Manuel Reina

The transcription factors Slug and Snail act as repressors of Claudin-1 expression in epithelial cells

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.

Erythropoietin protects the in vitro blood-brain barrier against VEGF-induced permeability.

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.

RGD Peptides and monoclonal antibodies, antagonists of αv-Integrin, enter the cells by independent endocytic pathways

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 αvβ3 was internalized in αv-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 αv 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.

The role of O-linked sugars in determining the very low density lipoprotein receptor stability or release from the cell.

The very low density lipoprotein receptor is a member of the low density lipoprotein receptor supergene family for which two isoforms have been reported, one lacking and the other containing an O‐linked sugar domain. In order to gain insight into their functionality, transient and stable transformants separately overexpressing previously cloned bovine variants were analyzed. We report evidence that the variant lacking the O‐linked sugar domain presented a rapid cleavage from the cell and that a large amino‐terminal very low density lipoprotein receptor fragment was released into the culture medium. As only minor proteolysis was involved in the other very low density lipoprotein receptor variant, the clustered O‐linked sugar domain may be responsible for blocking the access to the protease‐sensitive site(s). To test this hypothesis, a mutant Chinese hamster ovary cell line, ldlD, with a reversible defect in the protein O‐glycosylation, was used. The instability of the O‐linked sugar‐deficient very low density lipoprotein receptor on the cell surface was comparable to that induced by the proteolysis of the variant lacking the O‐linked sugar domain. Moreover, our data suggest that the O‐linked sugar domain may also protect the very low density lipoprotein receptor against unspecific proteolysis. Taken together, these results indicate that the presence of the O‐linked sugar domain may be required for the stable expression of the very low density lipoprotein receptor on the cell surface and its absence may be required for release of the receptor to the extracellular space. The exclusive expression of the variant lacking the O‐linked sugar domain in the bovine aortic endothelium opens new perspectives in the physiological significance of the very low density lipoprotein receptor.

Isolation and sequencing of a 28 kD glutelin-2 gene from maize. common elements in the 5′ flanking regions among zein and glutelin genes

Abstract Four 28 kD glutelin-2 genomic clones were selected from a partial Eco RI lambda sep 6 lac 5 maize genomic library. The complete nucleotide sequence of a 1.9 kilo base pairs (kb) EcoRI-HindIII fragment containing the coding region of a glutelin-2 gene, as well as its 5′ and 3′ flanking regions, was worked out. The most relevant characteristics of the gene are: (a) it has no introns; (b) putative TATA and CAAT boxes are found at positions −104 and ′141, respectively; (c) three polyadenilation signals are present in the 3′ region at position +743, +797 and +910. In comparison with the best fitting zein gene, more than 50% homology can be detected in long stretches of both 5′ and 3′ flanking regions, as well as in the signal peptide coding region. A number of common short conserved nucleotide sequences are found in glutelin-2 and zein genes upstream the CAAT box. The possibility for these sequences to be signals for the coordinated expression of these genes is discussed.

Localization of lipoprotein lipase to discrete areas of the guinea pig brain

Lipoprotein lipase is a key enzyme in lipoprotein metabolism present primarily in extrahepatic tissues with high turnover of fatty acids. Using immunocytochemistry we have explored where lipoprotein lipase is localized in guinea pig brain. The enzyme was found to be associated with neuronal cells and vascular endothelial surfaces. The distribution was strikingly uneven with intense reaction in some areas, and virtually no reaction in adjacent areas. The highest reactivity was in neocortex, in hippocampus, in Purkinje cells of the cerebellum and in some motor nuclei of the brainstem. The results suggest marked differences between individual brain areas in utilization of plasma lipoproteins.

Identification of a novel Ezrin-binding site in syndecan-2 cytoplasmic domain

ERM (Ezrin/Radixin/Moesin) proteins are crosslinkers between plasma membrane proteins and the actin cytoskeleton, thereby involved in the formation of cell adhesion sites. Earlier work showed that Ezrin links syndecan‐2 to the actin cytoskeleton. Here we provide evidence that the Ezrin N‐terminal domain binds to the syndecan‐2 cytoplasmic domain with an estimated K D of 0.71 μM and without the requirement of other proteins. We also studied the regions in the syndecan‐2 cytoplasmic domain implicated in the binding to Ezrin. By truncating the syndecan‐2 cytoplasmic domain and by oligopeptide competition assays we show that the Ezrin‐binding sequence is not located in the positively charged juxtamembrane region (RMRKK), but in the neighboring sequence DEGSYD. We therefore conclude that the consensus sequence for Ezrin binding is unique among membrane proteins, suggesting a distinct regulation.

Differential binding of platelet-derived growth factor isoforms to glycosaminoglycans

The platelet-derived growth factor (PDGF) family comprises disulfide-bonded dimeric isoforms and plays a key role in the proliferation and migration of mesenchymal cells. Traditionally, it consists of homo- and heterodimers of A and B polypeptide chains that occur as long (AL and BL) or short (AS and BS) isoforms. Short isoforms lack the basic C-terminal extension that mediates binding to heparin. In the present study, we show that certain PDGF isoforms bind in a specific manner to glycosaminoglycans (GAGs). Experiments performed with wild-type and mutant Chinese hamster ovary cells deficient in the synthesis of GAGs revealed that PDGF long isoforms bind to heparan sulfate and chondroitin sulfate, while PDGF short isoforms only bind to heparan sulfate. This was confirmed by digestion of cell surface GAGs with heparitinase and chondroitinase ABC and by incubation with sodium chloride to prevent GAG sulfation. Furthermore, exogenous GAGs inhibited the binding of long isoforms to the cell membrane more efficiently than that of short isoforms. Additionally, we performed surface plasmon resonance experiments to study the inhibition of PDGF isoforms binding to low molecular weight heparin by GAGs. These experiments showed that PDGF-AAL and PDGF-BBS isoforms bound to GAGs with the highest affinity. In conclusion, PDGF activity at the cell surface may depend on the expression of various cellular GAG species.

Mfn2 downregulation in excitotoxicity causes mitochondrial dysfunction and delayed neuronal death.

Mitochondrial fusion and fission is a dynamic process critical for the maintenance of mitochondrial function and cell viability. During excitotoxicity neuronal mitochondria are fragmented, but the mechanism underlying this process is poorly understood. Here, we show that Mfn2 is the only member of the mitochondrial fusion/fission machinery whose expression is reduced in in vitro and in vivo models of excitotoxicity. Whereas in cortical primary cultures, Drp1 recruitment to mitochondria plays a primordial role in mitochondrial fragmentation in an early phase that can be reversed once the insult has ceased, Mfn2 downregulation intervenes in a delayed mitochondrial fragmentation phase that progresses even when the insult has ceased. Downregulation of Mfn2 causes mitochondrial dysfunction, altered calcium homeostasis, and enhanced Bax translocation to mitochondria, resulting in delayed neuronal death. We found that transcription factor MEF2 regulates basal Mfn2 expression in neurons and that excitotoxicity‐dependent degradation of MEF2 causes Mfn2 downregulation. Thus, Mfn2 reduction is a late event in excitotoxicity and its targeting may help to reduce excitotoxic damage and increase the currently short therapeutic window in stroke.

Alpha v integrin antagonists induce the disassembly of focal contacts in melanoma cells

In recent years, several antagonists of alpha(v)beta3 have been used to develop therapeutic approaches to the treatment of melanoma neoplasia. We studied the effects of anti-alpha(v)-integrin-blocking antibodies on attached M21 melanoma cells, the cellular distribution of alpha(v)-integrin and the molecular organization of focal structures. Anti-alpha(v)-integrin-blocking antibodies 17E6 and LM609, and an anti-alpha(v)beta3-integrin antagonist peptide cRGD 85189 induced detachment of M21 melanoma cells cultured for 24 hours on various substrates. cRGD was the most effective antagonist, reducing the number of adherent cells by 80%, while 17E6 reduced adhesion by only 30%. Light- and electron microscopy revealed attached cells with a flat shape and well-formed actin cytoskeleton. After treatment, cells became rounded and detached from the culture dish. alpha(v)-Integrins and focal-contact proteins were observed at adhesion sites in focal structures by immunocytochemistry. After treatment, however, cell rounding was accompanied by disorganization of the actin filaments and redistribution of alpha(v)-integrins and most of the focal proteins studied, except vinculin and tensin. Our results indicate that treatment of M21 melanoma cells with a(v)-integrin antagonists disrupts the actin cytoskeleton, redistributes a(v)-integrin and induces molecular disassembly of focal contacts.