Cullin-3/KCTD10 complex is essential for K27-polyubiquitination of EIF3D in human hepatocellular carcinoma HepG2 cells.

Eukaryotic translation initiation factor 3 subunit D (EIF3D) binds to the 5'-cap of specific mRNAs, initiating their translation into polypeptides. From a pathological standpoint, EIF3D has been observed to be essential for cell growth in various cancer types, and cancer patients with high EIF3D mRNA levels exhibit poor prognosis, indicating involvement of EIF3D in oncogenesis. In this study, we found, by mass spectrometry, that Cullin-3 (CUL3)/KCTD10 ubiquitin (Ub) ligase forms a complex with EIF3D. We also demonstrated that EIF3D is K27-polyubiquitinated at the lysine 153 and 275 residues in a KCTD10-dependent manner in human hepatocellular carcinoma HepG2 cells. Similar to other cancers, high expression of EIF3D significantly correlated with poor prognosis in hepatocellular carcinoma patients, and depletion of EIF3D drastically suppressed HepG2 cell proliferation. These results indicate that EIF3D is a novel substrate of CUL3/KCTD10 Ub ligase and suggest involvement of K27-polyubiquitinated EIF3D in the development of hepatocellular carcinoma.

Cullin-3 and its adaptor protein ANKFY1 determine the surface level of integrin ß1 in endothelial cells.

Angiogenesis, the formation of new blood vessels from the pre-existing vasculature, is related to numerous pathophysiological events. We previously reported that a RING ubiquitin ligase complex scaffold protein, cullin-3 (CUL3), and one of its adaptor proteins, BAZF, regulated angiogenesis in the mouse retina by suppressing Notch signaling. However, the degree of inhibition of angiogenesis was made greater by CUL3 depletion than by BAZF depletion, suggesting other roles of CUL3 in angiogenesis besides the regulation of Notch signaling. In the present study, we found that CUL3 was critical for the cell surface level of integrin ß1, an essential cell adhesion molecule for angiogenesis in HUVECs. By siRNA screening of 175 BTBPs, a family of adaptor proteins for CUL3, we found that ANKFY1/Rabankyrin-5, an early endosomal BTBP, was also critical for localization of surface integrin ß1 and angiogenesis. CUL3 interacted with ANKFY1 and was required for the early endosomal localization of ANKFY1. These data suggest that CUL3/ANKFY1 regulates endosomal membrane traffic of integrin ß1. Our results highlight the multiple roles of CUL3 in angiogenesis, which are mediated through distinct CUL3-adaptor proteins.

Neddylated Cullin 3 is required for vascular endothelial-cadherin-mediated endothelial barrier function.

Vascular endothelial (VE)-cadherin, a major endothelial adhesion molecule, regulates vascular permeability, and increased vascular permeability has been observed in several cancers. The aim of this study was to elucidate the role of the NEDD8-Cullin E3 ligase, in maintaining barrier permeability. To this end, we investigated the effects of the inhibition of Cullin E3 ligases, by using inhibitors and knockdown techniques in HUVECs. Furthermore, we analyzed the mRNA and protein levels of the ligases by quantitative RT-PCR and Western blotting, respectively. The results revealed that NEDD8-conjugated Cullin 3 is required for VE-cadherin-mediated endothelial barrier functions. Treatment of HUVECs with MLN4924, a chemical inhibitor of the NEDD8-activating enzyme, led to high vascular permeability due to impaired cell-cell contact. Similar results were obtained when HUVECs were treated with siRNA directed against Cullin 3, one of the target substrates of NEDD8. Immunocytochemical staining showed that both treatments equally depleted VE-cadherin protein localized at the cell-cell borders. However, quantitative RT-PCR showed that there was no significant difference in the VE-cadherin mRNA levels between the treatment and control groups. In addition, cycloheximide chase assay revealed that the half-life of VE-cadherin protein was dramatically reduced by Cullin 3 depletion. Together, these findings suggest that neddylated Cullin 3 plays a crucial role in endothelial cell barrier function by regulating VE-cadherin.

Prognostic value of the ubiquitin ligase carboxyl terminus of the Hsc70-interacting protein in postmenopausal breast cancer.

The carboxyl terminus of the Hsc70-interacting protein (CHIP) is considered to induce the ubiquitination and degradation of several oncogenic proteins, and play a role in the inhibition of tumor progression and invasion under experimental conditions. However, the impact of CHIP expression on the prognosis of breast cancer patients has not yet been established. In this study, using an immunohistochemical method, 272 patients with invasive breast cancer were assessed for the expression of CHIP (graded scores 0-3) and the statuses of biomarkers, such as estrogen receptor (ER), progesterone receptor (PgR), and HER2. The relationships between the statuses of CHIP and biomarkers as well as clinical features were also evaluated, and that between the expression of CHIP and patient prognosis was analyzed. We revealed that the strong expression of CHIP correlated with positive ER (P < 0.001), positive PgR (P < 0.001), and negative HER2 (P = 0.02). In postmenopausal patients, relapse-free survival (RFS) was significantly better in the high CHIP group than in the low CHIP group (P = 0.042). In addition, RFS and cancer-specific survival (CSS) were significantly better in patients with ER-positive/CHIP score 3 tumors than in those with ER-negative/CHIP score 0 tumors (RFS: P = 0.038, CSS: P = 0.0098). The methylation status of CHIP gene promoter did not always account for the down-regulation of its expression. In conclusion, the overexpression of CHIP is a potent prognostic factor of a good prognosis in ER-positive breast cancer patients in the postmenopausal phase.

2-(4-Hydroxy-3-methoxyphenyl)-benzothiazole suppresses tumor progression and metastatic potential of breast cancer cells by inducing ubiquitin ligase CHIP.

Breast cancer is the most common malignancy among women and has poor survival and high recurrence rates for aggressive metastatic disease. Notably, triple-negative breast cancer (TNBC) is a highly aggressive cancer and there is no preferred agent for TNBC therapy. In this study, we show that a novel agent, 2-(4-hydroxy-3-methoxyphenyl)-benzothiazole (YL-109), has ability to inhibit breast cancer cell growth and invasiveness in vitro and in vivo. In addition, YL-109 repressed the sphere-forming ability and the expression of stem cell markers in MDA-MB-231 mammosphere cultures. YL-109 increased the expression of carboxyl terminus of Hsp70-interacting protein (CHIP), which suppresses tumorigenic and metastatic potential of breast cancer cells by inhibiting the oncogenic pathway. YL-109 induced CHIP transcription because of the recruitment of the aryl hydrocarbon receptor (AhR) to upstream of CHIP gene in MDA-MB-231 cells. Consistently, the antitumor effects of YL-109 were depressed by CHIP or AhR knockdown in MDA-MB-231 cells. Taken together, our findings indicate that a novel agent YL-109 inhibits cell growth and metastatic potential by inducing CHIP expression through AhR signaling and reduces cancer stem cell properties in MDA-MB-231 cells. It suggests that YL-109 is a potential candidate for breast cancer therapy.

Identification of a Novel Compound That Suppresses Breast Cancer Invasiveness by Inhibiting Transforming Growth Factor-ß Signaling via Estrogen Receptor α.

Breast cancer is the most frequently diagnosed cancer and the leading cause of death by cancer among females worldwide. An overwhelming majority of these deaths is because of metastasis. Estrogen stimulates and promotes growth of breast tumors, whereas transforming growth factor-beta (TGF-ß) signaling promotes invasion and metastasis. We previously reported that estrogen and estrogen receptor alpha (ERα) suppressed breast cancer metastasis by inhibiting TGF-ß signaling, whereas antiestrogens that suppress breast cancer growth, such as the selective ER modulator tamoxifen (TAM) or the pure antiestrogen fulvestrant (ICI 182,780), cannot suppress TGF-ß signaling or breast cancer invasiveness. Therefore, we predicted that a compound that inhibits TGF-ß signaling but does not facilitate ERα signaling would be ideal for suppressing breast cancer invasiveness and growth. In the present study, we identified an ideal candidate compound, N-23. Like estrogen, N-23 strongly decreased expression of TGF-ß/Smad target gene plasminogen activator inhibitor-1 (PAI-1), but it did not increase the expression of ERα target gene pS2. While estrogen decreased the levels of phosphorylated Smad2 and Smad3, N-23 had no effect. In addition, TGF-ß-dependent recruitment of Smad3 to the PAI-1 gene promoter was inhibited in the presence of estrogen or N-23. We also investigated the effects of N-23 on proliferation, migration, and invasion of breast cancer cells. In contrast to estrogen, N-23 inhibited the cellular proliferation of breast cancer cells. Moreover, we showed that N-23 suppressed the migration and invasion of breast cancer cells to the same extent as by estrogen. Taken together, our findings indicate that N-23 may be a candidate compound that is effective in inhibiting breast cancer progression.

Hepatic rRNA transcription regulates high-fat-diet-induced obesity.

Ribosome biosynthesis is a major intracellular energy-consuming process. We previously identified a nucleolar factor, nucleomethylin (NML), which regulates intracellular energy consumption by limiting rRNA transcription. Here, we show that, in livers of obese mice, the recruitment of NML to rRNA gene loci is increased to repress rRNA transcription. To clarify the relationship between obesity and rRNA transcription, we generated NML-null (NML-KO) mice. NML-KO mice show elevated rRNA level, reduced ATP concentration, and reduced lipid accumulation in the liver. Furthermore, in high-fat-diet (HFD)-fed NML-KO mice, hepatic rRNA levels are not decreased. Both weight gain and fat accumulation in HFD-fed NML-KO mice are significantly lower than those in HFD-fed wild-type mice. These findings indicate that rRNA transcriptional activation promotes hepatic energy consumption, which alters hepatic lipid metabolism. Namely, hepatic rRNA transcriptional repression by HFD feeding is essential for energy storage.

Estrogen and antiestrogens alter breast cancer invasiveness by modulating the transforming growth factor-ß signaling pathway.

In the later stages of breast cancer, estrogen receptor (ER)α-negative cancers typically have higher histological grades than ERα-positive cancers, and transforming growth factor (TGF)-ß promotes invasion and metastasis. Our previous study indicated that ERα inhibited TGF-ß signaling by inducing the degradation of Smad in an estrogen-dependent manner. In the present study, we report that the suppressive effects of ERα and estrogen on tumor progression are mediated by inhibiting TGF-ß signaling. Furthermore, we investigated the effects of antiestrogens such as ICI182,780 (ICI) or tamoxifen (TAM) on TGF-ß signaling and breast cancer invasiveness. The levels of total Smad and pSmad were reduced by estrogen, whereas ICI slightly increased them, and TAM had no effect. To investigate the effect of antiestrogens on breast cancer invasiveness, we generated highly migratory and invasive MCF-7-M5 cells. The migration and invasion of these cells were suppressed by the inhibitor of TGF-ß receptor kinase, SB-505124, and estrogen. However, antiestrogens did not suppress the migration and invasion of these cells. In addition, we screened TGF-ß target genes whose expression was reduced by estrogen treatment and identified four genes associated with breast cancer invasiveness and poor prognosis. The expression of these genes was not decreased by antiestrogens. These observations provide a new insight into estrogen function and the mechanisms underlying estrogen-mediated suppression of tumor progression.

Na,K-ATPase signal transduction triggers CREB activation and dendritic growth.

Dendritic growth is pivotal in the neurogenesis of cortical neurons. The sodium pump, or Na,K-ATPase, is an evolutionarily conserved protein that, in addition to its central role in establishing the electrochemical gradient, has recently been reported to function as a receptor and signaling mediator. Although a large body of evidence points toward a dual function for the Na,K-ATPase, few biological implications of this signaling pathway have been described. Here we report that Na,K-ATPase signal transduction triggers dendritic growth as well as a transcriptional program dependent on cAMP response element binding protein (CREB) and cAMP response element (CRE)-mediated gene expression, primarily regulated via Ca(2+)/calmodulin-dependent protein (CaM) kinases. The signaling cascade mediating dendritic arbor growth also involves intracellular Ca(2+) oscillations and sustained phosphorylation of mitogen-activated protein (MAP) kinases. Thus, our results suggest a novel role for the Na,K-ATPase as a modulator of dendritic growth in developing neurons.

Angiotensin II stimulates endothelial NO synthase phosphorylation in thoracic aorta of mice with abdominal aortic banding via type 2 receptor.

Abdominal aortic banding in mice induces upregulation of angiotensin II (Ang II) type 2 (AT2) receptors in the pressure-overloaded thoracic aorta. To clarify mechanisms underlying the vascular AT2 receptor-dependent NO production, we measured aortic levels of endothelial NO synthase (eNOS), eNOS phosphorylated at Ser633 and Ser1177, protein kinase B (Akt), and Akt phosphorylated at Ser473 in thoracic aortas of mice after banding. Total eNOS, both forms of phosphorylated eNOS, Akt, and phosphorylated Akt levels, as well as cGMP contents, were significantly increased 4 days after banding. The administration of PD123319 (an AT2 receptor antagonist) or icatibant (a bradykinin B2 receptor antagonist) abolished the banding-induced upregulation of both forms of phosphorylated eNOS, as well as elevation of cGMP, but did not affect the upregulation of eNOS, Akt, and phosphorylated Akt. In the in vitro experiments using aortic rings prepared from banded mice, Ang II produced significant increases in both forms of phosphorylated eNOS, as well as cGMP, and these effects were blocked by PD123319 and icatibant. Ang II-induced eNOS phosphorylation and cGMP elevation in aortic rings were inhibited by protein kinase A (PKA) inhibitors H89 and KT5720 but not by phosphatidylinositol 3-kinase inhibitors wortmannin and LY24002. The contractile response to Ang II was attenuated in aortic rings from banded mice via AT2 receptor, and this attenuation was blocked by PKA inhibitors. These results suggest that the activation of AT2 receptor by Ang II induces phosphorylation of eNOS at Ser633 and Ser1177 via a PKA-mediated signaling pathway, resulting in sustained activation of eNOS.

The lipopolysaccharide-induced up-regulation of bradykinin B2-receptor in the mouse heart is mediated by tumor necrosis factor-alpha and angiotensin II.

Our present study aimed to characterize the effects of lipopolysaccharide (LPS) on the expression of the bradykinin B2-receptor in the mouse heart, which may have a role in cardiac depression during sepsis. We found that LPS induced the up-regulation of B2-receptor mRNA in the heart in vivo and in cultured cardiac myocytes in vitro. Like LPS, tumor necrosis factor-alpha (TNF-alpha) but not interleukin (IL)-1-beta, IL-6 or endothelin-1 stimulated B2-receptor expression in cultured myocytes. The effect of LPS on the expression of B2-receptor mRNA was also mimicked in cardiac myocytes by Ang II via Ang II type 1 (AT1-) receptor. Losartan, an AT1-receptor antagonist, inhibited about 50% of the LPS-induced up-regulation of B2-receptor mRNA in the heart in vivo and in cultured cardiac myocytes in vitro. Furthermore, the up-regulation of B2-receptor mRNA by either LPS or Ang II in cultured myocytes was abolished by anti-TNF-alpha antibody. These results suggest that the up-regulation of cardiac B2-receptor expression by LPS is mediated through TNF-alpha, which is produced in the myocardium by two different mechanisms in an AT1-receptor-dependent and independent manners, implying the role of the cardiac kallikrein-kinin system in the development of cardiac dysfunction during sepsis.

Angiotensin type 2 receptor-mediated phosphorylation of eNOS in the aortas of mice with 2-kidney, 1-clip hypertension.

To evaluate the role of vascular angiotensin II (Ang II) type 2 (AT2) receptor in renovascular hypertension, we investigated expressions of AT2 receptor and endothelial nitric oxide synthase (eNOS) in thoracic aortas of mice with 2-kidney, 1-clip (2K1C) hypertension. The mRNA levels of AT2 receptor in aortas, but not those of AT1 and bradykinin B2 receptors, increased 14 days but not 42 days after clipping. The contractile response to Ang II (>0.1 micromol/L) was attenuated in aortic rings excised 14 days after clipping and was restored to that of rings from sham mice by antagonists of AT2 receptor (PD123319) and B2 receptor (icatibant). The aortic levels of total eNOS, phosphorylated eNOS at Ser1177 (p-eNOS), total Akt, and phosphorylated Akt at Ser473 (p-Akt) were increased in 2K1C mice on day 14, whereas only eNOS levels were increased on day 42. The aortic cGMP levels were 20-fold greater in 2K1C mice on day 14 compared with sham mice. Administration of nicardipine for 4 days before the excision of aortas 14 days after clipping not only reduced blood pressure but also decreased the aortic levels of eNOS, p-eNOS, Akt, p-Akt, and cGMP to sham levels, whereas the administration of PD123319 or icatibant to 2K1C mice decreased p-eNOS and cGMP to sham levels without affecting blood pressure and the levels of eNOS, Akt and p-Akt. These results suggest that vascular NO production is enhanced by increased eNOS phosphorylation via the activation of AT2 receptors in the course of 2K1C hypertension.

Stimulation of cyclic GMP production via AT2 and B2 receptors in the pressure-overloaded aorta after banding.

Abdominal aortic banding induces upregulation of the angiotensin II (Ang II) type-2 (AT2) receptor, thereby decreasing the contractile response to Ang II in the thoracic aorta of the rat. The aim of this study was to use a mouse model to clarify the mechanisms by which the banding elicits upregulation of the aortic AT2 receptor and the subsequent attenuation of Ang II responsiveness. Concomitantly with the elevation in blood pressure and plasma renin concentration after banding, AT2-receptor mRNA levels in the thoracic aorta rapidly increased in mice within 4 days. Upregulation of the AT2 receptor, as well as blood pressure elevation after banding, was abolished by losartan administration. The contractile response to Ang II was depressed in aortic rings of banding mice but not of sham mice, and was restored by either the AT2-receptor antagonist PD123319 or the bradykinin B2-receptor antagonist icatibant. cGMP content in the thoracic aorta of banding mice was 9-fold greater than that of sham mice, and the elevation was reduced to sham levels 1 hour after intravenous injection of PD123319 or icatibant. When aortic rings were incubated with Ang II, cGMP content increased in banding rings but not in sham rings; the pretreatment with PD123319 or icatibant inhibited Ang II-induced cGMP production. These results suggest that aortic banding induces upregulation of the AT2 receptor through increased circulating Ang II via the AT1 receptor, thereby activating a vasodilatory pathway in vessels through the AT2 receptor via the kinin/cGMP system.

Up-regulation of angiotensin II type 2 receptor in rat thoracic aorta by pressure-overload.

We have examined whether expression of angiotensin II (Ang II) type 1 (AT(1)) and/or type 2 (AT(2)) receptors are changed in thoracic aorta under pressure-overload by abdominal aortic banding in rats and determined whether their changes are accompanied by alteration in contractile response of thoracic aorta to Ang II. AT(2) receptor mRNA levels determined by reverse transcription-polymerase chain reaction or quantitative real-time polymerase chain reaction were increased by about 300% in aortas 4, 7, 14, and 28 days after banding without changes in AT(1) receptor mRNA levels. Contractile response of aortic rings to Ang II was decreased in thoracic aortas 7 days after banding, and AT(2) receptor antagonist PD123319 (1-[[4-(dimethulamino)-3-methylphenyl]methyl]-5-(diphenylacetyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-6-carboxylic acid ditrifluoroacetate) (10(-6) M) increased the Ang II responsiveness in pressure-loaded but not in sham rings. After removal of the endothelium or treatment with N(G)-nitro-L-arginine methyl ester (L-NAME), no differences were observed in Ang II responsiveness between sham and pressure-loaded rings. Either losartan (1 mg/kg/day i.p.) or candesartan (2 mg/kg/day p.o.) for 7 days after banding not only abolished the up-regulation of AT(2) receptor mRNA in aortas but also recovered their Ang II responsiveness. Basal cGMP levels were 2 times higher in pressure-loaded than in sham rings; both levels were not affected by Ang II (10(-7) M; 5 min), but greatly decreased by L-NAME (10(-4) M, 30 min). These results suggest that pressure-overload induces the up-regulation of AT(2) receptor expression in aortas via AT(1) receptor and thereby negatively modulates the vasoconstrictor sensitivity to Ang II, probably mediated by the mechanisms independent of the nitric oxide-cGMP system.