RESUMO
Single-cell RNA-seq has led to novel designations for mesenchymal cells associated with bone as well as multiple designations for what appear to be the same cell type. The main goals of this study were to increase the amount of single-cell RNA sequence data for osteoblasts and osteocytes, to compare cells from the periosteum to those inside bone, and to clarify the major categories of cell types associated with murine bone. We created an atlas of murine bone-associated cells by harmonizing published datasets with in-house data from cells targeted by Osx1-Cre and Dmp1-Cre driver strains. Cells from periosteal bone were analyzed separately from those isolated from the endosteum and trabecular bone. Over 100,000 mesenchymal cells were mapped to reveal 11 major clusters designated fibro-1, fibro-2, chondrocytes, articular chondrocytes, tenocytes, adipo-Cxcl12 abundant reticular (CAR), osteo-CAR, preosteoblasts, osteoblasts, osteocytes, and osteo-X, the latter defined in part by periostin expression. Osteo-X, osteo-CAR, and preosteoblasts were closely associated with osteoblasts at the trabecular bone surface. Wnt16 was expressed in multiple cell types from the periosteum but not in cells from endocortical or cancellous bone. Fibro-2 cells, which express markers of stem cells, localized to the periosteum but not trabecular bone in adult mice. Suppressing bone remodeling eliminated osteoblasts and altered gene expression in preosteoblasts but did not change the abundance or location of osteo-X or osteo-CAR cells. These results provide a framework for identifying bone cell types in murine single-cell RNA-seq datasets and suggest that osteoblast progenitors reside near the surface of remodeling bone.
Assuntos
Células-Tronco Mesenquimais , Osteoblastos , Osteócitos , Periósteo , Animais , Camundongos , Condrócitos/metabolismo , Condrócitos/citologia , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/citologia , Osteoblastos/metabolismo , Osteoblastos/citologia , Osteócitos/metabolismo , Osteócitos/citologia , Periósteo/citologia , Periósteo/metabolismo , Análise de Célula Única , Camundongos Endogâmicos C57BLRESUMO
The vitamin D endocrine system regulates mineral homeostasis through its activities in the intestine, kidney, and bone. Terminal activation of vitamin D3 to its hormonal form, 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3), occurs in the kidney via the cytochrome P450 enzyme CYP27B1. Despite its importance in vitamin D metabolism, the molecular mechanisms underlying the regulation of the gene for this enzyme, Cyp27b1, are unknown. Here, we identified a kidney-specific control module governed by a renal cell-specific chromatin structure located distal to Cyp27b1 that mediates unique basal and parathyroid hormone (PTH)-, fibroblast growth factor 23 (FGF23)-, and 1,25(OH)2D3-mediated regulation of Cyp27b1 expression. Selective genomic deletion of key components within this module in mice resulted in loss of either PTH induction or FGF23 and 1,25(OH)2D3 suppression of Cyp27b1 gene expression; the former loss caused a debilitating skeletal phenotype, whereas the latter conferred a quasi-normal bone mineral phenotype through compensatory homeostatic mechanisms involving Cyp24a1 We found that Cyp27b1 is also expressed at low levels in non-renal cells, in which transcription was modulated exclusively by inflammatory factors via a process that was unaffected by deletion of the kidney-specific module. These results reveal that differential regulation of Cyp27b1 expression represents a mechanism whereby 1,25(OH)2D3 can fulfill separate functional roles, first in the kidney to control mineral homeostasis and second in extra-renal cells to regulate target genes linked to specific biological responses. Furthermore, we conclude that these mouse models open new avenues for the study of vitamin D metabolism and its involvement in therapeutic strategies for human health and disease.
Assuntos
25-Hidroxivitamina D3 1-alfa-Hidroxilase/biossíntese , Calcitriol/metabolismo , Colecalciferol/metabolismo , Regulação Enzimológica da Expressão Gênica/fisiologia , Homeostase/fisiologia , Rim/metabolismo , 25-Hidroxivitamina D3 1-alfa-Hidroxilase/genética , Animais , Calcitriol/genética , Colecalciferol/genética , Fator de Crescimento de Fibroblastos 23 , Deleção de Genes , Camundongos , Especificidade de Órgãos/fisiologia , Vitamina D3 24-Hidroxilase/biossíntese , Vitamina D3 24-Hidroxilase/genéticaRESUMO
Receptor activator of NF-κB ligand (RANKL) is a TNF-like cytokine which mediates diverse physiological functions including bone remodeling and immune regulation. RANKL has been identified in atherosclerotic lesions; however, its role in atherosclerotic plaque development remains elusive. An enhancer located 75 kb upstream of the murine Rankl gene's transcription start site designated D5 is important for its calciotropic hormone- and cytokine-mediated expression. Here, we determined the impact of RANKL levels in atherosclerotic plaque development in the D5 enhancer-null (D5-/- ) mice in an atherogenic Apoe-/- background fed a high-fat diet (HFD). Rankl mRNA transcripts were increased in aortic arches and thoracic aortae of Apoe-/- mice; however, this increase was blunted in Apoe-/- ;D5-/- mice. Similarly, higher Rankl transcripts were identified in splenic T lymphocytes in Apoe-/- mice, and their levels were reduced in Apoe-/- ;D5-/- mice. When analyzed by micro-computed tomography (µCT), atherosclerotic plaque calcification was identified in six out of eight Apoe-/- mice, whereas only one out of eight Apoe-/- ;D5-/- mice developed plaque calcification after 12 weeks of HFD. However, following 18 weeks of HFD challenge, all of Apoe-/- and Apoe-/- ;D5-/- animals developed atherosclerotic plaque calcification. Likewise, atherosclerotic lesion sizes were site-specifically reduced in the aortic arch of Apoe-/- ;D5-/- mice at initial stage of atherosclerosis and this effect was diminished as atherosclerosis proceeded to a more advanced stage. Our data suggest that deletion of the RANKL D5 enhancer delays the progression of atherosclerotic plaque development and plaque calcification in hypercholesterolemic mice. This work provides important insight into RANKL's regulatory role in atherosclerosis. J. Cell. Biochem. 118: 4240-4253, 2017. © 2017 Wiley Periodicals, Inc.
Assuntos
Calcinose/metabolismo , Elementos Facilitadores Genéticos , Hipercolesterolemia/complicações , Placa Aterosclerótica/metabolismo , Ligante RANK/genética , Deleção de Sequência , Animais , Sequência de Bases , Calcinose/etiologia , Calcinose/genética , Dieta Hiperlipídica , Modelos Animais de Doenças , Progressão da Doença , Feminino , Camundongos , Camundongos Knockout , Placa Aterosclerótica/etiologia , Placa Aterosclerótica/genéticaRESUMO
Epidemiological and clinical data suggest adverse cardiovascular outcomes with respect to vitamin D deficiency. Here, we explored the effects of vitamin D in atherosclerotic plaque calcification in vivo by utilizing vitamin D receptor (Vdr)-deficient mice in an Apoe-/- background. Animals were fed a high-fat diet (HFD) for either 12 or 18 weeks and then examined for atherosclerotic plaque development. In order to prevent calcium deficiency, Vdr-/- and Apoe-/- ;Vdr-/- animals were fed a high-calcium rescue diet prior to initiation of the HFD feeding and supplemented with high-calcium water during HFD feeding. Although calcium supplementation improved bone mass in Vdr-/- and Apoe-/- ;Vdr-/- mice, neither strain was fully rescued. Systemic inflammatory responses observed in the absence of VDR were exaggerated in Apoe-/- mice. Whereas, hyperlipidemic profiles seen in Apoe-/- mice were ameliorated in the absence of VDR. Micro-computed tomography (µCT) analysis revealed that six out of eight Apoe-/- animals developed atherosclerotic plaque calcification following 12 weeks of HFD feeding and 100% of the mice developed plaque calcification after 18 weeks. In contrast, although atherosclerotic lesions were evident in Apoe-/- ;Vdr-/- mice at 12 and 18 weeks of HFD challenge, none of these animals developed plaque calcification at either time point. The active vitamin D hormone, 1,25(OH)2 D3 likely increased calcification in aortic smooth muscle cells perhaps by directly modulating expression of Alpl, Rankl, and Opg. Our data suggest that the absence of VDR inhibits atherosclerotic plaque calcification in hypercholesterolemic Apoe-/- mice, providing additional insight into the role of vitamin D in atherosclerotic plaque calcification. J. Cell. Biochem. 118: 1050-1064, 2017. © 2016 Wiley Periodicals, Inc.
Assuntos
Apolipoproteínas E/deficiência , Cálcio/administração & dosagem , Hipercolesterolemia/complicações , Placa Aterosclerótica/prevenção & controle , Receptores de Calcitriol/deficiência , Animais , Apolipoproteínas E/genética , Dieta Hiperlipídica/efeitos adversos , Modelos Animais de Doenças , Feminino , Hidroxicolecalciferóis/metabolismo , Hipercolesterolemia/induzido quimicamente , Camundongos , Camundongos Knockout , Placa Aterosclerótica/diagnóstico por imagem , Placa Aterosclerótica/genética , Receptores de Calcitriol/genética , Microtomografia por Raio-XRESUMO
Bone mass declines with age but the mechanisms responsible remain unclear. Here we demonstrate that deletion of a conditional allele for Atg7, a gene essential for autophagy, from osteocytes caused low bone mass in 6-month-old male and female mice. Cancellous bone volume and cortical thickness were decreased, and cortical porosity increased, in conditional knock-out mice compared with control littermates. These changes were associated with low osteoclast number, osteoblast number, bone formation rate, and wall width in the cancellous bone of conditional knock-out mice. In addition, oxidative stress was higher in the bones of conditional knock-out mice as measured by reactive oxygen species levels in the bone marrow and by p66(shc) phosphorylation in L6 vertebra. Each of these changes has been previously demonstrated in the bones of old versus young adult mice. Thus, these results demonstrate that suppression of autophagy in osteocytes mimics, in many aspects, the impact of aging on the skeleton and suggest that a decline in autophagy with age may contribute to the low bone mass associated with aging.
Assuntos
Fêmur/metabolismo , Vértebras Lombares/metabolismo , Osteócitos/fisiologia , Envelhecimento , Animais , Autofagia , Proteína 7 Relacionada à Autofagia , Densidade Óssea , Diferenciação Celular , Células Cultivadas , Feminino , Fêmur/diagnóstico por imagem , Fêmur/patologia , Vértebras Lombares/diagnóstico por imagem , Vértebras Lombares/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Associadas aos Microtúbulos/deficiência , Proteínas Associadas aos Microtúbulos/genética , Osteoblastos/fisiologia , Osteoclastos/fisiologia , Estresse Oxidativo , Radiografia , Espécies Reativas de Oxigênio/metabolismoRESUMO
Chaperone-mediated autophagy (CMA) is a lysosome-dependent degradation pathway that eliminates proteins that are damaged, partially unfolded, or targeted for selective proteome remodeling. CMA contributes to several cellular processes, including stress response and proteostasis. Age-associated increase in cellular stressors and decrease in CMA contribute to pathologies associated with aging in various tissues. CMA contributes to bone homeostasis in young mice. An age-associated reduction in CMA was reported in osteoblast lineage cells; however, whether declining CMA contributes to skeletal aging is unknown. Herein we show that cellular stressors stimulate CMA in UAMS-32 osteoblastic cells. Moreover, the knockdown of an essential component of the CMA pathway, LAMP2A, sensitizes osteoblasts to cell death caused by DNA damage, ER stress, and oxidative stress. As elevations in these stressors are thought to contribute to age-related bone loss, we hypothesized that declining CMA contributes to the age-associated decline in bone formation by sensitizing osteoblast lineage cells to elevated stressors. To test this, we aged male CMA-deficient mice and controls up to 24 months of age and examined age-associated changes in bone mass and architecture. We showed that lack of CMA did not alter age-associated decline in bone mineral density as measured by dual x-ray absorptiometry (DXA). Moreover, microCT analysis performed at 24 months of age showed that vertebral cancellous bone volume, cortical thickness, and porosity of CMA-deficient and control mice were similar. Taken together, these results suggest that reduction of CMA does not contribute to age-related bone loss.
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Production of the cytokine receptor activator of NFκB ligand (RANKL) by lymphocytes has been proposed as a mechanism by which sex steroid deficiency causes bone loss. However, there have been no studies that functionally link RANKL expression in lymphocytes with bone loss in this condition. Herein, we examined whether RANKL expression in either B or T lymphocytes contributes to ovariectomy-induced bone loss in mice. Mice harboring a conditional RANKL allele were crossed with CD19-Cre or Lck-Cre mice to delete RANKL in B or T lymphocytes, respectively. Deletion of RANKL from either cell type had no impact on bone mass in estrogen-replete mice up to 7 months of age. However, mice lacking RANKL in B lymphocytes were partially protected from the bone loss caused by ovariectomy. This protection occurred in cancellous, but not cortical, bone and was associated with a failure to increase osteoclast numbers in the conditional knock-out mice. Deletion of RANKL from T lymphocytes had no impact on ovariectomy-induced bone loss. These results demonstrate that lymphocyte RANKL is not involved in basal bone remodeling, but B cell RANKL does contribute to the increase in osteoclasts and cancellous bone loss that occurs after loss of estrogen.
Assuntos
Linfócitos B/metabolismo , Remodelação Óssea/imunologia , Osteoclastos/metabolismo , Osteoporose/metabolismo , Ligante RANK/metabolismo , Alelos , Animais , Linfócitos B/imunologia , Densidade Óssea/genética , Densidade Óssea/imunologia , Estrogênios/genética , Estrogênios/imunologia , Estrogênios/metabolismo , Feminino , Humanos , Camundongos , Camundongos Transgênicos , Osteoclastos/imunologia , Osteoporose/genética , Osteoporose/imunologia , Ovariectomia , Ligante RANK/genética , Ligante RANK/imunologia , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
Genetically modified mouse models have shaped our understanding of biological systems in both physiological and pathological conditions. For decades, mouse genome engineering has relied on transgenesis and spontaneous gene replacement in embryonic stem (ES) cells. While these technologies provided a wealth of knowledge, they remain imprecise and expensive to use. Recent advances in genome editing technologies such as the development of targetable nucleases, the improvement of delivery systems, and the simplification of targeting strategies now allow for the rapid, precise manipulation of the mouse genome. In this review article, we discuss novel methods and targeting strategies for the generation of mouse models for the study of bone and skeletal muscle biology.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Animais , Camundongos , Animais Geneticamente Modificados , Terapia Genética , Engenharia Genética/métodosRESUMO
Single-cell RNA sequencing has led to numerous novel designations for mesenchymal cell types associated with bone. Consequently, there are now multiple designations for what appear to be the same cell type. In addition, existing datasets contain relatively small numbers of mature osteoblasts and osteocytes and there has been no comparison of periosteal bone cells to those at the endosteum and trabecular bone. The main goals of this study were to increase the amount of single cell RNA sequence data for osteoblasts and osteocytes, to compare cells from the periosteum to those inside bone, and to clarify the major categories of cell types associated with murine bone. To do this, we created an atlas of murine bone-associated cells by harmonizing published datasets with in-house data from cells targeted by Osx1-Cre and Dmp1-Cre driver strains. Cells from periosteal bone were analyzed separately from those isolated from the endosteum and trabecular bone. Over 100,000 mesenchymal cells were mapped to reveal 11 major clusters designated fibro-1, fibro-2, chondrocytes, articular chondrocytes, tenocytes, adipo-CAR, osteo-CAR, pre-osteoblasts, osteoblasts, osteocytes, and osteo-X, the latter defined in part by Postn expression. Osteo-X, osteo-CAR, and pre-osteoblasts were closely associated with osteoblasts at the trabecular bone surface. Wnt16 was expressed in multiple cell types from the periosteum but not in any cells from endocortical or cancellous bone. Fibro-2 cells, which express markers of skeletal stem cells, localized to the periosteum but not trabecular bone in adult mice. Suppressing bone remodeling eliminated osteoblasts and altered gene expression in pre-osteoblasts but did not change the abundance or location of osteo-X or osteo-CAR cells. These results provide a framework for identifying bone cell types in murine single cell RNA sequencing datasets and suggest that osteoblast progenitors reside near the surface of remodeling bone.
RESUMO
Cre-mediated recombination is frequently used for cell type-specific loss of function (LOF) studies. A major limitation of this system is recombination in unwanted cell types. CRISPR interference (CRISPRi) has been used effectively for global LOF in mice. However, cell type-specific CRISPRi, independent of recombination-based systems, has not been reported. To test the feasibility of cell type-specific CRISPRi, we produced two novel knock-in mouse models that achieve gene suppression when used together: one expressing dCas9::KRAB under the control of a cell type-specific promoter and the other expressing a single guide RNA from a safe harbor locus. We then compared the phenotypes of mice in which the same gene was targeted by either CRISPRi or the Cre-loxP system, with cell specificity conferred by Dmp1 regulatory elements in both cases. We demonstrate that CRISPRi is effective for cell type-specific LOF and that it provides improved cell type-specificity compared to the Cre-loxP system.
RESUMO
Chaperone-mediated autophagy (CMA) is a protein degradation pathway that eliminates soluble cytoplasmic proteins that are damaged, incorrectly folded, or targeted for selective proteome remodeling. However, the role of CMA in skeletal homeostasis under physiological and pathophysiological conditions is unknown. To address the role of CMA for skeletal homeostasis, we deleted an essential component of the CMA process, namely Lamp2a, from the mouse genome. CRISPR-Cas9-based genome editing led to the deletion of both Lamp2a and Lamp2c, another Lamp2 isoform, producing Lamp2AC global knockout (L2ACgKO) mice. At 5 weeks of age female L2ACgKO mice had lower vertebral cancellous bone mass compared to wild-type (WT) controls, whereas there was no difference between genotypes in male mice at this age. The low bone mass of L2ACgKO mice was associated with elevated RANKL expression and the osteoclast marker genes Trap and Cathepsin K. At 18 weeks of age, both male and female L2ACgKO mice had lower vertebral cancellous bone mass compared to WT controls. The low bone mass of L2ACgKO mice was associated with increased osteoclastogenesis and decreased mineral deposition in cultured cells. Consistent with these findings, specific knockdown of Lamp2a in an osteoblastic cell line increased RANKL expression and decreased mineral deposition. Moreover, similar to what has been observed in other cell types, macroautophagy and proteasomal degradation were upregulated in CMA-deficient osteoblasts in culture. Thus, an increase in other protein degradation pathways may partially compensate for the loss of CMA in osteoblasts. Taken together, our results suggest that CMA plays a role in vertebral cancellous bone mass accrual in young adult mice and that this may be due to an inhibitory role of CMA on osteoclastogenesis or a positive role of CMA in osteoblast formation or function.
Assuntos
Autofagia , Osso Esponjoso/metabolismo , Proteína 2 de Membrana Associada ao Lisossomo/genética , Chaperonas Moleculares/genética , Osteoclastos/metabolismo , Coluna Vertebral/metabolismo , Animais , Calcificação Fisiológica , Feminino , Proteína 2 de Membrana Associada ao Lisossomo/metabolismo , Masculino , Camundongos , Camundongos Knockout , Chaperonas Moleculares/metabolismo , Tamanho do ÓrgãoRESUMO
The cytokine RANKL is essential for osteoclast formation during physiological and pathological bone resorption. RANKL also contributes to lymphocyte production, development of lymph nodes and mammary glands, as well as other biological activities. Transcriptional control of the Tnfsf11 gene, which encodes RANKL, is complex and involves distant regulatory regions. Nevertheless, cell culture studies suggest that an enhancer region near the transcription start site is involved in the control of Tnfsf11 expression by hormones such as 1,25-(OH)2 vitamin D3 and parathyroid hormone, as well as the sympathetic nervous system. To address the significance of this region in vivo, we deleted the sequence between -510 to -1413 bp, relative to Tnfsf11 exon 1, from mice using CRISPR-based gene editing. MicroCT analysis of the femur and fourth lumbar vertebra of enhancer knockout mice showed no differences in bone mass compared to wild type littermates at 5 weeks and 6 months of age, suggesting no changes in osteoclast formation. RNA extracted from the tibia, fifth lumbar vertebra, thymus, and spleen at 6 months of age also showed no reduction in Tnfsf11 mRNA abundance between these groups. However, maximal stimulation of Tnfsf11 mRNA abundance in cultured stromal cells by PTH was reduced approximately 40% by enhancer deletion, while stimulation by 1,25-(OH)2 vitamin D3 was unaffected. The abundance of B and T lymphocytes in the bone marrow did not differ between genotypes. These results demonstrate that the region between -510 and -1413 does not contribute to Tnfsf11 expression, osteoclast support, or lymphocyte production in mice under normal physiological conditions but may be involved in situations of elevated parathyroid hormone.
Assuntos
Densidade Óssea/fisiologia , Osteoclastos/fisiologia , Ligante RANK/genética , Animais , Sistemas CRISPR-Cas , Células Cultivadas , Feminino , Linfócitos/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Animais , Osteoclastos/citologia , Hormônio Paratireóideo/metabolismo , Regiões Promotoras Genéticas , Ligante RANK/metabolismo , Sequências Reguladoras de Ácido NucleicoRESUMO
Drawbacks of conditional gene deletion in mice include the need for extensive breeding and, often, a lack of cell type-specificity. CRISPR interference (CRISPRi) is an alternative approach for loss-of-function studies that inhibits expression by guiding a transcriptional repressor to the transcription start-site of target genes. However, there has been limited exploration of CRISPRi in mice. We tested the effectiveness of a single CRISPRi transgene broadly expressing a single guide RNA and a catalytically dead Cas9 fused to the KRAB repressor domain to suppress a well-characterized target gene, Tnfsf11. The phenotype of CRISPRi transgenic mice was compared to mice with germline deletion of Tnfsf11, which are osteopetrotic and do not form lymph nodes. High transgene expression mimicked gene deletion, with failure of lymph node development and classic signs of osteopetrosis such as high bone mass and failure of tooth eruption. Mice with low transgene expression were normal and mice with medium expression displayed an intermediate phenotype. Transgene expression in tissues from these mice correlated inversely with Tnfsf11 mRNA levels. These results demonstrate that a single CRISPRi transgene can effectively suppress a target gene in mice and suggest that this approach may be useful for cell type-specific loss-of-function studies.
Assuntos
Sistemas CRISPR-Cas/genética , Interferência de RNA , Transcrição Gênica , Animais , Vetores Genéticos/genética , Células HEK293 , Humanos , Lentivirus/genética , Camundongos , Camundongos Transgênicos , Ligante RANK/genética , RNA Guia de Cinetoplastídeos/genética , Transdução Genética , Transgenes/genéticaRESUMO
Fibroblast growth factor 23 (FGF23) is a bone-derived hormone involved in the control of phosphate (P) homeostasis and vitamin D metabolism. Despite advances, however, molecular details of this gene's regulation remain uncertain. In this report, we created mouse strains in which four epigenetically marked FGF23 regulatory regions were individually deleted from the mouse genome using CRISPR/Cas9 gene-editing technology, and the consequences of these mutations were then assessed on Fgf23 expression and regulation in vivo. An initial analysis confirmed that bone expression of Fgf23 and circulating intact FGF23 (iFGF23) were strongly influenced by both chronic dietary P treatment and acute injection of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. However, further analysis revealed that bone Fgf23 expression and iFGF23 could be rapidly upregulated by dietary P within 3 and 6 hours, respectively; this acute upregulation was lost in the FGF23-PKO mouse containing an Fgf23 proximal enhancer deletion but not in the additional enhancer-deleted mice. Of note, prolonged dietary P treatment over several days led to normalization of FGF23 levels in the FGF23-PKO mouse, suggesting added complexity associated with P regulation of FGF23. Treatment with 1,25(OH)2D3 also revealed a similar loss of Fgf23 induction and blood iFGF23 levels in this mouse. Finally, normal lipopolysaccharide (LPS) induction of Fgf23 expression was also compromised in the FGF23-PKO mouse, a result that, together with our previous report, indicates that the action of LPS on Fgf23 expression is mediated by both proximal and distal Fgf23 enhancers. These in vivo data provide key functional insight into the genomic enhancers through which Fgf23 expression is mediated.
Assuntos
Fatores de Crescimento de Fibroblastos/metabolismo , Regulação da Expressão Gênica , Animais , Osso e Ossos/metabolismo , Sistemas CRISPR-Cas , Calcitriol , Elementos Facilitadores Genéticos , Feminino , Fator de Crescimento de Fibroblastos 23 , Fatores de Crescimento de Fibroblastos/genética , Lipopolissacarídeos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosfatos/sangue , Regiões Promotoras GenéticasRESUMO
Fibroblast growth factor 23 (FGF23) production is regulated by both calciotropic hormones and inflammation. Consistent with this, elevated FGF23 levels are associated with inflammatory markers as well as parathyroid hormone (PTH) in various disease states, including chronic kidney disease (CKD). However, the molecular mechanisms underpinning Fgf23 transcription in response to these regulators are largely unknown. We therefore utilized chromatin immunoprecipitation followed by DNA sequencing (ChIP-seq) data from an osteocyte cell line to identify potential regulatory regions of the Fgf23 gene. Based on ChIP-seq analysis of enhancer-associated histone modifications, including H3K4 methylation and H3K9 acetylation, we discovered several potential enhancers for Fgf23, one of which was located 16kb upstream of the gene's transcriptional start site. Deletion of this putative enhancer from the mouse genome using CRISPR-Cas9 technology led to lower bone, thymus, and spleen expression of Fgf23 mRNA without altering circulating levels of the intact hormone, although as previously reported, only bone displayed significant basal expression. Nevertheless, lack of the -16kb enhancer blunted FGF23 upregulation in a tissue-specific manner by the acute inflammatory inducers lipopolysaccharide (LPS), interleukin-1-beta (IL-1ß), and tumor necrosis factor-alpha (TNFα) in bone, non-osseous tissues, and in circulation. Lack of the -16kb enhancer also inhibited PTH-induced bone Fgf23 mRNA. Moreover, the absence of this Fgf23 enhancer in an oxalate diet-induced murine CKD model prevented the early onset induction of osseous, renal, and thymic Fgf23 mRNA levels and led to a significant blunting of elevated circulating intact FGF23 levels. These results suggest that -16kb enhancer mediates the induction of Fgf23 by inflammation and PTH and facilitates the increase in FGF23 expression in a murine model of CKD. As exemplified herein, these Fgf23 enhancer-deleted mice will provide a unique model in which to study the role of FGF23 expression in inflammatory diseases.
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The vitamin D receptor (VDR) is the single known regulatory mediator of hormonal 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] in higher vertebrates. It acts in the nucleus of vitamin D target cells to regulate the expression of genes whose products control diverse, cell type-specific biological functions that include mineral homeostasis. In this Review we describe progress that has been made in defining new cellular sites of action of this receptor, the mechanisms through which this mediator controls the expression of genes, the biology that ensues, and the translational impact of this receptor on human health and disease. We conclude with a brief discussion of what comes next in understanding vitamin D biology and the mechanisms that underlie its actions.
Assuntos
Calcitriol , Regulação da Expressão Gênica/efeitos dos fármacos , Genômica/métodos , Receptores de Calcitriol , Pesquisa Translacional Biomédica/métodos , Animais , Calcitriol/genética , Calcitriol/metabolismo , Humanos , Receptores de Calcitriol/genética , Receptores de Calcitriol/metabolismoRESUMO
Receptor activator of nuclear factor-κB ligand (RANKL) is a tumor necrosis factor (TNF)-like cytokine that is necessary for osteoclast formation and survival. Elevated RANKL synthesis is associated with both increased osteoclast number and bone resorption. Earlier studies identified an enhancer 76 kb upstream of the Tnfsf11 transcriptional start site (TSS) termed RL-D5 or the distal control region (DCR) that modulates RANKL expression in response to PTH, 1,25(OH)2D3,, and an array of cytokines. Mice lacking RL-D5 exhibit high bone mass associated with decreased RANKL expression in bone, spleen, and thymus. In addition to RL-D5, genome-wide studies have identified 9 additional Tnfsf11 enhancers residing upstream of the gene's TSS, which provide RANKL cell type-specificity and responsiveness to local and systemic factors. ChIP-chip analyses has revealed inducible vitamin D receptor (VDR) and cAMP response element-binding protein (CREB) binding at an enhancer termed RL-D2 23 kb upstream of the Tnfsf11 TSS in osteoblastic ST2 cells. Herein, we use ChIP-seq analyses to confirm this finding and then delete this enhancer from the mouse genome to determine its physiological role in vivo. RL-D2(-/-) primary stromal cells showed decreased RANKL-induction by both forskolin and 1,25(OH)2D3 ex vivo. Consistent with this, the parathyroid hormone (PTH) induction of RANKL expression was significantly blunted in RL-D2(-/-) mice in vivo. In contrast, lack of RL-D2 had no effect on 1,25(OH)2D3 induction of RANKL in vivo. Similar to the results found in RL-D5(-/-) mice, lack of RL-D2 led to decreased skeletal RANKL expression, resulting in decreased osteoclast numbers and a progressive increase in bone mineral density. Lack of RL-D2 increased cancellous bone mass in femur and spine but did not alter femoral cortical bone thickness. These results highlight the role of distal enhancers in the regulation of RANKL expression by PTH and perhaps 1,25(OH)2D3 and suggest that the RL-D2 and RL-D5 enhancers contribute in either an additive or synergistic manner to regulate bone remodeling.
Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Osteoblastos/metabolismo , Hormônio Paratireóideo/farmacologia , Fenótipo , Ligante RANK/biossíntese , Elementos de Resposta , Animais , Calcitriol/farmacologia , Colforsina/farmacologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Camundongos , Camundongos Knockout , Tamanho do Órgão/efeitos dos fármacos , Tamanho do Órgão/genética , Osteoblastos/citologia , Ligante RANK/genética , Receptores de Calcitriol/genética , Receptores de Calcitriol/metabolismoRESUMO
Matrix metalloproteinase 13 (MMP13, collagenase-3) is a vital component for chondrocyte and osteoblast maturation, and is aberrantly expressed in numerous disease states. At the transcriptional level, Mmp13 is controlled by many different growth factors and hormones. Most notably, Mmp13 is regulated by the vitamin D hormone (1,25(OH)2D3), parathyroid hormone (PTH), and several cytokines. These activities occur through participation by the transcription factors VDR, RUNX2, FOS, JUN, and Osterix (OSX), respectively. Recently, we discovered that Mmp13 is regulated by elements quite distal to the transcriptional start site -10, -20, and -30kb upstream. These enhancers, along with minor contributions from the region proximal to the promoter, are responsible for the ligand inducible and, most strikingly, the basal activities of Mmp13 gene regulation. Here, we found that the actions of PTH and OSX do not occur through the -10kb VDR bound enhancer. Rather, the -30kb RUNX2 bound enhancer and the promoter proximal regions were essential for activity. Through RUNX2 deletion and OSX overexpression in cells, we showed a specific role for OSX in Mmp13 regulation. Finally, we created an in vivo CRISPR deleted -10kb enhancer mouse model. Despite normal bone density and growth, they fail to up-regulate Mmp13 in response to 1,25(OH)2D3. These data are consistent with those obtained through UAMS osteoblast cell culture and further define the specific roles of distal enhancers in the regulation of Mmp13.
Assuntos
Calcitriol/farmacologia , Elementos Facilitadores Genéticos , Metaloproteinase 13 da Matriz/genética , Osteoblastos/efeitos dos fármacos , Hormônio Paratireóideo/farmacologia , Fatores de Transcrição/genética , Animais , Linhagem Celular , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Feminino , Edição de Genes , Regulação da Expressão Gênica , Masculino , Metaloproteinase 13 da Matriz/metabolismo , Camundongos , Camundongos Knockout , Osteoblastos/citologia , Osteoblastos/metabolismo , Regiões Promotoras Genéticas , Receptores de Calcitriol/genética , Receptores de Calcitriol/metabolismo , Transdução de Sinais , Fator de Transcrição Sp7 , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
Autophagy maintains cell function and homeostasis by recycling intracellular components. This process is also required for morphological changes associated with maturation of some cell types. Osteoblasts are bone forming cells some of which become embedded in bone and differentiate into osteocytes. This transformation includes development of long cellular projections and a reduction in endoplasmic reticulum and mitochondria. We examined the role of autophagy in osteoblasts by deleting Atg7 using an Osterix1-Cre transgene, which causes recombination in osteoblast progenitors and their descendants. Mice lacking Atg7 in the entire osteoblast lineage had low bone mass and fractures associated with reduced numbers of osteoclasts and osteoblasts. Suppression of autophagy also reduced the amount of osteocyte cellular projections and led to retention of endoplasmic reticulum and mitochondria in osteocytes. These results demonstrate that autophagy in osteoblasts contributes to skeletal homeostasis and to the morphological changes associated with osteocyte formation.
Assuntos
Autofagia , Osso e Ossos/fisiologia , Osteoblastos/citologia , Osteócitos/citologia , Absorciometria de Fóton , Animais , Proteína 7 Relacionada à Autofagia/deficiência , Proteína 7 Relacionada à Autofagia/genética , Densidade Óssea , Células da Medula Óssea/citologia , Remodelação Óssea , Osso e Ossos/diagnóstico por imagem , Catalase/genética , Diferenciação Celular , Linhagem da Célula , Células Cultivadas , Retículo Endoplasmático/metabolismo , Feminino , Fraturas Ósseas/etiologia , Camundongos , Microscopia de Fluorescência , Mitocôndrias/metabolismo , Osteoblastos/metabolismo , Osteoclastos/citologia , Osteoclastos/metabolismo , Osteócitos/metabolismo , Osteogênese , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismoRESUMO
Insight into mechanisms that link the actions of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) to the regulation of gene expression has evolved extensively since the initial discovery of a nuclear protein known as the vitamin D receptor (VDR). Perhaps most important was the molecular cloning of this receptor which enabled its inclusion within the nuclear receptor gene family and further studies of both its structure and regulatory function. Current studies are now refocused on the vitamin D hormone's action at the genome, where VDR together with other transcription factors coordinates the recruitment of chromatin active coregulatory complexes that participate directly in the modification of gene output. These studies highlight the role of chromatin in the expression of genes and the dynamic impact of the epigenetic landscape that contextualizes individual gene loci thus influencing the VDR's transcriptional actions. In this chapter, we summarize advances made over the past few years in understanding vitamin D action on a genome-wide scale, focusing on overarching principles that have emerged at this level. Of particular significance is the finding that dynamic changes that occur to the genome during cellular differentiation at both genetic and epigenetic levels profoundly alter the ability of 1,25(OH)2D3 and its receptor to regulate gene expression. We address the broad impact of differentiation on specific epigenetic histone modifications that occur across the genome and the ability of the VDR to influence this activity at selected gene loci as well. These studies advance our understanding of not only vitamin D action but also of the complex and dynamic role played by the genome itself as a major determinant of VDR activity.