Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 33
Filtrar
1.
J Biol Chem ; 300(2): 105613, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38159855

RESUMO

Notch signaling plays a key regulatory role in bone remodeling and NOTCH2 enhances osteoclastogenesis, an effect that is mostly mediated by its target gene Hes1. In the present study, we explored mechanisms responsible for the enhanced osteoclastogenesis in bone marrow-derived macrophages (BMM) from Notch2tm1.1Ecan, harboring a NOTCH2 gain-of-function mutation, and control mice. Notch2tm1.1Ecan mice are osteopenic and have enhanced osteoclastogenesis. Bulk RNA-Seq and gene set enrichment analysis of Notch2tm1.1Ecan BMMs cultured in the presence of macrophage colony stimulating factor (M-CSF) and receptor activator of NF-κB ligand revealed enrichment of genes associated with enhanced cell metabolism, aerobic respiration, and mitochondrial function, all associated with osteoclastogenesis. These pathways were not enhanced in the context of a Hes1 inactivation. Analysis of single cell RNA-Seq data of pooled control and Notch2tm1.1Ecan BMMs treated with M-CSF or M-CSF and receptor activator of NF-κB ligand for 3 days identified 11 well-defined cellular clusters. Pseudotime trajectory analysis indicated a trajectory of clusters expressing genes associated with osteoclast progenitors, osteoclast precursors, and mature cells. There were an increased number of cells expressing gene markers associated with the osteoclast and with an unknown, albeit related, cluster in Notch2tm1.1Ecan than in control BMMs as well as enhanced expression of genes associated with osteoclast progenitors and precursors in Notch2tm1.1Ecan cells. In conclusion, BMM cultures display cellular heterogeneity, and NOTCH2 enhances osteoclastogenesis, increases mitochondrial and metabolic activity of osteoclasts, and affects cell cluster allocation in BMMs.


Assuntos
Osteoclastos , Osteogênese , Receptor Notch2 , Transcriptoma , Animais , Camundongos , Células da Medula Óssea/metabolismo , Diferenciação Celular/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Fator Estimulador de Colônias de Macrófagos/metabolismo , Camundongos Endogâmicos C57BL , Mutação , Osteoclastos/citologia , Osteoclastos/metabolismo , Osteogênese/genética , Receptor Ativador de Fator Nuclear kappa-B/metabolismo , Receptor Notch2/genética , Receptor Notch2/metabolismo , Fatores de Transcrição HES-1/metabolismo , Transcriptoma/genética
2.
J Biol Chem ; 299(12): 105372, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-37865314

RESUMO

Notch regulates the immune and inflammatory response and has been associated with the pathogenesis of osteoarthritis in humans and preclinical models of the disease. Notch2tm1.1Ecan mice harbor a NOTCH2 gain-of-function and are sensitized to osteoarthritis, but the mechanisms have not been explored. We examined the effects of tumor necrosis factor α (TNFα) in chondrocytes from Notch2tm1.1Ecan mice and found that NOTCH2 enhanced the effect of TNFα on Il6 and Il1b expression. Similar results were obtained in cells from a conditional model of NOTCH2 gain-of-function, Notch22.1Ecan mice, and following the expression of the NOTCH2 intracellular domain in vitro. Recombination signal-binding protein for immunoglobulin Kappa J region partners with the NOTCH2 intracellular domain to activate transcription; in the absence of Notch signaling it inhibits transcription, and Rbpj inactivation in chondrocytes resulted in Il6 induction. Although TNFα induced IL6 to a greater extent in the context of NOTCH2 activation, there was a concomitant inhibition of Notch target genes Hes1, Hey1, Hey2, and Heyl. Electrophoretic mobility shift assay demonstrated displacement of recombination signal-binding protein for immunoglobulin Kappa J region from DNA binding sites by TNFα explaining the increased Il6 expression and the concomitant decrease in Notch target genes. NOTCH2 enhanced the effect of TNFα on NF-κB signaling, and RNA-Seq revealed increased expression of pathways associated with inflammation and the phagosome in NOTCH2 overexpressing cells in the absence and presence of TNFα. Collectively, NOTCH2 has important interactions with TNFα resulting in the enhanced expression of Il6 and inflammatory pathways in chondrocytes.


Assuntos
Condrócitos , Osteoartrite , Receptor Notch2 , Fator de Necrose Tumoral alfa , Animais , Humanos , Camundongos , Condrócitos/citologia , Condrócitos/efeitos dos fármacos , Condrócitos/metabolismo , Imunoglobulinas , Interleucina-6/genética , Interleucina-6/metabolismo , Osteoartrite/genética , Osteoartrite/metabolismo , Receptor Notch2/genética , Receptor Notch2/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Inflamação , Modelos Animais de Doenças , Condrogênese , Transdução de Sinais/efeitos dos fármacos , Domínios Proteicos/imunologia , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/genética , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/metabolismo , Deleção de Genes , Regulação da Expressão Gênica/efeitos dos fármacos
3.
J Biol Chem ; 297(6): 101376, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34742737

RESUMO

Notch2tm1.1Ecan mice, which harbor a mutation replicating that found in Hajdu-Cheney syndrome, exhibit marked osteopenia because of increased osteoclast number and bone resorption. Hairy and enhancer of split 1 (HES1) is a Notch target gene and a transcriptional modulator that determines osteoclast cell fate decisions. Transcript levels of Hes1 increase in Notch2tm1.1Ecan bone marrow-derived macrophages (BMMs) as they mature into osteoclasts, suggesting a role in osteoclastogenesis. To determine whether HES1 is responsible for the phenotype of Notch2tm1.1Ecan mice and the skeletal manifestations of Hajdu-Cheney syndrome, Hes1 was inactivated in Ctsk-expressing cells from Notch2tm1.1Ecan mice. Ctsk encodes the protease cathepsin K, which is expressed preferentially by osteoclasts. We found that the osteopenia of Notch2tm1.1Ecan mice was ameliorated, and the enhanced osteoclastogenesis was reversed in the context of the Hes1 inactivation. Microcomputed tomography revealed that the downregulation of Hes1 in Ctsk-expressing cells led to increased bone volume/total volume in female mice. In addition, cultures of BMMs from CtskCre/WT;Hes1Δ/Δ mice displayed a decrease in osteoclast number and size and decreased bone-resorbing capacity. Moreover, activation of HES1 in Ctsk-expressing cells led to osteopenia and enhanced osteoclast number, size, and bone resorptive capacity in BMM cultures. Osteoclast phenotypes and RNA-Seq of cells in which HES1 was activated revealed that HES1 modulates cell-cell fusion and bone-resorbing capacity by supporting sealing zone formation. In conclusion, we demonstrate that HES1 is mechanistically relevant to the skeletal manifestation of Notch2tm1.1Ecan mice and is a novel determinant of osteoclast differentiation and function.


Assuntos
Diferenciação Celular/fisiologia , Osteoclastos/citologia , Receptor Notch2/metabolismo , Transdução de Sinais/fisiologia , Fatores de Transcrição HES-1/fisiologia , Animais , Feminino , Síndrome de Hajdu-Cheney/genética , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Receptor Notch2/genética
4.
J Biol Chem ; 296: 100583, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33774049

RESUMO

Notch receptors maintain skeletal homeostasis. NOTCH1 and 2 have been studied for their effects on bone remodeling. Although NOTCH3 plays a significant role in vascular physiology, knowledge about its function in other cellular environments, including bone, is limited. The present study was conducted to establish the function of NOTCH3 in skeletal cells using models of Notch3 misexpression. Microcomputed tomography demonstrated that Notch3 null mice did not have appreciable bone phenotypes. To study the effects of the NOTCH3 activation in the osteoblast lineage, BGLAP-Cre or Dmp1-Cre transgenics were crossed with RosaNotch3 mice, where the NOTCH3 intracellular domain is expressed following the removal of a loxP-flanked STOP cassette. Microcomputed tomography demonstrated that BGLAP-Cre;RosaNotch3 and Dmp1-Cre;RosaNotch3 mice of both sexes exhibited an increase in trabecular bone and in connectivity, with a decrease in cortical bone and increased cortical porosity. Histological analysis revealed a decrease in osteoclast number and bone resorption in trabecular bone and an increase in osteoclast number and void or pore area in cortical bone of RosaNotch3 mice. Bone formation was either decreased or could not be determined in Cre;RosaNotch3 mice. NOTCH3 activation in osteoblasts inhibited Alpl (alkaline phosphatase) and Bglap (osteocalcin) and induced Tnfsf11 (RANKL) and Tnfrsf11b (osteoprotegerin) mRNA, possibly explaining the trabecular bone phenotype. However, NOTCH3 induced Tnfsf11 and suppressed Tnfrsf11b in osteocytes, possibly explaining the cortical porosity. In conclusion, basal NOTCH3 is dispensable for skeletal homeostasis, whereas activation of NOTCH3 in osteoblasts/osteocytes inhibits osteoclastogenesis and bone resorption in cancellous bone but increases intracortical remodeling and causes cortical porosity.


Assuntos
Remodelação Óssea , Osteoblastos/metabolismo , Osteócitos/metabolismo , Receptor Notch3/metabolismo , Animais , Regulação da Expressão Gênica , Camundongos , Osteoblastos/citologia , Osteócitos/citologia
5.
PLoS Genet ; 15(6): e1008214, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31251738

RESUMO

Postpartum depression is a severe emotional and mental disorder that involves maternal care defects and psychiatric illness. Postpartum depression is closely associated with a combination of physical changes and physiological stress during pregnancy or after parturition in stress-sensitive women. Although postpartum depression is relatively well known to have deleterious effects on the developing fetus, the influence of genetic risk factors on the development of postpartum depression remains unclear. In this study, we discovered a novel function of T cell death-associated gene 51 (TDAG51/PHLDA1) in the regulation of maternal and depressive-like behavior. After parturition, TDAG51-deficient dams showed impaired maternal behavior in pup retrieving, nursing and nest building tests. In contrast to the normal dams, the TDAG51-deficient dams also exhibited more sensitive depressive-like behaviors after parturition. Furthermore, changes in the expression levels of various maternal and depressive-like behavior-associated genes regulating neuroendocrine factor and monoamine neurotransmitter levels were observed in TDAG51-deficient postpartum brain tissues. These findings indicate that TDAG51 plays a protective role against maternal care defects and depressive-like behavior after parturition. Thus, TDAG51 is a maternal care-associated gene that functions as a crucial regulator of maternal and depressive-like behavior after parturition.


Assuntos
Transtorno Depressivo/genética , Comportamento Materno , Parto/genética , Fatores de Transcrição/genética , Animais , Encéfalo/metabolismo , Transtorno Depressivo/fisiopatologia , Feminino , Regulação da Expressão Gênica/genética , Humanos , Camundongos , Camundongos Knockout , Neurotransmissores/genética , Parto/fisiologia , Gravidez
6.
J Biol Chem ; 295(12): 3952-3964, 2020 03 20.
Artigo em Inglês | MEDLINE | ID: mdl-31992595

RESUMO

Notch receptors play critical roles in cell-fate decisions and in the regulation of skeletal development and bone remodeling. Gain-of-function NOTCH2 mutations can cause Hajdu-Cheney syndrome, an untreatable disease characterized by osteoporosis and fractures, craniofacial developmental abnormalities, and acro-osteolysis. We have previously created a mouse model harboring a point 6955C→T mutation in the Notch2 locus upstream of the PEST domain, and we termed this model Notch2tm1.1Ecan Heterozygous Notch2tm1.1Ecan mutant mice exhibit severe cancellous and cortical bone osteopenia due to increased bone resorption. In this work, we demonstrate that the subcutaneous administration of Notch2 antisense oligonucleotides (ASO) down-regulates Notch2 and the Notch target genes Hes-related family basic helix-loop-helix transcription factor with YRPW motif 1 (Hey1), Hey2, and HeyL in skeletal tissue from Notch2tm1.1Ecan mice. Results of microcomputed tomography experiments indicated that the administration of Notch2 ASOs ameliorates the cancellous osteopenia of Notch2tm1.1Ecan mice, and bone histomorphometry analysis revealed decreased osteoclast numbers in Notch2 ASO-treated Notch2tm1.1Ecan mice. Notch2 ASOs decreased the induction of mRNA levels of TNF superfamily member 11 (Tnfsf11, encoding the osteoclastogenic protein RANKL) in cultured osteoblasts and osteocytes from Notch2tm1.1Ecan mice. Bone marrow-derived macrophage cultures from the Notch2tm1.1Ecan mice displayed enhanced osteoclastogenesis, which was suppressed by Notch2 ASOs. In conclusion, Notch2tm1.1Ecan mice exhibit cancellous bone osteopenia that can be ameliorated by systemic administration of Notch2 ASOs.


Assuntos
Síndrome de Hajdu-Cheney/patologia , Oligonucleotídeos Antissenso/metabolismo , Receptor Notch2/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Osso e Ossos/diagnóstico por imagem , Osso e Ossos/patologia , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Modelos Animais de Doenças , Feminino , Síndrome de Hajdu-Cheney/metabolismo , Macrófagos/citologia , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Músculo Esquelético/metabolismo , Oligonucleotídeos Antissenso/administração & dosagem , Osteoclastos/citologia , Osteoclastos/metabolismo , Osteogênese , Fenótipo , Mutação Puntual , Ligante RANK/genética , Ligante RANK/metabolismo , Receptor Notch2/antagonistas & inibidores , Receptor Notch2/genética
7.
J Biol Chem ; 294(39): 14203-14214, 2019 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-31371452

RESUMO

Hajdu Cheney syndrome (HCS) is characterized by craniofacial developmental abnormalities, acro-osteolysis, and osteoporosis and is associated with gain-of-NOTCH2 function mutations. A mouse model of HCS termed Notch2tm1.1Ecan harboring a mutation in exon 34 of Notch2 replicating the one found in HCS was used to determine whether the HCS mutation sensitizes the skeleton to the osteolytic effects of tumor necrosis factor α (TNFα). TNFα injected over the calvarial vault caused a greater increase in osteoclast number, osteoclast surface, and eroded surface in Notch2tm1.1Ecan mice compared with littermate WT controls. Accordingly, the effect of TNFα on osteoclastogenesis was greatly enhanced in cultures of bone marrow-derived macrophages (BMMs) from Notch2tm1.1Ecan mice when compared with the activity of TNFα in control cultures. TNFα induced the expression of Notch2 and Notch2 mutant mRNA by ∼2-fold, possibly amplifying the NOTCH2-dependent induction of osteoclastogenesis. The effect of TNFα on osteoclastogenesis in Notch2tm1.1Ecan mutants depended on NOTCH2 activation because it was reversed by anti-NOTCH2 negative regulatory region and anti-jagged 1 antibodies. The inactivation of Hes1 prevented the TNFα effect on osteoclastogenesis in the context of the Notch2tm1.1Ecan mutation. In addition, the induction of Il1b, but not of Tnfa and Il6, mRNA by TNFα was greater in Notch2tm1.1Ecan BMMs than in control cells, possibly contributing to the actions of TNFα and NOTCH2 on osteoclastogenesis. In conclusion, the HCS mutation enhances TNFα-induced osteoclastogenesis and the inflammatory bone-resorptive response possibly explaining the acro-osteolysis observed in affected individuals.


Assuntos
Síndrome de Hajdu-Cheney/genética , Mutação , Osteoclastos/metabolismo , Receptor Notch2/genética , Fator de Necrose Tumoral alfa/metabolismo , Animais , Células Cultivadas , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Osteoclastos/efeitos dos fármacos , Osteoclastos/patologia , Receptor Notch2/metabolismo , Fatores de Transcrição HES-1/metabolismo , Fator de Necrose Tumoral alfa/farmacologia
8.
J Cell Physiol ; 235(11): 8520-8532, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32329053

RESUMO

The present study defines the function of nuclear factor of activated T cells (NFAT)c1 and NFATc2 in osteoblast function in vivo and in vitro. Nfatc1loxP/loxP , Nfatc2loxP/loxP , and Nfatc1loxP/loxP ;Nfatc2loxP/loxP conditional mice were mated with BGLAP-Cre transgenics to inactivate Nfatc1 and Nfatc2 singly and in combination in osteoblasts. Microcomputed tomography demonstrated that male and female conditionally inactivated Nfatc1, Nfatc2 and dual Nfatc1;Nfatc2 mice had osteopenia at Lumbar 3 (L3) sites when compared to littermate controls. However, the Nfatc1 and Nfatc2 inactivation singly and in combination in Bglap-expressing osteoblasts did not result in an appreciable phenotype at femoral sites. Bone histomorphometry of L3 confirmed the osteopenic phenotype and demonstrated that Nfatc1;Nfatc2 inactivated male mice had a significant decrease in osteoblast number and in osteoblast surface and osteoid surface. The dual downregulation of Nfatc1 and Nfatc2 in bone marrow stromal cells caused a decrease in Alpl and Bglap expression, confirming a role of these transcription factors in osteoblast function. In conclusion, our studies reveal that NFATc1 and NFATc2 are necessary for optimal vertebral, but not femoral, bone homeostasis in vivo and osteoblast differentiation in vitro.


Assuntos
Homeostase/fisiologia , Fatores de Transcrição NFATC/metabolismo , Osteoblastos/metabolismo , Linfócitos T/metabolismo , Animais , Doenças Ósseas Metabólicas/metabolismo , Diferenciação Celular/genética , Ativação Linfocitária/fisiologia , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Osteogênese/genética , Microtomografia por Raio-X/métodos
9.
J Cell Physiol ; 235(1): 210-220, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31188489

RESUMO

Lateral meningocele syndrome (LMS), a genetic disorder characterized by meningoceles and skeletal abnormalities, is associated with NOTCH3 mutations. We created a mouse model of LMS (Notch3tm1.1Ecan ) by introducing a tandem termination codon in the Notch3 locus upstream of the proline (P), glutamic acid (E), serine (S) and threonine (T) domain. Microcomputed tomography demonstrated that Notch3tm1.1Ecan mice exhibit osteopenia. The cancellous bone osteopenia was no longer observed after the intraperitoneal administration of antibodies directed to the negative regulatory region (NRR) of Notch3. The anti-Notch3 NRR antibody suppressed the expression of Hes1, Hey1, and Hey2 (Notch target genes), and decreased Tnfsf11 (receptor activator of NF Kappa B ligand) messenger RNA in Notch3tm1.1Ecan osteoblast (OB) cultures. Bone marrow-derived macrophages (BMMs) from Notch3tm1.1Ecan mutants exhibited enhanced osteoclastogenesis in culture, and this was increased in cocultures with Notch3tm1.1Ecan OB. Osteoclastogenesis was suppressed by anti-Notch3 NRR antibodies in Notch3tm1.1Ecan OB/BMM cocultures. In conclusion, the cancellous bone osteopenia of Notch3tm1.1Ecan mutants is reversed by anti-Notch3 NRR antibodies.


Assuntos
Anormalidades Múltiplas/genética , Anormalidades Múltiplas/terapia , Anticorpos/uso terapêutico , Meningocele/genética , Meningocele/terapia , Receptor Notch3/imunologia , Animais , Osso e Ossos/anormalidades , Feminino , Predisposição Genética para Doença , Macrófagos/fisiologia , Masculino , Camundongos , Mutação , Osteoblastos/fisiologia , Microtomografia por Raio-X
10.
J Biol Chem ; 293(36): 14165-14177, 2018 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-30042232

RESUMO

Lateral meningocele syndrome (LMS) is a rare genetic disorder characterized by neurological complications and osteoporosis. LMS is associated with mutations in exon 33 of NOTCH3 leading to a truncated protein lacking sequences for NOTCH3 degradation and presumably causing NOTCH3 gain of function. To create a mouse model reproducing human LMS-associated mutations, we utilized CRISPR/Cas9 to introduce a tandem termination codon at bases 6691-6696 (ACCAAG→TAATGA) and verified this mutation (Notch3tm1.1Ecan ) by DNA sequencing of F1 mice. One-month-old male and female heterozygous Notch3tm1.1Ecan mice had cancellous and cortical bone osteopenia but exhibited no obvious neurological alterations, and histopathology of multiple organs revealed no abnormalities. Microcomputed tomography of these mutants revealed a 35-60% decrease in cancellous bone volume associated with a reduction in trabecular number and decreased connectivity. During maturation, cancellous and cortical bones were restored in female but not in male mice, which exhibited cancellous bone osteopenia at 4 months. Cancellous bone histomorphometry revealed increased osteoblast and osteocyte numbers and a modest increase in osteoclast surface and bone formation rate. Notch3tm1.1Ecan calvarial osteoblasts had increased proliferation and increased bone γ-carboxyglutamate protein (Bglap) and TNF superfamily member 11 (Tnfsf11) mRNA levels and lower Tnfrsf11b levels. Tnfsf11 mRNA was increased in osteocyte-rich femora from Notch3tm1.1Ecan mice. Cultures of bone marrow-derived macrophages from Notch3tm1.1Ecan mice revealed increased osteoclast formation, particularly in cocultures with osteoblasts from Notch3tm1.1Ecan mice. In conclusion, the Notch3tm1.1Ecan mutation causes osteopenia despite an increase in osteoblast proliferation and function and is associated with enhanced Tnfsf11 expression in osteoblasts and osteocytes.


Assuntos
Anormalidades Múltiplas/genética , Doenças Ósseas Metabólicas/etiologia , Mutação com Ganho de Função , Meningocele/genética , Receptor Notch3/genética , Animais , Doenças Ósseas Metabólicas/genética , Osso Esponjoso/patologia , Feminino , Macrófagos , Masculino , Meningocele/complicações , Camundongos , Osteoblastos/metabolismo , Osteoclastos , Osteócitos/metabolismo , Ligante RANK/metabolismo
11.
Am J Pathol ; 188(1): 149-159, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29037852

RESUMO

The neurogenic locus notch homolog protein (Notch)-2 receptor is a determinant of B-cell allocation, and gain-of-NOTCH2-function mutations are associated with Hajdu-Cheney syndrome (HCS), a disease presenting with osteoporosis and acro-osteolysis. We generated a mouse model reproducing the HCS mutation (Notch2HCS), and heterozygous global mutant mice displayed gain-of-Notch2 function. In the mutant spleen, the characteristic perifollicular rim marking the marginal zone (MZ), which is the interface between the nonlymphoid red pulp and the lymphoid white pulp, merged with components of the white pulp. As a consequence, the MZ of Notch2HCS mice occupied most of the splenic structure. To explore the mechanisms involved, lymphocyte populations from the bone marrow and spleen were harvested from heterozygous Notch2HCS mice and sex-matched control littermates and analyzed by flow cytometry. Notch2HCS mice had an increase in CD21/35highCD23- splenic MZ B cells of approximately fivefold and a proportional decrease in splenic follicular B cells (CD21/35intCD23+) at 1, 2, and 12 months of age. Western blot analysis revealed that Notch2HCS mutant splenocytes had increased phospho-Akt and phospho-Jun N-terminal kinase, and gene expression analysis of splenic CD19+ B cells demonstrated induction of Hes1 and Hes5 in Notch2HCS mutants. Anti-Notch2 antibodies decreased MZ B cells in control and Notch2HCS mice. In conclusion, Notch2HCS mutant mice have increased mature B cells in the MZ of the spleen.


Assuntos
Linfócitos B/citologia , Síndrome de Hajdu-Cheney/imunologia , Mutação , Receptor Notch2/genética , Baço/imunologia , Animais , Linfócitos B/metabolismo , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Síndrome de Hajdu-Cheney/genética , Tecido Linfoide/imunologia , Tecido Linfoide/metabolismo , Camundongos , Camundongos Transgênicos , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Baço/citologia , Baço/metabolismo
12.
Am J Pathol ; 188(6): 1430-1446, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29545197

RESUMO

Mice harboring Notch2 mutations replicating Hajdu-Cheney syndrome (Notch2tm1.1ECan) have osteopenia and exhibit an increase in splenic marginal zone B cells with a decrease in follicular B cells. Whether the altered B-cell allocation is responsible for the osteopenia of Notch2tm1.1ECan mutants is unknown. To determine the effect of NOTCH2 activation in B cells on splenic B-cell allocation and skeletal phenotype, a conditional-by-inversion (COIN) Hajdu-Cheney syndrome allele of Notch2 (Notch2[ΔPEST]COIN) was used. Cre recombination generates a permanent Notch2ΔPEST allele expressing a transcript for which sequences coding for the proline, glutamic acid, serine, and threonine-rich (PEST) domain are replaced by a stop codon. CD19-Cre drivers were backcrossed into Notch2[ΔPEST]COIN/[ΔPEST]COIN to generate CD19-specific Notch2ΔPEST/ΔPEST mutants and control Notch2[ΔPEST]COIN/[ΔPEST]COIN littermates. There was an increase in marginal zone B cells and a decrease in follicular B cells in the spleen of CD19Cre/WT;Notch2ΔPEST/ΔPEST mice, recapitulating the splenic phenotype of Notch2tm1.1ECan mice. The effect was reproduced when the NOTCH1 intracellular domain was induced in CD19-expressing cells (CD19Cre/WT;RosaNotch1/WT mice). However, neither CD19Cre/WT;Notch2ΔPEST/ΔPEST nor CD19Cre/WT;RosaNotch1/WT mice had a skeletal phenotype. Moreover, splenectomies in Notch2tm1.1ECan mice did not reverse their osteopenic phenotype. In conclusion, Notch2 activation in CD19-expressing cells determines B-cell allocation in the spleen but has no skeletal consequences.


Assuntos
Antígenos CD19/metabolismo , Linfócitos B/citologia , Síndrome de Hajdu-Cheney/patologia , Homeostase , Músculo Esquelético/citologia , Mutação , Receptor Notch2/fisiologia , Animais , Linfócitos B/metabolismo , Feminino , Síndrome de Hajdu-Cheney/genética , Síndrome de Hajdu-Cheney/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Esquelético/metabolismo
13.
J Biol Chem ; 292(29): 12232-12244, 2017 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-28592489

RESUMO

Individuals with Hajdu-Cheney syndrome (HCS) present with osteoporosis, and HCS is associated with NOTCH2 mutations causing deletions of the proline-, glutamic acid-, serine-, and threonine-rich (PEST) domain that are predicted to enhance NOTCH2 stability and cause gain-of-function. Previously, we demonstrated that mice harboring Notch2 mutations analogous to those in HCS (Notch2HCS) are severely osteopenic because of enhanced bone resorption. We attributed this phenotype to osteoclastic sensitization to the receptor activator of nuclear factor-κB ligand and increased osteoblastic tumor necrosis factor superfamily member 11 (Tnfsf11) expression. Here, to determine the individual contributions of osteoclasts and osteoblasts to HCS osteopenia, we created a conditional-by-inversion (Notch2COIN ) model in which Cre recombination generates a Notch2ΔPEST allele expressing a Notch2 mutant lacking the PEST domain. Germ line Notch2COIN inversion phenocopied the Notch2HCS mutant, validating the model. To activate Notch2 in osteoclasts or osteoblasts, Notch2COIN mice were bred with mice expressing Cre from the Lyz2 or the BGLAP promoter, respectively. These crosses created experimental mice harboring a Notch2ΔPEST allele in Cre-expressing cells and control littermates expressing a wild-type Notch2 transcript. Notch2COIN inversion in Lyz2-expressing cells had no skeletal consequences and did not affect the capacity of bone marrow macrophages to form osteoclasts in vitro In contrast, Notch2COIN inversion in osteoblasts led to generalized osteopenia associated with enhanced bone resorption in the cancellous bone compartment and with suppressed endocortical mineral apposition rate. Accordingly, Notch2 activation in osteoblast-enriched cultures from Notch2COIN mice induced Tnfsf11 expression. In conclusion, introduction of the HCS mutation in osteoblasts, but not in osteoclasts, causes osteopenia.


Assuntos
Doenças Ósseas Metabólicas/etiologia , Síndrome de Hajdu-Cheney/fisiopatologia , Mutação , Osteoblastos/metabolismo , Receptor Notch2/genética , Transdução de Sinais , Alelos , Animais , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Células da Medula Óssea/patologia , Células Cultivadas , Cruzamentos Genéticos , Feminino , Deleção de Genes , Síndrome de Hajdu-Cheney/imunologia , Síndrome de Hajdu-Cheney/metabolismo , Síndrome de Hajdu-Cheney/patologia , Humanos , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/patologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Osteoblastos/patologia , Osteoclastos/metabolismo , Osteoclastos/patologia , Domínios e Motivos de Interação entre Proteínas , Receptor Notch2/metabolismo , Regulação para Cima
14.
J Cell Biochem ; 119(7): 6016-6023, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29575203

RESUMO

Glucocorticoids in excess suppress osteoblast function and cause osteoporosis. We demonstrated that cortisol induces the expression of selected Notch receptors in osteoblasts, revealing a potential mechanism for the skeletal effects of glucocorticoids. However, it remains to be determined whether increased expression of Notch receptors results into enhanced signaling. Following activation of Notch, its intracellular domain (NICD) binds to the DNA-associated protein recombination signal binding protein for immunoglobulin kappa-J region (RBPJ) and induces the expression of target genes such as Hey1, Hey2, and HeyL. To determine whether glucocorticoids modulate Notch signaling in the skeleton, 1 month old wild-type mice were administered prednisolone or placebo and sacrificed after 72 h, and gene expression was analyzed in femoral bone. Prednisolone induced Tsc22d3, a glucocorticoid target gene, and suppressed Hey1 and HeyL expression, which is indicative of inhibited Notch receptor activity or direct Hey downregulation. To determine the mechanisms of Hey suppression, wild-type osteoblast-enriched cells were seeded on the Notch cognate ligand Delta-like (DLL)1 or transfected with constructs expressing the NOTCH1 NICD fragment and exposed to either cortisol or vehicle. Cortisol opposed the induction of mRNA and heterogeneous nuclear RNA for Hey1, Hey2, and HeyL by DLL1, but had no effect on mRNA stability, indicating that glucocorticoids inhibit Hey expression by transcriptional mechanisms. Transactivation studies and electrophoretic mobility shift assays revealed that cortisol did not oppose RBPJ-mediated transcription or RBPJ/DNA interactions, respectively. In conclusion, glucocorticoids suppress expression of Hey1, Hey2, and HeyL in osteoblasts by RBPJ-independent transcriptional mechanisms.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/antagonistas & inibidores , Proteínas de Ciclo Celular/antagonistas & inibidores , Regulação da Expressão Gênica/efeitos dos fármacos , Osteoblastos/metabolismo , Prednisolona/farmacologia , Receptor Notch1/metabolismo , Fatores de Transcrição/metabolismo , Animais , Glucocorticoides/farmacologia , Hidrocortisona/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Receptor Notch1/genética , Proteínas Repressoras/antagonistas & inibidores , Fatores de Transcrição/genética
15.
J Cell Biochem ; 119(11): 9334-9345, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30010214

RESUMO

Nuclear factor of activated T cells (NFAT) c2 is important for the immune response and it compensates for NFATc1 for its effects on osteoclastogenesis, but its role in this process is not established. To study the function of NFATc2 in the skeleton, Nfatc2loxP/loxP mice, where the Nfact2 exon 2 is flanked by loxP sequences, were created and mated with mice expressing the Cre recombinase under the control of the Lyz2 promoter. Bone marrow-derived macrophage (BMM) from Lyz2Cre/WT ;Nfatc2Δ/Δ mice cultured in the presence of macrophage-colony stimulating factor and receptor activator of NF-κB ligand exhibited a decrease in the number and size of osteoclasts and a smaller sealing zone when compared to BMMs from Nfatc2loxP/loxP littermate controls. Bone resorption was decreased in osteoclasts from Lyz2Cre/WT ;Nfatc2Δ/Δ mice. This demonstrates that NFATc2 is necessary for optimal osteoclast maturation and function in vitro. Male and female Lyz2Cre/WT ;Nfatc2Δ/Δ mice did not exhibit an obvious skeletal phenotype by microcomputed tomography (µCT) at either 1 or 4 months of age when compared to Nfatc2loxP/loxP sex-matched littermates. Bone histomorphometry confirmed the µCT results, and conditional 4-month-old Lyz2Cre/WT ;Nfatc2Δ/Δ mice did not exhibit changes in parameters of bone histomorphometry. In conclusion, NFATc2 is necessary for optimal osteoclastogenesis in vitro, but its downregulation in the myeloid lineage has no consequences in skeletal remodeling in vivo.


Assuntos
Fatores de Transcrição NFATC/metabolismo , Osteoclastos/citologia , Osteoclastos/metabolismo , Osteogênese/fisiologia , Animais , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Feminino , Immunoblotting , Masculino , Camundongos , Camundongos Mutantes , Fatores de Transcrição NFATC/genética , Osteogênese/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Microtomografia por Raio-X
16.
J Biol Chem ; 291(39): 20643-60, 2016 09 23.
Artigo em Inglês | MEDLINE | ID: mdl-27507811

RESUMO

The signaling pathway downstream of stimulation of receptor activator of nuclear factor κB (RANK) by RANK ligand is crucial for osteoclastogenesis. RANK recruits TNF receptor-associated factor 6 (TRAF6) to TRAF6-binding sites (T6BSs) in the RANK cytoplasmic tail (RANKcyto) to trigger downstream osteoclastogenic signaling cascades. RANKcyto harbors an additional highly conserved domain (HCR) that also activates crucial signaling during RANK-mediated osteoclastogenesis. However, the functional cross-talk between T6BSs and the HCR in the RANK signaling complex remains unclear. To characterize the cross-talk between T6BSs and the HCR, we screened TRAF6-interacting proteins using a proteomics approach. We identified Vav3 as a novel TRAF6 binding partner and evaluated the functional importance of the TRAF6-Vav3 interaction in the RANK signaling complex. We demonstrated that the coiled-coil domain of TRAF6 interacts directly with the Dbl homology domain of Vav3 to form the RANK signaling complex independent of the TRAF6 ubiquitination pathway. TRAF6 is recruited to the RANKcyto mutant, which lacks T6BSs, via the Vav3 interaction; conversely, Vav3 is recruited to the RANKcyto mutant, which lacks the IVVY motif, via the TRAF6 interaction. Finally, we determined that the TRAF6-Vav3 interaction resulting from cross-talk between T6BSs and the IVVY motif in RANKcyto enhances downstream NF-κB, MAPK, and NFATc1 activation by further strengthening TRAF6 signaling, thereby inducing RANK-mediated osteoclastogenesis. Thus, Vav3 is a novel TRAF6 interaction partner that functions in the activation of cooperative signaling between T6BSs and the IVVY motif in the RANK signaling complex.


Assuntos
Sistema de Sinalização das MAP Quinases/fisiologia , Complexos Multiproteicos/metabolismo , Osteoclastos/metabolismo , Proteínas Proto-Oncogênicas c-vav/metabolismo , Receptor Ativador de Fator Nuclear kappa-B/metabolismo , Fator 6 Associado a Receptor de TNF/metabolismo , Motivos de Aminoácidos , Linhagem Celular , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Complexos Multiproteicos/genética , Fatores de Transcrição NFATC/genética , Fatores de Transcrição NFATC/metabolismo , Osteoclastos/citologia , Proteínas Proto-Oncogênicas c-vav/genética , Receptor Ativador de Fator Nuclear kappa-B/genética , Fator 6 Associado a Receptor de TNF/genética , Ubiquitinação/fisiologia
17.
J Biol Chem ; 290(15): 9660-73, 2015 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-25716317

RESUMO

The signaling pathway downstream of TNF receptor (TNFR) is involved in the induction of a wide range of cellular processes, including cell proliferation, activation, differentiation, and apoptosis. TNFR-associated factor 2 (TRAF2) is a key adaptor molecule in TNFR signaling complexes that promotes downstream signaling cascades, such as nuclear factor-κB (NF-κB) and mitogen-activated protein kinase activation. TRAF-interacting protein (TRIP) is a known cellular binding partner of TRAF2 and inhibits TNF-induced NF-κB activation. Recent findings that TRIP plays a multifunctional role in antiviral response, cell proliferation, apoptosis, and embryonic development have increased our interest in exploring how TRIP can affect the TNFR-signaling pathway on a molecular level. In our current study, we demonstrated that TRIP is negatively involved in the TNF-induced inflammatory response through the down-regulation of proinflammatory cytokine production. Here, we demonstrated that the TRAF2-TRIP interaction inhibits Lys(63)-linked TRAF2 ubiquitination by inhibiting TRAF2 E3 ubiquitin (Ub) ligase activity. The TRAF2-TRIP interaction inhibited the binding of sphingosine 1-phosphate, which is a cofactor of TRAF2 E3 Ub ligase, to the TRAF2 RING domain. Finally, we demonstrated that TRIP functions as a negative regulator of proinflammatory cytokine production by inhibiting TNF-induced NF-κB activation. These results indicate that TRIP is an important cellular regulator of the TNF-induced inflammatory response.


Assuntos
Lisofosfolipídeos/metabolismo , Esfingosina/análogos & derivados , Fator 2 Associado a Receptor de TNF/metabolismo , Peptídeos e Proteínas Associados a Receptores de Fatores de Necrose Tumoral/metabolismo , Ubiquitina/metabolismo , Sítios de Ligação/genética , Citocinas/genética , Citocinas/metabolismo , Expressão Gênica , Células HEK293 , Células HeLa , Humanos , Immunoblotting , Lisina/genética , Lisina/metabolismo , NF-kappa B/metabolismo , Ligação Proteica , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Esfingosina/metabolismo , Fator 2 Associado a Receptor de TNF/genética , Peptídeos e Proteínas Associados a Receptores de Fatores de Necrose Tumoral/genética , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Ubiquitinação
18.
J Biol Chem ; 289(52): 35868-81, 2014 Dec 26.
Artigo em Inglês | MEDLINE | ID: mdl-25359771

RESUMO

Genetic mutations in osteoclastogenic genes are closely associated with osteopetrotic bone diseases. Genetic defects in OSTM1 (osteopetrosis-associated transmembrane protein 1) cause autosomal recessive osteopetrosis in humans. In particular, OSTM1 mutations that exclude the transmembrane domain might lead to the production of a secreted form of truncated OSTM1. However, the precise role of the secreted form of truncated OSTM1 remains unknown. In this study, we analyzed the functional role of truncated OSTM1 in osteoclastogenesis. Here, we showed that a secreted form of truncated OSTM1 binds to the cell surface of osteoclast (OC) precursors and inhibits the formation of multinucleated OCs through the reduction of cell fusion and survival. Truncated OSTM1 significantly inhibited the expression of OC marker genes through the down-regulation of the BLIMP1 (B lymphocyte-induced maturation protein 1)-NFATc1 (nuclear factor of activated T cells c1) axis. Finally, we demonstrated that truncated OSTM1 reduces lipopolysaccharide-induced bone destruction in vivo. Thus, these findings suggest that autosomal recessive osteopetrosis patients with an OSTM1 gene mutation lacking the transmembrane domain produce a secreted form of truncated OSTM1 that inhibits osteoclastogenesis.


Assuntos
Proteínas de Membrana/metabolismo , Fatores de Transcrição NFATC/metabolismo , Osteoclastos/fisiologia , Fatores de Transcrição/metabolismo , Animais , Reabsorção Óssea/imunologia , Reabsorção Óssea/metabolismo , Diferenciação Celular , Fusão Celular , Sobrevivência Celular , Células Cultivadas , Regulação para Baixo , Expressão Gênica , Lipopolissacarídeos/farmacologia , Masculino , Proteínas de Membrana/genética , Camundongos Endogâmicos C57BL , Osteoclastos/imunologia , Osteoporose/imunologia , Osteoporose/metabolismo , Fator 1 de Ligação ao Domínio I Regulador Positivo , Transdução de Sinais
19.
Vet Microbiol ; 297: 110193, 2024 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-39116640

RESUMO

Porcine epidemic diarrhea virus is attenuated upon adaptation to cell culture. Exclusively genomic mutations have been traced to the ORF3 gene of the laboratory strains. Previous attempts to express the protein were unsuccessful. We sought to express the ORF3 protein in both mammalian and bacteria cells as a prerequisite for investigation of the protein's role. For prokaryotic expression, two vector systems, pET28-a(+) and pGEX-4T-1 were constructed and expressed in Escherichia coli cells. For eukaryotic analyses, ORF3/pEGFP-C1 vector constructs were expressed in human embryonic, green monkey kidney and mouse fibrous cells. Intriguingly, there was minimal expression of the ORF3 gene. Following a documented hint that truncated ORF3 revealed higher expression, ORF3 gene was truncated. The simple modular architecture research tool analysis predicted two transmembrane domains between amino acid (aa) 41-63 and aa 76-98. Consequently, we generated two fragments; ORF-N (aa 1-98) inclusive of transmembrane domains and ORF3-C (aa 99-224). These truncated sequences were constructed as the whole gene here referred to as ORF3 wild type (wt). Coomassie blue stained gels revealed bands of ORF3-C expressed as a fusion protein of 17.5 and 39 kDa in pET28-a(+) and pGEX-4T-1 vectors, respectively. In contrast, ORF3-N was not. Additionally, ORF3-N induction decreased total cellular proteins suggesting inhibition of protein synthesis or metabolism. Solubility tests carried out at 30 °C, 25 °C and 18 °C showed that ORF3 formed inclusion bodies. Similar findings were observed in mammalian cells. Noteworthy, morphological distortions appeared in mammalian cells expressing ORF3 protein or its truncated mutants suggesting significance in host viability.


Assuntos
Vírus da Diarreia Epidêmica Suína , Animais , Vírus da Diarreia Epidêmica Suína/genética , Camundongos , Humanos , Suínos , Chlorocebus aethiops , Proteínas Virais/genética , Proteínas Virais/metabolismo , Fases de Leitura Aberta , Linhagem Celular , Escherichia coli/genética , Células HEK293
20.
J Clin Immunol ; 32(6): 1360-71, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22711011

RESUMO

PURPOSE: Osteoclasts (OCs) are multinucleated giant cells that resorb bone matrix. Accelerated bone destruction by OCs might cause several metabolic bone-related diseases, such as osteoporosis and inflammatory bone loss. D-pinitol (3-O-methyl-D-chiro-inositol) is a prominent component of dietary legumes and is actively converted to D-chiro-inositol, which is a putative insulin-like mediator. In this study, we analyzed the effect of D-chiro-inositol on OC differentiation. METHODS: To analyze the role of D-chiro-inositol on OC differentiation, we examined OC differentiation by the three types of osteoclastogenesis cultures with tartrate-resistant acid phosphatase (TRAP) staining and solution assay. Then, we carried out cell fusion assay with purified TRAP(+) mononuclear OC precursors. Finally, we analyzed the effect of D-chiro-inositol on OC maker expression in response to the regulation of nuclear factor of activated T cells c1 (NFATc1). RESULTS: We demonstrated that D-chiro-inositol acts as an inhibitor of receptor activator of NF-κB ligand-induced OC differentiation. The formation of multinucleated OCs by cell-cell fusion is reduced by treatment with D-chiro-inositol in a dose-dependent manner. In addition, we demonstrated that D-chiro-inositol inhibits the expression of several osteoclastogenic genes by down-regulating NFATc1. CONCLUSIONS: We have shown that D-chiro-inositol is negatively involved in osteoclastogenesis through the inhibition of multinucleated OC formation by cell-cell fusion. The expression of NFATc1 was significantly down-regulated by D-chiro-inositol in OCs and consequently, the expression of OC marker genes was significantly reduced. Hence, these results show that D-chiro-inositol might be a good candidate to treat inflammatory bone-related diseases or secondary osteoporosis in diabetes mellitus.


Assuntos
Regulação para Baixo/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Células Gigantes/efeitos dos fármacos , Inositol/farmacologia , Fatores de Transcrição NFATC/genética , Osteoclastos/efeitos dos fármacos , Ligante RANK/genética , Animais , Biomarcadores/metabolismo , Diferenciação Celular/efeitos dos fármacos , Fusão Celular , Linhagem Celular , Relação Dose-Resposta a Droga , Células Gigantes/patologia , Humanos , Inositol/análogos & derivados , Camundongos , Fatores de Transcrição NFATC/metabolismo , Osteoclastos/citologia , Osteoclastos/metabolismo , Ligante RANK/metabolismo , Estereoisomerismo
SELEÇÃO DE REFERÊNCIAS
Detalhe da pesquisa