RESUMO
Increasing evidence has demonstrated the important role of autophagy in skeletal homeostasis; however, the role of autophagy in craniofacial bone development and acquisition is largely unknown. In this study, we investigated the effect of autophagy suppression on craniofacial bone acquisition by deleting Fip200 or Atg5, two essential autophagy genes, using Osterix-Cre (Osx-Cre). We found that the Osx-Cre transgene mildly decreased the bone mass of parietal bone but not frontal bone, and did not affect cranial base bone mass in adult mice. In the cranial vault, Fip200 or Atg5 deletion similarly decreased 50% bone mass of neural crest-derived frontal bone; Atg5 deletion decreased 50% and Fip200 deletion decreased 30% bone mass of mesoderm-derived parietal bone. In the cranial base, Fip200 or Atg5 deletion similarly decreased 30% bone mass of neural crest-derived presphenoid bone; Atg5 deletion decreased 30% and Fip200 deletion decreased 16% bone mass of mesoderm-derive basioccipital bone. Lastly, we used doxycycline treatment to inhibit the Osx-Cre expression until 2 months of age and showed that postnatal Fip200 deletion led to cranial vault bone mass decrease in association with a small increase in both bone volume/tissue volume and tissue mineral density. Altogether, this study demonstrated the important role of autophagy in craniofacial bone acquisition during development and postnatal growth.
Assuntos
Autofagia/fisiologia , Desenvolvimento Ósseo/fisiologia , Ossos Faciais/crescimento & desenvolvimento , Crânio/crescimento & desenvolvimento , Animais , Camundongos , Camundongos TransgênicosRESUMO
The lateral habenula (LHb) is a well-established brain region involved in depressive disorders. Synaptic transmission of the LHb neurons is known to be enhanced by stress exposure; however, little is known about genetic modulators within the LHb that respond to stress. Using recently developed molecular profiling methods by phosphorylated ribosome capture, we obtained transcriptome profiles of stress responsive LHb neurons during acute physical stress. Among such genes, we found that KCNB1 (Kv2.1 channel), a delayed rectifier and voltage-gated potassium channel, exhibited increased expression following acute stress exposure. To determine the roles of KCNB1 on LHb neurons during stress, we injected short hairpin RNA (shRNA) against the kcnb1 gene to block its expression prior to stress exposure. We observed that the knockdown of KCNB1 altered the basal firing pattern of LHb neurons. Although KCNB1 blockade did not rescue despair-like behaviors in acute learned helplessness (aLH) animals, we found that KCNB1 knockdown prevented the enhancement of synaptic strength in LHb neuron after stress exposure. This study suggests that KCNB1 may contribute to shape stress responses by regulating basal firing patterns and neurotransmission intensity of LHb neurons.
RESUMO
Focal adhesion kinase (FAK) has important functions in bone homeostasis but its role in early osteoprogenitor cells is unknown. We show herein that mice lacking FAK in Dermo1-expressing cells exhibited low bone mass and decreased osteoblast number. Mechanistically, FAK-deficient early osteoprogenitor cells had decreased proliferation and significantly reduced mammalian/mechanistic target of rapamycin complex 1 (mTORC1) signaling, a central regulator of cell growth and proliferation. Furthermore, our data showed that the pharmacological inhibition of FAK kinase-dependent function alone was sufficient to decrease the proliferation and compromise the mineralization of early osteoprogenitor cells. In contrast to the Fak deletion in early osteoprogenitor cells, FAK loss in Col3.6 Cre-targeted osteoblasts did not cause bone loss, and Fak deletion in osteoblasts did not affect proliferation, differentiation, and mTORC1 signaling but increased the level of active proline-rich tyrosine kinase 2 (PYK2), which belongs to the same non-receptor tyrosine kinase family as FAK. Importantly, mTORC1 signaling in bone marrow stromal cells (BMSCs) was reduced if FAK kinase was inhibited at the early osteogenic differentiation stage. In contrast, mTORC1 signaling in BMSCs was not affected if FAK kinase was inhibited at a later osteogenic differentiation stage, in which, however, the concomitant inhibition of both FAK kinase and PYK2 kinase reduced mTORC1 signaling. In summary, our data suggest that FAK promotes early osteoprogenitor cell proliferation by enhancing mTORC1 signaling via its kinase-dependent function and the loss of FAK in osteoblasts can be compensated by the upregulated active PYK2. © 2020 American Society for Bone and Mineral Research.
Assuntos
Osteogênese , Animais , Proliferação de Células , Feminino , Quinase 1 de Adesão Focal/metabolismo , Quinase 2 de Adesão Focal/metabolismo , Proteína-Tirosina Quinases de Adesão Focal , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação , Transdução de SinaisRESUMO
The receptor activator of nuclear factor-κB (RANK) protein activates various protein kinase signaling cascades, including those involving NF-κB, mitogen-activated protein kinase (MAPK), and Bruton tyrosine kinase (Btk)/tyrosine-protein kinase Tec. However, the mechanism underlying the negative regulation of RANK by downstream signaling molecules remains unclear. Here, we report that Src homology 3 domain and cysteine-rich domain-containing protein 2 (STAC2) is a novel RANK ligand-inducible protein that negatively regulates RANK-mediated osteoclast formation. STAC2 physically interacts with RANK and inhibits the formation of the RANK signaling complex, which contains Grb-2-associated binder 2 (Gab2) and phospholipase Cγ2 (PLCγ2), thus leading to the suppression of RANK-mediated NF-κB and MAPK activation. Furthermore, STAC2 overexpression limits Btk/Tec-mediated PLCγ2 phosphorylation via the interaction between STAC2 and Btk/Tec. Taken together, our results reveal a novel mechanism whereby RANK signaling is restricted by its physical interaction with STAC2.
Assuntos
Proteínas do Tecido Nervoso/metabolismo , Receptor Ativador de Fator Nuclear kappa-B/metabolismo , Transdução de Sinais , Proteínas Adaptadoras de Transdução de Sinal , Tirosina Quinase da Agamaglobulinemia/metabolismo , Animais , Células da Medula Óssea/citologia , Diferenciação Celular/efeitos dos fármacos , Fator Estimulador de Colônias de Macrófagos/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/genética , Osteoclastos/citologia , Osteoclastos/metabolismo , Osteogênese/efeitos dos fármacos , Fosfolipase C gama/metabolismo , Fosfoproteínas/metabolismo , Fosforilação , Ligação Proteica , Proteínas Tirosina Quinases/metabolismo , Ligante RANK/farmacologia , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
A reduction in trabecular bone mass is often associated with an increase in marrow fat in osteoporotic bones. The molecular mechanisms underlying this inverse correlation are incompletely understood. Here, we report that mice lacking tuberous sclerosis 1 (Tsc1) in Osterix-expressing cells had a significant decrease in trabecular bone mass characterized by decreased osteoblastogenesis, increased osteoclastogenesis, and increased bone marrow adiposity in vivo. In vitro study showed that Tsc1-deficient bone marrow stromal cells (BMSCs) had decreased proliferation, decreased osteogenic differentiation, and increased adipogenic differentiation in association with the downregulation of Wnt/ß-catenin signaling. Mechanistically, TSC1 deficiency led to autophagy suppression and consequent Notch1 protein increase, which mediated the GSK3ß-independent ß-catenin degradation. Together, our results indicate that Tsc1 controls the balance between osteoblast and adipocyte differentiation of BMSCs. © 2018 American Society for Bone and Mineral Research.
Assuntos
Adipócitos/citologia , Autofagia , Diferenciação Celular , Osteoblastos/citologia , Receptores Notch/metabolismo , Transdução de Sinais , Proteína 1 do Complexo Esclerose Tuberosa/metabolismo , beta Catenina/metabolismo , Adipócitos/metabolismo , Adipogenia , Animais , Medula Óssea/metabolismo , Células da Medula Óssea/metabolismo , Osso Esponjoso/patologia , Regulação para Baixo , Fêmur/patologia , Deleção de Genes , Fator Estimulador de Colônias de Macrófagos/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Modelos Biológicos , Tamanho do Órgão , Osteoblastos/metabolismo , Osteogênese , Fator de Transcrição Sp7/metabolismo , Proteína 1 do Complexo Esclerose Tuberosa/deficiênciaRESUMO
The receptor activator of NF-κB (RANK) and immunoreceptor tyrosine-based activation motif (ITAM)-containing adaptors are essential factors involved in regulating osteoclast formation and bone remodeling. Here, we identify early estrogen-induced gene 1 (EEIG1) as a novel RANK ligand (RANKL)-inducible protein that physically interacts with RANK and further associates with Gab2, PLCγ2 and Tec/Btk kinases upon RANKL stimulation. EEIG1 positively regulates RANKL-induced osteoclast formation, likely due to its ability to facilitate RANKL-stimulated PLCγ2 phosphorylation and NFATc1 induction. In addition, an inhibitory peptide designed to block RANK-EEIG1 interaction inhibited RANKL-induced bone destruction by reducing osteoclast formation. Together, our results identify EEIG1 as a novel RANK signaling component controlling RANK-mediated osteoclast formation, and suggest that targeting EEIG1 might represent a new therapeutic strategy for the treatment of pathological bone resorption.
Assuntos
Células da Medula Óssea/metabolismo , Proteínas de Transporte/genética , Osteoclastos/metabolismo , Ligante RANK/genética , Receptor Ativador de Fator Nuclear kappa-B/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Reabsorção Óssea/genética , Reabsorção Óssea/metabolismo , Reabsorção Óssea/patologia , Proteínas de Transporte/metabolismo , Diferenciação Celular , Linhagem Celular , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Fatores de Transcrição NFATC/genética , Fatores de Transcrição NFATC/metabolismo , Osteoclastos/citologia , Osteoclastos/efeitos dos fármacos , Peptídeos/farmacologia , Fosfolipase C gama/genética , Fosfolipase C gama/metabolismo , Fosforilação/efeitos dos fármacos , Ligação Proteica , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Proteínas , Ligante RANK/metabolismo , Receptor Ativador de Fator Nuclear kappa-B/antagonistas & inibidores , Receptor Ativador de Fator Nuclear kappa-B/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Macrophage colony-stimulating factor (M-CSF) stimulation results in the production of reactive oxygen species (ROS) that participate in the proliferation of monocyte/macrophage. However, the molecular mechanisms whereby ROS modulate the signaling processes of M-CSF remain poorly defined. We report here that the redox-sensitive Src homology region 2 domain-containing phosphatase 1 (SHP1) is a critical regulator of M-CSF-mediated signaling in bone marrow monocyte/macrophage lineage cells (BMMs). Application of diphenylene iodonium (DPI) inhibited the responses of BMMs to M-CSF, including ROS production, cell proliferation, and phosphorylation of c-Fms as well as Akt kinase, but not of MAP kinases such as ERK, p38, and JNK. Dysregulation of SHP1 by overexpression or RNA interference in BMMs showed that SHP1 specifically regulates PI3 kinase (PI3K)/Akt signaling, but not MAP kinases in a redox-dependent manner, thereby regulating proliferation of BMMs through cyclins D1 and D2. These findings demonstrate that M-CSF-mediated ROS generation leads to SHP1 oxidation, which promotes cell proliferation through the PI3K/Akt-dependent signaling pathway.
Assuntos
Proliferação de Células , Fator Estimulador de Colônias de Macrófagos/metabolismo , Macrófagos/citologia , Monócitos/citologia , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Animais , Western Blotting , Células Cultivadas , Ciclina D/metabolismo , Vetores Genéticos , Peróxido de Hidrogênio/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Oniocompostos/farmacologia , Oxirredução , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Interferência de RNA , Receptor de Fator Estimulador de Colônias de Macrófagos/metabolismo , Retroviridae/patogenicidade , Transdução de Sinais , Especificidade por Substrato , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
A specific and sensitive fluorescence-based method was developed for the imaging of microbe-induced HOCl production. Furthermore, we demonstrate dual oxidase (DUOX)-mediated HOCl generation in the mucosa of live animals providing a novel insight into mucosal innate immunity.
Assuntos
Fenômenos Fisiológicos Bacterianos , Fungos/fisiologia , Ácido Hipocloroso/metabolismo , Imagem Molecular/métodos , Animais , Sobrevivência Celular , Drosophila melanogaster/imunologia , Drosophila melanogaster/microbiologia , Humanos , Imunidade Inata , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiologia , NADPH Oxidases/metabolismo , Neutrófilos/citologia , Neutrófilos/microbiologia , Fagossomos/metabolismo , Rodaminas/químicaRESUMO
Regulation of the formation and function of bone-resorbing osteoclasts (OCs) is a key to understanding the pathogenesis of skeletal disorders. Gene-targeting studies have shown that the RANK signaling pathway plays a critical role in OC differentiation and function. Although pharmaceutical blockade of RANK may be a viable strategy for preventing bone destruction, RANK is implicated in multiple biological processes. Recently, a cytoplasmic motif of RANK was identified that may be specifically involved in OC differentiation. Here, we developed a cell-permeable inhibitor termed the RANK receptor inhibitor (RRI), which targets this motif. The RRI peptide blocked RANKL-induced OC formation from murine bone marrow-derived macrophages. Furthermore, RRI inhibited the resorptive function of OCs and induced OC apoptosis. Treatment with the peptide impaired downstream signaling of RANK linked to Vav3, Rac1, and Cdc42 and resulted in disruptions of the actin cytoskeleton in differentiated OCs. In addition, RRI blocked inflammation-induced bone destruction and protected against ovariectomy-induced bone loss in mice. These data may be useful in the development of selective therapeutic agents for the treatment of osteoporosis and other bone diseases.