Disparate effects of serum on basal and evoked NFAT activity in primary astrocyte cultures

Abstract

In astrocytes, the Ca2+-dependent protein phosphatase calcineurin (CN) strongly regulates neuro-immune/inflammatory cascades through activation of the transcription factor, nuclear factor of activated T cells (NFAT). While primary cell cultures provide a useful model system for investigating astrocytic CN/NFAT signaling, variable results may arise both within and across labs because of differences in culture conditions. Here, we determined the extent to which serum and cell confluency affect basal and evoked astrocytic NFAT activity in primary cortical astrocyte cultures. Cells were grown to either ~50% or >90% confluency, pre-loaded with an NFAT-luciferase reporter construct, and maintained for 16 h in medium with or without 10% fetal bovine serum (FBS). NFAT-dependent luciferase expression was then measured 5 h after treatment with vehicle alone to assess basal NFAT activity, or with Ca2+ mobilizers and IL-1β to assess evoked activity. The results revealed significantly higher levels of basal NFAT activity in FBS-containing medium, regardless of cell confluency. Conversely, evoked NFAT activation was significantly lower in serum-containing medium, with an even greater inhibition observed in confluent cultures. Application of 10% FBS to serum-free astrocyte cultures quickly evoked a roughly seven-fold increase in NFAT activity that was significantly reduced by co-delivery of neutralizing agents for IL-1β, TNFα, and/or IFNγ, suggesting that serum occludes evoked NFAT activation through a cytokine-based mechanism. Together, the results demonstrate that the presence of serum and cell confluency have a major impact on CN/NFAT signaling in primary astrocyte cultures and therefore must be taken into consideration when using this model system.

Cooperative roles of NF-κB and NFAT4 in polyomavirus JC regulation at the KB control element

“…In an alternative scenario, another cytokine such as IL-1β, which evokes NFAT activity in primary astrocytes (Furman et al, 2010; Sama et al, 2009) and is also upregulated in the CNS in HIV/AIDS “cytokine storms” (Benveniste, 1994; Kaul et al, 2005) may be involved in activation of the NFAT pathway and may act synergistically with TNF-α/NF-κB signaling to activate JCV through the KB element. IL-6 is another cytokine that has been reported to be upregulated in the CNS in HIV/AIDS (Benveniste, 1994; Yeung et al, 1995) and can activate NFAT signaling (Diehl and Rincón, 2002).…”

Progesterone inhibition of voltage-gated calcium channels is a potential neuroprotective mechanism against excitotoxicity

“…This would include the inhibition of NFAT- and FasL-dependent apoptosis [22, 23]. Interestingly, NFAT has also been shown to be involved in the initiation of neuro-inflammation [41, 42], and thus progesterone may be neuroprotective via inhibition of this pathway as well. Interestingly, progesterone-mediated inhibition of CRE-dependent transcription would appear to be counterproductive, as CREB regulates a number of pro-survival genes.…”

Proteolysis of calcineurin is increased in human hippocampus during mild cognitive impairment and is stimulated by oligomeric Abeta in primary cell culture

“…Methods for eliciting and measuring NFAT-dependent transcriptional activation in primary cultures were very similar to those used previously by our group (Sama et al 2008; Abdul et al 2009; Furman et al 2010). At approximately 24 hr before treatment with oligomeric Aβ, 35 mm hippocampal culture dishes were infected (at a multiplicity of infection of 100) with recombinant adenovirus encoding an NFAT-dependent luciferase reporter construct (Ad-NFAT-Luc), provided by Dr. Jeff Molkentin at the University of Cincinnati.…”