Rac1/osmosensing scaffold for MEKK3 contributes
via phospholipase C-γ1 to activation of the
osmoprotective transcription factor NFAT5
Xiaoming Zhoua,1, Yuichiro Izumib, Maurice B. Burgb,1, and Joan D. Ferrarisb
aDepartment of Medicine, Uniformed Services University of the Health Sciences, Bethesda, MD 20814; andbSystems Biology Center, National Heart, Lung,
and Blood Institute, National Institutes of Health, Bethesda, MD 20892
Contributed by Maurice B. Burg, May 25, 2011 (sent for review March 17, 2011)
Separate reports that hypertonicity activates p38 via a Rac1–OSM–
MEKK3–MKK3–p38 pathway and that p38α contributes to activa-
tion of TonEBP/OREBP led us to the hypothesis that Rac1 might
activate TonEBP/OREBP via p38. The present studies examine that
possibility. High NaCl is hypertonic. We find that siRNA knockdown
of Rac1 reduces high NaCl-induced increase of TonEBP/OREBP tran-
scriptional activity (by reducing its transactivating activity but not
its nuclear localization). Similarly, siRNA knockdown of osmosens-
ing scaffold for MEKK3 (OSM) also reduces high NaCl-dependent
TonEBP/OREBP transcriptional and transactivating activities. Simul-
taneous siRNA knockdown of Rac1 and OSM is not additive in
reduction of TonEBP/OREBP transcriptional activity, indicating a
common pathway. However, siRNA knockdown of MKK3 does
not reduce TonEBP/OREBP transcriptional activity, although siRNA
knockdown of MKK6 does. Nevertheless, the effect of Rac1 on
TonEBP/OREBP is also independent of MKK6 because it occurs in
MKK6-null cells. Furthermore, we find that siRNA knockdown of
Rac1 or OSM actually increases activity (phosphorylation) of p38,
ratherthandecreasingit, aspreviouslyreported.Thus,the effectof
Rac1 on TonEBP/OREBP is independent of p38. We find instead that
γ1–null mouse embryonic fibroblast cells, catalytically active Rac1
does not increase TonEBP/OREBP transcriptional activity unless
PLC-γ1 is reconstituted. Similarly, dominant-negative Rac1 also
does not inhibit TonEBP/OREBP in PLC-γ1–null cells unless PLC-γ1
is reconstituted. We conclude that Rac1/OSM supports TonEBP/
in increased expression of osmoprotective genes (1). Hyperto-
nicity increases TonEBP/OREBP activity by increasing its abun-
dance(1),transactivating activity(1),nuclear localization (1),and
phosphorylation (2, 3). The regulatory increase of phosphoryla-
tion depends on both increased kinase activity and reduced
phosphatase activity (4, 5). The stress-activated MAP kinase p38
has been extensively studied in this regard, but understanding its
role has been complicated because hypertonicity activates two
different p38s, namely p38α (MAPK14) and p38δ (MAPK13),
which have opposite effects on TonEBP/OREBP activity: p38α
increases TonEBP/OREBP activity and p38δ decreases it (6).
Furthermore, the phosphospecific antibodies commonly used to
measure p38 activity do not directly distinguish between p38α
activity and p38δ activity, nor do some forms of inhibition that
have been used (6). In this article, we will continue to refer to
“p38” unless p38α and p38δ are distinguished. Hypertonicity
activates p38α via the upstream kinase MKK3 (MAP2K3) or
MKK6 (MAP2K6) (7), and p38α and MEKK3 (MAP3K3) con-
tribute to hypertonicity-induced activation of TonEBP/OREBP
(6, 8, 9). Thus, p38α contributes to TonEBP/OREBP activity.
Rac1 is a Rho GTPase in the Ras superfamily (10). It is a mo-
lecular switch that transmits diverse signals from cell-surface
ypertonicity (e.g., high NaCl) activates the transcription
factor TonEBP/OREBP (also known as NFAT5), resulting
receptors to intracellular targets, regulating many cellular activ-
ities, including gene transcription, cytoskeleton reorganization,
cell growth, migration, and oncogenesis (10). A link between
Rac1 and p38 was indicated in a report showing that Rac1 forms
a complex with osmosensing scaffold for MEKK3 (OSM),
MEKK3, and MKK3 that activates p38 in response to hyperto-
nicity (11). Also, Rac1 and p38α are reported to contribute to the
hypertonicity-induced increased expression of TonEBP/OREBP
mRNA (12). Putting this information together, it has been pro-
posed that Rac1 and OSM might contribute to activation of
TonEBP/OREBP via a MEKK3–MKK3–p38 pathway (13, 14).
The initial purpose of the present experiments was to test this
hypothesis directly. We confirm that Rac1/OSM contributes to
activation of TonEBP/OREBP, but the activation is via phos-
pholipase C-γ1 (PLC-γ1), not p38.
Effect of Rac1 and OSM on the Transcriptional Activity of TonEBP/
OREBP. We measured TonEBP/OREBP transcriptional activity in
HEK293 cells stably expressing a luciferase reporter contain-
ing the osmotic response elements (OREs) that are the target of
TonEBP/OREBP in the aldose reductase gene. Raising osmo-
lality from 290 to 500 mosmol/kg by adding NaCl for 16 h in-
creases the transcriptional activity of TonEBP/OREBP by 85-fold
significantly reduces TonEBP/OREBP transcriptional activity at
both 290 and 500 mosmol/kg (Fig. 1B). As a control, when the
DNA elements in the reporter are mutated to prevent TonEBP/
OREBP binding, reporter activity is much lower, is not affected
by osmolality, and is not affected by the siRNAs (Fig. 1C). Si-
multaneous knockdown of both Rac1 and OSM does not inhibit
TonEBP/OREBP transcriptional activity any more than knocking
act in the same pathway. We conclude that Rac1 and OSM con-
tribute to TonEBP/OREBP transcriptional activity.
Effect of Rac1 and OSM on the High NaCl-Induced Increase of TonEBP/
OREBP Transactivating Activity. High NaCl increases the trans-
activating activity of TonEBP/OREBP in HEK293 cells stably
expressing a reporter that contains the TonEBP/OREBP trans-
activation domain (Fig. 2 A and B). Reporter activity in this assay
depends on expression of the TonEBP/OREBP transactivation
domain but is independent of expression of native TonEBP/
OREBP. siRNA knockdown of Rac1 or OSM significantly re-
duces this high NaCl-induced increase of TonEBP/OREBP
Author contributions: X.Z., M.B.B., and J.D.F. designed research; X.Z. and Y.I. performed
research; X.Z., Y.I., M.B.B., and J.D.F. analyzed data; and X.Z., M.B.B., and J.D.F. wrote the
The authors declare no conflict of interest.
1To whom correspondence may be addressed. E-mail: firstname.lastname@example.org or maurice_burg@
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| July 19, 2011
| vol. 108
| no. 29
transactivating activity. We conclude that Rac1 and OSM con-
tribute to the high NaCl-induced increase of TonEBP/OREBP
Effect of Rac1 on High NaCl-Induced Nuclear Localization and Protein
Abundance of TonEBP/OREBP. High NaCl increases the nuclear to
cytoplasmic ratio of TonEBP/OREBP (Fig. 2C). However,
induced TonEBP/OREBP nuclear localization (Fig. 2C). In
contrast, siRNA-mediated knockdown of Rac1 significantly re-
duces TonEBP/OREBP protein abundance regardless of NaCl
concentration (Fig. 2D). We conclude that Rac1 contributes to
high NaCl-induced increase of TonEBP/OREBP transcriptional
activity by increasing its transactivating activity and protein
abundance but not its nuclear localization.
Effects of MKK3 and MKK6 on High NaCl-Induced Activation of
TonEBP/OREBP. To determine whether Rac1 and OSM activate
TonEBP/OREBP through a MEKK3–MKK3–p38 pathway, we
tested the effect of siRNA knockdown of MKK3. Knockdown of
MKK3 has no significant effect on TonEBP/OREBP transcrip-
tional activity even though it reduces high NaCl-induced phos-
phorylation of p38 (Fig. 3A), consistent with our previous finding
transcriptional activity even though it reduces phosphorylation
of p38 (15). On the other hand, MKK6 apparently is involved.
siRNA against MKK6 reduces both the high NaCl-induced in-
crease of TonEBP/OREBP transcriptional activity and the high
NaCl-induced increase of p38 phosphorylation (Fig. 3B). Domi-
nant-negative MKK6 has the same effect (Fig. 3C). We conclude
that MKK3 does not contribute to high NaCl-induced increase of
TonEBP/OREBP transcriptional activity but that MKK6 does.
Effects of MKK6 and p38 on High NaCl-Induced Increase of TonEBP/
OREBP Transactivating Activity and Nuclear Localization. siRNA
against MKK6 significantly reduces TonEBP/OREBP’s trans-
used a luciferase reporter containing three OREs in the context of the aldose
reductase gene to measure TonEBP/OREBP transcriptional activity. Cells were
transfected with Rac1 or OSM siRNAs for 32 h, then osmolality was increased
to 500 mosmol/kg (NaCl added) or left at 290 mosmol/kg for 16 h. (A) Specific
siRNAs against Rac1 and OSM greatly reduce the abundance of those pro-
teins. (B) Knockdown of either Rac1 or OSM significantly reduces TonEBP/
OREBP transcriptional activity, measured in HEK293 cells stably expressing
the ORE luciferase reporter. (C) The siRNAs have no significant effect when
the reporter contains OREs mutated to prevent binding by TonEBP/OREBP
(*P < 0.05 compared with respective control, n = 3). (D) Combined knock-
down of both Rac1 and OSM has no greater effect on TonEBP/OREBP tran-
scriptional activity than knocking them down individually (*P < 0.05
compared with control, n = 3).The Rac1 siRNA concentration was reduced (as
recommended by Invitrogen) by 60% from 33.3 nM to 13.3 nM to reduce its
effect sufficiently so that any additive effect of simultaneously knocking
down OSM could be seen.
Rac1 and OSM regulate TonEBP/OREBP transcriptional activity. We
OREBP transactivating activity and TonEBP/OREBP protein abundance but
not to TonEBP/OREBP nuclear localization. (A and B) As in Fig. 1, except that
we measured TonEBP/OREBP transactivating activity in HEK293 cells stably
expressing a yeast binary GAL4 reporter assay system. High NaCl increases
TonEBP/OREBP transactivating activity, and the increase is reduced signifi-
cantly by siRNA against either Rac1 (A) or OSM (B) (*P < 0.05 compared with
control at 500 mosmol/kg, n = 3). (C) HEK293 cells were transfected with
siRNA against Rac1 for 24 h at 290 mosmol/kg. After incubation for an ad-
ditional 24 h, the medium was changed for 30 min, either maintaining os-
molality at 290 mosmol/kg or increasing it to 500 mosmol/kg (NaCl added).
Proteins were extracted separately from cytoplasm (C) and nuclei (N) and
then analyzed by Western blotting. Note that the subcellular distributions of
β-tubulin (cytoplasmic marker) and Brg-1 (nuclear marker) are unaffected by
NaCl concentration or siRNA against Rac1, showing consistent separation of
cytoplasmic and nuclear proteins. siRNA against Rac1 does not affect nuclear
localization of TonEBP/OREBP. N:C, nuclear to cytoplasmic ratio. (D) HEK293
cells were transfected with Rac1 siRNA for 32 h, then osmolality was in-
creased to 500 mosmol/kg (NaCl added) or left at 290 mosmol/kg for 16 h
(*P < 0.05 compared with respective control, n = 3). Knockdown of Rac1
significantly reduces TonEBP/OREBP protein abundance.
Rac1 and OSM contribute to high NaCl-induced increase of TonEBP/
| www.pnas.org/cgi/doi/10.1073/pnas.1108107108Zhou et al.
activating activity (Fig. 3D) but does not affect its nuclear locali-
zation (Fig. 3E). Thus, MKK6 contributes to high NaCl-induced
TonEBP/OREBP transcriptional activity by increasing its trans-
activating activity, not its nuclear localization. Previous studies
have shown that p38 is a major target of MKK6 (7) and that both
dominant-negative p38α and the p38 inhibitor SB203580 reduce
TonEBP/OREBP transactivating activity (9). Thus, MKK6 ap-
parently increases TonEBP/OREBP transactivating activity via
p38α. That being so, the lack of an effect of MKK6 on TonEBP/
OREBP nuclear localization suggests that p38 is not involved in
high NaCl-induced nuclear localization of TonEBP/OREBP.
Consistent with this idea, the p38 inhibitor SB203580 does not
significantly affect high NaCl-induced TonEBP/OREBP nuclear
localization (Fig. 3F).
Effect of Rac1 and OSM on Phosphorylation of p38. siRNA-mediated
knockdown of OSM was reported to inhibit the increase of p38
phosphorylation caused by high sorbitol-induced hypertonicity in
HEK293 cells (11). We attempted to extend this observation to
OSM also inhibits increased phosphorylation of p38 when hy-
pertonicity is caused by adding NaCl. Contrary to ourexpectation,
we found that siRNA-mediated knockdown of OSM actually
osmolality is increased to 500 mosmol/kg by adding NaCl (Fig.
4A). siRNA against Rac1 has a similar effect (Fig. 4A). Given this
of sorbitol (11). Using the previously published protocol (11), we
found that siRNA knockdown of OSM or Rac1 increases phos-
phorylation of p38 both at 290 mosmol/kg and when osmolality is
increased to 500 mosmol/kg by adding sorbitol (Fig. S1). The ef-
fect of knockdown of Rac1 on phosphorylation of p38 is not me-
diated through Rac2 because we find no evidence that HEK293
cells express Rac2 protein (Fig. S2). We do not know why our
result (Fig. S1) is different fromthe previously published one(11).
The p38 inhibitor SB203580 significantly reduces TonEBP/
OREBP protein expression at 500 mosmol/kg but not at 300
mosmol/kg (Fig. 4B).We conclude that Rac1 and OSM do not
stimulate p38, but may actually inhibit it, and that the stimulation
of TonEBP/OREBP by Rac1 and OSM does not depend on p38.
Role of MKK6 in Activation of TonEBP/OREBP by Rac1. Having found
that TonEBP/OREBP activity depends on expression of both
Rac1 (Fig. 1B) and MKK6 (Fig. 3B), we tested to see whether
the effects are related. Lack of MKK6 [MKK6−/−mouse em-
bryonic fibroblast cells (MEFs)] does not prevent overexpression
of catalytically active Rac1 (caRac1) from increasing TonEBP/
OREBP transcriptional activity at 290 mosmol/kg (Fig. 4C).
caRac1 does not increase TonEBP/OREBP transcriptional ac-
tivity any further when NaCl is high (Fig. 4C), but that may be
because native Rac1 is already activated enough by high NaCl
in these cells to obscure the effect of additional activity from
caRac1. In addition, dominant-negative Rac1 reduces TonEBP/
OREBP transcriptional activity even in the absence of MKK6
expression (Fig. 4D). We conclude that the contribution of Rac1
to TonEBP/OREBP activity is independent of MKK6.
Role of PLC-γ1 in Activation of TonEBP/OREBP by Rac1. Not finding
that MAPK pathways are involved in the activation of TonEBP/
OREBP by Rac1, we turned our attention to PLC-γ1 based on
reports that PLC-γ1 contributes to activation of TonEBP/
OREBP (3) and that Rac1 can activate PLC-γ1 (16, 17). We
compared the effect of Rac1 in PLC-γ1–null MEFs to its effect in
the same cells in which PLC-γ1 is reconstituted (18). These cells
express approximately the same amount of TonEBP/OREBP
whether PLC-γ1 is reconstituted or not (Fig. 5A). caRac1 does
not increase TonEBP/OREBP transcriptional activity in the null
cells, but it does when PLC-γ1 is reconstituted (Fig. 5B). Simi-
TonEBP/OREBP transcriptional activity. (A) HEK293 cells stably expressing an
ORE-X luciferase reporter of TonEBP/OREBP activity were transfected with
control siRNA or siRNA against MKK3 for 32 h. Then the medium was
changed, either maintaining osmolality at 290 mosmol/kg or increasing it to
500 mosmol/kg (NaCl added) for 16 h before measuring luciferase activity.
(B) As in A, except siRNA was against MKK6 (*P < 0.05 compared with
control at 500 mosmol/kg, n = 3). (C) A conditional (Tet-On) vector
expressing dominant-negative MKK6-FLAG (dnMKK6) was cotransfected
together with an ORE-X reporter of TonEBP/OREBP transcriptional activity
into HEK293 cells for 16 h. Then expression of dnMKK6 was induced by
adding doxycycline for 24 h before changing the medium, either maintain-
ing osmolality at 290 mosmol/kg or increasing it to 500 mosmol/kg (NaCl
added) for 16 h (*P < 0.05 compared with control at 500 mosmol/kg, n = 3).
(D) MKK6 contributes to high NaCl-induced increase of TonEBP/OREBP
transactivating activity. As in B, except that TonEBP/OREBP transactivating
activity was measured in HEK293 cells stably expressing a yeast binary GAL4
reporter assay system (*P < 0.05 compared with respective control, n = 3). (E)
MKK6 does not contribute to high NaCl-induced nuclear localization of
TonEBP/OREBP. As in B, except that NaCl was increased for 30 min before
extracting cytoplasmic and nuclear proteins separately for Western blot
analysis of TonEBP/OREBP and calculating its nuclear to cytoplasmic ratio. (F)
p38 does not contribute to high NaCl-induced nuclear localization of
TonEBP/OREBP. HEK293 cells were preincubated with 0.1% DMSO (Control)
or 10 μM SB203580 in 0.1% DMSO for 60 min. Then the medium was
changed, either maintaining osmolality at 290 mosmol/kg with the inhibitor
or increasing it to 500 mosmol/kg with the inhibitor (NaCl added) for 30 min
before measuring the TonEBP/OREBP nuclear to cytoplasmic ratio.
MKK6, but not MKK3, contributes to high NaCl-induced increase of
Zhou et al.PNAS
| July 19, 2011
| vol. 108
| no. 29
larly, dominant-negative Rac1 does not inhibit TonEBP/OREBP
in PLC-γ1–null cells unless they are reconstituted with PLC-γ1
(Fig. 5C). We conclude that Rac1 activates TonEBP/OREBP
Rac1–OSM–MEKK3–MKK3–p38–TonEBP/OREBP? Our original pur-
pose was to directly test the hypothesis (13) that Rac1 contrib-
utes to TonEBP/OREBP activity via an OSM–MEKK3–MKK3–
p38 pathway (11). We find that knockdown of Rac1 or OSM
reduces TonEBP/OREBP activity, and knockdown of both Rac1
and OSM reduces TonEBP/OREBP activity similarly to each
alone (Fig. 1), indicating a common pathway. Thus, our data are
in accord with a tonicity-dependent physical association between
Rac1 and OSM (11). However, activation of TonEBP/OREBP is
not via a Rac1–OSM–MEKK3–MKK3–p38 pathway because
knockdown of MKK3 does not affect TonEBP/OREBP tran-
scriptional activity (Fig. 3) and knockdown of Rac1 or OSM
increases, rather than decreases, p38 activity (phosphorylation)
(Fig. 4A). The effects of Rac1 and p38 on TonEBP/OREBP
protein abundance differ in that siRNA knockdown of Rac1
reduces TonEBP/OREBP protein at both 300 and 500 mosmol/
kg (Fig. 2D), but the p38 inhibitor SB203580 decreases TonEBP/
OREBP abundance only at 500 mosmol/kg (Fig. 4B). The in-
crease of p38 phosphorylation when we knocked down Rac1 is
somewhat surprising because Rac1 generally activates p38 (19),
but there are other exceptions. For example, Rac1 contributes to
EGF-induced phosphorylation of p38 in papilloma cells but not
in normal laryngeal cells (20). Furthermore, similar to our
finding, knockdown of OSM in human endothelial cells increases
phosphorylation of p38 (21).
Rac1 is located in the nucleus as well as in the cytoplasm (22).
siRNA against Rac1 knocks it down more in the nucleus than in
the cytoplasm (Fig. 2C), probably because it both regulates and is
regulated by the cell cycle. Inhibition of Rac1 induces G1 cell-
cycle arrest (23), and Rac1 is excluded from the nucleus in early
G1 (24). This combination accounts for its disproportionate de-
crease in the nucleus when it is knocked down.
Rac1–PLC-γ1–TonEBP/OREBP. Having found that regulation of
TonEBP/OREBP by Rac1 is not mediated by MKK3 and p38,
we tested whether mediation is via PLC-γ1, which contributes to
regulation of TonEBP/OREBP (3), interacts directly with Rac1
(16, 25), and can be a downstream target of Rac1 (16, 17). In
addition, Rac1 often acts in combination with PLC-γ1 to regulate
cellular signaling events (16, 25, 26). In the present study, we find
that caRac1 and dominant-negative Rac1 do not affect TonEBP/
OREBP transcriptional activity in PLC-γ1–null MEFs unless
PLC-γ1 is reconstituted (Fig. 5). When PLC-γ1 is reconstituted,
caRac1 increases TonEBP/OREBP transcriptional activity, and
dominant-negative Rac1 reduces it (Fig. 5), similar to the result
in HEK293 cells (Fig. 4 C and D). We conclude that, because the
effect of caRac1 depends on expression of PLC-γ1, Rac1 must be
acting upstream of PLC-γ1 and not vice versa. It should be
(A) HEK293 cells were transfected with either Rac1 or OSM siRNA for 24 h.
Then 24 h later, the medium was changed, either maintaining osmolality at
290 mosmol/kg or increasing it to 500 mosmol/kg (NaCl added) for 30 min
before Western blot analysis of p38 and phospho-p38 (p38-P) (*P < 0.05
compared with respective control, n = 3). (B) SB203580 significantly reduces
TonEBP/OREBP protein abundance. HEK293 cells were preincubated with
0.1% DMSO (Vehicle) or 10 μM SB203580 in 0.1% DMSO for 60 min, then
osmolality was increased to 500 mosmol/kg with the inhibitor (NaCl added)
or left at 290 mosmol/kg with the inhibitor for 8 h (*P < 0.05, compared with
control at 500 mosmol/kg, n = 3). (C and D) Expression of Rac1 contributes to
transcriptional activity of TonEBP/OREBP independent of MKK6. (Upper)
Representative Western blots of expressed transfected MKK6, caRac1, or
dominant-negative Rac1 (dnRac1). (C) MKK6−/−MEFs, or the same cells re-
constituted with MKK6, were cotransfected with caRac1 or empty vector
(EV) and an ORE reporter for 32 h, then the medium was changed, either
maintaining osmolality at 290 mosmol/kg or increasing it to 500 mosmol/kg
(NaCl added) for 16 h before measuring TonEBP/OREBP transcriptional ac-
tivity (*P < 0.05 vs. EV, n = 3). (D) As in C, except cells were transfected with
dominant-negative Rac1 (*P < 0.05 vs. EV, n = 3).
siRNA knockdown of Rac1 or OSM increases phosphorylation of p38.
OREBP and Rac1 is equivalent in PLC-γ1+/+and PLC-γ1−/−MEFs. (B and C
Upper) Representative Western blots of expressed transfected PLC-γ1,
caRac1 or dominant-negative Rac1 (dnRac1). (B) PLC-γ1+/+and PLC-γ1−/−
MEFs were transfected with caRac1 plus ORE reporter for 32 h, then the
medium was changed, either maintaining osmolality at 290 mosmol/kg or
increasing it to 500 mosmol/kg (NaCl added) for 16 h before measuring
TonEBP/OREBP transcriptional activity (*P < 0.05 versus respective control,
n = 3). (C) As in B, except cells were transfected with dominant-negative
Rac1 (*P < 0.05 versus respective control, n = 3).
Rac1 activates TonEBP/OREBP via PLC-γ1. (A) Expression of TonEBP/
| www.pnas.org/cgi/doi/10.1073/pnas.1108107108 Zhou et al.
noted, however, that in other cells and circumstances, PLC-γ1
can act upstream of Rac1 (25).
p38 and TonEBP/OREBP. p38α contributes to hypertonicity-induced
activation of TonEBP/OREBP (9), but full understanding of this
interaction has been complicated by opposite effects of p38α
and p38δ; p38α increases TonEBP/OREBP activity and p38δ
decreases it, but the phosphospecific antibodies commonly used
to measure p38 activity do not directly distinguish between p38α
activity and p38δ activity (6). In addition, some of the ways of
inhibiting p38 inhibit both p38α and p38δ, resulting in no net ef-
fect on TonEBP/OREBP activity. For example, MKK3 is an up-
stream activator of p38, but, although dominant-negative MKK3
prevents high NaCl-induced phosphorylation of p38, it does not
inhibit the activation of TonEBP/OREBP (15). That result is
confirmed in the present experiments by siRNA knockdown of
MKK3 (Fig. 3A). Because MKK3 activates both p38α and p38δ
(7), inhibition of MKK3 reduces their opposing effects, resulting
in no net change of TonEBP/OREBP activity. Another example
is MAPK phosphatase 1 (MKP-1), which also inhibits both
p38α and p38δ without any effect on TonEBP/OREBP activity
(6). SB203580 is an often-used inhibitor of p38. It inhibits p38α
and p38β, but not p38δ (27, 28). SB203580 reduces high NaCl-
induced TonEBP/OREBP transcriptional activity, which was
interpreted as simply attributable to inhibition of a positive effect
of p38 (9, 29). However, the situation evidently is more compli-
cated. Inhibition of the hypertonicity-induced positive effect of
p38α by SB203580 also unmasks the accompanying hypertonicity-
induced negative effect of p38δ. Similarly, reduction of high
NaCl-induced TonEBP/OREBP transcriptional activity by dom-
Hypertonicity-Induced Activation of p38. Both MKK3 and MKK6
can activate p38 by phosphorylating it. However, although MKK6
is involved in high NaCl-induced increase of TonEBP/OREBP
transcriptional activity, MKK3 is not. Dominant-negative MKK6
reduces high NaCl-induced TonEBP/OREBP transcriptional ac-
tivityin mouseinnermedullarycollecting ductcells(30)assiRNA
does against MKK6 in HEK293 cells (Fig. 3B). Also, reconsti-
tution of MKK6 increases TonEBP/OREBP transcriptional ac-
tivity in MKK6-null cells (Fig. 4 C and D). In contrast, siRNA
against MKK3 does not reduce TonEBP/OREBP transcriptional
activity (Fig. 3A). MKK3 and MKK6 have different effects be-
cause they activate different p38s. Both of them activate p38α
(31), but MKK3 strongly activates p38δ in response to hyper-
osmolality, whereas MKK6 does not (32). As discussed above,
activation of p38α alone increases TonEBP/OREBP activity, but
activation of both p38α and p38δ does not because of their op-
Additional Considerations. In the present study, we find that a
Rac1–PLC-γ1 pathway contributes to regulation of TonEBP/
OREBP transactivating activity. We previously found that PLC-
γ1 regulates TonEBP/OREBP transactivating activity via its SH3
domain, independent of its lipase activity (3). In addition, PLC-γ1
also regulates TonEBP/OREBP in a way that apparently does not
involve Rac1. PLC-γ1, through its C-SH2 domain, regulates nu-
clear localization of TonEBP/OREBP, dependent on its lipase
activity (3). This activation evidently is independent of Rac1 be-
cause Rac1 does not contribute to TonEBP/OREBP nuclear lo-
calization (Fig. 2C). Phosphatidylinositol 3-kinase (PI3K) may be
an upstream activator of the Rac1–PLC-γ1–TonEBP/OREBP
pathway. PI3K increases high NaCl-dependent transactivating
activity of TonEBP/OREBP (33), and PI3K is known to activate
Rac1 in other contexts (34).
MKK6 (Fig. 3D), like p38α (9), increases TonEBP/OREBP
transactivating activity, but neither MKK6 nor p38α increases its
nuclear localization (Fig. 3 E and F). MEKK3 may be the up-
stream activator of the MKK6–p38α–TonEBP/OREBP pathway
because MEKK3 is known to regulate TonEBP/OREBP activity
(8) and to act upstream of MKK6 (35). However, this role
remains to be established. Also, at present there is no direct in-
dication of how p38α increases high NaCl-dependent TonEBP/
OREBP transactivating activity.
Rac1 activation by hyperosmotic stress was previously shown in
HEK293 (11) and LLC-PK1 cells (36). In both cases, the activa-
back to baseline and even below it, which raises the question of
whether the osmotically induced increase of Rac1 activity is re-
as a background—is needed for optimal TonEBP/OREBP stim-
ulation. The fact that dominant-negative and siRNA-mediated
knockdown of Rac1 reduce TonEBP/OREBP activity both at 300
and 500 mosmol/kg is consistent with the possibility that only
background activity of Rac1 is necessary. We cannot distinguish
between these possibilities but would emphasize that knockdown
of Rac1 reduces the high NaCl-induced increase of TonEBP/
OREBP activity (Figs. 1B, 4D, and 5C) and that even a transient
increase of Rac1 activity could initiate longer-lived down-
Finally, it is not surprising that Rac1 and p38α contribute
separately to high NaCl-induced activation of TonEBP/OREBP
because signaling networks often include parallel pathways. Ro-
bustness is a property that allows a system to maintain its func-
tions against internal and external perturbations, and redundancy
is a critical strategy for robustness (37). Other examples of ki-
nases that independently signal high NaCl-induced activation of
TonEBP/OREBP are Fyn and p38 (9).
Materials and Methods
Cells, Chemicals, and Antibodies. See SI Materials and Methods for details.
Plasmids, siRNAs, Transfections, and Luciferase Activity. Plasmids pcDNA3.1-
myc-caRac1 and pcDNA3.1-myc-dnRac1 were gifts from Andras Kapus (Uni-
of dominant-negative MKK6, pTRE2-Flag-dnMKK6, was provided by Norman
was purchased from Addgene. The human ORE-X, ORE, and mutated ORE
described previously (33). The two siRNAs against OSM, described previously
(11), were combined in equal amounts. siRNA against human Rac1 (5′-
GGUAUUUUACAGCACCAAUCUCCUUAG-3′ and 5′-Phos/AAGGAGAUUGGU-
GCUGUAAAAUACC-3′) was synthesized by Integrated DNA Technologies.
MKK3 and MKK6 siRNAs were purchased from Qiagen. We used Effectene,
according the manufacturer’s protocol (Qiagen), for transient transfection of
plasmids into HEK293 cells. Transfection of siRNAs and transfection of DNA
plasmids into MEFs were done with Lipofectamine 2000 in the recommended
ratio of siRNA or DNA to Lipofectamine 2000 (Invitrogen). All transfections
were done by adding cell suspensions to plated DNA or siRNA and trans-
TonEBP/OREBP Transactivating Activity and Nuclear Localization. TonEBP/
OREBP transactivating activity was analyzed in HEK293 cells stably expressing
a yeast binary GAL4 reporter assay system, as previously described (39). To
were extracted separately with NE-PER (Pierce). TonEBP/OREBP in each frac-
tion was measured by Western blot analysis, and nuclear to cytoplasmic ratio
that the ratio was affected by inadequate separation of nuclear and cyto-
cells were pooled and then split into dishes to receive medium change.
Statistics. Data are expressed as mean ± SEM. Analyses were performed by
paired t test with P < 0.05 considered significant.
ACKNOWLEDGMENTS. We thank Dr. Roger J. Davis for MKK6−/−MEFs,
Dr. Graham Carpenter for PLC-γ1+/+and PLC-γ1−/−MEFs, Dr. Andras Kapus
Zhou et al.PNAS
| July 19, 2011
| vol. 108
| no. 29
for Rac1 mutant constructs, and Dr. Norman P. Curthoys for pTRE2-Flag- Download full-text
dnMKK6 plasmid. This study was supported by the Intramural Research Pro-
gram of the National Heart, Lung, and Blood Institute, National Institutes of
Health, Department of Health and Human Services and by Grant CO83WU
from the Uniformed Services University of the Health Sciences, Department
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| www.pnas.org/cgi/doi/10.1073/pnas.1108107108Zhou et al.