Up-regulation of ALCAM expression in caveolin-rich microvessesls from HIV-infected drug (cocaine) abusers
Since the degree of neurologic deficit in HIV-infected individuals is strongly correlated with the number of activated macrophages and microglia within the basal ganglia, we verified the CD68-positive staining in postmortem brain tissues from HIV-, HIV+/no cocaine and HIV+/cocaine drug abusers. Due to the inherent difficulty of finding tissues from uni-drug abusers, we resorted to samples from poly-drug abusers that included a history of cocaine abuse. In order to establish clinical relevance of ALCAM expression in the context of HIV-1 infection with drug (cocaine) abusers, we examined ALCAM expression and double-labeling with CD68. As shown , the increased CD68 immunostaining seen in HIV+/cocaine drug abusers paralleled with up-regulation of ALCAM in the brain endothelium compared with HIV+/no cocaine or uninfected controls. This new finding suggested that ALCAM plays a role in promoting leukocyte infiltration across the BBB.
Cocaine-mediated induction of ALCAM expression in human brains
Further validation of up-regulated expression of ALCAM, sections from basal ganglia region of the brain were stained for both ALCAM and caveolin-1 (Cav-1, endothelial cell membrane marker). ALCAM expression, although weak in microvessels from both HIV- and HIV+/no cocaine individuals, was significantly enhanced in vessels from HIV+ /cocaine drug abusers. Merged images of ALCAM and Cav-1 staining confirmed co-localization of ALCAM on the surface of endothelial cells (Supplementary Fig.1
Cav-1 and ALCAM signal intensities in CNS blood vessels from HIV-(), HIV+/no cocaine () and HIV+/cocaine drug abusers () were quantified by recording x-y planar images (0.1 μm in thickness) on two distinct channels, followed by reconstruction of 3-μm z-stack images. ALCAM staining was significantly stronger in HIV+/cocaine drug abusers compared with the HIV-or the HIV+/no cocaine group (). Intriguingly, there was no significant change in the expression of other adhesion molecules such as ICAM-1 or VCAM-1 in the brains tissues in response to cocaine as shown in Supplementary Fig.2 & 3
, thus underscoring the specificity of ALCAM induction by cocaine.
ALCAM promotes monocyte adhesion and transmigration in mice treated with cocaine
To further validate cocaine-mediated induction of ALCAM in vivo, we examined the expression level of ALCAM in brain capillaries isolated from cocaine versus saline-treated mice. Following administration of cocaine, there was an increase in the expression level of ALCAM in isolated microvessels from cocaine-exposed mice compared with controls ().
Cocaine-mediated induction of ACLAM enhances monocyte adhesion and transmigration in vivo
Since ALCAM expression was increased in cocaine-treated mice, to validate the role of ALCAM in monocyte transmigration in vivo, mice were treated with cocaine, followed by tail vein injection of labeled mouse BMM and their detection in the brain. Of particular note, the distribution of labeled monocytes was primarily within the perivascular cuffs (See arrowheads in ), with some localization in the parenchyma (See arrows in ). Quantification of the brain sections revealed increased transmigration in the CNS of cocaine-treated mice compared with controls. Specificity of ALCAM action was further demonstrated in mice pre-treated with the neutralizing antibody to ALCAM. Monocyte transmigration in these mice was ameliorated lending further support to the role of ALCAM ().
Cocaine-mediated up-regulation of ALCAM protein in HBMECs
To better understand how cocaine regulates ALCAM expression, we examined the effects of cocaine on ALCAM induction in HBMECs. As an initial screening study to identify the effective concentration of cocaine on ALCAM expression, HBMECs were treated with varying concentrations of cocaine (1,10 and 100μM) with a maximal response at 10μM (Supplementary Fig. 4 A
). This concentration of cocaine was therefore chosen for all our further studies. The next step was to examine the time course of ALCAM induction. Cocaine-mediated induction of ALCAM was observed as early as 6 hours following treatment and was significantly up-regulated even at 24 hours post-treatment in HBMECs (shown in supplementary Fig. 4 B & C
). Furthermore, using flow cytometry it was demonstrated that under normal conditions HBMECs expressed almost equal levels of ALCAM and ICAM-1, and lower levels of VCAM-1 (). Intriguingly, activation of cells with cocaine elicited robust up-regulation of ALCAM, but a weaker induction of ICAM-1 ().
Cocaine-mediated induction of ALCAM expression and its translocation to lipid rafts
The next step was to examine the distribution of ALCAM on these cells. As evidenced by confocal microscopy (), following cocaine exposure, ALCAM immunoreactivity concentrated around the plasma membrane. Since ALCAM is present in cholesterol-enriched membrane microdomains (Cayrol et al., 2008
), as expected, ALCAM was primarily recruited to cholesterol-enriched membrane microdomains (fractions 4-5), positive for ganglioside marker of lipid raft, GM1 (). Following cocaine treatment, ALCAM expression in lipid raft fractions of HBMECs was increased significantly compared to controls ().
Engagement of sigma-1 receptor (σ-1R) is critical for cocaine-induced ALCAM expression
σ-1R belonging to the non-opioid receptor family bind to diverse classes of psychotropic drugs including cocaine (Hayashi and Su, 2003
). As shown by RT-PCR () and Western blotting (), HBMECs expressed σ-1R mRNA and protein. Cocaine-mediated induction of ALCAM was significantly attenuated by pre-treatment of cells with the σ-1R antagonist BD1047 (). Transfection of cells with σ-1R siRNA also resulted in significant knock-down of expression of σ-1R () with significant abrogation of cocaine-mediated induction of ALCAM expression ().
Engagement of σ-1R is critical for cocaine-mediated induction of ALCAM expression
Exposure of HBMECs to cocaine activates PDGF-βR
Engagement of σ-1R can regulate both the activation and signaling of tyrosine kinase receptors, including PDGF-βR, which in turn, regulate the expression of cell adhesion molecules (Lin et al., 2007
). We thus rationalized that cocaine/σ-1R dyad could result in activation of PDGF-βR, and subsequently, induction of ALCAM. Lysates from HBMECs treated with cocaine induced time-dependent phosphorylation of PDGF-βR at Tyr 751 (), that was abrogated by both STI571 () and BD1047 (). Pretreatment with STI571 () and transfection with PDGF-βR siRNA () attenuated ALCAM expression.
Cocaine-mediated induction of ALCAM involves PDGF-βR activation
These findings were further validated using human phospho-receptor tyrosine kinase (RTK) antibody arrays specific for PDGF-βR (Supplementary Table 1 online
). Cocaine specifically induced greater than 2-fold increase in phosphorylation of PDGF-βR at Tyr 751. On the other hand, phosphorylation of PDGF-βR (Tyr 1021 &741) and PDGF-αR (Tyr 849) were not affected by cocaine. Phosphorylation at Tyr 771 but not Tyr 1009 was also observed with cocaine treatment (Supplementary Fig.5
Interaction between σ-1R and PDGF- βR
To explore protein-protein interaction of the two receptors, GST-fusion protein containing PDGF-βR was synthesized and used as GST immobilized baits in the pull-down assay. σ-1R was readily precipitated by GST-PDGF-βR ().
Protein-protein interaction of σ-1R and PDGF-βR was further confirmed by FRET analysis. As shown in (left panels), CHO cells transfected with GFP and the σ-1R-RFP failed to demonstrate any FRET signal. In contrast, cells co-transfected with both PDGF-βR-GFP and σ-1R-RFP demonstrated enhanced protein-protein interaction as shown in (right panels).
To further assess this interaction, series of co-immunoprecipitations using lysates from cocaine-treated HBMECs were performed. σ-1R immunoreactive band was consistently seen in the protein precipitated by anti-PDGF-βR antibody () and reciprocally, PDGF-βR band was displayed in the σ-1R precipitates (). These data demonstrate an evident interaction between σ-1R and PDGF-βR in response to cocaine. Further validation of this interaction was performed using confocal microscopy. As shown in , there was a diffuse pattern of σ-1R expression in the cytoplasm of untreated HBMECs. Treatment with cocaine resulted in clustering and polarization of σ-1R with the cell membrane. A 1-μm z-stack reconstruction demonstrated co-localization of both σ-1R and PDGF-βR in the presence of cocaine (see merged image and ).
Cocaine-induced expression of ALCAM involves c-Src activation
Activation of c-Src has been documented as an upstream event of PDGF-βR activation (Lin et al., 2007
). We thus examined its involvement in cocaine-mediated activation of PDGF-βR and ALCAM induction. Treatment with cocaine resulted in increased c-Src phosphorylation, which was inhibited by Src tyrosine kinase inhibitor PP2, but not by its inactive ortholog PP3 (). Intriguingly, cocaine-stimulated PDGF-βR phosphorylation was also blocked by PP2, but not by PP3 (). In contrast, cocaine-induced activation of Src was not blocked by STI-571(), thereby suggesting the role of c-Src activation in transactivation of PDGF-βR. Pre-treatment with PP2 significantly blocked cocaine-induced expression of ALCAM, thus confirming the role of c-Src in this process ().
Cocaine-mediated induction of ALCAM involves activation of c-Src tyrosine kinase
Cocaine-induced expression of ALCAM involves MAPK (ERK1/2, JNK, p38) and PI3K-Akt pathways
MAPK kinase and PI3K-Akt pathways play critical roles in both cocaine (Yao et al., 2009a
) and PDGF-βR signaling (Lin et al., 2005
; Yao et al., 2009a
). We next examined the involvement of these pathways in cocaine-mediated induction of ALCAM. Treatment with cocaine resulted in time-dependent increase in phosphorylation of MAPK () and Akt pathways (). Pretreatment of cells with either MEK1/2 (U0126), JNK (SP600125), p38 (SB203580), or PI3K (LY294002) inhibitor resulted in amelioration of cocaine-mediated induction of ALCAM, thus underpinning their roles in the process (). Further validation of the Akt pathway in this process was confirmed by transfecting cells with either the WT or DN Akt, followed by treatment with cocaine. Cocaine-mediated induction of ALCAM was attenuated by the DN-Akt, but not the WT-Akt construct ().
Cocaine-mediated induction of ALCAM expression involves MAPKs and PI3K/Akt cell signaling pathways
Cocaine-mediated activation of MAPK and Akt pathways involves upstream mediators such as σ-1R, c-Src and PDGF-βR
Having determined that cocaine mediated translocation of the σ-1R to the cell membrane and, that subsequent activation of the c-Src, PDGF-βR, and MAPKs pathways were critical processes involved in the induction of ALCAM, we next sought to link σ-1R & PDGF-βR activation with the signal transduction pathways. HBMECs pretreated with either the σ-1R or PDGF-βR (antagonists and siRNAs) followed by treatment with cocaine, were assessed for activation of signaling pathways. Both σ-1R and PDGF-βR antagonists inhibited cocaine-mediated activation of MAPKs (ERK1/2, JNK and p38) and Akt (). In addition to the pharmacological approach, silencing using σ-1R () and PDGF-βR siRNAs () was also able to inhibit cocaine-mediated activation of these pathways. Similarly, we also found abrogation of cocaine-mediated activation of both MAPK and Akt pathways in cells pretreated with Src kinase inhibitor ().
Involvement of σ-1R, Src kinase and PDGF-βR in the regulation of MAPKs and PI3K/Akt cell signaling pathways
Cocaine-mediated induction of ALCAM involves NF-κB activation
Members of the NF-κB family are considered to play essential roles in both cocaine and PDGF-βR-mediated signaling (Huang et al., 2003
; Lin et al., 2005
). Treatment of HBMECs with cocaine resulted in translocation of NF-κB p65 into the nucleus (). Pretreatment of cells with either IκB kinase-2 inhibitor-SC514 () or mutant NF-κB adenovirus () abrogated cocaine-induced ALCAM expression, thereby underscoring the role of NF-κB p65 in cocaine-mediated induction of ALCAM.
Cocaine-mediated induction of ALCAM expression involves NF-κB activation
Next step was to examine whether there existed a link that could tie activation of σ-1R and PDGF-βR with NF-κB translocation. Both σ-1R () and PDGF-βR siRNAs () were able to inhibit cocaine-mediated activation of NF-κB. To further confirm the involvement of NF-κB binding of NF-κB to ALCAM promoter was explored using ChIP assays. These experiments revealed increased binding of NF-κB to the ALCAM promoter in HBMECs treated with cocaine ().
Cocaine induced monocyte adhesion on HBMECs and monocyte transmigration across the in vitro BBB model
To identify the functional relevance of up-regulated ALCAM, we examined the ability of cocaine-induced ALCAM to induce monocyte adhesion. Treatment of HBMECs with cocaine resulted in increase in both HIV-infected and uninfected monocyte adhesion (). To confirm the role of ALCAM in this process, monocyte adhesion was evaluated in the presence of blocking antibodies. ALCAM antibody significantly restricted monocyte adhesion compared with the control (), suggesting thereby that ALCAM was critical for monocyte adhesion.
Cocaine-mediated induction of ACLAM enhances monocyte adhesion and transmigration in vitro
Roles of σ-1R and PDGF-βR in this process were confirmed by pre-treating cells with either the σ-1R or PDGF-βR antagonist, both of which resulted in inhibition of monocyte adhesion (). Similarly, a link between cocaine-mediated adhesion of monocytes and activation of c-Src with the signaling pathways was also established as evidenced by the fact that Src kinase inhibitor abolished cocaine-mediated monocyte adhesion (). Inhibitors specific for MAPK, PI3K & IkB kinase also blocked cocaine-induced monocyte adhesion (), further supporting the notion that in HBMECs sequential activation of σ-1R/Src/PDGF-βR/MAPK/PI3K pathways results in up-regulation of ALCAM and monocyte adhesion.
Next, we examined the ability of cocaine to induce monocyte transmigration in an in vitro model of human BBB. Exposure of HBMECS to cocaine resulted in increased monocyte transmigration (). Pre-treatment of HBMECs with anti-ALCAM antibody significantly restricted monocyte migration compared with the isotype control, thereby underpinning the role of ALCAM in transmigration of monocytes.
Cocaine-mediated induction of ALCAM in mouse models of systemic inflammation and T-cell deficiency
To understand whether cocaine-mediated induction of ALCAM was dependent on systemic inflammation, two mouse models: a) systemic inflammation model of LPS injection and, b) T cell deficiency model using nude mice, were exposed to cocaine and examined for induction of ALCAM in the isolated brain microvessels. As shown in , administration of LPS resulted in induction of ALCAM expression compared with the control group without LPS exposure. This effect was potentiated in the presence of cocaine. Intriguingly, in T cell deficient mice, cocaine failed to induce ALCAM expression. These findings were also validated by confocal microscopy in sections of microvessels stained with ALCAM and caveolin-1 from the three groups of mice exposed to cocaine as shown in .
Effect of cocaine on induction of ALCAM expression in mouse models of systemic inflammation and T-cell deficiency