RESUMO
A goal of osteoporosis therapy is to restore lost bone with structurally sound tissue. Mice lacking the transcription factor nuclear matrix protein 4 (Nmp4, Zfp384, Ciz, ZNF384) respond to several classes of osteoporosis drugs with enhanced bone formation compared with wild-type (WT) animals. Nmp4-/- mesenchymal stem/progenitor cells (MSPCs) exhibit an accelerated and enhanced mineralization during osteoblast differentiation. To address the mechanisms underlying this hyperanabolic phenotype, we carried out RNA-sequencing and molecular and cellular analyses of WT and Nmp4-/- MSPCs during osteogenesis to define pathways and mechanisms associated with elevated matrix production. We determined that Nmp4 has a broad impact on the transcriptome during osteogenic differentiation, contributing to the expression of over 5,000 genes. Phenotypic anchoring of transcriptional data was performed for the hypothesis-testing arm through analysis of cell metabolism, protein synthesis and secretion, and bone material properties. Mechanistic studies confirmed that Nmp4-/- MSPCs exhibited an enhanced capacity for glycolytic conversion: a key step in bone anabolism. Nmp4-/- cells showed elevated collagen translation and secretion. The expression of matrix genes that contribute to bone material-level mechanical properties was elevated in Nmp4-/- cells, an observation that was supported by biomechanical testing of bone samples from Nmp4-/- and WT mice. We conclude that loss of Nmp4 increases the magnitude of glycolysis upon the metabolic switch, which fuels the conversion of the osteoblast into a super-secretor of matrix resulting in more bone with improvements in intrinsic quality.
Assuntos
Matriz Óssea/metabolismo , Células-Tronco Mesenquimais/metabolismo , Proteínas Associadas à Matriz Nuclear/genética , Osteoblastos/metabolismo , Osteogênese/genética , Fatores de Transcrição/genética , Animais , Calcificação Fisiológica/genética , Colágeno/genética , Colágeno/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Glicólise/genética , Camundongos , Camundongos Knockout , Osteoblastos/citologia , Osteoporose/metabolismo , RNA Mensageiro/metabolismoRESUMO
Modulation of the vitamin D receptor (VDR) with a ligand has the potential to be useful for the oral treatment of osteoporosis. One component of our lead generation strategy to identify synthetic ligands for VDR included a fragment based drug design approach. Screening of ligands in a VDR fluorescence polarization assay and a RXR/VDR conformation sensing assay resulted in the identification of multiple fragment hits (lean >0.30). These fragment scaffolds were subsequently evaluated for interaction with the VDR ligand binding domain using hydrogen-deuterium exchange (HDX) mass spectrometry. Significant protection of H/D exchange was observed for some fragments in helixes 3, 7, and 8 of the ligand binding domain, regions which are similar to those seen for the natural hormone VD3. The fragments appear to mimic the A-ring of VD3 thereby providing viable starting points for synthetic expansion.
Assuntos
Medição da Troca de Deutério , Compostos Orgânicos/farmacologia , Receptores de Calcitriol/metabolismo , Relação Dose-Resposta a Droga , Desenho de Fármacos , Ligantes , Espectrometria de Massas , Modelos Moleculares , Estrutura Molecular , Compostos Orgânicos/química , Relação Estrutura-AtividadeRESUMO
Autosomal dominant hypophosphatemic rickets (ADHR) is unique among the disorders involving Fibroblast growth factor 23 (FGF23) because individuals with R176Q/W and R179Q/W mutations in the FGF23 (176)RXXR(179)/S(180) proteolytic cleavage motif can cycle from unaffected status to delayed onset of disease. This onset may occur in physiological states associated with iron deficiency, including puberty and pregnancy. To test the role of iron status in development of the ADHR phenotype, WT and R176Q-Fgf23 knock-in (ADHR) mice were placed on control or low-iron diets. Both the WT and ADHR mice receiving low-iron diet had significantly elevated bone Fgf23 mRNA. WT mice on a low-iron diet maintained normal serum intact Fgf23 and phosphate metabolism, with elevated serum C-terminal Fgf23 fragments. In contrast, the ADHR mice on the low-iron diet had elevated intact and C-terminal Fgf23 with hypophosphatemic osteomalacia. We used in vitro iron chelation to isolate the effects of iron deficiency on Fgf23 expression. We found that iron chelation in vitro resulted in a significant increase in Fgf23 mRNA that was dependent upon Mapk. Thus, unlike other syndromes of elevated FGF23, our findings support the concept that late-onset ADHR is the product of gene-environment interactions whereby the combined presence of an Fgf23-stabilizing mutation and iron deficiency can lead to ADHR.
Assuntos
Raquitismo Hipofosfatêmico Familiar/genética , Fatores de Crescimento de Fibroblastos/genética , Deficiências de Ferro , Anemia Ferropriva/complicações , Animais , Raquitismo Hipofosfatêmico Familiar/fisiopatologia , Feminino , Fator de Crescimento de Fibroblastos 23 , Interação Gene-Ambiente , Glucuronidase/metabolismo , Hipofosfatemia/genética , Proteínas Klotho , Sistema de Sinalização das MAP Quinases , Masculino , Camundongos , Camundongos Transgênicos , Osteócitos/citologia , Osteomalacia/genética , Fenótipo , Estrutura Terciária de Proteína , RatosAssuntos
Medula Óssea/metabolismo , Osso e Ossos/metabolismo , Eritropoetina/farmacologia , Fatores de Crescimento de Fibroblastos/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Animais , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Células Cultivadas , Relação Dose-Resposta a Droga , Fator de Crescimento de Fibroblastos 23 , Fatores de Crescimento de Fibroblastos/sangue , Perfilação da Expressão Gênica , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Humanos , CamundongosRESUMO
The retinoic acid receptor-related orphan receptors alpha and gamma (RORalpha (NR1F1) and RORgamma (NR1F3)) are orphan nuclear receptors and perform critical roles in regulation of development, metabolism, and immune function. Cholesterol and cholesterol sulfate have been suggested to be RORalpha ligands, but the physiological significance is unclear. To date, no endogenous RORgamma ligands have been described. Here, we demonstrate that 7-oxygenated sterols function as high affinity ligands for both RORalpha and RORgamma by directly binding to their ligand-binding domains (K(i) approximately 20 nM), modulating coactivator binding, and suppressing the transcriptional activity of the receptors. One of the 7-oxygenated sterols, 7alpha-hydroxycholesterol (7alpha-OHC), serves as a key intermediate in bile acid metabolism, and we show that 7alpha-OHC modulates the expression of ROR target genes, including Glc-6-Pase and phosphoenolpyruvate carboxykinase, in an ROR-dependent manner. Furthermore, glucose output from hepatocytes is suppressed by 7alpha-OHC functioning as an RORalpha/gamma ligand. Thus, RORalpha and RORgamma are ligand-regulated members of the NR superfamily and may serve as sensors for 7-oxygenated sterols.
Assuntos
Hidroxicolesteróis/metabolismo , Membro 1 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismo , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismo , Animais , Linhagem Celular , Células Cultivadas , Imunoprecipitação da Cromatina , Células Hep G2 , Humanos , Espectrometria de Massas , Camundongos , Modelos Biológicos , Reação em Cadeia da Polimerase , Ligação Proteica/fisiologiaRESUMO
Atopic dermatitis (AD) is a chronic inflammatory skin disease that affects up to one in five children and millions of adults in developed countries. Clinically, AD skin lesions manifest as subacute and/or chronic lichenified eczematous plaques, which are often intensely pruritic and prone to secondary bacterial and viral infections. Despite the emergence of novel therapeutic agents, treatment options and outcomes for AD remain suboptimal. An improved understanding of AD pathogenesis may help improve patient outcomes. Dysregulated Th2-polarized skin inflammation and impaired skin barrier function interact to drive AD pathogenesis; however, much remains to be understood about the molecular mechanisms underlying this interplay. The current study used published clinical trial datasets to define a skin-related AD gene signature. This meta-analysis revealed significant reductions in IL1F7 transcripts (encodes IL-37) in AD patient samples. Reduced IL1F7 correlated with lower transcripts for key skin barrier function genes in the epidermal differentiation complex. Immunohistochemical analysis of normal (healthy) human skin specimens and an in vitro three-dimensional human skin model localized IL-37 protein to the epidermis. In comparison with normal human skin, IL-37 levels were decreased in AD patient skin. Addition of Th2 cytokines to the aforementioned in vitro three-dimensional skin model recapitulates key aspects of AD skin and was sufficient to reduce epidermal IL-37 levels. Image analysis also indicated close relationship between epidermal IL-37 and skin epidermal differentiation complex proteins. These findings suggest IL-37 is intimately linked to normal keratinocyte differentiation and barrier function and implicates IL-37 as a potential biomarker and therapeutic target for AD.
Assuntos
Dermatite Atópica/imunologia , Epiderme/patologia , Interleucina-1/metabolismo , Adulto , Azetidinas/uso terapêutico , Biópsia , Diferenciação Celular/imunologia , Dermatite Atópica/diagnóstico , Dermatite Atópica/tratamento farmacológico , Dermatite Atópica/patologia , Regulação para Baixo/imunologia , Epiderme/imunologia , Epiderme/metabolismo , Feminino , Perfilação da Expressão Gênica , Voluntários Saudáveis , Humanos , Queratinócitos/imunologia , Queratinócitos/metabolismo , Masculino , Pessoa de Meia-Idade , Purinas/uso terapêutico , Pirazóis/uso terapêutico , Índice de Gravidade de Doença , Sulfonamidas/uso terapêutico , Células Th2/imunologia , Células Th2/metabolismoRESUMO
The Th17 pathway has been implicated in autoimmune diseases. The retinoic acid receptor-related orphan receptor C2 (RORγt) is a master regulator of Th17 cells and controls the expression of IL-17A. RORγt is expressed primarily in IL-17A-producing lymphoid cells. Here we describe a virtual screen of the ligand-binding pocket and subsequent screen in a binding assay that identified the 1-benzyl-4',5'-dihydrospiro[piperidine-4,7'-thieno[2,3-c]pyran]-2'-carboxamide scaffold as a starting point for optimization of binding affinity and functional activity guided by structure-based design. Compound 12 demonstrated activity in a mouse PK/PD model and efficacy in an inflammatory arthritis mouse model that were used to define the level and duration of target engagement required for efficacy in vivo. Further optimization to improve ADME and physicochemical properties with guidance from simulations and modeling provided compound 22, which is projected to achieve the level and duration of target engagement required for efficacy in the clinic.
Assuntos
Ligantes , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismo , Tiofenos/química , Animais , Artrite/induzido quimicamente , Artrite/tratamento farmacológico , Artrite/patologia , Sítios de Ligação , Cristalografia por Raios X , Modelos Animais de Doenças , Desenho de Fármacos , Feminino , Meia-Vida , Humanos , Interleucina-17/genética , Interleucina-17/metabolismo , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Camundongos , Simulação de Dinâmica Molecular , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/química , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/genética , Ligação Proteica , Relação Estrutura-Atividade , Tiofenos/metabolismo , Tiofenos/farmacologia , Tiofenos/uso terapêuticoRESUMO
Vitamin D receptor (VDR) ligands are therapeutic agents for the treatment of psoriasis, osteoporosis, and secondary hyperparathyroidism. VDR ligands also show immense potential as therapeutic agents for autoimmune diseases and cancers of skin, prostate, colon, and breast as well as leukemia. However, the major side effect of VDR ligands that limits their expanded use and clinical development is hypercalcemia that develops as a result of the action of these compounds mainly on intestine. In order to discover VDR ligands with less hypercalcemia liability, we sought to identify tissue-selective VDR modulators (VDRMs) that act as agonists in some cell types and lack activity in others. Here, we describe LY2108491 and LY2109866 as nonsecosteroidal VDRMs that function as potent agonists in keratinocytes, osteoblasts, and peripheral blood mononuclear cells but show poor activity in intestinal cells. Finally, these nonsecosteroidal VDRMs were less calcemic in vivo, and LY2108491 exhibited more than 270-fold improved therapeutic index over the naturally occurring VDR ligand 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] in an in vivo preclinical surrogate model of psoriasis.
Assuntos
Acetatos/farmacologia , Sulfonatos de Arila/farmacologia , Receptores de Calcitriol/metabolismo , Tiofenos/farmacologia , Vitamina D/análogos & derivados , Vitamina D/farmacologia , Acetatos/síntese química , Acetatos/metabolismo , Animais , Sulfonatos de Arila/síntese química , Sulfonatos de Arila/metabolismo , Células CACO-2 , Calcitriol/metabolismo , Calcitriol/farmacologia , Proliferação de Células , Células Cultivadas , Neoplasias do Colo/metabolismo , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Feminino , Humanos , Hipercalcemia/metabolismo , Intestinos , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Ligantes , Camundongos , Camundongos Pelados , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Modelos Biológicos , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Psoríase/tratamento farmacológico , Ratos , Receptores de Calcitriol/agonistas , Transdução de Sinais , Especificidade da Espécie , Tiofenos/síntese química , Tiofenos/metabolismo , Transcrição Gênica , Células Tumorais Cultivadas , Vitamina D/síntese química , Vitamina D/metabolismoRESUMO
The nuclear hormone receptors liver X receptor alpha (LXRalpha) and LXRbeta function as physiological receptors for oxidized cholesterol metabolites (oxysterols) and regulate several aspects of cholesterol and lipid metabolism. Seladin-1 was originally identified as a gene whose expression was down-regulated in regions of the brain associated with Alzheimer's disease. Seladin-1 has been demonstrated to be neuroprotective and was later characterized as 3beta-hydroxysterol-Delta24 reductase (DHCR24), a key enzyme in the cholesterologenic pathway. Seladin-1 has also been shown to regulate lipid raft formation. In a whole genome screen for direct LXRalpha target genes, we identified an LXRalpha occupancy site within the second intron of the Seladin-1/DHCR24 gene. We characterized a novel LXR response element within the second intron of this gene that is able to confer LXR-specific ligand responsiveness to reporter gene in both HepG2 and human embryonic kidney 293 cells. Furthermore, we found that Seladin-1/DHCR24 gene expression is significantly decreased in skin isolated from LXRbeta-null mice. Our data suggest that Seladin-1/DHCR24 is an LXR target gene and that LXR may regulate lipid raft formation.
Assuntos
Doença de Alzheimer/genética , Proteínas de Ligação a DNA/genética , Proteínas do Tecido Nervoso/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/genética , Receptores Citoplasmáticos e Nucleares/genética , Animais , Encéfalo/metabolismo , Linhagem Celular , Proteínas de Ligação a DNA/metabolismo , Humanos , Íntrons , Receptores X do Fígado , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/metabolismo , Receptores Nucleares Órfãos , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Elementos de Resposta , Pele/metabolismoRESUMO
Type I human hepatic 3alpha-hydroxysteroid dehydrogenase (AKR1C4) plays a significant role in bile acid biosynthesis, steroid hormone metabolism, and xenobiotic metabolism. Utilization of a hidden Markov model for predictive modeling of nuclear hormone receptor response elements coupled with chromatin immunoprecipitation/microarray technology revealed a putative binding site in the AKR1C4 promoter for the nuclear hormone receptor known as liver X receptor alpha, (LXRalpha [NR1H3]), which is the physiological receptor for oxidized cholesterol metabolites. The putative LXRalpha response element (LXRE), identified by chromatin immunoprecipitation, was approximately 1.5 kilobase pairs upstream of the transcription start site. LXRalpha was shown to bind specifically to this LXRE and mediate transcriptional activation of the AKR1C4 gene, leading to increased AKR1C4 protein expression. These data suggest that LXRalpha may modulate the bile acid biosynthetic pathway at a unique site downstream of CYP7A1 and may also modulate the metabolism of steroid hormones and certain xenobiotics.
Assuntos
Proteínas de Ligação a DNA/biossíntese , Regulação da Expressão Gênica/fisiologia , Oxirredutases/biossíntese , Receptores Citoplasmáticos e Nucleares/biossíntese , Linhagem Celular , Colesterol 7-alfa-Hidroxilase/biossíntese , Colesterol 7-alfa-Hidroxilase/genética , Proteínas de Ligação a DNA/genética , Humanos , Receptores X do Fígado , Receptores Nucleares Órfãos , Oxirredutases/genética , Receptores Citoplasmáticos e Nucleares/genéticaRESUMO
The vitamin D receptor/retinoid X receptor-α heterodimer (VDRRXRα) regulates bone mineralization via transcriptional control of osteocalcin (BGLAP) gene and is the receptor for 1α,25-dihydroxyvitamin D3 (1,25D3). However, supra-physiological levels of 1,25D3 activates the calcium-regulating gene TRPV6 leading to hypercalcemia. An approach to attenuate this adverse effect is to develop selective VDR modulators (VDRMs) that differentially activate BGLAP but not TRPV6. Here we present structural insight for the action of a VDRM compared with agonists by employing hydrogen/deuterium exchange. Agonist binding directs crosstalk between co-receptors upon DNA binding, stabilizing the activation function 2 (AF2) surfaces of both receptors driving steroid receptor co-activator-1 (SRC1) interaction. In contrast, AF2 of VDR within VDRM:BGLAP bound heterodimer is more vulnerable for large stabilization upon SRC1 interaction compared with VDRM:TRPV6 bound heterodimer. These results reveal that the combination of ligand structure and DNA sequence tailor the transcriptional activity of VDR toward specific target genes.The vitamin D receptor/retinoid X receptor-α heterodimer (VDRRXRα) regulates bone mineralization. Here the authors employ hydrogen/deuterium exchange (HDX) mass spectrometry to study the conformational dynamics of VDRRXRα and give mechanistic insights into how VDRRXRα controls the transcriptional activity of specific genes.
Assuntos
DNA/química , Receptores de Calcitriol/química , Receptores de Calcitriol/metabolismo , DNA/genética , DNA/metabolismo , Medição da Troca de Deutério , Dimerização , Humanos , Hidrogênio , Ligantes , Espectrometria de Massas , Osteocalcina/genética , Osteocalcina/metabolismo , Ligação Proteica , Receptores de Calcitriol/genética , Receptores X de Retinoides/química , Receptores X de Retinoides/genética , Receptores X de Retinoides/metabolismo , Vitamina D/análogos & derivados , Vitamina D/metabolismoRESUMO
Combining anticatabolic agents with parathyroid hormone (PTH) to enhance bone mass has yielded mixed results in osteoporosis patients. Toward the goal of enhancing the efficacy of these regimens, we tested their utility in combination with loss of the transcription factor Nmp4 because disabling this gene amplifies PTH-induced increases in trabecular bone in mice by boosting osteoblast secretory activity. We addressed whether combining a sustained anabolic response with an anticatabolic results in superior bone acquisition compared with PTH monotherapy. Additionally, we inquired whether Nmp4 interferes with anticatabolic efficacy. Wild-type and Nmp4-/- mice were ovariectomized at 12 weeks of age, followed by therapy regimens, administered from 16 to 24 weeks, and included individually or combined PTH, alendronate (ALN), zoledronate (ZOL), and raloxifene (RAL). Anabolic therapeutic efficacy generally corresponded with PTH + RAL = PTH + ZOL > PTH + ALN = PTH > vehicle control. Loss of Nmp4 enhanced femoral trabecular bone increases under PTH + RAL and PTH + ZOL. RAL and ZOL promoted bone restoration, but unexpectedly, loss of Nmp4 boosted RAL-induced increases in femoral trabecular bone. The combination of PTH, RAL, and loss of Nmp4 significantly increased bone marrow osteoprogenitor number, but did not affect adipogenesis or osteoclastogenesis. RAL, but not ZOL, increased osteoprogenitors in both genotypes. Nmp4 status did not influence bone serum marker responses to treatments, but Nmp4-/- mice as a group showed elevated levels of the bone formation marker osteocalcin. We conclude that the heightened osteoanabolism of the Nmp4-/- skeleton enhances the effectiveness of diverse osteoporosis treatments, in part by increasing hyperanabolic osteoprogenitors. Nmp4 provides a promising target pathway for identifying barriers to pharmacologically induced bone formation.
Assuntos
Osso e Ossos/efeitos dos fármacos , Osso e Ossos/metabolismo , Difosfonatos/administração & dosagem , Imidazóis/administração & dosagem , Osteoporose/tratamento farmacológico , Hormônio Paratireóideo/administração & dosagem , Cloridrato de Raloxifeno/administração & dosagem , Animais , Reabsorção Óssea/tratamento farmacológico , Reabsorção Óssea/genética , Reabsorção Óssea/metabolismo , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Quimioterapia Combinada , Feminino , Camundongos , Camundongos Knockout , Proteínas Associadas à Matriz Nuclear/genética , Osteoporose/genética , Osteoporose/patologia , Fatores de Transcrição/genética , Ácido ZoledrônicoRESUMO
The farnesoid X receptor (FXR; NR1H4) is a nuclear hormone receptor that functions as the bile acid receptor. In addition to the critical role FXR plays in bile acid metabolism and transport, it regulates a variety of genes important in lipoprotein metabolism. We demonstrate that FXR also plays a role in carbohydrate metabolism via regulation of phosphoenolpyruvate carboxykinase (PEPCK) gene expression. Treatment of either H4IIE or MH1C1 rat hepatoma cell lines as well as primary rat or human hepatocytes with FXR agonists led to stimulation of PEPCK mRNA expression to levels comparable to those obtained with glucocorticoid receptor agonists. We examined the physiological significance of FXR agonist-induced enhancement of PEPCK expression in primary rat hepatocytes. In addition to inducing PEPCK expression in primary hepatocytes, FXR agonists stimulated glucose output to levels comparable to those observed with a glucocorticoid receptor agonist. Consistent with these observations, treatment of C57BL6 mice with GW4064 significantly increased hepatic PEPCK expression. Activation of FXR initiated a cascade involving induction of peroxisome proliferator-activated receptor alpha and TRB3 expression that is consistent with stimulation of PEPCK gene expression via interference with a pathway that may involve Akt-dependent phosphorylation of Forkhead/winged helix transcription factor (FOXO1). The FXR-peroxisome proliferator-activated receptor alpha-TRB3 pathway was conserved in rat hepatoma cell lines, mice, as well as primary human hepatocytes. Thus, in addition to its role in the regulation of lipid metabolism, FXR regulates carbohydrate metabolism.
Assuntos
Carboidratos/química , Proteínas de Ligação a DNA/fisiologia , Fatores de Transcrição/fisiologia , Animais , Ácidos e Sais Biliares/metabolismo , Metabolismo dos Carboidratos , Carcinoma Hepatocelular/metabolismo , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/metabolismo , Relação Dose-Resposta a Droga , Glucocorticoides/metabolismo , Glucose/metabolismo , Hepatócitos/metabolismo , Humanos , Immunoblotting , Isoxazóis/farmacologia , Metabolismo dos Lipídeos , Lipoproteínas/metabolismo , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Modelos Biológicos , PPAR alfa/metabolismo , Fosfoenolpiruvato Carboxiquinase (GTP)/fisiologia , Fosforilação , Pregnenodionas/farmacologia , RNA Mensageiro/metabolismo , Ratos , Receptores Citoplasmáticos e Nucleares , Fatores de Transcrição/metabolismoRESUMO
The vitamin D receptor (VDR) belongs to the thyroid hormone/retinoid receptor subfamily of nuclear receptors and functions as a heterodimer with retinoid X receptor (RXR). The RXR-VDR heterodimer, in contrast to other members of the class II nuclear receptor subfamily, is nonpermissive where RXR does not bind its cognate ligand, and therefore its role in VDR-mediated transactivation by liganded RXR-VDR has not been fully characterized. Here, we show a unique facet of the intermolecular RXR-VDR interaction, in which RXR actively participates in vitamin D3-dependent gene transcription. Using helix 3 and helix 12 mutants of VDR and RXR, we provide functional evidence that liganded VDR allosterically modifies RXR from an apo (unliganded)- to a holo (liganded)-receptor conformation, in the absence of RXR ligand. As a result of the proposed allosteric modification of RXR by liganded VDR, the heterodimerized RXR shows the "phantom ligand effect" and thus acquires the capability to recruit coactivators steroid receptor coactivator 1, transcriptional intermediary factor 2, and amplified in breast cancer-1. Finally, using a biochemical approach with purified proteins, we show that RXR augments the 1,25-dihydroxyvitamin D3-dependent recruitment of transcriptional intermediary factor 2 in the context of RXR-VDR heterodimer. These results confirm and extend the previous observations suggesting that RXR is a significant contributor to VDR-mediated gene expression and provide a mechanism by which RXR acts as a major contributor to vitamin D3-dependent transcription.
Assuntos
Receptores de Calcitriol/metabolismo , Receptores do Ácido Retinoico/metabolismo , Fatores de Transcrição/metabolismo , Ativação Transcricional , Vitamina D/análogos & derivados , Regulação Alostérica , Dimerização , Células HeLa , Histona Acetiltransferases , Humanos , Mutação/genética , Coativador 1 de Receptor Nuclear , Coativador 2 de Receptor Nuclear , Coativador 3 de Receptor Nuclear , Estrutura Terciária de Proteína , Receptores de Calcitriol/química , Receptores de Calcitriol/genética , Receptores do Ácido Retinoico/química , Receptores do Ácido Retinoico/genética , Receptores X de Retinoides , Fatores de Transcrição/química , Fatores de Transcrição/genética , Ativação Transcricional/efeitos dos fármacos , Vitamina D/farmacologiaRESUMO
Mechanistic understanding of the preferential homing of circulating tumor cells to bone and their perturbation on bone metabolism within the tumor-bone microenvironment remains poorly understood. Alteration in both transforming growth factor ß (TGFß) signaling and sphingolipid metabolism results in the promotion of tumor growth and metastasis. Previous studies using MDA-MB-231 human breast cancer-derived cell lines of variable metastatic potential were queried for changes in sphingolipid metabolism genes to explore correlations between TGFß dependence and bone metastatic behavior. Of these genes, only sphingosine kinase-1 (SPHK1) was identified to be significantly increased following TGFß treatment. Induction of SPHK1 expression correlated to the degree of metastatic capacity in these MDA-MB-231-derived cell lines. We demonstrate that TGFß mediates the regulation of SPHK1 gene expression, protein kinase activity and is critical to MDA-MB-231 cell viability. Furthermore, a bioinformatic analysis of human breast cancer gene expression supports SPHK1 as a hallmark TGFß target gene that also bears the genetic fingerprint of the basal-like/triple-negative breast cancer molecular subtype. These data suggest a potential new signaling axis between TGFß/SphK1 that may have a role in the development, prognosis or the clinical phenotype associated with tumor-bone metastasis.
RESUMO
PTH is an osteoanabolic for treating osteoporosis but its potency wanes. Disabling the transcription factor nuclear matrix protein 4 (Nmp4) in healthy, ovary-intact mice enhances bone response to PTH and bone morphogenetic protein 2 and protects from unloading-induced osteopenia. These Nmp4(-/-) mice exhibit expanded bone marrow populations of osteoprogenitors and supporting CD8(+) T cells. To determine whether the Nmp4(-/-) phenotype persists in an osteoporosis model we compared PTH response in ovariectomized (ovx) wild-type (WT) and Nmp4(-/-) mice. To identify potential Nmp4 target genes, we performed bioinformatic/pathway profiling on Nmp4 chromatin immunoprecipitation sequencing (ChIP-seq) data. Mice (12 w) were ovx or sham operated 4 weeks before the initiation of PTH therapy. Skeletal phenotype analysis included microcomputed tomography, histomorphometry, serum profiles, fluorescence-activated cell sorting and the growth/mineralization of cultured WT and Nmp4(-/-) bone marrow mesenchymal stem progenitor cells (MSPCs). ChIP-seq data were derived using MC3T3-E1 preosteoblasts, murine embryonic stem cells, and 2 blood cell lines. Ovx Nmp4(-/-) mice exhibited an improved response to PTH coupled with elevated numbers of osteoprogenitors and CD8(+) T cells, but were not protected from ovx-induced bone loss. Cultured Nmp4(-/-) MSPCs displayed enhanced proliferation and accelerated mineralization. ChIP-seq/gene ontology analyses identified target genes likely under Nmp4 control as enriched for negative regulators of biosynthetic processes. Interrogation of mRNA transcripts in nondifferentiating and osteogenic differentiating WT and Nmp4(-/-) MSPCs was performed on 90 Nmp4 target genes and differentiation markers. These data suggest that Nmp4 suppresses bone anabolism, in part, by regulating IGF-binding protein expression. Changes in Nmp4 status may lead to improvements in osteoprogenitor response to therapeutic cues.
Assuntos
Reabsorção Óssea/tratamento farmacológico , Linfócitos T CD8-Positivos/citologia , Proteínas Associadas à Matriz Nuclear/genética , Osteoporose/tratamento farmacológico , Hormônio Paratireóideo/uso terapêutico , Fatores de Transcrição/genética , Animais , Densidade Óssea/efeitos dos fármacos , Doenças Ósseas Metabólicas/prevenção & controle , Proteína Morfogenética Óssea 2/metabolismo , Reabsorção Óssea/genética , Linfócitos T CD8-Positivos/imunologia , Células Cultivadas , Mapeamento Cromossômico , Células-Tronco Embrionárias/citologia , Feminino , Terapia Genética , Humanos , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina/genética , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina/metabolismo , Células-Tronco Mesenquimais/citologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Osteogênese/efeitos dos fármacos , Osteoporose/genética , Ovariectomia , Ovário/cirurgiaRESUMO
Breast cancer is the most prevalent cancer among females worldwide leading to approximately 350,000 deaths each year. It has long been known that cancers preferentially metastasize to particular organs, and bone metastases occur in ~70% of patients with advanced breast cancer. Breast cancer bone metastases are predominantly osteolytic and accompanied by increased fracture risk, pain, nerve compression and hypercalcemia, causing severe morbidity. In the bone matrix, transforming growth factor-ß (TGF-ß) is one of the most abundant growth factors, which is released in active form upon tumor-induced osteoclastic bone resorption. TGF-ß, in turn, stimulates bone metastatic tumor cells to secrete factors that further drive osteolytic bone destruction adjacent to the tumor. Thus, TGF-ß is a crucial factor responsible for driving the feed-forward vicious cycle of cancer growth in bone. Moreover, TGF-ß activates epithelial-to-mesenchymal transition, increases tumor cell invasiveness and angiogenesis and induces immunosuppression. Blocking the TGF-ß signaling pathway to interrupt this vicious cycle between breast cancer and bone offers a promising target for therapeutic intervention to decrease skeletal metastasis. This review will describe the role of TGF-ß in breast cancer and bone metastasis, and pre-clinical and clinical data will be evaluated for the potential use of TGF-ß inhibitors in clinical practice to treat breast cancer bone metastases.
RESUMO
The skeleton is a preferred site for cancer metastasis. These bone metastases cause dysregulated bone remodeling and the associated morbidity of fractures, pain, hypercalcemia and catastrophic nerve compression syndromes. Transforming growth factor-ß (TGF-ß) is stored in mineralized bone matrix, and released and activated by osteoclastic bone resorption. Once activated, TGF-ß stimulates nearby metastatic tumor cells within the bone microenvironment to secrete factors that further drive osteolytic destruction of the bone. Therefore, TGF-ß and its signaling constitute a critical component driving the feed-forward vicious cycle of cancer growth in bone. Moreover, additional pro-tumorigenic activities attributed to TGF-ß include activation of epithelial-to-mesenchymal transition, increased tumor cell invasion, enhanced angiogenesis and various immunomodulatory properties. Blocking the TGF-ß signaling pathway to interrupt this vicious cycle and manipulate the bone microenvironment offers a promising area for therapeutic intervention to decrease skeletal metastasis and normalize bone homeostatic mechanisms. In this review, preclinical and clinical data are evaluated for the potential use of TGF-ß pathway inhibitors in clinical practice to treat bone metastases and its associated comorbidities.
RESUMO
Breast cancer is the most prevalent cancer among females worldwide. It has long been known that cancers preferentially metastasize to particular organs, and bone metastases occur in â¼70% of patients with advanced breast cancer. Breast cancer bone metastases are predominantly osteolytic and accompanied by bone destruction, bone fractures, pain, and hypercalcemia, causing severe morbidity and hospitalization. In the bone matrix, transforming growth factor-ß (TGF-ß) is one of the most abundant growth factors, which is released in active form upon tumor-induced osteoclastic bone resorption. TGF-ß, in turn, stimulates bone metastatic cells to secrete factors that further drive osteolytic destruction of the bone adjacent to the tumor, categorizing TGF-ß as a crucial factor responsible for driving the feed-forward vicious cycle of cancer growth in bone. Moreover, TGF-ß activates epithelial-to-mesenchymal transition, increases tumor cell invasiveness and angiogenesis and induces immunosuppression. Blocking the TGF-ß signaling pathway to interrupt this vicious cycle between breast cancer and bone offers a promising target for therapeutic intervention to decrease skeletal metastasis. This review will describe the role of TGF-ß in breast cancer and bone metastasis, and pre-clinical and clinical data will be evaluated for the potential use of TGF-ß inhibitors in clinical practice to treat breast cancer bone metastases.
RESUMO
The vitamin D receptor (VDR) functions as an obligate heterodimer in complex with the retinoid X receptor (RXR). These nuclear receptors are multidomain proteins, and it is unclear how various domains interact with one another within the nuclear receptor heterodimer. Here, we show that binding of intact heterodimer to DNA alters the receptor dynamics in regions remote from the DNA-binding domains (DBDs), including the coactivator binding surfaces of both co-receptors, and that the sequence of the DNA response element can determine these dynamics. Furthermore, agonist binding to the heterodimer results in changes in the stability of the VDR DBD, indicating that the ligand itself may play a role in DNA recognition. These data suggest a mechanism by which nuclear receptors show promoter specificity and have differential effects on various target genes, providing insight into the function of selective nuclear receptor modulators.