Jirouta Kitagaki

Cyclic depsipeptides as potential cancer therapeutics.

Cyclic depsipeptides are polypeptides in which one or more amino acid is replaced by a hydroxy acid, resulting in the formation of at least one ester bond in the core ring structure. Many natural cyclic depsipeptides possessing intriguing structural and biological properties, including antitumor, antifungal, antiviral, antibacterial, anthelmintic, and anti-inflammatory activities, have been identified from fungi, plants, and marine organisms. In particular, the potent effects of cyclic depsipeptides on tumor cells have led to a number of clinical trials evaluating their potential as chemotherapeutic agents. Although many of the trials have not achieved the desired results, romidepsin (FK228), a bicyclic depsipeptide that inhibits histone deacetylase, has been shown to have clinical efficacy in patients with refractory cutaneous T-cell lymphoma and has received Food and Drug Administration approval for use in treatment. In this review, we discuss antitumor cyclic depsipeptides that have undergone clinical trials and focus on their structural features, mechanisms, potential applications in chemotherapy, and pharmacokinetic and toxicity data. The results of this study indicate that cyclic depsipeptides could be a rich source of new cancer therapeutics.

FGF8 is essential for formation of the ductal system in the male reproductive tract

During development of the urogenital tract, fibroblast growth factor 8 (Fgf8) is expressed in mesonephric tubules, but its role in this tissue remains undefined. An evaluation of previously generated T-Cre-mediated Fgf8-deficient mice (T-Cre; Fgf8flox/Δ2,3 mice), which lack Fgf8 expression in the mesoderm, revealed that the cranial region of the Wolffian duct degenerated prematurely and the cranial mesonephric tubules were missing. As a result, the epididymis, vas deferens and efferent ductules were largely absent in mutant mice. Rarb2-Cre was used to eliminate FGF8 from the mesonephric tubules but to allow expression in the adjacent somites. These mutants retained the cranial end of the Wolffian duct and formed the epididymis and vas deferens, but failed to elaborate the efferent ductules, indicating that Fgf8 expression by the mesonephric tubules is required specifically for the formation of the ductules. Ret knockout mice do not form the ureteric bud, a caudal outgrowth of the Wolffian duct and progenitor for the collecting duct network in the kidney, but they do develop the cranial end normally. This indicates that Fgf8, but not Ret, expression is essential to the outgrowth of the cranial mesonephric tubules from the Wolffian duct and to the development of major portions of the sex accessory tissues in the male reproductive tract. Mechanistically, FGF8 functions upstream of Lhx1 expression in forming the nephron, and analysis of Fgf8 mutants similarly shows deficient Lhx1 expression in the mesonephric tubules. These results demonstrate a multifocal requirement for FGF8 in establishing the male reproductive tract ducts and implicate Lhx1 signaling in tubule elongation.

Effects of the proteasome inhibitor, bortezomib, on cytodifferentiation and mineralization of periodontal ligament cells

BACKGROUND AND OBJECTIVE The proteasome inhibitor, bortezomib, is known to induce osteoblastic differentiation in a number of cell lines, such as mesenchymal stem cells and osteoblastic precursor cells. As periodontal ligament (PDL) cells are multipotent, we examined whether bortezomib may induce the differentiation of PDL cells into hard-tissue-forming cells. MATERIAL AND METHODS A mouse PDL clone cell line, MPDL22 cells, was cultured in mineralization medium in the presence or absence of bortezomib. Expression of calcification-related genes and calcified-nodule formation were evaluated by real-time PCR and Alizarin Red staining, respectively. RESULTS Bortezomib increased the expression of calcification-related mRNAs, such as tissue nonspecific alkaline phosphatase isoenzyme (ALPase), bone sialoprotein (Bsp), runt-related transcription factor 2 (Runx2) and osteopontin, and calcified-nodule formation in MPDL22 cells. These effects were induced, in part, by increasing the cytosolic accumulation and nuclear translocation of β-catenin, leading to an increase in expression of bone morphogenetic protein (Bmp)-2, -4 and -6 mRNAs. In addition, bortezomib enhanced BMP-2-induced expression of Bsp and osteopontin mRNAs and increased calcified-nodule formation in MPDL22 cells. CONCLUSION Bortezomib induced cytodifferentiation and mineralization of PDL cells by enhancing the accumulation of β-catenin within the cytosol and the nucleus and increasing the expression of Bmp-2, -4 and -6 mRNAs. Moreover, bortezomib enhanced the BMP-2-induced cytodifferentiation and mineralization of PDL cells, suggesting that bortezomib may be efficacious for use in periodontal regeneration therapy.

A Putative Association of a Single Nucleotide Polymorphism in GPR126 with Aggressive Periodontitis in a Japanese Population.

Periodontitis is an inflammatory disease causing loss of tooth-supporting periodontal tissue. Disease susceptibility to the rapidly progressive form of periodontitis, aggressive periodontitis (AgP), appears to be influenced by genetic risk factors. To identify these in a Japanese population, we performed whole exome sequencing of 41 unrelated generalized or localized AgP patients. We found that AgP is putatively associated with single nucleotide polymorphism (SNP) rs536714306 in the G-protein coupled receptor 126 gene, GPR126 [c.3086 G>A (p.Arg1029Gln)]. Since GPR126 activates the cAMP/PKA signaling pathway, we performed cAMP ELISA analysis of cAMP concentrations, and found that rs536714306 impaired the signal transactivation of GPR126. Moreover, transfection of human periodontal ligament (HPDL) cells with wild-type or mutant GPR126 containing rs536714306 showed that wild-type GPR126 significantly increased the mRNA expression of bone sialoprotein, osteopontin, and Runx2 genes, while mutant GPR126 had no effect on the expression of these calcification-related genes. The increase in expression of these genes was through the GPR126-induced increase of bone morphogenic protein-2, inhibitor of DNA binding (ID) 2, and ID4 expression. These data indicate that GPR126 might be important in maintaining the homeostasis of periodontal ligament tissues through regulating the cytodifferentiation of HPDL cells. The GPR126 SNP rs536714306 negatively influences this homeostasis, leading to the development of AgP, suggesting that it is a candidate genetic risk factor for AgP in the Japanese population.

Sphingomyelin Phosphodiesterase 3 Enhances Cytodifferentiation of Periodontal Ligament Cells

Sphingomyelin phosphodiesterase 3 (Smpd3), which encodes neutral sphingomyelinase 2 (nSMase2), is a key molecule for skeletal development as well as for the cytodifferentiation of odontoblasts and alveolar bone. However, the effects of nSMase2 on the cytodifferentiation of periodontal ligament (PDL) cells are still unclear. In this study, the authors analyzed the effects of Smpd3 on the cytodifferentiation of human PDL (HPDL) cells. The authors found that Smpd3 increases the mRNA expression of calcification-related genes, such as alkaline phosphatase (ALPase), type I collagen, osteopontin, Osterix (Osx), and runt-related transcription factor (Runx)-2 in HPDL cells. In contrast, GW4869, an inhibitor of nSMase2, clearly decreased the mRNA expression of ALPase, type I collagen, and osteocalcin in HPDL cells, suggesting that Smpd3 enhances HPDL cytodifferentiation. Next, the authors used exome sequencing to evaluate the genetic variants of Smpd3 in a Japanese population with aggressive periodontitis (AgP). Among 44 unrelated subjects, the authors identified a single nucleotide polymorphism (SNP), rs145616324, in Smpd3 as a putative genetic variant for AgP among Japanese people. Moreover, Smpd3 harboring this SNP did not increase the sphingomyelinase activity or mRNA expression of ALPase, type I collagen, osteopontin, Osx, or Runx2, suggesting that this SNP inhibits Smpd3 such that it has no effect on the cytodifferentiation of HPDL cells. These data suggest that Smpd3 plays a crucial role in maintaining the homeostasis of PDL tissue.

Transcriptome Reveals Cathepsin K in Periodontal Ligament Differentiation

Periodontal ligaments (PDLs) play an important role in remodeling the alveolar bond and cementum. Characterization of the periodontal tissue transcriptome remains incomplete, and an improved understanding of PDL features could aid in developing new regenerative therapies. Here, we aimed to generate and analyze a large human PDL transcriptome. We obtained PDLs from orthodontic treatment patients, isolated the RNA, and used a vector-capping method to make a complementary DNA library from >20,000 clones. Our results revealed that 58% of the sequences were full length. Furthermore, our analysis showed that genes expressed at the highest frequencies included those for collagen type I, collagen type III, and proteases. We also found 5 genes whose expressions have not been previously reported in human PDL. To access which of the highly expressed genes might be important for PDL cell differentiation, we used real-time polymerase chain reaction to measure their expression in differentiating cells. Among the genes tested, the cysteine protease cathepsin K had the highest upregulation, so we measured its relative expression in several tissues, as well as in osteoclasts, which are known to express high levels of cathepsin K. Our results revealed that PDL cells express cathepsin K at similar levels as osteoclasts, which are both expressed at higher levels than those of the other tissues tested. We also measured cathepsin K protein expression and enzyme activity during cell differentiation and found that both increased during this process. Immunocytochemistry experiments revealed that cathepsin K localizes to the interior of lysosomes. Last, we examined the effect of inhibiting cathepsin K during cell differentiation and found that cathepsin K inhibition stimulated calcified nodule formation and increased the levels of collagen type I and osteocalcin gene expression. Based on these results, cathepsin K seems to regulate collagen fiber accumulation during human PDL cell differentiation into hard tissue-forming cells.

Expression pattern of zinc-finger transcription factor Odd-skipped related 2 in murine development and neonatal stage

The Odd-skipped gene, first identified as a Drosophila pair-rule zinc-finger transcription factor, plays an important role in Drosophila development. The mammalian homolog, Odd-skipped related 2 (Osr2), regulates limb, tooth, and kidney development in mouse embryos. However, the detailed expression pattern of Osr2 during neonatal development remains unclear. In this study, we investigated Osr2 expression patterns in mouse neonatal and embryo tissues using qPCR and in situ hybridization methods. First, we examined the tissue distribution of Osr2 by qPCR, and found it to be highly expressed in the uterus and moderately in the testes, small intestine, and prostate. That expression was also found in eye, kidney, placenta, lung, thymus, lymph node, stomach, and skeletal muscle tissues, and in all embryonic stages. On the other hand, Osr2 was not expressed in brain, heart, liver, or spleen samples. Next, we examined the tissue localization of Osr2 using in situ hybridization. Osr2 was found in the craniofacial region on E13.5, with notable expression in dental germ mesenchyme as well as the renal corpuscle on E17.5. As for neonatal tissues, Osr2 was expressed in the dental papilla, dental follicle, Harderian gland, nasal bone, eyelid dermis, synovial joint, and tibial subcutis. Our findings suggest that Osr2 functions in reproductive system organs, such as the uterus, testes, prostate, placenta, and ovaries. Furthermore, based on its expression in kidney, Harderian gland, eyelid dermis, and tibial subcutis tissues, this transcription factor may be involved in hormone synthesis and function. Together, our results demonstrate the role of Osr2 in postnatal development and embryogenesis.

Syntaxin 4a Regulates Matrix Vesicle-Mediated Bone Matrix Production by Osteoblasts

Osteoblasts secrete matrix vesicles and proteins to bone surfaces, but the molecular mechanisms of this secretion system remain unclear. The present findings reveal the roles of important genes in osteoblasts involved in regulation of extracellular matrix secretion. We especially focused on “soluble N‐ethylmaleimide‐sensitive factor (NSF) attachment protein receptor” (SNARE) genes and identified notable Syntaxin 4a (Stx4a) expression on the basolateral side of the plasma membrane of osteoblasts. Furthermore, Stx4a overexpression was found to increase mineralization by osteoblastic cells, whereas Stx4a knockdown reduced levels of mineralization. Also, BMP‐4 and IGF‐1 induced the localization of Stx4a to the basolateral side of the cells. To examine the function of Stx4a in osteoblasts, we generated osteoblast‐specific Stx4a conditional knockout mice, which demonstrated an osteopenic phenotype due to reduced matrix secretion. Bone mineral density, shown by peripheral quantitative computed tomography (pQCT), was reduced in the femur metaphyseal and diaphyseal regions of Stx4a osteoblast‐specific deficient mice, whereas bone parameters, shown by micro–computed tomography (μCT) and bone histomorphometric analysis, were also decreased in trabecular bone. In addition, primary calvarial cells from those mice showed decreased mineralization and lower secretion of matrix vesicles. Our findings indicate that Stx4a plays a critical role in bone matrix production by osteoblasts.

Identification of genetic risk factors of aggressive periodontitis using genomewide association studies in association with those of chronic periodontitis

To identify the genetic risk factors for aggressive periodontitis (AgP), it is important to understand the progression and pathogenesis of AgP. The purpose of this review was to summarize the genetic risk factors for AgP identified through a case-control genomewide association study (GWAS) and replication study. The initial studies to identify novel AgP risk factors were potentially biased because they relied on previous studies. To overcome this kind of issue, an unbiased GWAS strategy was introduced to identify genetic risk factors for various diseases. Currently, three genes glycosyltransferase 6 domain containing 1 (GLT6D1), defensin α1 and α3 (DEFA1A3), and sialic acid-binding Ig-like lectin 5 (SIGLEC5) that reach the threshold for genomewide significance have been identified as genetic risk factors for AgP through a case-control GWAS.