Shunji Itoh

MicroRNAs that target Ca 2+ transporters are involved in vascular smooth muscle cell calcification

The role of microRNAs (miRNAs) in vascular calcification is currently unclear. To examine how miRNAs are involved in vascular smooth muscle cell (VSMC) calcification, we explored the alteration of miRNAs in VSMC calcification in vitro and in vivo. Klotho homozygous mutant mice (kl/kl) display vascular calcification and have perturbations of calcium handling. We therefore hypothesized that the calcium perturbations in VSMCs could be mediated by miRNAs. Using an miRNA array analysis, we demonstrated that miRNAs are aberrantly expressed in the aortic media of 3-week-old kl/kl mice compared with wild-type (WT) mice. The expression levels of miR-135a*, miR-762, miR-714, and miR-712* in the aortic media of kl/kl mice were significantly higher than in WT mice. We used quantitative real-time reverse transcriptase polymerase chain reaction to further confirm that these miRNAs were increased in the aortic media of kl/kl mice and in cultured VSMCs treated with high phosphate and calcium. A search of the miRNA database indicated that the Ca2+ efflux proteins NCX1, PMCA1, and NCKX4 frequently appeared as potential targets of these miRNAs. The transfection of miRNA mimics into cultured VSMCs reduced the protein levels of each potential target. Conversely, miRNA inhibitors reduced phosphate and calcium-induced VSMC calcification. Furthermore, these inhibitors decreased the intracellular Ca2+ concentration in cultured VSMCs after treatment with phosphate and calcium. Our results suggest that increased expression of miR-135a*, miR-762, miR-714, and miR-712* in VSMCs may be involved in VSMC calcification by disrupting Ca2+ efflux proteins.

Trps1 Haploinsufficiency Promotes Renal Fibrosis by Increasing Arkadia Expression

Mutations in TRPS1 cause tricho-rhino-pharyngeal syndrome (TRPS). Trps1 is essential for nephron development, acting downstream of Bmp7. Because Bmp7 counteracts epithelial-to-mesenchymal transition (EMT) and reverses chronic renal injury, we examined the function of Trps1 in renal fibrosis. Immunohistochemistry revealed Trps1 expression in proximal tubular epithelial cells of mice. Unilateral ureteral obstruction reduced mRNA and protein expression of Trps1 in wild-type and heterozygous Trps1-knockout (Trps1(+/-)) mice. Trps1 haploinsufficiency promoted tubulointerstitial fibrosis via increased phosphorylation of Smad3 and decreased Smad7 protein. In primary culture, Trps1 deficiency promoted TGF-beta1-mediated EMT in proximal tubule cells. Trps1(+/-)-derived cells had higher levels of phosphorylated Smad3, and TGF-beta1 induced a time-dependent decrease in Smad7 protein in wild-type and Trps1(+/-) kidneys. In addition, compared with wild-type cells, Trps1(+/-) cells had double the amount of the E3 ubiquitin ligase Arkadia, and TGF-beta1 induced further Arkadia expression. Furthermore, knockdown of Arkadia inhibited TGF-beta1-induced EMT in Trps1(+/-) cells. Collectively, these data suggest that Trps1 haploinsufficiency enhances TGF-beta1-induced EMT and tubulointerstitial fibrosis by modulating the amount of Smad7 through Arkadia/ubiquitin-mediated degradation.

SNAIL induces epithelial-to-mesenchymal transition in a human pancreatic cancer cell line (BxPC3) and promotes distant metastasis and invasiveness in vivo

SNAIL, a potent repressor of E-cadherin expression, plays a key role in inducing epithelial-to-mesenchymal transition (EMT) in epithelial cells. During EMT, epithelial cells lose cell polarity and adhesion, and undergo drastic morphological changes acquiring highly migratory abilities. Although there is increasing evidence that EMT is involved in the progression of some human cancers, its significance in the progression of pancreatic cancer remains elusive. In Panc-1, a well-known human pancreatic cancer cell line in which EMT is triggered by TGF-β1 treatment, SNAIL and vimentin are highly expressed, whereas E-cadherin expression is scant. In contrast, another human pancreatic cancer cell line, BxPC3, in which SNAIL expression is not detected, has high levels of E-cadherin expression and does not undergo EMT upon TGF-β1 treatment. After transfecting the SNAIL gene into BxPC3, however, the cells undergo EMT with remarkable alterations in cell morphology and molecular expression patterns without the addition of any growth factors. Furthermore, in an orthotopic transplantation model using SCID mice, SNAIL-transfected BxPC3 displayed highly metastatic and invasive activities. In the immunohistochemical analysis of the tumor derived from the SNAIL-expressing BxPC3, alterations suggestive of EMT were observed in the invasive tumor front. SNAIL enabled BxPC3 to undergo EMT, endowing it with a highly malignant potential in vivo. These results indicate that SNAIL-mediated EMT may be relevant in the progression of pancreatic cancer, and SNAIL could be a molecular target for a pancreatic cancer intervention.

Trps1 Functions Downstream of Bmp7 in Kidney Development

During embryonic development, the mesenchyme of the lungs, gut, kidneys, and other tissues expresses Trps1, an atypical member of the GATA-type family of transcription factors. Our previous work suggested the possibility that Trps1 acts downstream of bone morphogenic protein 7 (Bmp7), which is essential for normal renal development. To examine the role of Trps1 during early renal development, we generated Trps1-deficient mice and examined their renal histology. Compared with wild-type mice, Trps1-deficient newborn mice had fewer tubules and glomeruli, an expanded renal interstitium, and numerous uninduced metanephric mesenchymal cells, which resulted in fewer nephrons. In wild-type kidneys, Trps1 expression was present in ureteric buds, cap mesenchyme, and renal vesicles, whereas Trps1 was virtually absent in Bmp7-deficient kidneys. Furthermore, Trps1-deficient kidneys had low levels of Pax2 and Wt1, which are markers of condensed mesenchymal cells, suggesting that a lack of Trps1 affects the differentiation of cap mesenchyme to renal vesicles. In cultured metanephric mesenchymal cells, Bmp7 induced Trps1 and E-cadherin and downregulated vimentin. Knockdown of Trps1 with small interference RNA inhibited this Bmp7-induced mesenchymal-to-epithelial transition. Last, whole-mount in situ hybridization of Wnt9b and Wnt4 demonstrated prolonged branching of ureteric buds and sparse cap mesenchyme in the kidneys of Trps1-deficient mice. Taken together, these findings suggest that normal formation of nephrons requires Trps1, which mediates mesenchymal-to-epithelial transition and ureteric bud branching during early renal development.

Trps1 plays a pivotal role downstream of Gdf5 signaling in promoting chondrogenesis and apoptosis of ATDC5 cells

Tricho‐rhino‐phalangeal syndrome (TRPS) is an autosomal dominant skeletal disorder caused by mutations of TRPS1. Based on the similar expression patterns of Trps1 and Gdf5, we hypothesized a possible functional interaction between these two molecules. Using a chondrogenic cell line (ATDC5), we investigated the association of Gdf5‐mediated signaling pathways with Trps1 and the phenotypic changes of ATDC5 cells due to over‐expression or suppression of Trps1. Treatment of cells with Gdf5 enhanced Trps1 protein levels and phosphorylation of p38 mitogen‐activated protein kinase (MAPK) in a dose‐dependent manner. Nuclear translocation of Trps1 was also induced by Gdf5. These effects were blocked by a dominant negative form of activin‐linked kinase 6 (dn‐Alk6) and by SB203580, an inhibitor of the p38 MAPK pathway. Conversely, Gdf5 expression was suppressed by the over‐expression of Trps1. Trps1‐overexpressing ATDC5 (O/E) cells differentiated into chondrocytes more quickly than mock‐infected control cells, whereas cells transfected with dn‐Alk6 showed slower differentiation. On the other hand, O/E cells showed an increase of apoptosis along with the up‐regulation of cleaved caspase 3 and down‐regulation of Bcl‐2, whereas dn‐Alk6 cells showed suppression of apoptosis. In conclusion, Trps1 acts downstream of the Gdf5 signaling pathway and promotes the differentiation and apoptosis of ATDC5 cells.

TNF-α deficiency accelerates renal tubular interstitial fibrosis in the late stage of ureteral obstruction

TNF-alpha and TGF-beta1 have a complementary relationship in fibrogenesis. This study was performed to investigate the role of TNF-alpha in renal tubular interstitial fibrosis. We compared the extent of renal tubular interstitial fibrosis after unilateral ureteral obstruction (UUO) between wild-type and TNF-alpha-deficient mice by using immunohistochemistry, enzyme-linked immunoassay, and the real-time polymerase chain reaction (PCR). In comparison with wild-type mice, there was no significant difference in the extent of renal fibrosis in the TNF-alpha-deficient mice at 2 weeks after UUO. By 4 weeks after UUO, however, fibrosis marked an increase in the TNF-alpha-deficient mice to exceed that in the wild-type mice. Immunohistochemistry, enzyme-linked immunoassay, and real-time PCR demonstrated an increase of extracellular matrix in the kidneys of TNF-alpha-deficient mice that was caused by upregulation of the expression of TGF-beta1 and Snail, which in turn resulted from an increase of infiltrating macrophages. Real-time PCR revealed an increase in expression of the TNF-alpha type 2 receptor at 4 weeks after UUO, which explained the difference in the extent of renal fibrosis between TNF-alpha-deficient and wild-type mice. In the chronic stage of renal fibrosis, TNF-alpha suppresses the infiltration of macrophages by inducing TNF-alpha type 2 receptor expression, resulting in the amelioration of fibrosis.

Trps1 deficiency enlarges the proliferative zone of growth plate cartilage by upregulation of Pthrp

We have reported that elongation of the columnar proliferative zone of long bone growth plates in Trps1-/- mice during the late fetal stage in the previous study [1]. Since expression of Trps1 protein was found to overlap with that of mRNAs for Indian hedgehog (Ihh), PTH/PTHrP receptor (PPR), and PTHrP, we hypothesized that Trps1 may inhibit the hypertrophic differentiation of chondrocytes by interacting with the Ihh/PTHrP feedback loop. To investigate whether Trps1 has a role in this Ihh/PTHrP feedback loop, we compared the growth plates of Trps1-/- mice and wild-type (Trps1+/+) mice. Immunohistochemistry showed that Trps1 protein was strongly expressed in the periarticular and prehypertrophic zones of the fetal growth plate in wild-type mice on embryonic day 18.5 (E18.5). On the other hand, Ihh, PPR, and PTHrP mRNAs were predominantly expressed in the prehypertrophic zone at this stage of development. While expression of Ihh and PPR by prehypertrophic chondrocytes was unaffected in the growth plates of Trps1-/- mice, the range of PTHrP expression was expanded toward the proliferating zone in these mice. Quantitative real-time PCR analysis demonstrated upregulation of PTHrP in the epiphyseal growth plates of Trps1-/- mice. Furthermore, promoter analysis combined with the chromatin immunoprecipitation (ChIP) assay demonstrated that direct binding of Trps1 to the PTHrP promoter suppressed the transcription of PTHrP. Finally, organ culture of E14.5 tibiae in the absence or the presence of Pthrp revealed that the proliferative zone of the tibial growth plate was elongated by culture with Pthrp compared to that of control tibiae. Taken together, these data provide the first genetic evidence that lack of Trps1 leads to overexpression of PTHrP, and that Trps1 is required to maintain the normal organization of chondrocytes in the growth plate.

Trps1 is necessary for normal temporomandibular joint development

Mutation of the human TRPS1 gene leads to trichorhinophalangeal syndrome (TRPS), which is characterized by an abnormal development of various organs including the craniofacial skeleton. Trps1 has recently been shown to be expressed in the jaw joints of zebrafish; however, whether Trps1 is expressed in the mammalian temporomandibular joint (TMJ), or whether it is necessary for TMJ development is unknown. We have analyzed (1) the expression pattern of Trps1 during TMJ development in mice and (2) TMJ development in Trps1 knockout animals. Trps1 is expressed in the maxillo-mandibular junction at embryonic day (E) 11.5. At E15.5, expression is restricted to the developing condylar cartilage and to the surrounding joint disc progenitor cells. In Trps1 knockout mice, the glenoid fossa of the temporal bone forms relatively normally but the condylar process is extremely small and the joint disc and cavities do not develop. The initiation of condyle formation is slightly delayed in the mutants at E14.5; however, at E18.5, the flattened chondrocyte layer is narrowed and most of the condylar chondrocytes exhibit precocious chondrocyte maturation. Expression of Runx2 and its target genes is expanded toward the condylar apex in the mutants. These observations underscore the indispensable role played by Trps1 in normal TMJ development in supporting the differentiation of disc and synoviocyte progenitor cells and in coordinating condylar chondrocyte differentiation.

Loss of Smad3 gives rise to poor soft callus formation and accelerates early fracture healing

Smad3 is an intracellular signaling molecule in the transforming growth factor β (TGF-β) pathway that serves as a regulator of chondrogenesis and osteogenesis. To investigate the role of the TGF-β/Smad3 signaling in the process of fracture healing, an open fracture was introduced in mouse tibiae, and the histology of the healing process was compared between wild-type (WT) and Smad3-null (KO) mice. In KO mice, the bone union formed more rapidly with less formation of cartilage in the callus and eventually the fracture was repaired more rapidly than in WT mice. Alkaline phosphatase staining showed that osteoblastic differentiation in the fracture callus was promoted in KO mice. Additionally, TRAP staining and the TUNEL assay revealed that the induction of osteoclasts and apoptotic cells was significantly promoted in the healing callus of KO mice. Sox9 expression clearly decreased at both mRNA and protein levels in the early stage of fracture in KO mice. In contrast, the expression of genes for osteogenesis and osteoclast formation increased from day 5 until day 14 post-fracture in KO mice compared to WT mice. From these results, we concluded that the loss of TGF-β/Smad3 signaling promoted callus formation by promoting osteogenesis and suppressing chondrogenesis, which resulted in faster fracture healing.

A novel oncogenic pathway by TLS–CHOP involving repression of MDA-7/IL-24 expression

Background:Translocated in liposarcoma-CCAAT/enhancer binding protein homologous protein (TLS–CHOP) (also known as FUS-DDIT3) chimeric oncoprotein is found in the majority of human myxoid liposarcoma (MLS), but its molecular function remains unclear.Methods:We knockdowned TLS–CHOP expression in MLS-derived cell lines by a specific small interfering RNA, and analysed the gene expression profiles with microarray.Results:TLS-CHOP knockdown inhibited growth of MLS cells, and induced an anticancer cytokine, melanoma differentiation-associated gene 7 (MDA-7)/interleukin-24 (IL-24) expression. However, double knockdown of TLS–CHOP and MDA-7/IL-24 did not inhibit MLS cell growth.Conclusion:Repression of MDA-7/IL-24 expression by TLS–CHOP is required for MLS tumour growth, and TLS–CHOP may become a promising therapeutic target for MLS treatment.