Currently many studies focus on TIMP-1 activity in many diseases, multiple tissues and cell types 
; 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 
. In these studies we aimed to identify signaling pathways responsible for IL-1β-induced astrocyte TIMP-1 expression. Our data indicate, for the first time, that the ERK1/2 pathway is essential for IL-1β-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-1β-mediated increases in astrocyte TIMP-1 mRNA in a dose-dependent manner up to 1 µM; at which point, it plateaus. p38K-selective inhibitors also reduced IL-1β-induced astrocyte TIMP-1 protein, suggesting the p38K pathway contributes to IL-1β-mediated increases in astrocyte TIMP-1. Concentrations of ERK1/2-selective inhibitor, U0126, as low as 0.1 µM significantly reduced IL-1β-mediated increases in astrocyte TIMP-1; however, 5–20 µM concentrations reduced TIMP-1 mRNA below basal levels. Inhibiting the ERK1/2 pathway is sufficient to block IL-1β-mediated increases in astrocyte TIMP-1 protein, indicating that the ERK1/2 pathway is critical for IL-1β-mediated increases in astrocyte TIMP-1. ERK1/2-selective inhibitors were most effective at blocking IL-1β-induced astrocyte TIMP-1 expression, at all doses tested. Blocking p38K activity diminishes IL-1β-mediated increases in astrocyte C/EBPβ. Interestingly, blocking ERK1/2 activation with 20 µM of U0126 may enhance IL-1β-induced C/EBPβ expression while at the same time blocking IL-1β-mediated increases in astrocyte TIMP-1 expression. Lastly, blocking ERK1/2 activation was shown to block IL-1β- or C/EBPβ-mediated increases in TIMP-1 promoter activity, suggesting ERK1/2 signals to key transcription factors necessary for C/EBPβ-mediated TIMP-1 promoter regulation. These data may shed light on how astrocytes regulate C/EBPβ 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 
. ERK1/2 and p38K regulate astrocyte morphology, cell death and extracellular matrix turnover 
. Furthermore, small molecule inhibitors of signal transduction pathways are being explored as treatment options for multiple diseases 
. At minimum, small molecule inhibitors are useful tools to identify pathways responsible for cellular processes 
. The p38K-selective inhibitor, SB203580, inhibited IL-1β-mediated increases in astrocyte TIMP-1 mRNA by 50% at doses as low as 5 µM and as high as 20 µM. Pretreatment with the 0.1-20 µM concentrations of ERK1/2-selective inhibitor, U0126, reduced IL-1β-mediated 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-1β-mediated increases in astrocyte TIMP-1 expression (Figure S2
). Crosstalk among MAPK proteins may account for multiple MAPK-selective inhibitors' effect on IL-1β-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 NFκB mediates IL-1β-induced gene expression in many cell types, including astrocytes. However, NFκB 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
. Wilczynska et al
. showed that NFκB is involved in IL-1β-induced astrocyte TIMP-1 expression 
. Here, we focused on elucidating the role of another transcription factor, C/EBPβ that is expressed in astrocytes and is associated with inflammatory responses. C/EBPβ is expressed in the brain of HIV-1-infected individuals and contributes to IL-1β-mediated increases in astrocyte TIMP-1 
. Therefore, it was surprising that ERK1/2-selective inhibitors did not reduce and may enhance IL-1β-mediated increases in astrocyte C/EBPβ expression while decreasing TIMP-1 expression. p38K-selective inhibitor, SB203580, decreased IL-1β-mediated increases in astrocyte C/EBPβ and TIMP-1 expression. The fact that ERK1/2 inhibition strongly blocks IL-1β-mediated increases in astrocyte TIMP-1 suggests ERK1/2 may activate one or more critical transcription factors that lay the framework for IL-1β-mediated increases in astrocyte TIMP-1, independent of C/EBPβ activity. Consistent with previous reports, IL-1β induces transcription from the TIMP-1 promoter region containing the upstream elements between −2200 and −2700 
. However, ERK1/2 inhibition was more effective than p38K inhibition at blocking IL-1β-induced astrocyte TIMP-1 expression; suggesting ERK1/2 may activate factors that bind the TIMP-1 promoter IL-1β-responsive element. Data suggest that ERK1/2 signaling can repress C/EBPβ activity through direct phosphorylation 
. In control human astrocytes ERK1/2 activity may affect C/EBPβ activity indirectly through regulation of binding partners. C/EBPβ may utilize some, or all, of the TIMP-1 basal promoter, exon 1 and 2 and five CCAAT sites 
. Our data show that IL-1β induces C/EBPβ nuclear localization in U0126-pretreated cells; suggesting other critical factors required for C/EBPβ increases in TIMP-1 promoter activity may be absent in these conditions. These data suggest that ERK1/2 activity is required for IL-1β- and C/EBPβ-mediated increases in astrocyte TIMP-1 expression.