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CXCR7 protein expression in human adult brain and differentiated neurons.

Shimizu S, Brown M, Sengupta R, Penfold ME, Meucci O - PLoS ONE (2011)

Bottom Line: Furthermore, confocal microscopy and binding assays in cultured neurons show that CXCR7 protein is mainly located into cytoplasm, while little to no protein expression is found on neuronal plasma membrane.Interestingly, specific CXCR7 ligands that inhibit CXCL12 binding to CXCR7 do not alter CXCR4-activated survival signaling (pERK/pAkt) in rat cortical neurons.Overall, these results implicate CXCR4 as the main CXCL12 signaling receptor on the surface of differentiated neurons and suggest that CXCR7 may interact with CXCR4 at the intracellular level, possibly affecting CXCR4 trafficking and/or coupling to other proteins.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Physiology, Drexel University College of Medicine, Philadelphia, Pennsylvania, United States of America.

ABSTRACT

Background: CXCR7 and CXCR4 are receptors for the chemokine CXCL12, which is involved in essential functions of the immune and nervous systems. Although CXCR7 transcripts are widely expressed throughout the central nervous system, little is known about its protein distribution and function in the adult brain. To evaluate its potential involvement in CXCL12/CXCR4 signaling in differentiated neurons, we studied CXCR7 protein expression in human brain and cultured neurons.

Methodology/principal findings: Immunohistochemistry and RT-PCR analyses of cortex and hippocampus from control and HIV-positive subjects provided the first evidence of CXCR7 protein expression in human adult neurons, under normal and pathological conditions. Furthermore, confocal microscopy and binding assays in cultured neurons show that CXCR7 protein is mainly located into cytoplasm, while little to no protein expression is found on neuronal plasma membrane. Interestingly, specific CXCR7 ligands that inhibit CXCL12 binding to CXCR7 do not alter CXCR4-activated survival signaling (pERK/pAkt) in rat cortical neurons. Neuronal CXCR7 co-localizes to some extent with the endoplasmic reticulum marker ERp29, but not with early/late endosome markers. Additionally, large areas of overlap are detected in the intracellular pattern of CXCR7 and CXCR4 expression.

Conclusions/significance: Overall, these results implicate CXCR4 as the main CXCL12 signaling receptor on the surface of differentiated neurons and suggest that CXCR7 may interact with CXCR4 at the intracellular level, possibly affecting CXCR4 trafficking and/or coupling to other proteins.

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CXCL12-induced phosphorylation of ERK or Akt in the presence of CXCR7                            ligands.(A) Rat cortical neurons were treated with the CXCR7 ligands CCX733 (100                            nM) or CCX754 (100 nM) 15 minutes before (and during) their exposure to                            vehicle or CXCL12 (20 nM, as indicated). Activation of survival pathways                            (i.e., ERK/Akt) induced by CXCL12 was measured by Western blot analysis                            using phosphospecific antibodies [30]. (B/C)                            Densitometric analysis showed no significant difference in pERK                            (analysis of variance [ANOVA]; NS, part B) or pAkt (ANOVA; NS,                            part C) bands after CCX733/754 treatment compared with CXCL12 alone.                            Total ERK and Akt were used for normalization of pERK and pAkt,                            respectively. Data represent the mean±SEM, as obtained in three                            independent experiments.
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pone-0020680-g007: CXCL12-induced phosphorylation of ERK or Akt in the presence of CXCR7 ligands.(A) Rat cortical neurons were treated with the CXCR7 ligands CCX733 (100 nM) or CCX754 (100 nM) 15 minutes before (and during) their exposure to vehicle or CXCL12 (20 nM, as indicated). Activation of survival pathways (i.e., ERK/Akt) induced by CXCL12 was measured by Western blot analysis using phosphospecific antibodies [30]. (B/C) Densitometric analysis showed no significant difference in pERK (analysis of variance [ANOVA]; NS, part B) or pAkt (ANOVA; NS, part C) bands after CCX733/754 treatment compared with CXCL12 alone. Total ERK and Akt were used for normalization of pERK and pAkt, respectively. Data represent the mean±SEM, as obtained in three independent experiments.

Mentions: The above results suggest limited to expression of CXCR7 on the neuronal surface. Thus, functional assays were performed to validate these observations with a different approach as well as to establish the potential contribution of even minimal CXCR7 expression to CXCR4 signaling. Rat neurons were exposed to CXCR7-specific ligands (CCX733 or CCX754, 100 nM) known to inhibit CXCL12 binding to CXCR7 [2], [8], [9], [25]. The goal was to determine whether treatment with the small-molecule compounds would affect the activation of intracellular pathways stimulated by CXCL12, specifically phosphorylation of ERK1/2 and Akt. As we previously demonstrated, activation of these pathways in neurons depends on CXCR4, involves Gi/o proteins, and ultimately promotes neuronal survival [30], [35], [39]. The results presented here show that CCX733 or CCX754, applied to pure neuronal cultures 15 minutes before and during CXCL12 treatment (20 nM, for indicated time), did not significantly alter CXCL12-induced stimulation of ERK1/2 and Akt (Figure 7). The effect of 100 nM CCX733/754 is reported here, but similar results were obtained in additional experiments using a wider range of CCX concentrations, i.e. from 10 nM to 1 µM (not shown). Also, the CXCR7 ligands did not induce ERK or Akt phosphorylation (nor Gi stimulation, not shown) in the absence of CXCL12 (not shown). These results are consistent with the lack of CXCR7 expression on the neuronal surface and with previous reports with similar compounds [21].


CXCR7 protein expression in human adult brain and differentiated neurons.

Shimizu S, Brown M, Sengupta R, Penfold ME, Meucci O - PLoS ONE (2011)

CXCL12-induced phosphorylation of ERK or Akt in the presence of CXCR7                            ligands.(A) Rat cortical neurons were treated with the CXCR7 ligands CCX733 (100                            nM) or CCX754 (100 nM) 15 minutes before (and during) their exposure to                            vehicle or CXCL12 (20 nM, as indicated). Activation of survival pathways                            (i.e., ERK/Akt) induced by CXCL12 was measured by Western blot analysis                            using phosphospecific antibodies [30]. (B/C)                            Densitometric analysis showed no significant difference in pERK                            (analysis of variance [ANOVA]; NS, part B) or pAkt (ANOVA; NS,                            part C) bands after CCX733/754 treatment compared with CXCL12 alone.                            Total ERK and Akt were used for normalization of pERK and pAkt,                            respectively. Data represent the mean±SEM, as obtained in three                            independent experiments.
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getmorefigures.php?uid=PMC3105114&req=5

pone-0020680-g007: CXCL12-induced phosphorylation of ERK or Akt in the presence of CXCR7 ligands.(A) Rat cortical neurons were treated with the CXCR7 ligands CCX733 (100 nM) or CCX754 (100 nM) 15 minutes before (and during) their exposure to vehicle or CXCL12 (20 nM, as indicated). Activation of survival pathways (i.e., ERK/Akt) induced by CXCL12 was measured by Western blot analysis using phosphospecific antibodies [30]. (B/C) Densitometric analysis showed no significant difference in pERK (analysis of variance [ANOVA]; NS, part B) or pAkt (ANOVA; NS, part C) bands after CCX733/754 treatment compared with CXCL12 alone. Total ERK and Akt were used for normalization of pERK and pAkt, respectively. Data represent the mean±SEM, as obtained in three independent experiments.
Mentions: The above results suggest limited to expression of CXCR7 on the neuronal surface. Thus, functional assays were performed to validate these observations with a different approach as well as to establish the potential contribution of even minimal CXCR7 expression to CXCR4 signaling. Rat neurons were exposed to CXCR7-specific ligands (CCX733 or CCX754, 100 nM) known to inhibit CXCL12 binding to CXCR7 [2], [8], [9], [25]. The goal was to determine whether treatment with the small-molecule compounds would affect the activation of intracellular pathways stimulated by CXCL12, specifically phosphorylation of ERK1/2 and Akt. As we previously demonstrated, activation of these pathways in neurons depends on CXCR4, involves Gi/o proteins, and ultimately promotes neuronal survival [30], [35], [39]. The results presented here show that CCX733 or CCX754, applied to pure neuronal cultures 15 minutes before and during CXCL12 treatment (20 nM, for indicated time), did not significantly alter CXCL12-induced stimulation of ERK1/2 and Akt (Figure 7). The effect of 100 nM CCX733/754 is reported here, but similar results were obtained in additional experiments using a wider range of CCX concentrations, i.e. from 10 nM to 1 µM (not shown). Also, the CXCR7 ligands did not induce ERK or Akt phosphorylation (nor Gi stimulation, not shown) in the absence of CXCL12 (not shown). These results are consistent with the lack of CXCR7 expression on the neuronal surface and with previous reports with similar compounds [21].

Bottom Line: Furthermore, confocal microscopy and binding assays in cultured neurons show that CXCR7 protein is mainly located into cytoplasm, while little to no protein expression is found on neuronal plasma membrane.Interestingly, specific CXCR7 ligands that inhibit CXCL12 binding to CXCR7 do not alter CXCR4-activated survival signaling (pERK/pAkt) in rat cortical neurons.Overall, these results implicate CXCR4 as the main CXCL12 signaling receptor on the surface of differentiated neurons and suggest that CXCR7 may interact with CXCR4 at the intracellular level, possibly affecting CXCR4 trafficking and/or coupling to other proteins.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Physiology, Drexel University College of Medicine, Philadelphia, Pennsylvania, United States of America.

ABSTRACT

Background: CXCR7 and CXCR4 are receptors for the chemokine CXCL12, which is involved in essential functions of the immune and nervous systems. Although CXCR7 transcripts are widely expressed throughout the central nervous system, little is known about its protein distribution and function in the adult brain. To evaluate its potential involvement in CXCL12/CXCR4 signaling in differentiated neurons, we studied CXCR7 protein expression in human brain and cultured neurons.

Methodology/principal findings: Immunohistochemistry and RT-PCR analyses of cortex and hippocampus from control and HIV-positive subjects provided the first evidence of CXCR7 protein expression in human adult neurons, under normal and pathological conditions. Furthermore, confocal microscopy and binding assays in cultured neurons show that CXCR7 protein is mainly located into cytoplasm, while little to no protein expression is found on neuronal plasma membrane. Interestingly, specific CXCR7 ligands that inhibit CXCL12 binding to CXCR7 do not alter CXCR4-activated survival signaling (pERK/pAkt) in rat cortical neurons. Neuronal CXCR7 co-localizes to some extent with the endoplasmic reticulum marker ERp29, but not with early/late endosome markers. Additionally, large areas of overlap are detected in the intracellular pattern of CXCR7 and CXCR4 expression.

Conclusions/significance: Overall, these results implicate CXCR4 as the main CXCL12 signaling receptor on the surface of differentiated neurons and suggest that CXCR7 may interact with CXCR4 at the intracellular level, possibly affecting CXCR4 trafficking and/or coupling to other proteins.

Show MeSH
Related in: MedlinePlus