonina/farmacologia , Primers do DNA/genética , Humanos , Líquido Intracelular/metabolismo , Osteossarcoma/genética , Receptores da Calcitonina/genética , Receptores da Calcitonina/metabolismo , Receptores de Peptídeo Relacionado com o Gene de Calcitonina/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Células Tumorais CultivadasRESUMO
Interaction of calcitonin gene-related peptide (CGRP) with its receptors leads to stimulation of adenylyl cyclase and/or phospholipase C (PLC). While regulation of adenylyl cyclase is thought to involve the G-protein Gs, it is not known whether activation of PLC results from coupling the receptor to Gq family proteins or whether beta gamma subunits released from receptor-activated Gs activate PLC. We used human bone cells OHS-4 bearing CGRP receptors in which CGRP activates only the PLC signaling pathway to determine how CGRP acts. CGRP increased the concentration of intracellular calcium ([Ca2+]i) within 5 s via a Ca2+ influx through voltage-gated calcium channels and by mobilizing calcium from the endoplasmic reticulum. The activation of effectors, like PLC coupled to G-proteins, is the early event in the pathway leading to inositol 1,4,5-trisphosphate formation, which is responsible for Ca2+ mobilization. Western blotting demonstrated a range of PLC-beta isoforms (beta1, beta3, beta4, but not beta2) and G-proteins (Galphaq/11 and Galphas). Only phospholipase C-beta1 is involved in the mobilization of Ca2+ from the endoplasmic reticulum of Fura-2-loaded confluent OHS-4 cells and the formation of inositol 1,4,5-trisphosphate by CGRP; PLC-gamma have no effect. Activation of PLC-beta1 by CGRP involves the Galphaq/11 subunit, which is insensitive to pertussis toxin, but not Gbeta gamma subunits. We therefore believe that CGRP causes the activation of two separate G-proteins.
Assuntos
Peptídeo Relacionado com Gene de Calcitonina/farmacologia , Proteínas de Ligação ao GTP/metabolismo , Isoenzimas/metabolismo , Fosfolipases Tipo C/metabolismo , Toxina Adenilato Ciclase , Anticorpos/farmacologia , Osso e Ossos/fisiologia , Cálcio/metabolismo , Linhagem Celular , Ativação Enzimática/fisiologia , Fura-2/análogos & derivados , Fura-2/metabolismo , Guanosina Trifosfato/farmacologia , Humanos , Isoenzimas/química , Toxina Pertussis , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfolipase C beta , Transdução de Sinais/fisiologia , Fatores de Virulência de Bordetella/farmacologiaRESUMO
OBJECTIVES: Evaluation of a protocol of intravesical BCG therapy using 75 mg of Pasteur strain BCG with 2 years of maintenance treatment, and a follow-up of up to 60 months. MATERIAL AND METHODS: 189 patients treated by transurethral resection (TUR) for a pTa (N = 80) or pT1 (N = 109) bladder tumour were included in the study. The local and general safety was excellent. We retrospectively compared this series to a group of patients treated by TUR alone (N = 42) another group treated with TUR and Mitomycin C (MMC) (N = 81). The 3 groups were statistically comparable. RESULTS: At 48 months, 62% of patients treated with BCG were recurrence-free, versus only 18% for patients treated with TUR alone and 38% for patients treated with TUR and MMC (p = 0.001). At 42 months, 11% of pT1 tumours treated with BCG had progressed to invasive carcinoma, and this progression occurred during the first 18 months in every case. In comparison, this progression was observed in 25% of pT1 tumours treated by TUR alone and 21% of tumours treated with TUR and MMC. CONCLUSIONS: Our study confirms the efficacy of our BCG protocol ro reduce the potential for recurrence and progression of superficial bladder tumours, despite reduction of the dose to 75 mg. It also suggests the superiority of BCG compared to MMC in terms of recurrence and progression.
Assuntos
Vacina BCG/uso terapêutico , Neoplasias da Bexiga Urinária/terapia , Análise Atuarial , Administração Intravesical , Adulto , Idoso , Idoso de 80 Anos ou mais , Antibióticos Antineoplásicos/uso terapêutico , Vacina BCG/administração & dosagem , Vacina BCG/classificação , Carcinoma/patologia , Cistoscopia , Progressão da Doença , Intervalo Livre de Doença , Endoscopia , Feminino , Seguimentos , Humanos , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Mitomicina/uso terapêutico , Invasividade Neoplásica , Recidiva Local de Neoplasia/patologia , Estadiamento de Neoplasias , Estudos Retrospectivos , Segurança , Neoplasias da Bexiga Urinária/tratamento farmacológico , Neoplasias da Bexiga Urinária/patologia , Neoplasias da Bexiga Urinária/cirurgiaRESUMO
There is general agreement that calcitonin (CT) inhibits bone resorption by its effects on osteoclast function. CT was also found to have direct effects on osteoblast-like cells. In this study, we investigated the expression of CT and calcitonin gene-related peptide (CGRP), the two peptides encoded by the CT/CGRP gene, in human osteosarcoma cell lines and in normal human trabecular osteoblastic cells (HOB), and we studied the modulation of CT/CGRP gene expression by dibutyryl cyclic adenosine monophosphate ((Bu)2, cAMP), a cAMP analog. We first detected by Northern blot hybridization the presence of CT and CGRP mRNAs in different osteosarcoma cell lines (OHS-4, MG-63, Saos-2, HOS-TE85) and HOB cells. In the steady state, OHS-4 cells express slightly more CT and CGRP mRNAs than other cell lines or normal human osteoblasts, in parallel with messengers of differentiated osteoblasts, such as osteocalcin (OC) and alkaline phosphatase (ALP). OHS-4 cells also express CT and CGRP proteins, as demonstrated by immunocytochemistry. Stimulation of OHS-4 cells with 1 mM (Bu)2 cAMP induced a significant increase in mRNA levels for CT (x 2.5) and CGRP (x 3), as determined by a semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) procedure. The involvement of a transcriptional mechanism in this effect was evidenced by nuclear run-off transcription assay. In addition, (Bu)2 cAMP increased OC (x 4) and ALP (x 3) mRNA levels in OHS-4 cells. These effects were observed at 24 h and were maximal at 48 h, indicating that (Bu)2, cAMP induced cell differentiation and increased the transcription of the CT/CGRP gene in OHS-4 osteoblast-like cells. The results indicate that human osteosarcoma cells and primary human osteoblastic cells express CT and CGRP mRNA and proteins, and that (Bu)2 cAMP, an activator of protein kinase A, induces up-regulation of osteoblastic phenotypic genes and enhances CT and CGRP gene transcription, indicating that induction of osteoblastic differentiation by (Bu)2 cAMP is associated with enhanced expression of CT and CGRP in human osteoblastic cells.