RESUMO
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
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
Multiple sclerosis (MS) is a chronic debilitating disease, with lowest incidence in equatorial regions and highest incidence in temperate regions. This relationship is believed to be related to sunlight or UV light exposure. Recent evidence with experimental autoimmune encephalomyelitis (EAE), an animal model of MS, established that this suppression is not mediated by vitamin D production. UV is comprised of three general wave bands: UVC (100-280nm), UVB (280-320nm) and UVA (320-400nm). In the present study we used four lamps that emit different wavelengths of UV: (1) broad band UVB (BB-UVB: 280-320nm); (2) narrow band UVB (NB-UVB: 300-315nm); (3) broad band UVA (BB-UVA: 300-400nm); and (4) long wavelength UVA (UVA-1: 340-400nm). The effect of these light sources was studied in vitamin D-sufficient C57BL/6 mice. The NB-UVB largely accounted for the suppression and delay of onset of EAE by BB-UVB. In contrast, UVA-1 failed to suppress EAE severity at low (â¼2.5KJ/m(2)), medium (â¼5.0KJ/m(2)) and high (â¼10.0KJ/m(2)) doses. Serum calcium and 25-(OH)D3 levels were unchanged after both NB-UVB and UVA-1 treatments. The results demonstrate that NB-UVB (300-315nm) is largely responsible for light-induced suppression of EAE and its effect is not via production of vitamin D.