Department of Pediatrics, University of Michigan Medical Center, Ann Arbor, Michigan 48109-0656
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ABSTRACT |
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The
ras-related protein Rho p21 regulates
various actin-dependent functions, including smooth muscle contraction.
However, the precise mechanism of action of Rho p21 is still not clear. We report here that Rho A is a key regulator of agonist-induced contractile effects in rabbit colonic smooth muscle. Endothelin-1 and
C2 ceramide were used. Both seem
to activate phosphoinositide 3-kinase (PI 3-kinase) through G protein
and pp60src, respectively.
Immunoprecipitation and immunoblotting revealed one form of 21-kDa Rho
A that translocated from the cytosol to the membrane in response to
stimulation by either endothelin
(107 M) or ceramide
(10
7 M) (~30% increase
at 30 s that was sustained at 4 min). The translocation of Rho A to the
membrane was confirmed by immunostaining. The translocation of Rho A
was inhibited by Clostridium
botulinum C3 exoenzyme, which ADP
ribosylated Rho A, but was not inhibited by the
pp60src inhibitor herbimycin A or
by the protein kinase C (PKC) inhibitor calphostin C, suggesting that
Rho A may be upstream of pp60src
and PKC or may belong to a different pathway than these proteins. Both
ceramide- and endothelin-induced PI 3-kinase activation was inhibited
by C3 exoenzyme pretreatment. However, the C3 exoenzyme inhibited
endothelin- but not ceramide-induced mitogen-activated protein kinase
phosphorylation, indicating that Rho regulates ceramide- and
endothelin-induced contraction through different pathways. Furthermore,
the dominant negative form of Rho (N19Rho) inhibited the actin binding
protein, 27-kDa heat shock protein (HSP27), reorganization in response
to ceramide and endothelin observed under confocal microscopy.
signal transduction; ceramide; endothelin; actin
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INTRODUCTION |
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THE LOW-MOLECULAR-WEIGHT GTP-binding protein Rho p21 is a member of the wide-spread superfamily of ras-related proteins, which can be divided into three subfamilies: Rho, Rac, and Cdc42 (45). These proteins function by utilizing a guanine nucleotide-binding and hydrolyzing cycle (5, 12). The Rho sequences were originally isolated from an Aplysia cDNA library (28) and were used subsequently to identify three homologues in mammals (Rho A, Rho B, and Rho C) (7) and the yeast Saccharomyces cerevisiae (29). The evidence to date indicates that Rho regulates the cytoskeletal system, particularly actin-dependent functions, such as cell motility (43), formation of stress fibers and focal adhesions (35), and smooth muscle contraction (15).
The modes of activation and action of Rho p21 have not been identified. Similar to other G proteins, Rho p21 cycles between two interconvertible forms, GDP- and GTP-bound forms. In the cytoplasm, Rho is in the GDP-bound form, presumably the inactive form, complexed with Rho-guanine nucleotide dissociation inhibitor (Rho-GDI). Rho receives upstream signals through their regulators and passes the signals to their downstream targets. It is activated by conversion to GTP-Rho by two possible mechanisms (45): 1) GDP-Rho first dissociates from Rho-GDI and is then activated by its stimulatory guanine nucleotide factor (GEF), resulting in the formation of GTP-Rho, or 2) GDP-Rho complexed with Rho-GDI is activated directly by a stimulatory GEF, resulting in the formation of GTP-Rho. It has been demonstrated that the Rho-Rho GDI system plays an important role in temporal and spatial determination of the actin cytoskeletal control (38).
Stimulation of smooth muscle cells by specific agonists induces
Ca2+ mobilization and activation
of myosin light chain (MLC) kinase, which subsequently phosphorylates
MLC and activates the myosin ATPase. The cascade of events described
above leads to contraction of smooth musle (41) and interaction of
actin and myosin for stress fiber formation in nonmuscle cells (9).
However, because the cytosolic concentration of
Ca2+ is not always proportional to
the extent of MLC phosphorylation and the force of contraction, it has
been proposed that there may be an additional mechanism to regulate the
Ca2+ sensitivity of both processes
(6). Because agonists induce MLC phosphorylation and contraction in
permeablilized smooth muscle at submaximal concentrations of
Ca2+ in a GTP-dependent manner, a
GTP-binding protein is thought to regulate the receptor-mediated
sensitization of MLC phosphorylation to
Ca2+ (24). The small GTPase Rho is
implicated here in the enhancement of
Ca2+ sensitivity of smooth muscle
contraction by GTP (15). In permeabilized smooth muscle cells, the
nonhydrolyzable GTP analog guanosine 5'-O-(3-thiotriphosphate)
(GTPS) increases MLC phosphorylation by inhibiting dephosphorylation
of MLC presumably by activation of Rho (34). GTP-Rho then presumably
binds to specific targets and thereby exerts its biological functions
(33, 34).
We have investigated how Rho A modulates agonist-induced signal transduction cascades in smooth muscle contraction and how it modulates actin binding proteins. We present evidence that Rho A is present in smooth muscle cells and that it is activated during ceramide- and endothelin-induced phosphoinositide 3-kinase (PI 3-kinase)-mediated sustained contraction. We also show that Rho A may regulate smooth muscle contraction through different pathways via reorganization of the actin binding protein, 27-kDa heat shock protein (HSP27).
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MATERIALS AND METHODS |
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Materials
The following reagents were purchased. Monoclonal mouse anti-Rho A and polyclonal rabbit anti-Rho A IgG were from Santa Cruz Biotechnology (Santa Cruz, CA); monoclonal mouse anti-PI 3-kinase NH2-terminal-SH2 domain antibody was from Upstate Biotechnology (Lake Placid, NY); polyclonal rabbit phosphospecific p44/p42 mitogen-activated protein (MAP) kinase antibody was from New England Biolabs (Beverly, MA); FITC-conjugated Affinipure F(ab')2 donkey anti-mouse IgG and lissamine rhodamine sulfonyl chloride (LRSC)-conjugated Affinipure F(ab')2 sheep anti-mouse IgG were purchased from Jackson Immunoresearch Laboratories (West Grove, PA); a monoclonal mouse anti-HSP27 (IgG) used was as previously described (3); C2 ceramide was from Matreya (Pleasant Gap, PA); and endothelin-1 was from Peninsula Laboratories (Belmont, CA). Clostridium botulinum C3 exoenzyme was from Biomol Research Laboratories (Plymouth Meeting, PA); collagenase type II was purchased from Worthington Biochemical (Freehold, NJ); herbimycin A was from Calbiochem (La Jolla, CA). Calphostin C was from Kamiya Biomedical (Thousand Oaks, CA). Wortmannin, soybean trypsin inhibitor (STI), poly-L-lysine, creatinine phosphate, creatine phosphokinase, and ATP were obtained from Sigma Chemical (St. Louis, MO); analytic silica gel 60-precoated glass TLC plates were obtained from Merck (Darmstadt, Germany); protein G-Sepharose was from Pharmacia Biotech (Uppsala, Sweden); [Methods
Isolation of smooth muscle cells from rabbit rectosigmoid. Smooth muscle cells from rabbit rectosigmoid were isolated as described previously (3). Briefly, the internal anal sphincter from anesthetized New Zealand White rabbits, consisting of the most distal 3 mm of the circular muscle layer, ending at the junction of skin and mucosa, was removed by sharp dissection. A 5-cm length of the rectosigmoid orad to the junction was dissected and digested to yield isolated smooth muscle cells. The tissue was incubated for two successive 1-h periods at 31°C in 15 ml of HEPES (pH 7.4) containing (in mM) 115 NaCl, 5.7 KCl, 2.0 KH2PO4, 24.6 HEPES, 1.9 CaCl2, 0.6 MgCl2, 5.6 glucose containing 0.1% (wt/vol) collagenase (150 U/mg; Worthington CLS type II), 0.01 (wt/vol) STI, and 0.184 (wt/vol) DMEM. After the end of the second enzymatic incubation period, the medium was filtered through 500-µm Nitex mesh. The partially digested tissue left on the filter was washed four times with 10 ml of collagenase-free buffer solution. Tissue was then transferred into 15 ml of fresh collagenase-free buffer solution, and cells were gently dispersed. After a hemocytometric cell count, the harvested cells were resuspended in collagenase-free HEPES buffer (pH 7.4). Each rectosigmoid yielded 10-20 × 106 cells.
Particulate fractions. Isolated smooth muscle cells were counted on a hemocytometer and diluted with HEPES buffer as needed. Cells were then treated with agonist and/or antagonist for the indicated periods. After the treatment, the cells were washed twice with buffer A (in mM: 150 NaCl, 16 Na2HPO4, 4 NaH2PO4, and 1 sodium orthovanadate, pH 7.4) and sonicated in buffer B (1 mM Na3VO4, 1 mM NaF, 2 mM PMSF, 5 mM EDTA, 1 mM Na4MoO4, 1 mM dithiothreitol, 20 mM NaH2PO4, 20 mM Na2HPO4, 20 mM Na4P2O7 · 10 H2O, 50 µl/ml DNAse/RNAse, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 10 µg/ml pepstatin A, and 10 µg/ml antipain, pH 7.4). The cell sonicates were centrifuged at 100,000 g for 60 min. The supernatant material from the high-speed centrifugation was collected as soluble cytosolic fraction. The pellet material was resuspended by sonication twice for 30 s in the lysis buffer plus 1% Triton X-100 and collected as soluble particulate fraction. The protein content was determined by Bio-Rad protein assay reagent.
Preparation of permeable smooth muscle cells. In experiments involving preincubation of smooth muscle cells with Clostridium botulinum C3 exoenzyme, which does not readily pass across the cell membrane, muscle cells were made permeable without affecting their overall function (2). The partially digested muscle tissue was washed with 50 ml of a "cytosolic" enzyme-free medium (cytosolic buffer) of the following composition (in mM): 20 NaCl, 100 KCl, 5.0 MgSO4, 0.96 NaH2PO4, 25.0 NaHCO3, 1.0 EGTA, and 0.48 CaCl2. The medium contained 2% BSA and was equilibrated with 95% O2-5% CO2 to maintain a pH of 7.2. Muscle cells were allowed to disperse spontaneously in this medium and were harvested by filtration on 500-µM Nitex mesh. Isolated cells were permeabilized by incubation for 3 min in saponin (75 µg/ml). The cell suspension was centrifuged down and resuspended in the cytosolic buffer containing antimycin A (10 µM), ATP (1.5 µM), and an ATP-regenerating system consisting of creatinine phosphate (5 µM) and creatine phosphokinase (10 U/ml). The permeabilized cells were allowed to rest in a 95% O2-5% CO2 environment before the experiments.
Immunoprecipitation and immunoblotting using monoclonal anti-Rho A antibody. Each sample (400-500 ug protein) obtained as described above was subjected to immunoprecipitation with monoclonal anti-Rho A antibody overnight at a ratio of 1:250. The protein G-Sepharose beads were then added and rocked for 2 h. The beads were washed in TBS twice and boiled in 2× Laemmli sample buffer with 2-mercaptoethanol. The samples were subjected to 12.5% SDS-PAGE and electrophoretically transferred to nitrocellulose membrane. Immunoblotting was performed using mouse monoclonal anti-Rho A antibody (1:100 dilution) as primary antibody. The membrane was reacted with peroxidase-conjugated goat anti-mouse IgG (1:3,000 dilution) for 1 h. The enzymes on the membrane were visualized with ECL substrates.
Measurement of PI 3-kinase activity.
Whole cell lysates were obtained as described above. Equal amounts of
protein were subjected to immunoprecipitation with anti-PI 3-kinase
antibody (specific to the p85 subunit). The substrates were prepared by extracting the lipid mixture of 10 µg of
phosphatidylserine and 10 µg phosphoinositol in 1 ml methanol and
chloroform (1:1), followed by 1 h sonication in 10 ml kinase assay
buffer. The immunoprecipitates were then reacted with 10 ml lipid
substrate mixture and 10 µl of 20 µM
[-32P]ATP cocktail
(2 µl [
-32P]ATP
with 8 µl of 104 µM ATP) in 15 µl kinase assay buffer for 10 min
at 30°C. The reaction was then stopped by adding 100 µl 1 N HCl.
The radioactive-labeled product
L-
-phosphatidylinositol 4-monophosphate was extracted in 160 µl methanol and chloroform (1:1,
vol/vol). The lipid extract was resolved by TLC using
CHCl3, MeOH,
NH4OH, and distilled
H2O (45:35:1.5:8.5,
vol/vol). The TLC plates were dried and exposed to Kodak
XOMAT LS film at 70°C for 1-2 days. Duplicates were also made
for each reaction, as well as negative controls, by replacing the
substrates with kinase assay buffer.
Measurement of contraction. Aliquots consisting of 2.5 × 104 cells in 0.5 ml of medium were added to 0.1 ml of a solution containing the test agents, which included agonists or combinations of inhibitors and agonists. The reaction was interrupted at 4 min by the addition of 0.1 ml of acrolein at a final concentration of 1%. Individual cell length was measured by computerized image micrometry. The average length of cells in the control state or after addition of test agents was obtained from 50 cells encountered randomly in successive microscopic fields. The contractile response is defined as the decrease in the average length of the 50 cells and is expressed as the absolute change or percent change from control length (3).
Cell culture and confocal imaging of HSP27 and Rho A. CELL CULTURE. The rabbit rectosigmoid was removed and washed in PBS with penicillin/streptomycin and ethanol twice. The mucosa and serosa were carefully removed, and the circular smooth muscle layer was washed three times in PBS with penicillin/streptomycin. The smooth muscle layer was digested with HEPES buffer containing 0.1% (wt/vol) collagenase (150 U/mg, Worthington CLS type II) at 37°C with 5% CO2 for 2 h. At the end of the enzymatic incubation period, the tissue was washed in PBS with penicillin/streptomycin and transferred into DMEM supplemented with 10% FBS. The cell suspension was filtered by Nitex 500-µm mesh. The cell suspension was collected in the medium and transferred to six-well plates coated with collagen IV, which were then cultured in a humidified 5% CO2 incubator for 7-14 days before they reached confluency.
COVERSLIP PREPARATION. Glass coverslips were washed in 70% ethanol for 30 min with gentle agitation. They were allowed to air dry, placed in six-well plates, and irradiated with ultraviolet light for 30 min. The coverslips were then coated with 0.5% (wt/vol) poly-L-lysine and allowed to air dry. The coverslips were placed in a six-well tissue culture plate. At the end of the second enzymatic digestion, the digested tissue was washed, titrated, and filtered as described previously. The dispersed cells were collected in DMEM with 10% FBS medium and transferred to the six-well tissue culture plate and allowed to settle for 2 days on the poly-L-lysine-coated coverslips in a humidified 5% CO2 environment. CELL FIXATION. Cells were fixed with 3.5% formaldehyde in PBS for 10 min. The fixative was removed, and the cells were washed twice with 100 mM glycine buffer, pH 7.4, for 5 min, followed by one wash with PBS. The cells were then permeabilized by adding 3 ml of the permeabilization solution with Triton X-100 to each coverslip for 10 min. After permeabilization, the cells were rinsed three times with 3-ml aliquots of PBS. DUAL LABELING FOR RHO A AND HSP27. Dual labeling of Rho A and HSP27 was performed in either resting or contracted smooth muscle cells. A previously described protocol was used (46). After isolation, cells were cultured in DMEM with 10% FBS for 2 days on coverslips coated with poly-L-lysine. The cells were treated with C2 ceramide or endothelin-1, permeabilized, and fixed. Cells were incubated for 1 h with normal goat serum, followed by three 10-min washes in PBS. The cells were incubated for 1 h with the first primary antibody, a monoclonal mouse anti-HSP27 (1:50), together with the second primary antibody, a polyclonal rabbit anti-Rho A (1:50), followed by three 10-min washes in PBS. Subsequently, the cells were incubated with the first secondary antibody, a 1:200 dilution of Affinipure F(ab')2 donkey anti-mouse IgG FITC, together with the second secondary antibody, a 1:100 dilution of Affinipure F(ab')2 sheep anti-rabbit RITC, for 1 h, followed by three 10-min washes in PBS. Finally, the cells on the coverslip were mounted on a slide with DABCO mounting medium and sealed with Aqua Mount. The following controls were also performed: 1) FITC only, 2) RITC only, 3) FITC with first primary antibody, 4) RITC with first primary antibody, 5) FITC with second primary antibody, and 6) RITC with second primary antibody. The excitation for the fluorescent probes was as follows: FITC excitation at 492 nm and emission at 520 nm and LRSC excitation at 570 nm and emission at 590 nm. The confocal images were obtained on the Bio-Rad 600 confocal imaging system. TRANSIENT TRANSFECTION. When the cells in culture reached ~65-70% confluency, they were washed with serum-free DMEM twice. We gently mixed 10 µl lipofectamine and 3 µg myc-tagged N19Rho plasmid DNA with 200 µl DMEM, incubating the mixture at room temperature for 45 min. Then the mixture was added to the cells together with 1.8 ml DMEM for each P60 plate. The cells were cultured at 37°C for 2 h with 5% CO2. The medium containing the lipofectamine and DNA was removed, and fresh DMEM with 10% FBS was added to the cells. After two days, the cells were subjected to immunofluorescent labeling. ![]() |
RESULTS |
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C2 Ceramide- and Endothelin-1-Induced Activation and Translocation of Rho A in Colonic Smooth Muscle Cells
Both endothelin-1 and C2 ceramide can induce sustained smooth muscle contraction. Endothelin-1, a potent vasoconstrictor, acts through G protein (4), whereas C2 ceramide, a sphingomyelin breakdown product, seems to act through nonreceptor tyrosine kinase pp60src kinase (19). Immunoprecipitation followed by immunoblotting using mouse monoclonal anti-Rho A antibody detected a single form of Rho A in the whole cell lysate of isolated smooth muscle cells in rabbit rectosigmoid, with a relative molecular mass of 21 kDa (Fig. 1). Rho GTPases are thought to be associated with Rho-GDI in the cytosol, as inactive forms, and therefore must translocate to the plasma membrane (where they would presumably meet a GEF) to be activated (13). Gong et al. (10) have demonstrated that 1) Rho A can translocate from the cytosolic to the particulate fractions on stimulation by agonists such as GTP
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C3 Exoenzyme Inhibited Rho A Translocation to the Membrane and Decreased PI 3-Kinase Activity
Rho A is unique among the ras-related GTPases, since it is a specific substrate for ADP ribosylation catalyzed by the C3 exoenzyme from Clostridium botulinum (1, 32). C3 exoenzyme selectively modifies Rho A at the asparagine residue 41 and inhibits its biological function, presumably by interfering with its interaction with downstream targets (40). Because C3 exoenzyme is not cell permeable, we permeabilized the cells with saponin to examine the effects of C3 exoenzyme in the sustained contraction of smooth muscle cells induced by endothelin-1 and C2 ceramide. Preincubation of permeabilized smooth muscle cells with Rho A-specific C3 exoenzyme (2 ng/5,000,000 cells) for 20 min, followed by incubation with C2 ceramide or endothelin-1 for 30 s or 4 min at 37°C, resulted in decreased Rho A translocation to the membrane fractions (Fig. 4), which further confirmed that translocation is required for Rho A biological activity in smooth muscle contraction. We also examined PI 3-kinase activity, which in platelets requires the involvement of Rho in rapid thrombin-induced cytoskeletal reorganization (47). We found increased PI 3-kinase activity on ceramide and endothelin stimulation. However, after C3 exoenzyme treatment, it was found that C3 exoenzyme blocked the increased PI 3-kinase activity in response to both C2 ceramide and endothelin-1 (Fig. 5). These data suggest that Rho A in smooth muscle cells is an upstream regulator of PI 3-kinase activation.
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C3 Exoenzyme Inhibits Colonic Smooth Muscle Contraction
We have previously shown that both C2 ceramide (10Rho A May Exert Its Effects on the Cytoskeleton via PI 3-Kinase through Different Pathways
In an attempt to identify the pathways involved in Rho A activation, we used different antagonists to identify the possible upstream and downstream molecules related to Rho A function in cytoskeletal reorganization in smooth muscle contraction. Our data show that herbimycin A (3 × 10
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Rho A Activation is Correlated with Redistribution of HSP27
We have previously reported that the low-molecular-weight HSP27, which has been shown to be an actin filament binding protein (31), is involved (3) and cotranslocates with MAP kinase (46) in the PKC-mediated sustained contraction of rabbit colonic smooth muscle contraction. Here we investigated HSP27 fluorescent distribution under confocal microscopy in relation to Rho A activation in either resting or contracted rabbit rectosigmoid smooth muscle cells. On stimulation with C2 ceramide or endothelin-1, there was a clear translocation of Rho A to the cell membrane (Fig. 8, left), while HSP27 redistributed itself in the region of the surface membrane and formed traverse bands across the cells (Fig. 8, middle). The areas of strong colocalization on the cell edge (indicated by yellow dots) show that the pattern of colocalization is similar to that seen with redistribution of Rho A after contraction (Fig. 8, right). This result suggests that HSP27 translocates with Rho A during cell contraction and implicates the possible interaction between these two proteins. To further assess the relationship between Rho A and HSP27, we transfected the cells with the dominant negative form of Rho, N19Rho plasmid (Fig. 9), which inactivates Rho. HSP27 aligned itself along the axis of the cells, and there was no translocation of HSP27 that was seen in normal control cells (Fig. 10). These data suggest that Rho A may regulate actin structure through the PI 3-kinase-mediated HSP27 reorganization and translocation.
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DISCUSSION |
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Ca2+ and MLC phosphorylation are key regulators of the dynamic reorganization of actin filaments. Because the contraction-to-Ca2+ ratio is not always proportional, the Ca2+- and calmodulin-dependent MLC kinase pathway cannot solely account for the Ca2+ sensitivity (6, 27, 42). In past years, evidence accumulated that the ras-related small GTP binding protein Rho is another important signaling element that mediates various actin-dependent cytoskeletal functions, including smooth muscle contraction (45). However, its roles in different signal transduction cascades may vary depending on cell type.
Rho has been shown to play pivotal roles in Ca2+ sensitization (23, 27). Several Rho targets have been identified, including protein kinase N, Rho-associated kinase (Rho kinase), and the myosin-binding subunit of myosin phosphatase (22). It has been proposed that Rho activates Rho kinase, which in turn phosphorylates MLC by inhibiting myosin phosphatase (22, 23, 27). Here, we attempted to study Rho functions in the sustained contraction of rabbit rectosigmoid smooth muscle cells. Our data indicated that Rho plays an important role in the signal transduction modulating rabbit colon smooth muscle contractions, on stimulation by agonists such as endothelin-1, a known potent vasoconstrictor, as well as by C2 ceramide, which is a breakdown product of sphingomyelin hydrolysis.
Ceramide is an important regulatory molecule implicated in various biological processes in response to stress and cytokines. We have previously shown that the sustained contraction induced by the peptide agonist bombesin is accompanied by an increase in sphingolipid-derived ceramide. Ceramide produced in the cell acts as an intracellular messenger. Ceramide induces a sustained contraction of smooth muscle cells through a pathway that involves the activation of MAP kinase (39). Thus ceramide could be an important mediator of contraction and could account for the sustained contraction observed in circular smooth muscle cells from the rabbit rectosigmoid.
Endothelin-1 is a vasoconstrictor peptide originally derived from
endothelial cells functioning as a local regulator of vascular tone and
has been reported to possess a wide variety of other biological
activities. Recent studies indicate the presence of endothelin-like
immunoreactivity, endothelin-1 mRNA, and endothelin receptors in colon
(20, 44). Evidence suggested that endothelin is a neuropeptide in the
human intestine with binding sites on neural plexuses and mucosa,
implying a role in the modulation of intestinal motility and secretion
(20). Binding of endothelin-1 to its heterotrimeric G protein-coupled
receptors stimulates various signaling cascades involving phospholipase
C-, phospholipase D, PKC, tyrosine kinases,
Ca2+- and calmodulin-dependent
kinase, and Ras. It has also been show that Rho activation is critical
for the endothelin-1-induced nuclear signaling (21). Endothelin-1 could
also cause translocation of Rho A to cell membrane in Swiss 3T3
fibroblasts (8). Furthermore, the Rho A inhibitor C3 exoenzyme could
inhibit endothelin-induced cytoskeletal actin reorganization in
cultured astrocytes (25) and tyrosine phosphorylation of p125 focal
adhesion kinase and paxillin in Swiss 3T3 cells (36), suggesting the
possible role of Rho in endothelin signaling pathways in smooth muscle contraction.
We have previously shown that endothelin-1 induces a sustained G protein-mediated contraction in smooth muscle cells of the colon (4) whereas C2 ceramide, a sphingolipid metabolite produced in smooth muscle cells, induces sustained contraction through activation of cytoplasmic tyrosine kinase of the Src family (19). When stimulated with endothelin-1 or C2 ceramide, Rho A was observed evidently, by both Western blots and immunostaining data, to translocate from the cytosol to membrane. This translocation was blocked by the Rho A-specific inhibitor Clostridium botulinum C3 exoenzyme by ADP ribosylation of Rho A at the asparagine residue 41, perhaps by preventing Rho from interacting with its target molecule (40). It has been shown that, on receptor activation, Rho A translocates from the cytosol to the plasma membrane in certain cell types after the subsequent dynamic actin-cytoskeletal reorganization. When preincubating the cells with C3 exoenzyme, we observed that the contraction induced by ceramide and endothelin was inhibited. The data above strongly suggest that both endothelin-1 and C2 ceramide could exert their contractile effects via Rho A activation.
The possible interrelationship between Rho A and other serine/threonine kinases or tyrosine kinases is more complex. Our data show that the pp60src inhibitor herbimycin A was not able to inhibit Rho A translocation from the cytosol to the membrane, which suggested that Rho A activation by endothelin and ceramide is either independent or upstream of these tyrosine kinases. It has been suggested that Rho A is regulating Ca2+ sensitivity in smooth muscle via the PKC/MAP kinase pathway or through a PKC-mediated effect on MLC phosphatase (15, 16). After preincubation with the PKC inhibitor calphostin C, the data indicate that both ceramide- and endothelin-induced Rho A translocation is not inhibited, suggesting that Rho A is at least upstream of PKC. This view was also supported by the observation that Rho inhibitors block PKC translocation and activation in endothelial and epithelial cells, suggesting a Rho A requirement for PKC activation/translocation (14). We have also shown that both endothelin and ceramide can increase PI 3-kinase activity, with subsequent production of lipid to mediate actin cytoskeleton or interact with Rho A. It has previously been shown that Rho may regulate PI 3-kinase activity in Swiss 3T3 cells as well as platelets, suggesting that PI 3-kinase is downstream of Rho A (26, 47), although PI 3-kinase can be either upstream or downstream of the Rho family depending on the system (37). In our experiments, when smooth muscle cells were preincubated with Clostridium botulinum C3 exoenzyme, which selectively ADP ribosylates the Rho A in asparagine residue 41, the translocation of Rho A was inhibited, as was endothelin- and ceramide-induced PI 3-kinase activity. On the basis of these observations, we propose that ceramide and endothelin both activate Rho A, which in turn activates PI 3-kinase. However, it is of interest to understand whether Rho activates PI 3-kinase through a serine/threonine PKC pathway or via a tyrosine kinase pathway. Alternatively, ceramide and endothelin may also transduce their signals downstream of Rho A through different pathways to activate PI 3-kinase. It has been shown that ceramide and endothelin can phosphorylate p42 and p44 MAP kinase; our data also suggested that Rho A may regulate PI 3-kinase-mediated smooth muscle contraction induced by ceramide through pp60src, whereas endothelin directly activates MAP kinase cascade. The precise complex mechanism is yet to be determined.
Actin binding proteins play a key role in shaping the actin cytoskeleton. To further understand how Rho A protein affects actin filament dynamics induced by agonists in smooth muscle contraction, we assessed the correlation between Rho A and the low-molecular-weight HSP27 identified as an actin binding protein. HSP27 is expressed in a variety of tissues in the absence of stress and has been suggested to have a phosphorylation-activated homeostatic function at the actin cytoskeleton level. Phosphorylation of HSP27 has been shown to increase in response to diverse stimuli, including phorbol esters and Ca2+ ionophores. The degree of phosphorylation varies in response to different stimuli. HSP27 has also been suggested to be involved in contraction of intestinal smooth muscle. We have previously shown that HSP27 plays an integral role in the orientation or activation of the contractile machinery necessary to maintain a sustained contraction in rabbit gastrointestinal smooth muscle (46). HSP27 distribution during contraction of smooth muscle is not well understood. We previously reported that HSP27 could colocalize with MAP kinase during contraction. It has been reported that inhibition of Rho A by C3 exoenzyme could block endothelin-induced cytoskeletal actin reorganization in cultured astrocytes (25). In cells transiently transfected with the dominant negative Rho A, we observed that ceramide or endothelin-induced redistribution of HSP27 disappeared, which suggested that Rho A may exert its effects on cytoskeletal reorganization via HSP27. Moreover, the requirement of Rho A for HSP27 redistribution suggests the possible interaction between these cytoskeletal proteins in sustained smooth muscle contraction.
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ACKNOWLEDGEMENTS |
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We thank Xuehui Su, M.S., for technical assistance and Erin McDaid-Kelly for preparing the manuscript. We also thank Professor A. Hall for the kind gift of Rho-N19 plasmid.
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FOOTNOTES |
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This study was supported by National Institutes of Health Grant RO1-DK-42876.
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. §1734 solely to indicate this fact.
Address for reprint requests: K. N. Bitar, Univ. of Michigan Medical School, 1150 W. Medical Center Dr., A520D, MSRB I, Ann Arbor, MI 48109-0656.
Received 18 April 1998; accepted in final form 27 August 1998.
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