Heat shock protein 90 regulates stabilization rather than activation of soluble guanylate cyclase.

Heat shock protein 90 regulates sta
of soluble guan
Pavel I. Nedvetskya,1, Sabine Meurerb, N
Helmut Mu¨llera, Harald
a Rudolf-Buchheim-lnstitute for Pharmacol
b Department of Pharmacology and Centre for Vascular Hea
Melbourne, Clayton Campus, Wellington Ro
Received 27 August 2007; revised 4 Decemb
e 26 D
icha
term activity regulation.
� 2007 Federation of European Biochemical Societies. Pub-
signaling.
aimed to study the role of hsp90 in both activity regulation and
including 0.5 mg/mL collagenase for 10 min at 37 �C. PPAECs were
FEBS Letters 582 (2008) 327–331sGC, soluble guanylate cyclase; SMCs, smooth muscle cellsHeat shock protein 90 (hsp90) is a ubiquitously expressed
ATP-dependent chaperone required for the stability and func-
tion of a range of proteins [14]. Both endothelial NO synthase
cultured in M199 supplemented with 15% FCS, 100 U/mL penicillin
and 100 lg/mL streptomycin. Confluent cells from passages 2–3 were
used for experiments. Porcine smooth muscle cells (SMCs) were iso-
lated by incubation of the aortic inner surface with collagenase
(0.5 mg/ml in PBS; 30 min at 37 �C) after removal of endothelial cells.
SMCs were cultured in DMEM supplemented with 20% FCS, 100 U/
mL penicillin and 100 lg/mL streptomycin, and passages 2–12 were
used for experiments.
2.3. sGC expression in Sf9 cells
Expression of glutathione-S-transferase (GST)-tagged human sGC
(a1b1 isoform) in Sf9 cells and protein purification by affinity chroma-
tography were described earlier [18].
2.4. Western blotting
For immunodetection of sGC subunits, cells were washed with PBS
and lysed in Roti-Load sample buffer (Carl Roth, Karlsruhe, Ger-
many) preheated to 95 �C and boiled for an additional 10 min. Protein
Abbreviations: cGMP, cyclic GMP; eNOS, endothelial NO synthase;
GA, geldanamycin; hsp90, heat shock protein 90; NO, nitric oxide;
PPAECs, porcine pulmonary artery endothelial cells; RD, radicicol;
*Corresponding author. Fax: +61 3 990 55729.
E-mail address: harald.schmidt@med.monash.edu.au
(H.H.H.W. Schmidt).
1Pressent address: Forschungsinstitut fu¨r Molekulare Pharmakologie,
Campus Berlin-Buch, Robert-Ro¨ssle-Strasse 10, D-13125 Berlin,
Germany.lished by Elsevier B.V. All rights reserved.
Keywords: Soluble guanylate cyclase; cGMP; hsp90;
Proteasome; Nitric oxide
1. Introduction
NO-sensitive soluble guanylate cyclase (sGC), the best-
established nitric oxide (NO) receptor, is a heme containing
ab heterodimer. Binding of NO to the ferrous heme moiety re-
sults in the formation of the second messenger cyclic GMP
(cGMP) [1] in the blood vessel wall and neurons [2–4]. Beyond
NO stimulation, the regulation of sGC activity by its redox
state [5,6], subcellular localization [7], and reversible protein–
protein interactions with adaptor proteins or chaperones
[8–13], may all contribute to the fine-tuning of the NO/cGMPAvailable onlin
Edited by M
Abstract Endothelium-derived nitric oxide (NO) activates the
heterodimeric heme protein soluble guanylate cyclase (sGC) to
form cGMP. In different disease states, sGC levels and activity
are diminished possibly involving the sGC binding chaperone,
heat shock protein 90 (hsp90). Here we show that prolonged
hsp90 inhibition in different cell types reduces protein levels of
both sGC subunits by about half, an effect that was prevented
by the proteasome inhibitor MG132. Conversely, acute hsp90
inhibition affected neither basal nor NO-stimulated sGC activity.
Thus, hsp90 is a molecular stabilizer for sGC tonically prevent-
ing proteasomal degradation rather than having a role in short-0014-5793/$32.00 � 2007 Federation of European Biochemical Societies. Pu
doi:10.1016/j.febslet.2007.12.025protein stability of sGC in different cell types.
2. Materials and methods
2.1. Materials
Collagenase CLS Type II was obtained from Biochrom (Berlin, Ger-
many); ECL� detection reagents from Amersham (Vienna, Austria);
geldanamycin, radicicol, bradykinin, A23187, DEA/NO, DRB,
MG132, IBMX, and zaprinast were from Alexis (Lausen, Switzerland).
All other reagents were from Sigma (St. Louis, MO).
2.2. Cell isolation and culture
PC12 cells were cultured in RPMI 1640 supplemented with 10%
FCS. Porcine pulmonary artery endothelial cells (PPAECs) were iso-
lated enzymatically by incubation of the inner surface of porcine pul-
monary arteries with phosphate-buffered saline (PBS; 10 mM
Na2HPO4, 1.8 mM KH2PO4, 140 mM NaCl, 2.7 mM KCl, pH 7.4)bilization rather than activation
ylate cyclase
ils Opitzb, Tatiana Y. Nedvetskayaa,
H.H.W. Schmidtb,*
ogy, University of Gießen, Germany
lth, School of Biomedical Sciences, Monash University,
ad, Building 13e, VIC 3800, Australia
er 2007; accepted 12 December 2007
ecember 2007
el R. Bubb
(eNOS) and sGC a1b1 interact with hsp90 [10,11,15,16] sug-
gesting a signaling complex. Binding of hsp90 to eNOS en-
hances its activation and thus endothelium-dependent
relaxation of blood vessels [16]. The relevance of the interac-
tion between hsp90 and sGC is less clear. NO-induced activa-
tion of sGC in endothelial cells and vasorelaxation in rat aorta
and mesenteric arteries has been reported to be unaffected
[16,17] whilst in cultured vascular smooth muscle and endothe-
lial cells the hsp90 inhibitor geldanamycin (GA) appears to
acutely affect sGC activity [10] similar to eNOS [15,16]. Alter-
natively, sGC protein stability has also been reported to be af-
fected by hsp90 [11,12] suggesting different mechanisms of
action for hsp90 with respect to eNOS and sGC. We thereforeblished by Elsevier B.V. All rights reserved.

concentration from each sample was determined and equal amounts of
protein (20 lg) were loaded on a gel. Samples were subjected to SDS–
PAGE and analyzed by Western blotting as described previously
[18,19]. Immunoreactive bands were quantified using an Kodak Ima-
ger Station 440 CF.
2.5. cGMP determination
To measure sGC activity, PPAECs (1.5–2 · 106) were treated with
drugs for timepoints as indicated. Thirty minutes prior sGC stimula-
tion, phosphodiesterase inhibitors, 1 mM IBMX and 100 lM zapri-
added. Cells were scraped and after removing of ethanol by vacuum
centrifugation, pellet was resuspended in assay buffer and cGMP was
determined using an enzyme immunoassay kit (Biotrend, Cologne,
Germany).
2.6. Data analysis and statistics
Data are expressed as means ± S.E.M. of n independent experi-
ments. Statistical calculations and curve fitting was done with Graph-
Pad Prism 3.0b software package (Graphpad Software, San Diego,
CA, USA). To compare multiple data sets, one-way (Fig. 5) or two-
way (Fig. 1) ANOVA with Bonferoni post-test was used. Statistical
significance was set at *P < 0.05 or **P < 0.01.
3. Results
3.1. No acute effect of hsp90 inhibition on sGC activity in intact
cells
To study the role of hsp90 in the short-term activity regula-
tion of sGC we used the hsp90 inhibitor geldanamycin (GA), a
benzoquinone ansamycin antibiotic, which binds to the ATP
pocket of the chaperone. Primary porcine pulmonary artery
endothelial cells (PPAECs) were incubated in the absence or
presence of GA for 1 h followed by stimulation either with
the NO donor and direct sGC activator, DEA/NO, or the
eNOS activators, calcium ionophore A23187 and bradykinin,
respectively (Fig. 1). GA did not alter DEA/NO- or A23187-
induced but almost completely abolished bradykinin-induced
cGMP elevation. Thus, hsp90 is required for receptor-medi-
ated eNOS activation but not for the acute downstream signal-
ing of NO to sGC. However, after chronic treatment of
Fig. 1. Short-term effects of hsp90 inhibition on sGC activity. Porcine
pulmonary artery endothelial cells (PPAECs) were treated with (black
and grey columns) or without (white columns) 1 lM GA for the time as
indicated followed by stimulation for 5 min with 250 lM DEA/NO,
0.5 lM A23187 or for 15 min with 1 lM bradykinin. cGMP levels were
measured. Data are means ± S.E.M. of three independent experiments
each performed in triplicates.
328 P.I. Nedvetsky et al. / FEBS Letters 582 (2008) 327–331nast, were added. cGMP production was stimulated with 250 lM
DEA/NO or 0.5 lM A23187 for 5 min, or with 1 lM bradykinin for
15 min. Cells were washed with PBS and 1 ml of 80% ethanol wasFig. 2. Protein levels of sGC are markedly reduced in the presence of hsp9
geldanamycin (GA; A) or radicicol (RD; B) for 24 h. Alternatively, cells were
Cell lysates were analyzed by Western blotting using antibodies specific to a
bands were quantified. Data represent means ± S.E.M. from four independe0 inhibitors. PPAECs were incubated with various concentrations of
incubated with 0.3 lM GA (C) or RD (D) for timepoints as indicated.
1 or b1 subunit of sGC (A–D) and actin (A and B). Immunoreactive
nt experiments performed in triplicate expressed as percent of control.PPAECs with GA (24 h), DEA/NO-induced cGMP produc-
tion was reduced by about 65% (Fig. 1), suggesting either a de-
layed effect on activity or an effect on sGC protein levels.

3.2. Long-term hsp90 inhibition down-regulates sGC protein in
different cell types
GA inhibits the hsp90-mediated maturation/refolding reac-
ment the 40% reduction in sGC levels caused by GA (Fig. 4A).
In addition, inhibitors of protein synthesis, emetine and cyclo-
heximide, did also not significantly affect sGC protein levels
[5]. With respect to sGC stability, it is well established that
co-expression of both sGC subunits is required to form an ac-
tive heterodimeric enzyme [24,25]. As hsp90 interacts with sGC
[10,11], we wished to examine whether hsp90 inhibitors can
prevent sGC heterodimer formation. In order to allow for
rapid separation of sGC heterodimers from monomeric sub-
units, we switched to an Sf9 cell system overexpressing GST-
tagged sGCa1 together with non-tagged sGCb1. Only the a1
subunit had an affinity tag and co-elution of both subunits
Fig. 3. Inhibition of hsp90 decreases sGC protein levels in PC12 and
smooth muscle cells. PC12 cells or smooth muscle cells were incubated
in the presence or absence of 0.3 lM GA. Cell lysates were analyzed by
Western blotting using a mixture of antibodies specific to a1 or b1
subunit of sGC (anti-a1b1).
P.I. Nedvetsky et al. / FEBS Letters 582 (2008) 327–331 329tion, and often results in the degradation of hsp90 client pro-
teins. To test whether the delayed effect of GA on sGC
activity was due to a decrease in sGC stability and protein lev-
els, Western blot analysis was performed using subunit specific
antibodies followed by densitometrical analysis. Indeed GA
led to a concentration-dependent down-regulation of both
sGC protein subunits, a1 and b1, with a maximal effect at
0.3 lM (Fig. 2A). The time-course of this effect was monopha-
sic with a parallel, 60–70% reduction of both subunits after
24 h and a half-maximal effect after 4–6 h (Fig. 2C). A struc-
turally distinct hsp90 inhibitor, radicicol (RD), showed effects
similar to GA (Fig. 2B and D). While RD had no significant
effect on actin levels, GA-induced a slight decrease, which
was however lower than that on sGC (Fig. 2A and B). To fur-
ther exclude that the effects of GA were due to its quinone
backbone or ROS (reactive oxygen species) generation [20]
we used pyrogallol (3 lM, 24 h) and hydrogen peroxide
(100 lM up to 24 h), neither of which mimicked the effects
of hsp90 inhibitors (not shown). To further exclude that GA-
induced down-regulation of sGC protein is restricted to endo-
thelial cells, experiments were performed with porcine smooth
muscle cells (SMCs) and neuronal cell line PC12 leading to
similar results (Fig. 3).
3.3. Hsp90 neither modulates de novo sGC expression nor
subunit heterodimerization
Having established that hsp90 inhibition affects protein sta-
bility of both sGC subunits, we wished to explore the underly-
ing mechanisms in more detail. Hsp90 is a molecular
chaperone participating in many cellular signal transduction
and gene expression pathways [21–23]. To rule out that our ob-Fig. 5. Hsp90 inhibitors targets sGC to proteasomal degradation. PPAECs
0.3 lM GA for 24 h. After cell lysis, protein levels of a1 (A) and b1 (B) were a
of three independent experiments performed in triplicates.served effects on sGC protein levels in cells treated with GA or
RD are rather indirect and due to transcriptional effects and
altered sGC expression, we pre-treated PPAECs with the tran-
scription inhibitor 5,6-dichloro-1-b-D-ribofuranosyl benzimid-
azole (DRB) in the absence or presence of GA for 24 h. DRB
had no significant effect on sGC protein levels nor did it aug-
Fig. 4. Effects of hsp90 inhibitors are not connected to the de novo
sGC synthesis. (A) PPAECs were treated with 0.3 lM GA, 100 lM
DRB, or the combination of both compounds for 24 h. Cells were
lysed and proteins analyzed by Western blotting using specific
antibodies to a1 or b1 followed by densitometrical quantification of
immunoreactive bands. Data are means ± S.E.M. of four independent
experiments each performed in triplicates. (B) Sf9 cells overexpressing
GST-a1 and b1 in the absence (left panel) or presence (right panel) of
1 lM RD for 24 h were lysed followed by purification of sGC using
GST-affinity chromatography. Samples of elution fractions were
analyzed by Western blotting with anti-a1b1.were incubated with 1 lM MG132 (A and B) alone or together with
nalyzed by Western blotting and quantified. Data are means ± S.E.M.

sGC, could be coupled to eNOS via hsp90 [10]. Furthermore, it
has been shown that sGC interacts directly with the chaperone
919–925.
330 P.I. Nedvetsky et al. / FEBS Letters 582 (2008) 327–331hsp70 in a cGMP-promoting manner [13]. We are only begin-
ning to understand how the NO/cGMP pathway may be ef-
fected in different disease states [5,27].
In agreement with published data [11,12], long-term inhibi-
tion of hsp90 results in a time-dependent, monophasic loss
of both sGC subunits in endothelial, smooth muscle and neu-
ronal PC12 cells. Two chemically unrelated inhibitors of hsp90
showed very similar effects, suggesting specificity. GA can in-
crease ROS production in cells [10,20]. However, we excluded
that GA was acting via its quinone backbone or ROS genera-
tion, which is in line with published data showing that low con-
centrations of GA as used by us do not increase ROS [10].
Moreover, chronic treatment of rat aortic smooth muscle cells
with 150 lM of the ROS, hydrogen peroxide, does not de-
crease sGC protein levels [28].
With respect to sGC, hsp90 inhibition was rather acting by
destabilization and subsequent proteasomal protein degrada-
tion, as neither inhibition of transcription nor of protein syn-
thesis had a similar effect to hsp90 inhibition. Our datademonstrates intracellular heterodimerization. Treatment of
Sf9 cells with radicicol (Fig. 4B) or geldanamycin (data not
shown) had no effect on sGC heterodimerization as the elution
profile of both subunits did not significantly alter under these
experimental conditions. Thus, neither endothelial sGC gene
expression nor protein heterodimerization is affected by
hsp90 inhibition.
3.4. Inhibition of proteasomes prevents hsp90 inhibitor-induced
sGC degradation
To investigate whether hsp90 protects sGC from proteaso-
mal degradation PPAECs were incubated with the widely used
proteasome inhibitor MG132 in the absence or presence of GA
for 24 h. MG132 alone results in a slight but significant de-
crease in sGC as observed by others [11]. More importantly,
MG132 effectively prevented a further GA-induced decrease
in sGC protein levels (Fig. 5). Thus, we conclude that inhibi-
tion of hsp90 targets sGC to a tonic proteasomal protein deg-
radation pathway.
4. Discussion
Here we demonstrate that short-term inhibition of hsp90
leaves sGC activity unaffected whereas long-term inhibition
targets sGC to proteasomal degradation. These data are con-
sistent with the lack of effect of hsp90 inhibitors on acute vaso-
relaxation [16,17] and define a more chronic role of hsp90 on
sGC within NO/cGMP signaling. This contrasts then to the
clearly acute activity modulation of eNOS by hsp90. With
the recent discovery of a pathophysiologically significant redox
regulation and oxidation-induced degradation of sGC [5] such
effects may become important and disease relevant mecha-
nisms both in cardiovascular and in neuronal cells.
A growing body of evidences suggests that proteins involved
in regulation of NO-dependent signaling are organized in com-
plexes rather than distributed separately within the cell [7].
More than 20 proteins were found to interact with NOS di-
rectly or indirectly and form well-organized dynamic protein
network, which regulates the NO production (for review see
[26]). Recently, it has been demonstrated that the NO receptor,[3] Lucas, K.A., Pitari, G.M., Kazerounian, S., Ruiz-Stewart, I.,
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H.H.H.W. and Stasch, J.P. (2006) NO-independent stimulators
and activators of soluble guanylate cyclase: discovery and
therapeutic potential. Nat. Rev. Drug. Discov. 5, 755–768.
[7] Zabel, U. et al. (2002) Calcium-dependent membrane association
sensitizes soluble guanylyl cyclase to nitric oxide. Nat. Cell. Biol.
4, 307–311.
[8] Russwurm, M., Wittau, N. and Koesling, D. (2001) Guanylyl
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[10] Venema, R.C. et al. (2003) Novel complexes of guanylate cyclase
with heat shock protein 90 and nitric oxide synthase. Am. J.
Physiol. Heart Circ. Physiol. 285, H669–H678.
[11] Papapetropoulos, A., Zhou, Z., Gerassimou, C., Yetik, G.,
Venema, R.C., Roussos, C., Sessa, W.C. and Catravas, J.D.
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of the domains mediating binding. Mol. Pharmacol. 68, 1133–
1141.support the hypothesis that sGC rapidly undergoes protein
degradation via the proteasome pathway if not stabilized by
hsp90. Why this decrease remains submaximal remains unclear
and may reflect distinct subcellular pools of sGC or post-trans-
lational modifications that may affect sGC�s susceptibility [7].
We could not find evidence for hsp90 playing a role in the
acute and direct regulation of sGC activity. Bradykinin-in-
duced cGMP accumulation was completely prevented by the
hsp90 inhibitor. However, bradykinin acts through receptor-
mediated eNOS activation, a hsp90-dependent event [15].
Receptor-independent calcium ionophore-induced eNOS/sGC
activation, presumably not involving hsp90, or direct sGC acti-
vation by the NO donor, DEA/NO, were not affected. Indeed,
Venema and others have demonstrated that stimulation of
endothelial cells with bradykinin results in the formation of
an eNOS/sGC/hsp90 complexes and that this [10] and the bra-
dykinin-induced activation of eNOS [15] are blocked by GA.
Taken together, our data argue against a general require-
ment of hsp90 to activate sGC by endogenously generated or
exogenously added NO. The requirement for hsp90 so far
seems to reside solely in preventing a tonic degradation tone
of a substantial cellular fraction of sGC mediated via the pro-
teasome pathway in different cell types. It will be of interest
whether a lack of functional hsp90/sGC interaction explains
some of the disease conditions where lower levels of sGC
and pathological NO-cGMP signalling have been observed
[27,29–32].
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