Loss of HACE1 promotes colorectal cancer cell migration via upregulation of YAP1

Colorectal cancer (CRC) is the third-leading cause of cancer mortality worldwide. HACE1 function as a tumor-suppressor gene and is downregulated in several kinds of cancers. However, the distribution and clinical significance of HACE1 in CRC is still not clarified. In this study, we found that the HACE1 expression is greatly downregulated in CRC tissues and cell lines. Moreover, the HACE1 expression was significantly associated with inhibition of CRC cell proliferation, metastasis, and invasion. HACE1 inhibited epithelial–mesenchymal transition in CRC cells. Furthermore, we found that HACE1 altered the protein expression of the Hippo pathway by downregulation of YAP1. HACE1 suppresses the invasive ability of CRC cells by negatively regulating the YAP1 pathway. Our data indicates that HACE1 directly targets YAP1 and induces downregulation of YAP1, thereby increasing the activity of the Hippo pathway. In summary, these findings demonstrated that HACE1–YAP1 axis had an important part in the CRC development and progression.     

The E3 ligase HACE1 is a critical chromosome 6q21 tumor suppressor involved in multiple cancers

Transformation and cancer growth are regulated by the coordinate actions of oncogenes and tumor suppressors. Here, we show that the novel E3 ubiquitin ligase HACE1 is frequently downregulated in human tumors and maps to a region of chromosome 6q21 implicated in multiple human cancers. Genetic inactivation of HACE1 in mice results in the development of spontaneous, late-onset cancer. A second hit from either environmental triggers or genetic heterozygosity of another tumor suppressor, p53, markedly increased tumor incidence in a Hace1-deficient background. Re-expression of HACE1 in human tumor cells directly abrogates in vitro and in vivo tumor growth, whereas downregulation of HACE1 via siRNA allows non-tumorigenic human cells to form tumors in vivo. Mechanistically, the tumor-suppressor function of HACE1 is dependent on its E3 ligase activity and HACE1 controls adhesion-dependent growth and cell cycle progression during cell stress through degradation of cyclin D1. Thus, HACE1 is a candidate chromosome 6q21 tumor-suppressor gene involved in multiple cancers.     

The E3 ubiquitin-ligase HACE1 catalyzes the ubiquitylation of active Rac1

Rac1 small GTPase controls essential aspects of cell biology and is a direct target of numerous bacterial virulence factors. The CNF1 toxin of pathogenic Escherichia coli addresses Rac1 to ubiquitin-proteasome system (UPS). We report the essential role of the tumor suppressor HACE1, a HECT-domain containing E3 ubiquitin-ligase, in the targeting of Rac1 to UPS. HACE1 binds preferentially GTP-bound Rac1 and catalyzes its polyubiquitylation. HACE1 expression increases the ubiquitylation of Rac1, when the GTPase is activated by point mutations or by the GEF-domain of Dbl. RNAi-mediated depletion of HACE1 blocks the ubiquitylation of active Rac1 and increases GTP-bound Rac1 cellular levels. HACE1 antagonizes cell isotropic spreading, a hallmark of Rac1 activation, and is required for endothelial cell monolayer invasion by bacteria. Together, these data establish the role of the HACE1 E3 ubiquitin-ligase in controlling Rac1 ubiquitylation and activity.     

Gsh-1, an orphan Hox gene, is required for normal pituitary development.

The anterior pituitary regulates the function of multiple organ systems as well as body growth, and in turn is controlled by peptides released by the hypothalamus. We find that mutation of the Gsh-1 homeobox gene results in pleiotropic effects on pituitary development and function. Homozygous mutants exhibit extreme dwarfism, sexual infantilism and significant perinatal mortality. The mutant pituitary is small in size and hypocellular, with severely reduced numbers of growth hormone- and prolactin-producing cells. Moreover, the pituitary content of a subset of pituitary hormones, including growth hormone, prolactin and luteinizing hormone, is significantly decreased. The hypothalamus, although morphologically normal, is also perturbed in mutants. The gsh-1 gene is shown to be essential for growth hormone-releasing hormone (GHRH) gene expression in the arcuate nucleus of the hypothalamus. Further, sequence and electrophoretic mobility shift data suggest the Gsh-1 and GHRH genes as potential targets regulated by the Gsh-1-encoded protein. The mutant phenotype indicates a critical role for Gsh-1 in the genetic hierarchy of the formation and function of the hypothalamic-pituitary axis.     

Expression of retinaldehyde dehydrogenase 1 in the anterior pituitary glands of adult rats

Retinoic acid (RA) plays a critical role in cell growth and tissue development and is also a regulatory factor of pituitary function. However, whether RA is generated in the pituitary gland and plays a role as a paracrine and/or autocrine factor is generally unknown. RA is synthesized from retinoids through oxidation processes. Dehydrogenases that catalyze the oxidation of retinal to RA are members of the retinaldehyde dehydrogenase (RALDH) family. Recently, we demonstrated that RALDH2 and RALDH3, but not RALDH1, were expressed in the developing anterior pituitary gland of rats, but the expression of RALDHs in the adult pituitary gland was not determined. Therefore, we have now examined the expression of RALDH1, RALDH2, and RALDH3 mRNAs in the pituitary gland of adult rats. Analysis by quantitative real-time polymerase chain reaction of adult pituitary glands has revealed a high level of RALDH1 mRNA but not of RALDH2 mRNA or RALDH3 mRNA. We have also detected mRNA expression for RALDH1 in the anterior pituitary gland by in situ hybridization with digoxigenin-labeled cRNA probes. Double-staining for RALDH1 mRNA and pituitary hormones or S-100 protein, a marker of folliculo-stellate cells (FS-cells), has revealed RALDH1 mRNA expression in a portion of prolactin-producing cells, marginal layer cells, and FS-cells. Our results suggest that RA is generated in the adult anterior pituitary gland, and that it may act locally on pituitary cells. This work was supported by a Grant-in-Aid for Young Scientists (B) from the Japanese Ministry of Education, Culture, Sports, Science, and Technology (18790149) and by the Foundation of Growth Science.     

Interleukin-4 is a potent mitogen for capillary endothelium

Interleukin-4 (IL-4) is a mitogen for both microvascular (human adrenal capillary, HACE) and large vessel (human umbilical vein, HUVEC) endothelial cells. Comparison of growth promotion by IL-4 to that by the potent endothelial mitogen fibroblast growth factor (FGF) showed the activity of IL-4 on HACE cells to be strong (50% of that with FGF) but on HUVEC's weak (12% of that with FGF). Growth stimulation was characterised by both 3H-thymidine incorporation and by cell number, and was maximal at 1 nM IL-4. The presence of IL-4 receptors on HACE cells and HUVEC's was confirmed by specific binding of radioiodinated IL-4. Scatchard analysis confirmed a single high affinity binding receptor on both HACE cells (Kd = 80 pM, 358 receptors/cell) and HUVEC's (Kd = 88 pM, 2,580 receptors/cell). Potent activity on capillary as opposed to large vessel endothelium places IL-4 in a unique position amongst endothelial mitogens.     

Isolation and properties in culture of human adrenal capillary endothelial cells

The isolation of human adrenal capillary endothelial (HACE) cells without resort to fluorescence activated cell sorting is described, together with their properties in culture. HACE cells were isolated by plating collagenase digests at high dilution in the presence of endothelial cell growth supplement, followed by clonal selection of endothelial colonies. HACE cells exhibit a typical endothelial 'cobblestone' morphology at confluence and formed 'tubes' when seeded onto 'Matrigel'. They are positive for human MHC1, and the endothelial markers ENDOCAM (CD31) and weakly CD34, they also take up dil-acetyl low density lipoprotein but are negative for Factor VIII. Their growth is strongly stimulated by FGF and inhibited by TGF-beta I. Like their much studied bovine counterparts they are robust in culture, retaining the properties described up to senescence. HACE cells provide a readily available alternative to human umbilical vein endothelial cells in that they are easily isolated pure and in quantity. They should be particularly useful in studies where human capillary, as opposed to large vessel endothelium, is required.     

新的生长激素释放激素类似肽Pro-hGHRH(1-44)-Gly-Gly-Cys的重组和比较活性*

目的:为了寻找高活性和长半衰期生的GHRH类似肽。方法:通过使用独特的酸敏感水解位点Asp-Pro的原核表达系统,构建了新的Pro-hGHRH(1-44)-Gly-Gly-Cys类似肽。通过重组细菌裂解、包含体洗涤、乙醇分级沉淀、酸水解、SP-Sephadex C-25和Sephadex G-10柱层析等技术,纯化了高纯度的Pro-hGHRH(1-44)-Gly-Gly-Cys肽。通过使用SDS-PAGE、离子化质谱、雌大鼠垂体和人流产胎儿垂体,测定了多肽的纯度、分子量、生长激素释放活性。结果:Pro-hGHRH(1-44)-Gly-Gly-Cys肽分子量5373Da与实际值吻合,0.1~10 μg/ml的肽剂量不论是对人垂体还是大鼠垂体都增加了垂体生长激素的释放,大鼠垂体生长激素的释放具有剂量依赖性。与标准的hGHRH(1-40)肽比较,新的类似肽有较高的GH释放活性。结果也显示了,Pro-GHRH(1-44)-Gly-Gly-Cys与Pro-hGHRH(1-44)肽的GH释放活性无统计学差异。结论：新的类似肽有较好的生长激素释放活性、功能选择性和种属特异性。     

高原脑水肿患者血浆的蛋白质组学研究

目的：高原脑水肿是急性高原病的重症表现之一,其发病机制尚不完全清楚。本实验目的是应用蛋白质组学方法研究高原脑水肿发病时血浆蛋白质表达的变化,为研究高原脑水肿发病分子机制以及高原脑水肿的预防、诊断及治疗提供重要的分子标志物。方法：应用二维凝胶电泳（2D）结合质谱的方法比较了一例高原脑水肿（HACE）患者与高原肺水肿（HAPE）和轻型急性高原反应（mAMS）患者血浆蛋白表达的差异,对差异蛋白进行鉴定,最后用ELISA方法检测载脂蛋白E含量。结果：我们检测到HACE与HAPE患者血浆比较有6个差异蛋白质点,HACE与mAMS患者血浆比较也有6个差异蛋白质点,质谱鉴定结果显示两组比较中都有载脂蛋白E含量变化,ELISA检测结果与二维凝胶电泳结果一致。结论：本实验首次将蛋白质组学技术应用到高原脑水肿研究中,并且发现了可能与高原脑水肿发病密切相关的载脂蛋白E,该蛋白对于研究高原脑水肿发病分子机制以及疾病的预防、诊断及治疗的意义有待进一步研究。     

The novel pituitary polypeptide 7B2 is a highly-conserved protein coexpressed with proopiomelanocortin

In the amphibian intermediate pituitary gland the biosynthetic activity for production of the precursor protein proopiomelanocortin (POMC) can be physiologically manipulated; POMC synthesis is high in animals adapted to a black background and low in white-adapted animals. In order to study genes associated with POMC gene expression we applied a differential hybridization technique involving screening of a pituitary cDNA library with probes derived from RNA of inactive and physiologically activated intermediate pituitary cells of the amphibian Xenopus laevis. A differentially hybridizing Xenopus pituitary cDNA clone encoded the novel polypeptide 7B2. This Mr-21,000 secretory granule-associated protein of unknown function is shown to be highly conserved between Xenopus and human (83% amino acid sequence similarity). Conserved segments within the 7B2 structure included the N-terminal portion, three pairs of basic amino acids which are potential recognition sites for proteolytic enzymes, and three regions sharing similarity with putative GTP-binding domains. Levels of 7B2 mRNA were about 3% of POMC mRNA levels in Xenopus pituitary glands. In the intermediate pituitary the amount of both POMC and 7B2 mRNA was much higher in black-adapted toads than in white-adapted animals. These physiologically-induced changes in POMC and 7B2 mRNA levels were not found in the anterior pituitary. We conclude that the POMC and 7B2 genes are coexpressed and that modulation of the activity of these genes is tissue-specific.     

PDK1 deficiency in POMC-expressing cells reveals FOXO1-dependent and -independent pathways in control of energy homeostasis and stress response

Insulin- and leptin-stimulated phosphatidylinositol-3 kinase (PI3K) activation has been demonstrated to play a critical role in central control of energy homeostasis. To delineate the importance of pathways downstream of PI3K specifically in pro-opiomelanocortin (POMC) cell regulation, we have generated mice with selective inactivation of 3-phosphoinositide-dependent protein kinase 1 (PDK1) in POMC-expressing cells (PDK1(DeltaPOMC) mice). PDK1(DeltaPOMC) mice initially display hyperphagia, increased body weight, and impaired glucose metabolism caused by reduced hypothalamic POMC expression. On the other hand, PDK1(DeltaPOMC) mice exhibit progressive, severe hypocortisolism caused by loss of POMC-expressing corticotrophs in the pituitary. Expression of a dominant-negative mutant of FOXO1 specifically in POMC cells is sufficient to ameliorate positive energy balance in PDK1(DeltaPOMC) mice but cannot restore regular pituitary function. These results reveal important but differential roles for PDK1 signaling in hypothalamic and pituitary POMC cells in the control of energy homeostasis and stress response.     

Cofactor CLIM2 promotes the repressive action of LIM homeodomain transcription factor Lhx2 in the expression of porcine pituitary glycoprotein hormone α subunit gene

We have cloned a porcine orthologue of cofactor CLIM2 (Ldb1/NLI) from the porcine pituitary cDNA library by protein–protein interaction with the Yeast Two-Hybrid System using porcine Lhx2 as a bait protein. Porcine CLIM2 shows a high identity (99%) in the dimerization domain, nuclear localization signal and LIM binding domain with those of man and mouse. The expression of CLIM2 gene in the anterior pituitary lobe was detected during the porcine fetal and postnatal period by RT-PCR analysis, suggesting that this protein is constitutively expressing and plays a basic role in the anterior pituitary. Transfection assay to the pituitary tumor derived LβT2 cells, and the Chinese hamster ovary cells demonstrated that CLIM2 acts as a corepressor of the porcine Lhx2 function. Interestingly, CLIM2 alone apparently repressed the high level of αGSU gene expression in LβT2 cells. These data suggest that CLIM2 is a basic factor in the pituitary development and function, and plays the role of repressor to modify the function of Lhx2 on the αGSU gene expression.