Disruption of Astrocyte STAT3 Signaling Decreases
Mitochondrial Function and Increases Oxidative Stress In
Theodore A. Sarafian1*, Cindy Montes1, Tetsuya Imura1, Jingwei Qi1, Giovanni Coppola2, Daniel H.
Geschwind2, Michael V. Sofroniew1
1Department of Neurobiology, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California, United States of America, 2Department of
Neurology, The Semel Institute for Neuroscience and Human Behavior, David Geffen School of Medicine, University of California Los Angeles, California, United States of
Background: Astrocytes exert a wide variety of functions in health and disease and respond to a wide range of signaling
pathways, including members of the Janus-kinase signal transducers and activators of transcription (Jak-STAT) family. We
have recently shown that STAT3 is an important regulator of astrocyte reactivity after spinal cord injury in vivo .
Methodology/Principal Findings: Here, we used both a conditional gene deletion strategy that targets the deletion of
STAT3 selectively to astrocytes (STAT3-CKO), and a pharmacological inhibitor of JAK-2, AG490, in cultured astrocytes in vitro,
to investigate potential functions and molecules influenced by STAT3 signaling in relation to mitochondrial function and
oxidative stress. Our findings show that the absence of STAT3 signaling in astrocytes leads to (i) increased production of
superoxide anion and other reactive oxygen species and decreased level of glutathione, (ii) decreased mitochondrial
membrane potential and decreased ATP production, and (iii) decreased rate of cell proliferation. Many of the differences
observed in STAT3-CKO astrocytes were distinctly altered by exposure to rotenone, suggesting a role for complex I of the
mitochondrial electron transport chain. Gene expression microarray studies identified numerous changes in STAT3-CKO
cells that may have contributed to the identified deficits in cell function.
Conclusions/Significance: Taken together, these STAT3-dependent alterations in cell function and gene expression have
relevance to both reactive gliosis and to the support and protection of surrounding cells in neural tissue.
Citation: Sarafian TA, Montes C, Imura T, Qi J, Coppola G, et al. (2010) Disruption of Astrocyte STAT3 Signaling Decreases Mitochondrial Function and Increases
Oxidative Stress In Vitro. PLoS ONE 5(3): e9532. doi:10.1371/journal.pone.0009532
Editor: Colin Combs, University of North Dakota, United States of America
Received September 24, 2009; Accepted February 8, 2010; Published March 10, 2010
Copyright: ? 2010 Sarafian et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by National Institutes of Health grants 5R01NS057624 (MVS) and DA021580 (TS) and by the Adelson Medical Research
Foundation (www.adelsonfoundation.org) (Adelson Program in Neural Rehabilitation and Repair). The funders had no role in study design, data collection and
analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com
Astrocytes play many essential roles in the healthy central nervous
system (CNS), including regulation of blood flow, provision of energy
and maintenance of the extracellular balance of ions, fluids and
transmitters [2,3]. In addition, astrocytes are primary responders to
disease, where they exert important tissue defense mechanisms or
where their dysfunction may be involved in disease pathology [4–7].
Astrocytes are able to take part in this broad range of activities in part
by being able to respond to a plethora of extra- and intra-cellular
signaling mechanisms that regulate their functions and molecular
mechanisms that regulate astrocyte activities is of interest in
understanding normal function in the healthy CNS, understanding
disease mechanisms and identifying potential novel therapeutic targets.
Many molecules have been implicated as triggers of astrogliosis,
including a broad group of growth factors and cytokines that signal
through members of the Janus-kinase signal transducers and
activators of transcription (Jak-STAT) signaling family [1,10,11].
Using a transgenic conditional gene deletion strategy, we have
recently shown that one intracellular member of this family, STAT3,
is a particularly important regulator of astrocyte reactivity after spinal
cord injury in vivo, such that conditional deletion of STAT3 signaling
from astrocytes attenuated reactive astrogliosis and disrupted scar
formation, which was associated with increased inflammation,
increased lesion size and decreased recovery of motor function .
In this study, we used both a genetic conditional deletion strategy and
pharmacological inhibition of STAT3 to assess whether STAT3
deficient astrocytes have impaired function that may be detrimental
to the astrocytes and ultimately to surrounding CNS tissue.
Materials and Methods
Generation of mice deficient in STAT3 expression selectively
in astrocytes (STAT3-CKO) using glial fibrillary acidic protein
PLoS ONE | www.plosone.org1 March 2010 | Volume 5 | Issue 3 | e9532
(GFAP) promoter-directed Cre/loxP technology was described
previously . Mice were genotyped for Cre recombinase (Cre)
and loxP sequence by DNA isolation from liver sections using the
Qiagen DNAeasy kit (Valencia, CA) followed by PCR and agarose
gel electrophoresis as described previously. Experiments were
performed according to protocols approved by the Chancellor’s
Animal Research Committee of the Office for Protection of
Research Subjects at the University of California, Los Angeles.
Culture media and trypsin were obtained from HyClone (Logan,
UT). Versene was purchased from Gibco (Gaithersburg, MD).
Fluorescent probes dichlorofluoresceine-diacetate (DCF), dihy-
droethidine (HE), 5,59,6,69- tetrachloro-1,19,3,39-tetraethylbenzi-
midazolylcarbocyanin iodide (JC-1), MitoSOX Red, MitoTracker
Green, monochlorobimane (MCB), and propidium iodide (PI)
were obtained from Molecular Probes (Eugene, OR). The Cell-
Titre-Glo luminescence viability assay kit was from Promega
(Madison, WI). Primary antibody to GFAP was from Dako (rabbit,
was from Cell Signaling Technology, Inc. (rabbit 1:500, Danvers,
MA) to S100 was from QED Bioscience, Inc. (sheep, San Diego,
CA), to p-p38, pJNK, and pErk MAP kinases were from Santa Cruz
Biotechnology (rabbit 1:1000, Santa Cruz, CA) and to actin and
glutamine synthetase were from Sigma (rabbit 1:4000, St. Louis,
MO). Conjugated secondary antibodies to rabbit, goat, and donkey
IgG were from Bio Rad (1:1000, Hercules, CA). All other reagents
were from Sigma.
Astrocyte cell cultures were prepared from 2-3-day-old STAT3
conditional knock-out (STAT3-CKO) mice and from littermate
CRE-negative controls (STAT3 +/+) by a modification of the
procedure of McCarthy and De Vellis . Cerebral cortices from
individual mice were isolated and, after removal of meninges,
placed in 10614 cm Stomacher bags with 2 ml culture medium
(DMEM/Ham’s F12 with 4.5 g/l glucose, 15 mM HEPES, 2 mM
Glutamine, 10% fetal bovine serum and 1% Penicillin/Strepto-
mycin). The tissue was disaggregated manually by finger
compression for 3 min, triturated with 12 passes through a 1 ml
pipetter and filtered through a 100 mm nylon cell strainer. Cells
were then centrifuged at 800 rpm for 5 min, resuspended in
culture medium and transferred to one T-75 culture flask. Media
were replaced twice weekly. Upon reaching 90% confluence, cells
were passaged (1:3 in surface area) in order to generate sufficient
cell number. For passage, cells were washed three times with
phosphate-buffered saline (PBS) and incubated with 4 ml Versene
(0.02% EDTA in PBS) for 20 min at 37u C. Then 1.5 ml of 0.05%
trypsin and 0.02% EDTA were added and incubated 8 min at 37u
C. Following addition of 1.5 ml trypsin neutralizing solution
(Clonetics/Lonza), cells were collected by centrifugation (800g,
4 min) and counted with a hemocytometer.
Upon reaching 70–90% confluence, cells cultured in T75 flasks
were used for gene expression array studies and in multi-well
plates for all other experiments. Levels of GFAP and STAT3
expression did not change significantly in STAT3 +/+ cells during
the culture periods used in these studies. Comparisons between
STAT3 +/+ and STAT3-CKO for glutathione and ROS levels
did not change as a function of days in vitro or between passages 2
Astrocyte cultures were prepared in 48-well plates and fixed
with formalin. Cells were stained with anti-GFAP (1:2000), anti
S100b (1:1000) or anti BrdU (1:6000) followed by AlexaFluor-
tagged secondary antibodies Alexa 488 (green) or Alexa 568 (red).
Cells stained for BrdU were pretreated with 2M HCl for 30 min
and rinsed three times with PBS. Images were recorded by
fluorescence microscopy (Zeiss, Oberkochen, Germany).
Western Blot Staining
Cells cultured in 12-well plates were washed three times with
PBS and extracted with RIPA lysis buffer containing 0.8 mM
aprotinin, 20 mM leupeptin, 10 mM pepstatin A, 2 mM phenyl-
methylsulfonyl fluoride, 20 mM NaF and 1 mM sodium orthova-
nadate. Following protein measurement using The Bio-Rad DC
protein assay, 25 mg protein per well was applied to 4–12%
gradient SDS polyacrylamide gels (Invitrogen). Electrophoresis
was run at 100–130V for 2.5 hr followed by transfer to Hybond-P
membranes (Amersham Biosciences) for 1.5 hr at 30V. Mem-
branes were then blocked with 5% dry milk powder in Tris-
buffered saline with 0.05% Tween 20 and stained with 1:500
dilutions of various antibodies. Following secondary antibody
staining, membranes were exposed to ECL chemiluminescent
reagent (Amersham Biosciences - GE Health Care Bio-Sciences,
Piscataway, NJ) and exposed to Kodak XAR5 film.
Hydroethidine Assay for Superoxide
Cells in 96-well plates were washed with 200 ml of Krebs Ringer
buffer (KR: 25 mM HEPES, pH 7.4, 125 mM NaCl, 5 mM KCl,
1.2 mM KH2PO4, 5 mM NaHCO3, 6 mM glucose, 1.2 mM
MgSO4and 1 mM CaCl2). Rotenone, or carbonyl cyanide p-
trifluoromethoxy phenylhydrazone (FCCP) or DMSO vehicle
(0.1%) was added in KR to the appropriate rows [13–16]. In select
wells PBS was used to produce glucose-starvation. The plate was
incubated for 30 minutes whereupon toxins were removed and
wells washed once with 200 ml of Krebs Ringer. 10 mM HE in KR
or PBS was then applied and fluorescence was read at Ex=530,
Em=595 over a 30 minute period.
Measurement of Reactive Oxygen Species, Glutathione,
and Total Cell Number
Analysis of astrocyte reactive oxygen species (ROS) production
wasperformedbya modificationofpreviouslydescribed procedures
. Cells were washed twice with 200 ml of Krebs Ringer buffer
KR. 20 mg/ml DCF-DA was added to the wells in 100ml of KR and
the plate was sealed with mylar tape for 20 minutes. The plate was
then washed twice more with 200 ml and 100 ml of KR and toxins
added in 100 ml KR. Fluorescence readings were taken every
15 minutes for 1 hour at Ex=485 and Em=530. Then KR
containing the toxins was removed and replaced with100 ml 40 mM
MCB in KR to determine glutathione (GSH) levels. The cells were
incubated with the MCB for 20 minutes at 37uC and fluorescence
readings taken at Ex=390, Em=460. Ten ml of 0.5 mM PI was
added to the wells and, after incubation for 15 minutes at room
temperature, red fluorescence was read at Ex=535, Em=590. Ten
ml of 1.6 mM digitonin was then added to each well and incubated
for 20 minutes at room temperature. PI fluorescence measurement
was repeated to quantify total cell number which was used to
normalize ROS and GSH levels. For each probe used, subtracted
background values were obtained from wells containing fluorescent
probe without cells.
Mitochondrial Membrane Potential, ROS, and Mass
Mitochondrial membrane potential was measured by using the
fluorescent probe JC-1 as described previously . Rotenone,
antimycin A, or FCCP were added to wells in a 96-well plate.
Then 1 mg/ml JC-1 in culture media was added and the plate was
incubated at 37u in a CO2incubator for 1 hour. Red and green
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fluorescence measurements were taken at 2, 30, and 60 minutes
using Ex=485, Em=530 for green and Ex=530, Em=590 for
red. Following subtraction of blank values, red/green fluorescence
ratios were calculated for each well using data from 60 min
incubation. These data were compared with those from 2 and
30 min to verify appropriate time-dependent changes.
Mitochondrial-specific ROS was measured using MitoSOX
Red. Astrocytes cultured in 96-well plates were exposed to 4 mM
MitoSOX Red in culture media containing toxins or DMSO
vehicle control. In order to measure total mitochondrial mass,
parallel wells contained 0.1 mM MitoTracker Green which
produces a green fluorescence independent of mitochondrial
membrane potential. Following 2 hr incubation in a CO2
incubator, green and red fluorescence was measured as described
for JC-1. Background fluorescence was determined from wells
containing probes without cells and subtracted from respective red
and green fluorescence values. Red fluorescence values were
normalized to mitochondrial mass represented by averaged
MitoTracker Green fluorescence values from respective STAT3
+/+ or CKO cells.
STAT3 +/+ and STAT3-CKO cells were grown in 96-well
plates to 90% confluence. Media was removed from the wells and
50 ml of firefly extract from the CellTitre-Glo Viabiltiy assay kit
(Promega) was added to each well. The plate was shaken for
3 minutes and samples were transferred to a white 96-well plate.
The wells were rinsed with 50 ml of 10 mM Tris-HCl pH 7.4 and
the rinse was added to the samples in the white plate. ATP
standards from 2 to 400 pmole were used to generate a standard
curve. Luminescence was measured with a Molecular Devices
Spectra Max Gemini EM plate reader in top-read mode using
SoftMax Pro v5 Software.
Cells in 96-well were washed three times with 200 ml KR. 50 ml
of 1M NaOH were added to each well and the plate shaken for
3 minutes. 50 ml of 1M HCl was added to each well. Each well
was mixed well with a pipette before 20 ml in duplicate were taken
from each well and placed in a new 96-well plate. IgG was used as
protein standard which included 20 ml of 1 M NaOH/HCl mix
(1:1). 20 ml of Bio-Rad Coomassie Blue reagent was added to
standards and samples. The plate was read on the SLT Spectra
plate reader at 620nm wavelength.
Cell Proliferation Assay
Astrocytes cultured to passage 2 over a period of 3 weeks were
plated into 96-well culture plates at a density of 56103/well. N-
acetylcysteine (0.5 mM) or 1 mM deferroxamine mesylate in
sterile H2O were added 1, 3, and 5 days after plating. After 1, 4,
and 7 days in culture, cell number was quantified using propidium
iodide in the presence of digitonin as described above. Values
obtained after 1 day were subtracted from values after 7 days to
derive the relative increase in cell number. GSH levels were
assayed with monchlorobimane after 4 days as described above.
Gene Expression Micro Array Studies
Total RNA was isolated from astrocytes cultured in T-75 flasks
(P3, 70–80% confluence) using the Qiagen RNeasy Kit protocol
for adherent cells. RNA yields ranged from 1 to 13 mg and had an
A260/280 ratio .1.75. Four replicates were run per condition, for
a total of 8 arrays. RNA quantity was assessed with Nanodrop
(Nanodrop Technologies) and quality with the Agilent Bioanalyzer
(Agilent Technologies). Total RNA (200 ng) was amplified,
biotinylated and hybridized on Illumina Mouse Mouseref-8
Expression Beadchips v1.1, querying the expression of ,22,000
Refseq transcripts, as per manufacturer’s protocol. Slides were
scanned using Illumina BeadStation and signal extracted using
Illumina BeadStudio software (Illumina, San Diego CA). Raw data
was analyzed using Bioconductor packages (www.bioconductor.
org, ). Low level quality-control analysis was performed using
several indices, including inter-array Pearson correlation, cluster-
ing based on variance, and the mean absolute deviation (MAD)
using the top 1000 most variant probes . Data were
normalized using quantile normalization. Analysis of differential
expression was performed using a linear model fitting (LIMMA
package, ). After linear model fitting, a Bayesian estimate of
differential expression was calculated and the threshold for
statistical significance was set at p,0.005 (Bayesian modified
t-test). Differentially expressed genes were classified according to
gene ontology, using Bioconductor packages and online tools
(DAVID/EASE, http://david.abcc.ncifcrf.gov/, WebGestalt, http://
genereg.ornl.gov/webgestalt/). In DAVID, levels 3 and 4 of
molecular function, biological process and cellular localization were
selected. Literature data mining for co-occurrence of gene names
and keywords of interest (e.g., oxidative stress, mitochondria etc.)
was performed using Chilibot (www.chilibot.net). Pathway analysis
was carried out using Ingenuity Pathway Analysis (Ingenuity
Data from in vitro cellular assays were analyzed by 2-way
ANOVA with Bonferroni post-hoc test using Prism Graphpad
software. Cell proliferation assays were analyzed by 1-way
ANOVA with Tukey’s post hoc test.
Characterization of STAT3-CKO Astrocyte Cell Cultures
We have previously demonstrated the specificity of the STAT3-
CKO transgenic model for targeting STAT3 gene deletion to
astrocytes by (i) analyzing Cre mediated activation of reporter
gene expression at the single cell level in vivo and (ii) analyzing the
selective deletion of STAT3 and pSTAT3 from astrocytes in vivo
and in vitro . For the present study, we confirmed and extended
these observations by characterizing in various ways primary
astrocyte cultures prepared from STAT3-CKO mice.
To evaluate the effects of STAT3-CKO on the appearance and
various molecular expression profiles of astrocytes in vitro, we used
primary astrocyte cultures that are over 95% GFAP-expressing
cells [21,22]. Primary astroctye cell cultures prepared from
perinatal STAT3-CKO mice grew well under standard conditions
and had an appearance under phase-contrast microscopy similar
to that of cells from littermate control mice negative for Cre
expression. STAT3-CKO astrocytes exhibited a moderately
reduced expression of GFAP, but expressed S100b or glutamine
synthetase at normal levels as detected by immunocytochemistry
(Figs. 1A,B) and Western blotting (Fig. 1C). In agreement with our
previous report , STAT3-CKO cultures exhibited almost no
detectable pSTAT3, whereas pSTAT3 was present in control
(STAT3 +/+) cultures (Fig. 1C). These findings demonstrate that
(i) STAT3 is activated and signaling in STAT3 +/+ astrocytes
under basal culture conditions that contain serum, and (ii) our
Cre-loxP model for STAT3-CKO effectively deleted signaling via
STAT3. We also looked for potential effects of STAT3-CKO on
other signaling pathways, and found no detectable differences in
the levels of p-p38, pErk and pJnk MAP kinases between control
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and STAT3-CKO astrocytes (Fig. 1C). We therefore focused the
present studies on the effects of STAT3-CKO on astrocyte
functions under standard culture conditions in serum that are
associated with a basal constitutive activation of STAT3.
STAT3-CKO Alters Functions Related to Oxidative Stress
and Antioxidant Defense
We next compared astrocyte properties contributing to
oxidative stress and antioxidant defense. Superoxide (O22) levels
were assessed using the fluorescent probe, HE. Superoxide
generation over a 30-minute period was 56% greater in STAT3-
CKO astrocytes relative to STAT3 +/+ astrocytes (p,0.02) under
control culture conditions (Fig. 2A). The complex I inhibitor,
rotenone, significantly increased superoxide production in both
STAT3 +/+ and STAT3-CKO astrocytes, but did so to a lesser
extent in STAT3-CKO astrocytes as compared to STAT3 +/+
astrocytes. Similar results were obtained with the mitochondrial
ROS were measured using DCF. This probe fluoresces on
reaction with H2O2, hydroxyl radical, nitiric oxide and peroxyni-
trite, but does not react with superoxide . ROS levels were
significantly higher by 15% in STAT3-CKO astrocytes relative to
STAT3 +/+ astrocytes (p,0.05) under control culture conditions
(Fig. 2B). Rotenone significantly and markedly increased ROS by
73% in STAT3-CKO astrocytes relative to values under control
conditions (p,0.001) and had no significant effect on ROS in
STAT3 +/+ astrocytes. Menadione, a generator of ROS via redox
cycling [24,25], significantly and markedly increased ROS by over
90% in both STAT3 +/+ and STAT3-CKO astrocytes relative
to values under control conditions (p,0.001) and eliminated
the significant difference between the two cell types. Glucose
starvation by transfer of cells from control culture conditions to
PBS eliminated the increased ROS generation by STAT3-CKO
astrocytes relative to STAT3 +/+ astrocytes during a one-hour
Glutathione (GSH) is a major component of cellular antioxidant
defense. To assess GSH levels, we used the fluorescent probe,
MCB. GSH levels were significantly lower by 30% in STAT3-
CKO astrocytes relative to STAT3 +/+ astrocytes (Fig. 2A). We
next compared GSH levels after subjecting astrocytes to glucose
starvation or different forms of oxidative stress. Glucose starvation
and H2O2both moderately reduced GSH levels by about 20–35%
in both STAT3 +/+ and STAT3-CKO astrocytes relative to
control conditions, while retaining the significant reductions in
STAT3-CKO astrocytes as compared to STAT3 +/+ astrocytes.
Menadione significantly and markedly decreased GSH by 50% in
Figure 1. Immunofluorescence and Western blot staining of enriched astrocyte cell cultures derived from neonatal forebrain. (A,B)
Single channel and merged images of double labeling immunofluorescence show that in control cultures (A1–A3) essentially all astrocytes express
both GFAP and S100b, whereas in STAT3-CKO cultures (B1–B3) most astrocytes do not express detectable levels of GFAP but do express S100b. (C)
Western blotting of primary astrocyte cultures shows markedly reduced expression of pSTAT3 and GFAP, but not of phosphorylated MAP kinases or
glutamine synthetase (Gl Syn), in STAT3-CKO cultures as compared with controls. Equivalent amounts of total protein were applied to each lane.
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STAT-3 +/+ astrocytes, and by 90% in STAT3-CKO astrocytes
(p,0.001) relative to control culture conditions.
Absence of STAT3 Impairs Astrocyte Mitochondrial
Function and ATP Production
We next looked for potential effects of STAT3-CKO on
astrocyte cell energetics. Mitochondrial membrane potential was
assessed with the dual wavelength fluorescent probe, JC-1. The
ratio of red/green JC-1 fluorescence was significantly lower by
25% in STAT3-CKO astrocytes relative to STAT3 +/+ astrocytes
(p,0.001) under control culture conditions (Fig. 3A). We then
compared the effects of inhibitors of mitochondrial functions.
Rotenone, a selective inhibitor of complex I, decreased mitochon-
drial membrane potential in STAT3 +/+ astrocytes but not in
STAT3-CKO astrocytes, thereby eliminating the significant
difference between the two cell types. Antimycin A, a selective
inhibitor of complex III, significantly decreased mitochondrial
membrane potential in both STAT3 +/+ and STAT3-CKO
astrocytes, while retaining a significant relative reduction in
STAT3-CKO astrocytes as compared to STAT3 +/+ astrocytes.
FCCP, a mitochondrial uncoupler, significantly and markedly
decreased mitochondrial membrane potential in both STAT3 +/+
and STAT3-CKO astrocytes by over 75%, and eliminated the
significant difference between the two cell types.
We also assessed ATP levels which were significantly lower by
10% in STAT3-CKO astrocytes relative to STAT3 +/+ astrocytes
(p,0.05)undercontrolculture conditions (Fig.3B). ATP levelswere
significantly decreased to varying degrees by rotenone, antimycin A
and FCCP inbothSTAT3 +/+ and STAT3-CKO astrocytes, while
in all cases retaining a significant relative reduction in STAT3-
CKO astrocytes as compared to STAT3 +/+ astrocytes.
In order to determine if mitochondria contributed to the elevated
ROS observed in STAT3-CKO cells, the fluorescent probe
MitoSOX Red was used. Unlike DCF and hydroethidine, MitoSOX
Red selectively fluoresces in and is retained by mitochondria as a
function of ROS generation [26,27]. For these studies, parallel
mitochondrial mass, as this probe selectively stains mitochondria
independent of mitochondrial membrane potential or ROS
generation . While apparent mitochondrial mass was ,15%
lower in STAT3-CKO astrocytes compared with STAT3 +/+ (see
inset Fig 3C), mitochondrial ROS was ,35% higher in STAT3-
The increased mitochondrial ROS in STAT3-CKO cells was also
observed in the presence of mitochondrial inhibitors rotenone,
FCCP, and antimycin A. The relative difference between STAT3 +/
A and FCCP than by rotenone.
Cell Proliferation Is Decreased in STAT3-CKO Astrocyte
STAT3 signaling has been implicated in regulating cell pro-
liferation [29,30]. We looked for influences of STAT3-CKO on
Figure 2. Oxidative stress studies using fluorescent probes.
Cortical astrocytes cultured at passage 2 or 3 were assayed in the
presence of 25 mM rotenone, 25 mM FCCP, 100 mM menadione, PBS, or
0.5% DMSO (vehicle control). (A) Superoxide generation was measured
using the fluorescent probe HE as described in Materials and Methods
and was consistently higher in STAT3-CKO cells. Data, expressed as per
cent of STAT3 +/+ control, represent means of 7–8 determinations 6
SEM. * p,0.05 compared with STAT3 +/+. (B) Generation of ROS was
measured using DCF. Values are expressed as DCF fluorescence after
1 hr incubationnormalizedtototalcellnumber derivedby PIfluorescence
determinations 6 SEM. * p,0.01 compared with STAT3 +/+ cells and
^ p,0.05 compared with corresponding vehicle-treated control by 2-way
ANOVA with Bonferroni post-hoc test. (C) Glutathione (GSH) levels were
measured using 40 mM MCB after 30 min incubation at 37 u. Relative
fluorescence values were normalized to total cell number and represent
means of 6 determinations 6 SEM. * p,0.001 compared with corres-
ponding STAT-3 +/+ cells and ^ p,0.05 compared with corresponding
control using 2-way ANOVA with Bonferroni post-hoc test.
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primary astrocyte proliferation in several ways. Qualitatively we
noted that STAT3-CKO cells required longer to reach confluence
after passage and plating, suggesting a slower rate of proliferation.
To evaluate this observation quantitatively, we first compared the
number of S100b-positive astrocytes that incorporated BrdU
administered as a pulse and found that about 3.5% of STAT3-
CKO astrocytes incorporated BrdU over a 6 hour pulse, which
was significantly lower and roughly half of the 7% of STAT3 +/+
astrocytes that incorporated BrdU over the same time period
Cell proliferation was also quantified using a propidium iodide
fluorescence assay. Cell numbers which were similar one day after
plating was ,35% lower in STAT3-CKO cell cultures compared
with STAT3 +/+ after 7 days (Fig. 4D). Since oxidative stress is
known to suppress cell division, we sought to determine if the
observed higher levels of ROS in STAT3-CKO cells played a role
in the lower proliferation rate. To address this issue, the
proliferation assay was repeated in the presence of the antioxidant,
N-acetylcysteine. Inclusion of 0.5 mM N-acetylcysteine in the
medium elevated cellular GSH levels by 10% in STAT3 +/+ cells
and by 34% in STAT3-CKO cells, producing levels of GSH
similar to that in STAST3 +/+ cells. Despite these increases in
GSH level, proliferation rate was not increased in either cell type.
Similar results were obtained with 0.25 mM deferroxamine
mesylate, and iron-chelating anti-oxidant (data not shown).
Effects of Pharmacologic Inhibition of STAT3 Activation In
Vitro Using AG490
We next compared the effects of STAT3-CKO with the
pharmacological blockade of the STAT3 signaling pathway in
astrocyte cultures prepared from wild-type mice using AG490, an
inhibitor of Jak2 kinase. Dose response studies indicated that
AG490 over a concentration range of 10 to 100 mM caused no
increase in cell death measured with propidium iodide (data not
shown). AG490 was used for subsequent studies at 25 mM, a
concentration previously shown to prevent STAT3 tyrosine
phosphorylation in vascular smooth muscle cells . Treatment
of wild-type astrocyte cultures with 25 mM AG490 suppressed
STAT3 phosphorylation under basal conditions (with serum) as
well as in response to added Il-6 (Fig. 5A). In addition, treatment
of wild-type astrocyte cultures with AG490 reproduced changes in
various cell functions observed in STAT3-CKO astrocytes.
Astrocyte proliferation in vitro was significantly and markedly
attenuated in wild-type astrocytes continuously exposed to AG490
(Fig. 5B). Mitochondrial membrane potential was significantly
decreased by 30% in wild-type astrocytes exposed to AG490 for
2 hours (Fig. 5C). Exposure to the oxidative stress of H2O2
significantly and markedly reduced mitochondrial membrane
potential in both control and AG490-treated astrocytes by over
60%, eliminating the significant difference between the two cell
types (Fig. 5C). GSH levels were significantly lower by 35% in
wild-type astrocytes exposed to AG490 for 2 hours (Fig. 5D).
Exposure to H2O2significantly reduced GSH levels by about 15%
in both untreated and AG490-treated astrocytes relative to levels
Figure 3. Analysis of mitochondrial function. Mitochondrial
membrane potential (A), ATP levels (B), and mitochondrial ROS (C) of
cortical astrocytes. Cells treated with the mitochondrial inhibitors 25 mM
rotenone, 10 mM antimycin A (Ant A)or 25 mM FCCP. (A) Cells cultured in
48-well plates were first exposed to theinhibitors or vehicle control(0.5%
DMSO) for 1 hr prior to addition of 1mM JC-1. After an additional hour of
incubation in a CO2incubator, both red (Ex=530, Em=590 nm) and
green (Ex=485, Em=530 nm) fluorescence was measured. After
background subtraction, the ratio of red to green fluorescence was
calculated as a measure of mitochondrial membrane potential. Values
represent means of 16 determinations 6 SEM. * p,0.05 compared with
STAT3 +/+ using 2-way ANOVA with Bonferroni post-hoc test. ^ p,0.05
compared with control. (B) ATPlevels were measured using the Promega
CellTitre-Glo luminescence assay and a standard curve for ATP
quantification. Values represent means of 6 determinations 6 SEM. The
experiment was repeated twice with similar results. (C) Mitochondrial-
specific ROS assays were performed in 96-well culture plates as described
in Materials and Methods. Values represent means of 60 determinations
6 SEM. * p,0.05 compared with STAT3 +/+ using 2-way ANOVA with
Bonferroni post-hoc test. ^ p,0.05 compared with vehicle-treated
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under control conditions, thereby retaining the significant
reductions in AG490-treated astrocytes as compared to untreated
astrocytes (Fig. 5D).
Effects of STAT3-CKO on Astrocyte Gene Expression
To identify candidate molecules regulated or influenced by
STAT3 signaling that might be involved with mechanisms related
to mitochondrial function and the response to oxidative stress we
studied global gene expression profiles using microarrays compar-
ing astrocytes from STAT3-CKO mice with astrocytes from
STAT3 +/+ mice. Cells were cultured to passage 3 over a period
of 4–6 weeks and four biological replicates per condition were
performed. Over 1200 genes exhibited statistically significant
(p,0.005) increases or decreases in expression levels of 50% or
more. As expected, and in agreement with Western data, GFAP
mRNA was decreased nearly 5-fold in STAT3-CKO cells, as was
the related intermediate filament nestin (Table 1).
As relates to the other experiments in this study, differences
were observed in expression of genes involved cell cycle control
and proliferation (Table 1), mitochondrial function (Table 2),
oxidative stress and oxidative defense (Table 3), and apoptosis
(Table 4). The majority of mitochondrial genes impacted were
negatively regulated in the absence of STAT3 signaling, including
NADH dehydrogenase NDUFS4 (1.4-fold decrease), MAP kinase
10 (2.1-fold decrease) and PARK7 (1.4-fold decrease) (Table 2).
Notable among the gene expression differences were a number of
genes involved in oxidative stress and antioxidant defense.
STAT3-CKO cells displayed lower levels of mRNA for peroxir-
edoxin 5 (1.4-fold), peroxiredoxin 6 (2.1-fold), glutathione
reductase (1.5-fold) and metallothione 2 (3.5-fold) compared with
STAT3 +/+ cells (Table 3). Conversely glutathione synthetase,
glutathione peroxidase 7, NADPH quinine oxidoreductase, and
glutathione-S-transferase A3 mRNA were elevated (1.3-fold, 2.8-
fold, 7.5-fold and 4.9-fold, respectively). mRNA for superoxide
dismutases (SOD) 1 and 2 were not significantly different in
STAT3-CKO cells. mRNA for SOD3 was 2-fold lower in
Figure 4. Astrocyte cell proliferation analyzed by BrdU
incorporation and propidium iodide fluorescence. Merged
images of double labeling immunofluorescence for S100b and BrdU
(A,B) and graph (C) of cell counts show that significantly fewer S100b
expressing astrocytes are dividing and labeled with BrdU in STAT3-CKO
(B) compared with littermate control (A) cultures (n=3 per group,
* p,0.01 t-test). (D) Cells were cultured to passage 2 over a period of 3
weeks and plated into 96-well plates at a density of 56103/well. 0.5 mM
N-actetylcysteine (NAC) was added 1, 3, and 5 days after plating. Cell
number and GSH assays were performed as described in Materials and
Methods. * p,0.01 compared with STAT3 +/+ control using one-way
ANOVA with Tukey’s post-hoc test.
Figure 5. Effect of AG490 on cortical astrocyte cell function and
STAT-3 phosphorylation. Cells from wild-type black C6 mice were
pretreated for 2 hr with 25 mM AG490 followed by 1 hr exposure to
10 ng/ml interleukin 6 (IL-6) or 100 mm H2O2. (A) AG490 (AG)
suppressed both basal and IL-6-stimulated STAT-3 phosphorylation.
25 mg of total protein was applied to each lane. (B) Prolonged AG490
exposure suppressed cell proliferation and reduced cell number after 1
day in vitro. Cell number was assessed by relative fluorescence of
propidium iodide in the presence of 160 mM digitonin (C, D)
Measurement of mitochondrial membrane potential using JC-1 and
reduced GSH using MCB. Values represent means of 4 determinations 6
SEM. These studies were repeated twice with similar results.
* p,0.05 comparing AG490-treated with untreated cells. ^ p,0.05
comparing H2O2- treated with corresponding control cells by 2-way
ANOVA with Bonferroni post-hoc test.
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STAT3-CKO cells although statistical significance was not quite
attained n=4 (p,0.11). Catalase levels were low and comparable
in the two cell types. In addition several apoptosis-related genes
were affected. Bcl-2 was reduced 2-fold while caspases 6 and 8
were elevated (50% and 30%, respectively) (Table 4).
Previous studies with transgenic mice expressing a conditional
deletion of the STAT-3 gene in astroglial cells demonstrated the
role of STAT-3 in regulating astrogliosis . While astrogliogen-
esis resulted in normal astrocyte numbers and morphology in these
mice, cellular response to injury was significantly altered. In
addition to GFAP synthesis, nestin and vimentin were under-
expressed and cells demonstrated impaired ability to regulate
inflammation. These findings raised the possibility that intrinsic
properties of STAT3-CKO astrocytes diminished their capacity to
withstand stress and their ability to protect neurons. In this study
we sought to determine the effects of STAT3 deletion on astroglial
mitochondrial functions and on oxidative stress response and
One established function of astrocytes is to limit the oxidative
stress of ROS generation resulting from the high rate of neuronal
oxygen consumption. Astroglial protection from oxidative stress
has been documented with excitotoxicity [32–34], neuropatho-
logical disorders (eg., Alzheimer’s, Parkinson’s, ALS) [35–37],
autoimmune diseases (eg., multiple sclerosis) [38,39], and heavy
metal [40,41] or chemical [42,43] neurotoxicity. A common tool
used for detection of ROS is the cell-permanent fluorescent probe
DCF-DA which becomes oxidized and fluorogenic in the presence
of H2O2, OHN, peroxinitrite and other oxidants . Using this
method ROS was 23% higher in STAT3-CKO cells compared
with STAT3 +/+ cells. This pattern was not observed in the
absence of glucose and was strongly enhanced in the presence of
25 mM rotenone, suggesting differential sensitivity of complex I of
the electron transport chain in the two cell types. However, DCF-
DA fails to detect the superoxide anion. Using the probe, HE,
which specifically detects superoxide anion , we observed 66%
higher basal levels of superoxide production in STAT3-CKO cells.
Rotenone increased superoxide production two-fold but attenuat-
ed the relative difference between STAT3 +/+ and STAT3-CKO
cells to 25%, further suggesting that STAT3 may ultimately
impact complex I of the mitochondrial electron transport chain.
This STAT3-dependent difference in superoxide generation was
also diminished with the uncoupling agent, FCCP, suggesting a
dependence on the mitochondrial membrane potential gradient.
One possible explanation for the elevation and O22would be
lower levels of superoxide dismutase in STAT3-CKO cells.
STAT3 has been shown to up-regulate expression of MnSOD
(SODII) in hepatocytes , cardiomyocytes , and hippo-
campal neurons . In support of this hypothesis, gene
expression array studies suggested that there may be small
decreases in mRNA for SOD enzymes in the STAT3-CKO cells.
Elevated levels of NADPH quinone dehydrogenase (7.6-fold),
GSH peroxidase 7 (2.5-fold) and GSH synthetase (30%) in
STAT3-CKO cells are indicative of ARE promoter activation and
chronic oxidative stress. However, several other genes normally
associated with the ARE pathway such as hemoxygenase I and
peroxiredoxin 6 were not elevated. Decreased expression of
peroxiredoxins, metallothionein 2, and GSH reductase may be
contributing factors to lower levels of reduced GSH and increased
Numerous indicators of mitochondrial function were diminished
in STAT3-CKO cells. Apparent mitochondrial mass, as revealed by
potential and cellular ATP levels were also lower. These abnormal-
ities suggested to us that mitochondria may play a contributing role
to the elevation in ROS in STAT3-CKO cells. Evidence for this
effect was provided by studies using MitoSOX Red. When
normalized to mitochondrial mass, mitochondrial-specific ROS
Table 1. Gene expression differences: cytoskeletal and cell cycle proteins.
AbrevAccess. NMProteinCKO(+ +/+ +) CKO EffectFold Change p value
GFAP 010277.1 Glial Fibrillary Acidic Protein 8.7a
Nes 016701.2Nestin 10.512.4
CCND 007631.1Cyclin D1 11.412.4
Skp1A001543.2 S-phase kinase-associated protein 111.3 11.8
CDKN009877.1 Cyclin dependant kinase inhibitor 2A13.1 12.3 0.83
alog2-transformed absolute RNA expression level.
Table 2. Gene expression differences: mitochondrial proteins.
Abrev Access. NMProtein(CKO)(+ +/+ +) CKO Effect Fold Changep value
MAPK10009158 Mitogen-activated Protein Kinase 10 7.3a
BACE2 019517.2Beta-site API-cleaving enzyme 27.58.3
PRDX5 012021.1 Peroxiredoxin 514.2 14.8
PARK7 020569.1Parkinson Disease 713.814.3
NDUFS4 010887.1 NADH Dehydrogenase13.513.9
GPX7024198.1GPX 7 11.9 10.4
alog2-transformed absolute RNA expression level.
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was 38% higher in STAT3-CKO cells. Antimycin A potentiated the
difference between STAT3 +/+ and STAT3-CKO cells and
produced an ,6-fold greater stimulation of mitochondrial ROS
than did rotenone.
Gene expression array studies also identified decreased
expression of NDUFS4, a subunit of NADH dehydrogenase of
complex I of the mitochondrial electron transport chain. Since
complex I is a source of ROS generation, abnormal function of
this site could account for both increased production of superoxide
anion and diminished ATP production [49,50]. Further evidence
for a role of astrocyte STAT3 in mitochondrial function is
provided by studies of cell energetics. Using JC-1, mitochondrial
membrane potential was found to be 25% lower in STAT3-CKO
cells compared with STAT3 +/+ cells. This mitochondrial effect
was accompanied by a 10% decrease in cellular ATP. The
dissimilarity in membrane potential values was eliminated by
rotenone exposure, as this agent did not change mitochondrial
membrane potential in STAT3-CKO cells while lowering it by
,25% in STAT3 +/+ cells. Antimycin A, however, caused a 70%
reduction in mitochondrial membrane potential in STAT3-CKO
cells and had a more potent effect on ATP level and mitochondrial
ROS generation relative to rotenone. These findings again
suggested that the STAT3 effect
on mitochondria was dependent on mitochondrial complex I
function. Recent studies have identified a direct interaction
between STAT3 and mitochondria in cells from heart and liver
[51,52]. This interaction appeared to be dependent on serine
phosphorylation of STAT3 and independent of transcriptional
activity of the STAT3 protein. Mitochondrial respiration was
reduced via inhibition of activities of complexes I and II of the
electron transport chain. Our present observations of increased
superoxide generation and decreased mitochondrial function
modified by rotenone in STAT3-CKO cells support this novel
mechanism of STAT3 action in cortical astrocytes.
Table 3. Gene expression differences: oxidative stress and defense proteins.
Abrev Access. NMProtein(CKO)(+ +/+ +) CKO EffectFold Changep value
Mt 2 008630.1 Metallothioneine 210.1a
IDH2 173011.1Isocitrate Dehydrogenase a (NADP+) 10.1 11.3
OPLAH 153122.1 5-oxoprolinase (ATP Hydrolyzing)8.6 9.8
PRDX6 007453.2 Peroxiredoxin 610.711.8
SOD3 011435.2 Superoxide Dismutase 38.59.5
GPX4008162 Glutathione Peroxidase 414.7 15.3
GSR 010344.3Glutathione Reductase8.4 9.0
NFE2L1008686.2 Nuclear Factor -like 1 13.713.9
SOD2013671.2 Superoxide Dismutase 2 12.012.1
SOD1 011434.1Superoxide Dismutase 1 13.7 13.7
GSS 008180.1GSH Synthetase10.1 9.7
c-Glutamyltransferase-like Activity 17.9 7.20.0003
GPX 7 024198.1GSH Peroxidase 711.9 10.40.0026
ANPEP 008486,1Alanyl (Membrane) Aminopeptidase 9.88.3 0.0046
GSTA3010356.2 Gl Glutathione S-transferase A311.4 9.1
Ptges022415.2 Prostaglandin E Synthetase10.2 7.6
NQO1008706.1NAD(P)H Dehydrogenase Quinone 112.19.2
Ptgis008968.2Prostagland I (prostacyclin) Synthetase 13.88.7
alog2-transformed absolute RNA expression level.
Table 4. Gene expression differences: apoptosis.
AbrevAccess. NMProtein(CKO)(+ +/+ +) CKO EffectFold Changep value
BCL2009741.2B-cell CLL/Lymphoma 29.6a
TXNDC1028338.1 Thioredoxin Domain Containing 19.49.8
TNFRSf6 007987.1Fas (TNF Receptor Superfamily, 6)10.011.1
CASP6009811.2Caspase 611.611.0 0.60.0011
DAPK1029653.1Death-Associated Protein Kinase 1 220.127.116.11 0.0001
TNFRSf22023680.2 Fas (TNF Receptor Superfamily, 22)12.110.6 1.5
alog2-transformed absolute RNA expression level.
STAT3 In Vitro
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Results from studies using the Jak-2 kinase inhibitor, AG490,
with enriched cortical astrocyte cell cultures confirmed the notion
that prevention of STAT3 activation alters cellular function and
compromises cell defense capabilities. Two-hr exposure to 25 mM
AG490 suppressed basal and Il-6-induced STAT3 phosphoryla-
tion and lowered astrocyte GSH levels, mitochondrial membrane
potential and rate of cell proliferation. The magnitudes of these
effects were similar to those observed when comparing astrocytes
from STAT3-CKO and control STAT3 +/+ mice. Unlike the
STAT3-CKO model, wherein STAT3 activation was absent
selectively in the astrocyte lineage throughout pre- and postnatal
development, AG490-treated cells were acutely deprived of
activated STAT3 for only two hr after many days of normal
activity. Our results suggest that the duration of STAT3 inhibition
in normal, unstressed cells was of little consequence for the effects
on mitochondrial and cellular defense properties examined in this
The present studies identify several abnormalities in astrocytes
lacking the STAT3 gene. Mitochondria displayed lower mass and
were less efficient in maintaining their membrane potential and
producing ATP. Elevated rates of superoxide generation in
STAT3-CKO cells and loss of GSH are indicative of oxidative
stress. Finally, lower rates of DNA synthesis and cell proliferation
were observed. The decreased cell proliferation was not corrected
by antioxidants and likely resulted from altered expression of cell
cycle control and apoptosis regulatory genes. These defects would
likely compromise the ability of astrocytes to promote gliosis and to
protect neurons .
The authors wish to thank Rose Korsak and Ara Darakjian for technical
assistance and manuscript preparation.
Conceived and designed the experiments: TAS DHG MVS. Performed the
experiments: TAS CM TI JQ GC. Analyzed the data: TAS CM GC MVS.
Contributed reagents/materials/analysis tools: TAS MVS. Wrote the
paper: TAS MVS.
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