This increase in mitochondrial mass was associated with an increase in the mRNA levels of mitochondrial biogenesis transcription factors: nuclear res-piratory factor-1 and -2b, as well a
Trang 1biogenesis in primary bovine aortic endothelial cells
Ian T Struewing, Corey D Barnett, Tao Tang and Catherine D Mao
Graduate Center for Nutritional Sciences, University of Kentucky, Lexington, USA
Lithium is commonly used as a therapeutic agent in
the treatment of bipolar disorder (or maniac
depres-sion) [1], and as a mimic of Wnt signaling both in vivo
and in vitro [2] The beneficial effects of lithium in
the treatment of bipolar disorder are thought to be
due to a combination of activation of the Wnt⁄ b-catenin
signaling pathway, via inhibition of the glycogen
syn-thase kinase-3b (GSK3b) [3], and depletion of the
intracellular inositol pool via the inhibition of various
enzymes in the phosphoinositide pathways, for example, the rate-limiting enzyme inositol monophospha-tase 1 (IMPase-1) [4,5] In addition, evidence suggests that lithium is neuroprotective and is beneficial in the treatment of ischemia–reperfusion injuries and neuro-degenerative diseases, including Alzheimer’s, Parkin-son’s and Huntington’s disease [6] For Huntington’s disease, it has been proposed that lithium increases the degradation of aggregated Hungtintin-mutated
Keywords
cell signaling; CREB; FOXO; gene
expression; mitochondria
Correspondence
C D Mao, Graduate Center for Nutritional
Sciences, University of Kentucky, 900
Limestone Street, Lexington, KY 40536,
USA
Fax: +1 859 257 3646
Tel: +1 859 323 4933, Ext 81377
E-mail: cdmao2@uky.edu
(Received 14 January 2007, revised 13
March 2007, accepted 23 March 2007)
doi:10.1111/j.1742-4658.2007.05809.x
Lithium is a therapeutic agent commonly used to treat bipolar disorder and its beneficial effects are thought to be due to a combination of activa-tion of the Wnt⁄ b-catenin pathway via inhibition of glycogen synthase kin-ase-3b and depletion of the inositol pool via inhibition of the inositol monophosphatase-1 We demonstrated that lithium in primary endothelial cells induced an increase in mitochondrial mass leading to an increase in ATP production without any significant change in mitochondrial efficiency This increase in mitochondrial mass was associated with an increase in the mRNA levels of mitochondrial biogenesis transcription factors: nuclear res-piratory factor-1 and -2b, as well as mitochondrial transcription factors A and B2, which lead to the coordinated upregulation of oxidative phos-phorylation components encoded by either the nuclear or mitochondrial genome These effects of lithium on mitochondrial biogenesis were inde-pendent of the inhibition of glycogen synthase kinase-3b and indeinde-pendent
of inositol depletion Also, expression of the coactivator PGC-1a was increased, whereas expression of the coactivator PRC was not affected Lithium treatment rapidly induced a decrease in activating Akt-Ser473 phosphorylation and inhibitory Forkhead box class O (FOXO1)-Thr24 phosphorylation, as well as an increase in activating c-AMP responsive ele-ment binding (CREB)-Ser133 phosphorylation, two mechanisms known to control PGC-1a expression Together, our results show that lithium induces mitochondrial biogenesis via CREB⁄ PGC-1a and FOXO1 ⁄ PGC-1a cas-cades, which highlight the pleiotropic effects of lithium and reveal also novel beneficial effects via preservation of mitochondrial functions
Abbreviations
BAEC, bovine aortic endothelial cell; COX, cytochrome c oxidase; CREB, c-AMP responsive element binding; DCF, 2¢-7¢-dichlorofluorescein; FOXO, Forkhead box class O; GSK3, glycogen synthase kinase; IMP, inositol monophosphatase; LPS, lipopolysaccharide; MTP, mitochondria transmembrane potential; NRF, nuclear respiratory factor; OXPHOS, oxidative phosphorylation; PGC, peroxisome proliferators-activated receptor-gamma coactivator; PRC, PGC-1a-related coactivator; TFAM, transcription factor A mitochondria; TFB, transcription factor B; UCP, uncoupling protein.
Trang 2proteins via autophagy in an IMPase-1-dependent manner
[7] By contrast, the preconditioning and protective
effects of lithium in brain and heart
ischemia–reper-fusion injury models appear to depend upon the
inhi-bition of GSK3b [8,9] Although much attention has
been paid to the inhibitory effects of lithium on
GSK3b and IMPase-1 activity, lithium acts as a
com-petitive inhibitor of numerous Mg2+-dependent
fac-tors, transporters and enzymes, including a key
glycolytic enzyme, phosphoglucomutase [10] Such a
wide spectrum of potential targets is consistent with
the narrow range of lithium doses that can be used
therapeutically in the absence of toxicity and with
lim-ited side effects [1]
GSK3b plays a pivotal role in the canonical Wnt⁄
b-catenin signaling pathway by phosphorylating and
targeting b-catenin to the proteasomal degradation
pathway in the absence of Wnt signals In the presence
of Wnt signals, GSK3b becomes phosphorylated on
Ser9 and is inactivated, allowing the cytosolic stabilization
and nuclear translocation of b-catenin In the nucleus,
b-catenin interacts with the TCF⁄ LEF transcription
factors and activates the transcription of genes involved
in cell proliferation and adhesion [11] Lithium, via
inhibition of GSK3b, increases b-catenin⁄ TCF
tran-scriptional activity and induces proliferation in various
tumor cell lines [2,12] By contrast, lithium did not
induce proliferation or activation of the transcriptional
activity of b-catenin⁄ TCF complexes in primary bovine
aortic endothelial cells (BAEC), but rather induced
G2⁄ M cell-cycle arrest leading to a cell senescence-like
phenotype [13] In these cells, lithium treatment
activa-ted the tumor suppressor p53 resulting in increased
expression of the cyclin-dependent kinase inhibitor
p21cip at both the mRNA and protein levels; this was
found to be independent of depletion of the
intracellu-lar inositol pool [13] Simiintracellu-larly, an increase in p21cip
protein stability was also seen in human endothelial
cells in response to lithium and this was shown to be
dependent upon inhibition of GSK3b [14] The
antipro-liferative effects of lithium have also been seen in other
primary cells, including human umbilical vein
endothel-ial cells [15], vascular smooth muscle cells [13], lens
epi-thelial cells [16], and some tumor cell lines such as B16
melanoma [17] and P19 embryonal carcinoma cells [18]
Both GSK3b and p53 have been shown to localize
to mitochondria GSK3b was localized to the
mito-chondria of cerebellar cells [19] and enrichment of
act-ive GSK3b in mitochondria and nuclei has also been
observed in neuronal cells [20] In the immortalized
neuronal cell line SH-SY5Y, mitochondrial and active
GSK3b was shown to interact with p53 and thereby
promote the pro-apoptotic activities of p53 [21]
By contrast, activation⁄ inhibition of GSK3b was shown to control glycolysis–oxidative phosphorylation (OXPHOS) coupling and apoptosis in HeLa tumor cells via the release⁄ binding of hexokinase II from the mitochondria outer membrane [22] However, these effects appear to be cell-type dependent, because lithium had the opposite effect in B16 melanoma cells in association with a cell-cycle arrest [17] Recently, a novel p53 target has been identified, synthesis of cyto-chrome oxidase 2 (SCO2), which is responsible for bio-genesis of the cytochrome oxidase complex in the inner mitochondrial membrane and thus links the tumor suppressor p53 with the control of energy metabolism and glycolysis switching in tumor cells [23] Although lithium has been shown to affect glycolysis via direct inhibition of phosphoglucomutase [10], its effects on mitochondrial energy metabolism and biogenesis have not been addressed
Mitochondrial biogenesis is highly orchestrated, and involves signal cross-talk between the nucleus and mitochondria leading to the coordinated regulation of gene expression [24,25] The mitochondrial genome encodes only 13 OXPHOS components; the other OXPHOX components and all the factors required for assembly of the OXPHOX complexes and for replica-tion and transcripreplica-tion of the mitochondrial genome are encoded by the nuclear genome The nuclear transcrip-tion factors, nuclear respiratory factor (NRF)-1 and -2,
in conjunction with the coactivators peroxisome prolif-erators activated receptor-c coactivator (PGC)-1a and PGC-1a-related coactivator (PRC), control the expres-sion of nuclear-encoded OXPHOX genes and transcrip-tion factor A mitochondria (TFAM), transcriptranscrip-tion factor B (TFB)-1 and TFB-2 These mitochondrial transcription factors in turn control transcription and replication of the mitochondrial genome [25]
Under physiological conditions, changes in mitoch-ondrial biogenesis and mass within cells and tissues reflect changes in energy demand and hormonal status, and depend mainly on changes in the activity and⁄ or expression of the transcription factors NRF1 and NRF2 and coactivator PGC-1a [25,26] By contrast, under pathological conditions, such as oxidative stress [27], an increase in mitochondrial biogenesis can occur
as a compensatory mechanism in response to mito-chondrial dysfunction and damage, however, ATP pro-duction is usually impaired Mitochondrial dysfunction
or loss is a common characteristic of many neuro-degenerative diseases such as Alzheimer’s and Parkin-son’s [28], and has also been seen in postmortem brain biopsies from subjects with bipolar disorder [29] Mutated huntingtin protein, responsible for the devel-opment of Huntington’s disease, was shown to cause
Trang 3mitochondrial dysfunction by interfering with
cAMP-responsive element binding (CREB) transcription
fac-tor-dependent regulation of PGC-1a expression [30]
Inversely, increasing the expression of PGC-1a was
shown to be neuroprotective in the mutated-hungtintin
transgenic mouse model of Huntington disease [30]
Interestingly, the preconditioning effects of various
substances and factors in brain, heart and vessel
ischemic–reperfusion injuries also seem to be mediated
in part by an increase in mitochondrial biogenesis and
preservation of mitochondrial function [31,32] The
protective effects of lithium, both during
precondition-ing in heart and brain ischemic–reperfusion injury
models [8,9], and in neurodegenerative disease models
[6], have been studied only in relation to the apoptotic
function of mitochondria, and not their energy
homeo-static function
In this study, we show that lithium increases
mitoch-ondrial biogenesis in BAEC leading to an increase in
ATP production Unexpectedly, this novel effect of
lithium was independent of the inhibition of GSK3b
and of inositol depletion Moreover, our results reveal that lithium treatment affects two cascades known to converge to the upregulation of PGC-1a expression by CREB and Forkhead box class O (FOXO1) trans-cription factors and hence to increase mitochondrial biogenesis
Results
Lithium increases mitochondrial mass in BAEC BAEC were treated with 10 mm lithium, a dose com-monly used to achieve GSK3b inhibition and activa-tion of Wnt⁄ b-catenin signaling [33], and the levels of ATP were measured using a standard luminescent luciferin⁄ luciferase assay Lithium treatment resulted in
a significant 1.4 ± 0.1- and 1.49 ± 0.2-fold increase in ATP production at 24 and 36 h, respectively (P < 0.01), whereas treatment with 1 mm valproate, another mood stabilizer [33], had a weaker effect, with
a 1.23 ± 0.1-fold increase at both 24 and 36 h of
0 0.25 0.5 0.75 1 1.25 1.5 1.75 2
* *
*
A
*
0 0.5 1 1.5 2
10 m M Na
5 m M Li
10 m M Li
1 m M VPA
B
12S/TFAM Cytb/ATPbeta CR/RRS1 0
0.25 0.5 0.75 1 1.25 1.5 1.75
*
C
Fig 1 Lithium increases mitochondrial mass in BAEC (A) Lithium
increases ATP production BAEC were plated 8 h prior to treatment
with either 10 m M NaCl (Na), 10 m M LiCl (Li) or 1 m M Na-valproate
(VPA) for 24 or 36 h After cell lysis, levels of ATP per lg of protein
were determined as described in Experimental procedures The
graph represents mean ± SEM of the fold increase in treated
ver-sus control NaCl-treated cells obtained in 6–8 independent
experi-ments performed in duplicate (B) Lithium increases the
mitochondria membrane potential BAEC were treated with the
indicated doses of NaCl, LiCl or VPA for 36 h prior to staining with
500 n M of Mitotracker-CMXRos for 45 min The accumulation of
Mitotracker-CMXRos in active mitochondria was determined by
measuring the fluorescence intensity at kexc550⁄ k em590 and the cell
number was quantified subsequently using CyQuant staining and
measurement of the fluorescence intensity at kexc485⁄ k em535 The
MTP levels were corrected for the variation in cell number and the
results are expressed as relative levels with the control
NaCl-trea-ted cells equal to 1 The graph represents mean ± SEM obtained in
eight independent experiments preformed in triplicate (C) Lithium
increases mitochondrial mass in BAEC BAEC were treated with
10 m M NaCl (Na), 10 m M LiCl (Li) or 1 m M valproate (VPA) for 36 h
prior to isolation of total DNA Levels of mitochondrial DNA and
lev-els of nuclear DNA were quantified by real-time PCR with specific
bovine primers for the mitochondrial encoded genes, cytochrome b
(cyt-b) and 12S rRNA, as well as for the mitochondria genome
control region (CR) and for the nuclear encoded genes: TFAM,
ribosome biogenesis regulator-1 (RRS1) and ATP synthase-b
(ATP-beta) The ratio of mitochondrial DNA to nuclear DNA was
determined for each treatment and for each of the pair of
MitDNA ⁄ NuDNA: 12S ⁄ TFAM, Cytb ⁄ ATPb and CR ⁄ RRS1 Results
are expressed in relative levels with the control NaCl equal to 1.
Mean ± SEM obtained from 3–4 independent experiments are
reported in the graph In all cases, after Student’s t-test analysis,
the results were considered significant at P < 0.05 (*).
Trang 4treatment (P < 0.05) (Fig 1A) Because lithium was
shown to inhibit various enzymes of the glycolytic and
tricarboxylic acid pathways and to decrease ATP
pro-duction via glycolysis [17,34], we first tested whether
lithium increases ATP production via changes in
mito-chondrial activity and⁄ or mass in BAEC The
mito-chondria transmembrane potential (MTP) is a marker
of mitochondrial OXPHOS activity that can be
assessed using fluorescent probes accumulating in
mitochondria depending on the MTP such as
Mito-tracker-CMXRos [35] In this study, the fluorescent
probes, nonyl acridine orange and Mitotracker-Green,
usually used for mitochondria staining independent of
MTP, were also sensitive to the uncoupler carbonyl
cyanide 3-chlorophenylhydrazone (not shown) and
thus were not used to directly determine mitochondrial
mass per cell Also, we have previously shown that
BAEC treated with lithium were arrested in the G2⁄ M
phase and displayed a reduced cell number compared
with sodium-treated cells [13] Therefore, cell number
was determined using CYQUANT staining and
fluor-escence quantification, and MTP levels were corrected
by the cell number The increase in ATP production
induced by lithium was associated with a significant,
1.33 ± 0.05-fold, increase in the relative MTP levels
per cell in BAEC treated for 36 h (P < 0.05), whereas
valproate had no significant effect (Fig 1B) Because
an increase in MTP can reflect either an increase in
mitochondrial mass or an increase in the efficiency of
mitochondrial OXPHOS, we determined the effects of
lithium on mitochondrial mass using a real-time
PCR-based assay Relative levels of mitochondrial DNA
versus nuclear DNA were determined using three
dif-ferent genes encoded by the mitochondrial genome and
three encoded by the nuclear genome to avoid bias of
differential efficiency of amplification between primer
sets As shown in Fig 1C, lithium treatment increased
significantly the relative levels of mitochondrial DNA,
about 1.35 ± 0.14, 1.4 ± 0.07 and 1.49 ± 0.07-fold
for 12S⁄ TFAM, cytochrome b ⁄ ATP synthase-b and
CR⁄ RRS1 MitDNA ⁄ NuDNA pairs, respectively, which
indicated an increase of the mitochondrial mass,
whereas VPA had no significant effect Taken together,
these results show that lithium treatment in BAEC
increases mitochondrial mass significantly, leading to
an increase in ATP production without changes in
mit-ochondrial efficiency This lithium-induced increase in
mitochondrial mass was not accompanied by any
signi-ficant changes in mitochondrial morphology or
distri-bution, as shown by immunofluorescence microscopy
of BAEC stained with Mitotracker-Deep Red (Fig 2)
Mitochondria in lithium-treated BAEC were found
around the nucleus and protrusion, as in control cells,
repeated treatments with 50 lm of the NO donor, DETA-NO, a known inducer of mitochondrial biogen-esis [36], also increased the mitochondrial mass but the mitochondria were mainly perinuclear (Fig 2)
Lithium increases mitochondrial biogenesis markers
To determine whether the increase in mitochondrial mass was due to an increase in mitochondrial bio-genesis, the mRNA levels of various OXPHOS compo-nents encoded by either the nuclear genome or the mitochondrial genome were assessed using real-time RT-PCR As shown in Fig 3, after 24 or 36 h treat-ment of BAEC with lithium, the mRNA levels of mitochondrial-encoded genes such as cytochrome oxid-ase II (1.65 ± 0.07-fold, complex IV), ATP synthoxid-ase subunit-6 (2.5 ± 0.32-fold, complex V), and cyto-chrome b (3.4 ± 0.4-fold, complex III) were increased
Fig 2 Lithium does not affect mitochondrial distribution in BAEC BAEC were grown on glass chamber slides and treated with
10 m M NaCl, 10 m M LiCl or the NO donor DETA-NO for 72 h prior
to addition of 100 n M Mitotracker-Deep Red633 for 45 min at
37 C After removal of the staining solution, fresh medium was added for 10 min incubation prior to cell fixation in 3.7% formalde-hyde and slide mounting Immunofluorescence confocal images of several fields (n ¼ 10) were taken and representative images are shown (scale bars: 20 lm).
Trang 5significantly, however, no significant difference was
observed between the two treatment times Similarly,
36 h lithium treatment led to a significant increase in
the RNA levels of nuclear-encoded genes such as
cyto-chrome oxidase VIc and VIa by 1.85 ± 0.08 and
1.56 ± 0.1-fold, respectively (complex IV), and
ATP-synthase subunit-b by 2.66 ± 0.57-fold (complex V),
whereas the mRNA levels of cytochrome c and
mito-chondria DNA polymerase were increased slightly, by
1.3 ± 0.04 and 1.33 ± 0.1-fold, respectively,
with-out reaching statistical significance (Fig 3) Levels of
uncoupling protein 2 (UCP2) mRNA were also
signifi-cantly increased after 36 h lithium treatment
1.4 ± 0.1 fold, which was similar to the increase
observed for the mitochondrial biogenesis markers
This coordinated increase in mRNA levels for the
mit-ochondrial biogenesis markers and UCP2 is in
agree-ment with the unaffected OXPHOS efficiency observed
after lithium treatment (Fig 1) To confirm that the
increase in mRNA was accompanied by an increase in
protein levels, we assessed the expression of the ATP
synthase-b protein by immunoblotting A significant,
1.7 ± 0.3-fold, increase in ATP synthase-b was
observed after lithium treatment, although valproate
had no effect (Fig 3C) Our results show that lithium
increased the expression of mitochondrial biogenesis
markers in a coordinated fashion, although the effects
of lithium on mRNA levels for mitochondrial-encoded genes are stronger than the effects on the expression of nuclear-encoded genes This may be due to an increase
in mitochondrial mass (Fig 1) and⁄ or a greater increase in the expression of mitochondrial transcrip-tion factors
Lithium increases mRNA levels for transcription factors involved in mitochondrial biogenesis Expression of OXPHOS genes encoded by the mito-chondrial genome is under the control of specific tran-scription factors: TFAM, TFB1 and TFB2, whose expression, as well as expression of the nuclear-enco-ded OXPHOS genes, is mainly under the control of the NRF1 and NRF2 transcription factors [25]
Fig 3 Lithium increases the mRNA levels of oxidative
phosphoryla-tion components BAEC were treated with either 10 m M NaCl or
10 m M LiCl for the indicated times prior to RNA extraction, and the
levels of target mRNAs were quantified using real-time PCR as
described in Experimental procedures Levels of target mRNAs
were corrected for variation of the mRNA levels with the internal
control rpL30 and for each target gene the ratio of the corrected
level obtained in LiCl-treated versus NaCl-treated cells was
deter-mined The fold induction of the target mRNA is reported in the
graph in (A) for OXPHOS genes encoded by the mitochondrial
gen-ome: cytochrome oxidase subunit II (COX-II), ATP synthase-6 and
cytochrome b (cyt-b), and in the graph in (B) for OXPHOS genes
encoded by the nuclear genome: ATP synthase-b, cytochrome
oxid-ase subunits VIa and VIc (COX VIa and VIc), cytochrome c (cyt-c)
and mitochondria DNA polymerase (MitDNA polymerase) Mean ±
SEM values obtained from 3–5 independent experiments are
repor-ted in the graphs and the results were considered significant at
P < 0.05 (*) (Student’s t-test) (C) Lithium increases the level of
ATP synthase-b protein BAEC were treated for 36 h with 10 m M
NaCl, 10 m M LiCl or 1 m M VPA prior to cell lysis and western blot
analysis of the levels of ATP synthase-b and the loading control
a-tubulin The intensity of the protein bands was evaluated by
den-sitometry and the ratio of ATP synthase-b intensity to a-tubulin
intensity was determined for each treatment A representative
experiment is shown and the mean ± SEM results obtained from
seven independent experiments are reported in the graph Results
were considered significant at P < 0.05 (*) (Student’s t-test).
A
0 1 2 3 4 5
**
Fold increase of mRNA levels of nuclear enclosed genes Fold increase of mRNA levels of mitochondrial enclosed genes
*
0 0.25 0.5 0.75 1 1.25 1.5 1.75 2
*
Ratio α-Tubulin
ATP
C
0 1 2 3 4 5
24h
0 1 2 3 4 5
36h
*
B
β
ATP
0 1 2 3 4 5
24h 36h
*
Trang 6c-Myc, a b-catenin target gene [37], has also been
shown to increase the levels and activity of TFAM
[38] Therefore, we tested the mRNA levels of all these
transcription factors in response to lithium A
signifi-cant increase in mRNA levels for TFAM and TFB2
was seen after 24 h lithium treatment, 2.2 ±
0.5-and 1.7 ± 0.2-fold, respectively, 0.5-and these effects
were even more pronounced at 36 h with a 4.4 ±
1.1-and 4.1 ± 1-fold increase, respectively (Fig 4A)
Among the nuclear transcription factors, only NRF2b
mRNA levels were increased significantly at 24 h, by
1.6 ± 0.14-fold (Fig 4A) After 36 h lithium treatment, NRF2b mRNA levels were further increased by 3.3 ± 0.5-fold, whereas mRNA levels of NRF1 and c-myc were increased only approximately twofold Thus, the increased expression of the mitochondrial markers observed at 24 h (Fig 2) was mainly associ-ated with increased expression of TFAM and TFB2,
as well as of NRF2b
Redox-dependent activation of NRF1 and NRF2 is known to mediate the increase in expression of the mitochondrial biogenesis transcription factors observed
in response to various oxidative stresses such as lipopolysacharride (LPS) treatment [27] Therefore, we assessed the effects of short lithium treatments on intracellular levels of H2O2 using a 5- (and 6)-chloro-methyl-2¢-7¢-dichlorodihydrofluorescein diacetate
(CM-H2DCFDA) probe, which is deacetylated by cellular esterase and oxidized in the presence of H2O2 to give a fluorescent 2¢-7¢-dichlorofluorescein (DCF) compound Treatment of BAEC with 10 mm lithium for between
30 min and 2 h had no significant effect on intra-cellular H2O2 levels, whereas treatment with 1 lm LPS resulted in a small but significant increase after 2 h treatment ( 1.36 ± 0.1-fold; Fig 4B) Longer lithium treatments resulted in a decrease in peroxide produc-tion (not shown) Therefore, lithium-induced mito-chondrial biogenesis was not due to a compensatory mechanism following mitochondrial damage induced
by an increase in oxidative stress
Lithium effects on mitochondrial biogenesis are partially dependent on inositol depletion Lithium is a competitive inhibitor of various enzymes
of the inositol pathway including the limiting enzyme IMPase-1, resulting in a marked depletion of the intra-cellular inositol pool that can be restored by the addi-tion of myo-inositol [5] We tested whether lithium was able to increase the expression of mitochondrial bio-genesis markers after pretreatment with 1 mm myo-inositol As shown in Fig 5A, addition of 1 mm myo-inositol attenuated the effects of lithium with a 20–25% decrease in mRNA levels for TFAM, cyto-chrome b and ATP synthase-6, although it did not sig-nificantly affect these levels in NaCl-treated cells However, the changes between LiCl + myo-inositol-treated cells and LiCl-myo-inositol-treated cells were not statistically significant using one-way anova To further assess the involvement of inositol depletion in lithium-induced mitochondrial biogenesis, lithium-dependent changes
in mitochondrial mass were monitored in the absence
or presence of 1 mm myo-inositol pretreatment As shown in Fig 5B, myo-inositol pretreatment did not
0
1
2
3
4
5
6
24h 36h
*
*
*
*
*
*
*
*
A
0
0.2
0.4
0.6
0.8
1
1.2
1.4
*
*
B
Fig 4 Lithium increases the mRNA levels of transcription factors
involved in the control of mitochondrial biogenesis in the absence
of oxidative stress (A) Lithium increases the mRNA levels of
mitochondrial biogenesis transcription factors BAEC were treated
for the indicated times with either 10 m M NaCl as a control or
10 m M LiCl prior to RNA extraction, and the levels of target mRNAs
were quantified using real-time RT-PCR The graph represents
mean ± SEM results obtained from five independent experiments.
(B) Lithium does not induce oxidative stress in BAEC Equal
num-bers of BAEC were treated for the indicated times with 10 m M
NaCl as control, 10 m M LiCl or 1 l M LPS as positive control for
oxidative stress prior to staining with 2.5 l M CM-H 2 DCFDA for
30 min in the dark The fluorescence intensities were measured at
kexc485⁄ k em535 Mean ± SEM results obtained from 4–6
independ-ent experimindepend-ents performed in triplicate are reported in the graphs.
Results were considered significant at P < 0.05 (*) (Student’s
t-test).
Trang 7affect the lithium-dependent increase of mitochondrial
mass as determined by the ratio of MitDNA⁄ NuDNA
These results indicated that myo-inositol pretreatment
did not prevent lithium-induced mitochondrial
bio-genesis
Lithium effects on mitochondrial biogenesis are
independent of GSK3b inhibition
Active GSK3b has been shown to be localized in
mito-chondria [20], we also examined whether the effects of
lithium on the expression of mitochondrial biogenesis
markers were dependent upon GSK3b inhibition by comparing the effects of two other unrelated inhibitors
of GSK3b, valproate and indirubin-3¢-monoxime Val-proate has been shown to indirectly inhibit GSK3b [39], although its activation of the Wnt⁄ b-catenin sign-aling pathway in various cells appears to depend mainly on histone deacetylases 2 inhibition [40] Among these various inhibitors, only lithium treatment led to a significant increase in the mRNA levels of mito-chondrial-encoded genes, ATP synthase-6 and cyto-chrome b, nuclear-encoded genes, ATP synthase-b, cytochrome oxidase VIc and TFAM (Fig 6A) By contrast, indirubin had no effect, whereas valproate increased, 1.5-fold, mRNA levels for the nuclear-encoded genes cytochrome oxidase VIc and TFAM (Fig 6A) These results were in agreement with a weak
or lack of effect of valproate on ATP production and mitochondrial mass, respectively (Fig 1) To further rule out a role for GSK3b in the lithium-dependent increase in mitochondrial biogenesis markers, BAEC were transfected with wild-type GSK3b, constitutive active S9A-GSK3b and the inactive kinase-dead K85A-GSK3b for 36 h prior to the analysis of mito-chondrial and nuclear gene expression If the effects of lithium on mitochondrial biogenesis were dependent
on GSK3b inhibition, expression of the catalytic inactive K86R-GSK3b form should also increase expression of the mitochondrial biogenesis markers However, mRNA levels of the nuclear genes ATP syn-thase-b, cytochrome oxidase VIc and TFAM, and of the mitochondrial genes ATP synthase-6 and cyto-chrome b, were not significantly affected by expression
of any forms of GSK3b including the inactive K86R-GSK3b form (Fig 6B) By contrast, levels of interleu-kin-8 mRNA were increased 3.6 ± 1.3 fold in response to expression of the inactive K85-GSK3b, as expected for a target gene of GSK3b inhibition [41] Our results with the various inhibitors of GSK3b and expression of the inactive form of GSK3b indicate that the lithium-induced increase of mitochondrial biogen-esis is independent of GSK3b inhibition
Lithium increases cell size in the absence of Akt activation
Of the various inhibitors used in this study, only lith-ium led to a significant increase in cell size and spread-ing in BAEC (Fig 7A), as well as cell-cycle arrest [13] Although activation of the Akt pathway via Akt phos-phorylation on Ser473 and Akt-dependent inactivation
of GSK3b by phosphorylation on Ser9 have been implicated in skeletal and cardiac hypertrophy [42,43], lithium treatment was associated with a decrease in
ATP synthase−−β COX-VIc
ATP
synthase-6
0
1
2
3
4
5
6 Na Na + myo-inositol Li Li + myo-inositol
*
*
*
*
*
*
*
*
A
0
0.25
0.5
0.75
1
1.25
1.5
1.75
NaCl LiCl LiCl +
myo-inositol NaCl +
myo-inositol
B
Fig 5 Maintenance of the inositol pool had minimal effects on
lith-ium-induced mitochondrial biogenesis After pretreatment with
1 m M myo-inositol to maintain the intracellular inositol pool, BAEC
were treated for 36 h with either 10 m M NaCl or 10 m M LiCl prior
to either RNA extraction (A) or DNA isolation (B) (A) mRNA levels
of the indicated genes were determined using real-time PCR.
Mean ± SEM results obtained from six independent experiments
are reported in the graphs Results were considered significant at
P < 0.05 (*) (one-way ANOVA followed by posthoc Bonferroni’s
test) (B) Mitochondrial mass was determined from the ratio
mitochondrial DNA ⁄ nuclear DNA using real-time PCR as described
in Experimental procedures Mean ± SEM results obtained from
three independent experiments are reported in the graph Results
were considered significant at P < 0.05 (*) (Student’s t-test).
Trang 8Akt-S473 phosphorylation rather than an increase in
BAEC As shown in Fig 7B, lithium had no
signifi-cant effect on inhibitory GSK3b-S9 phosphorylation
early during treatment, between 5 and 30 min, but
increased it significantly at later times with a twofold
increase at 24 h These results are in agreement with the effects of chronic lithium treatment on GSK3b-S9 phosphorylation in various cell lines, including
neuron-al cells [44] However, in BAEC, lithium decreased activating Akt-S473 phosphorylation significantly, about twofold, as early as 30 min into treatment (Fig 7B), and this decrease persisted over 24 (Fig 7B) and 36 h (not shown) This decrease in active Akt was consequently associated with a decrease at 6 h in the transcription factor FOXO1 phosphorylation on Thr24, an Akt substrate site in vivo [45] (Fig 7B) Val-proate, like lithium, did not induce an increase but rather a decrease in Akt-S473 phosphorylation, although the latter occurred later after 24 h treatment (Fig 7B) Similarly, although to a lesser extent than lithium, valproate increased inhibitory GSK3b-S9 phosphorylation By contrast, indirubin induced rapid disappearance of both the activating Akt-S473 and the inhibitory GSK3b-S9 phosphorylations at treatment periods between 5 min and 24 h, compared with con-trol-treated cells (Fig 7B) Therefore, valproate and lithium appeared to induce similar changes in the Akt⁄ FOXO1 signaling cascade in primary BAEC except for stronger and faster effects with lithium
Lithium increases the expression of PGC-1a
in BAEC The finding that the Akt⁄ FOXO1 cascade is affected
by lithium in BAEC prompted us to investigate the effects of lithium on the expression of PGC-1a as it has previously been shown that activation of Akt led
to downregulation of PGC-1a expression via nuclear exclusion of FOXO1 in skeletal muscle cells [46] Lev-els of PGC1-a mRNA, as well as those of the related coactivator PRC, were determined using real-time PCR after BAEC treatments (Fig 8A) As expected for valproate, a short branched-chain fatty acid, there was a strong increase in the mRNA levels of PGC-1a,
3.1 ± 0.7-fold at 36 h treatment (Fig 8A) Indeed, these results, although novel for valproate, are consis-tent with the regulation of PGC-1a expression by nutrient availability and, in particular, by various fatty acids [26,47] Surprisingly, lithium treatment was also associated with an increase in PGC-1a mRNA levels,
2.6 ± 0.6-fold, although no statistically significant change in PRC expression was observed (Fig 8A) In addition to being regulated by FOXO1 transcription factor, PGC-1a expression is also upregulated by act-ive CREB transcription factor [26] Lithium is a well-known inducer of CREB activation via an increase in activating CREB-S133 phosphorylation in neuronal cells [48] Therefore, we tested the effects of lithium on
ATP synthase–ββ COX-VIc
ATP
synthase-6
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ATP
synthase-6
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Control WT S9A K85R
IL-8
C
PCR3 Wt S9A K85R
Phospho-S9
GSK3β
His-Tag
GSK3β
Fig 6 Lithium effects on mitochondrial biogenesis are independent
of GSK3b inhibition BAEC were either treated for 36 h with various
known inhibitors of GSK3b, 10 m M LiCl, 1 m M VPA, and 5 l M
indirubin (IND) (A) or transfected for 36 h with either
wildtype-GSK3b (WT), the constitutive active S9A-wildtype-GSK3b (S9A) or the
inac-tive K85R-GSK3b (K85R) prior to RNA extraction mRNA levels of
the indicated genes were determined by real-time PCR Levels of
IL-8 mRNAs were determined as a control for the dominant effects
of the inactive K85R-GSK3b Mean ± SEM results obtained from
four independent experiments are reported in the graphs Results
were considered significant at P < 0.05 (*) (Student’s t-test).
(C) The expression of the histidine-tagged GSK3b proteins and their
Ser9 phosphorylation status were controlled by western blotting.
Trang 9CREB-S133 phosphorylation in BAEC and found that
treatment with lithium, or valproate, for 8 h
signifi-cantly induced activating CREB-S133 phosphorylation
by 2- and 1.5-fold, respectively, compared with
NaCl-treated cells (Fig 8B) These results suggest that
lithium increases PGC-1a expression via at least two
mechanisms: activation of FOXO1 and CREB
Discussion
Lithium is commonly used to treat bipolar disorder [1]
and recent evidence suggests that it might also be
beneficial in the treatment of neurodegenerative
dis-eases [6] However, the mechanisms involved in both
the beneficial effects and side effects of lithium are not
fully identified We report a novel effect of lithium at
doses commonly used to inhibit GSK3b activity and mimic Wnt signaling [33] In primary endothelial cells, lithium treatment triggered an increase in mitochond-rial mass and ATP production without changing mitochondrial efficiency This increase in mitochondrial biogenesis correlated with the upregulation of key master controllers of mitochondrial biogenesis: tran-scription factors NRF1 and NRF2b and coactivator PGC-1a [25,26] In addition, we showed that two different signaling cascades known to regulate PGC-1a expression, inactivation of Akt [46] and activation of CREB [26], were triggered by lithium treatment
An increase in mitochondrial biogenesis has been described in numerous physiological conditions as an adaptive mechanism during muscle exercise, calorie restriction, hormone treatment and cell
differenti-A
Akt
ββ-actin phosphoS473-Akt
Treatments
B
PhosphoS473-AKT/AKT
0 0.5 1 1.5 2 2.5 3
3.5
Li VPA
PhosphoS9-GSK/GSK
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PhosphoT24-FOXO1/FOXO1
0 0.5 1 1.5 2 2.5
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C
24 h
Fig 7 Lithium increases BAEC cell size and affects Akt ⁄ FOXO1 signaling cascade (A) Lithium increases the spreading and size of BAEC BAEC were plated for 12 h prior to being treated with 10 m M NaCl, 10 m M LiCl, 1 m M VPA or 5 l M indirubin for 36 h Phase-contrast micro-scope images were taken and representative images are shown for each treatment (B) Lithium increases the inhibitory phosphorylation of GSK3b on Ser9 in absence of Akt activation BAEC were treated as indicated in (A) for 5 min, 30 min, 2 h and 24 h prior to cell harvesting and analysis of GSK3b and Akt phosphorylations using immunoblotting with specific phospho-S9-GSK3b and phospho-S473-Akt antibodies Akt-dependent phosphorylation of FOXO1 on Thr24 was also studied in parallel Total levels of GSK3b, Akt and FOXO1 were used to normal-ize for changes in expression and b-actin was used as loading control A representative experiment is shown and the fold changes obtained after lithium treatment from four independent experiments are reported in the graphs.
Trang 10ation, as well as in various pathological situations to
compensate for mitochondrial dysfunction or damage
[24,25] It is possible that lithium as a potential
com-petitive inhibitor of some Mg2+-dependent
mito-chondrial enzymes and transporters, might induce
mitochondrial biogenesis in response to mitochondrial
dysfunction However, the kinetics of the increase in
mitochondrial mass and ATP production observed
between 18 and 36 h of treatment are incompatible with initial mitochondrial dysfunction (Fig 1) Simi-larly, the kinetics are incompatible with a compensa-tory mechanism following mitochondrial oxidative damage because, in this case, an initial decrease in both mitochondrial mass and ATP production would
be expected prior to the recovery phase Moreover, treatment of BAEC with lithium did not increase intra-cellular levels of hydrogen peroxide (Fig 4B), which would be indicative of oxidative stress
Also consistent with the absence of lithium-induced oxidative stress in BAEC, the distribution of mitochon-dria within cells was not altered compared with sodium-treated cells, apart from an increase in cell and mitochondrion size (Fig 2) The distribution of mito-chondria within cells is mainly dependent on movement along microtubules and changes in the cell cycle [49] Lithium treatment increases microtubule stabilization
in a GSK3b-dependent manner [50,51] Lithium also affects the cell cycle, although in a different manner depending on the cell type In particular, lithium indu-ces G2⁄ M cell-cycle arrest in several cell types, inclu-ding BAEC [13,18] The microtubule polymerizing agent, taxol, has been shown to induce both an increase
in mitochondrial biogenesis and G2⁄ M cell-cycle arrest
in the human 143B osteosarcoma cell line, but unlike lithium, these changes were associated with an abnor-mal distribution of mitochondria around the nucleus [52] By contrast, mitochondrial DNA replication starts
at the G1⁄ S phase transition, whereas mitochondrial biogenesis peaks in the G2⁄ M phase, allowing equal distribution of mitochondria between the two daughter cells during cytokinesis [49,53] Thus, the lithium-induced cell-cycle arrest in G2⁄ M might be sufficient to explain lithium-induced mitochondrial biogenesis Our results also showed that this lithium-dependent increase in mitochondrial biogenesis in BAEC was associated with an increase in mRNA levels for coacti-vator PGC-1a but not coacticoacti-vator PRC (Fig 8A) This
is consistent with the cell-cycle arrest induced by lith-ium Indeed, regulation of PRC expression is mainly dependent on cell-proliferation status, i.e increased
in the presence of growth factors and decreased in contact-inhibited cells [26] However, regulation of PGC-1a expression during cell differentiation is well documented, as are the effects of lithium on cell differ-entiation PGC-1a expression increases during regener-ative skeletal myogenesis as the cells grow, fuse and acquire contractile functions [54], and both lithium and Wnt signaling activate myogenic differentiation in cell-culture systems and the muscle regeneration model [55,56] By contrast, activation of the canonical Wnt⁄ b-catenin signaling pathway in highly differentiated
*
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8 h
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Treatments
phosphoS133-CREB
phosphoS133-ATF1
CREB
β-actin
0
0.5
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1.5
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Li VPA
PhosphoS133-CREB/CREB
8 h
Fig 8 Lithium increases the expression of the coactivator PGC-1a.
(A) Lithium increases the levels of PGC-1a mRNA BAEC were
trea-ted for 36 h with 10 m M NaCl, 10 m M LiCl, 1 m M VPA or 5 l M
indirubin prior to RNA extraction and the levels of PGC-1a and PRC
mRNAs were quantified using real-time RT-PCR The graph
repre-sents mean ± SEM results obtained from five independent
experi-ments (B) Lithium increases the levels of phospho-S133-CREB.
BAEC were treated for 8 h with 10 m M NaCl, 10 m M LiCl, 1 m M
VPA or 5 l M indirubin prior to cell lysis and the levels of
phospho-S133-CREB and total CREB were analyzed using immunoblotting
with b-actin as the loading control A representative experiment is
shown the fold changes obtained after lithium treatment in three
independent experiments are reported.