Division of Nephrology and Department of Cell and Developmental Biology, Oregon Health and Science University and the Portland Veterans Affairs Medical Center, Portland, Oregon 97201
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Novel protein kinase C
(PKC) isoforms PKC and PKC
have recently been implicated in
signaling by hypertonic stress. We investigated the role of the
putative PKC
inhibitor rottlerin on tonicity-dependent gene
regulation. In the renal medullary mIMCD3 cell line, rottlerin blocked
tonicity-dependent transcription of a tonicity enhancer (TonE)-driven
luciferase reporter gene, as well as tonicity-dependent transcription
of the physiological tonicity effector gene aldose reductase, but not
urea-dependent transcription. Consistent with these data,
rottlerin inhibited tonicity-dependent expression of TonE
binding protein (TonEBP) at the mRNA and protein levels. Another
inhibitor of both novel and conventional PKC isoforms, GF-109203X, suppressed TonEBP-dependent transcription but failed to
influence tonicity-inducible TonEBP expression. Global PKC downregulation with protracted phorbol ester treatment, however, failed
to influence tonicity-dependent signaling, arguing against a
PKC
-dependent mechanism of rottlerin action in this model. In
addition, hypertonic stress failed to induce phosphorylation of PKC
.
Furthermore, in a PC-12 cell model with a comparable degree of
tonicity-dependent transcription, constitutive overexpression of
dominant negative-acting PKC
or PKC
effectively decreased tonicity signaling to extracellular signal-regulated kinase activation, as expected, but failed to influence TonE-dependent
transcription. TonE-dependent transcription, however, remained
rottlerin sensitive in this PC-12 cell model. In the aggregate,
these data indicate that rottlerin dramatically inhibits
tonicity-dependent TonEBP expression and TonE-dependent transcription
but, despite its reputed mode of action, does so through a
PKC
-independent pathway.
hypertonicity; inner medullary collecting duct; renal; kidney; signal transduction
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
THE 11 PROTEIN KINASE C (PKC) ISOFORMS INCLUDE conventional
(,
I,
II, and
), novel (
,
,
, and
), and atypical (
and
) isoforms (27).
Through a combination of predominantly inhibitor-based and PKC
downregulation studies, several groups have broadly implicated PKC
activation in hypertonic stress signaling and in the acquisition of the
hypertonically stressed phenotype (16, 20, 21, 33, 36);
other groups, however, have failed to observe PKC dependence or
activation in this setting (2, 9, 10). In a recent and
thorough series of experiments, Zhuang et al. (45)
described a role for the novel PKC isoforms PKC
and PKC
in
osmotic activation of the extracellular signal-regulated kinase (ERK),
mitogen-activated protein kinase (MAPK) (45). In their
fibroblastic 3T3 cell model, hypertonic stress induced membrane
translocation of PKC
and PKC
, as well as the conventional PKC
isoform. Studies with the inhibitors of novel (n)PKC, rottlerin
and GF-109203X underscored a role for PKC
and PKC
in
tonicity-dependent ERK activation (45).
In addition to activating the ERKs, however, hypertonic stress activates the p38 and c-jun N-terminal-directed kinase (JNK) families of MAPKs through activation of their respective upstream activators (reviewed in Ref. 18). The role of these events in the downstream activation (41) of the tonicity enhancer binding protein (TonEBP), the ubiquitous tonicity-responsive transcription factor of higher eukaryotes (26), and in TonEBP-dependent transcription (19, 32) remains incompletely understood. TonEBP was extensively and elegantly characterized in the present physiological context by Kwon et al. (reviewed in Ref 13) and cloned independently as TonEBP (26), nuclear factor of activated T cells-5 (23), and osmotic response element binding protein (17). In response to hypertonic stress, TonEBP upregulation occurs at the mRNA (40) and protein levels (26), as well as at the level of subcellular localization (41). Signaling events influencing expression and translocation of TonEBP are receiving increasing attention. Signaling through the p38 MAPK (32) and proteasomal function (41) have been implicated in tonicity-dependent transcription or tonicity-dependent TonEBP expression; however, urea, the renal medullary solute, blocks tonicity-dependent expression of TonEBP independently of these pathways (38). Downstream effectors of TonEBP in the renal epithelium, physiologically stressed in vivo by dramatic fluctuations in ambient tonicity, include the sorbitol-synthesizing enzyme aldose reductase and transporters of osmotically active solutes (reviewed in Ref. 3).
We investigated the role of novel PKC and PKC
isoforms in
tonicity-dependent gene regulation and observed that, whereas the
purportedly PKC
-specific inhibitor rottlerin (12)
potently inhibited the tonicity-dependent increase in TonEBP expression at the mRNA and protein levels and inhibited TonEBP-dependent transcription of both a TonE-driven luciferase reporter gene and the
physiological TonEBP effector aldose reductase, it did so in a
PKC
-independent fashion.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
General methods.
Cell culture and solute treatment were performed as previously
described (30, 37). The following inhibitors and stimuli (purchased from Calbiochem unless otherwise indicated) were used: 200 mM urea (Sigma); 100 mM NaCl (200 mosmol/kgH2O; Sigma); 100 nM epidermal growth factor; 100 nM 12-O-tetradecanoylphorbol
13-acetate (TPA; Sigma); 10-50 µM rottlerin; 30-300 nM
Go-6983, 10 µM GF-109203X, 1 µM Go-6976, and 50 µM SB-203580.
Cells receiving pretreatment with an inhibitor or solute (i.e., urea)
remained exposed to the pretreatment compound for the duration of the
experiment until determination of the experimental end point. Depicted
data represent means ± SE unless indicated; statistical
significance is assigned to P < 0.05 (Excel;
Microsoft). PC-12 cell lines expressing dominant negative-acting PKC
isoforms were kindly provided by Drs. Thomas McMahon and Robert O. Messing and were previously described (15). Cell lines
were as follows: vector-transfected, originally designated 10-C;
DN-, originally designated DND-21; and DN-
, originally designated
DNE-1.
TonEBP and aldose reductase RNase protection assay. Total cellular RNA was prepared using the TriZol reagent (Life Technologies) in accordance with the manufacturer's directions. Murine TonEBP and aldose reductase partial cDNAs were previously described (38). Plasmids were linearized with XhoI, gel-purified (QIAEX II kit; Qiagen), reconstituted in diethylpyrocarbonate-treated H2O, and used for biotinylated antisense riboprobe preparation with biotin RNA labeling mix and T7 polymerase (Boehringer Mannheim). Hybridization of probe (800 pg) with total RNA (15 µg) was performed at 45°C (RPA II kit; Ambion); subsequent digestion with RNase A and T1 was performed in solution at 37°C. Detection was achieved with the BrightStar BioDetect nonisotopic detection kit (Ambion).
Reporter gene assay.
The plasmid BGT-2X-Luc, based on the TonE element
(25), has been previously described
(37). Cells were transfected with the plasmid of
interest (as well as a lacZ expression plasmid under the
control of the cytomegalovirus long terminal repeat) via
electroporation; luciferase and -galactosidase activities were
quantitated at 6 h of treatment as previously described
(6) except that, for the latter, a Luminescent
-Galactosidase Detection Kit II (Clontech) was used in accordance
with the manufacturer's directions.
Immunoblotting.
Immunoblotting, using commercially available anti-phospho-ERK and
anti-phospho-p38 (Cell Signaling Technologies) was performed as
previously described (42).
Anti-phospho-(Thr505) PKC was obtained from Cell
Signaling Technologies and used in accordance with the manufacturer's
directions. Rabbit polyclonal anti-TonEBP (anti-N-terminal NFAT5) was
kindly provided by Drs. Cristina Lopez-Rodriguez and Anjana Rao
(23) and used at 1:600 dilution in 0.075% Tween 20. For
TonEBP immunoblotting, a combined mechanical and detergent-based lysis
system (7) was employed to ensure nuclear disruption.
RT-PCR.
RT-PCR was performed essentially as previously described, using primers
selected to be specific for PKC, PKC
, PKC
, and PKC
isoforms
and the PKC-related kinase-1 (PRK1). Primers were chosen from
variable regions that lacked significant homology with other known PKC
isoforms. For each isoform depicted (see Fig. 9), a product of only the
anticipated size was generated. Optimization for each isoform was
performed by varying buffer composition and pH over known increments
for a total of 12 individual reactions/sample (OptiPrime; Stratagene)
in accordance with the manufacturer's directions. Primer pairs for PKC
isoforms (Integrated DNA Technologies, Coralville, IA) were as follows
(all are listed 5' to 3'): PKC
, 5'-GGA TTG CAA GCA GTC TAT GCG-3'
and TCA CTT GGT TCA AGG CCT CAG; PKC
, 5'-TTG TCC ACA AGC GAT GTC
ATG-3' and TGA CTT GGA TCG GTC GTC TTC; PKC
, 5'-AGA CAT GTG TGG CCT
GCA CC-3' and TCG ATG ACA GGC TTA AGG TCC; PKC
, 5'-AAG CGT CGT CCA GTC TAG GTC-3' and CCT CGC TCA TGA AGA TAA TTC AG; and PRK1, 5'-CAA CGA
CGA AGT TCG CTA TCC-3' and AAG CAC ACT CCA GTC CAG ATG C. Starting
material was total RNA harvested from mIMCD3 cells (TriZol; Life Technologies).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Because Zhuang et al. (45) observed dependence of
elements of tonicity-mediated signaling on PKC in murine
fibroblasts, we examined whether other aspects of tonicity signaling
also require this pathway. In the best-studied model of
tonicity-dependent gene regulation, transcription proceeds through
upregulated interaction of TonEBP with its cognate DNA consensus
element within the 5'-flanking region of a tonicity-responsive gene
(reviewed in Ref. 13). mIMCD3 cells were transiently
transfected with BGT-2X-Luc (44), an expression
plasmid harboring a luciferase reporter gene under the control of two
tandem repeats of the BGT TonE enhancer element (35) (Fig.
1A). As anticipated and
consistent with the data of others, hypertonic stress increased
TonE-dependent transcription by approximately eightfold; this effect
was markedly inhibited by pretreatment of cells with the
PKC
-specific inhibitor rottlerin (12) and the nPKC and
conventional (c)PKC inhibitor GF-109203X (Fig. 1B).
Cytomegalovirus long terminal repeat-driven
-galactosidase reporter
gene activity, used for normalization, was inhibited by <20% in the
presence of either NaCl or rottlerin. We were unable to assess the
effect of these inhibitors on basal and tonicity-dependent transcription from an enhancerless thymidine kinase promoter because this construct exhibited no detectable activity in the present model.
Therefore, the effect of these inhibitors on urea-inducible transcription of the immediate-early gene Egr-1 (Fig.
1C) was examined in parallel. Consistent with earlier
observations (6), urea increased Egr-1 reporter
gene activity by approximately sixfold; however, neither GF-109203 nor
rottlerin significantly inhibited this effect (Fig. 1D),
underscoring the specificity of the effect on tonicity-dependent
signaling. Next, similar studies were performed in the more
physiological context of tonicity-dependent (and TonEBP-dependent) transcription of the physiological osmotic stress effector aldose reductase (Fig. 2). Hypertonic stress
(100 mM NaCl × 6 h) markedly increased aldose reductase
expression at the mRNA level, and this effect was substantially
inhibited by pretreatment with rottlerin. GF-109203X failed to
block the effect of hypertoncicity, as did the specific inhibitor
of cPKC isoforms Go-6976. These data implicated an inhibitory
effect of rottlerin on TonEBP action, and this pathway was examined
further.
|
|
The effect of rottlerin on tonicity-dependent TonEBP expression at the
mRNA level was examined via an RNase protection assay, as previously
described (38). Consistent with our earlier data and those
of others, hypertonic stress increased TonEBP mRNA expression severalfold; this effect was almost completely blocked by rottlerin (Fig. 3A). GF-109203X,
however, appeared to be ineffective in this assay. The effect of these
nPKC inhibitors on TonEBP expression at the protein level was explored
in parallel. As anticipated and again consistent with the data of
others (26, 40), hypertonic stress modestly increased
TonEBP protein abundance in whole cell lysates (Fig. 3B).
Again, strikingly similar to the mRNA expression data, rottlerin
markedly inhibited basal and tonicity-inducible TonEBP expression,
whereas GF-109203X failed to do so.
|
PKC and PKC
have been implicated in signaling to ERK activation
by hypertonicity (45). Because the putative PKC
inhibitor rottlerin blocked NaCl signaling to TonE-dependent
transcription, the effect of another maneuver designed to abrogate
PKC
was examined. Phorbol ester activates all classic and nPKC
isoforms, but not the atypical PKC
and PKC
isoforms
(28). Protracted treatment with TPA has been used to
downregulate all of the TPA-responsive PKC isoforms; this
treatment effectively downregulated tonicity-dependent signaling to ERK activation in 3T3 cells (45). In
the mIMCD3 cell model, hypertonic NaCl markedly increased ERK
phosphorylation, as previously shown by us and others (2,
43), but protracted pretreatment with TPA failed to inhibit this
effect (Fig. 4A). In similar
fashion, the effect of PKC downregulation on tonicity-dependent transcription was examined. It was anticipated that this
rottlerin-sensitive process should be sensitive to PKC downregulation.
Unexpectedly, yet consistent with the lack of effect of PKC
downregulation on tonicity-dependent ERK activation, prolonged TPA
treatment failed to significantly influence TonE-mediated transcription
(Fig. 4B). As a positive control to confirm PKC
downregulation (5), the effect of urea on transcription of
the Egr-1 gene was substantially suppressed by this maneuver
(Fig. 4B).
|
Activation of the p38 MAPK has been implicated in TonEBP-dependent
transcription, and a role for PKC in tonicity-inducible ERK
activation has been suggested by data obtained in nonrenal cells
(45). For these reasons, the effect of rottlerin
pretreatment on tonicity-dependent ERK and p38
activation/phosphorylation was examined in mIMCD3 cells. Rottlerin
exhibited a negligible effect on ERK and p38 activation under basal
conditions (Fig. 5, A and B). ERK activation by hypertonicity was in large part
sensitive to rottlerin, whereas ERK activation by the phorbol ester TPA and by the peptide mitogen epidermal growth factor was not (Fig. 5A). MAPK p38 was substantially activated only by hypertonic
stress; TPA and epidermal growth factor produced no consistently
demonstrable effect (Fig. 5B). Rottlerin pretreatment only
very modestly inhibited p38 activation in response to hypertonic
stress. It was concluded in preliminary fashion that rottlerin was not
inhibiting TonEBP signaling via an effect on p38. To corroborate these
data, the effect of rottlerin was compared with that of the p38
inhibitor SB-203580 with respect to inhibition of
tonicity-dependent TonEBP-mediated transcription. In mIMCD3 cells
transiently transfected with BGT-2X-Luc, the effect of
SB-203580 was very modest whereas the effect of rottlerin was again
substantial (Fig. 5C). In the aggregate, these data
indicated that rottlerin was not operating through inhibition of a
p38-dependent pathway in the present context.
|
Data from Zhuang et al. (45) support a role for
PKC in tonicity signaling. Our own data suggested that PKC
might
be involved, as it is a rottlerin-sensitive process, whereas other data
showed that tonicity signaling was insensitive to PKC downregulation. Additional studies were undertaken to determine the role of PKC
activation in the tonicity-inducible transcription. A series of PC-12
cell lines were used that constitutively overexpress dominant negative-acting truncation mutants of nPKC
and nPKC
isoforms or empty vector alone. These cell lines exhibited dramatically downregulated PKC
and PKC
activity in other experimental contexts (8, 14, 15, 34). Initially, a degree of hypertonic stress was established at which tonicity-dependent transcription could be
demonstrated in this PC-12 model. Hypertonic stress (800 mosmol/kgH2O NaCl) increased ERK dual phosphorylation at 15 min of treatment in untransfected PC-12 cells (Fig.
6A),
consistent with earlier observations (24). A comparable
degree of induction was observed in vector-transfected PC-12 cells;
however, in the PC-12 cell lines harboring dominant negative PKC
and
PKC
, there was a marked diminution in the effect of hypertonicity in
the absence of an effect on basal levels of ERK activation (Fig.
6A). There was no effect on p38 activation in response to
hypertonicity. It was concluded that the dominant negative PKC isoforms
were effectively inhibiting nPKC-dependent signaling in this model,
consistent with the findings of Zhuang et al. (45) in the
3T3 cell line.
|
Next, the role of nPKC in tonicity-dependent transcription, in contrast
to ERK activation, was examined in this validated PC-12 cell model.
Cells were transfected with a luciferase reporter gene, under the
control of tandem copies of the TonE enhancer element cloned upstream
of a thymidine kinase promoter, and then exposed to hypertonic NaCl for
6 h. In both wild-type PC-12 cells and in PC-12 cells stably
transfected with vector alone, tonicity-dependent transcription was
increased approximately six- to sevenfold relative to control (Fig.
6B), consistent with observations with this construct in
other cell lines (38). Interestingly, in the cell lines
expressing dominant negative PKC and PKC
, there was no inhibitory
effect on TonE-mediated transcription (expressed as fold-induction), arguing against a role for nPKC in this process. (Of note, there was
substantial variation in the basal level of TonEBP-dependent transcription between cell lines whereas the fold induction remained unchanged.) To corroborate that rottlerin was effective in this model
as well, both wild-type and dominant negative PKC
-expressing PC-12 cell lines were exposed to hypertonicity, in the presence or
absence of rottlerin pretreatment. Again, as in the mIMCD3 cell model,
rottlerin effectively abrogated tonicity-dependent transcription in
both wild-type and dominant negative nPKC-expressing PC-12 cell lines
(Fig. 6C).
Phosphorylation accompanies activation of PKC isoforms; the effect of
hypertonic stress on phosphorylation of PKC (Thr505) was
examined in the mIMCD3 cell model as an additional correlate of PKC
activation (22). Although TPA treatment increased PKC
phosphorylation in a time-dependent fashion, no effect of NaCl treatment relative to time 0 could be demonstrated (Fig.
7).
|
In the aggregate, these data strongly supported the absence of a role
for PKC in tonicity signaling to ERK activation and TonE-dependent
transcription in renal medullary cells. Atypical isoforms PKC
and
PKC
(PKC
is the murine homolog of PKC
) are insensitive to
downregulation with protracted TPA treatment; it was therefore
hypothesized that one of these isoforms may be involved in
rottlerin-sensitive tonicity signaling. RT-PCR analysis was performed
with poly-A+ RNA prepared from mIMCD3 cells to determine
which isoforms were expressed in these cells. PKC
, PKC
, PKC
,
and PKC
, as well as the related kinase PRK1, were all expressed in
renal medullary cells (Fig. 8). Of the
atypical isoforms, only PKC
has been widely studied. In addition,
only PKC
, PKC
, and PKC
appear to be widely expressed.
Furthermore, because PKC
is not downregulated by protracted TPA
treatment and does not necessarily undergo translocation in the setting
of activation, it was a reasonable candidate for mediating the effect
of hypertonicity on TonE-dependent transcription. The inhibitor Go-6983
is reported to inhibit PKC
(11); however, this compound
failed to abrogate the effect of hypertonic NaCl on tonicity-dependent
transcription by reporter gene assay (data not shown). The only PKC
isoform unaffected by Go-6983 is PKCµ (11), more
commonly known as protein kinase D (PKD) (31). The
cPKC-directed inhibitor Go-6976 is also a potent PKCµ inhibitor (11), yet it failed to influence TonEBP-dependent
transcription (Fig. 2 and data not shown).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Recent evidence supports a role for PKC in hypertonic stress
signaling. Zhuang et al. (45) described the sensitivity of tonicity-inducible ERK activation to both the putative
PKC
-specific inhibitor rottlerin and to PKC downregulation
(45). In addition, membrane translocation of the PKC
isoform was detected. Although no other data have implicated nPKC
isoforms per se, others have described an inhibitory effect of
PKC downregulation (affecting both novel and conventional
isoforms) on hypertonic signaling to ERK activation (16, 20, 21,
33, 36). We attempted to extend this observation
to transcriptional events engendered by hypertonic stress and
uncovered a discrepancy between rottlerin sensitivity and PKC
dependence. Rottlerin dramatically suppressed TonEBP-dependent
transcription, apparently by inhibiting the tonicity-dependent increment in TonEBP expression at the mRNA and protein level. Interestingly, another PKC
inhibitor, GF-109203, also suppressed tonicity-mediated transcription but failed to abrogate TonEBP expression. Most importantly, through a combination of biochemical and
dominant-negative approaches, hypertonicity-inducible TonEBP-mediated transcription was found to be independent of PKC
or PKC
activation.
It is puzzling that two inhibitors with purportedly similar mechanisms of action should operate at distinct loci in the same signaling pathway. It is possible that the effect of rottlerin and GF-109203X overlap in terms of TonEBP-dependent transcription but that rottlerin exhibits an additional and independent effect on TonEBP expression. GF-109203X reportedly exhibits greater potency with respect to conventional PKC isoforms than novel ones; nonetheless, inhibitors of classic PKC (e.g., Go-6976 and PKC downregulation with protracted TPA treatment) failed to influence TonEBP-dependent signaling. It is also possible that regulation of the TonE-dependent reporter might differ in some respects from that of the native aldose reductase gene.
Beyond PKC, present data also refute a role for any known PKC
isoforms in mediating tonicity-dependent transcription in renal medullary cells. PKC downregulation with prolonged phorbol ester treatment failed to influence TonEBP-dependent signaling; hence, the
cPKC (
,
I,
II, and
) and nPKC (
,
,
,
) isoforms are unlikely to be involved. The atypical
isoforms PKC
and PKC
may be TPA insensitive, and both are
expressed in mIMCD3 cells (data herein and Ref. 4); the
former isoform, unlike TonEBP-dependent transcription, is sensitive to
Go-6983, whereas few data are available regarding PKC
translocation
or inhibitor sensitivity. PKCµ (PKD) should be sensitive to Go-6976,
whereas TonEBP-dependent transcription is not. PRK1, the related
kinase, is expressed in mIMCD3 cells; however, as for PKC
, limited
data are available with respect to pharmacological inhibition. In the
aggregate, these data argue against a role for any PKC isoform in this
process in renal medullary cells, although involvement of PKC
or a
PKC-related kinase cannot be fully excluded on the basis of available
data and reagents. This is in marked contrast to the observations
of Zhuang et al. (45), who noted, in the fibroblastic 3T3
cell model, that both rottlerin and protracted phorbol ester treatment
abrogated tonicity-dependent signaling to ERK activation, findings
consistent with bona fide involvement of PKC.
These observations with rottlerin extend the small list of
pharmacological inhibitors known to influence TonEBP-dependent signal
transduction. Activation of the p38 MAPK has been variably implicated
in TonEBP expression or function (19, 32) as has proteasomal processing (41); inhibition of these pathways
abrogates TonEBP-dependent signaling. We have shown that pretreatment
with the membrane-permeant renal medullary solute urea (200 mM), but not the permeant solute glycerol, blocks hypertonic stress-inducible TonEBP mRNA expression and TonEBP-dependent transcription
(38). The mechanism through which rottlerin is acting in
the present context, independently of PKC, is unclear. Rottlerin
blocks TonEBP mRNA expression at an early time point and, because the
tonicity-dependent increase in TonEBP mRNA is not a consequence of
altered mRNA stability (40), rottlerin would appear to
block TonEBP transcription. No data are available with regard to
signaling events related to TonEBP transcription per se.
Although rottlerin does not appear to mediate its effect on
tonicity-dependent signaling through PKC, we demonstrated expression of
PKC, PKC
, PKC
, and PKC
, as well as the related PRK1 kinase, in the mIMCD3 murine inner medullary cell line. These data are consistent with and extend the observations of others. Ostlund et al.
(29) detected renal expression of PKC
,
PKC
, PKC
, and PKC
via a combination of approaches. Aristimuno
and Good (1) noted immunodetectable PKC
,
PKC
II, PKC
, PKC
, and PKC
in the inner stripe of
the outer medulla and medullary thick ascending limb, as well as
agonist-inducible membrane translocation of PKC
. Chou et al.
(4), in contrast, examining inner medullary collecting duct suspensions, noted agonist-inducible translocation of only the
PKC
isoform. In this latter model, which likely bears the closest
relation to the mIMCD3 cell culture system, expression of PKC
,
PKC
, PKC
, PKC
, and PKC
was demonstrated via immunoblotting (4).
In summary, the putative PKC-specific inhibitor, rottlerin,
blocks tonicity-dependent transcription and tonicity-dependent expression of the TonEBP transcription factor, but does so in a
PKC
-independent fashion. These data establish a potentially useful
pharmacological inhibitor of TonEBP expression and TonEBP-dependent signaling and call into question the presumed specificity of rottlerin action.
![]() |
ACKNOWLEDGEMENTS |
---|
The authors thank Thomas McMahon and Robert O. Messing for the PC-12 stable transfectants and Cristina Lopez-Rodriguez and Anjana Rao for the anti-NFAT5/TonEBP antiserum.
![]() |
FOOTNOTES |
---|
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-52494 and by the Department of Veterans Affairs.
Address for reprint requests and other correspondence: D. M. Cohen, Mailcode PP262, Oregon Health Sciences Univ., 3314 S.W. US Veterans Hospital Rd., Portland, OR 97201 (E-mail: cohend{at}ohsu.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
10.1152/ajprenal.00303.2001
Received 25 September 2001; accepted in final form 1 November 2001.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Aristimuno, PC,
and
Good DW.
PKC isoforms in rat medullary thick ascending limb: selective activation of the delta-isoform by PGE2.
Am J Physiol Renal Physiol
272:
F624-F631,
1997
2.
Berl, T,
Siriwardana G,
Ao L,
Butterfield LM,
and
Heasley LE.
Multiple mitogen-activated protein kinases are regulated by hyperosmolality in mouse IMCD cells.
Am J Physiol Renal Physiol
272:
F305-F311,
1997
3.
Burg, MB,
Kwon ED,
and
Kultz D.
Regulation of gene expression by hypertonicity.
Annu Rev Physiol
59:
437-455,
1997[ISI][Medline].
4.
Chou, CL,
Rapko SI,
and
Knepper MA.
Phosphoinositide signaling in rat inner medullary collecting duct.
Am J Physiol Renal Physiol
274:
F564-F572,
1998
5.
Cohen, DM,
Gullans SR,
and
Chin WW.
Urea signaling in cultured murine inner medullary collecting duct (mIMCD3) cells involves protein kinase C, inositol 1,4,5-trisphosphate (IP3), and a putative receptor tyrosine kinase.
J Clin Invest
97:
1884-1889,
1996
6.
Cohen, DM,
Gullans SR,
and
Chin WW.
Urea-inducibility of Egr-1 in murine inner medullary collecting duct cells is mediated by the serum response element and adjacent Ets motifs.
J Biol Chem
271:
12903-12908,
1996
7.
Dmitrieva, N,
Kultz D,
Michea L,
Ferraris J,
and
Burg M.
p53 Activation by hypertonicity in renal inner medullary epithelial cells (mIMCD3) protects them from apoptosis (Abstract).
J Biol Chem
275:
18243,
2000
8.
Gerstin, EH, Jr,
McMahon T,
Dadgar J,
and
Messing RO.
Protein kinase C delta mediates ethanol-induced up-regulation of L-type calcium channels.
J Biol Chem
273:
16409-16414,
1998
9.
Good, DW.
Hyperosmolality inhibits bicarbonate absorption in rat medullary thick ascending limb via a protein-tyrosine kinase-dependent pathway.
J Biol Chem
270:
9883-9889,
1995
10.
Grinstein, S,
Mack E,
and
Mills GB.
Osmotic activation of the Na+/H+ antiport in protein kinase C-depleted lymphocytes.
Biochem Biophys Res Commun
134:
8-13,
1986[ISI][Medline].
11.
Gschwendt, M,
Dieterich S,
Rennecke J,
Kittstein W,
Mueller HJ,
and
Johannes FJ.
Inhibition of protein kinase C µ by various inhibitors. Differentiation from protein kinase C isoenzymes.
FEBS Lett
392:
77-80,
1996[ISI][Medline].
12.
Gschwendt, M,
Muller HJ,
Kielbassa K,
Zang R,
Kittstein W,
Rincke G,
and
Marks F.
Rottlerin, a novel protein kinase inhibitor.
Biochem Biophys Res Commun
199:
93-98,
1994[ISI][Medline].
13.
Handler, JS,
and
Kwon HM.
Transcriptional regulation by changes in tonicity.
Kidney Int
60:
408-411,
2001[ISI][Medline].
14.
Hundle, B,
McMahon T,
Dadgar J,
Chen CH,
Mochly-Rosen D,
and
Messing RO.
An inhibitory fragment derived from protein kinase C epsilon prevents enhancement of nerve growth factor responses by ethanol and phorbol esters.
J Biol Chem
272:
15028-15035,
1997
15.
Hundle, B,
McMahon T,
Dadgar J,
and
Messing RO.
Overexpression of epsilon-protein kinase C enhances nerve growth factor-induced phosphorylation of mitogen-activated protein kinases and neurite outgrowth.
J Biol Chem
270:
30134-30140,
1995
16.
Itoh, T,
Yamauchi A,
Miyai A,
Yokoyama K,
Kamada T,
Ueda N,
and
Fujiwara Y.
Mitogen-activated protein kinase and its activator are regulated by hypertonic stress in Madin-Darby canine kidney cells.
J Clin Invest
93:
2387-2392,
1994[ISI][Medline].
17.
Ko, BC,
Turck CW,
Lee KW,
Yang Y,
and
Chung SS.
Purification, identification, and characterization of an osmotic response element binding protein.
Biochem Biophys Res Commun
270:
52-61,
2000[ISI][Medline].
18.
Kultz, D,
and
Burg MB.
Intracellular signaling in response to osmotic stress.
In: Cell Volume Regulation, edited by Lang F.. Basel: Karger, 1998, p. 94-109.
19.
Kultz, D,
Garcia-Perez A,
Ferraris JD,
and
Burg MB.
Distinct regulation of osmoprotective genes in yeast and mammals. Aldose reductase osmotic response element is induced independent of p38 and stress-activated protein kinase/Jun N-terminal kinase in rabbit kidney cells.
J Biol Chem
272:
13165-13170,
1997
20.
Kwon, HM,
Itoh T,
Rim JS,
and
Handler JS.
The MAP kinase cascade is not essential for transcriptional stimulation of osmolyte transporter genes.
Biochem Biophys Res Comm
213:
975-979,
1995[ISI][Medline].
21.
Larsen, AK,
Jensen BS,
and
Hoffmann EK.
Activation of protein kinase C during cell volume regulation in Ehrlich mouse ascites tumor cells.
Biochim Biophys Acta
1222:
477-482,
1994[ISI][Medline].
22.
Le Good, JA,
Ziegler WH,
Parekh DB,
Alessi DR,
Cohen P,
and
Parker PJ.
Protein kinase C isotypes controlled by phosphoinositide 3-kinase through the protein kinase PDK1.
Science
281:
2042-2045,
1998
23.
Lopez-Rodriguez, C,
Aramburu J,
Rakeman AS,
and
Rao A.
NFAT5, a constitutively nuclear NFAT protein that does not cooperate with Fos and Jun.
Proc Natl Acad Sci USA
96:
7214-7219,
1999
24.
Matsuda, S,
Kawasaki H,
Moriguchi T,
Gotoh Y,
and
Nishida E.
Activation of protein kinase cascades by osmotic shock.
J Biol Chem
270:
12781-12786,
1995
25.
Miyakawa, H,
Woo SK,
Chen CP,
Dahl SC,
Handler JS,
and
Kwon HM.
Cis- and trans-acting factors regulating transcription of the BGT1 gene in response to hypertonicity.
Am J Physiol Renal Physiol
274:
F753-F761,
1998
26.
Miyakawa, H,
Woo SK,
Dahl SC,
Handler JS,
and
Kwon HM.
Tonicity-responsive enhancer binding protein, a rel-like protein that stimulates transcription in response to hypertonicity.
Proc Natl Acad Sci USA
96:
2538-2542,
1999
27.
Mochly-Rosen, D,
and
Kauvar LM.
Pharmacological regulation of network kinetics by protein kinase C localization.
Semin Immunol
12:
55-61,
2000[ISI][Medline].
28.
Nishizuka, Y.
Intracellular signaling by hydrolysis of phospholipids and activation of protein kinase C.
Science
258:
607-614,
1992[ISI][Medline].
29.
Ostlund, E,
Mendez CF,
Jacobsson G,
Fryckstedt J,
Meister B,
and
Aperia A.
Expression of protein kinase C isoforms in renal tissue.
Kidney Int
47:
766-773,
1995[ISI][Medline]. [Erratum. Kidney Int 47: June 1995, p. 1838].
30.
Rauchman, MI,
Nigam S,
Delpire E,
and
Gullans SR.
An osmotically tolerant inner medullary collecting duct cell line from an SV40 transgenic mouse.
Am J Physiol Renal Fluid Electrolyte Physiol
265:
F416-F424,
1993
31.
Ron, D,
and
Kazanietz MG.
New insights into the regulation of protein kinase C and novel phorbol ester receptors.
FASEB J
13:
1658-1676,
1999
32.
Sheikh-Hamad, D,
Di Mari J,
Suki WN,
Safirstein R,
Watts BA, III,
and
Rouse D.
p38 kinase activity is essential for osmotic induction of mRNAs for HSP70 and transporter for organic solute betaine in Madin-Darby canine kidney cells.
J Biol Chem
273:
1832-1837,
1998
33.
Soleimani, M,
Singh G,
Dominguez JH,
and
Howard RL.
Long-term high osmolality activates Na(+)-H+ exchange and protein kinase C in aortic smooth muscle cells.
Circ Res
76:
530-535,
1995
34.
Solem, M,
McMahon T,
and
Messing RO.
Protein kinase A regulates inhibition of N- and P/Q-type calcium channels by ethanol in PC12 cells.
J Pharmacol Exp Ther
282:
1487-1495,
1997
35.
Takenaka, M,
Preston AS,
Kwon HM,
and
Handler JS.
The tonicity-sensing element that mediates increased transcription of the betaine transporter gene in response to hyperosmotic stress.
J Biol Chem
269:
29379-29381,
1994
36.
Terada, Y,
Tomita K,
Homma MK,
Nonoguchi H,
Yang T,
Yamada T,
Yuasa Y,
Krebs E,
Sasaki S,
and
Marumo F.
Sequential activation of Raf-1 kinase, mitogen-activated protein (MAP) kinase kinase, MAP kinase, and S6 kinase by hyperosmolality in renal cells.
J Biol Chem
269:
31296-31301,
1994
37.
Tian, W,
Boss GR,
and
Cohen DM.
Ras signaling in the inner medullary cell response to urea and NaCl.
Am J Physiol Cell Physiol
278:
C372-C380,
2000
38.
Tian, W,
and
Cohen DM.
Urea inhibits hypertonicity-inducible tonicity enhancer binding protein (TonEBP) expression and action.
Am J Physiol Renal Physiol
280:
F904-F912,
2001
39.
Tsai-Morris, C,
Cao X,
and
Sukhatme VP.
5' Flanking sequence and genomic structure of Egr-1, a murine mitogen inducible zinc finger encoding gene.
Nucleic Acids Res
16:
8835-8846,
1988[Abstract].
40.
Woo, SK,
Dahl SC,
Handler JS,
and
Kwon HM.
Bidirectional regulation of tonicity-responsive enhancer binding protein in response to changes in tonicity.
Am J Physiol Renal Physiol
278:
F1006-F1012,
2000
41.
Woo, SK,
Maouyo D,
Handler JS,
and
Kwon HM.
Nuclear redistribution of tonicity-responsive enhancer binding protein requires proteasome activity.
Am J Physiol Cell Physiol
278:
C323-C330,
2000
42.
Yang, XY,
Zhang Z,
and
Cohen DM.
ERK activation by urea in renal inner medullary cells.
Am J Physiol Renal Physiol
277:
F176-F185,
1999
43.
Zhang, Z,
and
Cohen DM.
NaCl but not urea activates p38 and jun kinase in mIMCD3 murine inner medullary cells.
Am J Physiol Renal Fluid Electrolyte Physiol
271:
F1234-F1238,
1996
44.
Zhang, Z,
Yang XY,
Soltoff SP,
and
Cohen DM.
PI3K signaling in the murine kidney inner medullary cell response to urea.
Am J Physiol Renal Physiol
278:
F155-F164,
2000
45.
Zhuang, S,
Hirai SI,
and
Ohno S.
Hyperosmolality induces activation of cPKC and nPKC, a requirement for ERK1/2 activation in NIH/3T3 cells.
Am J Physiol Cell Physiol
278:
C102-C109,
2000