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1.
Int J Mol Sci ; 21(8)2020 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-32316324

RESUMO

To identify proteins that cooperate with cellular communication network factor 2 (CCN2), we carried out GAL4-based yeast two-hybrid screening using a cDNA library derived from the chondrocytic cell line HCS-2/8. Rab14 GTPase (Rab14) polypeptide was selected as a CCN2-interactive protein. The interaction between CCN2 and Rab14 in HCS-2/8 cells was confirmed using the in situ proximity ligation assay. We also found that CCN2 interacted with Rab14 through its IGFBP-like domain among the four domains in CCN2 protein. To detect the colocalization between CCN2 and Rab14 in the cells in detail, CCN2, wild-type Rab14 (Rab14WT), a constitutive active form (Rab14CA), and a dominant negative form (Rab14DN) of Rab14 were overexpressed in monkey kidney-tissue derived COS7 cells. Ectopically overexpressed Rab14 showed a diffuse cytosolic distribution in COS7 cells; however, when Rab14WT was overexpressed with CCN2, the Rab14WT distribution changed to dots that were evenly distributed within the cytosol, and both Rab14 and CCN2 showed clear colocalization. When Rab14CA was overexpressed with CCN2, Rab14CA and CCN2 also showed good localization as dots, but their distribution was more widespread within cytosol. The coexpression of Rab14DN and CCN2 also showed a dotted codistribution but was more concentrated in the perinuclear area. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis revealed that the reduction in RAB14 or CCN2 mRNA by their respective siRNA significantly enhanced the expression of ER stress markers, BIP and CHOP mRNA in HCS-2/8 chondrocytic cells, suggesting that ER and Golgi stress were induced by the inhibition of membrane vesicle transfer via the suppression of CCN2 or Rab14. Moreover, to study the effect of the interaction between CCN2 and its interactive protein Rab14 on proteoglycan synthesis, we overexpressed Rab14WT or Rab14CA or Rab14DN in HCS-2/8 cells and found that the overexpression of Rab14DN decreased the extracellular proteoglycan accumulation more than the overexpression of Rab14WT/CA did in the chondrocytic cells. These results suggest that intracellular CCN2 is associated with Rab14 on proteoglycan-containing vesicles during their transport from the Golgi apparatus to endosomes in chondrocytes and that this association may play a role in proteoglycan secretion by chondrocytes.

2.
J Cell Biochem ; 2020 Feb 17.
Artigo em Inglês | MEDLINE | ID: mdl-32065439

RESUMO

Adipocyte differentiation is regulated by several transcription factors such as the CCAAT/enhancer-binding proteins (C/EBPs) and peroxisome proliferator-activated receptor-γ (PPARγ). Here, we demonstrate that low-intensity pulsed ultrasound (LIPUS) suppressed differentiation into mature adipocytes via multiple signaling pathways. When C3H10T1/2, a mesenchymal stem cell line, was treated with LIPUS (3.0 MHz, 60 mW/cm2 ) for 20 minutes once a day for 4 days during adipogenesis, and both the number of lipid droplets and the gene expression of PPARγ and C/EBPα were significantly decreased. Furthermore, LIPUS treatment decreased the phosphorylation of the insulin receptor and also that of Akt and ERK1/2, which are located downstream of this receptor. Next, we showed that LIPUS suppressed the gene expression of angiotensinogen (AGT), which is an adipokine produced by mature adipocytes, as well as that of angiotensin-converting enzyme 1 (ACE1) and angiotensin receptor type 1 (AT1 R) during adipogenesis of pre-adipogenic 3T3-L1 cells. Next, the translocation of Yes-associated protein (YAP) into the nucleus of 3T3-L1 cells was promoted by LIPUS, leading to upregulation of CCN family protein 2 (CCN2), a cellular communication network factor. Moreover, forced expression of CCN2 in 3T3-L1 cells decreased PPARγ gene expression, but it did not increase alkaline phosphatase and osterix gene expression. Finally, gene silencing of CCN2 in C3H10T1/2 cells diminished the effect of LIPUS on the gene expression of PPARγ and C/EBPα. These findings suggest that LIPUS suppressed adipogenesis through inhibition of insulin signaling and decreased PPARγ expression via increased CCN2 production, resulting in a possible decrease of mature adipocytes.

3.
Biochem Biophys Res Commun ; 524(2): 398-404, 2020 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-32007268

RESUMO

Neuroendocrine carcinoma of small cell type (SCNEC) is a rare pathological subtype in cervical cancer, which has a worse prognosis than other histological cell types. Due to its low incidence and the lack of experimental platforms, the molecular characteristics of SCNEC in the cervix remain largely unknown. Using the cancer tissue-originated spheroid (CTOS) method-an ex vivo 3D culture system that preserves the differentiation status of the original tumors-we established a panel of CTOS lines of SCNEC. We demonstrated that xenograft tumors and CTOSs, respectively, exhibited substantial intra-tumor and intra-CTOS variation in the expression levels of chromogranin A (CHGA), a neuroendocrine tumor marker. Since hypoxia affects differentiation in various tumors and in stem cells, we also investigated how hypoxia affected neuroendocrine differentiation of SCNEC of the uterine cervix. In the CTOS line cerv21, hypoxia suppressed expression of the neuroendocrine markers CHGA and synaptophysin (SYP). Flow cytometry analysis using CD99 (a membrane protein marker of SCNEC) revealed decreased CD99 expression in a subset of cells under hypoxic conditions. These expression changes were attenuated by HIF-1α knockdown, and by a Notch inhibitor, suggesting that these molecules played a role in the regulation of neuroendocrine differentiation. The examined SCNEC markers were suppressed under hypoxia in multiple CTOS lines. Overall, our present results indicated that neuroendocrine differentiation in SCNEC of the uterus is a variable phenotype, and that hypoxia may be one of the factors regulating the differentiation status.

4.
Int J Mol Sci ; 21(5)2020 Feb 25.
Artigo em Inglês | MEDLINE | ID: mdl-32106563

RESUMO

Retrotransposons are genetic elements that copy and paste themselves in the host genome through transcription, reverse-transcription, and integration processes. Along with their proliferation in the genome, retrotransposons inevitably modify host genes around the integration sites, and occasionally create novel genes. Even now, a number of retrotransposons are still actively editing our genomes. As such, their profound role in the evolution of mammalian genomes is obvious; thus, their contribution to mammalian skeletal evolution and development is also unquestionable. In mammals, most of the skeletal parts are formed and grown through a process entitled endochondral ossification, in which chondrocytes play central roles. In this review, current knowledge on the evolutional, physiological, and pathological roles of retrotransposons in mammalian chondrocyte differentiation and cartilage development is summarized. The possible biological impact of these mobile genetic elements in the future is also discussed.

5.
Sci Rep ; 9(1): 10913, 2019 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-31358778

RESUMO

In this study, we investigated the effect of CCN2 (cellular communication network factor 2), previously termed connective tissue growth factor, deposited in bone matrix on osteoclastogenesis and osteoblast differentiation. To mimic the bone matrix environment, osteocytic MLO-Y4 cells had been embedded in collagen-gel with recombinant CCN2 (rCCN2), and mouse macrophage-like RAW264.7 cells were inoculated on the gel and treated with receptor activator of NF-κB ligand (RANKL). NFATc1 and cathepsin K (CTSK) productions were more increased in the combination of RAW264.7 and MLO-Y4 cells treated with rCCN2 than the combination without rCCN2. Next, we isolated an osteocyte-enriched population of cells and osteoclast progenitor cells from wild type and tamoxifen-inducible Ccn2-deficient (KO) mice and performed similar analysis. NFATc1 and CTSK productions were decreased in the KO osteocyte-enriched population at 6 months after the tamoxifen injection, regardless of the origin of the osteoclast progenitor cells. Interestingly, CTSK production was rather increased in KO osteocytes at 1 year after the injection. Finally, the combination of osteoblastic MC3T3-E1 and MLO-Y4 cells in rCCN2-containing bone matrix revealed the up-regulation of osteoblastic marker genes. These findings suggest that CCN2 supplied by osteocytes regulates both osteoclastogenesis and osteoblast differentiation.

6.
Am J Obstet Gynecol ; 221(3): 241.e1-241.e6, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31075244

RESUMO

BACKGROUND: Smartphones recently have been applied in the medical setting. However, the literature evaluating the utility of smartphones in gynecologic oncology is limited. OBJECTIVE: To evaluate the utility of a smartphone in the detection of uterine cervical lesions in patients with abnormal cervical cytology. STUDY DESIGN: Seventy-five women with abnormal cervical cytology were enrolled. Two doctors independently inspected the uterine cervix by using smartphone or colposcopy. Images were captured using acetic acid, and biopsies were taken as standard-of-care procedures. The diagnostic performance of the smartphone for cervical intraepithelial neoplasm 1 or worse and cervical intraepithelial neoplasm 2 or worse were evaluated, and the kappa value was calculated to determine the chance corrected agreement of the histologic diagnoses based on the smartphone and colposcopic findings. RESULTS: There was a substantial agreement between histologic diagnoses based on the smartphone and colposcopic findings, with a kappa value of 0.67 (95% confidence interval, 0.43-0.90). The sensitivity, specificity, positive predictive value, and negative predictive value of the smartphone in the diagnosis of cervical intraepithelial neoplasm 1 or worse were 0.89 (95% confidence interval, 0.79-0.96), 0.33 (95% confidence interval, 0.08-0.70), 0.91 (95% confidence interval, 0.81-0.97), and 0.30 (95% confidence interval, 0.07-0.65), respectively. The sensitivity, specificity, positive predictive value, and negative predictive value in the diagnosis of cervical intraepithelial neoplasm 2 or worse were 0.92 (95% confidence interval, 0.81-0.98), 0.24 (95% confidence interval, 0.09-0.45), 0.71 (95% confidence interval, 0.58-0.81), and 0.60 (95% confidence interval, 0.26-0.88), respectively. CONCLUSION: We found that there was a substantial agreement between the histologic diagnoses based on the smartphone and colposcopic findings. The smartphone seems to be useful and may be an alternative to colposcopy.


Assuntos
Adenocarcinoma in Situ/diagnóstico , Carcinoma de Células Escamosas/diagnóstico , Neoplasia Intraepitelial Cervical/diagnóstico , Colposcopia , Smartphone , Neoplasias do Colo do Útero/diagnóstico , Adenocarcinoma in Situ/patologia , Adulto , Carcinoma de Células Escamosas/patologia , Neoplasia Intraepitelial Cervical/patologia , Feminino , Seguimentos , Humanos , Pessoa de Meia-Idade , Sensibilidade e Especificidade , Centros de Atenção Terciária , Neoplasias do Colo do Útero/patologia , Esfregaço Vaginal
7.
Poult Sci ; 98(10): 4327-4337, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31111951

RESUMO

Consumer preference for slow-growing broiler chickens is rising because of increased demand for high-quality poultry products. Korat chicken (KRC) is a slow-growing chicken generated in Thailand. A goal of the KRC breeding program is to produce meat with a low purine content to benefit an aging population, without interfering with growth performance. Thus, this study aimed to investigate the effects of genes encoding melanocortin 4 receptor (MC4R), calpain 1 (CAPN1), and adenylosuccinate lyase (ADSL) on body weight, muscle fiber, and content of purine and its derivatives (i.e., adenine, guanine, hypoxanthine, and xanthine), to develop molecular markers for breeding programs. Genotypes of MC4R, CAPN1, and ADSL were obtained from 583 KRCs by PCR-single-strand conformation polymorphism. The body weight and purine contents of the KRCs were measured every 2 wk until the KRCs reached market weight at 10 wk of age. A significant association between the MC4R genotype and body weight at 2, 4, and 10 wk of age was detected. KRC possessing the BB genotype of CAPN1 showed significantly heavier body weight at 6 wk of age and guanine content at 4 wk of age, and a smaller muscle fiber diameter in the breast muscle at 10 wk of age, compared with those of the other genotypes. In addition, high expression levels of the CAPN1 and ADSL genes were detected in the breast muscle at 2 wk of age. Although higher purine contents were detected at a young age, no significant associations with the MC4R, CAPN1, and ADSL genes were detected. Our results indicate that MC4R and CAPN1 could be used as genetic markers for growth and meat quality in the slow-growing chicken breeding program.


Assuntos
Proteínas Aviárias/genética , Peso Corporal/genética , Galinhas/fisiologia , Purinas/metabolismo , Adenilossuccinato Liase/genética , Adenilossuccinato Liase/metabolismo , Animais , Proteínas Aviárias/metabolismo , Calpaína/genética , Calpaína/metabolismo , Galinhas/genética , Galinhas/crescimento & desenvolvimento , Feminino , Marcadores Genéticos , Genótipo , Masculino , Carne/análise , Polimorfismo Conformacional de Fita Simples , Receptor Tipo 4 de Melanocortina/genética , Receptor Tipo 4 de Melanocortina/metabolismo
8.
J Endocrinol ; 2019 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-30889551

RESUMO

Endochondral ossification, including bone growth and other metabolic events, is regulated by circadian rhythms. Herein, we provide evidence that melatonin has a direct effect on the circadian rhythm of chondrocytes. We detected mRNA expression of the genes which encode the melatonin-synthesizing enzymes AANAT (arylalkylamine N-acetyltransferase) and HIOMT (hydroxyindole O-methyltransferase), as well as the melatonin receptors MT1 and MT2 in mouse primary chondrocytes and cartilage. Production of melatonin was confirmed by mass spectrometric analysis of primary rat and chick chondrocytes. Addition of melatonin to primary mouse chondrocytes caused enhanced cell growth and increased expression of Col2a1, Aggrecan, and Sox9, but inhibited Col10a1 expression in primary BALB/c mouse chondrocytes. Addition of luzindole, an MT1 and MT2 antagonist, abolished these effects. These data indicate that chondrocytes produce melatonin, which regulates cartilage growth and maturation via the MT1 and MT2 receptors. Kinetic analysis showed that melatonin caused rapid upregulation of Aanat, Mt1, Mt2, and Pthrp expression, followed by Sox9 and Ihh. Furthermore, expression of the clock gene Bmal1 was induced, while that of Per1 was downregulated. Chronobiological analysis of synchronized C3H mouse chondrocytes revealed that melatonin induced the cyclic expression of Aanat and modified the cyclic rhythm of Bmal1, Mt1, and Mt2. In contrast, Mt1 and Mt2 showed different rhythms from Bmal1 and Aanat, indicating the existence of different regulatory genes. Our results indicate that exogenous and endogenous melatonin work in synergy in chondrocytes to adjust rhythmic expression to the central suprachiasmatic nucleus clock.

9.
J Cell Commun Signal ; 13(1): 113-118, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30232710

RESUMO

Extracellular molecules coordinate the multiple signaling pathways spatiotemporally to exchange information between cells during development. Understanding the regulation of these signal molecule-dependent pathways elucidates the mechanism of intercellular crosstalks. CCN2/CTGF is one of the CCN family members that binds BMP2, fibronectin, aggrecan, FGFR2 - regulating cartilage and bone formation, angiogenesis, wound repair etc. Tsukushi (TSK), which belongs to the Small Leucine-Rich Proteoglycan (SLRP) family, binds nodal/Vg1/TGF-ß1, BMP4/chordin, Delta, FGF8, Frizzled4, and is involved in the early body formation, bone growth, wound healing, retinal stem cell regulation etc. These two secreted molecules are expressed in similar tissues and involved in several biological events by functioning as extracellular signaling modulators. Here, we examine the molecular interaction between CCN2 and TSK biochemically. Co-precipitation assay and Surface Plasmon Resonance measurement showed their direct binding with the Kd value 15.3 nM. Further, the Solid-phase Binding Assay indicated that TSK binds to IGFBP and CT domains of CCN2. Our data suggest that CCN2 and TSK exert their function together in the body formation.

10.
J Cell Commun Signal ; 13(2): 193-207, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30460593

RESUMO

Menisci are a pair of crescent-shaped fibrocartilages, particularly of which their inner region of meniscus is an avascular tissue. It has characteristics similar to those of articular cartilage, and hence is inferior in healing. We previously reported that low-intensity pulsed ultrasound (LIPUS) treatment stimulates the production of CCN2/CTGF, a protein involved in repairing articular cartilage, and the gene expression of major cartilage matrices such as type II collagen and aggrecan in cultured chondrocytes. Therefore, in this present study, we investigated whether LIPUS has also favorable effect on meniscus cells and tissues. LIPUS applied with a 60 mW/cm2 intensity for 20 min stimulated the gene expression and protein production of CCN2 via ERK and p38 signaling pathways, as well as gene expression of SOX9, aggrecan, and collagen type II in human inner meniscus cells in culture, and slightly stimulated the gene expression of CCN2 and promoted the migration in human outer meniscus cells in culture. LIPUS also induced the expression of Ccn2, Sox9, Col2a1, and Vegf in rat intact meniscus. Furthermore, histological evaluations showed that LIPUS treatment for 1 to 4 weeks promoted healing of rat injured lateral meniscus, as evidenced by better and earlier angiogenesis and extracellular matrix synthesis. The data presented indicate that LIPUS treatment might prevent meniscus from degenerative change and exert a reparative effect on injured meniscus via up-regulation of repairing factors such as CCN2 and that it might thus be useful for treatment of an injured meniscus as a non-invasive therapy.

11.
J Bone Miner Metab ; 37(2): 199-205, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29667005

RESUMO

The antagonist-specific regulation in tissue engineering constitutes important attempts to achieve an improved and rapid bone regeneration by controlling the natural biological response of the natural body growth factors. L51P is molecularly engineered bone morphogentic protein-2 (BMP-2) variant with a substitution of the 51st leucine with a proline residue. L51P is deficient in BMP receptor binding, but maintains its structure and affinity for inhibitory proteins such as noggin, chordin, and gremlin. These modifications convert the BMP-2 variant L51P into a receptor-inactive inhibitor of BMP antagonists. This current approach may prevent the uncontrolled bone overgrowth using high concentration of BMPs and thus regulates the possible growth factor's high-dose side effects. Exploring of L51P biological functions is required to broad our understanding of BMP mutant biological functions and their potential clinical applications. The progress of L51P researches would hopefully lead to the development of multiple applications for using the L51P in bone and fracture healing disorders.


Assuntos
Proteína Morfogenética Óssea 2/metabolismo , Proteínas de Transporte/metabolismo , Proteínas Mutantes/metabolismo , Animais , Receptores de Proteínas Morfogenéticas Ósseas Tipo I , Humanos , Ligação Proteica , Transdução de Sinais
12.
J Obstet Gynaecol Res ; 45(3): 679-685, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30565810

RESUMO

AIM: The use of less radical surgery for early stage cervical cancer has often been discussed. To better determine eligible candidates for less radical surgery, we investigated the risk factors for parametrial involvement (PI). METHODS: The study included 193 patients with International Federation of Gynecology and Obstetrics (FIGO) stage IB cervical cancer who were treated with radical hysterectomy and pelvic lymphadenectomy between 2008 and 2014. The patients were divided into two groups according to whether or not the parametrium was involved pathologically. The two groups were compared with regards to clinical and histopathological variables. RESULTS: Univariate analysis showed that International Federation of Gynecology and Obstetrics stage, clinical tumor size, depth of stromal invasion, lymphovascular space invasion and pelvic lymph node metastasis were significantly associated with PI (P < 0.05 each). Multivariate analysis showed pelvic lymph node metastasis was an independent risk factor for PI (odds ratio, 10.70; [95% confidence interval, 3.02-48.08]; P = 0.0006). All patients with clinical tumor size less than or equal to 2 cm and negative for pelvic lymph node metastasis had no PI. CONCLUSION: Cervical cancer with the tumor less than or equal to 2 cm and negative for pelvic lymph node metastasis seldom has PI. These patients are good candidates for less radical surgery.


Assuntos
Carcinoma de Células Escamosas/patologia , Linfonodos/patologia , Metástase Linfática/patologia , Neoplasias do Colo do Útero/patologia , Adulto , Idoso , Carcinoma de Células Escamosas/cirurgia , Feminino , Humanos , Histerectomia , Excisão de Linfonodo , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Estudos Retrospectivos , Neoplasias do Colo do Útero/cirurgia , Adulto Jovem
13.
Case Rep Obstet Gynecol ; 2018: 3096468, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30402311

RESUMO

Venous thrombophlebitis is an uncommon cause of fever and lower abdominal pain during the early postpartum period. It mostly occurs in the right ovarian vein, and computed tomography (CT) is useful for diagnosis. We present a case of thrombophlebitis of the renal capsular vein. A 27-year-old postpartum woman presented with right lower abdominal pain and fever unresponsive to antibiotics. Contrast CT showed a ring-enhancing mass in the right retroperitoneum, which was distinct from the right ovarian vein. Exploratory laparoscopy revealed a retroperitoneal hematoma and normal appendix. Reconstruction of CT images revealed that the mass was connected to the right renal capsular vein. Anticoagulation therapy improved the patient's symptoms. Postpartum thrombophlebitis can occur at locations other than the ovarian vein, such as the renal capsular vein. If a retroperitoneal mass is discovered during puerperium, a thorough investigation of the mass's continuity with surrounding vessels is essential to avoid unnecessary surgery.

14.
Medicines (Basel) ; 5(4)2018 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-30388792

RESUMO

Anti-inflammatory agents have been widely used to ameliorate severe inflammatory symptoms of a number of diseases, and such therapeutics are particularly useful for diseases with intolerable pain without significant mortality. A typical example of this is a disease known as stomatitis; although stomatitis itself is not a life-threatening disease, it severely impairs the individual's quality of life, and thus a standard therapeutic strategy for it has already been established. The topical application of a bioactive agent is quite easy, and a strong anti-inflammatory agent can be used without significant adverse effects. In contrast, natural products with relatively mild bioactivity are used for systemic intervention. However, new aspects of classical drugs used in these established therapeutic methods have recently been discovered, which is expanding the utility of these compounds to other oral diseases such as osteoarthritis of temporomandibular joints (TMJ-OA). In this review article, after summarizing the general concept and pathobiology of stomatitis, its established therapeutics are explained. Thereafter, recent advances in the research into related compounds, which is uncovering new biological functions of the agents used therein, are introduced. Indeed, regenerative therapeutics for TMJ-OA may be developed with the classical compounds currently being used.

15.
Int J Gynecol Cancer ; 28(9): 1751-1757, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30358701

RESUMO

OBJECTIVE: The phenotypic and pathological features of small cell cervical carcinoma (SMCC) and small small cell lung cancer (SCLC) are very similar; thus, the chemotherapy regimens used for the rare SMCC have been routinely based on regimens used for common SCLC. We set out to explore the protein expression profile similarities between these 2 cancers to prove that linking their therapeutic regimens is justified, with a secondary aim of finding tumor-specific proteins to use as additional biomarkers for more accurate diagnosis of SMCC, and potentially to use as therapeutic targets. METHODS: Protein expression analysis was performed for 3 cases of SMCC and 1 example each of SCLC, mucinous adenocarcinoma of the cervix (MACC), lung mucinous adenocarcinoma (MACL), and squamous cell carcinoma of the cervix (SCC). We used cancer tissue-originated spheroids (CTOS) and isobaric tags for relative and absolute quantitation (iTRAQ)-based comprehensive and quantitative protein expression profile analysis. Expression in corresponding clinical samples was verified by immunohistochemistry. RESULTS: Rather than organ of origin-specific patterns, the SMCC and SCLC samples revealed remarkably similar protein expression profiles-in agreement with their matching tumor pathology phenotypes. Sixteen proteins were expressed at least 2-fold higher in both small cell carcinomas (SMCC and SCLC) than in MACC or SCC. Immunohistochemical analysis confirmed higher expression of creatine kinase B-type in SMCC, compared with MACC and SCC. CONCLUSIONS: We demonstrate a significant overlapping similarity of protein expression profiles of lung and cervical small cell carcinomas despite the significant differences in their organs of origin.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma de Células Pequenas/metabolismo , Neoplasias Pulmonares/metabolismo , Proteínas de Neoplasias/biossíntese , Neoplasias do Colo do Útero/metabolismo , Linhagem Celular Tumoral , Cromatografia Líquida , Feminino , Humanos , Imuno-Histoquímica , Proteômica/métodos , Espectrometria de Massas em Tandem
16.
Molecules ; 23(7)2018 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-29937485

RESUMO

Recent studies suggest that the commensal microbiota affects not only host energy metabolism and development of immunity but also bone remodeling by positive regulation of osteoclast activity. However, the mechanism of regulation of bone cells by the commensal microbiota has not been elucidated. In this study, 8-week-old specific pathogen-free (SPF) and germ-free (GF) mice were compared in terms of alveolar bones and primary osteoblasts isolated from calvarias. Micro-CT analysis showed that SPF mice had larger body size associated with lower bone mineral density and bone volume fraction in alveolar bones compared with GF mice. Greater numbers of osteoclasts in alveolar bone and higher serum levels of tartrate-resistant acid phosphatase 5b were observed in SPF mice. Tissue extracts from SPF alveolar bone showed higher levels of cathepsin K, indicating higher osteoclast activity. SPF alveolar extracts also showed elevated levels of γ-carboxylated glutamic acid⁻osteocalcin as a marker of mature osteoblasts compared with GF mice. Polymerase chain reaction (PCR) array analysis of RNA directly isolated from alveolar bone showed that in SPF mice, expression of mRNA of osteocalcin, which also acts as an inhibitor of bone mineralization, was strongly enhanced compared with GF mice. Cultured calvarial osteoblasts from SPF mice showed reduced mineralization but significantly enhanced expression of mRNAs of osteocalcin, alkaline phosphatase, insulin-like growth factor-I/II, and decreased ratio of osteoprotegerin/receptor activator of nuclear factor-kappa B ligand compared with GF mice. Furthermore, PCR array analyses of transcription factors in cultured calvarial osteoblasts showed strongly upregulated expression of Forkhead box g1. In contrast, Gata-binding protein 3 was strongly downregulated in SPF osteoblasts. These results suggest that the commensal microbiota prevents excessive mineralization possibly by stimulating osteocalcin expression in osteoblasts, and enhances both osteoblast and osteoclast activity by regulating specific transcription factors.


Assuntos
Remodelação Óssea/genética , Microbioma Gastrointestinal/fisiologia , Vida Livre de Germes , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Osteogênese/genética , Simbiose/fisiologia , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Animais , Biomarcadores/metabolismo , Catepsina K/genética , Catepsina K/metabolismo , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Fator de Transcrição GATA3/genética , Fator de Transcrição GATA3/metabolismo , Regulação da Expressão Gênica , Fator de Crescimento Insulin-Like I/genética , Fator de Crescimento Insulin-Like I/metabolismo , Masculino , Camundongos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Osteoblastos/citologia , Osteoblastos/microbiologia , Osteocalcina/genética , Osteocalcina/metabolismo , Osteoclastos/citologia , Osteoclastos/microbiologia , Osteoprotegerina/genética , Osteoprotegerina/metabolismo , Ligante RANK/genética , Ligante RANK/metabolismo
17.
J Cell Commun Signal ; 12(1): 245-252, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29129024

RESUMO

The CCN family consists of 6 genes in the mammalian genome and produces multifunctional proteins involved in a variety of biological processes. Recent reports indicate the profound roles of CCN2 in energy metabolism in chondrocytes, and Ccn2 deficiency is known to alter the expression of 2 other family members including Ccn3. However, almost nothing is known concerning the regulation of the CCN family genes by energy metabolism. In order to gain insight into this critical issue, we initially and comprehensively evaluated the effect of inhibition of glycolysis on the expression of all of the CCN family genes in chondrocytic cells. Upon the inhibition of a glycolytic enzyme, repression of CCN2 expression was observed, whereas CCN3 expression was conversely induced. Similar repression of CCN2 was conferred by the inhibition of aerobic ATP production, which, however, did not induce CCN3 expression. In contrast, glucose starvation significantly enhanced the expression of CCN3 in those cells. The results of a reporter gene assay using a molecular construct containing a CCN3 proximal promoter revealed a dose-dependent induction of the CCN3 promoter activity by the glycolytic inhibitor in chondrocytic cells. These results unveiled a critical role of glycolytic activity in the regulation of CCN2 and CCN3, which activity mediated the mutual regulation of these 2 major CCN family members in chondrocytes.

18.
J Cell Physiol ; 233(6): 4825-4840, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29150954

RESUMO

A vast number of long-noncoding RNAs (lncRNA) are found expressed in human cells, which RNAs have been developed along with human evolution. However, the physiological functions of these lncRNAs remain mostly unknown. In the present study, we for the first time uncovered the fact that one of such lncRNAs plays a significant role in the differentiation of chondrocytes and, possibly, of osteoblasts differentiated from mesenchymal stem cells, which cells eventually construct the human skeleton. The urothelial cancer-associated 1 (UCA1) lncRNA is known to be associated with several human malignancies. Firstly, we confirmed that UCA1 was expressed in normal human chondrocytes, as well as in a human chondrocytic cell line; whereas it was not detected in human bone marrow mesenchymal stem cells (hBMSCs). Of note, although UCA1 expression was undetectable in hBMSCs, it was markedly induced along with the differentiation toward chondrocytes, suggesting its critical role in chondrogenesis. Consistent with this finding, silencing of the UCA1 gene significantly repressed the expression of chondrogenic genes in human chondrocytic cells. UCA1 gene silencing and hyper-expression also had a significant impact on the osteoblastic phenotype in a human cell line. Finally, forced expression of UCA1 in a murine chondrocyte precursor, which did not possess a UCA1 gene, overdrove its differentiation into chondrocytes. These results indicate a physiological and important role of this lncRNA in the skeletal development of humans, who require more sustained endochondral ossification and osteogenesis than do smaller vertebrates.


Assuntos
Condrócitos/metabolismo , Condrogênese , Osteoblastos/metabolismo , Osteogênese , RNA Longo não Codificante/metabolismo , Células-Tronco/metabolismo , Animais , Desdiferenciação Celular , Linhagem Celular Tumoral , Senescência Celular , Condrogênese/genética , Evolução Molecular , Regulação da Expressão Gênica no Desenvolvimento , Células HEK293 , Humanos , Osteogênese/genética , Fenótipo , Primatas , RNA Longo não Codificante/genética , Transdução de Sinais
19.
PLoS One ; 12(11): e0188014, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29145495

RESUMO

Serotonin (5-hydroxytryptamine: 5-HT) is recognized as a neurotransmitter in the central nerve system and as a regulator of systemic blood pressure in the peripheral tissues. Recently, it was reported that 5-HT2 receptors (5-HT2Rs) were expressed in cartilage tissues lacking both vessels and neurons, suggesting possible novel functions of 5-HT during cartilage development and regeneration. Our previous data indicated that CCN family protein 2/connective tissue growth factor (CCN2/CTGF) plays a central role in cartilage development and regeneration. Therefore, the aim of this study was to investigate the effect of 5-HT on the production of CCN2 in chondrocytes. Firstly, we showed that the mRNAs of 5-HT2R subtypes 5-HT2AR and 5-HT2BR, were expressed in a human chondrocytic cell line, HCS-2/8; however, 5-HT2CR mRNA was not detected. In addition, exogenously added 5-HT did not affect the 5-HT2AR and 5-HT2BR expressions. Next, we demonstrated that CCN2 production was increased by treatment with a 5-HT2AR agonist and the combination of 5-HT and 5-HT2BR antagonist. In contrast, treatment with a 5-HT2BR agonist and the combination of 5-HT and 5-HT2AR antagonist decreased CCN2 production. Furthermore, we showed that phosphorylation of Akt and p38 MAPK were increased by treatment with 5-HT2AR agonist, and that phosphorylation of PKCε, PKCζ, ERK1/2 and JNK were increased by treatment with 5-HT2BR agonist. Finally, we found that 5-HT2AR was localized in the growth plate, whereas 5-HT2BR was localized in the articular cartilage. These findings suggest that 5-HT promotes CCN2 production through the 5-HT2AR in growth plates, and that it represses CCN2 production through the 5-HT2BR in articular cartilage for harmonized development of long bones.


Assuntos
Condrócitos/metabolismo , Fator de Crescimento do Tecido Conjuntivo/biossíntese , Receptor 5-HT2A de Serotonina/fisiologia , Receptor 5-HT2B de Serotonina/fisiologia , Serotonina/fisiologia , Animais , Cálcio/metabolismo , Cartilagem Articular/metabolismo , Linhagem Celular Tumoral , Fator de Crescimento do Tecido Conjuntivo/genética , Expressão Gênica , Lâmina de Crescimento/metabolismo , Humanos , Transporte de Íons , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação , Proteínas Quinases/metabolismo , Receptor 5-HT2A de Serotonina/metabolismo , Receptor 5-HT2B de Serotonina/metabolismo , Transdução de Sinais
20.
Clin Calcium ; 27(12): 1697-1703, 2017.
Artigo em Japonês | MEDLINE | ID: mdl-29179163

RESUMO

Bone remodeling has important roles in the functions of bone tissues, such as supporting the body and mineral storage. Osteocytes, which are the most abundant cells in bone tissues, detect the mechanical loading and regulate both bone formation by osteoblasts and bone resorption by osteoclasts. However, its mechanism is still unknown. In this review, we summarize how osteocytes detect mechanical stress and discuss the potential role of CCN2/CTGF as a novel bone remodeling factor produced by osteocytes.


Assuntos
Remodelação Óssea , Osteócitos/fisiologia , Animais , Osso e Ossos/metabolismo , Fator de Crescimento do Tecido Conjuntivo/metabolismo , Humanos , Estresse Mecânico
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