Publications by authors named "Satoshi Kubota"

171 Publications

Association of growth hormone and insulin-like growth factor I genotype with body weight, dominance of body weight, and mRNA expression in Korat slow-growing chickens.

Anim Biosci 2021 Mar 10. Epub 2021 Mar 10.

School of Animal Technology and Innovation, Institute of Agricultural Technology, Suranaree University of Technology, Nakhon Ratchasima, 30000, Thailand.

Objective: Growth hormone (GH) and insulin-like growth factor I (IGF-I) play a critical role in animal growth rates. We aimed to investigate the effect of GH and IGF-I genotypes on body weight (BW), dominance, and gene expression in slow-growing chickens at different ages.

Methods: A total of 613 Korat chickens (KRs) were bred and divided into three groups by genotype - A1A1, A1A3, and A3A3 for GH and AA, AC, and CC for IGF-I. Chickens were weighed every two weeks, and liver and breast muscle tissues were collected at 10 weeks of age. Genetic parameters of KRs were estimated using ASReml software. GH and IGF-I mRNA levels were measured by quantitative PCR. Significant differences between traits were analyzed using the generalized linear model.

Results: A significant effect of GH genotypes on BW was found at most ages, and the A1A1 genotype had the highest value of BW. Compared with the A3A3 genotype, the A1A1 and A1A3 genotypes showed a higher dominance effect at 0 and 2 weeks, and genotype A1A1 had the highest value of dominance at 8 weeks of age. A difference in GH mRNA levels between genotypes was detected in breast muscle at 6 weeks and in the liver tissue at 2 weeks. In the case of IGF-I gene, the AA genotype had the highest BW at the beginning of life. Significant differences in BW dominance were found at 2 weeks. However, IGF-I mRNA levels were not different among genotypes in both breast muscles and liver tissues.

Conclusion: Our results revealed that GH and IGF-I influence growth, but may not be involved in heterosis. GH can be used as a marker gene in selection programs for growth because the homozygous genotype (A1A1) had the highest BW at all ages. IGF-I is not a useful marker gene for selection programs.
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http://dx.doi.org/10.5713/ab.20.0729DOI Listing
March 2021

Risk factors of throwing elbow injuries during pitching Analyzed by simulation using human musculoskeletal model in youth baseball pitcher.

J Shoulder Elbow Surg 2021 Mar 3. Epub 2021 Mar 3.

Department of Orthopedic Sports Medicine, Yokohama Minami Kyosai Hospital, Kanagawa, Japan.

Background: Pitching mechanics are believed to be risk factors for throwing elbow injury. Thus, a prospective study of abnormal mechanics in youth baseball players is needed. This study aimed to analyze the ulnar collateral ligament (UCL) during normal pitching using Software for Interactive Musculoskeletal Modeling (SIMM) for analysis, and investigate the risk parameters of throwing elbow injuries in youth baseball players. We hypothesized that excessive UCL force during pitching was a risk factor for throwing elbow injuries in this population.

Methods: In this cohort study, youth baseball pitchers (aged 9-11 years) were instructed to throw a ball into a netted target. Using an SIMM musculoskeletal model, we analyzed the force of the anterior band of the anterior oblique ligament (AOL_AB), posterior band of the AOL (AOL_PB), and elbow varus moment during pitching (foot-contact to ball release). We calculated the integral of each force of the AOL_AB and AOL_PB during pitching, and summarized to establish an impulse at the medial epicondyle. Each participant was followed for 12 months to assess the occurrence of throwing elbow injury.

Results: Eighteen pitchers (28.1%) reported throwing elbow injuries in the throwing arm during the 12-month follow-up period. The results of this study showed that the maximum AOL_PB force and the impulse at the medial epicondyle were risk factors for throwing elbow injuries. The maximum AOL_PB force was significantly higher in the throwing elbow injury group than in the uninjured group 59.4±17.8 N vs 47.1±17.5 N (P=0.014). The impulse at the medial epicondyle was also significantly different (11.1±4.0 N・s in the throwing elbow injury group versus 8.3±4.4 N・s in the uninjured group, P=0.025).results CONCLUSIONS: Increasing the AOL_PB force or the impulse at the medial epicondyle may increase the risk of throwing elbow injuries in youth baseball pitchers. It may be possible to reduce injury risk by focusing on ways to decrease AOL_PB load and cumulative stress on the medial epicondyle throughout the throwing motion, while still maintaining high levels of ball velocity.
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http://dx.doi.org/10.1016/j.jse.2021.01.039DOI Listing
March 2021

RFX1-mediated CCN3 induction that may support chondrocyte survival under starved conditions.

J Cell Physiol 2021 Mar 3. Epub 2021 Mar 3.

Department of Biochemistry and Molecular Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan.

Cellular communication network factor (CCN) family members are multifunctional matricellular proteins that manipulate and integrate extracellular signals. In our previous studies investigating the role of CCN family members in cellular metabolism, we found three members that might be under the regulation of energy metabolism. In this study, we confirmed that CCN2 and CCN3 are the only members that are tightly regulated by glycolysis in human chondrocytic cells. Interestingly, CCN3 was induced under a variety of impaired glycolytic conditions. This CCN3 induction was also observed in two breast cancer cell lines with a distinct phenotype, suggesting a basic role of CCN3 in cellular metabolism. Reporter gene assays indicated a transcriptional regulation mediated by an enhancer in the proximal promoter region. As a result of analyses in silico, we specified regulatory factor binding to the X-box 1 (RFX1) as a candidate that mediated the transcriptional activation by impaired glycolysis. Indeed, the inhibition of glycolysis induced the expression of RFX1, and RFX1 silencing nullified the CCN3 induction by impaired glycolysis. Subsequent experiments with an anti-CCN3 antibody indicated that CCN3 supported the survival of chondrocytes under impaired glycolysis. Consistent with these findings in vitro, abundant CCN3 production by chondrocytes in the deep zones of developing epiphysial cartilage, which are located far away from the synovial fluid, was confirmed in vivo. Our present study uncovered that RFX1 is the mediator that enables CCN3 induction upon cellular starvation, which may eventually assist chondrocytes in retaining their viability, even when there is an energy supply shortage.
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http://dx.doi.org/10.1002/jcp.30348DOI Listing
March 2021

Evaluation of CRISPR Diversity in the Human Skin Microbiome for Personal Identification.

mSystems 2021 Feb 2;6(1). Epub 2021 Feb 2.

National Research Institute of Police Science, Kashiwa, Chiba, Japan.

The highly personalized human skin microbiome may serve as a viable marker in personal identification. Amplicon sequencing resolution using 16S rRNA cannot identify bacterial communities sufficiently to discriminate between individuals. Thus, novel higher-resolution genetic markers are required for forensic purposes. The clustered regularly interspaced short palindromic repeats (CRISPRs) are prokaryotic genetic elements that can provide a history of infections encountered by the bacteria. The sequencing of CRISPR spacers may provide phylogenetic information with higher resolution than other markers. However, using spacer sequencing for discrimination of personal skin microbiome is difficult due to limited information on CRISPRs in human skin microbiomes. It remains unclear whether personal microbiome discrimination can be achieved using spacer diversity or which CRISPRs will be forensically relevant. We identified common CRISPRs in the human skin microbiome via metagenomic reconstruction and used amplicon sequencing for deep sequencing of spacers. We successfully reconstructed 24 putative CRISPR arrays using metagenomic data sets. A total of 1,223,462 reads from three CRISPR arrays revealed that spacers in the skin microbiome were highly personalized, and conserved repeats were commonly shared between individuals. These individual specificities observed using CRISPR typing were confirmed by comparing the CRISPR diversity to microbiome diversity assessed using 16S rRNA amplicon sequencing. CRISPR typing achieved 95.2% accuracy in personal classification, whereas 16S rRNA sequencing only achieved 52.6%. These results suggest that sequencing CRISPRs in the skin microbiome may be a more powerful approach for personal identification and ecological studies compared to conventional 16S rRNA sequencing. Microbial community diversity analysis can be utilized to characterize the personal microbiome that varies between individuals. CRISPR sequences, which reflect virome structure, in the human skin environment may be highly personalized similar to the structures of individual viromes. In this study, we identified 24 putative CRISPR arrays using a shotgun metagenome data set of the human skin microbiome. The findings of this study expand our understanding of the nature of CRISPRs by identifying novel CRISPR candidates. We developed a method to efficiently determine the diversity of three CRISPR arrays. Our analysis revealed that the CRISPR spacer diversity in the human skin microbiome is highly personalized compared with the microbiome diversity assessed by 16S rRNA sequencing, providing a new perspective on the study of the skin microbiome.
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http://dx.doi.org/10.1128/mSystems.01255-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7857535PMC
February 2021

Therapeutic benefit of lymphadenectomy for older patients with urothelial carcinoma of the upper urinary tract: a propensity score matching study.

Jpn J Clin Oncol 2021 Jan 13. Epub 2021 Jan 13.

Department of Urology, Tokyo Women's Medical University. 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-0054, Japan.

Objectives: Regional lymphadenectomy for urothelial carcinoma of the upper urinary tract is sometimes avoided in older patients to reduce surgical burden. We aimed to evaluate the therapeutic impact of lymphadenectomy in older patients undergoing curative therapy for upper urinary tract urothelial carcinoma.

Methods: The patients with urothelial carcinoma of the upper urinary tract older than 75 years at the time of surgery and without lymph node or distant metastasis who underwent curative therapy at two tertiary hospitals between 1994 and 2019 were retrospectively analyzed. Complete-lymphadenectomy was performed as per our protocol. Cancer-specific survival, overall survival and metastasis-free survival after surgery were evaluated between complete-lymphadenectomy and no/incomplete-lymphadenectomy groups before and after 1:1 propensity score matching.

Results: The original cohort included 150 patients (median age, 80.71 years), and complete-lymphadenectomy was performed in 42 (28.00%) patients. Patients in complete-lymphadenectomy group were younger and less likely to be aged >80 years (both, P < 0.0001). After matching, 30 patients were allocated to each group and the ages were comparable (78.58 vs. 77.48 years, P = 0.1738). High-grade perioperative complication rates did not differ between groups both before and after matching. Cancer-specific survival, overall survival and metastasis-free survival were significantly longer in the complete-lymphadenectomy group both before and after matching (all, P < 0.05).

Conclusions: This study suggests that complete-lymphadenectomy may provide therapeutic benefits for older patients. The decision to perform complete-lymphadenectomy must be based on the patient's physical condition, rather than his/her chronological age.
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http://dx.doi.org/10.1093/jjco/hyaa256DOI Listing
January 2021

Bipartite regulation of cellular communication network factor 2 and fibroblast growth factor 1 genes by fibroblast growth factor 1 through histone deacetylase 1 and fork head box protein A1.

J Cell Commun Signal 2021 Mar 4;15(1):81-91. Epub 2021 Jan 4.

Department of Biochemistry and Molecular Dentistry, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, 2-5-1 Shikata-cho, Kita-ku, Okayama, 700-8525, Japan.

Fibroblast growth factor 1 (FGF-1) is the first FGF family member, and it induces proliferation of fibroblasts and other types of the cells. However, recent studies are uncovering unexpected functions of this molecule. Our previous study redefined this growth factor as a catabolic molecule produced in cartilage upon metabolic insult. Indeed, FGF-1 was found to repress the gene expression of cellular communication network factor 2 (CCN2), which protects and regenerates cartilage, amplifying its own production through positive feedback regulation. In the present study, we investigated the molecular mechanism of this bipartite CCN2 repression and FGF1 activation by FGF-1 in chondrocytes. Repression of CCN2 and induction of FGF1 in human chondrocytic cells were both partly abolished by valproic acid, an inhibitor of histone deacetylase 1 (HDAC1), indicating the involvement of chromatin remodeling by histone acetylation in this system. In contrast, RNA degradation analysis suggested no contribution of post-transcriptional regulation of the mRNA stability to the effects conferred by FGF-1. Suspecting a regulation by a specific transcription factor, we next sought a candidate in silico from a large dataset. As a result, we found fork head box protein A1 (FOXA1) as the transcription factor that bound to both CCN2 and FGF1 loci. Functional analysis demonstrated that FOXA1 silencing significantly attenuated the CCN2 repression and FGF1 induction caused by FGF1. These findings collectively indicate that the bipartite regulation by FGF-1 is enabled by the combination of chromatin remodeling by HDACs and transcriptional modulation by FOXA1 with unknown transcriptional coactivators of opposite functionalities.
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http://dx.doi.org/10.1007/s12079-020-00600-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7905003PMC
March 2021

[Safe Handling Method for Splitting Azathioprine Tablets].

Yakugaku Zasshi 2021 ;141(1):125-133

Department of Clinical Pharmacy, Research and Education Center for Clinical Pharmacy, School of Pharmacy, Kitasato University.

The immunosuppressant azathioprine (AZA) is classified as a hazardous drug. AZA contamination during tablet-splitting increases exposure risk. However, there is no study on contamination and exposure during AZA tablet splitting and dispensing. AZA tablet splitting and dispensing methods were classified based on whether tweezers are used during splitting and packaging. In Dispensing Method (1), no tweezers were used in either step. In Dispensing Method (2), no tweezers were used during tablet splitting, but were used during packaging. In Dispensing Method (3), tweezers were used in both steps. After AZA half-tablet split-dispensing, we quantified the adherent AZA removed from the tools, packaging machines, and dispensing counters by three consecutive wipings with water-dampened polypropylene cloths. A large amount of AZA adhered to the gloves used in Dispensing Methods (1) and (2), wherein tablets were placed with gloved hands, compared with Dispensing Method (3), wherein tablets were held with tweezers. Thus, the gloves must be replaced before touching the packaging paper during the final step. After three consecutive wipings, AZA was not detected at most of the sites in the third round. Thus, we recommend that (1) AZA tablet splitting should be performed while wearing gloves, (2) the gloves should be changed before packaging the half tablets, and (3) the tools, packaging machines, and dispensing counters should be wiped twice or thrice with a water-dampened cloth after dispensing.
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http://dx.doi.org/10.1248/yakushi.20-00106DOI Listing
February 2021

Roles of CCN2 as a mechano-sensing regulator of chondrocyte differentiation.

Jpn Dent Sci Rev 2020 Nov 9;56(1):119-126. Epub 2020 Oct 9.

Department of Biochemistry and Molecular Dentistry, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama 700-8525, Japan.

Cellular communication network factor 2 (CCN2) is a cysteine-rich secreted matricellular protein that regulates various cellular functions including cell differentiation. CCN2 is highly expressed under several types of mechanical stress, such as stretch, compression, and shear stress, in mesenchymal cells including chondrocytes, osteoblasts, and fibroblasts. In particular, CCN2 not only promotes cell proliferation and differentiation of various cells but also regulates the stability of mRNA of TRPV4, a mechanosensitive ion channel in chondrocytes. Of note, CCN2 behaves like a biomarker to sense suitable mechanical stress, because CCN2 expression is down-regulated when chondrocytes are subjected to excessive mechanical stress. These findings suggest that CCN2 is a mechano-sensing regulator. CCN2 expression is regulated by the activation of various mechano-sensing signaling pathways, e.g., mechanosensitive ion channels, integrin-focal adhesion-actin dynamics, Rho GTPase family members, Hippo-YAP signaling, and G protein-coupled receptors. This review summarizes the characterization of mechanoreceptors involved in CCN2 gene regulation and discusses the role of CCN2 as a mechano-sensing regulator of mesenchymal cell differentiation, with particular focus on chondrocytes.
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http://dx.doi.org/10.1016/j.jdsr.2020.07.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7560579PMC
November 2020

CCN3 (NOV) Drives Degradative Changes in Aging Articular Cartilage.

Int J Mol Sci 2020 Oct 13;21(20). Epub 2020 Oct 13.

Department of Biochemistry and Molecular Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama 700-8525, Japan.

Aging is a major risk factor of osteoarthritis, which is characterized by the degeneration of articular cartilage. CCN3, a member of the CCN family, is expressed in cartilage and has various physiological functions during chondrocyte development, differentiation, and regeneration. Here, we examine the role of CCN3 in cartilage maintenance. During aging, the expression of mRNA in mouse primary chondrocytes from knee cartilage increased and showed a positive correlation with and mRNA. Increased accumulation of CCN3 protein was confirmed. To analyze the effects of CCN3 , either primary cultured human articular chondrocytes or rat chondrosarcoma cell line (RCS) were used. Artificial senescence induced by HO caused a dose-dependent increase in gene and CCN3 protein expression, along with enhanced expression of and mRNA and proteins, as well as SA-β gal activity. Overexpression of CCN3 also enhanced promoter activity via . Accordingly, the addition of recombinant CCN3 protein to the culture increased the expression of and mRNAs. We have produced cartilage-specific CCN3-overexpressing transgenic mice, and found degradative changes in knee joints within two months. Inflammatory gene expression was found even in the rib chondrocytes of three-month-old transgenic mice. Similar results were observed in human knee articular chondrocytes from patients at both mRNA and protein levels. These results indicate that CCN3 is a new senescence marker of chondrocytes, and the overexpression of CCN3 in cartilage may in part promote chondrocyte senescence, leading to the degeneration of articular cartilage through the induction of p53 and p21.
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http://dx.doi.org/10.3390/ijms21207556DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7593953PMC
October 2020

Regulation of cellular communication network factor 2 (CCN2) in breast cancer cells via the cell-type dependent interplay between CCN2 and glycolysis.

J Oral Biosci 2020 09 11;62(3):280-288. Epub 2020 Aug 11.

Department of Biochemistry and Molecular Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Kita-ku, Okayama, 700-8525, Japan. Electronic address:

Objectives: Anti-osteoclastic treatments for breast cancer occasionally cause medication-related osteonecrosis of the jaw. Moreover, elevated glycolytic activity, which is known as the Warburg effect, is usually observed in these breast cancer cells. Previously, we found that cellular communication network factor 2 (CCN2) production and glycolysis enhanced each other in chondrocytes. Here, we evaluated the interplay between CCN2 and glycolysis in breast cancer cells, as we suspected a possible involvement of CCN2 in the Warburg effect in highly invasive breast cancer cells.

Methods: Two human breast cancer cell lines with a distinct phenotype were used. Glycolysis was inhibited by using 2 distinct compounds, and gene silencing was performed using siRNA. Glycolysis and the expression of relevant genes were monitored via colorimetric assays and quantitative RT-PCR, respectively.

Results: Although CCN2 expression was almost completely silenced when treating invasive breast cancer cells with a siRNA cocktail against CCN2, glycolytic activity was not affected. Notably, the expression of glycolytic enzyme genes, which was repressed by CCN2 deficiency in chondrocytes, tended to increase upon CCN2 silencing in breast cancer cells. Inhibition of glycolysis, which resulted in the repression of CCN2 expression in chondrocytic cells, did not alter or strongly enhanced CCN2 expression in the invasive and non-invasive breast cancer cells, respectively.

Conclusions: High CCN2 expression levels play a critical role in the invasion and metastasis of breast cancer. Thus, a collapse in the intrinsic repressive machinery of CCN2 due to glycolysis may induce the acquisition of an invasive phenotype in breast cancer cells.
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http://dx.doi.org/10.1016/j.job.2020.07.001DOI Listing
September 2020

CTGF/CCN2 facilitates LRP4-mediated formation of the embryonic neuromuscular junction.

EMBO Rep 2020 08 17;21(8):e48462. Epub 2020 Jun 17.

Division of Neurogenetics, Center for Neurological Diseases and Cancer, Nagoya University Graduate School of Medicine, Nagoya, Japan.

At the neuromuscular junction (NMJ), lipoprotein-related receptor 4 (LRP4) mediates agrin-induced MuSK phosphorylation that leads to clustering of acetylcholine receptors (AChRs) in the postsynaptic region of the skeletal muscle. Additionally, the ectodomain of LRP4 is necessary for differentiation of the presynaptic nerve terminal. However, the molecules regulating LRP4 have not been fully elucidated yet. Here, we show that the CT domain of connective tissue growth factor (CTGF/CCN2) directly binds to the third beta-propeller domain of LRP4. CTGF/CCN2 enhances the binding of LRP4 to MuSK and facilitates the localization of LRP4 on the plasma membrane. CTGF/CCN2 enhances agrin-induced MuSK phosphorylation and AChR clustering in cultured myotubes. Ctgf-deficient mouse embryos (Ctgf ) have small AChR clusters and abnormal dispersion of synaptic vesicles along the motor axon. Ultrastructurally, the presynaptic nerve terminals have reduced numbers of active zones and mitochondria. Functionally, Ctgf embryos exhibit impaired NMJ signal transmission. These results indicate that CTGF/CCN2 interacts with LRP4 to facilitate clustering of AChRs at the motor endplate and the maturation of the nerve terminal.
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http://dx.doi.org/10.15252/embr.201948462DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7403661PMC
August 2020

Knockout of MMP3 Weakens Solid Tumor Organoids and Cancer Extracellular Vesicles.

Cancers (Basel) 2020 05 16;12(5). Epub 2020 May 16.

Department of Dental Pharmacology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama 700-8525, Japan.

The tumor organoid (tumoroid) model in three-dimensional (3D) culture systems has been developed to reflect more closely the in vivo tumors than 2D-cultured tumor cells. Notably, extracellular vesicles (EVs) are efficiently collectible from the culture supernatant of gel-free tumoroids. Matrix metalloproteinase (MMP) 3 is a multi-functional factor playing crucial roles in tumor progression. However, roles of MMP3 within tumor growth and EVs have not unveiled. Here, we investigated the protumorigenic roles of MMP3 on integrities of tumoroids and EVs. We generated MMP3-knockout (KO) cells using the CRISPR/Cas9 system from rapidly metastatic LuM1 tumor cells. Moreover, we established fluorescent cell lines with palmitoylation signal-fused fluorescent proteins (tdTomato and enhanced GFP). Then we confirmed the exchange of EVs between cellular populations and tumoroids. LuM1-tumoroids released large EVs (200-1000 nm) and small EVs (50-200 nm) while the knockout of MMP3 resulted in the additional release of broken EVs from tumoroids. The loss of MMP3 led to a significant reduction in tumoroid size and the development of the necrotic area within tumoroids. MMP3 and CD9 (a category-1 EV marker tetraspanin protein) were significantly down-regulated in MMP3-KO cells and their EV fraction. Moreover, CD63, another member of the tetraspanin family, was significantly reduced only in the EVs fractions of the MMP3-KO cells compared to their counterpart. These weakened phenotypes of MMP3-KO were markedly rescued by the addition of MMP3-rich EVs or conditioned medium (CM) collected from LuM1-tumoroids, which caused a dramatic rise in the expression of MMP3, CD9, and Ki-67 (a marker of proliferating cells) in the MMP3-null/CD9-low tumoroids. Notably, MMP3 enriched in tumoroids-derived EVs and CM deeply penetrated recipient MMP3-KO tumoroids, resulting in a remarkable enlargement of solid tumoroids, while MMP3-null EVs did not. These data demonstrate that EVs can mediate molecular transfer of MMP3, resulting in increasing the proliferation and tumorigenesis, indicating crucial roles of MMP3 in tumor progression.
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http://dx.doi.org/10.3390/cancers12051260DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7281240PMC
May 2020

Roles of Interaction between CCN2 and Rab14 in Aggrecan Production by Chondrocytes.

Int J Mol Sci 2020 Apr 16;21(8). Epub 2020 Apr 16.

Advanced Research Center for Oral and Craniofacial Sciences, Okayama University Dental School/Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama 700-8525, Japan.

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 or mRNA by their respective siRNA significantly enhanced the expression of ER stress markers, and 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.
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http://dx.doi.org/10.3390/ijms21082769DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7215643PMC
April 2020

Retrotransposons Manipulating Mammalian Skeletal Development in Chondrocytes.

Int J Mol Sci 2020 Feb 25;21(5). Epub 2020 Feb 25.

Department of Biochemistry and Molecular Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama 700-8525, Japan.

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.
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http://dx.doi.org/10.3390/ijms21051564DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7084347PMC
February 2020

Suppression of adipocyte differentiation by low-intensity pulsed ultrasound via inhibition of insulin signaling and promotion of CCN family protein 2.

J Cell Biochem 2020 Feb 17. Epub 2020 Feb 17.

Department of Biochemistry and Molecular Dentistry, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, Japan.

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/cm ) 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 (AT 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.
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http://dx.doi.org/10.1002/jcb.29680DOI Listing
February 2020

Dedifferentiation of neuroendocrine carcinoma of the uterine cervix in hypoxia.

Biochem Biophys Res Commun 2020 04 30;524(2):398-404. Epub 2020 Jan 30.

Department of Biochemistry, Osaka International Cancer Institute, Osaka, Japan; Department of Clinical Bio-resource Research and Development, Graduate School of Medicine Kyoto University, Kyoto, Japan. Electronic address:

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.
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http://dx.doi.org/10.1016/j.bbrc.2020.01.024DOI Listing
April 2020

Roles of matricellular CCN2 deposited by osteocytes in osteoclastogenesis and osteoblast differentiation.

Sci Rep 2019 07 29;9(1):10913. Epub 2019 Jul 29.

Advanced Research Center for Oral and Craniofacial Sciences, Okayama University Dental School, Okayama, Japan.

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.
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http://dx.doi.org/10.1038/s41598-019-47285-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6662664PMC
July 2019

Effects of the MC4R, CAPN1, and ADSL genes on body weight and purine content in slow-growing chickens.

Poult Sci 2019 Oct;98(10):4327-4337

School of Animal Technology and Innovation, Institute of Agricultural Technology, Suranaree University of Technology, Nakhon Ratchasima, 30000, Thailand.

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.
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http://dx.doi.org/10.3382/ps/pez262DOI Listing
October 2019

Histologic correlation between smartphone and coloposcopic findings in patients with abnormal cervical cytology: experiences in a tertiary referral hospital.

Am J Obstet Gynecol 2019 09 7;221(3):241.e1-241.e6. Epub 2019 May 7.

Department of Obstetrics and Gynecology, Osaka University Graduate School of Medicine, Suita, Osaka, Japan.

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.
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http://dx.doi.org/10.1016/j.ajog.2019.04.039DOI Listing
September 2019

Circadian production of melatonin in cartilage modifies rhythmic gene expression.

J Endocrinol 2019 Mar 1. Epub 2019 Mar 1.

T Hattori, Department of Biochemistry and Molecular Dentistry, Okayama University Graduate School of Medicine Dentistry and Pharmaceutical Sciences, Okayama, Japan.

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.
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http://dx.doi.org/10.1530/JOE-19-0022DOI Listing
March 2019

Retrospective analysis for predictors of parametrial involvement in IB cervical cancer.

J Obstet Gynaecol Res 2019 Mar 22;45(3):679-685. Epub 2018 Nov 22.

Department of Obstetrics and Gynecology, Osaka University Graduate School of Medicine, Osaka, Japan.

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.
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http://dx.doi.org/10.1111/jog.13855DOI Listing
March 2019

Possible reparative effect of low-intensity pulsed ultrasound (LIPUS) on injured meniscus.

J Cell Commun Signal 2019 Jun 20;13(2):193-207. Epub 2018 Nov 20.

Advanced Research Center for Oral and Craniofacial Sciences (ARCOCS), Okayama University Dental School/Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Kita-ku, Okayama, 700-8525, Japan.

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/cm 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.
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http://dx.doi.org/10.1007/s12079-018-0496-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6498275PMC
June 2019

Thrombophlebitis of the Right Renal Capsular Vein during the Early Postpartum Period.

Case Rep Obstet Gynecol 2018 9;2018:3096468. Epub 2018 Oct 9.

Department of Obstetrics and Gynecology, Osaka Saiseikai Nakatsu Hospital, 2-10-39 Shibata, Kita-ku, Osaka 530-0012, Japan.

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.
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http://dx.doi.org/10.1155/2018/3096468DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6198557PMC
October 2018

New Functions of Classical Compounds against Orofacial Inflammatory Lesions.

Medicines (Basel) 2018 Nov 1;5(4). Epub 2018 Nov 1.

Department of Biochemistry and Molecular Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama 700-8558, Japan.

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.
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http://dx.doi.org/10.3390/medicines5040118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6313344PMC
November 2018

Small Cell Carcinomas of the Uterine Cervix and Lung: Proteomics Reveals Similar Protein Expression Profiles.

Int J Gynecol Cancer 2018 11;28(9):1751-1757

Department of Obstetrics and Gynecology, Osaka University Graduate School of Medicine.

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.
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http://dx.doi.org/10.1097/IGC.0000000000001354DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6221385PMC
November 2018

CCN2/CTGF binds the small leucine rich proteoglycan protein Tsukushi.

J Cell Commun Signal 2019 Mar 20;13(1):113-118. Epub 2018 Sep 20.

Advanced Research Center for Oral and Craniofacial Sciences, Okayama University Dental School/Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, 2-5-1, Shikata-cho, Okayama, 700-8525, Japan.

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-β1, 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.
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http://dx.doi.org/10.1007/s12079-018-0487-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6381378PMC
March 2019

Commensal Microbiota Enhance Both Osteoclast and Osteoblast Activities.

Molecules 2018 06 23;23(7). Epub 2018 Jun 23.

Department of Preventive Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Kita-ku, Okayama 700-8558, Japan.

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 , 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 , and decreased ratio of compared with GF mice. Furthermore, PCR array analyses of transcription factors in cultured calvarial osteoblasts showed strongly upregulated expression of . In contrast, 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.
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http://dx.doi.org/10.3390/molecules23071517DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6100304PMC
June 2018

The BMP-2 mutant L51P: a BMP receptor IA binding-deficient inhibitor of noggin.

J Bone Miner Metab 2019 Mar 17;37(2):199-205. Epub 2018 Apr 17.

Physiological Chemistry II, Theodor-Boveri-Institute for Biocenter of Würzburg University, Würzburg, Germany.

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.
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http://dx.doi.org/10.1007/s00774-018-0925-0DOI Listing
March 2019

[A Case of Successful CDDP plus CPT-11 Therapy for Recurrent Carcinomatous Lymphangiosis Occurring during Postoperative Adjuvant Therapy for Gastric Cancer].

Gan To Kagaku Ryoho 2017 Nov;44(12):1895-1897

Dept. of Surgery, Hiroshima City Hiroshima Citizens Hospital.

The case involved a 67-year-old man. Type 2 gastric cancer in the body of stomach was discovered, and the patient was referred to this department, where distal gastrectomy with Roux-en-Y reconstruction was carried out. The pathological classification was pT2N2H0P0CY0M0, pStage II B, and S-1 administration was started as postoperative adjuvant therapy. After 10 months of administration, a chest computed tomography(CT)scan revealed fine nodular shadows and irregular thickening of the alveolar septa in both lungs, a finding that was judged to be carcinomatous lymphangiosis. CDDP plus CPT- 11 therapy was subsequently started. Chest CT scan after 2 courses of administration showed the disappearance of the carcinomatous lymphangiosis. However, peritoneal metastasis was noted immediately below the abdominal wall. After completing 6courses of administration, the recurrence of peritoneal metastasis disappeared, and the administration of chemotherapy was terminated. There was no subsequent recurrence, and the patient remains alive today, 6years after the surgery. In the present case, the CT scan did not show clear mediastinal or hilar lymph node enlargement, but nodular shadows were noted at the periphery of the lung field, which were thought to be carcinomatous lymphangiosis as a result of haematogenous or anterograde metastasis into the lungs.
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November 2017

[The role of osteocytes in bone remodeling.]

Clin Calcium 2017;27(12):1697-1703

Advanced Research Center for Oral and Craniofacial Sciences, Okayama University Dental School, Okayama, Japan.

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.
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http://dx.doi.org/CliCa171216971703DOI Listing
April 2018