RESUMO
Skeletal muscle regeneration is mediated by myoblasts that undergo epigenomic changes to establish the gene expression program of differentiated myofibers. mSWI/SNF chromatin remodeling enzymes coordinate with lineage-determining transcription factors to establish the epigenome of differentiated myofibers. Bromodomains bind to acetylated lysines on histone N-terminal tails and other proteins. The mutually exclusive ATPases of mSWI/SNF complexes, BRG1 and BRM, contain bromodomains with undefined functional importance in skeletal muscle differentiation. Pharmacological inhibition of mSWI/SNF bromodomain function using the small molecule PFI-3 reduced differentiation in cell culture and in vivo through decreased myogenic gene expression, while increasing cell cycle-related gene expression and the number of cells remaining in the cell cycle. Comparative gene expression analysis with data from myoblasts depleted of BRG1 or BRM showed that bromodomain function was required for a subset of BRG1- and BRM-dependent gene expression. Reduced binding of BRG1 and BRM after PFI-3 treatment showed that the bromodomain is required for stable chromatin binding at target gene promoters to alter gene expression. Our findings demonstrate that mSWI/SNF ATPase bromodomains permit stable binding of the mSWI/SNF ATPases to promoters required for cell cycle exit and establishment of muscle-specific gene expression.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Cromatina/genética , DNA Helicases/genética , Desenvolvimento Muscular/genética , Proteínas Nucleares/genética , Fatores de Transcrição/genética , Adenosina Trifosfatases/genética , Animais , Compostos Azabicíclicos/farmacologia , Diferenciação Celular/genética , Montagem e Desmontagem da Cromatina/genética , Proteínas de Ligação a DNA/genética , Histonas/genética , Humanos , Músculo Esquelético/citologia , Músculo Esquelético/crescimento & desenvolvimento , Piridinas/farmacologia , Fatores de Transcrição/antagonistas & inibidoresRESUMO
Mammalian SWI/SNF (mSWI/SNF) complexes are ATP-dependent chromatin remodeling enzymes that are critical for normal cellular functions. mSWI/SNF enzymes are classified into three sub-families based on the presence of specific subunit proteins. The sub-families are Brm- or Brg1-associated factor (BAF), ncBAF (non-canonical BAF), and polybromo-associated BAF (PBAF). The biological roles for the different enzyme sub-families are poorly described. We knocked down the expression of genes encoding unique subunit proteins for each sub-family, Baf250A, Brd9, and Baf180, which mark the BAF, ncBAF, and PBAF sub-families, respectively, and examined the requirement for each in myoblast differentiation. We found that Baf250A and the BAF complex were required to drive lineage-specific gene expression. KD of Brd9 delayed differentiation. However, while the Baf250A-dependent gene expression profile included myogenic genes, the Brd9-dependent gene expression profile did not, suggesting Brd9 and the ncBAF complex indirectly contributed to differentiation. Baf180 was dispensable for myoblast differentiation. The results distinguish between the roles of the mSWI/SNF enzyme sub-families during myoblast differentiation.
Assuntos
Cromatina , Proteínas Cromossômicas não Histona , Humanos , Animais , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Montagem e Desmontagem da Cromatina/genética , Mioblastos/metabolismo , Mamíferos/metabolismoRESUMO
Three named cell types degrade and remove skeletal tissues during growth, repair, or disease: osteoclasts, chondroclasts, and septoclasts. A fourth type, unnamed and less understood, removes nonmineralized cartilage during development of secondary ossification centers. "Osteoclasts," best known and studied, are polykaryons formed by fusion of monocyte precursors under the influence of colony stimulating factor 1 (CSF)-1 (M-CSF) and RANKL. They resorb bone during growth, remodeling, repair, and disease. "Chondroclasts," originally described as highly similar in cytological detail to osteoclasts, reside on and degrade mineralized cartilage. They may be identical to osteoclasts since to date there are no distinguishing markers for them. Because osteoclasts also consume cartilage cores along with bone during growth, the term "chondroclast" might best be reserved for cells attached only to cartilage. "Septoclasts" are less studied and appreciated. They are mononuclear perivascular cells rich in cathepsin B. They extend a cytoplasmic projection with a ruffled membrane and degrade the last transverse septum of hypertrophic cartilage in the growth plate, permitting capillaries to bud into it. To do this, antiangiogenic signals in cartilage must give way to vascular trophic factors, mainly vascular endothelial growth factor (VEGF). The final cell type excavates cartilage canals for vascular invasion of articular cartilage during development of secondary ossification centers. The "clasts" are considered in the context of fracture repair and diseases such as arthritis and tumor metastasis. Many observations support an essential role for hypertrophic chondrocytes in recruiting septoclasts and osteoclasts/chondroclasts by supplying VEGF and RANKL. The intimate relationship between blood vessels and skeletal turnover and repair is also examined.
Assuntos
Desenvolvimento Ósseo , Condrócitos/patologia , Doença , Neovascularização Patológica/patologia , Osteoclastos/patologia , Cicatrização , Animais , HumanosRESUMO
Mammalian MBNL (muscleblind-like) proteins are regulators of alternative splicing and have been implicated in myotonic dystrophy, the most common form of adult onset muscular dystrophy. MBNL3 functions as an inhibitor of muscle differentiation and is expressed in proliferating muscle precursor cells but not in differentiated skeletal muscle. Here we demonstrate that MBNL3 regulates the splicing pattern of the muscle transcription factor myocyte enhancer factor 2 (Mef2) by promoting exclusion of the alternatively spliced ß-exon. Expression of the transcriptionally more active (+)ß isoform of Mef2D was sufficient to overcome the inhibitory effects of MBNL3 on muscle differentiation. These data suggest that MBNL3 antagonizes muscle differentiation by disrupting Mef2 ß-exon splicing. MBNL3 regulates Mef2D splicing by directly binding to intron 7 downstream of the alternatively spliced exon in the pre-mRNA. The RNA binding activity of MBNL3 requires the CX(7)CX(4-6)CX(3)H zinc finger domains. Using a cell culture model of myotonic dystrophy and myotonic dystrophy patient tissue, we have evidence that expression of CUG expanded RNAs can lead to an increase in MBNL3 expression and a decrease in Mef2D ß-exon splicing. These studies suggest that elevating MBNL3 activity in myogenic cells could lead to muscle degeneration disorders such as myotonic dystrophy.
Assuntos
Proteínas de Transporte/metabolismo , Fatores de Regulação Miogênica/metabolismo , RNA/metabolismo , Processamento Alternativo , Animais , Diferenciação Celular , Linhagem Celular , Éxons , Imuno-Histoquímica/métodos , Fatores de Transcrição MEF2 , Camundongos , Modelos Genéticos , Músculo Esquelético/citologia , Músculo Esquelético/metabolismo , Distrofia Miotônica/metabolismo , Distrofia Miotônica/patologia , Proteínas de Ligação a RNA , Retroviridae/metabolismoRESUMO
Calcineurin (Cn) is a calcium-activated serine/threonine protein phosphatase that is broadly implicated in diverse cellular processes, including the regulation of gene expression. During skeletal muscle differentiation, Cn activates the nuclear factor of activated T-cell (NFAT) transcription factor but also promotes differentiation by counteracting the negative influences of protein kinase C beta (PKCß) via dephosphorylation and activation of Brg1, an enzymatic subunit of the mammalian SWI/SNF ATP-dependent chromatin remodeling enzyme. Here we identified four major temporal patterns of Cn-dependent gene expression in differentiating myoblasts and determined that Cn is broadly required for the activation of the myogenic gene expression program. Mechanistically, Cn promotes gene expression through direct binding to myogenic promoter sequences and facilitating the binding of Brg1, other SWI/SNF subunit proteins, and MyoD, a critical lineage determinant for skeletal muscle differentiation. We conclude that the Cn phosphatase directly impacts the expression of myogenic genes by promoting ATP-dependent chromatin remodeling and formation of transcription-competent promoters.
Assuntos
Calcineurina/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Perfilação da Expressão Gênica/métodos , Redes Reguladoras de Genes , Mioblastos/citologia , Fatores de Transcrição/metabolismo , Animais , Diferenciação Celular , Linhagem Celular , Montagem e Desmontagem da Cromatina , DNA Helicases/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Redes Reguladoras de Genes/efeitos dos fármacos , Camundongos , Proteína MyoD , Mioblastos/química , Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas/efeitos dos fármacos , Tacrolimo/farmacologiaRESUMO
It has been shown that cyclic GMP (cGMP) modulates the inflammatory responses of macrophages, but the underlying molecular mechanisms are still poorly understood. Looking for proteins potentially regulated by cGMP in rat peritoneal macrophages (PMs), in this study we analyzed expression and activity of cGMP-hydrolyzing and cGMP-regulated phosphodiesterases (PDEs). It was found that freshly isolated peritoneal exudate macrophages (PEMs) express enzymes belonging to families PDE1-3, PDE5, PDE10, and PDE11. Analysis of substrate specificity, sensitivity to inhibitors, and subcellular localization showed that PDE2 and PDE3 are the main cGMP-regulated PDE isoforms in PEMs. The profile of PDE expression was altered by maintaining PEMs in culture and treatment with bacterial endotoxin (LPS). After 24 h culture, PDE5 was not present and the levels of PDE2, PDE3, and PDE11 were markedly decreased. However, their expression and activity was recovered after treatment of cultured cells with LPS. A similar pattern of changes was observed for the expression of TNFalpha, but not for guanylyl cyclase A (GC-A). LPS up-regulated PDE expression also in resident peritoneal macrophages (RPMs), although not all PDEs present in PEMs were detected in RPMs. Taken together, our results show that in rat PMs expression of cGMP-dependent PDEs positively correlates with the activation state of cells. Moreover, the fact that most of these PDEs hydrolyze also cAMP indicates that cGMP can play a role of potent regulator of cAMP signaling in macrophages.
Assuntos
3',5'-GMP Cíclico Fosfodiesterases/genética , 3',5'-GMP Cíclico Fosfodiesterases/metabolismo , Lipopolissacarídeos/farmacologia , Macrófagos Peritoneais/efeitos dos fármacos , Macrófagos Peritoneais/enzimologia , Regulação para Cima/efeitos dos fármacos , 3',5'-GMP Cíclico Fosfodiesterases/antagonistas & inibidores , Animais , AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Proteínas Quinases Dependentes de GMP Cíclico/genética , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Guanilato Ciclase/genética , Guanilato Ciclase/metabolismo , Isoenzimas/antagonistas & inibidores , Isoenzimas/genética , Isoenzimas/metabolismo , Ativação de Macrófagos/efeitos dos fármacos , Masculino , Inibidores de Fosfodiesterase/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Especificidade por Substrato/efeitos dos fármacos , Fator de Necrose Tumoral alfa/genética , Regulação para Cima/genéticaRESUMO
The nitric oxide/soluble guanylyl cyclase/cGMP-dependent protein kinase (NO/sGC/PKG) cascade has been shown to affect important functions of circulating neutrophils. We demonstrate that neutrophils isolated from rats treated intraperitoneally with peptone protease cannot use this signaling pathway. Although PKG was detected at both the mRNA and protein levels in peripheral blood neutrophils (PBNs) of control rats, it was expressed neither in PBNs nor in peritoneal exudate neutrophils (PENs) of provoked rats. Also, mRNA of the alpha and beta chains of heterodimeric sGC was present in PBNs, but absent in PENs. Consistently, PBNs responded to activators of sGC with cGMP synthesis, while PENs did not. These results showed that neutrophils recruited by a provoking agent lost PKG and, in the case of PENs, also sGC and thus the capacity to respond to NO with cGMP signaling. We speculate that such downregulation of the sGC/PKG pathway is likely a result of the high activity of inducible NO synthase observed in inflammatory neutrophils.
Assuntos
Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Guanilato Ciclase/metabolismo , Neutrófilos/enzimologia , Receptores Citoplasmáticos e Nucleares/metabolismo , Animais , Células Cultivadas , Proteínas Quinases Dependentes de GMP Cíclico/genética , Guanilato Ciclase/genética , Masculino , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Óxido Nítrico/biossíntese , Óxido Nítrico Sintase Tipo II/metabolismo , Peptonas/farmacologia , Ratos , Ratos Wistar , Receptores Citoplasmáticos e Nucleares/genética , Transdução de Sinais , Guanilil Ciclase SolúvelRESUMO
Histone acetyltransferases of the MYST family are recruited to chromatin by BRPF scaffolding proteins. We explored functional consequences and the therapeutic potential of inhibitors targeting acetyl-lysine dependent protein interaction domains (bromodomains) present in BRPF1-3 in bone maintenance. We report three potent and selective inhibitors: one (PFI-4) with high selectivity for the BRPF1B isoform and two pan-BRPF bromodomain inhibitors (OF-1, NI-57). The developed inhibitors displaced BRPF bromodomains from chromatin and did not inhibit cell growth and proliferation. Intriguingly, the inhibitors impaired RANKL-induced differentiation of primary murine bone marrow cells and human primary monocytes into bone resorbing osteoclasts by specifically repressing transcriptional programs required for osteoclastogenesis. The data suggest a key role of BRPF in regulating gene expression during osteoclastogenesis, and the excellent druggability of these bromodomains may lead to new treatment strategies for patients suffering from bone loss or osteolytic malignant bone lesions.
Assuntos
Células da Medula Óssea/fisiologia , Proteínas de Transporte/metabolismo , Diferenciação Celular/fisiologia , Osteoclastos/fisiologia , Animais , Proteínas de Transporte/genética , Biologia Computacional , Humanos , Modelos Moleculares , Família Multigênica , Análise Serial de Proteínas , Conformação Proteica , Domínios Proteicos , Células-TroncoRESUMO
Plekhm1 is a large, multi-modular, adapter protein implicated in osteoclast vesicle trafficking and bone resorption. In patients, inactivating mutations cause osteopetrosis, and gain-of-function mutations cause osteopenia. Investigations of potential Plekhm1 interaction partners by mass spectrometry identified TRAFD1 (FLN29), a protein previously shown to suppress toll-like receptor signaling in monocytes/macrophages, thereby dampening inflammatory responses to innate immunity. We mapped the binding domains to the TRAFD1 zinc finger (aa 37-60), and to the region of Plekhm1 between its second pleckstrin homology domain and its C1 domain (aa 784-986). RANKL slightly increased TRAFD1 levels, particularly in primary osteoclasts, and the co-localization of TRAFD1 with Plekhm1 also increased with RANKL treatment. Stable knockdown of TRAFD1 in RAW 264.7 cells inhibited resorption activity proportionally to the degree of knockdown, and inhibited acidification. The lack of acidification occurred despite the presence of osteoclast acidification factors including carbonic anhydrase II, a3-V-ATPase, and the ClC7 chloride channel. Secretion of TRAP and cathepsin K were also markedly inhibited in knockdown cells. Truncated Plekhm1 in ia/ia osteopetrotic rat cells prevented vesicle localization of Plekhm1 and TRAFD1. We conclude that TRAFD1, in association with Plekhm1/Rab7-positive late endosomes-early lysosomes, has a previously unknown role in vesicle trafficking, acidification, and resorption in osteoclasts.
Assuntos
Ácidos/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Glicoproteínas de Membrana/metabolismo , Osteoclastos/metabolismo , Animais , Proteínas Relacionadas à Autofagia , Diferenciação Celular , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , Osteoclastos/citologia , Ligação Proteica , RNA Mensageiro/genética , RatosRESUMO
The fusion of monocyte/macrophage lineage cells into fully active, multinucleated, bone resorbing osteoclasts is a complex cell biological phenomenon that utilizes specialized proteins. OC-STAMP, a multi-pass transmembrane protein, has been shown to be required for pre-osteoclast fusion and for optimal bone resorption activity. A previously reported knockout mouse model had only mononuclear osteoclasts with markedly reduced resorption activity in vitro, but with paradoxically normal skeletal micro-CT parameters. To further explore this and related questions, we used mouse ES cells carrying a gene trap allele to generate a second OC-STAMP null mouse strain. Bone histology showed overall normal bone form with large numbers of TRAP-positive, mononuclear osteoclasts. Micro-CT parameters were not significantly different between knockout and wild type mice at 2 or 6 weeks old. At 6 weeks, metaphyseal TRAP-positive areas were lower and mean size of the areas were smaller in knockout femora, but bone turnover markers in serum were normal. Bone marrow mononuclear cells became TRAP-positive when cultured with CSF-1 and RANKL, but they did not fuse. Expression levels of other osteoclast markers, such as cathepsin K, carbonic anhydrase II, and NFATc1, were not significantly different compared to wild type. Actin rings were present, but small, and pit assays showed a 3.5-fold decrease in area resorbed. Restoring OC-STAMP in knockout cells by lentiviral transduction rescued fusion and resorption. N- and C-termini of OC-STAMP were intracellular, and a predicted glycosylation site was shown to be utilized and to lie on an extracellular loop. The site is conserved in all terrestrial vertebrates and appears to be required for protein stability, but not for fusion. Based on this and other results, we present a topological model of OC-STAMP as a 6-transmembrane domain protein. We also contrast the osteoclast-specific roles of OC- and DC-STAMP with more generalized cell fusion mechanisms.
Assuntos
Fusão Celular , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Osteoclastos/citologia , Osteoclastos/metabolismo , Fosfatase Ácida/metabolismo , Alelos , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Biomarcadores/metabolismo , Reabsorção Óssea/patologia , Sobrevivência Celular , Sequência Conservada , Fêmur/metabolismo , Fêmur/patologia , Regulação da Expressão Gênica , Glicosilação , Células HEK293 , Humanos , Isoenzimas/metabolismo , Lentivirus/metabolismo , Proteínas de Membrana/deficiência , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Animais , Dados de Sequência Molecular , Osteoclastos/enzimologia , Osteogênese , Fosfatase Ácida Resistente a Tartarato , Transdução GenéticaRESUMO
Intact rat peritoneal macrophages (rPM) treated with 3-isobutyl-1-methylxanthine (IBMX), an inhibitor of phosphodiesterases (PDEs), accumulated more cGMP than untreated cells. A PDE activity toward [(3)H]cGMP was detected in the soluble and particulate fractions of rPM. The hydrolysis of cGMP was Ca(2+)/calmodulin-independent but increased in the presence of cGMP excess. Similar results were obtained when [(3)H]cAMP was used as a substrate. The hydrolytic activity towards both nucleotides was inhibited in the presence of IBMX. Therefore, the PDEs of families 2, 5, 10 and 11 are potential candidates for cGMP hydrolysis in the rPM. They may not only regulate the cGMP level in a feedback-controlled way but also link cGMP-dependent pathways with those regulated by cAMP.
Assuntos
GMP Cíclico/metabolismo , Macrófagos Peritoneais/metabolismo , 1-Metil-3-Isobutilxantina/farmacologia , Animais , Fator Natriurético Atrial/farmacologia , Cálcio/metabolismo , Calmodulina/metabolismo , Células Cultivadas , GMP Cíclico/farmacologia , Hidrólise , Macrófagos Peritoneais/efeitos dos fármacos , Masculino , Inibidores de Fosfodiesterase/farmacologia , Diester Fosfórico Hidrolases/metabolismo , Ratos , Ratos Wistar , SolubilidadeRESUMO
The aim of our studies was to establish which enzymes constitute the "cGMP pathway" in rat and guinea pig peritoneal macrophages (PM). We found that in guinea pig PM synthesis of the nucleotide was significantly enhanced in response to activators of soluble guanylyl cyclase (sGC) and it was only slightly stimulated by specific activators of particulate guanylyl cyclases (pGC). In contrast, rat PM responded strongly to atrial natriuretic peptide (ANP), the activator of pGC type A. The rat cells synthesized about three-fold more cGMP than an equal number of the guinea pig cells. The activity of phosphodiesterases (PDE) hydrolyzing cGMP was apparently regulated by cGMP itself in PM of both species and again it was higher in the rat cells than in those isolated from guinea pig. However, guinea pig PM revealed an activity of Ca(2+)/calmodulin-dependent PDE1, which was absent in the rat cells. Using Western blotting analysis we were unable to detect the presence of cGMP-dependent protein kinase 1 (PKG1) in PM isolated from either species. In summary, our findings indicate that particulate GC-A is the main active form of GC in the rat PM, while in guinea pig macrophages the sGC activity dominates. Since the profiles of the PDE activities in rat and guinea pig PM are also different, we conclude that the mechanisms regulating cGMP metabolism in PM are species-specific. Moreover, our results suggest that targets for cGMP other than PKG1 should be present in PM of both species.
Assuntos
Fator Natriurético Atrial/metabolismo , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Guanilato Ciclase/metabolismo , Macrófagos Peritoneais/metabolismo , Animais , Células Cultivadas , Nucleotídeo Cíclico Fosfodiesterase do Tipo 1 , Cobaias , Masculino , Diester Fosfórico Hidrolases/metabolismo , Ratos , Ratos Wistar , Frações SubcelularesRESUMO
A key goal of molecular/cell biology/biotechnology is to identify essential genes in virtually every physiological process to uncover basic mechanisms of cell function and to establish potential targets of drug therapy combating human disease. This article describes a semester-long, project-oriented molecular/cellular/biotechnology laboratory providing students, within a framework of bone cell biology, with a modern approach to gene discovery. Students are introduced to the topics of bone cells, bone synthesis, bone resorption, and osteoporosis. They then review the theory of microchip gene arrays, and study microchip array data generated during the differentiation of bone-resorbing osteoclasts in vitro. The class selects genes whose expression increases during osteoclastogenesis, and researches them in small groups using web-based bioinformatics tools. Students then go to a biotechnology company website to find and order small inhibitory RNAs (siRNAs) designed to "knockdown" expression of the gene of interest. Students then learn to transfect these siRNAs into osteoclasts, stimulate the cells to differentiate, assay osteoclast differentiation in vitro, and measure specific gene expression using real-time PCR and immunoblotting. Specific siRNA knockdown resulting in a decrease in osteoclastogenesis is indicative of a gene's physiological relevance. The results are analyzed statistically and presented to the class in groups. In the past 2 years, students identified several genes essential for optimal osteoclast differentiation, including Myo1d. The students hypothesize that the myo1d protein functions in osteoclasts to deliver important proteins to the cell surface via vesicular transport along microfilaments. Student response to the new course was overwhelmingly positive.