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The dorsal and anal fins can vary widely in position and length along the anterior-posterior axis in teleost fishes. However, the molecular mechanisms underlying the diversification of these fins remain unknown. Here, we used genetic approaches in zebrafish and medaka, in which the relative positions of the dorsal and anal fins are opposite, to demonstrate the crucial role of hox genes in the patterning of the teleost posterior body, including the dorsal and anal fins. By the CRISPR-Cas9-induced frameshift mutations and positional cloning of spontaneous dorsalfinless medaka, we show that various hox mutants exhibit the absence of dorsal or anal fins, or a stepwise posterior extension of these fins, with vertebral abnormalities. Our results indicate that multiple hox genes, primarily from hoxc-related clusters, encompass the regions responsible for the dorsal and anal fin formation along the anterior-posterior axis. These results further suggest that shifts in the anterior boundaries of hox expression which vary among fish species, lead to diversification in the position and size of the dorsal and anal fins, similar to how modulations in Hox expression can alter the number of anatomically distinct vertebrae in tetrapods. Furthermore, we show that hox genes responsible for dorsal fin formation are different between zebrafish and medaka. Our results suggest that a novel mechanism has occurred during teleost evolution, in which the gene network responsible for fin formation might have switched to the regulation downstream of other hox genes, leading to the remarkable diversity in the dorsal fin position.
Assuntos
Nadadeiras de Animais , Genes Homeobox , Proteínas de Homeodomínio , Oryzias , Peixe-Zebra , Animais , Oryzias/genética , Peixe-Zebra/genética , Genes Homeobox/genética , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Padronização Corporal/genética , Proteínas de Peixes/genética , Proteínas de Peixes/metabolismoRESUMO
The vertebral column is a characteristic structure of vertebrates. Genetic studies in mice have shown that Hox-mediated patterning plays a key role in specifying discrete anatomical regions of the vertebral column. Expression pattern analyses in several vertebrate embryos have provided correlative evidence that the anterior boundaries of Hox expression coincide with distinct anatomical vertebrae. However, because functional analyses have been limited to mice, it remains unclear which Hox genes actually function in vertebral patterning in other vertebrates. In this study, various zebrafish Hox mutants were generated for loss-of-function phenotypic analysis to functionally decipher the Hox code responsible for the zebrafish anterior vertebrae between the occipital and thoracic vertebrae. We found that Hox genes in HoxB- and HoxC-related clusters participate in regulating the morphology of the zebrafish anterior vertebrae. In addition, medaka hoxc6a was found to be responsible for anterior vertebral identity, as in zebrafish. Based on phenotypic similarities with Hoxc6 knockout mice, our results suggest that the Hox patterning system, including at least Hoxc6, may have been functionally established in the vertebral patterning of the common ancestor of ray-finned and lobe-finned fishes.
Assuntos
Padronização Corporal , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio , Coluna Vertebral , Proteínas de Peixe-Zebra , Peixe-Zebra , Animais , Peixe-Zebra/genética , Peixe-Zebra/embriologia , Coluna Vertebral/embriologia , Padronização Corporal/genética , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo , Genes Homeobox/genética , Oryzias/genética , Oryzias/embriologia , CamundongosRESUMO
We encountered a pediatric case of cubitus varus deformity with a sheared olecranon fracture in an 8-year-old boy who underwent corrective osteotomy without relevant medical history. The patient fell, resulting in a sheared olecranon fracture. He underwent a closed reduction and casting. As the displacement slightly increased within a week, we followed him without secondary reduction to expect remodeling. No remodeling occurred; corrective osteotomy was performed one-year post-injury for a marked cubitus varus deformity. At 2.5 years after corrective osteotomy, little difference existed in the carrying angle (CA) and varus angulation (VA) of the proximal ulna than that of the contralateral side, without pain or limited range of motion. The acceptable displacement range for pediatric forearm fractures is <1 cm shortening and 15° angular deformation in patients under 10 years old, and 10° angular deformation in older children. Here, the deformity of the ulna in the coronal plane did not remodel. Proximal forearm deformity can be accurately evaluated in flexion contracture elbows by measuring VA. Ulnar osteotomies are commonly performed on Monteggia fractures to reduce the radial head, and the osteotomy site is at the center of the deformity of the diaphysis. Corrective osteotomy for cubitus varus deformity after supracondylar humerus fracture improves function and cosmetic appearance, with good clinical results. In addition, it could prevent cubitus varus deformity from causing posterolateral rotatory instability. The coronal-plane deformity of the proximal ulnar was not expected to remodel. We recommended early accurate reduction and consideration of additional internal fixation for preventing re-displacement. Corrective osteotomy for cubitus varus deformity of the proximal ulna was an effective treatment.
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Approximately 1.14 billion smokers worldwide are at risk of developing tumors, cardiovascular diseases and respiratory diseases. Smoking cessation is the first choice of health care; however, the disease should be attenuated in individuals who never stop smoking, which escalates medical costs. Therefore, alternative options are needed to manage the social burden. The present study proposed an alternative method to prevent such diseases by inhalation of ß-caryophyllene (BCP). A placebo-targeted, dose-searching, double-blind, parallel-group comparative study was conducted on 19 subjects. The BCP intervention was performed using a flavor capsule inserted in a cigarette filter. The primary endpoint was the reducibility of brachial-ankle pulse wave velocity (baPWV). The secondary endpoints were confirmation of the bioavailability of BCP inhalation with cigarette smoke, confirmation of the effect of BCP inhalation on respiratory function, and association between respiratory function and blood concentration and baPWV reduction. The BCP concentration in the blood reached 4 ng/ml in the BCP 15% group 10 min after inhalation. The baPWV decreased in BCP-inhaling subjects whose initial baPWV was >1,300 cm/sec. The correlation analyses revealed that the higher the forced expiratory volume in 1 sec, the better the transition of baPWV. Inhaled BCP with cigarette smoke could reduce the baPWV and the risk of cardiovascular diseases in smokers. These findings indicated that with the introduction of BCP capsule-cigarettes in the future, smokers will be able to take care of their health, which may help reduce national medical costs. BCP microcapsules placed in cigarette wrapping paper may possibly reduce the risk of sidestream smoke and contribute to improved public health. This clinical research was retrospectively registered in the University Hospital Medical Information Network (UMIN)-Clinical Trials Registry with the following identifications: UMIN000048510 and UMIN000048512 on August 15, 2022.
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Multiple subcutaneous extensor tendon ruptures in more than the ulnar three fingers sometimes occur in patients with rheumatoid arthritis accompanied by wrist deformity. In these situations, the flexor digitorum superficialis tendon of the middle finger (the FDS3 tendon) and that of the ring finger (the FDS4 tendon) are used for the transferred tendon (modified Boyes' procedure). Here, we treated two patients with rheumatoid arthritis, whose extensor tendons of more than three fingers were ruptured, using the modified Boyes' procedure. Case 1 had ruptures in four fingers (index through little), and Case 2 had ruptures in three fingers (middle through little). The FDS3 and FDS4 tendons were passed subcutaneously around the radial side of the wrist to the extensor sides and interlaced with the distal stump of the ruptured tendons. Switching of the finger movement was achieved smoothly in both cases. The post-operative evaluation showed an extension lag of -15° for the index finger 0° for the middle through the little fingers in Case 1, and 0° for the middle finger and -5° for the ring and little fingers in Case 2. The average post-operative extension lag was -3.5°. However, median nerve palsy occurred in both cases, and it gradually recovered. Stretching of the nerve by the correction of the wrist deformity and increased pressure in the carpal tunnel were supposed to be causes of this palsy. Modified Boyes' procedure is a useful method for more than three ulnar finger extensor tendon ruptures; however, post-operative median nerve palsy should be considered.
Assuntos
Artrite Reumatoide , Doenças Musculares , Humanos , Tendões/cirurgia , Artrite Reumatoide/complicações , Artrite Reumatoide/diagnóstico , Artrite Reumatoide/cirurgia , Dedos , Paralisia/complicaçõesRESUMO
ß-caryophyllene (BCP) is a volatile bicyclic sesquiterpenoid found in essential oils obtained from several spices such as black pepper, oregano, basil, rosemary, cinnamon, and clove. BCP is a selective agonist of cannabinoid receptor 2 (CB2 receptor), and orally administered BCP exhibits various biological activities, including anti-inflammatory, antioxidant, and neuroprotective effects. However, it is still unclear how volatile BCP affects living organisms. We previously reported that inhaled BCP is transferred to sera and organs in mice; additionally, metabolomic analysis revealed inhaled BCP affect the dynamics of metabolites in the livers of mice. These data suggest that inhaled BCP may affect several biological activities by stimulating biological systems. In this study, we evaluated the effects of BCP inhalation on nicotine-induced degeneration of the aortic wall. In the group of mice which inhaled volatile BCP, nicotine-induced increases in elastic fiber degradation and matrix metalloproteinase-2 (MMP-2)-positive areas were attenuated. In addition, BCP improved the nicotine-induced stiffness of aortae and vulnerability to aortic rupture. In cultured aortae, the suppressive effects of BCP were inhibited by the CB2 receptor inhibitor AM630. These results suggest that inhaled BCP is incorporated into the aortic wall and prevents nicotine-induced degeneration of the aorta via a CB2 receptor-dependent pathway.
Assuntos
Nicotina , Sesquiterpenos , Animais , Aorta , Metaloproteinase 2 da Matriz , Camundongos , Sesquiterpenos Policíclicos , Receptor CB2 de Canabinoide , Sesquiterpenos/farmacologiaRESUMO
In this study, we fabricated two different microneedles (MNs) - semi-hollow and bird-bill - to overcome the limitations of solid and coated MNs, respectively. The two MN arrays were developed using a general injection molding process to obtain high-quality MNs with uniform shape. The semi-hollow and bird-bill MNs could penetrate the micropores of swine skin up to depths of 178.5 ± 27.6 µm and 232.1 ± 51.3 µm, respectively. When the semi-hollow MNs were used for the transdermal delivery of insulin in diabetic rats, it was observed that the blood glucose concentration (BGC) decreased remarkably within 30 min, and the desired effect of insulin was maintained for an additional 3 h after the removal of insulin from the skin surface. The bird-bill MN was able to load a coating gel at a maximum capacity of 3.20 ± 0.21 mg per MN array, and the BGC continued to decrease significantly after MN application for up to 2-6 h. In summary, we fabricated semi-hollow and bird-bill MN arrays using the injection molding method; these can be mass produced and are capable of effectively producing micro-holes in the stratum corneum. The two MN arrays could provide effective transdermal delivery of large-molecular-weight drugs such as insulin.
Assuntos
Diabetes Mellitus Experimental , Preparações Farmacêuticas , Administração Cutânea , Animais , Diabetes Mellitus Experimental/tratamento farmacológico , Sistemas de Liberação de Medicamentos , Insulina , Microinjeções , Agulhas , Ratos , PeleRESUMO
Vertebrate Hox clusters are comprised of multiple Hox genes that control morphology and developmental timing along multiple body axes. Although results of genetic analyses using Hox-knockout mice have been accumulating, genetic studies in other vertebrates have not been sufficient for functional comparisons of vertebrate Hox genes. In this study, we isolated all of the seven hox cluster loss-of-function alleles in zebrafish using the CRISPR-Cas9 system. Comprehensive analysis of the embryonic phenotype and X-ray micro-computed tomography scan analysis of adult fish revealed several species-specific functional contributions of homologous Hox clusters along the appendicular axis, whereas important shared general principles were also confirmed, as exemplified by serial anterior vertebral transformations along the main body axis, observed in fish for the first time. Our results provide insights into discrete sub/neofunctionalization of vertebrate Hox clusters after quadruplication of the ancient Hox cluster. This set of seven complete hox cluster loss-of-function alleles provide a formidable resource for future developmental genetic analysis of the Hox patterning system in zebrafish.
Assuntos
Genes Homeobox/genética , Família Multigênica , Peixe-Zebra/genética , Peixe-Zebra/fisiologia , Animais , Sistemas CRISPR-Cas , Desenvolvimento Embrionário/genética , Evolução Molecular , Feminino , Duplicação Gênica , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Mutação , Esqueleto/anatomia & histologia , Esqueleto/crescimento & desenvolvimento , Especificidade da Espécie , Microtomografia por Raio-X , Peixe-Zebra/embriologiaRESUMO
Members of the enhancer of split- and hairy-related protein (SHARP) family, SHARP-1 and SHARP-2, are basic helix-loop-helix transcriptional repressors and belong to the clock genes. In this study, an effect of retinoic acid (RA) on the SHARP family gene expression in the differentiated cells was examined. RA rapidly and temporarily induced the SHARP-2 mRNA expression in hepatic H4IIE cells. Then, whether the SHARP-2 mRNA expression is altered by dexamethasone (Dex), insulin, and the combination of RA and Dex or RA and insulin was examined. Dex had different effects on the expression of SHARP-2 mRNA in the presence or absence of RA. Then, the molecular mechanisms were investigated using inhibitors of various signaling molecules. The RA-induction of SHARP-2 mRNA level was mainly inhibited by LY294002, staurosporine, and actinomycin D, respectively. Finally, whether RA acts on the transcriptional regulatory region of the SHARP-2 gene was analysed using luciferase reporter gene assay. At least two RA-responsive regions were mapped at the nucleotide sequences between -3,700 and -1,600 of the SHARP-2 gene. In addition, this effect was dependent on the RA receptor and retinoid X receptor. Thus, we conclude that RA stimulated transcription of the SHARP-2 gene via multiple pathways.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Proteínas de Homeodomínio/genética , Transcrição Gênica/efeitos dos fármacos , Tretinoína/farmacologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Linhagem Celular , Dexametasona/farmacologia , Células Hep G2 , Hepatócitos/metabolismo , Proteínas de Homeodomínio/metabolismo , Humanos , Insulina/farmacologia , RNA Mensageiro/genética , Ratos , Receptores do Ácido Retinoico/metabolismo , Receptores X de Retinoides/metabolismo , Transdução de Sinais/efeitos dos fármacos , Tretinoína/metabolismoRESUMO
Melatonin plays physiological roles in various critical processes, including circadian rhythms, oxidative stress defenses, anti-inflammation responses, and immunity; however, the current understanding of the role of melatonin in hepatic glucose metabolism is limited. In this study, we examined whether melatonin affects gene expression of the key gluconeogenic enzyme, phosphoenolpyruvate carboxykinase (PEPCK). We found that melatonin treatment increased PEPCK mRNA levels in rat highly differentiated hepatoma (H4IIE) cells and primary cultured hepatocytes. In addition, we found that melatonin induction was synergistically enhanced by dexamethasone, whereas it was dominantly inhibited by insulin. We also report that the effect of melatonin was blocked by inhibitors of mitogen-activated protein kinase/extracellular signal-regulated protein kinase (MAPK/ERK), RNA polymerase II, and protein synthesis. Furthermore, the phosphorylated (active) forms of ERK1 and ERK2 (ERK1/2) increased 15 min after melatonin treatment. We performed luciferase reporter assays to show that melatonin specifically stimulated promoter activity of the PEPCK gene. Additional reporter analysis using 5'-deleted constructs revealed that the regulatory regions responsive to melatonin mapped to two nucleotide regions, one between -467 and -398 nucleotides and the other between -128 and +69 nucleotides, of the rat PEPCK gene. Thus, we conclude that melatonin induces PEPCK gene expression via the ERK1/2 pathway at the transcriptional level, and that induction requires de novo protein synthesis.
Assuntos
Hepatócitos/metabolismo , Melatonina/farmacologia , Fosfoenolpiruvato Carboxiquinase (ATP)/genética , Animais , Masculino , Fosfoenolpiruvato Carboxiquinase (ATP)/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Células Tumorais CultivadasRESUMO
The rat enhancer of split- and hairy-related protein (SHARP)-1 genes encode insulin-inducible transcriptional repressors. A longevity gene, sirtuin 1 (SIRT1) encodes protein deacetylase. These play an important role in regulating hepatic glucose metabolism. In this study, to evaluate a correlation with these gene expressions, we examined whether SIRT1 effects on expression of the SHARP-1 gene by a treatment with a SIRT1 inhibitor or activator in rat H4IIE hepatoma cells. Whereas the SIRT1 inhibitor increased the level of SHARP-1 mRNA, the SIRT1 activator decreased it. Next, whether SHARP-1 effect on the transcriptional activity of the human SIRT1 gene using luciferase reporter assays was determined. Promoter activity of the SIRT1 gene was specifically repressed by SHARP-1. Further reporter analysis using 5'- deleted or mutated constructs revealed that an E box sequence (5'-CACGTG-3') of the SIRT1 gene promoter was required for the inhibitory effect of SHARP-1. Thus, we conclude that expressions between the SHARP-1 and the SIRT1 genes show a negative correlation and that SHARP-1 represses transcription of the SIRT1 gene.
RESUMO
In the zebrafish segmentation clock, hairy/enhancer of split-related genes her1, her7, and hes6 encodes components of core oscillators. Since the expression of cyclic genes proceeds rapidly in the presomitic mesoderm (PSM), these hairy-related mRNAs are subject to strict post-transcriptional regulation. In this study, we demonstrate that inhibition of the CCR4-NOT deadenylase complex lengthens poly(A) tails of hairy-related mRNAs and increases the amount of these mRNAs, which is accompanied by defective somite segmentation. In transgenic embryos, we show that EGFP mRNAs with 3'UTRs of hairy-related genes exhibit turnover similar to endogenous mRNAs. Our results suggest that turnover rates of her1, her7, and hes6 mRNAs are differently regulated by the CCR4-NOT deadenylase complex possibly through their 3'UTRs in the zebrafish PSM.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Regulação da Expressão Gênica no Desenvolvimento , RNA Mensageiro/genética , Somitos/metabolismo , Fatores de Transcrição/genética , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/genética , Animais , Animais Geneticamente Modificados , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Relógios Biológicos , Padronização Corporal/genética , Exorribonucleases/genética , Exorribonucleases/metabolismo , Mesoderma/embriologia , Mesoderma/metabolismo , Processamento Pós-Transcricional do RNA , RNA Mensageiro/metabolismo , Somitos/embriologia , Fatores de Transcrição/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/metabolismoRESUMO
We performed whole exome or genome sequencing in eight multiply affected families with ostensibly isolated congenital anosmia. Hypothesis-free analyses based on the assumption of fully penetrant recessive/dominant/X-linked models obtained no strong single candidate variant in any of these families. In total, these eight families showed 548 rare segregating variants that were predicted to be damaging, in 510 genes. Three Kallmann syndrome genes (FGFR1, SEMA3A, and CHD7) were identified. We performed permutation-based analysis to test for overall enrichment of these 510 genes carrying these 548 variants with genes mutated in Kallmann syndrome and with a control set of genes mutated in hypogonadotrophic hypogonadism without anosmia. The variants were found to be enriched for Kallmann syndrome genes (3 observed vs. 0.398 expected, p = 0.007), but not for the second set of genes. Among these three variants, two have been already reported in genes related to syndromic anosmia (FGFR1 (p.(R250W)), CHD7 (p.(L2806V))) and one was novel (SEMA3A (p.(T717I))). To replicate these findings, we performed targeted sequencing of 16 genes involved in Kallmann syndrome and hypogonadotrophic hypogonadism in 29 additional families, mostly singletons. This yielded an additional 6 variants in 5 Kallmann syndrome genes (PROKR2, SEMA3A, CHD7, PROK2, ANOS1), two of them already reported to cause Kallmann syndrome. In all, our study suggests involvement of 6 syndromic Kallmann genes in isolated anosmia. Further, we report a yet unreported appearance of di-genic inheritance in a family with congenital isolated anosmia. These results are consistent with a complex molecular basis of congenital anosmia.
Assuntos
Síndrome de Kallmann/genética , Transtornos do Olfato/congênito , DNA Helicases/genética , Proteínas de Ligação a DNA/genética , Proteínas da Matriz Extracelular/genética , Feminino , Hormônios Gastrointestinais/genética , Humanos , Masculino , Proteínas do Tecido Nervoso/genética , Neuropeptídeos/genética , Transtornos do Olfato/genética , Transtornos do Olfato/patologia , Linhagem , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética , Semaforina-3A/genética , Sequenciamento do ExomaRESUMO
Leukocyte telomere length and serum levels of high-molecular-weight adiponectin and dehydroepiandrosterone-sulfate (DHEA-S) were assessed in association with nutrition and performance status (PS) in Japanese centenarians. Twenty-three centenarians (five men, 18 women) were classified according to their PS 1 (nearly fully ambulatory, n = 2), 2 (in bed less than 50% of daytime, n = 10), 3 (in bed greater than 50%, n = 6), and 4 (completely bedridden, n = 5). Leukocyte telomere length was determined by the hybridization protection assay, and the adiponectin and DHEA-S levels were measured by chemiluminescent enzyme immunoassay. Among variables of PS, body mass index (BMI), albumin, adiponectin, DHEA-S, and telomere length, there were significant correlations between PS and albumin (r = -.694, p < .01), between telomere length and BMI (r = .522, p < .05), between adiponectin and BMI (r = -.574, p < .01), and between DHEA-S and albumin (r = .530, p < .01). When excluding two cancer-bearing centenarians with short telomere, telomere length significantly correlated with PS (r = -.632, p < .01). It was indicated that the short leukocyte telomere was associated with poor PS and cancer development and that the adiponectin or DHEA-S was associated with adiposity or nutritional status. Despite a small number of subjects, these biomarkers seemed to reflect distinct aspects of longevity in Japanese centenarians.
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The efficacy and safety of botulinum toxin B (BTX-B) for treatment of Raynaud's phenomenon and digital ulcers in patients with systemic sclerosis was assessed. A total of 45 patients with systemic sclerosis who had Raynaud's phenomenon were blinded and divided randomly into 4 groups: a no-treatment control group, and 3 treatment groups, using 250, 1,000 or 2,000 international units (U) of BTX-B injections in the hand with more severe symptoms. Four weeks after injection, pain/numbness visual analogue scale scores and Raynaud's score in the groups treated with 1,000 and 2,000 U BTX-B were significantly lower than in the control group and the group treated with 250 U BTX-B. These beneficial effects were sustained until 16 weeks after the single injection. At 4 weeks after injection skin temperature recovery in the group treated with 2,000 U BTX-B was significantly improved. The numbers of digital ulcers in the groups treated with 1,000 and 2,000 U BTX-B were significantly lower than in the control group. In conclusion, 1,000 and 2,000 U BTX-B injections significantly suppressed the activity of Raynaud's phenomenon and digital ulcers in patients with SSc without serious adverse events.
Assuntos
Inibidores da Liberação da Acetilcolina/administração & dosagem , Toxinas Botulínicas Tipo A/administração & dosagem , Doença de Raynaud/tratamento farmacológico , Escleroderma Sistêmico/complicações , Úlcera Cutânea/tratamento farmacológico , Inibidores da Liberação da Acetilcolina/efeitos adversos , Idoso , Toxinas Botulínicas Tipo A/efeitos adversos , Feminino , Humanos , Injeções , Japão , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Doença de Raynaud/diagnóstico , Doença de Raynaud/etiologia , Doença de Raynaud/fisiopatologia , Método Simples-Cego , Temperatura Cutânea/efeitos dos fármacos , Úlcera Cutânea/diagnóstico , Úlcera Cutânea/etiologia , Úlcera Cutânea/fisiopatologia , Fatores de Tempo , Resultado do Tratamento , Cicatrização/efeitos dos fármacosRESUMO
The rat enhancer of split- and hairy-related protein-2 (SHARP-2) is an insulin-inducible transcription factor which represses transcription of the rat phosphoenolpyruvate carboxykinase gene. In this study, a regulatory mechanism of the SHARP-2 mRNA level by insulin was analyzed. Insulin rapidly induced the level of SHARP-2 mRNA. This induction was blocked by inhibitors for phosphoinositide 3-kinase (PI 3-K), protein kinase C (PKC), and mammalian target of rapamycin (mTOR), actinomycin D, and cycloheximide. Whereas an adenovirus infection expressing a dominant negative form of atypical PKC lambda (aPKCλ) blocked the insulin-induction of the SHARP-2 mRNA level, insulin rapidly activated the mTOR. Insulin did not enhance transcriptional activity from a 3.7 kb upstream region of the rat SHARP-2 gene. Thus, we conclude that insulin induces the expression of the rat SHARP-2 gene at the transcription level via both a PI 3-K/aPKCλ- and a PI 3-K/mTOR- pathways and that protein synthesis is required for this induction.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Proteínas de Homeodomínio/genética , Insulina/farmacologia , Transdução de Sinais/efeitos dos fármacos , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/biossíntese , Linhagem Celular Tumoral , Proteínas de Homeodomínio/biossíntese , Isoenzimas/genética , Proteína Quinase C/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Serina-Treonina Quinases TOR/metabolismo , Transcrição Gênica/efeitos dos fármacosRESUMO
Raynaud's phenomenon is frequently observed in systemic sclerosis (SSc) patients, and cold- or stress-induced norepinephrine (NE) has been speculated to be associated with vasoconstriction. Objective was to elucidate the role of NE in fibrosis in SSc. IL-6 is a potent stimulator of collagen production in fibroblasts. NE enhanced IL-6 production and proliferation more significantly in SSc fibroblasts than in normal fibroblasts. Furthermore, the production of IL-6 and phosphorylation of p38 in SSc fibroblasts was enhanced by adrenergic receptor (AR)ß agonist, isoproterenol, but not ARα agonist, oxymetazoline. ARß blocker, propranolol, inhibited NE-induced IL-6 production and phosphorylation of p38 in SSc fibroblasts. NE-induced IL-6 was significantly inhibited by p38 inhibitor, SB203580, suggesting that NE-induced phosphorylation of p38 via ARß enhances IL-6 production in SSc fibroblasts. NE-induced phosphorylation of ERK1/2 via ARα inhibited IL-6 production in SSc fibroblasts. Combined treatment with NE and endothelin-1 resulted in an additive increase in IL-6 production in SSc fibroblasts. NE-induced IL-6/IL-6 receptor trans-signaling increased the production of collagen type I in SSc fibroblasts, and both propranolol and SB203580 inhibited NE-induced collagen production. These results suggest that cold exposure and/or emotional stress-induced NE might contribute to the skin fibrosis via potentiation of IL-6 production from fibroblasts in SSc.
Assuntos
Fibroblastos/metabolismo , Norepinefrina/efeitos adversos , Escleroderma Sistêmico/metabolismo , Feminino , Fibroblastos/patologia , Fibrose , Humanos , Interleucina-6/biossíntese , Masculino , Norepinefrina/farmacologia , Fosforilação/efeitos dos fármacos , Escleroderma Sistêmico/patologia , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Resistin is a cytokine inducing insulin resistance in mice. We previously identified single nucleotide polymorphisms (SNPs) at -420 (rs1862513) and -358 (rs3219175) located in the human resistin gene (RETN) promoter as strong determinants for circulating resistin in the Japanese population. The objective was to identify additional functional variants for circulating resistin. We conducted a genome-wide association study in 448 Japanese subjects. A peak association signal was found on chromosome 19 where RETN is located. The top-hit SNP was SNP -358 G>A, followed by rs1423096 C>T, SNP -420 C>G, and rs10401670 C>T (P = 5.39×10-47, 1.81×10-22, 2.09×10-16, and 9.25×10-15, respectively). Meta-analysis including another two independent general Japanese populations showed that circulating resistin was most strongly associated with SNP-358, followed by SNP-420, rs1423096, and rs10401670. Rs1423096 and rs10401670 were located in the 3'-region of RETN and were in strong linkage disequilibrium. Although these SNPs were also in linkage disequilibrium with the promoter SNPs, conditional and haplotype association analyses identified rs1423096 and rs10401670 as independent determinants for circulating resistin. Functionally, nuclear proteins specifically recognized T but not C at rs10401670 as evidenced by an electrophoretic mobility shift assay. The promoter activity of a luciferase reporter with T at either rs1423096 or rs10401670 was lower than that with C in THP-1 human monocytes. Therefore, rs1423096 and rs10401670, in addition to SNP-420 and SNP-358, were identified as possible functional variants affecting circulating resistin by the genome-wide search in the Japanese population.
Assuntos
Povo Asiático/genética , Estudo de Associação Genômica Ampla , Polimorfismo de Nucleotídeo Único/genética , Resistina/sangue , Resistina/genética , Idoso , Cromossomos Humanos Par 19/genética , Feminino , Genes Reporter , Haplótipos/genética , Humanos , Desequilíbrio de Ligação/genética , Luciferases/metabolismo , Masculino , Pessoa de Meia-Idade , Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas , Reprodutibilidade dos TestesRESUMO
Atypical progeroid syndrome (APS), including atypical Werner syndrome (AWS), is a disorder of premature ageing caused by mutation of the lamin A gene, the same causal gene involved in Hutchinson-Gilford syndrome (HGS). We previously reported the first Japanese case of APS/AWS with a LMNA mutation (p.D300N). Recently, it has been reported that UVA induced abnormal truncated form of lamin A, called progerin, as well as HGS-like abnormal nuclear structures in normal human fibroblasts, being more frequent in the elderly, suggesting that lamin A may be involved in the regulation of photoageing. The objective of this study was to elucidate the sensitivity to cell damage induced by oxidative stress or UVA in fibroblasts from APS/AWS patient. Using immunofluorescence staining and flow cytometry analysis, the amount of early apoptotic cells and degree of intra-cellular reactive oxygen species (ROS) generation were higher in H2 02 - or UVA-treated APS/AWS fibroblasts than in normal fibroblasts, suggesting that repeated UV exposure may induce premature ageing of the skin in APS/AWS patients and that protecting against sunlight is possibly important for delaying the emergence of APS/AWS symptoms. In addition, we demonstrated that H2 O2 -, or UVA-induced apoptosis and necrosis in normal and APS/AWS fibroblasts were enhanced by farnesyltransferase inhibitor (FTI) treatment, indicating that FTI might not be useful for treating our APS/AWS patient.