María Dolores Gutiérrez-López

The tetraspanin CD9 inhibits the proliferation and tumorigenicity of human colon carcinoma cells

The implication of the tetraspanin CD9 in cancer has received much recent attention and an inverse correlation between CD9 expression and the metastatic potential and cancer survival rate has been established for different tumor types. In contrast to the well‐established role of CD9 in metastasis, very little is known about the involvement of this tetraspanin in the process of development of primary tumors. In the present study, we present evidence on the implication of CD9 in colon carcinoma tumorigenesis. We report here that ectopic expression of CD9 in colon carcinoma cells results in enhanced integrin‐dependent adhesion and inhibition of cell growth. Consistently with these effects, treatment of these cells with anti‐CD9‐specific antibodies resulted in (i) increased β1 integrin‐mediated cell adhesion through a mechanism involving clustering of integrin molecules rather than altered affinity; (ii) induction of morphological changes characterized by the acquisition of an elongated cell phenotype; (iii) inhibition of cell proliferation with no significant effect on cell survival; (iv) increased expression of membrane TNF‐α, and finally (v) inhibition of the in vivo tumorigenic capacity in nude mice. In addition, through the use of selective blockers of TNF‐α, we have demonstrated that this cytokine partly mediates the antiproliferative effects of CD9. These results clearly establish for the first time a role for CD9 in the tumorigenic process.

The sheddase activity of ADAM17/TACE is regulated by the tetraspanin CD9

ADAM17/TACE is a metalloproteinase responsible for the shedding of the proinflammatory cytokine TNF-α and many other cell surface proteins involved in development, cell adhesion, migration, differentiation, and proliferation. Despite the important biological function of ADAM17, the mechanisms of regulation of its metalloproteinase activity remain largely unknown. We report here that the tetraspanin CD9 and ADAM17 partially co-localize on the surface of endothelial and monocytic cells. In situ proximity ligation, co-immunoprecipitation, crosslinking, and pull-down experiments collectively demonstrate a direct association between these molecules. Functional studies reveal that treatment with CD9-specific antibodies or neoexpression of CD9 exert negative regulatory effects on ADAM17 sheddase activity. Conversely, CD9 silencing increased the activity of ADAM17 against its substrates TNF-α and ICAM-1. Taken together, our results show that CD9 associates with ADAM17 and, through this interaction, negatively regulates the sheddase activity of ADAM17.

A study on the effect of JNK inhibitor, SP600125, on the disruption of blood–brain barrier induced by methamphetamine

Methamphetamine (METH) is a widely consumed drug with high abuse potential. Studies in animals have shown that the drug produces dopaminergic neurotoxicity following both single high-dose and repeated low-dose administration. In addition, METH produces an increase in matrix metalloproteinase expression and loss of BBB integrity. We have examined the effect of repeated low-dose METH on MMP-9/2 expression and activity and laminin expression and the role of MMPs and JNK 1/2 phosphorylation on the changes induced by the drug in BBB integrity. Mice were given METH (4 mg/kg, i.p., three times separated by 3 h) and killed at different times after the last dose. Striatal MMP-9/2 activity was determined by zymography and expression of MMPs, laminin and phosphorylated JNK 1/2 was determined by western blot. BBB integrity was determined by IgG immunoreactivity. SP600125 and BB-94 were used to inhibit JNK and MMPs respectively. METH increased striatal MMP-9 expression and activity, IgG immunoreactivity and p-JNK 1/2 expression and decreased laminin expression. Increased IgG immunoreactivity colocalized with areas of greater MMP-9 activity. JNK inhibition prevented METH-induced changes in MMP-9 activity, laminin degradation and BBB leakage. BB-94 also prevented laminin degradation and BBB leakage. The decrease in BBB integrity induced by METH is mediated by the JNK pathway which activates MMP-9 causing degradation of laminin and BBB leakage.

Evidence that MDMA ('ecstasy') increases cannabinoid CB2 receptor expression in microglial cells: role in the neuroinflammatory response in rat brain.

J. Neurochem. (2010) 10.1111/j.1471‐4159.2010.06578.x

ALCAM/CD166 adhesive function is regulated by the tetraspanin CD9

ALCAM/CD166 is a member of the immunoglobulin superfamily of cell adhesion molecules (Ig-CAMs) which mediates intercellular adhesion through either homophilic (ALCAM–ALCAM) or heterophilic (ALCAM–CD6) interactions. ALCAM-mediated adhesion is crucial in different physiological and pathological phenomena, with particular relevance in leukocyte extravasation, stabilization of the immunological synapse, T cell activation and proliferation and tumor growth and metastasis. Although the functional implications of ALCAM in these processes is well established, the mechanisms regulating its adhesive capacity remain obscure. Using confocal microscopy colocalization, and biochemical and functional analyses, we found that ALCAM directly associates with the tetraspanin CD9 on the leukocyte surface in protein complexes that also include the metalloproteinase ADAM17/TACE. The functional relevance of these interactions is evidenced by the CD9-induced upregulation of both homophilic and heterophilic ALCAM interactions, as reflected by increased ALCAM-mediated cell adhesion and T cell migration, activation and proliferation. The enhancement of ALCAM function induced by CD9 is mediated by a dual mechanism involving (1) augmented clustering of ALCAM molecules, and (2) upregulation of ALCAM surface expression due to inhibition of ADAM17 sheddase activity.

Disruption of blood–brain barrier integrity in postmortem alcoholic brain: preclinical evidence of TLR4 involvement from a binge-like drinking model

Inflammatory cytokines and reactive oxygen species are reported to be involved in blood–brain barrier (BBB) disruption. Because there is evidence that ethanol (EtOH) induces release of free radicals, cytokines and inflammatory mediators we examined BBB integrity and matrix metalloproteinase (MMP) activity in postmortem human alcoholic brain and investigated the role of TLR4 signaling in BBB permeability in TLR4‐knockout mice under a binge‐like EtOH drinking protocol.

Changes in interleukin-1 signal modulators induced by 3,4-methylenedioxymethamphetamine (MDMA): regulation by CB2 receptors and implications for neurotoxicity

Background3,4-Methylenedioxymethamphetamine (MDMA) produces a neuroinflammatory reaction in rat brain characterized by an increase in interleukin-1 beta (IL-1β) and microglial activation. The CB2 receptor agonist JWH-015 reduces both these changes and partially protects against MDMA-induced neurotoxicity. We have examined MDMA-induced changes in IL-1 receptor antagonist (IL-1ra) levels and IL-1 receptor type I (IL-1RI) expression and the effects of JWH-015. The cellular location of IL-1β and IL-1RI was also examined. MDMA-treated animals were given the soluble form of IL-1RI (sIL-1RI) and neurotoxic effects examined.MethodsDark Agouti rats received MDMA (12.5 mg/kg, i.p.) and levels of IL-1ra and expression of IL-1RI measured 1 h, 3 h or 6 h later. JWH-015 (2.4 mg/kg, i.p.) was injected 48 h, 24 h and 0.5 h before MDMA and IL-1ra and IL-1RI measured. For localization studies, animals were sacrificed 1 h or 3 h following MDMA and stained for IL-1β or IL-1RI in combination with neuronal and microglial markers. sIL-1RI (3 μg/animal; i.c.v.) was administered 5 min before MDMA and 3 h later. 5-HT transporter density was determined 7 days after MDMA injection.ResultsMDMA produced an increase in IL-ra levels and a decrease in IL-1RI expression in hypothalamus which was prevented by CB2 receptor activation. IL-1RI expression was localized on neuronal cell bodies while IL-1β expression was observed in microglial cells following MDMA. sIL-1RI potentiated MDMA-induced neurotoxicity. MDMA also increased IgG immunostaining indicating that blood brain-barrier permeability was compromised.ConclusionsIn summary, MDMA produces changes in IL-1 signal modulators which are modified by CB2 receptor activation. These results indicate that IL-1β may play a partial role in MDMA-induced neurotoxicity.

Increased interleukin-1β levels following low dose MDMA induces tolerance against the 5-HT neurotoxicity produced by challenge MDMA

BackgroundPreconditioning is a phenomenon by which tolerance develops to injury by previous exposure to a stressor of mild severity. Previous studies have shown that single or repeated low dose MDMA can attenuate 5-HT transporter loss produced by a subsequent neurotoxic dose of the drug. We have explored the mechanism of delayed preconditioning by low dose MDMA.MethodsMale Dark Agouti rats were given low dose MDMA (3 mg/kg, i.p.) 96 h before receiving neurotoxic MDMA (12.5 mg/kg, i.p.). IL-1β and IL1ra levels and 5-HT transporter density in frontal cortex were quantified at 1 h, 3 h or 7 days. IL-1β, IL-1ra and IL-1RI were determined between 3 h and 96 h after low dose MDMA. sIL-1RI combined with low dose MDMA or IL-1β were given 96 h before neurotoxic MDMA and toxicity assessed 7 days later.ResultsPretreatment with low dose MDMA attenuated both the 5-HT transporter loss and elevated IL-1β levels induced by neurotoxic MDMA while producing an increase in IL-1ra levels. Low dose MDMA produced an increase in IL-1β at 3 h and in IL-1ra at 96 h. sIL-1RI expression was also increased after low dose MDMA. Coadministration of sIL-1RI (3 μg, i.c.v.) prevented the protection against neurotoxic MDMA provided by low dose MDMA. Furthermore, IL-1β (2.5 pg, intracortical) given 96 h before neurotoxic MDMA protected against the 5-HT neurotoxicity produced by the drug, thus mimicking preconditioning.ConclusionsThese results suggest that IL-1β plays an important role in the development of delayed preconditioning by low dose MDMA.

3,4-Methylenedioxymethamphetamine (MDMA, ecstasy) produces edema due to BBB disruption induced by MMP-9 activation in rat hippocampus

ABSTRACT The recreational drug of abuse, 3,4‐methylenedioxymethamphetamine (MDMA, ecstasy) disrupts blood‐brain barrier (BBB) integrity in rats through an early P2X7 receptor‐mediated event which induces MMP‐9 activity. Increased BBB permeability often causes plasma proteins and water to access cerebral tissue leading to vasogenic edema formation. The current study was performed to examine the effect of a single neurotoxic dose of MDMA (12.5 mg/kg, i.p.) on in vivo edema development associated with changes in the expression of the perivascular astrocytic water channel, AQP4, as well as in the expression of the tight‐junction (TJ) protein, claudin‐5 and Evans Blue dye extravasation in the hippocampus of adult male Dark Agouti rats. We also evaluated the ability of the MMP‐9 inhibitor, SB‐3CT (25 mg/kg, i.p.), to prevent these changes in order to validate the involvement of MMP‐9 activation in MDMA‐induced BBB disruption. The results show that MDMA produces edema of short duration temporally associated with changes in AQP4 expression and a reduction in claudin‐5 expression, changes which are prevented by SB‐3CT. In addition, MDMA induces a short‐term increase in both tPA activity and expression, a serine‐protease which is involved in BBB disruption and upregulation of MMP‐9 expression. In conclusion, this study provides evidence enough to conclude that MDMA induces edema of short duration due to BBB disruption mediated by MMP‐9 activation. HIGHLIGHTSClaudin‐5 decrease and Evans Blue extravasation indicate BBB disruption after MDMA.MDMA‐induced edema was visualized in vivo by magnetic resonance.MMP‐9 inhibition by SB‐3CT prevents edema formation and BBB disruption.Biphasic changes in AQP4 could be related to edema formation and resolution.

Increasing kynurenine brain levels reduces ethanol consumption in mice by inhibiting dopamine release in nucleus accumbens

&NA; Recent research suggests that ethanol (EtOH) consumption behaviour can be regulated by modifying the kynurenine (KYN) pathway, although the mechanisms involved have not yet been well elucidated. To further explore the implication of the kynurenine pathway in EtOH consumption we inhibited kynurenine 3‐monooxygenase (KMO) activity with Ro 61‐8048 (100 mg/kg, i.p.), which shifts the KYN metabolic pathway towards kynurenic acid (KYNA) production. KMO inhibition decreases voluntary binge EtOH consumption and EtOH preference in mice subjected to “drinking in the dark” (DID) and “two‐bottle choice” paradigms, respectively. This effect seems to be a consequence of increased KYN concentration, since systemic KYN administration (100 mg/kg, i.p.) similarly deters binge EtOH consumption in the DID model. Despite KYN and KYNA being well‐established ligands of the aryl hydrocarbon receptor (AhR), administration of AhR antagonists (TMF 5 mg/kg and CH‐223191 20 mg/kg, i.p.) and of an agonist (TCDD 50 &mgr;g/kg, intragastric) demonstrates that signalling through this receptor is not involved in EtOH consumption behaviour. Ro 61‐8048 did not alter plasma acetaldehyde concentration, but prevented EtOH‐induced dopamine release in the nucleus accumbens shell. These results point to a critical involvement of the reward circuitry in the reduction of EtOH consumption induced by KYN and KYNA increments. PNU‐120596 (3 mg/kg, i.p.), a positive allosteric modulator of &agr;7‐nicotinic acetylcholine receptors, partially prevented the Ro 61‐8048‐induced decrease in EtOH consumption. Overall, our results highlight the usefulness of manipulating the KYN pathway as a pharmacological tool for modifying EtOH consumption and point to a possible modulator of alcohol drinking behaviour. Graphical abstract Figure. No caption available. HighlightsKMO inhibition, by Ro 61‐8048, increases kynurenine and kynurenic acid concentration in both plasma and limbic forebrain and reduces voluntary binge ethanol consumption and preference in mice.Ro 61‐8048 prevents ethanol‐induced dopamine release in nucleus accumbens.AhR signalling is not involved in the reduction of ethanol consumption induced by KMO inhibition.Ro 61‐8048 does not modify plasma acetaldehyde concentration.