No abstract

Claudin-1 is an integral membrane protein component of tight junctions. The Snail family of transcription factors are repressors that play a central role in the epithelial-mesenchymal transition, a process that occurs during cancer progression. Snail and Slug members are direct repressors of E-cadherin and act by binding to the specific E-boxes of its proximal promoter. In the present study, we demonstrate that overexpression of Slug or Snail causes a decrease in transepithelial electrical resistance. Overexpression of Slug and Snail in MDCK (Madin-Darby canine kidney) cells down-regulated Claudin-1 at protein and mRNA levels. In addition, Snail and Slug are able to effectively repress human Claudin-1-driven reporter gene constructs containing the wild-type promoter sequence, but not those with mutations in two proximal E-box elements. We also demonstrate by band-shift assay that Snail and Slug bind to the E-box motifs present in the human Claudin-1 promoter. Moreover, an inverse correlation in the levels of Claudin-1 and Slug transcripts were observed in breast cancer cell lines. E-box elements in the Claudin-1 promoter were found to play a critical negative regulatory role in breast cancer cell lines that expressed low levels of Claudin-1 transcript. Significantly, in invasive human breast tumours, high levels of Snail and Slug correlated with low levels of Claudin-1 expression. Taken together, these results support the hypothesis that Claudin-1 is a direct downstream target gene of Snail family factors in epithelial cells.

Beta-catenin is a multifunctional protein involved in cell-cell adhesion and Wnt signal transduction. Beta-catenin signaling has been proposed to act as inducer of cell proliferation in different tumors. However, in some developmental contexts and cell systems beta-catenin also acts as a positive modulator of apoptosis. To get additional insights into the role of beta-catenin in the regulation of the cell cycle and apoptosis, we have analyzed the levels and subcellular localization of endogenous beta-catenin and its relation with adenomatous polyposis coli (APC) during the cell cycle in S-phase-synchronized epithelial cells. Beta-catenin levels increase in S phase, reaching maximum accumulation at late G2/M and then abruptly decreasing as the cells enter into a new G1 phase. In parallel, an increased cytoplasmic and nuclear localization of beta-catenin and APC is observed during S and G2 phases. In addition, strong colocalization of APC with centrosomes, but not beta-catenin, is detected in M phase. Interestingly, overexpression of a stable form of beta-catenin, or inhibition of endogenous beta-catenin degradation, in epidermal keratinocyte cells induces a G2 cell cycle arrest and leads to apoptosis. These results support a role for beta-catenin in the control of cell cycle and apoptosis at G2/M in normal and transformed epidermal keratinocytes.

We report the identification of a novel human gene, designated p619, that encodes a polypeptide of 4861 amino acid residues, one of the largest human proteins known to date. The p619 protein contains two regions of seven internal repeats highly related to the cell cycle regulator RCC1, a guanine nucleotide exchange factor for the small GTP binding protein, Ran. In addition, p619 possesses seven beta‐repeat domains characteristic of the beta‐subunit of heterotrimeric G proteins, three putative SH3 binding sites, seven polar amino acid‐rich regions, a putative leucine zipper and a carboxy‐terminal HECT domain characteristic of E3 ubiquitin‐protein ligases. p619 is expressed ubiquitously in mouse and human tissues and overexpressed in several human tumor cell lines. Subcellular localization studies indicate that p619 is located in the cytosol and in the Golgi apparatus. Localization of p619 in the Golgi is altered by Brefeldin A. The carboxy‐terminal RCC1‐like domain of p619 interacts specifically with myristoylated ARF1, a small GTP binding protein also located in the Golgi. Moreover, the second RCC1‐like motif located at the amino‐terminus of p619 stimulates guanine nucleotide exchange on ARF1 and on members of the related Rab proteins, but not on other small GTP binding proteins such as Ran or R‐Ras2/TC21. These observations suggest that p619 is a Brefeldin A‐sensitive Golgi protein that functions as a guanine nucleotide exchange factor for ARF1 and, possibly, for members of the Rab family of proteins.

Lipoprotein lipase (LPL, E.C. 3.3.1.34) is the enzyme responsible for hydrolysis of triacylglycerols in plasma lipoproteins, making the fatty acids available for use by subjacent tissues. LPL is functional at the surface of endothelial cells, but it is not clear which cells synthesize the enzyme and what its distribution is within tissues and vessels. We have searched for specific cell expression of the LPL gene by in situ hybridization using a RNA probe and for the corresponding protein distribution by immunocytochemistry on cryosections of some LPL-producing tissues of guinea pigs. In white and brown adipose tissues, heart and skeletal muscle, and lactating mammary gland, there was positive hybridization for LPL mRNA over all members of the major cell types, indicating that mature and immature adipocytes, muscle cells, and mammary epithelial cells are main sources of LPL. In large vessels, LPL expression was detected in some smooth muscle cells in the media layer. There was no positive hybridization for LPL mRNA over endothelial cells in any of the tissues studied, but there was immunoreaction for LPL protein at endothelial surfaces of all blood vessels. In the kidney, there was strong immunofluorescence at the vascular endothelium, particularly in the glomeruli, but little or no LPL mRNA was detected in the surrounding cells. These observations suggest that in some tissues LPL is synthesized by parenchymal cells and spreads along the vascular mesh. Transfer to the vascular endothelium is, however, not the only route taken by LPL. In the mammary gland most of the enzyme protein appeared to be secreted, partly in association with milk fat droplets.(ABSTRACT TRUNCATED AT 250 WORDS)

The blood-brain barrier (BBB) ensures the homeostasis of the brain microenvironment, mostly through complex tight junctions between brain endothelial cells that prevent the passage of hydrophilic molecules from blood to brain and vice versa. A recent study has shown in vivo that systemic administration of erythropoietin (Epo) protects against brain injury. Using an in vitro model of the bovine BBB, we observed that the expression of the Epo receptor is modulated by its ligand and hypoxic stimuli such as vascular endothelial growth factor (VEGF) treatment. In addition, Epo protects against the VEGF-induced permeability of the BBB, decreases the levels of endothelial nitric oxide synthase and restores junction proteins. The kinetic transport experiments revealed the capacity of Epo to cross the in vitro BBB in a saturable and specific way. Our results suggest a new mechanism for Epo-induced neuroprotection, in which circulating Epo controls and maintains the BBB through an Epo receptor signalling pathway and the re-establishment of cell junctions.

SUMMARY:Cyclic synthetic peptides containing the arginine-glycine-aspartate motif (cRGD) and monoclonal antibodies (mAbs) targeted for individual integrins have been developed as potential therapeutic drugs for the treatment of several diseases. We showed that a cRGD peptide targeted for ␣ v ␤ 3 was internalized in ␣ v -integrin expressing and nonexpressing melanoma cells by an integrin independent fluid-phase endocytosis pathway that does not alter the number of functional integrin receptors at the cell surface. In contrast, a blocking mAb directed to ␣ v was internalized by an integrin-dependent endocytosis pathway that reduced the number of functional integrin receptors at the cell surface. We prove that melanoma cells pretreated with the mAb do not readhere to the substrate, whereas cells pretreated with cRGD peptide retain their readhesion capacity. Given the growing importance of RGD peptides, knowledge of these cellular mechanisms is required to improve the development of antiangiogenic and anti-inflammatory drugs. (Lab Invest 2001, 81:1615-1626.

After incubation with glucose a dramatic change in the intracellular distribution of glycogen synthase was observed in rat hepatocytes. Confocal laser scanning microscopy showed that glycogen synthase existed diffusely in the cytosol of control cells, whereas in cells incubated with glucose it accumulated at the cell periphery. Colocalization analysis between glycogen synthase immunostaining and actin filaments showed that the change in glycogen synthase distribution induced by glucose correlated with a marked increase in the co-distribution of the two proteins, indicating that, in response to glucose, glycogen synthase moves to the actin-rich area close to the membrane. When glycogen synthase was immunostained with rabbit anti-(glycogen synthase) and Protein A-colloidal gold, few particles were observed close to the membrane in control cells. In contrast, in cells incubated with glucose most of the gold particles were found near the membrane, confirming that glycogen synthase had moved to the cell cortex. Furthermore, in agreement with the glycogen synthase distribution, glycogen deposition appeared to be more active at the periphery of the cell.