Heparanase regulates esophageal keratinocyte differentiation through nuclear translocation and heparan sulfate cleavage

Heparanase is an endo-beta-glucuronidase that specifically cleaves heparan sulfate (HS) chains. Heparanase is involved in the process of metastasis and angiogenesis through the degradation of HS chains of the extracellular matrix and cell surface. Recently, we demonstrated that heparanase was localized in the cell nucleus of normal esophageal epithelium and esophageal cancer, and that its expression was correlated with cell differentiation. However, the nuclear function of heparanase remains unknown. To elucidate the role of heparanase in esophageal epithelial differentiation, primary human esophageal cells were grown in monolayer as well as organotypic cultures, and cell differentiation was induced. Expression of heparanase, HS, involucrin, and p27 was determined by immunostaining and Western blotting. SF4, a novel pharmacological inhibitor, was used to specifically inhibit heparanase activity. Upon esophageal cell differentiation, heparanase was translocated from the cytoplasm to the nucleus. Such translocation of heparanase appeared to be associated with the degradation of HS chains in the nucleus and changes in the expression of keratinocyte differentiation markers such as p27 and involucrin, whose induction was inhibited by SF4. Furthermore, these in vitro observations agreed with the expression pattern of heparanase, HS, involucrin, cytokeratin 13, and p27 in normal esophageal epithelium. Nuclear translocation of heparanase and its catalytic cleavage of HS may play a critical role in the differentiation of esophageal epithelial cells. Our study provides a novel insight into the role of heparanase in an essential differentiation process.     

Heparanase expression and localization in different types of human lung cancer

Background

Heparanase is the only known mammalian glycosidase capable of cleaving heparan sulfate chains. The expression of this enzyme has been associated with tumor development because of its ability to degrade extracellular matrix and promote cell invasion.

Methods

We analyzed heparanase expression in lung cancer samples to understand lung tumor progression and malignancy. Of the samples from 37 patients, there were 14 adenocarcinomas, 13 squamous cell carcinomas, 5 large cell carcinomas, and 5 small cell carcinomas. Immunohistochemistry was performed to ascertain the expression and localization of heparanase.

Results

All of the tumor types expressed heparanase, which was predominantly localized within the cytoplasm and nucleus. Significant enzyme expression was also observed in cells within the tumor microenvironment, such as fibroblasts, epithelial cells, and inflammatory cells. Adenocarcinomas exhibited the strongest heparanase staining intensity and the most widespread heparanase distribution. Squamous cell carcinomas, large cell carcinomas, and small cell carcinomas had a similar subcellular distribution of heparanase to adenocarcinomas but the distribution was less widespread. Heparanase expression tended to correlate with tumor node metastasis (TNM) staging in non-small cell lung carcinoma.

Conclusion

In this study, we showed that heparanase was localized to the cytoplasm and nucleus of tumor cells and to cells within the microenvironment in different types of lung cancer. This enzyme exhibited a differential distribution based on the type of lung tumor.General significanceElucidating the heparanase expression patterns in different types of lung cancer increased our understanding of the crucial role of heparanase in lung cancer biology. This article is part of a Special Issue entitled Matrix-mediated cell behaviour and properties.     

CD38 is constitutively expressed in the nucleus of human hematopoietic cells

CD38 is a type II glycoprotein that acts both as a bifunctional enzyme, responsible for the synthesis and hydrolysis of cyclic ADP-ribose, and as a signal-transducing surface receptor. Although CD38 was originally described as a plasma membrane molecule, several reports indicate that CD38 is expressed in the nucleus, even in cells known to be CD38 surface-negative. In this study, firstly we investigated the presence of nuclear CD38 by immunofluorescence and confocal microscopy using a panel of hematopoietic cell lines that exhibit different levels of CD38 plasma membrane expression. Our second aim was to explore the relationship between the nuclear and plasma membrane forms of CD38 in human cell lines which represent discrete early maturation stages of the human lymphoid and myeloid compartments. Our results indicate that CD38 is constitutively present in the nucleus of cells belonging to distinct lineages. Furthermore, nuclear CD38 appears to be independent of the plasma membrane pool. The presence of nuclear CD38 during different stages of hematopoietic differentiation suggests that it may play a role in the control of nuclear Ca(2+) homeostasis and NAD levels.     

Syndecan-1 and FGF-2, but Not FGF Receptor-1, Share a Common Transport Route and Co-Localize with Heparanase in the Nuclei of Mesenchymal Tumor Cells

Syndecan-1 forms complexes with growth factors and their cognate receptors in the cell membrane. We have previously reported a tubulin-mediated translocation of syndecan-1 to the nucleus. The transport route and functional significance of nuclear syndecan-1 is still incompletely understood. Here we investigate the sub-cellular distribution of syndecan-1, FGF-2, FGFR-1 and heparanase in malignant mesenchymal tumor cells, and explore the possibility of their coordinated translocation to the nucleus. To elucidate a structural requirement for this nuclear transport, we have transfected cells with a syndecan-1/EGFP construct or with a short truncated version containing only the tubulin binding RMKKK sequence. The sub-cellular distribution of the EGFP fusion proteins was monitored by fluorescence microscopy. Our data indicate that syndecan-1, FGF-2 and heparanase co-localize in the nucleus, whereas FGFR-1 is enriched mainly in the perinuclear area. Overexpression of syndecan-1 results in increased nuclear accumulation of FGF-2, demonstrating the functional importance of syndecan-1 for this nuclear transport. Interestingly, exogenously added FGF-2 does not follow the route taken by endogenous FGF-2. Furthermore, we prove that the RMKKK sequence of syndecan-1 is necessary and sufficient for nuclear translocation, acting as a nuclear localization signal, and the Arginine residue is vital for this localization. We conclude that syndecan-1 and FGF-2, but not FGFR-1 share a common transport route and co-localize with heparanase in the nucleus, and this transport is mediated by the RMKKK motif in syndecan-1. Our study opens a new perspective in the proteoglycan field and provides more evidence of nuclear interactions of syndecan-1.     

Histidine-rich glycoprotein binds heparanase and regulates its enzymatic activity and cell surface interactions

Heparanase, an endo-β-d-glucuronidase, is involved in numerous normal physiological and pathological processes, such as inflammation, wound healing and tumour metastasis/angiogenesis, through its ability to mediate the degradation of heparan sulfate, a key structural component of the extracellular matrix and on the surface of cells. Identifying endogenous molecules that can regulate heparanase activity will aid the understanding of its molecular function in health and disease and provide the potential for development of novel anti-cancer and anti-inflammatory therapeutics. The ability of the extracellular heparanase to tether onto cell surface heparan sulfate proteoglycans and other receptor(s), such as the cation-independent mannose-6-phosphate receptor, is key to its activation, function and uptake into intracellular compartments. Here we describe experiments demonstrating that a relatively abundant plasma glycoprotein, histidine-rich glycoprotein, directly interacts with platelet-derived heparanase and enhances its enzymatic activity. The findings in this study also show that histidine-rich glycoprotein interferes with heparanase binding to cell surface receptors, particularly heparan sulfate proteoglycans. Thus, the interaction between histidine-rich glycoprotein and heparanase can potentially regulate the role of heparanase in a variety of physiological and pathological conditions.     

The cell adhesion receptor carcinoembryonic antigen-related cell adhesion molecule 1 regulates nucleocytoplasmic trafficking of DNA polymerase delta-interacting protein 38

The homophilic cell-cell adhesion receptor CEACAM1 (carcinoembryonic antigen-related cell adhesion molecule 1, CD66a) acts as a regulator of contact-dependent cell survival, differentiation, and growth. It is involved in the control of proliferation in hematopoietic and epithelial cells and can act as a tumor suppressor. In this study, we identify DNA polymerase delta-interacting protein 38 (PDIP38) as a novel binding partner for CEACAM1-L and CEACAM1-S. We show that PDIP38 can occur in the nucleus, in the cytoplasm and at the plasma membrane in NBT-II, IEC18, RBE, and HeLa cells and that the distribution in NBT-II cells is influenced by the confluency of the cells. We also demonstrate that the interaction of CEACAM1 and PDIP38 is of functional importance in NBT-II cells, which co-express the long and the short CEACAM1 isoform. In subconfluent, proliferating NBT-II cells, perturbation of CEACAM1 by antibody clustering induces increased binding to PDIP38 and results in rapid recruitment of PDIP38 to the plasma membrane. The same treatment of confluent, quiescent NBT-II cells leads to a different response, i.e. translocation of PDIP38 to the nucleus. Together, our data show that PDIP38 can shuttle between the cytoplasmic and the nuclear compartments and that its subcellular localization is regulated by CEACAM1, implicating that PDIP38 may constitute a novel downstream target of CEACAM1 signaling.     

Subcellular Localization of p44/WDR77 Determines Proliferation and Differentiation of Prostate Epithelial Cells

The molecular mechanism that controls the proliferation and differentiation of prostate epithelial cells is currently unknown. We previously identified a 44-kDa protein (p44/wdr77) as an androgen receptor-interacting protein that regulates a set of androgen receptor target genes in prostate epithelial cells and prostate cancer. In this study, we found that p44 localizes in the cytoplasm of prostate epithelial cells at the early stage of prostate development when cells are proliferating, and its nuclear translocation is associated with cellular and functional differentiation in adult prostate tissue. We further demonstrated that cytoplasmic p44 protein is essential for proliferation of prostate epithelial cells, whereas nuclear p44 is required for cell differentiation and prostate- specific protein secretion. These studies suggest a novel mechanism by which proliferation and differentiation of prostate epithelial cells are controlled by p44’s location in the cell.     

Subnuclear localization and differentiation-dependent increased expression of DGK-zeta in C2C12 mouse myoblasts

Diacylglycerol kinases (DGKs) catalyze phosphorylation of diacylglycerol (DG) to yield phosphatidic acid (PA). Previous evidence has shown that the nucleus contains several DGK isoforms. In this study, we have analyzed the expression and subnuclear localization of DGK-zeta employing C2C12 mouse myoblasts. Immunocytochemistry coupled to confocal laser scanning microscopy showed that both endogenous and green fluorescent protein-tagged overexpressed DGK-zeta localized mostly to the nucleus. In contrast, overexpressed DGK-alpha, -beta, -delta, and -iota did not migrate to the nucleus. DGK-zeta was present in the nuclear speckle domains, as also revealed by immuno-electron microscopy analysis. Moreover, DGK-zeta co-localized and interacted with phosphoinositide-specific phospholipase Cbeta1 (PLCbeta1), that is involved in inositide-dependent signaling pathways important for the regulation of cell proliferation and differentiation. Furthermore, we report that DGK-zeta associated with nuclear matrix, the fundamental organizing principle of the nucleus where many cell functions take place, including DNA replication, gene expression, and protein phosphorylation. Nuclear DGK-zeta increased during myogenic differentiation of C2C12 cells, while DGK-zeta down-regulation by siRNA markedly impaired differentiation. Overall, our findings further support the importance of speckles and nuclear matrix in lipid-dependent signaling and suggest that nuclear DGK-zeta might play some fundamental role during myogenic differentiation of C2C12 cells.     

Compartmentalization of the splicing machinery in plant cell nuclei

The cell nucleus is a membrane-surrounded organelle that contains numerous compartments in addition to chromatin. Compartmentalization of the nucleus is now accepted as an important feature for the organization of nuclear processes and for gene expression. Recent studies on nuclear organization of splicing factors in plant cells provide insights into the compartmentalization of the plant cell nuclei and conservation of nuclear compartments between plants and metazoans.     

Nuclear localization of leukotriene A4 hydrolase in type II alveolar epithelial cells in normal and fibrotic lung

Leukotriene A4 (LTA4) hydrolase catalyzes the final step in leukotriene B4 (LTB4) synthesis. In addition to its role in LTB4 synthesis, the enzyme possesses aminopeptidase activity. In this study, we sought to define the subcellular distribution of LTA4 hydrolase in alveolar epithelial cells, which lack 5-lipoxygenase and do not synthesize LTA4. Immunohistochemical staining localized LTA4 hydrolase in the nucleus of type II but not type I alveolar epithelial cells of normal mouse, human, and rat lungs. Nuclear localization of LTA4 hydrolase was also demonstrated in proliferating type II-like A549 cells. The apparent redistribution of LTA4 hydrolase from the nucleus to the cytoplasm during type II-to-type I cell differentiation in vivo was recapitulated in vitro. Surprisingly, this change in localization of LTA4 hydrolase did not affect the capacity of isolated cells to convert LTA4 to LTB4. However, proliferation of A549 cells was inhibited by the aminopeptidase inhibitor bestatin. Nuclear accumulation of LTA4 hydrolase was also conspicuous in epithelial cells during alveolar repair following bleomycin-induced acute lung injury in mice, as well as in hyperplastic type II cells associated with fibrotic lung tissues from patients with idiopathic pulmonary fibrosis. These results show for the first time that LTA4 hydrolase can be accumulated in the nucleus of type II alveolar epithelial cells and that redistribution of the enzyme to the cytoplasm occurs with differentiation to the type I phenotype. Furthermore, the aminopeptidase activity of LTA4 hydrolase within the nucleus may play a role in promoting epithelial cell growth.     

Tumorhead distribution to cytoplasmic membrane of neural plate cells is positively regulated by Xenopus p21-activated kinase 1 (X-PAK1)

Tumorhead (TH) regulates neural plate cell proliferation during Xenopus early development, and gain or loss of function prevents neural differentiation. TH shuttles between the nuclear and cytoplasmic/cortical cell compartments in embryonic cells. In this study, we show that subcellular distribution of TH is important for its functions. Targeting TH to the cell cortex/membrane potentiates a TH gain of function phenotype and results in neural plate expansion and inhibition of neuronal differentiation. We have found that TH subcellular localization is regulated, and that its shuttling between the nucleus and the cell cortex/cytoplasm is controlled by the catalytic activity of p21-activated kinase, X-PAK1. The phenotypes of embryos that lack, or have excess, X-PAK1 activity mimic the phenotypes induced by loss or gain of TH functions, respectively. We provide evidence that X-PAK1 is an upstream regulator of TH and discuss potential functions of TH at the cell cortex/cytoplasmic membrane and in the nucleus.     

抑癌基因PTEN在鼻咽癌细胞株中表达的研究

目的:检测人鼻咽癌细胞株中PTEN表达情况,探讨鼻咽癌细胞中PTEN表达与细胞分化程度的关系。方法:进行细胞株的培养,采用流式细胞仪和共聚焦显微镜检测方法对细胞中PTEN的表达进行定位定量检测。结果:两种细胞株均有PTEN的表达,表达强度和分布与分化程度有关,分化越好,表达越高,流式细胞仪检测PTEN在细胞株中表达强弱顺序为CNE1>CNE2,阳性表达细胞数CNE1>CNE2差异有统计学意义(P<0.01);激光共聚焦扫描显微镜检测PTEN主要表达在细胞核和细胞浆,分布与分化程度有关,细胞核表达强度CNE1相似文献