RESUMO
Intervertebral disk (IVD) degeneration is a prevalent health problem that is highly linked to back pain. To understand the disease and tissue response to therapies, ex vivo whole IVD organ culture systems have recently been introduced. The goal of this work was to develop and validate the design of a whole spinal segment culturing system that loads the disk in complex loading similar to the in vivo condition, while preserving the adjacent endplates and vertebral bodies. The complex loading applied to the spinal segment (flexion-extension (FE), bilateral bending, and compression) was achieved with three pneumatic cylinders rigidly attached to a triangular loading platform. A culture container housed the spinal segment and was attached to the loading mechanism, which allowed for loading of the spinal segment. The dynamic bioreactor was able to achieve physiologic loading conditions with 100 N of applied compression and approximately 2-4 N · m of applied torque. The function of the bioreactor was validated through testing of bovine caudal IVDs with intact endplates and vertebral bodies that were isolated within 2 hrs of death and cultured for 14 days. The resulting IVD cell viability following 14 days of loading was much higher than unloaded control IVDs. The loading system accurately mimicked FE, bilateral bending, and compression motions seen during daily activities. The results indicate that this complex dynamic bioreactor may be appropriate for extended preclinical testing of vertebral-mounted spinal devices and therapies.
Assuntos
Reatores Biológicos , Técnicas de Cultura de Órgãos/métodos , Coluna Vertebral , Animais , Fenômenos Biomecânicos , Bovinos , Sobrevivência Celular , Desenho de Equipamento , Disco Intervertebral/citologia , Técnicas de Cultura de Órgãos/instrumentação , Coluna Vertebral/citologiaRESUMO
Background: Metabolic phenotypes are the result of an intricate interplay between multiple factors, including diet, genotype, and the gut microbiome. Per-Arnt-Sim (PAS) kinase is a nutrient-sensing serine/threonine kinase, whose absence (PASK-/-) protects against triglyceride accumulation, insulin resistance, and weight gain on a high-fat diet; conditions that are associated with dysbiosis of the gut microbiome. Methods: Herein, we report the metabolic effects of the interplay of diet (high fat high sugar, HFHS), genotype (PASK-/-), and microbiome (16S sequencing). Results: Microbiome analysis identified a diet-induced, genotype-independent forked shift, with two discrete clusters of HFHS mice having increased beta and decreased alpha diversity. A "lower" cluster contained elevated levels of Firmicutes, Bacteroidetes, Actinobacteria, Proteobacteria and Defferibacteres, and was associated with increased weight gain, glucose intolerance, triglyceride accumulation, and decreased claudin-1 expression. Genotypic effects were observed within the clusters, lower cluster PASK-/- mice displayed increased weight gain and decreased triglyceride accumulation, whereas upper PASK-/- were resistant to decreased claudin-1. Conclusions: These results confirm previous reports that PAS kinase deficiency can protect mice against the deleterious effects of diet, and they suggest that microbiome imbalances can override protection. In addition, these results support a healthy diet for beneficial microbiome maintenance and suggest microbial culprits associated with metabolic disease.
RESUMO
BACKGROUND: Bone morphogenetic proteins (BMPs) contribute to many different aspects of development including mesoderm formation, heart development, neurogenesis, skeletal development, and axis formation. They have previously been recognized only as secreted growth factors, but the present study detected Bmp2, Bmp4, and Gdf5/CDMP1 in the nuclei of cultured cells using immunocytochemistry and immunoblotting of nuclear extracts. RESULTS: In all three proteins, a bipartite nuclear localization signal (NLS) was found to overlap the site at which the proproteins are cleaved to release the mature growth factors from the propeptides. Mutational analyses indicated that the nuclear variants of these three proteins are produced by initiating translation from downstream alternative start codons. The resulting proteins lack N-terminal signal peptides and are therefore translated in the cytoplasm rather than the endoplasmic reticulum, thus avoiding proteolytic processing in the secretory pathway. Instead, the uncleaved proteins (designated nBmp2, nBmp4, and nGdf5) containing the intact NLSs are translocated to the nucleus. Immunostaining of endogenous nBmp2 in cultured cells demonstrated that the amount of nBmp2 as well as its nuclear/cytoplasmic distribution differs between cells that are in M-phase versus other phases of the cell cycle. CONCLUSIONS: The observation that nBmp2 localization varies throughout the cell cycle, as well as the conservation of a nuclear localization mechanism among three different BMP family members, suggests that these novel nuclear variants of BMP family proteins play an important functional role in the cell.
Assuntos
Proteína Morfogenética Óssea 2/metabolismo , Proteína Morfogenética Óssea 4/metabolismo , Núcleo Celular/metabolismo , Sequência de Aminoácidos , Animais , Proteína Morfogenética Óssea 2/análise , Proteína Morfogenética Óssea 2/genética , Proteína Morfogenética Óssea 4/análise , Proteína Morfogenética Óssea 4/genética , Divisão Celular , Linhagem Celular , Códon de Iniciação , Fator 5 de Diferenciação de Crescimento/análise , Fator 5 de Diferenciação de Crescimento/metabolismo , Camundongos , Dados de Sequência Molecular , Transdução de SinaisRESUMO
We have previously shown that the anti-proliferative effect of retinoic acid in human breast cancer cell line MCF-7 is dependent on HES-1 expression. Here we show that retinoic acid induces HES-1 expression via upregulation of transcription factor SOX9. By expressing a dominant negative form of SOX9, disrupting endogenous SOX9 activity, the retinoic acid-induced HES-1 mRNA expression was inhibited. We found an enhancer regulating HES-1 expression: two SOX9 binding sites upstream of the HES-1 gene that were capable of binding SOX9 in vitro. By performing chromatin immunoprecipitation, we showed that SOX9 binding to the HES-1 enhancer was induced by retinoic acid in vivo. In reporter assays, transfection of a SOX9 expression plasmid increased the activity of the HES-1 enhancer. The enhancer responded to retinoic acid; furthermore, the expression of a dominant negative SOX9 abolished this response. Taken together, we present here a novel transcriptional mechanism in regulating hormone-dependent cancer cell proliferation.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/biossíntese , Neoplasias da Mama/genética , Regulação Neoplásica da Expressão Gênica/genética , Proteínas de Homeodomínio/biossíntese , Fatores de Transcrição SOX9/metabolismo , Antineoplásicos/farmacologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/efeitos dos fármacos , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Western Blotting , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Ensaio de Desvio de Mobilidade Eletroforética , Elementos Facilitadores Genéticos/genética , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteínas de Homeodomínio/efeitos dos fármacos , Proteínas de Homeodomínio/genética , Humanos , Imunoprecipitação , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição SOX9/efeitos dos fármacos , Fatores de Transcrição SOX9/genética , Fatores de Transcrição HES-1 , Transfecção , Tretinoína/farmacologiaRESUMO
The transcription factor Lc-Maf, which is a splice variant of c-Maf, is expressed in cartilage undergoing endochondral ossification and participates in the regulation of type II collagen through a cartilage-specific Col2a1 enhancer element. Type XXVII and type XI collagens are also expressed in cartilage during endochondral ossification, and so enhancer/reporter assays were used to determine whether Lc-Maf could regulate cartilage-specific enhancers from the Col27a1 and Col11a2 genes. The Col27a1 enhancer was upregulated over 4-fold by Lc-Maf, while the Col11a2 enhancer was downregulated slightly. To confirm the results of these reporter assays, rat chondrosarcoma (RCS) cells were transiently transfected with an Lc-Maf expression plasmid, and quantitative RT-PCR was performed to measure the expression of endogenous Col27a1 and Col11a2 genes. Endogenous Col27a1 was upregulated 6-fold by Lc-Maf overexpression, while endogenous Col11a2 was unchanged. Finally, in situ hybridization and immunohistochemistry were performed in the radius and ulna of embryonic day 17 mouse forelimbs undergoing endochondral ossification. Results demonstrated that Lc-Maf and Col27a1 mRNAs are coexpressed in proliferating and prehypertrophic regions, as would be predicted if Lc-Maf regulates Col27a1 expression. Type XXVII collagen protein was also most abundant in prehypertrophic and proliferating chondrocytes. Others have shown that mice that are null for Lc-Maf and c-Maf have expanded hypertrophic regions with reduced ossification and delayed vascularization. Separate studies have indicated that Col27a1 may serve as a scaffold for ossification and vascularization. The work presented here suggests that Lc-Maf may affect the process of endochondral ossification by participating in the regulation of Col27a1 expression.
Assuntos
Condrócitos/fisiologia , Colágenos Fibrilares/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Osteogênese/fisiologia , Proteínas Proto-Oncogênicas c-maf/metabolismo , Animais , Sequência de Bases , Osso e Ossos/citologia , Osso e Ossos/metabolismo , Osso e Ossos/fisiologia , Condrócitos/citologia , Colágeno Tipo XI/genética , Colágeno Tipo XI/metabolismo , Elementos Facilitadores Genéticos , Colágenos Fibrilares/genética , Membro Anterior/anatomia & histologia , Membro Anterior/fisiologia , Genes Reporter , Camundongos , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-maf/genética , RatosRESUMO
The transcription factor SOX9 plays a critical role in chondrogenesis as well as in sex determination. Previous work has suggested that SOX9 functions as a DNA-dependent dimer when it activates genes involved in chondrogenesis, but functions as a monomer to activate genes involved in sex determination. We present evidence herein for a third binding configuration through which SOX9 can activate transcription. We have identified four separate SOX consensus sequences in a COL9A1 collagen gene enhancer. The sites are arranged as two pairs, and each pair is similar to previously discovered dimeric SOX9 binding sites. Increasing the spacing between the pairs of sites eliminated enhancer activity in chondrocytic cells, as did the mutation of any one of the four sites. The COL9A1 enhancer is ordinarily inactive in 10T1/2 cells, but cotransfection with a SOX9 expression plasmid was sufficient to activate the enhancer, and mutation of any one of the four sites reduced responsiveness to SOX9 overexpression. These results suggest a novel mechanism for transcriptional activation by SOX9, in which two SOX9 dimers that are bound at the two pairs of sites are required to interact with one another, either directly or indirectly, in order to produce a functional transcriptional activation complex.
Assuntos
Colágeno Tipo IX/genética , Elementos Facilitadores Genéticos , Proteínas de Grupo de Alta Mobilidade/metabolismo , Fatores de Transcrição/metabolismo , Ativação Transcricional , Animais , Sequência de Bases , Sítios de Ligação , Linhagem Celular Tumoral , Sequência Consenso , Dimerização , Humanos , Dados de Sequência Molecular , Ratos , Fatores de Transcrição SOX9RESUMO
We previously identified a nuclear variant of bone morphogenetic protein 2 (BMP2), named nBMP2, that is translated from an alternative start codon. Decreased nuclear localization of nBMP2 in the nBmp2NLStm mouse model leads to muscular, neurological, and immune phenotypes-all of which are consistent with aberrant intracellular calcium (Ca2+) response. Ca2+ response in these mice, however, has yet to be measured directly. Because a prior study suggested impairment of macrophage function in nBmp2NLStm mutant mice, bone marrow derived (BMD) macrophages and splenic macrophages were isolated from wild type and nBmp2NLStm mutant mice. Immunocytochemistry revealed that nuclei of both BMD and splenic macrophages from wild type mice contain nBMP2, while the protein is decreased in nuclei of nBmp2NLStm mutant macrophages. Live-cell Ca2+ imaging and engulfment assays revealed that Ca2+ response and phagocytosis in response to bacterial supernatant are similar in BMD macrophages isolated from naïve (uninfected) nBmp2NLStm mutant mice and wild type mice, but are deficient in splenic macrophages isolated from mutant mice after secondary systemic infection with Staphylococcus aureus, suggesting progressive impairment as macrophages respond to infection. This direct evidence of impaired Ca2+ handling in nBMP2 mutant macrophages supports the hypothesis that nBMP2 plays a role in Ca2+ response.
Assuntos
Proteína Morfogenética Óssea 2/biossíntese , Cálcio/metabolismo , Núcleo Celular/metabolismo , Expressão Gênica , Macrófagos/metabolismo , Proteínas Nucleares/biossíntese , Animais , Proteína Morfogenética Óssea 2/genética , Núcleo Celular/genética , Camundongos , Camundongos Transgênicos , Proteínas Nucleares/genética , Infecções Estafilocócicas/genética , Infecções Estafilocócicas/metabolismo , Staphylococcus aureus/metabolismoRESUMO
The disproportionate micromelia (Dmm) mouse has a mutation in the C-propeptide coding region of the Col2a1 gene that causes lethal dwarfism when homozygous (Dmm/Dmm) but causes only mild dwarfism observable approximately 1-week postpartum when heterozygous (Dmm/+). The purpose of this study was 2-fold: first, to analyze and quantify morphological changes that precede the expression of mild dwarfism in Dmm/+ animals, and second, to compare morphological alterations between Dmm/+ and Dmm/Dmm fetal cartilage that may correlate with the marked skeletal differences between mild and lethal dwarfism. Light and electron transmission microscopy were used to visualize structure of chondrocytes and extracellular matrix (ECM) of fetal rib cartilage. Both Dmm/+ and Dmm/Dmm fetal rib cartilage had significantly larger chondrocytes, greater cell density, and less ECM per unit area than +/+ littermates. Quantitative RT-PCR showed a decrease in aggrecan mRNA in Dmm/+ vs +/+ cartilage. Furthermore, the cytoplasm of chondrocytes in Dmm/+ and Dmm/Dmm cartilage was occupied by significantly more distended rough endoplasmic reticulum (RER) compared with wild-type chondrocytes. Fibril diameters and packing densities of +/+ and Dmm/+ cartilage were similar, but Dmm/Dmm cartilage showed thinner, sparsely distributed fibrils. These findings support the prevailing hypothesis that a C-propeptide mutation could interrupt the normal assembly and secretion of Type II procollagen trimers, resulting in a buildup of proalpha1(II) chains in the RER and a reduced rate of matrix synthesis. Thus, intracellular entrapment of proalpha1(II) seems to be primarily responsible for the dominant-negative effect of the Dmm mutation in the expression of dwarfism.
Assuntos
Cartilagem/patologia , Colágeno Tipo II/genética , Nanismo/patologia , Agrecanas/metabolismo , Animais , Animais Recém-Nascidos , Cartilagem/embriologia , Cartilagem/crescimento & desenvolvimento , Condrócitos/metabolismo , Condrócitos/patologia , Nanismo/embriologia , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Heterozigoto , Homozigoto , Camundongos , Camundongos Mutantes , MutaçãoRESUMO
A critical area of emphasis for science educators is the identification of effective means of teaching and engaging undergraduate students. Personal microbiome analysis is a means of identifying the microbial communities found on or in our body. We hypothesized the use of personal microbiome analysis in the classroom could improve science education by making courses more applied and engaging for undergraduate students. We determined to test this prediction in three Brigham Young University undergraduate courses: Immunology, Advanced Molecular Biology Laboratory, and Genomics. These three courses have a two-week microbiome unit and students during the 2016 semester students could submit their own personal microbiome kit or use the demo data, whereas during the 2017 semester students were given access to microbiome data from an anonymous individual. The students were surveyed before, during, and after the human microbiome unit to determine whether analyzing their own personal microbiome data, compared to analyzing demo microbiome data, impacted student engagement and interest. We found that personal microbiome analysis significantly enhanced the engagement and interest of students while completing microbiome assignments, the self-reported time students spent researching the microbiome during the two week microbiome unit, and the attitudes of students regarding the course overall. Thus, we found that integrating personal microbiome analysis in the classroom was a powerful means of improving student engagement and interest in undergraduate science courses.
Assuntos
Alergia e Imunologia/educação , Atitude , Genômica/educação , Microbiota , Biologia Molecular/educação , Estudantes , Humanos , UniversidadesRESUMO
Diabetes and the related disease metabolic syndrome are epidemic in the United States, in part due to a shift in diet and decrease in physical exercise. PAS kinase is a sensory protein kinase associated with many of the phenotypes of these diseases, including hepatic triglyceride accumulation and metabolic dysregulation in male mice placed on a high-fat diet. Herein we provide the first characterization of the effects of western diet (high-fat high-sugar, HFHS) on Per-Arnt-Sim kinase mice (PASK-/-) and the first characterization of both male and female PASK-/- mice. Soleus muscle from the PASK-/- male mice displayed a 2-fold higher oxidative phosphorylation capacity than wild type (WT) on the normal chow diet. PASK-/- male mice were also resistant to hepatic triglyceride accumulation on the HFHS diet, displaying a 2.7-fold reduction in hepatic triglycerides compared to WT mice on the HFHS diet. These effects on male hepatic triglyceride were further explored through mass spectrometry-based lipidomics. The absence of PAS kinase was found to affect many of the 44 triglycerides analyzed, preventing hepatic triglyceride accumulation in response to the HFHS diet. In contrast, the female mice showed resistance to hepatic triglyceride accumulation on the HFHS diet regardless of genotype, suggesting the effects of PAS kinase may be masked.
Assuntos
Respiração Celular , Dieta Ocidental , Gorduras na Dieta/efeitos adversos , Açúcares da Dieta/efeitos adversos , Fígado/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Triglicerídeos/sangue , Animais , Translocador Nuclear Receptor Aril Hidrocarboneto/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Diabetes Mellitus/metabolismo , Dieta , Dieta Hiperlipídica , Gorduras na Dieta/administração & dosagem , Gorduras na Dieta/metabolismo , Açúcares da Dieta/administração & dosagem , Feminino , Genótipo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Esquelético/metabolismo , Proteínas Serina-Treonina Quinases/deficiência , Proteínas Serina-Treonina Quinases/genética , Proteínas Repressoras/metabolismoRESUMO
Obesity is associated with a high prevalence of mood disorders such as anxiety and depression. Both stress and high fat diet can alter the gut microbiota and contribute to obesity. To examine the interrelationships between obesity, stress, gut microbiota and mood disorders, obesity was induced in mice using a high fat diet, and the mice were subsequently stressed using a chronic unpredictable mild stress protocol. During the experiment, the composition of the gut microbiota was analyzed by 16 S rRNA gene high-throughput sequencing, and anxiety-like behaviors were measured. The results revealed distinct gender differences in the impacts of obesity and stress on anxiety-like behaviors, activity levels, and composition of the gut microbiota. Male mice were more vulnerable to the anxiogenic effects of the high fat diet, and obese male mice showed decreased locomotion activity in response to stress whereas obese female mice did not. In females, stress caused the gut microbiota of lean mice to more closely resemble that of obese mice. Taken together, these results suggest the importance of considering gender as a biological variable in studies on the role of gut microbiota in obesity-related mood disorders.
Assuntos
Comportamento Animal , Dieta Hiperlipídica , Microbioma Gastrointestinal , Estresse Fisiológico , Animais , Biologia Computacional/métodos , Feminino , Masculino , Metagenoma , Metagenômica/métodos , Camundongos , Fatores SexuaisRESUMO
The novel nuclear protein nBMP2 is synthesized from the BMP2 gene by translational initiation at an alternative start codon. We generated a targeted mutant mouse, nBmp2NLStm, in which the nuclear localization signal (NLS) was inactivated to prevent nuclear translocation of nBMP2 while still allowing the normal synthesis and secretion of the BMP2 growth factor. These mice exhibit abnormal muscle function due to defective Ca2+ transport in skeletal muscle. We hypothesized that neurological function, which also depends on intracellular Ca2+ transport, could be affected by the loss of nBMP2. Age-matched nBmp2NLStm and wild type mice were analyzed by immunohistochemistry, behavioral tests, and electrophysiology to assess nBMP2 expression and neurological function. Immunohistochemical staining of the hippocampus detected nBMP2 in the nuclei of CA1 neurons in wild type but not mutant mice, consistent with nBMP2 playing a role in the hippocampus. Mutant mice showed deficits in the novel object recognition task, suggesting hippocampal dysfunction. Electrophysiology experiments showed that long-term potentiation (LTP) in the hippocampus, which is dependent on intracellular Ca2+ transport and is thought to be the cellular equivalent of learning and memory, was impaired. Together, these results suggest that nBMP2 in the hippocampus impacts memory formation.
Assuntos
Proteína Morfogenética Óssea 2/genética , Proteína Morfogenética Óssea 2/metabolismo , Região CA1 Hipocampal/metabolismo , Memória/fisiologia , Processamento Alternativo , Animais , Proteína Morfogenética Óssea 2/química , Região CA1 Hipocampal/fisiologia , Cálcio/metabolismo , Núcleo Celular/metabolismo , Códon de Iniciação , Potenciação de Longa Duração , Masculino , Camundongos , Mutação , Sinais de Localização NuclearRESUMO
Expression of the type XI collagen gene Col11a2 is directed to cartilage by at least three chondrocyte-specific enhancer elements, two in the 5' region and one in the first intron of the gene. The three enhancers each contain two heptameric sites with homology to the Sox protein-binding consensus sequence. The two sites are separated by 3 or 4 bp and arranged in opposite orientation to each other. Targeted mutational analyses of these three enhancers showed that in the intronic enhancer, as in the other two enhancers, both Sox sites in a pair are essential for enhancer activity. The transcription factor Sox9 binds as a dimer at the paired sites, and the introduction of insertion mutations between the sites demonstrated that physical interactions between the adjacently bound proteins are essential for enhancer activity. Additional mutational analyses demonstrated that although Sox9 binding at the paired Sox sites is necessary for enhancer activity, it alone is not sufficient. Adjacent DNA sequences in each enhancer are also required, and mutation of those sequences can eliminate enhancer activity without preventing Sox9 binding. The data suggest a new model in which adjacently bound proteins affect the DNA bend angle produced by Sox9, which in turn determines whether an active transcriptional enhancer complex is assembled.
Assuntos
Condrócitos/metabolismo , DNA/metabolismo , Elementos Facilitadores Genéticos/genética , Proteínas de Grupo de Alta Mobilidade/genética , Fatores de Transcrição/genética , Células 3T3 , Animais , Sequência de Bases , Sítios de Ligação/genética , Condrócitos/citologia , Colágeno Tipo XI/genética , DNA/genética , Ensaio de Desvio de Mobilidade Eletroforética , Proteínas de Grupo de Alta Mobilidade/metabolismo , Luciferases/genética , Luciferases/metabolismo , Camundongos , Camundongos Transgênicos , Mutação , Oligonucleotídeos/genética , Oligonucleotídeos/metabolismo , Ligação Proteica , Ratos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Sequências Reguladoras de Ácido Nucleico/genética , Fatores de Transcrição SOX9 , Fatores de Transcrição/metabolismo , Transcrição Gênica , Ativação Transcricional , Células Tumorais CultivadasRESUMO
The most recently discovered collagen gene, COL27A1, codes for type XXVII collagen. The COL27A1 gene is strongly expressed in developing cartilage and weakly expressed in many other tissue types. The present study was undertaken to identify transcriptional regulatory mechanisms that govern the expression of COL27A1 in cartilage, and in particular to determine whether SOX9, a key regulator of chondrogenesis, could activate COL27A1. The first intron of COL27A1 was examined to identify sites with homology to the Sox consensus sequence (A)/(T)(A)/(T)CAA(A)/(T)G. Three 50-bp regions that contained paired Sox sites arranged in opposite orientation to each other and separated by 3 or 4 bp were targeted for further analysis. The elements were tested by transient transfection of reporter plasmids, and two of the three elements showed enhancer activity in chondrocytic cells. The same two elements bound SOX9 in electrophoretic mobility shift assays (EMSA). They were not transcriptionally active in fibroblasts, but cotransfection with a SOX9 expression plasmid resulted in activation. The independent mutation of either Sox site in a pair prevented SOX9 binding to the enhancers in EMSA experiments, indicating that SOX9 binds these enhancers only as a dimer. Mutation of either site in a pair also abolished enhancer activity in chondrocytes, indicating that dimeric binding of SOX9 is required for transcriptional activation of the two new enhancers. In summary, these results suggest that SOX9 may play an important role in the transcriptional activation of the newest collagen gene, COL27A1.
Assuntos
Elementos Facilitadores Genéticos/fisiologia , Colágenos Fibrilares/genética , Proteínas de Grupo de Alta Mobilidade/fisiologia , Fatores de Transcrição/fisiologia , Animais , Sequência de Bases , Linhagem Celular Tumoral , Eletroforese em Gel de Poliacrilamida , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Humanos , Íntrons , Dados de Sequência Molecular , Mutação , Ratos , Fatores de Transcrição SOX9RESUMO
Sox9 is a transcription factor that is critical for chondrogenesis, testis determination, and development of several other organs in vertebrates. Thus the levels of Sox9 protein and its activity may be tightly regulated. Here we show that inhibitors of the 26S proteasome increase both the levels of Sox9 protein and its transcriptional activity measured with Col2a1 promoter/enhancer construct in RCS cells and C3H10T1/2 cells. Indeed, in intact cells ubiquitination assays indicate that Sox9 is multiply ubiquitinated. The K398A mutation, which was introduced in a potential ubiquitin-binding site, increases the stability of Sox9 protein and its transcriptional activity of Col2a1, Col11a2, and AMH promoter/enhancer constructs without affecting the subcellular localization and the DNA binding efficiency of Sox9. Pulse-chase experiments show that the increased Sox9 levels resulting from treatment with the MG132 proteasome inhibitor or from the K398A mutation produce stabilization of the protein. Our in vitro studies indicate that the ubiquitin-proteasome proteolytic system degrades Sox9 and regulates its transcriptional activity.
Assuntos
Proteínas de Grupo de Alta Mobilidade/genética , Proteínas de Grupo de Alta Mobilidade/metabolismo , Complexo de Endopeptidases do Proteassoma/química , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Ubiquitina/química , Animais , Sítios de Ligação , Western Blotting , Células COS , Linhagem Celular Tumoral , Células Cultivadas , Condrócitos/metabolismo , Colágeno Tipo II/genética , Elementos Facilitadores Genéticos , Proteínas de Grupo de Alta Mobilidade/química , Humanos , Camundongos , Camundongos Endogâmicos C3H , Microscopia de Fluorescência , Mutação , Regiões Promotoras Genéticas , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma , Estrutura Terciária de Proteína , Ratos , Fatores de Transcrição SOX9 , Fatores de Tempo , Fatores de Transcrição/química , Transcrição Gênica , Transfecção , Ubiquitina/metabolismoRESUMO
The Col11a2 gene codes for alpha2(XI), a subunit of type XI collagen that is a critical component of the cartilage extracellular matrix. The 5' regulatory region of Col11a2 was subjected to deletional analysis to detect any regulatory element in addition to the two known chondrocyte-specific enhancer elements B/C and D/E. Deletion of the region from -342 to -242 bp reduced transcriptional activity to less than 50% of wild-type, but the sequence showed no independent ability to increase transcription from a minimal promoter. When cloned downstream of the D/E enhancer, however, a subsection of the sequence nearly doubled transcriptional activity and produced an additional 3-fold activation in response to RA (retinoic acid). A 6-bp direct repeat, separated by 4 bp (a DR-4 element) near the 5'-end of this region, was found to be essential for its activity, and was further shown to bind the RA X receptor beta in electrophoretic mobility-shift assays. The present study has revealed a novel RA-response element in Col11a2 that does not interact directly with the promoter, but instead requires the D/E enhancer to mediate transcriptional activation. Proteins bound at the enhancer, therefore, would be expected to affect the transcriptional response to RA. Such a system of regulation, particularly if found to be operating in other cartilage genes, could explain the conflicting responses RA produces in chondrocytes under different experimental conditions.
Assuntos
Colágeno Tipo XI/genética , Elementos Facilitadores Genéticos/fisiologia , Elementos de Resposta/fisiologia , Ativação Transcricional/fisiologia , Tretinoína/fisiologia , Animais , Sequência de Bases , Condrócitos/metabolismo , Proteínas de Ligação a DNA/metabolismo , Camundongos , Ratos , Receptor X Retinoide beta/metabolismo , Deleção de Sequência , Células Tumorais CultivadasRESUMO
Chronic low back pain (LBP) is a prevalent global problem, which is often correlated with degenerative disc disease. The development and use of good, relevant animal models of the spine may improve treatment options for this condition. While no animal model is capable of reproducing the exact biology, anatomy, and biomechanics of the human spine, the quality of a particular animal model increases with the number of shared characteristics that are relevant to the human condition. The purpose of this study was to investigate the camelid (specifically, alpaca and llama) cervical spine as a model of the human lumbar spine. Cervical spines were obtained from four alpacas and four llamas and individual segments were used for segmental flexibility/biomechanics and/or morphology/anatomy studies. Qualitative and quantitative data were compared for the alpaca and llama cervical spines, and human lumbar specimens in addition to other published large animal data. Results indicate that a camelid cervical intervertebral disc (IVD) closely approximates the human lumbar disc with regard to size, spinal posture, and biomechanical flexibility. Specifically, compared with the human lumbar disc, the alpaca and llama cervical disc size are approximately 62%, 83%, and 75% with regard to area, depth, and width, respectively, and the disc flexibility is approximately 133%, 173%, and 254%, with regard to range of motion (ROM) in axial-rotation, flexion-extension, and lateral-bending, respectively. These results, combined with the clinical report of disc degeneration in the llama lower cervical spine, suggest that the camelid cervical spine is potentially well suited for use as an animal model in biomechanical studies of the human lumbar spine.
RESUMO
UNLABELLED: The giant panda evolved from omnivorous bears. It lives on a bamboo-dominated diet at present, but it still retains a typical carnivorous digestive system and is genetically deficient in cellulose-digesting enzymes. To find out whether this endangered mammalian species, like other herbivores, has successfully developed a gut microbiota adapted to its fiber-rich diet, we conducted a 16S rRNA gene-based large-scale structural profiling of the giant panda fecal microbiota. Forty-five captive individuals were sampled in spring, summer, and late autumn within 1 year. Significant intraindividual variations in the diversity and structure of gut microbiota across seasons were observed in this population, which were even greater than the variations between individuals. Compared with published data sets involving 124 gut microbiota profiles from 54 mammalian species, these giant pandas, together with 9 captive and 7 wild individuals investigated previously, showed extremely low gut microbiota diversity and an overall structure that diverged from those of nonpanda herbivores but converged with those of carnivorous and omnivorous bears. The giant panda did not harbor putative cellulose-degrading phylotypes such as Ruminococcaceae and Bacteroides bacteria that are typically enriched in other herbivores, but instead, its microbiota was dominated by Escherichia/Shigella and Streptococcus bacteria. Members of the class Clostridia were common and abundant in the giant panda gut microbiota, but most of the members present were absent in other herbivores and were not phylogenetically related with known cellulolytic lineages. Therefore, the giant panda appears not to have evolved a gut microbiota compatible with its newly adopted diet, which may adversely influence the coevolutionary fitness of this herbivore. IMPORTANCE: The giant panda, an endangered mammalian species endemic to western China, is well known for its unique bamboo diet. Unlike other herbivores that have successfully evolved anatomically specialized digestive systems to efficiently deconstruct fibrous plant matter, the giant panda still retains a gastrointestinal tract typical of carnivores. We characterized the fecal bacterial communities from a giant panda population to determine whether this animal relies on its symbiotic gut microbiota to cope with the complex carbohydrates that dominate its diet, as is common in other herbivores. We found that the giant panda gut microbiota is low in diversity and highly variable across seasons. It also shows an overall composition typical of bears and entirely differentiated from other herbivores, with low levels of putative cellulose-digesting bacteria. The gut microbiota of this herbivore, therefore, may not have well adapted to its highly fibrous diet, suggesting a potential link with its poor digestive efficiency.
Assuntos
Fezes/microbiologia , Microbioma Gastrointestinal , Ursidae/microbiologia , Animais , Animais de Zoológico , China , Análise por Conglomerados , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Ribossômico/química , DNA Ribossômico/genética , Dados de Sequência Molecular , Filogenia , RNA Ribossômico 16S/genética , Estações do Ano , Análise de Sequência de DNARESUMO
We recently identified a nuclear variant of the BMP2 growth factor, called nBMP2. In an effort to understand the function of this variant protein, we generated a mouse line in which BMP2 is expressed and functions normally, but nBMP2 is excluded from the nucleus. This novel mutation allows the study of nBMP2 without compromising BMP2 function. To determine whether nBMP2 plays a role in immune function, we performed a series of experiments in which we compared mouse survival, organ weights, immune cells numbers, and bacterial load in wild type and nBmp2NLS(tm) mice following primary and secondary challenges with Staphylococcus aureus. Following primary challenge with S. aureus, wild type and nBmp2NLS(tm) mice showed no differences in survival or bacterial load and generated similar numbers and types of leukocytes, although mutant spleens were smaller than wild type. Secondary bacterial challenge with S. aureus, however, produced differences in survival, with increased mortality seen in nBmp2NLS(tm) mice. This increased mortality corresponded to higher levels of bacteremia in nBmp2NLS(tm) mice and to a reduced enlargement of mutant spleens in response to the secondary infection. Together, these results suggest that the recently described nuclear variant of BMP2 is necessary for efficient secondary immune responses.
Assuntos
Proteína Morfogenética Óssea 2/imunologia , Mutação , Proteínas Nucleares/imunologia , Infecções Estafilocócicas/imunologia , Staphylococcus aureus/imunologia , Animais , Proteína Morfogenética Óssea 2/genética , Humanos , Camundongos , Camundongos Mutantes , Proteínas Nucleares/genética , Infecções Estafilocócicas/genética , Infecções Estafilocócicas/patologiaRESUMO
Animal models have historically provided an appropriate benchmark for understanding human pathology, treatment, and healing, but few animals are known to naturally develop intervertebral disc degeneration. The study of degenerative disc disease and its treatment would greatly benefit from a more comprehensive, and comparable animal model. Alpacas have recently been presented as a potential large animal model of intervertebral disc degeneration due to similarities in spinal posture, disc size, biomechanical flexibility, and natural disc pathology. This research further investigated alpacas by determining the prevalence of intervertebral disc degeneration among an aging alpaca population. Twenty healthy female alpacas comprised two age subgroups (5 young: 2-6 years; and 15 older: 10+ years) and were rated according to the Pfirrmann-grade for degeneration of the cervical intervertebral discs. Incidence rates of degeneration showed strong correlations with age and spinal level: younger alpacas were nearly immune to developing disc degeneration, and in older animals, disc degeneration had an increased incidence rate and severity at lower cervical levels. Advanced disc degeneration was present in at least one of the cervical intervertebral discs of 47% of the older alpacas, and it was most common at the two lowest cervical intervertebral discs. The prevalence of intervertebral disc degeneration encourages further investigation and application of the lower cervical spine of alpacas and similar camelids as a large animal model of intervertebral disc degeneration.