Department of Biomolecular Science and Department of Urology, Fukushima Medical University School of Medicine, Fukushima 960-1295, Japan
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ABSTRACT |
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Mechanical stretch has been implicated in phenotypic changes as an adaptive response to stretch stress physically loaded in bladder smooth muscle cells (BSMCs). To investigate stretch-induced signaling, we examined the mitogen-activated protein kinase (MAPK) family using rat primary BSMCs. When BSMCs were subjected to sustained mechanical stretch using collagen-coated silicon membranes, activation of c-Jun NH2-terminal kinase (JNK) was most relevant among three subsets of MAPK family members: the activity was elevated from 5 min after stretch and peaked at 10 min with an 11-fold increase. Activation of p38 was weak compared with that of JNK, and ERK was not activated at all. JNK activation by mechanical stretch was totally dependent on extracellular Ca2+ and inhibited by Gd3+, a blocker of stretch-activated (SA) ion channels. Nifedipine and verapamil, inhibitors for voltage-dependent Ca2+ channels, had no effect on this JNK activation. Moreover, none of the inhibitors pertussis toxin, genistein, wortmannin, or calphostin C affected stretch-induced JNK activation, indicating that G protein-coupled and tyrosine kinase receptors are unlikely to be involved in this JNK activation. On the other hand, W-7, a calmodulin inhibitor, and cyclosporin A, a calcineurin inhibitor, prevented JNK activation by stretch. These results suggest a novel pathway for stretch-induced activation of JNK in BSMCs: mechanical stretch evokes Ca2+ influx via Gd3+-sensitive SA Ca2+ channels, resulting in JNK activation under regulation in part by calmodulin and calcineurin.
stretch-activated ion channel; calmodulin; cyclosporin A; c-Jun NH2-terminal kinase
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INTRODUCTION |
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BLADDER OUTLET OBSTRUCTION is a common disorder resulting from benign prostate hyperplasia, urethral stricture disease, and congenital anomaly. Although the etiology of bladder obstruction is still obscure, in most instances excessive mechanical overload might be involved in the pathogenesis. In human obstructed bladder, sustained stretch stress causes bladder wall remodeling, such as changes in both composition and amount of extracellular matrix and smooth muscle cells (7, 16, 37). An in vivo rat model with partial urethral ligature reveals smooth muscle hypertrophy and extracellular matrix deposition in the bladder wall, which are quite similar to bladder obstruction in humans (20, 21, 23, 35). However, little is known about the mechanisms by which mechanical stretch of the bladder smooth muscle produces intracellular signals leading to nuclear events, including expression of specific genes such as cyclooxygenase-2 (COX-2) and heparin-binding epidermal growth factor (HB-EGF) (27-30).
Recent studies have identified some of the intracellular signaling pathways that mediate the biological effects evoked by mechanical stimulation in vitro. The mitogen-activated protein kinases (MAPKs) constitute a family of serine/threonine kinases that mediate the transduction of external stimuli into intracellular signals that regulate cell growth and differentiation. These include the extracellular signal-regulated kinase (ERK) pathway that is stimulated in response to mechanical stress of many cell types (11, 12, 15, 17, 18, 22). Some reports indicated that another MAPK family member, c-Jun NH2-terminal kinase (JNK), is activated by mechanical stretch in cardiac fibroblast cells (22), vascular smooth muscle cells (9), cardiac myocytes (17), and mesangial cells (12). JNK was shown to bind to a specific region within a transactivation domain of c-Jun and to phosphorylate serine residues 63 and 73 of c-Jun (4). In addition, JNK also phosphorylated transcription factors such as ATF-2 (8) and Elk-1 (36) and increased their transcription activating potential. p38 kinase (31), a member of the MAPK family, also plays an important role in cellular responses to various kinds of stress (32) such as ultraviolet stress (19). In the present study, we examined early signaling events provoked by sustained mechanical stretch using primary bladder smooth muscle cells (BSMCs) cultured on deformable silicon dishes and demonstrated a novel pathway to activate JNK in response to mechanical stretch in BSMCs.
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MATERIALS AND METHODS |
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Cell culture. The primary cultures of rat BSMCs were isolated and maintained as described (29). In brief, bladders were obtained from Sprague-Dawley rats weighing 250-300 g, and smooth muscle layers were dissected from epithelium and other extraneous tissues. Smooth muscle layer minced into 1- to 3-mm pieces was thoroughly washed with sterile phosphate-buffered saline (PBS) and incubated at 37°C for 4 h in medium 199 (GIBCO) containing 20% fetal bovine serum (FBS, Boehringer Mannheim), 0.125 mg/ml elastase (type III, Sigma), and 2.5 mg/ml collagenase (type I, Sigma). The resulting tissue suspension was triturated several times with a pipette and filtered through a 100-µm cell strainer (Falcon). Cells were recovered by centrifugation, and the pellet was resuspended in a growth medium consisting of medium 199, 20% FBS, 100 U/ml penicillin, and 0.1 mg/ml streptomycin (GIBCO). Cells were grown on 100-mm culture dishes (Falcon) and maintained in a humidified 5% CO2-95% air atmosphere at 37°C with a change of medium every 3 days. All experiments were performed on cells between passages 2 and 10.
Application of mechanical stretch. BSMCs harvested from 100-mm dishes were seeded on six-well silicon elastomer-bottomed culture plates that had been coated with collagen type I (Bioflex, Flexcell, McKeesport, PA). After achieving 90% confluency, cells were subjected to sustained mechanical stretch using a controlled vacuum unit (Flexercell strain unit FX-3000, Flexcell) initially described by Banes et al. (2). The intensity of stretch was changed from 5 to 25% elongation by applying a vacuum at 15-20 kPa because BSMCs were detached from silicon membrane when strains over 25% were loaded. These procedures were carried out in a humidified incubator with 5% CO2-95% air at 37°C.
Protein extraction.
BSMCs treated by stretch were washed with ice-cold PBS and harvested
with a cell scraper. Cells were recovered by centrifugation at 3,000 rpm for 1 min and solubilized in a lysis buffer consisting of 20 mM
Tris · HCl (pH 7.5), 1% Nonidet P-40, 1 mM EDTA, 50 mM NaF, 50 mM sodium -glycerophosphate, 0.05 mM Na3VO4,
10 µg/ml leupeptin, and 100 µM phenylmethylsulfonyl fluoride. After
incubating on ice for 15 min, cell extracts were centrifuged at 15,000 rpm for 5 min. The resultant supernatant was collected and used as lysate after protein determination by Bradford assay (Bio-Rad).
In-gel kinase assay.
For in-gel kinase assay of JNK, cell extracts (0.2 mg of protein) were
resolved on 10% SDS-polyacrylamide gels containing 40 µg/ml of
glutathione S-transferase (GST)-c-JUN (amino
acids 1-79) copolymerized in separating gel
(24). After electrophoresis, the gels were washed twice
with 100 ml of 20% 2-propanol and 20 mM HEPES-NaOH, pH 7.5, and twice
with buffer A (20 mM HEPES-NaOH, 5 mM -mercaptoethanol)
at room temperature. Gels were then incubated for 1 h in 100 ml of
buffer A containing 6 M urea, which was followed by serial
incubations in buffer A containing 0.05% Tween 20 and either 3, 1.5, or 0.75 M urea. Gels were washed several times with
buffer A containing 0.05% Tween 20 at 4°C and incubated
in a reaction buffer [20 mM HEPES-NaOH, pH 7.6, 20 mM
MgCl2, 20 mM
-glycerophosphate, 0.1 mM
Na3VO4, 2 mM dithiothreitol (DTT), 50 µM ATP,
and 5 µCi/ml of [
-32P]ATP (Amersham)] at 37°C for
1 h. The reaction was stopped by washing with 5% TCA and 1%
sodium pyrophosphate. After several more washes, gels were dried, and
the protein bands were detected by autoradiography.
In vitro kinase assay.
For in vitro kinase assays, cell extracts containing 0.2 mg of protein
were incubated with 1.0 µg of rabbit polyclonal antibody against
JNK-1 or p38 (Santa Cruz Biotechnology) for 4 h at 4°C with
rotation. Immune complex was recovered by incubating with protein
G-Sepharose (Zymed) for 1 h at 4°C. The resultant
immunoprecipitates were thoroughly washed and suspended in a reaction
buffer (25 mM HEPES-NaOH, pH 7.5, 10 mM magnesium acetate, 50 µM
ATP). Kinase reactions were carried out by incubating with
[-32P]ATP (50 µCi/ml) and with 1.0 µg of either
GST-c-JUN (amino acids 1-79) or PHAS-1 (Stratagene)
for 30 min at 30°C. The reaction products were resolved on 12.5%
(for JNK-1) or 14% (for p38) SDS-polyacrylamide gels and visualized by autoradiography.
Western blotting.
Cell lysates were resolved in a 10% SDS-polyacrylamide gel and
electrotransferred to a nitrocellulose membrane. Immunodetection of
JNK1, p38, and ERK1 was done using -JNK1,
-p38, and
-ERK1 antibodies (Santa Cruz Biotechnology), respectively. Incubation with an
anti-rabbit secondary antibody conjugated to horseradish peroxidase was
followed by chemiluminescence detection (Amersham Pharmacia Biotech).
Other chemicals. Ionomycin, gadolinium chloride (GdCl3), genistein, nifedipine, pertussis toxin (PTX), verapamil, and wortmannin were purchased from Sigma; KN-93 was from Calbiochem-Novabiochem; N-(6-aminohexyl)-5-chloro-1- naphthalenesulfonamide (W-7) was from Seikagaku; and 1,2-bis-(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester) (BAPTA-AM), calphostin C, cyclosporin A (CsA), 12-myristate 13-acetate (PMA), and ryanodine were from Wako Chemicals.
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RESULTS |
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Activation of MAPK family proteins in BSMCs exposed to mechanical
stretch.
Among MAPK family members, JNK and ERK activities were measured by
in-gel kinase assay using GST-c-Jun and MBP as substrates, respectively. p38 activity was determined by in vitro kinase assay using PHAS-1 as a substrate. When BSMCs were exposed to mechanical stretch for various periods, the activities of both JNK1 and JNK2 were
enhanced from 5 min after starting stretch and peaked at 10 min; an
11-fold increase was detected with stretch of 15% elongation for 10 min (Fig. 1A). The effect of
intensity of stretch on the activity of JNK was also examined.
Activation of JNK was observed from stretch with 10% elongation and
reached a plateau at 15% elongation (Fig.
2). Similarly, the activity of p38 was
enhanced by mechanical stretch in a time-dependent manner with a peak
level at 5 min; the level was about fourfold that in nonstimulating control (Fig. 1B). Like JNK, activation of p38 was observed
from stretch with 10% elongation and reached a plateau at 15%
elongation (Fig. 2). On the other hand, ERK1 and ERK2 did not appear to
be appreciably activated by mechanical stretch (Fig. 1C).
The significant activation of ERK was not detected at any time points
and at any intensity from 10 to 25%, while the activity was enhanced
by ultraviolet irradiation.
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Activation of JNK is dependent on extracellular
Ca2+.
Because JNK was activated the most prominently among the three MAPK
members in response to mechanical stretch, the activity of JNK was used
as an indicator of intracellular events induced by mechanical stress.
On the basis of the above results, we employed a condition of stretch
with 15% elongation for 10 min for the following assessments. Previous
reports have demonstrated that an increase of intracellular
Ca2+ is important for activation of JNK (1,
33). Therefore, we examined the contribution of extracellular
Ca2+ to the mechanical stress-induced activation of JNK
using HEPES-buffered saline solution containing various concentrations
of CaCl2 (Ca-HBS). When BSMCs suspended in this Ca-HBS were
exposed to mechanical stretch, activation of JNK by mechanical stretch
was totally dependent on extracellular Ca2+ concentrations
(Fig. 3A). In addition, when
BSMCs were suspended in HBS containing BAPTA-AM, a membrane-permeable
Ca2+ chelator, mechanical stretch-induced JNK activation
was abrogated in a concentration-dependent manner (Fig. 3B).
On the other hand, ryanodine, an inhibitor of intracellular
Ca2+ stores (Fig. 3C), had no effect on the JNK
activation, suggesting that Ca2+ does not come from the
release from intracellular stores.
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Stretch-activated ion channels are required for JNK activation. It has been demonstrated by patch-clamp experiments that mechanical stretch-activated (SA) ion channels are involved in increasing of the intracellular Ca2+ concentration in some cell types (3, 25, 38) and that Gd3+ is a specific inhibitor for this SA ion channel (3). Thus we tested the effect of Gd3+ on the SA JNK activation. When BSMCs were treated with 50 µM GdCl3, the stretch-induced activation of JNK was completely abrogated (Fig. 3D). In contrast, blockers of voltage-gated Ca2+ ion channels, nifedipine and verapamil, did not have any inhibitory effect on the stretch-induced JNK activation (Fig. 3E). These results indicate that an increase of intracellular Ca2+ via SA ion channels, rather than voltage-gated channels, is required for JNK activation.
Effects of signaling inhibitors on JNK activation.
We tested a number of pharmacological agents in a search for the
intracellular signaling pathways leading to activation of JNK in BSMCs
by mechanical stretch. BSMCs were pretreated with genistein, a tyrosine
kinase inhibitor, wortmannin, a phosphatidylinositol 3-kinase
inhibitor, PTX, a G protein-coupled receptor inhibitor, or calphostin
C, a protein kinase C (PKC) inhibitor, for 30 min and then exposed to
stretch with 15% elongation for 10 min. None of these inhibitors could
affect the stretch-induced JNK activation (Fig.
4).
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DISCUSSION |
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Mechanical overload is possibly involved in remodeling of bladder wall, and such stretch stress may cause phenotypic changes in BSMCs. However, little is known about signaling mechanisms induced by static stress in smooth muscle cells. To understand intracellular events activated by mechanical stretch, we examined MAPK family members using primary cultures of BSMCs maintained on collagen-coated silicon membranes with a vacuum-operated stretch-inducing device. The results demonstrate the following. 1) Mechanical stretch mainly activates JNK in BSMCs. p38 was moderately activated, but ERK was not activated at all. 2) Mechanical stretch evokes an influx of extracellular Ca2+, which is required for this JNK activation. 3) Gd3+-sensitive SA ion channels and CaM are possibly involved in the regulation of this JNK pathway. Although the molecular basis and activation mechanism of nonselective types of SA ion channels are still obscure, an important role for these channels has been proposed in Xenopus oocytes (38), myocytes (3), and vascular endothelial cells (25). However, there is no direct evidence to support a pathway by which an increase in intracellular Ca2+ induced by mechanical stretch could activate JNK. We observed that 1) elimination of extracellular Ca2+ abrogated stretch-induced JNK activation (Fig. 3A); 2) Gd3+ (Fig. 3D) and BAPTA-AM (Fig. 3B), but not ryanodine, nifedipine, and verapamil (Fig. 3, C and E), inhibited this JNK activation; and 3) Ca2+ influx was induced by G protein- and tyrosine kinase-free mechanisms (Fig. 4). These results suggest a hypothesis, i.e., stretch stress induces BSMCs to open Gd3+-sensitive SA ion channels by unknown mechanisms, resulting in Ca2+ influx, which leads to an increase in the intracellular Ca2+ concentration. The sustained or oscillating high Ca2+ levels activate JNK. It has been demonstrated that in endothelial cells, mechanical stretch caused a transient Ca2+ increase within a few seconds via SA ion channels that declined to the initial basal Ca2+ concentration in 100 s (25). Alternatively, the involvement of Ca2+ in JNK activation has been reported in some experiments (1, 33, 39). However, these two events have never merged in one pathway because it requires some minutes for stretch-induced JNK activation to occur, while the intracellular Ca2+ event occurs in seconds. In fact, in our BSMCs system, a single cycle of stretch-relax stimulation, as well as static stretch up to 1 min, did not cause JNK activation (data not shown). These findings suggest that a sustained or oscillating intracellular Ca2+ concentration may be required for stretch-induced JNK activation.
Previous studies have indicated that increased intracellular Ca2+ mediates protein tyrosine phosphorylation, such as focal adhesion kinase p125FAK and p130CAS in vascular endothelial cells (26), and Ca2+-dependent activation of receptor tyrosine kinase, such as EGF receptor in vascular smooth muscle cells (15). However, these signaling pathways should not contribute to stretch-induced JNK activation in BSMCs because tyrosine kinase inhibitor and PKC inhibitor did not affect JNK activation (Fig. 4). Thus we next tested CaM-dependent pathways. The Ca2+/CaM complex binds to and modulates the activities of multiple key signal transducing enzymes, including CaM kinase and Ca2+/CaM-dependent Ca2+ phosphatase, calcineurin. As a calmodulin inhibitor, W-7 attenuated JNK activation evoked by mechanical stretch or ionomycin (Fig. 5, A and C), a Ca2+/CaM-dependent signaling pathway associated with JNK activation. On the other hand, KN-93 had no effect on the activation (Fig. 5B). These results suggest that other CaM kinases (e.g.. CaM kinase IV), rather than CaM kinase II, are associated with stretch-induced activation of JNK.
We demonstrated here that Ca2+ influx is essential for stretch-induced JNK activation and that ERK is not activated by mechanical stretch. It is of interest that these observations are quite different from previous findings obtained from cardiomyocytes (17). Komuro and co-workers (17) observed in cardiomyocytes that EGTA has small effects on the increase in stretch-induced JNK activation. Furthermore, ERK is activated by mechanical stretch but at a faster time course than the activation of JNK. Tissue differences explain this discrepancy. That is, upstream signaling pathways for JNK and ERK activation would be different between BSMCs and cardiomyocytes.
There have been reports that activation of JNK by mechanical stretch induces expression of immediate early genes in cardiomyocytes (17) and vascular smooth muscle cells (15). JNK activation leads to phosphorylation of c-Jun (4), which is a component of the transcription factor AP-1. Thus it is thought that JNK activation by mechanical stretch regulates gene expression through AP-1 activation (12, 17). In this context, specific gene products including HB-EGF are critical; they act as a mitogen for smooth muscle cells (6, 10), and AP-1 is an essential element in their transcriptional activation in response to a mechanical stretch stimulus (27). Moreover, these changes in the gene expression system might induce phenotypic changes in BSMCs, possibly resulting in remodeling of the bladder wall. The effect of CsA is quite interesting. It is well known that CsA functions as an inhibitor of calcineurin, a ubiquitously expressed phosphatase that is activated by Ca2+ and Ca2+/CaM. However, recent findings have indicated that CsA inhibits the Ca2+-dependent activation of JNK in lymphocytes (13, 14, 34) and an in vivo model of mouse heart (5), which are similar to our results (Fig. 3E). These observations suggest the idea that CsA acts as a blocker of Ca2+ ion channels rather than an inhibitor of calcineurin.
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FOOTNOTES |
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Address for reprint requests and other correspondence: Y. Homma, Dept. of Biomolecular Science, Fukushima Medical Univ. School of Medicine, 1 Hikariga-oka, Fukushima 960-1295, Japan (E-mail: yoshihom{at}fmu.ac.jp).
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.
Received 18 October 2000; accepted in final form 7 June 2001.
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