RESUMO
Denosumab is an anti-RANKL Ab that potently suppresses bone resorption, increases bone mass, and reduces fracture risk. Discontinuation of denosumab causes rapid rebound bone resorption and bone loss, but the molecular mechanisms are unclear. We generated humanized RANKL mice and treated them with denosumab to examine the cellular and molecular conditions associated with rebound resorption. Denosumab potently suppressed both osteoclast and osteoblast numbers in cancellous bone in humanized RANKL mice. The decrease in osteoclast number was not associated with changes in osteoclast progenitors in bone marrow. Long-term, but not short-term, denosumab administration reduced osteoprotegerin (OPG) mRNA in bone. Localization of OPG expression revealed that OPG mRNA is produced by a subpopulation of osteocytes. Long-term denosumab administration reduced osteocyte OPG mRNA, suggesting that OPG expression declines as osteocytes age. Consistent with this, osteocyte expression of OPG was more prevalent near the surface of cortical bone in humans and mice. These results suggest that new osteocytes are an important source of OPG in remodeling bone and that suppression of remodeling reduces OPG abundance by reducing new osteocyte formation. The lack of new osteocytes and the OPG they produce may contribute to rebound resorption after denosumab discontinuation.
Assuntos
Reabsorção Óssea , Osteócitos , Humanos , Camundongos , Animais , Osteócitos/metabolismo , Denosumab/farmacologia , Denosumab/uso terapêutico , Denosumab/metabolismo , Osteoprotegerina/genética , Osteoprotegerina/metabolismo , Osteoclastos/metabolismo , Reabsorção Óssea/metabolismoRESUMO
We identified a family with a UMOD gene mutation (C106F) resulting in glomerular inflammation and complement deposition. To determine if the observed phenotype is due to immune system activation by mutant uromodulin, a mouse strain with a homologous cysteine to phenylalanine mutation (C105F) in the UMOD gene was generated using CRISPR-Cas9 gene editing and the effect of this mutation on mononuclear phagocytic cells was examined. Mutant mice developed high levels of intracellular and secreted aggregated uromodulin, resulting in anti-uromodulin antibodies and circulating uromodulin containing immune complexes with glomerular deposition and kidney fibrosis with aging. F4/80+ and CD11c+ kidney cells phagocytize uromodulin. Differential gene expression analysis by RNA sequencing of F4/80+ phagocytic cells revealed activation of the activating transcription factor 5 (ATF5)-mediated stress response pathway in mutant mice. Phagocytosis of mutant uromodulin by cultured dendritic cells resulted in activation of the endoplasmic reticulum stress response pathway and markers of cell inactivation, an effect not seen with wild-type protein. Mutant mice demonstrate a twofold increase in T-regulatory cells, consistent with induction of immune tolerance, resulting in decreased inflammatory response and improved tissue repair following ischemia-reperfusion injury. The C105F mutation results in autoantibodies against aggregated misfolded protein with immune complex formation and kidney fibrosis. Aggregated uromodulin may induce dendritic cell tolerance following phagocytosis through an unfolded protein/endoplasmic reticulum stress response pathway, resulting in decreased inflammation following tissue injury.
Assuntos
Autoimunidade/imunologia , Estresse do Retículo Endoplasmático/imunologia , Retículo Endoplasmático/metabolismo , Fagócitos/imunologia , Uromodulina/metabolismo , Animais , Modelos Animais de Doenças , Fibrose/metabolismo , Fibrose/patologia , Rim/imunologia , Rim/patologia , Nefropatias/imunologia , Nefropatias/patologia , Camundongos , Fenótipo , Resposta a Proteínas não Dobradas/imunologia , Uromodulina/genética , Uromodulina/imunologiaRESUMO
Osteoprotegerin (OPG) inhibits the ability of receptor activator of nuclear factor κB (NF-κB) ligand (RANKL) to stimulate the differentiation, activity, and survival of bone-resorbing osteoclasts. Genetic studies in mice show that osteocytes are an important source of RANKL, but the cellular sources of OPG are unclear. We use conditional deletion of Tnfrsf11b, which encodes OPG, from different cell populations to identify functionally relevant sources of OPG in mice. Deletion from B lymphocytes and osteocytes, two cell types commonly thought to supply OPG, has little or no impact on bone mass. By contrast, deletion of Tnfrsf11b from osteoblasts increases bone resorption and reduces bone mass to an extent similar to germline deletion, demonstrating that osteoblasts are an essential source of OPG. These results suggest that, in addition to producing new bone matrix, osteoblasts also play an active role in terminating the resorption phase of the bone remodeling cycle by suppressing RANKL activity.
Assuntos
Osteoblastos/metabolismo , Osteoclastos/metabolismo , Osteócitos/metabolismo , Osteoprotegerina/metabolismo , Animais , Remodelação Óssea , Diferenciação Celular , Humanos , CamundongosRESUMO
Receptor activator of NFkB ligand (RANKL) is a TNF-family cytokine required for osteoclast formation, as well as immune cell and mammary gland development. It is produced as a membrane-bound protein that can be shed to form a soluble protein. We created mice harboring a sheddase-resistant form of RANKL, in which soluble RANKL is undetectable in the circulation. Lack of soluble RANKL does not affect bone mass or structure in growing mice but reduces osteoclast number and increases cancellous bone mass in adult mice. Nonetheless, the bone loss caused by estrogen deficiency is unaffected by the lack of soluble RANKL. Lymphocyte number, lymph node development, and mammary gland development are also unaffected by the absence of soluble RANKL. These results demonstrate that the membrane-bound form of RANKL is sufficient for most functions of this protein but that the soluble form does contribute to physiological bone remodeling in adult mice.
Assuntos
Reabsorção Óssea/metabolismo , Osteoclastos/citologia , Osteoclastos/metabolismo , Ligante RANK/metabolismo , Animais , Estrogênios/metabolismo , Feminino , Humanos , Linfonodos/metabolismo , Glândulas Mamárias Humanas/citologia , Glândulas Mamárias Humanas/metabolismo , Camundongos , OvariectomiaRESUMO
The recent identification of profilin1 mutations in 25 familial ALS cases has linked altered function of this cytoskeleton-regulating protein to the pathogenesis of motor neuron disease. To investigate the pathological role of mutant profilin1 in motor neuron disease, we generated transgenic lines of mice expressing human profilin1 with a mutation at position 118 (hPFN1G118V). One of the mouse lines expressing high levels of mutant human PFN1 protein in the brain and spinal cord exhibited many key clinical and pathological features consistent with human ALS disease. These include loss of lower (ventral horn) and upper motor neurons (corticospinal motor neurons in layer V), mutant profilin1 aggregation, abnormally ubiquitinated proteins, reduced choline acetyltransferase (ChAT) enzyme expression, fragmented mitochondria, glial cell activation, muscle atrophy, weight loss, and reduced survival. Our investigations of actin dynamics and axonal integrity suggest that mutant PFN1 protein is associated with an abnormally low filamentous/globular (F/G)-actin ratio that may be the underlying cause of severe damage to ventral root axons resulting in a Wallerian-like degeneration. These observations indicate that our novel profilin1 mutant mouse line may provide a new ALS model with the opportunity to gain unique perspectives into mechanisms of neurodegeneration that contribute to ALS pathogenesis.
Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Encéfalo/metabolismo , Mutação de Sentido Incorreto , Profilinas/biossíntese , Medula Espinal/metabolismo , Substituição de Aminoácidos , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/patologia , Animais , Encéfalo/patologia , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Transgênicos , Profilinas/genética , Medula Espinal/patologiaRESUMO
The cytokine receptor activator of nuclear factor kappa B ligand (RANKL), encoded by the Tnfsf11 gene, is essential for osteoclastogenesis and previous studies have shown that deletion of the Tnfsf11 gene using a Dmp1-Cre transgene reduces osteoclast formation in cancellous bone by more than 70%. However, the Dmp1-Cre transgene used in those studies leads to recombination in osteocytes, osteoblasts, and lining cells making it unclear whether one or more of these cell types produce the RANKL required for osteoclast formation in cancellous bone. Because osteoblasts, osteocytes, and lining cells have distinct locations and functions, distinguishing which of these cell types are sources of RANKL is essential for understanding the orchestration of bone remodeling. To distinguish between these possibilities, we have now created transgenic mice expressing the Cre recombinase under the control of regulatory elements of the Sost gene, which is expressed in osteocytes but not osteoblasts or lining cells in murine bone. Activity of the Sost-Cre transgene in osteocytes, but not osteoblast or lining cells, was confirmed by crossing Sost-Cre transgenic mice with tdTomato and R26R Cre-reporter mice, which express tdTomato fluorescent protein or LacZ, respectively, only in cells expressing the Cre recombinase or their descendants. Deletion of the Tnfsf11 gene in Sost-Cre mice led to a threefold decrease in osteoclast number in cancellous bone and increased cancellous bone mass, mimicking the skeletal phenotype of mice in which the Tnfsf11 gene was deleted using the Dmp1-Cre transgene. These results demonstrate that osteocytes, not osteoblasts or lining cells, are the main source of the RANKL required for osteoclast formation in remodeling cancellous bone.
Assuntos
Remodelação Óssea , Divisão Celular , Osteoclastos/citologia , Osteócitos/metabolismo , Ligante RANK/metabolismo , Animais , Densidade Óssea , Camundongos , Camundongos Transgênicos , Osteoblastos/metabolismo , Ligante RANK/genética , Recombinação GenéticaRESUMO
Receptor activator of NF-κB ligand (RANKL) is a TNFα-like cytokine that is produced by a diverse set of lineage-specific cells and is involved in a wide variety of physiological processes that include skeletal remodeling, lymph node organogenesis, mammary gland development, and thermal regulation. Consistent with these diverse functions, control of RANKL expression is accomplished in a cell-specific fashion via a set of at least 10 regulatory enhancers that are located up to 170 kb upstream of the gene's transcriptional start site. Here we examined the in vivo consequence of introducing a contiguous DNA segment containing these components into a genetically deleted RANKL null mouse strain. In contrast to RANKL null littermates, null mice containing the transgene exhibited normalized body size, skeletal development, and bone mass as well as normal bone marrow cavities, normalized spleen weights, and the presence of developed lymph nodes. These mice also manifested normalized reproductive capacity, including the ability to lactate and to produce normal healthy litters. Consistent with this, the transgene restored endogenous-like RANKL transcript levels in several RANKL-expressing tissues. Most importantly, restoration of RANKL expression from this segment of DNA was fully capable of rescuing the complex aberrant skeletal and immune phenotype of the RANKL null mouse. RANKL also restored appropriate levels of B220+ IgM+ and B220+ IgD+ B cells in spleen. Finally, we found that RANKL expression from this transgene was regulated by exogenously administered 1,25(OH)2 D3 , parathyroid hormone (PTH), and lipopolysaccharide (LPS), thus recapitulating the ability of these same factors to regulate the endogenous gene. These findings fully highlight the properties of the Tnfsf11 gene locus predicted through previous in vitro dissection. We conclude that the mouse Tnfsf11 gene locus identified originally through unbiased chromatin immunoprecipitation with DNA microarray (ChIP-chip) analysis contains the necessary genetic information to direct appropriate tissue-specific and factor-regulated RANKL expression in vivo.
Assuntos
DNA/genética , Ligante RANK/deficiência , Ligante RANK/genética , Sequências Reguladoras de Ácido Nucleico/genética , Transcrição Gênica , Animais , Linfócitos B/citologia , Linfócitos B/efeitos dos fármacos , Linfócitos B/metabolismo , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Colecalciferol/farmacologia , Lâmina de Crescimento/efeitos dos fármacos , Lâmina de Crescimento/metabolismo , Humanos , Lipopolissacarídeos/farmacologia , Tecido Linfoide/metabolismo , Camundongos , Camundongos Transgênicos , Especificidade de Órgãos/efeitos dos fármacos , Osteoclastos/efeitos dos fármacos , Osteoclastos/metabolismo , Osteogênese/efeitos dos fármacos , Hormônio Paratireóideo/farmacologia , Fenótipo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Linfócitos T/citologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Transcrição Gênica/efeitos dos fármacos , TransgenesRESUMO
The syndrome of hereditary 1,25-dihydroxyvitamin D-resistant rickets (HVDRR) is a genetic disease of altered mineral homeostasis due to mutations in the vitamin D receptor (VDR) gene. It is frequently, but not always, accompanied by the presence of alopecia. Mouse models that recapitulate this syndrome have been prepared through genetic deletion of the Vdr gene and are characterized by the presence of rickets and alopecia. Subsequent studies have revealed that VDR expression in hair follicle keratinocytes protects against alopecia and that this activity is independent of the protein's ability to bind 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. In the present study, we introduced into VDR-null mice a human VDR (hVDR) bacterial artificial chromosome minigene containing a mutation that converts leucine to serine at amino acid 233 in the hVDR protein, which prevents 1,25(OH)2D3 binding. We then assessed whether this transgene recreated features of the HVDRR syndrome without alopecia. RT-PCR and Western blot analysis in one strain showed an appropriate level of mutant hVDR expression in all tissues examined including skin. The hVDR-L233S mutant failed to rescue the aberrant systemic and skeletal phenotype characteristic of the VDR null mouse due to the inability of the mutant receptor to activate transcription after treatment with 1,25(OH)2D3. Importantly, however, neither alopecia nor the dermal cysts characteristic of VDR-null mice were observed in the skin of these hVDR-L233S mutant mice. This study confirms that we have created a humanized mouse model of HVDRR without alopecia that will be useful in defining additional features of this syndrome and in identifying potential novel functions of the unoccupied VDR.
Assuntos
Alopecia/genética , Modelos Animais de Doenças , Raquitismo Hipofosfatêmico Familiar/genética , Camundongos Transgênicos , Receptores de Calcitriol/genética , Alopecia/complicações , Alopecia/patologia , Substituição de Aminoácidos , Animais , Resistência a Medicamentos/genética , Raquitismo Hipofosfatêmico Familiar/complicações , Raquitismo Hipofosfatêmico Familiar/patologia , Humanos , Leucina/genética , Camundongos , Camundongos Endogâmicos C57BL , Serina/genética , Vitamina D/análogos & derivados , Vitamina D/farmacologiaRESUMO
The biological actions of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) are mediated by the vitamin D receptor (VDR), which is expressed in numerous target tissues in a cell type-selective manner. Recent studies using genomic analyses and recombineered bacterial artificial chromosomes (BACs) have defined the specific features of mouse and human VDR gene loci in vitro. In the current study, we introduced recombineered mouse and human VDR BACs as transgenes into mice and explored their expression capabilities in vivo. Individual transgenic mouse strains selectively expressed BAC-derived mouse or human VDR proteins in appropriate vitamin D target tissues, thereby recapitulating the tissue-specific expression of endogenous mouse VDR. The mouse VDR transgene was also regulated by 1,25(OH)2D3 and dibutyryl-cAMP. When crossed into a VDR-null mouse background, both transgenes restored wild-type basal as well as 1,25(OH)2D3-inducible gene expression patterns in the appropriate tissues. This maneuver resulted in the complete rescue of the aberrant phenotype noted in the VDR-null mouse, including systemic features associated with altered calcium and phosphorus homeostasis and disrupted production of parathyroid hormone and fibroblast growth factor 23, and abnormalities associated with the skeleton, kidney, parathyroid gland, and the skin. This study suggests that both mouse and human VDR transgenes are capable of recapitulating basal and regulated expression of the VDR in the appropriate mouse tissues and restore 1,25(OH)2D3 function. These results provide a baseline for further dissection of mechanisms integral to mouse and human VDR gene expression and offer the potential to explore the consequence of selective mutations in VDR proteins in vivo.
Assuntos
Cromossomos Artificiais Bacterianos/genética , Cromossomos Artificiais Bacterianos/metabolismo , Regulação da Expressão Gênica , Receptores de Calcitriol/genética , Receptores de Calcitriol/metabolismo , Animais , Western Blotting , CMP Cíclico/análogos & derivados , CMP Cíclico/metabolismo , Feminino , Humanos , Camundongos , Camundongos Transgênicos , Fenótipo , Reação em Cadeia da Polimerase Via Transcriptase ReversaAssuntos
Linfotoxina-alfa/imunologia , Nódulos Linfáticos Agregados/crescimento & desenvolvimento , Baço/crescimento & desenvolvimento , Timo/crescimento & desenvolvimento , Animais , Linfócitos B/citologia , Linfócitos B/imunologia , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , História do Século XX , Linfotoxina-alfa/genética , Camundongos , Camundongos Mutantes , Nódulos Linfáticos Agregados/citologia , Nódulos Linfáticos Agregados/imunologia , Baço/citologia , Baço/imunologia , Timo/citologia , Timo/imunologiaRESUMO
Aging increases oxidative stress and osteoblast apoptosis and decreases bone mass, whereas forkhead box O (FoxO) transcription factors defend against oxidative stress by activating genes involved in free radical scavenging and apoptosis. Conditional deletion of FoxO1, FoxO3, and FoxO4 in 3-month-old mice resulted in an increase in oxidative stress in bone and osteoblast apoptosis and a decrease in the number of osteoblasts, the rate of bone formation, and bone mass at cancellous and cortical sites. The effect of the deletion on osteoblast apoptosis was cell autonomous and resulted from oxidative stress. Conversely, overexpression of a FoxO3 transgene in mature osteoblasts decreased oxidative stress and osteoblast apoptosis and increased osteoblast number, bone formation rate, and vertebral bone mass. We conclude that FoxO-dependent oxidative defense provides a mechanism to handle the oxygen free radicals constantly generated by the aerobic metabolism of osteoblasts and is thereby indispensable for bone mass homeostasis.
Assuntos
Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Osteoblastos/metabolismo , Estresse Oxidativo , Animais , Apoptose , Proteínas de Ciclo Celular , Células Cultivadas , Proteína Forkhead Box O1 , Proteína Forkhead Box O3 , Deleção de Genes , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Osteoblastos/citologia , Transgenes , Regulação para CimaRESUMO
Osteocytes, former osteoblasts buried within bone, are thought to orchestrate skeletal adaptation to mechanical stimuli. However, it remains unknown whether hormones control skeletal homeostasis through actions on osteocytes. Parathyroid hormone (PTH) stimulates bone remodeling and may cause bone loss or bone gain depending on the balance between bone resorption and formation. Herein, we demonstrate that transgenic mice expressing a constitutively active PTH receptor exclusively in osteocytes exhibit increased bone mass and bone remodeling, as well as reduced expression of the osteocyte-derived Wnt antagonist sclerostin, increased Wnt signaling, increased osteoclast and osteoblast number, and decreased osteoblast apoptosis. Deletion of the Wnt co-receptor LDL related receptor 5 (LRP5) attenuates the high bone mass phenotype but not the increase in bone remodeling induced by the transgene. These findings demonstrate that PTH receptor signaling in osteocytes increases bone mass and the rate of bone remodeling through LRP5-dependent and -independent mechanisms, respectively.
Assuntos
Remodelação Óssea/fisiologia , Osso e Ossos/anatomia & histologia , Osso e Ossos/fisiologia , Osteócitos/fisiologia , Receptor Tipo 1 de Hormônio Paratireóideo/genética , Receptores de Hormônios Paratireóideos/fisiologia , Animais , Proteínas da Matriz Extracelular/genética , Humanos , Camundongos , Camundongos Transgênicos , Osteoblastos/citologia , Osteoclastos/citologia , Fosfoproteínas/genética , Transdução de Sinais/fisiologiaRESUMO
Aldosterone classically promotes unidirectional transepithelial sodium transport, thereby regulating blood volume and blood pressure. Recently, both clinical and experimental studies have suggested additional, direct roles for aldosterone in the cardiovascular system. To evaluate aldosterone activation of cardiomyocyte mineralocorticoid receptors, transgenic mice overexpressing 11beta-hydroxysteroid dehydrogenase type 2 in cardiomyocytes were generated using the mouse alpha-myosin heavy chain promoter. This enzyme converts glucocorticoids to receptor-inactive metabolites, allowing aldosterone occupancy of cardiomyocyte mineralocorticoid receptors. Transgenic mice were normotensive but spontaneously developed cardiac hypertrophy, fibrosis, and heart failure and died prematurely on a normal salt diet. Eplerenone, a selective aldosterone blocker, ameliorated this phenotype. These studies confirm the deleterious consequences of inappropriate activation of cardiomyocyte mineralocorticoid receptors by aldosterone and reveal a tonic inhibitory role of glucocorticoids in preventing such outcomes under physiological conditions. In addition, these data support the hypothesis that aldosterone blockade may provide additional therapeutic benefit in the treatment of heart failure.