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

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.

Figure 1

Engagement of platelet-derived growth factor receptor (PDGF-R) is critical for PDGF-BB induced monocyte chemoattractant protein 1 (MCP-1) expression in astrocytes. (A) Human primary as well as A172 primary astrocytes express both PDGF-α and PDGF-β receptors. Untreated human primary and A172 astrocytes were subjected to flow cytometric analyses using antibodies against human PDGF-αR (CD140α) and PDGF-βR (CD140β). (B,C) PDGF-BB-mediated induction of MCP-1 mRNA and protein expression in human primary and A172 astrocytes. (B) Human primary and A172 astrocytes were exposed to PDGF-BB for 6 h. Total RNA isolated from human A172 astrocytes was subjected to real-time reverse-transcriptase polymerase chain reaction (RT-PCR) analysis using primers for human MCP-1. (C) Supernatant fluid from astrocytes treated with PDGF-BB for 24 h were assessed for release of chemokine MCP-1 using the human MCP-1 enzyme-linked immunosorbent assay (ELISA) kit. (D) Total RNA from A172 cells transfected with either nonsense (Non) or PDGF-Rβ short interfering (si)RNAs was subjected to RT-PCR analysis using primers specific for PDGF-Rβ. (E) siRNA, but not Non siRNA inhibited PDGF-BB-mediated induction of MCP-1 RNA. (F) Supernatants from A172 cells transfected with either Non or siRNAs were subjected to MCP-1 ELISA assay. PDGF-Rβ siRNA, but not Non siRNA inhibited PDGF-BB-mediated induction of MCP-1 protein. **P <0.01, ***P <0.001 versus control group.

Figure 2

HIV-1 induces platelet-derived growth factor (PDGF)-B and monocyte chemoattractant protein 1 (MCP-1) mRNA and protein expression in astrocytes. (A) HIV-1 LAI induced PDGF-B chain and MCP-1 mRNA expression in human A172 astrocytes. Total RNA isolated from human A172 astrocytes was subjected to real-time reverse-transcriptase polymerase chain reaction (RT-PCR) analysis using primers specific for human PDGF-B, MCP-1 and 18S. (B) HIV-1 LAI induces PDGF-BB protein expression in human A172 astrocytes. Protein lysates isolated from astrocytes exposed to HIV-1 were subjected to western blot analysis and analyzed for expression of PDGF-BB protein. (C) HIV-1 LAI induces MCP-1 mRNA levels. Total RNA isolated from human A172 astrocytes was subjected to RT-PCR analysis using primers specific for human MCP-1 and 18S. (D) HIV-1 LAI induces MCP-1 protein expression in human A172 astrocytes. Supernatants from astrocytes exposed to HIV-1 for 24 h were subjected to MCP-1 enzyme-linked immunosorbent assay (ELISA) analysis. All the data are presented as mean ± SD of three individual experiments. *P <0.05, **P <0.01, ***P <0.001 versus control group; ##P <0.01 versus HIV-1-treated group.

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.

Figure 4

Platelet-derived growth factor (PDGF)-BB-mediated induction of monocyte chemoattractant protein 1 (MCP-1) expression involves mitogen-activated protein kinases (MAPKs) and phosphatidylinositol 3-kinase (PI3K)/Akt cell signaling pathways. (A) Western blot analysis of time-dependent activation of extracellular-signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), P38 and Akt by PDGF-BB. (B) Inhibition of the ERK, JNK, p38 and Akt pathways by mitogen-activated protein (MAP) kinase kinase (MEK)1/2 (U0126), JNK (SP600125), p38 (SB20358) and PI3K (LY294002) inhibitors resulted in amelioration of PDGF-BB-mediated induction of MCP-1 expression in astrocytes. (C) Transfection with dominant-negative (DN)-MEK but not wild-type (WT)-MEK resulted in abrogation of PDGF-BB-mediated induction of MCP-1. (D) Transduction with DN-Akt but not WT-Akt also resulted in abrogation of PDGF-BB-mediated induction of MCP-1. All the data are presented as mean ± SD of three individual experiments. *P <0.05, **P <0.01, ***P <0.001 versus control group; #P <0.05, ###P <0.001 versus PDGF-BB-treated group.

Figure 5

Involvement of platelet-derived growth factor (PDGF)-Rβ in the regulation of mitogen-activated protein kinases (MAPKs) and phosphatidylinositol 3-kinase (PI3K)/Akt cell signaling pathways. (A) Whole cell lysates from A172 cells transfected with either nonsense (Non) or PDGF-Rβ short interfering (si)RNAs were subjected to immunoblot analysis using antibodies specific for MAPKs and PI3K/Akt signaling. PDGF-Rβ siRNA, but not Non siRNA inhibited PDGF-BB-mediated phosphorylation of extracellular-signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), p38 and Akt pathways. All the data are presented as mean ± SD of three individual experiments. **P <0.01, ***P <0.001 versus control group, ##P <0.01, ###P <0.001 versus PDGF-BB-treated group.

Figure 6

Platelet-derived growth factor (PDGF)-BB-mediated induction of monocyte chemoattractant protein 1 (MCP-1) expression involves nuclear factor κB (NFκB) activation. (A) Exposure of A172 cells to PDGF-BB resulted in a time-dependent increase in phosphorylation of the p65 subunit of NFκB in the nuclear cellular fraction. Reciprocally, PDGF-BB exposure resulted in a time-dependent increase in inhibitor of κBα (IκBα) phosphorylation in the cytosolic cellular fraction of A172 astrocytes. (B) Pretreatment with the IκBα inhibitor, SC514 resulted in inhibition of PDGF-BB-mediated induction of MCP-1. (C) Transduction with mutant-NFκB but not wild-type (WT)-NFκB resulted in abrogation of PDGF-BB-mediated induction of MCP-1. All the data are presented as mean ± SD of three individual experiments. ***P <0.001 versus control group, #P <0.05, ###P <0.001 versus PDGF-BB-treated group. (D) Schematic illustration of NFκB binding consensus sequence on the promoter region of MCP-1. (E) Chromatin immunoprecipitation (ChIP) assay demonstrating PDGF-BB-mediated binding of p65NFκB to the MCP-1 promoter.

Figure 7

Mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K)/Akt cell signaling pathways lie upstream of platelet-derived growth factor (PDGF)-BB induced nuclear factor κB (NFκB) in astrocytes. The nuclear fractions of A172 cells transfected with wild-type (WT) or dominant-negative (DN) forms of mitogen-activated protein (MAP) kinase kinase (MEK) and Akt were subjected to western blot analysis for NFκB. (A) Transfection with DN-MEK but not WT-MEK resulted in abrogation of PDGF-BB-mediated induction of NFκB phosphorylation. (B) Transduction with DN-Akt but not WT-Akt also resulted in abrogation of PDGF-BB-mediated induction of NFκB phosphorylation. All the data are presented as mean ± SD of three individual experiments. ***P <0.001 versus control group, ###P <0.001 versus PDGF-BB-treated group.

Figure 8

Platelet-derived growth factor (PDGF)-BB-induced monocyte transmigration and permeability is mediated by monocyte chemoattractant protein 1 (MCP-1). Human brain microvascular endothelial cells (HBMECs) were exposed for 24 h to supernatants from A172 cells treated with PDGF-BB with or without STI-571. MCP-1 neutralizing antibody was added to PDGF-BB-treated media prior to HBMEC exposure for 24 h. Human monocytes were added to the top transwell for 24 h followed by assessment of transmigration. Exposure of HBMECs to supernatants from A172 cells treated with PDGF-BB resulted in increased permeability and enhanced monocyte transmigration, which was abrogated by MCP-1 neutralizing antibody. All the data are presented as mean ± SD of three individual experiments. ***P <0.001 versus control group; ##P <0.01, ###P <0.001 versus PDGF-BB-treated group.