(Hypertension. 2001;37:160.)
© 2001 American Heart Association, Inc.
Scientific Contributions |
Fluid Shear Stress Reduces 11ß-Hydroxysteroid Dehydrogenase Type 2
From the Division of Nephrology/Hypertension, University of Berne, Berne, Switzerland.
Correspondence to Markus G. Mohaupt, MD, University Hospital Berne, Division of Nephrology/Hypertension, 3010 Berne, Switzerland. E-mail markus.mohaupt{at}insel.ch
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Abstract—In pregnancy, invading trophoblasts represent the inner vascular border of maternal spiral arteries and are exposed to elevated shear stress (ss) in hypertensive disorders. Intracellular cortisol availability is regulated by 11ß-hydroxysteroid dehydrogenases (11ß-HSDs), thus determining body fluid volume and vascular responses. The impact of ss on 11ß-HSD2 activity was studied in the human JEG-3 cell line, a model for trophoblasts. JEG-3 cells do not express 11ß-HSD1; however, 11ß-HSD2 message and activity are measured via cortisol/cortisone conversion in cell lysates, and both are reduced by ss. The reduction in 11ß-HSD2 activity via ss is dose dependent and completely reversible after the discontinuation of ss. cAMP-dependent protein kinase A activation increased the 11ß-HSD2 activity yet did not prevent the ss response. The ss response was completely protein kinase C independent. The mitogen-activated protein kinase kinase inhibitor PD-098059 enhanced 11ß-HSD2 activity in static conditions yet only ameliorated the ss effect. Cytochalasin D disrupts focal adhesion (FA)-cytoskeleton interactions and abolished the ss-induced tyrosine phosphorylation of FA kinase dose-dependently, thus maintaining 11ß-HSD2 activity. The 11ß-HSD2 activity was only partially restored by the tyrosine kinase inhibitor genistein; however, herbimycin A almost completely abolished the ss effect on 11ß-HSD2 activity. In conclusion, JEG-3 cells express 11ß-HSD2, which is downregulated by ss. Regulatory mechanisms involve transcriptional control and require intact FA-cytoskeleton signaling and phosphorylation of FA kinase. Thus, ss adds to an enhanced intracellular availability of cortisol, which may ultimately support a vasoconstrictive vascular response.
Key Words: 11ß-hydroxysteroid dehydrogenase • shear stress • cortisol • hypertension • focal adhesion • cytoskeleton
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Introduction |
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Intracellular cortisol availability is regulated via 11ß-hydroxysteroid dehydrogenase enzymes (11ß-HSDs). In contrast to the 11ß-HSD1 enzyme, the NAD-dependent 11ß-HSD2 enzyme has only oxidative activity, converting cortisol to cortisone at a lower Km value.1 2 3
Intracellular cortisol availability determines vascular responses and total body fluid volume. An increased fluid load characterizes several diseases, such as steroid-dependent hypertension or preeclampsia (PE). PE is a widespread disease that affects up to 10% of first pregnancies. In PE and steroid-dependent hypertension,4 5 low fetal birthweight is often present, thus increasing morbidity rates. In fetal growth retardation, enhanced cortisol availability is present due to reduced 11ß-HSD2 activity,6 accompanied by elevated blood flow resistance and increased shear stress (ss) in the uteroplacental circulation.7 8
An increased cortisol availability within the vascular wall, a known
target for glucocorticoids and mineralocorticoids, could be essential
for the response to vasoactive mediators, such as the pressor hormones
angiotensin II (Ang II) and 
-adrenergic
agonists.9 Glucocorticoids, such as
dexamethasone, have been shown (1) to stimulate Ang II type
1A (AT1A) receptor expression via glucocorticoid
responsive elements in the gene promotor,10 11 (2) to
enhance AT1 receptor binding of Ang II in
vascular smooth muscle cells,12 and (3) to reduce
AT2 receptor expression,13 all of
which could also explain the sensitization to Ang II in PE.
In pregnancy, cytotrophoblasts penetrate maternal spiral arterioles as far as the myometrial segments, thus increasing the diameter of the vessels. However, in PE, endovascular invasion by trophoblasts is reduced to superficial portions of the uterine spiral arteries, leaving these vessels narrow with a high blood flow resistance.14 15 Recently, evidence was provided that invasive cytotrophoblasts failed to mimic a vascular adhesion phenotype in PE. Despite the fact that the functional consequences of this abnormality are not known, it was speculated that they affect endovascular invasion and uterine arteriole remodeling and may even interfere with focal adhesion (FA) regulation. This may have detrimental effects on blood flow to the maternal-fetal interface.16
The human choriocarcinoma cell line JEG-3 serves as a model to investigate properties of trophoblasts. This cell line has been characterized to produce steroid hormones and vasoactive compounds17 18 19 and to express 11ß-HSD2 under static conditions.20
Blood flow along endovascular surfaces is associated with ss, which is sensed and transduced into biochemical signals via multiple pathways, such as integrins.21 Intracellular signaling of ss is conferred via phosphorylation of FA kinases (FAKs),22 23 consecutively activating numerous downstream events and finally leading to events such as altered transcriptional activity.
This study was performed to elucidate whether 11ß-HSD activity is regulated in trophoblasts by mechanical forces and to identify signaling events involved in mediation of the ss response.
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Methods |
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Materials
Plastic material for cell culture was from Becton Dickinson Labware and from Corning. Cortisol, glycyrrhetinic acid, minimal essential medium Eagle (MEME), transferrin, 8'-bromo-cAMP, lipopolysaccharide (Escherichia coli serotype O26:B6), interferon-
(IFN-), tumor necrosis factor-
(TNF-), interleukin-1ß (IL-1ß), and
phorbol-12-myristate-13-acetate (PMA) were purchased from Sigma
Chemical. GF109203X, PD-098059, forskolin, genistein, herbimycin A, and
cytochalasin D were from Calbiochem-Novabiochem. NAD and RNase
inhibitor were from Boehringer-Mannheim.
[3H]Cortisol (2.33
TBq/mmol) and the ECL components were from Amersham International.
Triton X-100 was purchased from Merck. The TLC plates (silica gel
60F254) were from Macherey-Nagel. FCS was from Biological Industries.
Primers were from Microsynth.
Cell Culture
Two different human choriocarcinoma cell lines, JEG-3 (HTB-36;
American Type Culture Collection) and CB2572 (European Collection of
Cell Cultures), were evaluated with comparable results. The JEG-3 cell
line was used for subsequent experiments and grown in MEME supplemented
with 10% FCS, 2 mmol/L glutamate, 100 U/mL penicillin, and 100
µg/mL streptomycin; passaged with trypsin/EDTA; plated in round cell
culture bottles; grown to confluence; and washed twice with PBS (pH 7.4
at 37°C) before the experiments were started. The ss was obtained
after transfer to an incubator, where an unidirectional flow
environment was created at 37°C for up to 48 hours with varying fluid
ss equal to a maximum of 0.05 dyne/mm2. At the
completion of each experiment, cells were again washed twice with
ice-cold PBS, and subsequent protocols were followed.
Assay for 11ß-HSD Activity in Cell Homogenates
Cells were homogenized on ice in a Tenbroeck glass
homogenizer in sucrose buffer (250 mmol/L sucrose,
2 mmol/L EDTA, 20 mmol/L Tris-HCl, pH 7.5) and
centrifuged (2 minutes at 1500g at 4°C), and the
supernatant was stored at -20°C. Protein content was determined with
the Bradford protein assay (Bio-Rad). The cortisol/cortisone conversion
was used to measure oxidation at C11 by 11ß-HSD2. In brief, according
to Leckie et al with minor modifications,24 cell
homogenates were incubated with 5 nCi
[3H]cortisol, 10 nmol/L cortisol, and 200
µmol/L NAD in sucrose buffer. The reaction was stopped by the
addition of 1 mg/mL unlabeled cortisol and cortisone in methanol and
TLC developed in chloroform-methanol (90:10 v/v). Steroids were located
with UV light, excised, and counted in a scintillation counter
(TRI-CARB 200GA; United Technologies, Packard). Activity of the
11ß-HSD1 enzyme was determined in the presence of 1% Triton X-100,
as previously described.25 Specific activity was expressed
as picomoles per milligram of protein per hour.
Reverse Transcription-Polymerase Chain Reaction of Human 11ß-HSD1
and -HSD2
After reverse transcription (RT) [1 µg total RNA,
oligo(dT)18; GIBCO BRL], homology-based
polymerase chain reaction (PCR) (30 cycles, annealing at 56°C) was
performed in a total volume of 50 µL with 10 mmol/L Tris-HCl (pH
8.3), 50 mmol/L KCl, 1.5 mmol/L MgCl2,
and specific primers (10 pmol). PCR products were size-separated on
ethidium bromide–stained agarose gels. Primer sequences for 11ß-HSD2
(GenBank accession number NM-000196) were sense at position 830 to 853
and antisense at position 1299 to 1278, and for 11ß-HSD1 (GenBank
accession number NM-005525.1), the sequences were sense at position 165
to 189 and antisense at position 885 to 861.
Northern Blot Hybridization Analysis of 11ß-HSD2
Transcripts
Total RNA (20 µg) was electrophorectically size-fractionated
as described previously.26 27 The resulting filters were
hybridized for 1 hour at 60°C with a digoxin-labeled
(Boehringer-Mannheim) human 11ß-HSD2 cDNA probe. The blots
were exposed to x-ray film (Kodak Biomax MR; Sigma) at room temperature
for varying amounts of time to establish linearity of the obtained
signals. To control RNA transfer and content, filters were stained with
bromophenol blue (0.04% in 500 mmol/L Na acetate; pH 5.5).
Quantification of 11ß-HSD2 Transcripts by TaqMan
Analysis
TaqMan primers and probe sequences for human GAPDH (GenBank
accession number J04038) were sense position 686 to 700,
antisense position 751 to 731, and probe FAM-TAMRA–labeled at position
706 to 728, and for human 11ß-HSD2 (GenBank accession number
NM-000196), the sequences were sense position 539 to 562, antisense
position 608 to 591, and probe FAM-TAMRA–labeled at position 564 to
585. RT of 1 µg total RNA in the presence of
oligo(dT)12–18 primer and 200 U Moloney murine
leukemia virus-reverse transcriptase (both GIBCO BRL) was followed by
PCR performed in a Perkin-Elmer ABI Prism 7700 Thermal Cycler Sequence
Detection System with ABI Prism PrimerExpress Software. The 11ß-HSD2
PCR product was normalized by the GAPDH PCR product.
Immunoprecipitation of FAK With Immunoblot
Analysis of FAK Tyrosine Phosphorylation
Cell lysates (500 µg protein) (in 50 mmol/L HEPES, pH
7.4, 150 mmol/L NaCl, 1% Triton X-100, 0.1% SDS, 1 mmol/L
EGTA, 1.5 mmol/L MgCl2, 10% glycerol,
1 mmol/L NaVO3, proteinase
inhibitor mix; Roche Diagnostics GmbH) were
precleared with activated protein A-Sepharose (Sigma) and
incubated with protein A-Sepharose and anti-FAK antibody (mouse,
polyclonal; Transduction Laboratories) overnight at 4°C. After
several washes with 0.1% Triton X-100, 50 mmol/L Tris-HCl, pH
7.4, 300 mmol/L NaCl, 5 mmol/L EDTA, and 0.02% (wt/vol)
sodium azide, the precipitates were subjected to Western blot
analysis as described earlier with the following
modifications28 : the primary antibody (anti-PY20, mouse,
monoclonal, 1:1000; Transduction Laboratories) detected with a
horseradish peroxidase–conjugated goat anti-mouse IgG antibody (1:2000
Jackson ImmunoResearch Laboratories).
Statistical Analysis
Mean±SEM values were determined. To test for statistically
significant differences, Student’s t test or ANOVA was
used. Significance was assigned at P<0.05. For 11ß-HSD
activity assays, the reaction was repeated 
4 times with different
protein amounts within each individual experiment.
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Results |
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Cultured JEG-3 Cells Displayed Endothelial Markers and 11ß-HSD2 Activity
Cultured JEG-3 cells expressed the endothelial cell surface makers CD31 and vWf (data not shown). Endothelial NO synthase (eNOS) was ss dependent expressed as detected on Western blot analysis (data not shown).
Expression of 11ß-HSD Isoforms in Cultured JEG-3 Cells
PCR products were detected in confluent JEG-3 cells at the
expected size of 469 bp for 11ß-HSD2, whereas no 11ß-HSD1 mRNA was
detectable. Full-size transcripts of 11ß-HSD2 mRNA were demonstrated
by Northern blot analysis at a size of 
1.9 kb as indicated
by the ribosomal bands29 (Figure 1).
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The ss Reduced 11ß-HSD2 mRNA Expression in Cultured JEG-3
Cells
With TaqMan RT-PCR reduced steady state levels were detected after
24 hours of ss (36% of static control, n=6), as well as by Northern
blot analysis after 48 hours of ss (49±5% of static control,
n=9), suggesting a transcriptional regulation of 11ß-HSD2 by ss
(Figure 1).
11ß-HSD Enzyme Activity of Cultured JEG-3 Cells Under Static and
ss Conditions
In JEG-3 cell homogenates, 11ß-HSD2 enzyme
activity was present, converting
[3H]cortisol to
[3H]cortisone (Figure 2). This conversion was inhibited by
glycyrrhetinic acid (10 µmol/L). The addition of Triton X-100 to
the 11ß-HSD assay mixture abolished the enzyme activity, indicating
that the activity was attributable to 11ß-HSD2 and not to
11ß-HSD1.25 These results are in line with other reports
on 11ß-HSD2 activity in cultured JEG-3 cells.20 30 31
Time course experiments up to 48 hours indicated a significant
reduction in 11ß-HSD2 activity in response to ss after 16 hours
(Figure 3A). 11ß-HSD1 activity remained
at background levels (data not shown). The reduction in 11ß-HSD2
activity depended on the dose of ss applied (Figure 3B). The ss
effect was reversible within 8 hours to baseline activity on abrogation
of a preceding exposure to 24 hours of ss (Figure 3C).
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The ss Responses of 11ß-HSD2 Activity Are Independent of Protein
Kinase C Signaling
Without ss, PMA-induced29 stimulation of protein
kinases C (PKCs) resulted in an almost 2-fold increase (181±34% of
static control) in 11ß-HSD2 activity compared with untreated cells.
Inhibition of PKCs by GF109203X did not affect 11ß-HSD2 activity
(103±31% of static control), yet neither GF109203X nor PMA treatment
could abolish the effect of ss on 11ß-HSD2 activity, which would
argue against a role of the PKC pathway in the ss-dependent regulation
of 11ß-HSD2 activity (Figure 4).
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The ss Responses of 11ß-HSD2 Activity Are Independent of
cAMP-Sensitive Protein Kinase A Signaling
Protein kinase A (PKA) activation with
forskolin32 resulted in an almost 4-fold increase in
11ß-HSD2 activity in static conditions. This was dose-dependently
imitated by the addition of 8'-bromo-cAMP. The addition of forskolin or
of 8'-bromo-cAMP slightly ameliorated the ss effect on 11ß-HSD2
activity (Figure 5), with baseline
11ß-HSD2 activity being reached; however, the 4-fold increase in
activity seen under static condition was prevented. This suggests that
PKA activation very effectively controls 11ß-HSD2 activity only under
static conditions but not the ss response.
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Mitogen-Activated Protein Kinase Kinase Is Not a Major
Participant of FA Downstream Signaling in Response to ss to
Control 11ß-HSD2 Activity
To elucidate the role of the MP1-scaffolded
mitogen-activated protein kinase (MAPK) cascade in 11ß-HSD2
ss responses, cells were incubated with the MAPK kinase (MAPKK)
inhibitor PD-098059.33 Without ss, PD-098059
led to an enhanced 11ß-HSD2 activity of 191±27% of control values.
The ss response of 11ß-HSD2 activity was only ameliorated by
PD-098059, yet not abolished, confirming no major role for
MAPK-dependent signaling under ss conditions (Figure 6). Dependency of the ss response on the
MAPKK activation would require the phosphorylation of
MEK1/2. In support of our data on 11ß-HSD2 activity, we found
no detectable phosphorylated MEK1/2 under control
conditions in the total protein lysates of JEG-3 cells; however, a
prominent signal was visible after 45 minutes of incubation with
cytokines (IFN-, IL-1ß, TNF-). In contrast, only a weak
signal was detectable after exposure to ss (data not shown).
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The ss Response of 11ß-HSD2 Requires Cytoskeleton Integrity and
Phosphorylation of FAK
To disrupt actin filament assembly,34 hence
abolishing ss-induced phosphorylation and activation of
FAK, cytochalasin D was applied. Cytochalasin D35 did not
affect 11ß-HSD2 activity in treated cells (96±18% of static
control). In the presence of ss, coincubation with cytochalasin D
almost completely inhibited the response to ss (87±10% of static
control at 2.5x10-6 mol/L) (Figure 7A) dose-dependently; however, at
cytochalasin D concentrations of 10-5 mol/L, the
cells showed signs of toxicity. To further support our findings, we
preincubated the cells for 90 minutes with cytochalasin D. Again,
concentrations of 10-5 mol/L were toxic to the
cells, and preincubation at a concentration of
2.5x10-6 mol/L completely abolished (93±15%
of static control) the effect of ss on 11ß-HSD2 activity. This
recovery was found to be dose dependent (at 10-6
mol/L, only 56±5% of static control) (Figure 7B). In agreement
herewith, the exposure of cultured JEG-3 cells to ss initiated an FA
signal that resulted in a tyrosine phosphorylation of
FAK, which was inhibited in the presence of cytochalasin D (Figure 7C).
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The phosphorylation of FAK is dependent on tyrosine kinases. Inhibition of this phosphorylation should inactivate FA signaling and preserve 11ß-HSD2 enzyme activity in the presence of ss. To support the role of FAK tyrosine phosphorylation as the transmitting signal for 11ß-HSD2 regulation, we applied the tyrosine kinase inhibitors genistein and herbimycin A.22 36 Genistein only partially returned the 11ß-HSD2 activity, whereas herbimycin A significantly abolished the ss effect (80±5%) (Figure 8). This strongly suggested a signal transduction that relied on phosphorylation of FAK and an intact cytoskeleton interaction during activation of FAs by ss.
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Glucocorticoid actions are determined by the concentration of unbound extracellular steroids and the intracellular availability of these steroid hormones. Extracellular steroids are provided either by endogenous adrenal secretion or by exogenous donation.37 In contrast, the intracellular access of steroids to their cognate receptor is regulated by 11ß-HSD enzymes.38
The regulation of 11ß-HSD enzymes has a recognized impact on arterial hypertension. These enzymes play an important role in the volume homeostasis and in the potentiation of vasoconstrictor responses, as has been demonstrated in vivo and in vitro with enhanced cortisol or mineralocorticoid availability.9 10 11 12 13
Thus, in fetal growth retardation, excessive cortisol availability, next to its direct fetal consequences,39 may limit placental substrate delivery via a reduced maternal blood supply with increased blood flow resistance and elevated ss, as is present in the uteroplacental circulation of preeclamptic women.
We hypothesized that ss reduces 11ß-HSD2 activity in the vascular compartment, such as in trophoblast cells, which line uterine spiral arteries. Consecutively, the increased intracellular cortisol availability would support an augmented vascular tone with elevated maternal blood pressure.
To investigate this hypothesis, we exploited the immortalized human choriocarcinoma cell line JEG-3. Trophoblasts in vivo undergo a comprehensive transformation of their adhesion molecule repertoire so as to mimic that of endothelial cells, such as VE-cadherin.16 We provide evidence that JEG-3 cells reveal endothelial phenotypic properties, such as expression of the typical endothelial markers CD31 and vWf immunohistochemically, as well as ss-dependent eNOS protein expression. In line with earlier findings by Pasquarette et al,20 we demonstrated 11ß-HSD2, but not 11ß-HSD1, activity in cultured JEG-3 cells. Because invading trophoblasts represent the inner vascular border in maternal spiral arteries, they are subjected to viscous drag of varying sizes depending on the location within the vascular bed. Continuously present in vivo, ss was applied to the cultured JEG-3 cells as to mimic these conditions. Several experiments were conducted to establish and control the shear conditions. The ss present in our model was comparable to that in other models with respect to induction of eNOS expression in response to ss.40
We observed a profound reduction in 11ß-HSD2 activity in response to ss. This would allow an enhanced cortisol availability within the cellular environment subsequent to increased mechanotransduction signals.
The time course experiments revealed a time-dependent reduction of 11ß-HSD2 activity, which could very well coincide with transcriptional regulatory events. Changes in cumulative mRNA responsible for changes in cellular 11ß-HSD2 activity have also been observed by others.20 29
The ss effect on 11ß-HSD2 activity was reversible within 8 hours and depended on the amount of ss applied, both of which strongly argue for an important physiological role of this observation.
Sp1 has been implicated as essential for 11ß-HSD2 mRNA transcription in JEG-3 cells.41 Because MAPKs lead to an activation of Sp1,42 the ss response could be signaled through the MP1-scaffolded MAPK cascade.
The activation of PKA is known to downregulate the kinase activity of Raf.32 In confirmation of earlier studies, 11ß-HSD2 activity was elevated by the addition of forskolin and 8'-bromo-cAMP under static conditions.20 29 However, the ss response of 11ß-HSD2 was only ameliorated by PKA activation, thus arguing against a relevant role of Raf activation.
Stimulation of the PKC pathway, another known modulator of Raf activity, increased 11ß-HSD2 activity under static conditions. Reports on the effect of PMA on 11ß-HSD are not uniform; for example, 11ß-HSD2 activity has been found to be unchanged in cultured placental trophoblasts29 or diminished in cultured LLC-PK1 cells (C.D. Heiniger, unpublished data, 1999). No relationship was observed between the PKC pathway and the ss effect on 11ß-HSD2 activity.
To further elucidate the role of the MP1-scaffolded MAPK pathway in ss,
we investigated the influence of MAPKK, which may also be
activated via non–Raf-related mechanisms43 and
which responds to the specific inhibitor
PD-098059.44 45 Even under static conditions, blockade of
MEK by PD-098059 enhanced 11ß-HSD2 activity; thus,
activators of MEK may be operative that provide a certain
basal activity. The ss response of 11ß-HSD2 was only ameliorated,
suggesting that the related signaling only in part depends on the MAPK
cascade. This is supported by the only minor expression of
phosphorylated MEK1/2 on ss compared with the profound
expression during cytokine (IFN-, IL-1ß, TNF-)
exposure.
The ss activates a number of intracellular signals, such as de novo transcription of growth factors (platelet-derived growth factor, transforming growth factor-ß)46 47 or vasoconstrictors such as endothelin.48 Integrins, which are mechanosensors of ss, prompt tyrosine kinase–related intracellular signals49 ; however, several conditions have to be fulfilled for signaling: integrin aggregation, integrin presence, tyrosine kinase activity with phosphorylation of FAK, and actin cytoskeletal integrity, with the latter 2 addressed in this study. Integrins mediate a focal accumulation of cytoskeletal molecules, including F-actin.50 Modifications of the microtubule cytoskeleton have been shown to evoke downstream signaling, including MP1-scaffolded MAPK cascade-independent responses.51 A role for cytoskeleton-mediated signaling was established for the conditions of our model with the demonstration of tyrosine phosphorylation of FAK in response to ss, as has been shown previously by Li et al.22 Tyrosine phosphorylation of FAK was abolished with the disruption of cytoskeleton integrity by cytochalasin D coincubation. The 11ß-HSD2 activity was preserved on preincubation and coincubation with cytochalasin D despite significant morphological alterations in cell shape. The effect of ss on 11ß-HSD2 activity was also investigated with 2 different tyrosine kinase inhibitors: herbimycin A and genistein.22 36 Genistein binding to ATP-binding sites of tyrosine kinases only partially inhibited the ss effect, whereas herbimycin A, by attacking critical sulfhydryl groups, completely inhibited the effect, thus supporting a role for FAK tyrosine phosphorylation in shear-related FA signals in 11ß-HSD2 regulation.
These results suggest that ss exerts an effect on 11ß-HSD2 activity and transcription via an FA-related mechanism that only partially depends on the MP1-scaffolded MAPK pathway. FA signaling required an intact cytoskeleton and tyrosine phosphorylation of FAK. With these data, evidence is provided that an increasing number of signaling events exert a sum effect on downstream regulators, such as transcription factors, regulating 11ß-HSD2 activity and transcription.
In conclusion, the regulation of 11ß-HSD2 by ss via activation of FA could be an important factor in the regulation and perpetuation of high blood pressure in PE. It may be associated with an enhanced local responsiveness to vasoconstrictors but also with an altered regulation of cortisol-dependent enzyme systems, such as the inducible NOS.52 The increased intracellular cortisol availability, with its several effects on Ang II responsiveness, could also explain the paradoxical sensitization to Ang II in PE, which is in contrast to that during a normal pregnancy. The physiological impact of the ss-dependent regulation on the intracellular cortisol availability will require future in vivo studies.
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Acknowledgments |
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This work was supported in part by grant 3200-055869.98/1 for scientific research from the Swiss National Science Foundation.
Received May 22, 2000; first decision June 21, 2000; accepted June 30, 2000.
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