CiteULike: jyuh's Fukami

Visualizing Spatiotemporal Dynamics of Multicellular Cell-Cycle Progression

Asako Sakaue-Sawano — 2008-02-08T16:32:00-00:00

Cell, Vol. 132, No. 3. (8 February 2008), pp. 487-498.

Summary The cell-cycle transition from G1 to S phase has been difficult to visualize. We have harnessed antiphase oscillating proteins that mark cell-cycle transitions in order to develop genetically encoded fluorescent probes for this purpose. These probes effectively label individual G1 phase nuclei red and those in S/G2/M phases green. We were able to generate cultured cells and transgenic mice constitutively expressing the cell-cycle probes, in which every cell nucleus exhibits either red or green fluorescence. We performed time-lapse imaging to explore the spatiotemporal patterns of cell-cycle dynamics during the epithelial-mesenchymal transition of cultured cells, the migration and differentiation of neural progenitors in brain slices, and the development of tumors across blood vessels in live mice. These mice and cell lines will serve as model systems permitting unprecedented spatial and temporal resolution to help us better understand how the cell cycle is coordinated with various biological events.

PIASy controls ubiquitination-dependent proteasomal degradation of Ets-1.

T Nishida — 2008-04-19T14:07:44-00:00

The Biochemical journal, Vol. 405, No. 3. (1 August 2007), pp. 481-488.

The ETS transcription factor Ets-1 (E26 transformation-specific-1) plays a critical role in many physiological processes including angiogenesis, haematopoietic development and tumour progression. Its activity can be regulated by post-translational modifications, such as phosphorylation. Recently, we showed that Ets-1 is a target for SUMO (small ubiquitin-like modifier) modification and that PIASy [protein inhibitor of activated STAT (signal transducer and activator of transcription) Y], a specific SUMO-E3 ligase for Ets-1, represses Ets-1-dependent transcription. In the present study, we demonstrated that Ets-1 is degraded by the proteasome and that overexpression of PIASy increased the stability of endogenous and ectopically expressed Ets-1 protein by preventing proteasomal degradation. Moreover, knockdown of the endogenous PIASy expression by RNA interference reduced the protein level of endogenous Ets-1. The proteasome inhibitor MG132 reversed this effect. Deletion analysis showed that the TAD (transcriptional activation domain), which has been identified as the interaction domain with PIASy, was also required for Ets-1 ubiquitination and proteasomal degradation. However, the Ets-1 stabilization by PIASy was not due to reduced ubiquitination of Ets-1. Our results suggested that PIASy controls Ets-1 function, at least in part, by inhibiting Ets-1 protein turnover via the ubiquitin-proteasome system.

Repression of E1AF transcriptional activity by sumoylation and PIASy.

T Nishida — 2008-04-19T14:07:51-00:00

Biochemical and biophysical research communications, Vol. 360, No. 1. (17 August 2007), pp. 226-232.

E1AF is a member of the Ets transcriptional factor family, and it plays a crucial role in tumor metastasis. However, the molecular mechanisms regulating its activity are not well characterized. In this study, we show that E1AF is sumoylated at four lysine residues, both in vivo and in vitro. Replacement of these lysines by arginine enhanced the transcriptional activity of E1AF, suggesting that sumoylation negatively regulates E1AF activity. We further demonstrated that PIASy enhanced sumoylation of E1AF as a specific SUMO-E3 ligase. In addition, PIASy repressed the transcriptional activity of both the wild-type and sumoylation defective mutants. However, the C342A mutant of PIASy, which abrogates SUMO-E3 ligase activity, had a significantly decreased ability to repress E1AF activity. Taken together, our results indicate that PIASy negatively regulates E1AF-mediated transcription by both E1AF sumoylation in a dependent and independent fashion.

PIASy-mediated repression of the Ets-1 is independent of its sumoylation.

T Nishida — 2008-04-19T14:08:31-00:00

Biochemical and biophysical research communications, Vol. 345, No. 4. (14 July 2006), pp. 1536-1546.

The transcription factor Ets-1 is involved in many physiological processes, including angiogenesis, hematopoietic development, and tumor progression, and its activity can be regulated by interactions with other proteins and post-translational modifications, such as phosphorylation. Here, we show that Ets-1 is a target for SUMO modification both in vivo and in vitro. Mutational analysis reveals that sumoylation of Ets-1 occurs at two lysine residues at amino acid positions 15 and 227, which lie within previously identified synergy control motifs. Replacement of sumoylation site lysines with arginine or overexpression of SENP1, a desumoylation enzyme, enhances the transactivation ability of Ets-1. Furthermore, we identify PIASy as a novel interaction partner and a specific SUMO-E3 ligase of Ets-1. PIASy represses the Ets-1-dependent transcription, and its repression is independent of the sumoylation status of Ets-1, but it is dependent on the sumoylation of other factors.

Olmesartan blocks advanced glycation end products (AGEs)-induced angiogenesis in vitro by suppressing receptor for AGEs (RAGE) expression.

S Yamagishi — 2008-01-16T02:06:09-00:00

Microvasc Res, Vol. 75, No. 1. (January 2008), pp. 130-134.

We have previously shown that advanced glycation end products (AGEs)-their receptor (RAGE) interaction elicits angiogenesis through autocrine production of vascular endothelial growth factor (VEGF), thus suggesting the active involvement of the AGEs-RAGE system in proliferative diabetic retinopathy (PDR). Since the crosstalk between the AGEs-RAGE and the renin-angiotensin system has also been proposed in the pathogenesis of PDR, we investigated here whether olmesartan, an angiotensin II type 1 receptor blocker, inhibited the AGEs-elicited angiogenesis in vitro by suppressing the NF-kappaB-mediated RAGE expression. Olmesartan significantly inhibited the AGEs-induced NF-kappaB promoter activity and RAGE gene expression in cultured microvascular endothelial cells (ECs). Further, olmesartan was found to block the AGEs-induced up-regulation of VEGF mRNA levels and consequent increase in DNA synthesis in ECs. These results demonstrated for the first time that olmesartan inhibited the AGEs signaling to angiogenesis by suppressing RAGE expression in ECs. Our present study suggests that blockade of the renin-angiotensin system by olmesartan may play a protective role against PDR by attenuating the deleterious effects of AGEs via down-regulation of RAGE.

Inhibition of NADPH oxidase prevents AGE mediated damage in diabetic nephropathy through a protein kinase C-alpha dependent pathway.

Vicki Thallas-Bonke — 2007-10-27T03:43:57-00:00

Diabetes (24 October 2007)

Objective: Excessive production of reactive oxygen species (ROS) via NADPH oxidase has been implicated in the pathogenesis of diabetic nephropathy. Since NADPH oxidase activation is closely linked to other putative pathways, its interaction with changes in PKC and increased advanced glycation was examined. Research Design and Methods: Streptozotocin diabetic or non-diabetic Sprague Dawley rats were followed for 32 wks with groups randomised to no treatment or the NADPH oxidase assembly inhibitor apocynin (15 mg/kg/day: wk 16-32). Complementary in vitro studies were performed in which primary rat mesangial cells, in the presence and absence of AGE-BSA, were treated with either apocynin or the PKC-alpha inhibitor Ro-32-0432. Results: Apocynin attenuated diabetes-associated increases in albuminuria and glomerulosclerosis. Circulating, renal cytosolic and skin collagen-associated AGE levels in diabetic rats were not reduced by apocynin. Diabetes-induced translocation of PKC, specifically PKC-alpha to renal membranes, was associated with increased NADPH dependent superoxide production and elevated renal, serum and urinary VEGF concentrations. In both diabetic rodents and in AGE-treated mesangial cells, blockade of NADPH oxidase or PKC-alpha attenuated cytosolic superoxide, PKC activation and increased VEGF. Finally renal extracellular matrix accumulation of fibronectin and collagen IV was decreased by apocynin. Conclusions: In the context of these and previous findings by our group we conclude that activation of NADPH oxidase via phosphorylation of PKC-alpha is downstream of the AGE-RAGE interaction in diabetic renal disease and may provide a novel therapeutic target for diabetic nephropathy.

AGEs activate mesangial TGF-beta-Smad signaling via an angiotensin II type I receptor interaction.

K Fukami — 2007-07-25T01:11:50-00:00

Kidney Int, Vol. 66, No. 6. (December 2004), pp. 2137-2147.

BACKGROUND: The renin-angiotensin system (RAS) and the accumulation of advanced glycation end products (AGEs) have been implicated in the pathogenesis of diabetic nephropathy. Whether there is a functional interaction between the RAS and AGEs in diabetic nephropathy is not known. In this study, we investigated whether AGEs could activate autocrine angiotensin II (Ang II) signaling and subsequently induce transforming growth factor-beta (TGF-beta)-Smad signaling in cultured rat mesangial cells. METHODS: The intracellular formation of reactive oxygen species (ROS) was detected using the fluorescent probe CM-H2DCFDA. Ang II was measured by radioimmunoassay. TGF-beta released into media was quantitatively analyzed in an enzyme-linked immunosorbent assay (ELISA). Smad2, p27(Kip1) (p27), fibronectin, and receptor for AGEs (RAGE) protein expression were determined by Western blot analysis. TGF-beta-inducible promoter activity was analyzed by a luciferase assay. DNA synthesis was evaluated by 5-bomo-2'-deoxyuridine (BrdU) incorporation and de novo protein synthesis was determined by [3H]leucine incorporation. RESULTS: AGEs increased intracellular ROS generation in mesangial cells, and this effect was significantly inhibited by an antiserum against RAGE. AGEs also were found to stimulate Ang II production in a time- and dose-dependent manner, which was completely prevented by an antioxidant, N-acetylcysteine (NAC). AGE-induced TGF-beta overproduction was completely blocked by candesartan, an Ang II type 1 receptor (AT1R) antagonist. Both candesartan and neutralizing antibody against TGF-beta completely prevented AGEs-induced Smad2 phosphorylation and TGF-beta-inducible promoter activity. Furthermore, AGEs were found to inhibit DNA synthesis and to stimulate de novo protein synthesis and fibronectin production in association with up-regulation of p27. All of these phenomena were completely prevented by candesartan or a polyclonal antibody against TGF-beta. CONCLUSION: The present study suggests that AGE-RAGE-mediated ROS generation activates TGF-beta-Smad signaling and subsequently induces mesangial cell hypertrophy and fibronectin synthesis by autocrine production of Ang II. This pathway may provide an important link between metabolic and haemodynamic factors in promoting the development and progression of diabetic nephropathy.

Plasminogen activator inhibitor-1 production is pathogenetic in experimental murine diabetic renal disease

Lassila — 2007-07-02T16:51:36-00:00

Diabetologia, Vol. 50, No. 6. (June 2007), pp. 1315-1326.