Extracellular Regulated Kinase 1/2 Signaling Is a Critical Regulator of Interleukin-1β-Mediated Astrocyte Tissue Inhibitor of Metalloproteinase-1 Expression

Astrocytes are essential for proper central nervous system (CNS) function and are intricately involved in neuroinflammation. Despite evidence that immune-activated astrocytes contribute to many CNS pathologies, little is known about the inflammatory pathways controlling gene expression. Our laboratory identified altered levels of tissue inhibitor of metalloproteinase (TIMP)-1 in brain lysates from human immunodeficiency virus (HIV)-1 infected patients, compared to age-matched controls, and interleukin (IL)-1β as a key regulator of astrocyte TIMP-1. Additionally, CCAAT enhancer binding protein (C/EBP)β levels are elevated in brain specimens from HIV-1 patients and the transcription factor contributes to astrocyte TIMP-1 expression. In this report we sought to identify key signaling pathways necessary for IL-1β-mediated astrocyte TIMP-1 expression and their interaction with C/EBPβ. Primary human astrocytes were cultured and treated with mitogen activated protein kinase-selective small molecule inhibitors, and IL-1β. TIMP-1 and C/EBPβ mRNA and protein expression were evaluated at 12 and 24 h post-treatment, respectively. TIMP-1 promoter-driven luciferase plasmids were used to evaluate TIMP-1 promoter activity in inhibitor-treated astrocytes. These data show that extracellular regulated kinase (ERK) 1/2-selective inhibitors block IL-1β-induced astrocyte TIMP-1 expression, but did not decrease C/EBPβ expression in parallel. The p38 kinase (p38K) inhibitors partially blocked both IL-1β-induced astrocyte TIMP-1 expression and C/EBPβ expression. The ERK1/2-selective inhibitor abrogated IL-1β-mediated increases in TIMP-1 promoter activity. Our data demonstrate that ERK1/2 activation is critical for IL-1β-mediated astrocyte TIMP-1 expression. ERK1/2-selective inhibition may elicit a compensatory response in the form of enhanced IL-1β-mediated astrocyte C/EBPβ expression, or, alternatively, ERK1/2 signaling may function to moderate IL-1β-mediated astrocyte C/EBPβ expression. Furthermore, p38K activation contributes to IL-1β-induced astrocyte TIMP-1 and C/EBPβ expression. These data suggest that ERK1/2 signals downstream of C/EBPβ to facilitate IL-1β-induced astrocyte TIMP-1 expression. Astrocyte ERK1/2 and p38K signaling may serve as therapeutic targets for manipulating CNS TIMP-1 and C/EBPβ levels, respectively.


Introduction
Astrocytes are essential cells of the central nervous system (CNS) and are subject to the perturbations coinciding with neural pathologies, including human immunodeficiency virus (HIV)-1associated neurocognitive disorders (HAND) [1,2,3]. During HAND, HIV-1-infected monocytes infiltrate the CNS where they disseminate viral particles, cytokines and other stimulatory molecules [4]. Cytokines and viral toxins produced in this inflamed environment may bring about deleterious changes in astrocyte gene expression [4,5]. Dysfunctional astrocytes compromise optimal maintenance of the blood brain barrier, glutamate reuptake and the matrix metalloproteinase (MMP): tissue inhibitor of metalloproteinase (TIMP) balance [6,7,8,9,10,11]. In the CNS astrocytes are major producers of TIMP-1 [5,12,13], a multifunc-tional glycoprotein that regulates extracellular matrix processing and cell growth/apoptosis [14,15,16]. TIMP-1 is expressed in multiple tissues, by various cell types and plays roles in angiogenesis, neurogenesis, metastasis and other physiological processes by binding MMPs to inhibit their function [17,18,19,20]. TIMP-1 displays antiapoptotic activity independent of MMPbinding function; this phenomenon has led to a search for a definite TIMP-1 receptor [21]. TIMP-1 affects neuronal development by altering dendrite outgrowth [16]. These intriguing functions, along with TIMP-1 being the inducible form and highly prevalent in disease, are currently being studied in the context of cancer, ischemia, Alzheimer's disease and HIV-1associated neurocognitive disorders (HAND) [17,22,23,24].
In the context of HIV-1 infection, TIMP-1 levels are reduced in HAD patients when compared with age-matched controls [5,34]. Additionally, human astrocytes treated with inflammatory cytokines [interleukin (IL)-1b or tumor necrosis factor-a] initially demonstrated increased TIMP-1 expression; however, production regresses during chronic stimulation with IL-1b [34]. Identifying the key pathways initiating IL-1b-induced astrocyte TIMP-1 expression may provide a target for restoring CNS TIMP-1. In this study, we used a combination of small molecule mitogen activated protein kinase (MAPK)-selective inhibitors and IL-1b to explore the role of p38K and ERK1/2 signal transduction pathways in IL-1b-induced astrocyte TIMP-1 expression. We employed reporter and overexpression plasmids along with MAPK-selective inhibitors to study how the IL-1b pathway connects C/EBPb to astrocyte TIMP-1 expression. Herein, we report ERK1/2 may provide a therapeutic target to restore CNS TIMP-1 levels. Furthermore, manipulating C/EBPb expression, along with that of its binding partners, NFkB, AP-1, or others, may be a promising route to manipulating CNS TIMP-1 levels.

Isolation, cultivation, and activation of human astrocytes
Human astrocytes were isolated from first-and early secondtrimester aborted specimens obtained from the Birth Defects Laboratory, University of Washington, Seattle, in full compliance with the ethical guidelines of the NIH. The institutional review boards of both the Universities of Washington and North Texas Health Science Center approved the collection of human tissues for research. The Birth Defects Laboratory obtained written consent from all tissue donors.
Astrocytes were isolated from specimens as originally described by Gardner [34]. Activation of astrocytes was achieved by applying IL-1b for various time intervals. IL-1b is a prototypical inflammatory cytokine expressed during HAD; making IL-1b an excellent choice for studying human astrocyte function during neuroinflammation [35]. We empirically tested IL-1b dosage to determine a concentration that promotes maximal activation of astrocytes, 20 ng/ml. In mouse models infected with IL-1bexpressing adenovirus, IL-1b levels reach 10 ng/mg total protein 7 days post-injection and a mean of 41 ng/mg throughout the striatum [36,37]. Accordingly, these data provide relevant implications for astrocyte function in many pathologies involving neuroinflammation. All experimental conditions were analyzed in multiple replicates, and all results were confirmed in at least two astrocyte donors.
RNA Isolation and real-time reverse transcription polymerase chain reaction (RT 2 PCR) Astrocyte RNA was extracted (RNeasy plus mini kit, Qiagen, Alameda, CA) and reverse-transcribed into cDNA as per the manufacturer's instructions (High Capacity cDNA Reverse Transcription Kit, Life Technologies Corp., Carlsbad, CA). TaqManH 5' nuclease RT 2 PCR assays were performed using a StepOnePlus system (Life Tech). The following TaqManH gene expression assay primers were used: TIMP-1 (Life Tech, C/N: Hs99999139_m1), C/EBPb (C/N: Hs00270923_s1) and glyceraldehyde phosphate dehydrogenase (GAPDH; C/N: 4310859). The reactions were carried out at 48uC for 30 min, 95uC for 10 min, followed by 40 cycles of 95uC for 15 s and 60uC for 1 min. Transcripts were quantified by the comparative C T method, and are represented as fold-change of control [42]. Samples were analyzed in triplicate.

Western Blot
Astrocytes were cultured as adherent monolayers in 75 cm 2 flasks at a density of 8610 6 cells per flask. The following day, cells were treated with MAPK-selective inhibitors (SB203580 and U0126) for one h and then IL-1b (20 ng/ml) for 24 h. Alternately, for western blot detection of protein phosphorylation status, astrocytes were activated for 30 min with IL-1b. Cells were lysed; total cell extracts were isolated using mammalian protein extraction reagent (Thermo Fisher Scientific, Waltham, MA, USA) or nuclear extracts were isolated using nuclear extraction reagent (Sigma-Aldrich Inc., St. Louis, MO). Equal amounts of protein (15 mg/lane) were resolved by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently transferred to a polyvinyl diflouride membrane using i-Blot (Life Tech). The membrane was incubated in anti-C/EBPb (C-19, Santa Cruz), p38K, phosphorylated (P)-p38K, ERK and P-ERK1/2 at a dilution of 1:200 (Cell Signaling Inc., Boston, MA, USA). Blots were then incubated in secondary antibody at a dilution of 1:5,000. b-actin (Sigma) or lamin A/C (Cell Signaling), as a loading control. The western blot was visualized with supersignal chemiluminescent substrate (Thermo Fisher) and band intensities were quantified by densitometry analysis (ProteinSimple, Santa Clara, CA, USA).
Measurement of TIMP-1 protein and 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay TIMP-1 levels in astrocyte supernatants were measured using a commercially available enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems, Minneapolis, MN). The MTT assay was performed at appropriate time points according to the method originally described by Manthrope [43]. The ELISA determinations yielded quantities of protein in units of ng/ml and were normalized to MTT values.

Transfection of primary human astrocytes with luciferase reporter constructs
Three TIMP-1-luc promoter plasmid (pTIMP-1-luc) constructs were used to measure TIMP-1 promoter activity in transfected astrocytes. The 24200/+96 and 21718/+96 portions of the TIMP-1 sequence were cloned into the pGL3-basic reporter vector (Promega, Madison, WI) and were kindly provided by Dr. Ian Clark at the University of East Anglia, UK [44].
Astrocytes were cultured as adherent monolayers in a 48-well plate at a density of 0.15610 6 cells per well. The following day, cells were transfected with 1.5 mg of total DNA using Lipofectamine TM reagent as per the manufacturer's instructions (Life Tech). Total DNA consisted of a mixture of the pTIMP-1luc and the pGL3-driven Renilla luciferase (pGL3-RL, Promega) plasmids. Twenty-four h post-transfection media was aspirated from selected wells and replaced with media supplemented with MAPK-selective inhibitors at 20 mM for 24 h with or without 20 ng/ml IL-1b. At two days post-transfection promoter activity was measured as luciferase activity in cell extracts using the Dual-GloH luciferase assay system per the manufacturers instructions (Promega). Luciferase activity was determined as a ratio of firefly to Renilla luciferase using the a Glomax luminimeter (Promega). Renilla luciferase activity was used as an internal control. All experimental conditions were analyzed in multiple replicates, and all results were confirmed in at least two astrocyte donors.

Immunocytochemistry
Astrocytes were cultured as adherent monolayers in a 48-well plate at a density of 0.1610 6 cells per well. The following day, cells were treated with 20 mM concentration of SB203580 or U0126 for 1 h and then with IL-1b for 24 h. Cells were fixed with cold acetone:methanol (1:1) for 30 mins at 220uC. Cells were then blocked in phosphate buffered saline (PBS) with 2% bovine serum albumin for 1 h at room temperature. Cells were incubated in PBS (2% bovine serum albumin, 0.1% triton X) plus C/EBPbantibody at 1:500 and glial fibrillary acidic protein (GFAP) at 1:600 for 8 h at 4uC. Cells were washed with PBS and then incubated with Alexa Fluor secondary antibody (Life Tech, 1:800) for 1 h at room temperature. Micrographs were taken on a Nikon Eclipse T i . (Nikon Inc., Melville, NY, USA).

Statistical Analyses
Results were analyzed with GraphPad Prism 5.0 (GraphPad Software, Inc., La Jolla, CA) using one-way analysis of variance with Newman Keul's post-test for multiple comparisons. Significance was set at p,0.05 and data represent mean values 6 standard error of the mean. Data presented are representative of a minimum of three independent experiments with two or more independent donors. In all figures, statistical significance is represented as * p,0.05, ** p,0.01, and *** p,0.001, significance indicates versus control unless indicated by bar.
As a transcription factor, C/EBPb is localized to nuclei where it can affect gene transcription. We pretreated astrocytes with inhibitors and then IL-1b for 24 h, fixed and colocalized GFAP as an astrocyte-specific marker (green) with C/EBPb (red) in control and activated human astrocytes. In control cells, GFAP is present throughout the cell body of the astrocytes. Control astrocytes have a larger cell body ( Figure 3A), whereas activated astrocytes have extensive processes protruding from their soma and denser staining of GFAP ( Figure 3B-D). Low levels of C/EBPb are present in the nuclei of control human astrocytes ( Figure 3A) compared with activated astrocytes (Figure 3B), where the red signal from the nuclei is markedly enhanced. Low levels of C/ EBPb are present in the nuclei of SB203580-pretreated astrocytes ( Figure 3C) compared to those treated with IL-1b alone ( Figure 3B) or U0126-pretreated astrocytes ( Figure 3D). Intense red signal is detected in nuclei of U0126-pretreated astrocytes ( Figure 3D). Combined with the mRNA and protein expression data represented earlier, these data suggest that blocking p38K activation decreases IL-1b-induced astrocyte C/EBPb nuclear expression.

Discussion
Currently many studies focus on TIMP-1 activity in many diseases, multiple tissues and cell types [16,17,45]; however, the regulation of TIMP-1 expression is as important as its activity in the tissue microenvironment. As previously mentioned, data suggest multiple signal transduction pathways may regulate TIMP-1 expression depending upon stimuli and the expressing cell [46]. In these studies we aimed to identify signaling pathways responsible for IL-1b-induced astrocyte TIMP-1 expression. Our data indicate, for the first time, that the ERK1/2 pathway is essential for IL-1b-mediated increases in astrocyte TIMP-1 and ERK1/2-targeted pharmacological inhibitors will prove sufficient in abrogating astrocyte-TIMP-1 upregulation. In these studies, we show that the p38K-selective inhibitors partially, but significantly, reduce IL-1b-mediated increases in astrocyte TIMP-1 mRNA in a dose-dependent manner up to 1 mM; at which point, it plateaus. p38K-selective inhibitors also reduced IL-1b-induced astrocyte TIMP-1 protein, suggesting the p38K pathway contributes to IL-1b-mediated increases in astrocyte TIMP-1. Concentrations of ERK1/2-selective inhibitor, U0126, as low as 0.1 mM significantly reduced IL-1b-mediated increases in astrocyte TIMP-1; however, 5-20 mM concentrations reduced TIMP-1 mRNA below basal levels. Inhibiting the ERK1/2 pathway is sufficient to block IL-1bmediated increases in astrocyte TIMP-1 protein, indicating that the ERK1/2 pathway is critical for IL-1b-mediated increases in astrocyte TIMP-1. ERK1/2-selective inhibitors were most effective at blocking IL-1b-induced astrocyte TIMP-1 expression, at all doses tested. Blocking p38K activity diminishes IL-1b-mediated increases in astrocyte C/EBPb. Interestingly, blocking ERK1/2 activation with 20 mM of U0126 may enhance IL-1b-induced C/ EBPb expression while at the same time blocking IL-1b-mediated increases in astrocyte TIMP-1 expression. Lastly, blocking ERK1/ 2 activation was shown to block IL-1b-or C/EBPb-mediated increases in TIMP-1 promoter activity, suggesting ERK1/2 signals to key transcription factors necessary for C/EBPb-mediated TIMP-1 promoter regulation. These data may shed light on how astrocytes regulate C/EBPb and TIMP-1 expression during neuroinflammation by identifying essential and auxiliary pathways.
Inflammatory stimuli lead to rapid phosphorylation of MAPK, p38K and ERK1/2 in astrocytes, initiating a cascade of signal transduction events that ultimately influence cellular function [47]. ERK1/2 and p38K regulate astrocyte morphology, cell death and extracellular matrix turnover [38,39,40]. Furthermore, small molecule inhibitors of signal transduction pathways are being explored as treatment options for multiple diseases [41]. At minimum, small molecule inhibitors are useful tools to identify pathways responsible for cellular processes [48]. The p38Kselective inhibitor, SB203580, inhibited IL-1b-mediated increases in astrocyte TIMP-1 mRNA by 50% at doses as low as 5 mM and as high as 20 mM. Pretreatment with the 0.1-20 mM concentrations of ERK1/2-selective inhibitor, U0126, reduced IL-1bmediated increases in astrocyte TIMP-1 mRNA expression to basal levels. Additional p38K-(SB202190) and ERK1/2-(PD98059) selective inhibitors were similarly able to block IL-1bmediated increases in astrocyte TIMP-1 expression ( Figure S2). Crosstalk among MAPK proteins may account for multiple MAPK-selective inhibitors' effect on IL-1b-mediated increases in astrocyte TIMP-1. Alternatively p38K and ERK1/2 may phosphorylate, to different degrees, the same or different transcription factors that bind to astrocyte TIMP-1 promoter. In either case, we add TIMP-1, a multifunctional molecule of major importance to CNS health and disease, to the list of astrocyte genes critically regulated by the ERK1/2 pathway.
It is well established that NFkB mediates IL-1b-induced gene expression in many cell types, including astrocytes. However, NFkB activity alone may not account for the massive changes in gene expression following immune stimulation; indeed, p65 and p50 associate with multiple factors, including C/EBP's, to regulate gene transcription [49]. Wilczynska et al. showed that NFkB is involved in IL-1b-induced astrocyte TIMP-1 expression [32]. Here, we focused on elucidating the role of another transcription factor, C/EBPb that is expressed in astrocytes and is associated with inflammatory responses. C/EBPb is expressed in the brain of HIV-1-infected individuals and contributes to IL-1b-mediated increases in astrocyte TIMP-1 [33]. Therefore, it was surprising that ERK1/2-selective inhibitors did not reduce and may enhance IL-1b-mediated increases in astrocyte C/EBPb expression while decreasing TIMP-1 expression. p38K-selective inhibitor, SB203580, decreased IL-1b-mediated increases in astrocyte C/ EBPb and TIMP-1 expression. The fact that ERK1/2 inhibition strongly blocks IL-1b-mediated increases in astrocyte TIMP-1 suggests ERK1/2 may activate one or more critical transcription factors that lay the framework for IL-1b-mediated increases in astrocyte TIMP-1, independent of C/EBPb activity. Consistent with previous reports, IL-1b induces transcription from the TIMP-1 promoter region containing the upstream elements between  . ERK1/2-selective inhibitor, U0126, overrides IL-1b-and C/EBPb-mediated increases in TIMP-1 promoter activity. Astrocytes were cultured in 48-well plates for 24 h and then cotransfected with TIMP-1 promoter-driven luciferase plasmids (B, 24200/+96 or C 21718/+96) and internal control pGL3-RL. (A) The 24200/+96 promoter harbors an IL-1b responsive element, while the 21718/+96 is the basal TIMP-1 promoter. Both promoters possess a predicted CCAAT-binding site prior to the start site [44]. Cells were allowed to recover for 24 h and then treated with MAPKselective inhibitors (SB203580, 20 mM; and U0126, 20 mM) for 1 h prior to activation with IL-1b (20 ng/ml) for 24 h. Total cell lysates were collected 24 h post-inhibitor treatment and assayed for luciferase expression. Data represent mean values 6 standard error of the mean in at least three independent experiments in at least two independent donors ( * p,0.05, ** p,0.01, *** p,0.001; significance indicates versus untreated unless indicated by bar). doi:10.1371/journal.pone.0056891.g004 22200 and 22700 [32]. However, ERK1/2 inhibition was more effective than p38K inhibition at blocking IL-1b-induced astrocyte TIMP-1 expression; suggesting ERK1/2 may activate factors that bind the TIMP-1 promoter IL-1b-responsive element. Data suggest that ERK1/2 signaling can repress C/EBPb activity through direct phosphorylation [49]. In control human astrocytes ERK1/2 activity may affect C/EBPb activity indirectly through regulation of binding partners. C/EBPb may utilize some, or all, of the TIMP-1 basal promoter, exon 1 and 2 and five CCAAT sites [44]. Our data show that IL-1b induces C/EBPb nuclear localization in U0126-pretreated cells; suggesting other critical factors required for C/EBPb increases in TIMP-1 promoter activity may be absent in these conditions. These data suggest that ERK1/2 activity is required for IL-1b-and C/EBPb-mediated increases in astrocyte TIMP-1 expression.

Conclusions
TIMP-1 expression has been studied in several cell types, but astrocyte TIMP-1 regulation is not completely understood [14,24,50]. Wilczynska et al. showed NFkB and p38K are involved in IL-1b-induced astrocyte TIMP-1 expression; however, our studies suggest, though p38K may be involved, ERK1/2 is critical for basal and IL-1b-activated astrocyte TIMP-1 expression ( Figure 5) [32]. Recently it was reported that TIMP-1 activates the AKT pathway in fibroblasts, indicating the molecule initiates further cascading of signal transduction, presumably playing an important role in response to inflammation [51]. Given the multiple functions of TIMP-1, restoring TIMP-1 levels in HAND patients may prevent or reverse deleterious effects of an MMP:TIMP imbalance and TIMP-1 deficiency. Knowledge of astrocyte TIMP-1 regulation may lead to identification of novel pharmacological targets, and ultimately therapies for HAND patients. Figure S1 MAPK-selective inhibitors block IL-1b-induced astrocyte p38K and ERK1/2 phosphorylation. (A and C) Astrocytes were treated with of p38K-and ERK1/2selective inhibitors (SB203580 and U0126, respectively) for 1 h, and then IL-1b (20 ng/ml). Total protein extracts were isolated 30 min post-IL-1b treatment, resolved by SDS-PAGE and then immunoblotted for p38K, P-p38K, ERK/2 and P-ERK1/2. (B and D) Phosphorylated and nonphosphorylated isoforms' band intensity was analyzed by densitometry analysis. Data presented are representative of a minimum of three independent experiments with two independent donors (n = 2), ( * p,0.05; significance indicates versus untreated unless indicated by bar). (TIF) Figure S2 p38K-and ERK1/2-selective small molecule inhibitors alter IL-1b-mediated astrocyte TIMP-1 and C/EBPb expression. (A and C) Astrocytes were treated with the p38K-selective inhibitor, SB202190, for 1 h, and then IL-1b (20 ng/ml) for 12 h. Total RNA was collected and reverse transcribed. TIMP-1, C/EBPb and GAPDH transcripts were quantified by real-time PCR. (B and D) Astrocytes were pretreated with the ERK1/2-selective inhibitor, PD98059, for 1 h, and then IL-1b (20 ng/ml) for 12 h. Total RNA was collected and reverse transcribed. TIMP-1, C/EBPb and GAPDH transcripts were quantified by real-time PCR. Data presented are representative of a minimum of three independent experiments with two or more independent donors ( * p,0.05, *** p,0.001; significance indicates versus untreated unless indicated by bar). (TIF) Figure 5. ERK1/2 is the predominant signaling pathway facilitating IL-1b-mediated astrocyte TIMP-1 expression. Neuroinflammation activates bystander microglia and astrocytes to secrete IL-1b. Astrocytes respond to IL-1b by secreting TIMP-1. In this study we use a panel of small molecule pathway-selective inhibitors to delineate the signal transduction mechanism facilitating IL-1b-induced astrocyte TIMP-1 expression. p38K-and ERK1/2-selective inhibitors reduced and completely blocked IL-1b-induced astrocyte TIMP-1 mRNA and protein expression, respectively. U0126, the ERK1/2-selective inhibitor, also blocked TIMP-1 promoter activity. doi:10.1371/journal.pone.0056891.g005