👤 Kazu Matsumoto

Nedd4-interacting protein 2 (NDFIP2) has three transmembrane domains and interacts with multiple Nedd4 family ubiquitin ligases through polyprolinetyrosine (PY) motifs located in its N-terminal cytoplasmic domain. It has been postulated that NDFIP2 acts as an adaptor for the ubiquitylation of substrates with Nedd4 ubiquitin ligase. However, whether NDFIP2 promotes or inhibits the ubiquitylation of Nedd4 substrates is still under debate. We show here that although NDFIP2 is detected in the Golgi/trans-Golgi network (TGN) area, it is rapidly delivered to and degraded in lysosomes with its half-life ca. 1.5 h. Intriguingly, knockdown (KD) of NDFIP2 with small interfering RNA (siRNA) impaired both the formation and function of gap junctions. Indeed, KD of NDFIP2 destabilized the gap junction protein connexin43 that contains PY motif. In support of this, overexpression of NDFIP2 stabilized connexin43 and enhanced the formation of gap junctions. Furthermore, the PY motifs of NDFIP2, which are required for its interaction with Nedd4, Atrophin-1 interacting protein (AIP) 4 (AIP4)/Itch, and AIP2/WWP2, were necessary for the targeting of NDFIP2 to lysosomes and/or the stability of connexin43 and gap junctions. Collectively these findings suggest that NDFIP2 may inhibit the Nedd4-dependent ubiquitylation of membrane proteins containing PY motifs, such as connexin43, in a competitive manner. Show less

During human embryogenesis, neural crest cells migrate to the anterior chamber of the eye and then differentiate into the inner layers of the cornea, the iridocorneal angle, and the anterior portion of the iris. When proper development does not occur, this causes iridocorneal angle dysgenesis and intraocular pressure (IOP) elevation, which ultimately results in developmental glaucoma. Here, we show that heparan sulfate (HS) deficiency in mouse neural crest cells causes anterior chamber dysgenesis, including corneal endothelium defects, corneal stroma hypoplasia, and iridocorneal angle dysgenesis. These dysfunctions are phenotypes of the human developmental glaucoma, Peters anomaly. In the neural crest cells of mice embryos, disruption of the gene encoding exostosin 1 (Ext1), which is an indispensable enzyme for HS synthesis, resulted in disturbed TGF-beta2 signaling. This led to reduced phosphorylation of Smad2 and downregulated expression of forkhead box C1 (Foxc1) and paired-like homeodomain transcription factor 2 (Pitx2), transcription factors that have been identified as the causative genes for developmental glaucoma. Furthermore, impaired interactions between HS and TGF-beta2 induced developmental glaucoma, which was manifested as an IOP elevation caused by iridocorneal angle dysgenesis. These findings suggest that HS is necessary for neural crest cells to form the anterior chamber via TGF-beta2 signaling. Disturbances of HS synthesis might therefore contribute to the pathology of developmental glaucoma. Show less

The molecular mechanism of thyroid hormone (TH) effects to fatty acid metabolism in liver is yet to be clear. The carbohydrate response element-binding protein (ChREBP) as well as sterol response element-binding protein (SREBP)-1c plays a pivotal role in hepatic lipogenesis. Both SREBP-1c and ChREBP are target genes of liver X receptors (LXRs). Because LXRs and TH receptors (TRs) cross talk mutually in many aspects of transcription, we examined whether TRs regulate the mouse ChREBP gene expression. In the current study, we demonstrated that TH up-regulated mouse ChREBP mRNA and protein expression in liver. Run-on and luciferase assays showed that TH and TR-beta1 positively regulated the ChREBP gene transcription. The mouse ChREBP gene promoter contains two direct repeat-4 sites (LXRE1 and LXRE2) and EMSAs demonstrated that LXR-alpha and TR-beta1 prefer to bind LXRE1 and LXRE2, respectively. The direct repeat-4 deletion and LXRE2 mutants of the promoter deteriorate the positive regulation by TR-beta1, indicating that LXRE2 is functionally important for the regulation. We also showed that human ChREBP gene expression and promoter activities were up-regulated by TH. These data suggest that ChREBP mRNA expression is positively regulated by TR-beta1 and TH at the transcriptional level in mammals. This novel observation indicates that TH fine-tunes hepatic lipogenesis via regulating SREBP-1c and ChREBP gene expression reciprocally. Show less

The liver X receptors (LXR-alpha and -beta) are nuclear oxysterol receptors that play pivotal roles in regulating the expression of genes involved in cholesterol transport and metabolism. Recently, several groups have reported that the LXRs also regulate adrenal steroidogenesis. However, the roles of LXRs in the hypothalami-pituitary-adrenal axis, especially whether they regulate proopiomelanocortin (POMC) gene expression in the pituitary, remain to be elucidated. In this report, we demonstrate that LXR mRNA is expressed in the pituitary and that at the protein level, LXR-alpha is dominantly expressed. Next, we show that the LXR agonist TO901317 (TO) increased POMC mRNA levels and the number of cells immunostained with anti-ACTH antibody in the mouse pituitary. We also confirmed that TO elevated plasma ACTH and serum corticosterone levels in vivo and increased the total tissue content of immunoreactive ACTH in the pituitary. TO activated the rat POMC gene promoter (-706/+64 bp) in GH3 and AtT-20 cells. Silencing of LXR-alpha mRNA expression in GH3 cells with small interfering RNA specific to LXR-alpha caused a loss of promoter activity induced by the LXR ligand, suggesting that LXR-alpha directly regulates the POMC gene promoter. EMSAs also demonstrated that the retinoid X receptor-alpha/LXR-alpha heterodimer bound to the region between -73 and -52 bp in the rat POMC gene promoter, and this site was responsible for the induction by TO, as confirmed by chromatin immunoprecipitation assays using AtT-20 cells. Our findings provide the first evidence that LXR-alpha positively regulates the POMC gene promoter at the transcriptional level and suggest LXR-alpha to be a coordinator for cross talk between lipid metabolism and neuroendocrinology. Show less

Vitreous samples collected in retinopathic surgeries have diverse properties, making proteomics analysis difficult. We report a cluster analysis to evade this difficulty. Vitreous and subretinal fluid samples were collected from 60 patients during surgical operation of non-proliferative diabetic retinopathy, proliferative diabetic retinopathy, proliferative vitreoretinopathy, and rhegmatogenous retinal detachment. For controls we collected vitreous fluid from patients of idiopathic macular hole, epiretinal, and from a healthy postmortem donor. Proteins from these samples were subjected to quantitative proteomics using two-dimensional gel electrophoresis. We selected 105 proteins robustly expressed among ca 400 protein spots and subjected them to permutation test. By using permutation test analysis we observed unique variations in the expression of some of these proteins in vitreoretinal diseases when compared to the control and to each other: 1) the levels of inflammation-associate proteins such as AAT, APOA4, ALB, and TF were significantly higher in all four types of vitreoretinal diseases, and 2) each vitreoretinal disease elevates a unique set of proteins which can be interpreted based on the pathology of retinopathy. Our protocol will be effective for the study of protein expression in other types of clinical samples of diverse property. Show less

There is increasing evidence that heparan sulfate (HS) plays an essential role in various axon guidance processes. These observations, however, have not addressed whether HS is required cell autonomously as an axonal coreceptor or as an environmental factor that modulates the localization of guidance molecules in the terrain in which growing axons navigate. Here we demonstrate that netrin-1-mediated commissural axon guidance requires cell-autonomous expression of HS in commissural neurons in vivo. We used the Wnt1-Cre transgene to drive region-specific ablation of Ext1, which encodes an enzyme essential for HS synthesis, in the dorsal part of the spinal cord. Remarkably, Wnt1-Cre-mediated ablation of Ext1 causes commissural axon pathfinding defects that share similarities with those of Netrin-1-deficient and DCC (deleted in colorectal cancer)-deficient mice. Neither Ext1-deficient dorsal spinal cord explants nor wild-type explants in which HS expression was ablated could extend axons in response to netrin-1. Intracellular signaling downstream of netrin-1 and DCC was defective in Ext1-deficient commissural neurons and in DCC-transfected HEK293T cells from which HS was removed. These results demonstrate that the expression of HS by commissural neurons is essential for these neurons to transduce netrin-1 signals, thus providing evidence for a cell-autonomous role of HS in netrin-1/DCC-mediated axon guidance. Show less

Embryonic stem (ES) cells are highlighted as promising cell sources for regenerative medicine. Here, we focused on providing the platform that forced ES cells to reproduce the vascular organization process, leading to efficiency and safety evaluation as preclinical testing of biological agents. Murine ES cell-derived embryoid bodies on matrigel, but not collagen or gelatin, could be differentiated into sprouting blood vessels without the addition of growth factors. The expression of endothelial cell marker CD31 and smooth muscle marker alpha-smooth muscle actin was partially colocalized and started to increase 7 days after culture on matrigel, accompanied by the induction of a number of growth factors, such as vascular endothelial growth factor, fibroblast growth factor-2, hepatocyte growth factor, transforming growth factor-beta, and angiopoietin-1. Moreover, notch-related genes, such as Del1 or Del4 (delta-like 1/4) and hey1 or hey2 (hairy/enhancer of split related TRPW motif 1/2), were upregulated in a similar time course. The treatment of neutralizing antibodies against these growth factors failed to inhibit the differentiation into the sprouting blood vessels, whereas arginine-glycine-aspartic peptide, a selective inhibitor for the alphavbeta3-integrins, did inhibit differentiation. An anticancer drug to inhibit angiogenesis, TNP-470, also blocked the vascular formation in this model. ES cells could reproduce the vascular organization process on the biosynthetic scaffolds, such as matrigel, without the addition of growth factors. In the future, a human ES-based tissue model would be an optional tool for the screening of pharmaceutical drugs for vascular disease. Show less

Three G1 cyclins, CLN1, CLN2, and CLN3, have been identified in the budding yeast Saccharomyces cerevisiae. G1 cyclins are essential, albeit functionally redundant, rate-limiting activators of cell cycle initiation. We have isolated dosage-dependent suppressor genes (designated HMD genes) of the mating defect caused by CLN3-2, a dominant mutation in CLN3, HMD2 and HMD3 are identical to STE4 and STE5, respectively, HMD1 is an essential gene that encodes a protein containing a putative RNA binding domain. Overproduction of HMD1 results in a relatively specific reduction in the level of the CLN3 or CLN3-2 transcript. This reduction occurs subsequent to transcription initiation of CLN3 since overexpression of HMD1 did not affect expression of a heterologous transcript from the CLN3 promoter but did result in a reduction of CLN3 transcript expressed from a heterologous promoter. HMD1 has at least one essential role independent of its effect on CLN3 since HMD1 remains essential for viability in the absence of a functional CLN3 gene. Show less

The GPA1 gene of S. cerevisiae encodes a G alpha subunit that plays a positive role in the transduction of signals stimulating recovery from pheromone-induced cell cycle arrest. The GPA1Val50 mutation, in which Gly-50 is replaced by valine, causes hyperadaptation to pheromone. However, GPA1Val50 cells do not recover from division arrest in the absence of both CLN1 and CLN3, which encode G1 cyclins, indicating that the recovery-promoting activity of GPA1Val50 requires the function of G1 cyclins. An sgv1 mutation suppresses the hyperadaptive response caused by GPA1Val50 and also confers cold- and temperature-sensitive growth. The SGV1 gene encodes an apparent protein kinase homologous to CDC28/cdc2 kinase: SGV1 is 42% identical to CDC28. The activated mutation, CLN3-2, partially suppresses the growth defect of sgv1, suggesting that the SGV1 and CLN3 proteins may act in the same growth control pathway. Show less