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Platelet-derived growth factor (PDGF)-BB-mediated induction of monocyte chemoattractant protein 1 in human astrocytes: implications for HIV-associated neuroinflammation.

Bethel-Brown C, Yao H, Hu G, Buch S - J Neuroinflammation (2012)

Bottom Line: Furthermore, treating astrocytes with the human recombinant PDGF-BB protein significantly increased the production and release of MCP-1 at both the RNA and protein levels.PDGF-BB-mediated release of MCP-1 was critical for increased permeability in an in vitro BBB model as evidenced by blocking antibody assays.Since MCP-1 is linked to disease severity, understanding its modulation by PDGF-BB could aid in understanding the proinflammatory responses in HAND.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, NE 68198, USA.

ABSTRACT
Chemokine (C-C motif) ligand 2, also known as monocyte chemoattractant protein 1 (MCP-1) is an important factor for the pathogenesis of HIV-associated neurocognitive disorders (HAND). The mechanisms of MCP-1-mediated neuropathogenesis, in part, revolve around its neuroinflammatory role and the recruitment of monocytes into the central nervous system (CNS) via the disrupted blood-brain barrier (BBB). We have previously demonstrated that HIV-1/HIV-1 Tat upregulate platelet-derived growth factor (PDGF)-BB, a known cerebrovascular permeant; subsequently, the present study was aimed at exploring the regulation of MCP-1 by PDGF-BB in astrocytes with implications in HAND. Specifically, the data herein demonstrate that exposure of human astrocytes to HIV-1 LAI elevated PDGF-B and MCP-1 levels. Furthermore, treating astrocytes with the human recombinant PDGF-BB protein significantly increased the production and release of MCP-1 at both the RNA and protein levels. MCP-1 induction was regulated by activation of extracellular-signal-regulated kinase (ERK)1/2, c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein (MAP) kinases and phosphatidylinositol 3-kinase (PI3K)/Akt pathways and the downstream transcription factor, nuclear factor κB (NFκB). Chromatin immunoprecipitation (ChIP) assays demonstrated increased binding of NFκB to the human MCP-1 promoter following PDGF-BB exposure. Conditioned media from PDGF-BB-treated astrocytes increased monocyte transmigration through human brain microvascular endothelial cells (HBMECs), an effect that was blocked by STI-571, a tyrosine kinase inhibitor (PDGF receptor (PDGF-R) blocker). PDGF-BB-mediated release of MCP-1 was critical for increased permeability in an in vitro BBB model as evidenced by blocking antibody assays. Since MCP-1 is linked to disease severity, understanding its modulation by PDGF-BB could aid in understanding the proinflammatory responses in HAND. These results suggest that astrocyte activation by PDGF-BB exaggerates monocyte recruitment into the brain via MCP-1 and underscores the critical role astrocytes play in HAND.

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Platelet-derived growth factor (PDGF)-BB induces monocyte chemoattractant protein 1 (MCP-1) mRNA and protein expression in human astrocytes. (A,B) Time-dependence of PDGF-BB-mediated induction of MCP-1 mRNA expression in human A172 astrocytes. Total RNA isolated from human A172 astrocytes was subjected to reverse-transcriptase polymerase chain reaction (RT-PCR) and real-time RT-PCR analysis using primers for human MCP-1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and 18S, respectively. PDGF-BB-mediated induction of MCP-1 mRNA expression peaked at 6 h and decline thereafter. (C) Time-dependence of PDGF-BB-mediated induction of MCP-1 protein expression in human A172 astrocytes. Supernatant fluid from human A172 cells treated with PDGF-BB for various time points were assessed for release of chemokine MCP-1 using the human MCP-1 enzyme-linked immunosorbent assay (ELISA) kit. PDGF-BB treatment resulted in a time-dependent induction of MCP-1 expression. All the data are presented as mean ± SD of three individual experiments. **P <0.01, ***P <0.001 versus control group; sfm = serum-free media.
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Figure 3: Platelet-derived growth factor (PDGF)-BB induces monocyte chemoattractant protein 1 (MCP-1) mRNA and protein expression in human astrocytes. (A,B) Time-dependence of PDGF-BB-mediated induction of MCP-1 mRNA expression in human A172 astrocytes. Total RNA isolated from human A172 astrocytes was subjected to reverse-transcriptase polymerase chain reaction (RT-PCR) and real-time RT-PCR analysis using primers for human MCP-1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and 18S, respectively. PDGF-BB-mediated induction of MCP-1 mRNA expression peaked at 6 h and decline thereafter. (C) Time-dependence of PDGF-BB-mediated induction of MCP-1 protein expression in human A172 astrocytes. Supernatant fluid from human A172 cells treated with PDGF-BB for various time points were assessed for release of chemokine MCP-1 using the human MCP-1 enzyme-linked immunosorbent assay (ELISA) kit. PDGF-BB treatment resulted in a time-dependent induction of MCP-1 expression. All the data are presented as mean ± SD of three individual experiments. **P <0.01, ***P <0.001 versus control group; sfm = serum-free media.

Mentions: All experiments involving the treatment of cells with exogenous PDGF-BB protein were conducted under serum-free conditions since PDGF promoter is known to have serum response elements [28,29]. To investigate the role of PDGF on MCP-1 expression, A172 cells were treated with recombinant PDGF-BB (20 ng/ml) for the indicated times and mRNA levels were assessed by RT-PCR and real-time RT-PCR. The concentration of PDGF-BB used was based on previous studies [25,30]. Following exposure of A172 astrocytes to PDGF-BB, there was a time-dependent upregulation of MCP-1 mRNA compared with untreated cells (Figure 3A) with a peak induction at 6 h post treatment (4.5-fold) and a subsequent downregulation at 12 h. Similar results were obtained via real-time PCR (Figure 3B).


Platelet-derived growth factor (PDGF)-BB-mediated induction of monocyte chemoattractant protein 1 in human astrocytes: implications for HIV-associated neuroinflammation.

Bethel-Brown C, Yao H, Hu G, Buch S - J Neuroinflammation (2012)

Platelet-derived growth factor (PDGF)-BB induces monocyte chemoattractant protein 1 (MCP-1) mRNA and protein expression in human astrocytes. (A,B) Time-dependence of PDGF-BB-mediated induction of MCP-1 mRNA expression in human A172 astrocytes. Total RNA isolated from human A172 astrocytes was subjected to reverse-transcriptase polymerase chain reaction (RT-PCR) and real-time RT-PCR analysis using primers for human MCP-1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and 18S, respectively. PDGF-BB-mediated induction of MCP-1 mRNA expression peaked at 6 h and decline thereafter. (C) Time-dependence of PDGF-BB-mediated induction of MCP-1 protein expression in human A172 astrocytes. Supernatant fluid from human A172 cells treated with PDGF-BB for various time points were assessed for release of chemokine MCP-1 using the human MCP-1 enzyme-linked immunosorbent assay (ELISA) kit. PDGF-BB treatment resulted in a time-dependent induction of MCP-1 expression. All the data are presented as mean ± SD of three individual experiments. **P <0.01, ***P <0.001 versus control group; sfm = serum-free media.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3526410&req=5

Figure 3: Platelet-derived growth factor (PDGF)-BB induces monocyte chemoattractant protein 1 (MCP-1) mRNA and protein expression in human astrocytes. (A,B) Time-dependence of PDGF-BB-mediated induction of MCP-1 mRNA expression in human A172 astrocytes. Total RNA isolated from human A172 astrocytes was subjected to reverse-transcriptase polymerase chain reaction (RT-PCR) and real-time RT-PCR analysis using primers for human MCP-1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and 18S, respectively. PDGF-BB-mediated induction of MCP-1 mRNA expression peaked at 6 h and decline thereafter. (C) Time-dependence of PDGF-BB-mediated induction of MCP-1 protein expression in human A172 astrocytes. Supernatant fluid from human A172 cells treated with PDGF-BB for various time points were assessed for release of chemokine MCP-1 using the human MCP-1 enzyme-linked immunosorbent assay (ELISA) kit. PDGF-BB treatment resulted in a time-dependent induction of MCP-1 expression. All the data are presented as mean ± SD of three individual experiments. **P <0.01, ***P <0.001 versus control group; sfm = serum-free media.
Mentions: All experiments involving the treatment of cells with exogenous PDGF-BB protein were conducted under serum-free conditions since PDGF promoter is known to have serum response elements [28,29]. To investigate the role of PDGF on MCP-1 expression, A172 cells were treated with recombinant PDGF-BB (20 ng/ml) for the indicated times and mRNA levels were assessed by RT-PCR and real-time RT-PCR. The concentration of PDGF-BB used was based on previous studies [25,30]. Following exposure of A172 astrocytes to PDGF-BB, there was a time-dependent upregulation of MCP-1 mRNA compared with untreated cells (Figure 3A) with a peak induction at 6 h post treatment (4.5-fold) and a subsequent downregulation at 12 h. Similar results were obtained via real-time PCR (Figure 3B).

Bottom Line: Furthermore, treating astrocytes with the human recombinant PDGF-BB protein significantly increased the production and release of MCP-1 at both the RNA and protein levels.PDGF-BB-mediated release of MCP-1 was critical for increased permeability in an in vitro BBB model as evidenced by blocking antibody assays.Since MCP-1 is linked to disease severity, understanding its modulation by PDGF-BB could aid in understanding the proinflammatory responses in HAND.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, NE 68198, USA.

ABSTRACT
Chemokine (C-C motif) ligand 2, also known as monocyte chemoattractant protein 1 (MCP-1) is an important factor for the pathogenesis of HIV-associated neurocognitive disorders (HAND). The mechanisms of MCP-1-mediated neuropathogenesis, in part, revolve around its neuroinflammatory role and the recruitment of monocytes into the central nervous system (CNS) via the disrupted blood-brain barrier (BBB). We have previously demonstrated that HIV-1/HIV-1 Tat upregulate platelet-derived growth factor (PDGF)-BB, a known cerebrovascular permeant; subsequently, the present study was aimed at exploring the regulation of MCP-1 by PDGF-BB in astrocytes with implications in HAND. Specifically, the data herein demonstrate that exposure of human astrocytes to HIV-1 LAI elevated PDGF-B and MCP-1 levels. Furthermore, treating astrocytes with the human recombinant PDGF-BB protein significantly increased the production and release of MCP-1 at both the RNA and protein levels. MCP-1 induction was regulated by activation of extracellular-signal-regulated kinase (ERK)1/2, c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein (MAP) kinases and phosphatidylinositol 3-kinase (PI3K)/Akt pathways and the downstream transcription factor, nuclear factor κB (NFκB). Chromatin immunoprecipitation (ChIP) assays demonstrated increased binding of NFκB to the human MCP-1 promoter following PDGF-BB exposure. Conditioned media from PDGF-BB-treated astrocytes increased monocyte transmigration through human brain microvascular endothelial cells (HBMECs), an effect that was blocked by STI-571, a tyrosine kinase inhibitor (PDGF receptor (PDGF-R) blocker). PDGF-BB-mediated release of MCP-1 was critical for increased permeability in an in vitro BBB model as evidenced by blocking antibody assays. Since MCP-1 is linked to disease severity, understanding its modulation by PDGF-BB could aid in understanding the proinflammatory responses in HAND. These results suggest that astrocyte activation by PDGF-BB exaggerates monocyte recruitment into the brain via MCP-1 and underscores the critical role astrocytes play in HAND.

Show MeSH
Related in: MedlinePlus