1Department of Medicine, Seoul National University, Bundang Hospital, Seoungnam, Gyeronggi-Do 463-707; 3Department of Medicine, Seoul National University College of Medicine and Liver Research Institute, Seoul, Korea 110-799; 2Department of Medicine, Rhode Island Hospital, and Division of Biology and Medicine, Brown University Medical School, Providence, Rhode Island 02903; and 4Smooth Muscle Research Group, University of Calgary, Calgary, Alberta, Canada T2N 4N1
Submitted 12 September 2003 ; accepted in final form 1 April 2004
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ABSTRACT |
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protein kinase C; myosin light chain kinase; phosphatase; integrin-linked kinase
MLCP is subject to complex regulation. The holoenzyme is a trimer of a catalytic subunit of 3738 kDa, a 110- to 130-kDa myosin targeting subunit (MYPT1), and a 20-kDa subunit of unknown function. The best characterized mechanisms of inhibition of MLCP involve phosphorylation of MYPT1, which is accompanied by a decrease in phosphatase activity (20, 43, 46), and interaction of phosphorylated CPI-17 with the catalytic subunit of the phosphatase (51). CPI-17 is a 17-kDa cytosolic protein that becomes a potent inhibitor of type 1 protein phosphatase when phosphorylated at Thr38 (18). MYPT1 can be phosphorylated by various kinases, including Rho-associated kinase (ROK) (20, 36), MYPT1 kinase [a zipper-interacting protein (ZIP)-like kinase (44)], Raf-1 (9), and integrin-linked kinase (ILK) (46). The inhibitory phosphorylation site in MYPT1 is Thr695 (20), although phosphorylation at other sites may also have an inhibitory effect (46). Phosphorylation of CPI-17 at Thr38 is catalyzed by protein kinase C (PKC) (18), ROK (40), protein kinase N (23), MYPT1 kinase (44), and ILK (13). Inhibition of MLCP activity plays an important role in "Ca2+ sensitization" of smooth muscle contraction, i.e., agonist-induced increase in force or contraction without a change in [Ca2+]i (59).
It is becoming increasingly clear that, while smooth muscle contraction in some cases is regulated by the balance between MLCK and MLCP activities, exceptions occur. This is exemplified by comparison of the circular smooth muscles of the esophagus (ESO) and the lower esophageal sphincter (LES) of the cat, which fulfill quite different physiological roles. ESO is normally relaxed and exhibits a brief and powerful phasic contraction in response to stimulation of cholinergic nerves induced by swallowing. LES, however, maintains spontaneous myogenic tone and relaxes in response to stimulation of nonadrenergic, noncholinergic neurons. Previous work has suggested that fundamental differences exist in the signal transduction pathways involved in excitation-contraction coupling in these two smooth muscles (54, 55) for the initial phase of contraction (47). In both cases, contractile stimuli such as acetylcholine (ACh) trigger an increase in MLC phosphorylation (54). In the case of LES, both phosphorylation and contraction are inhibited by CaM and MLCK inhibitors and are unaffected by PKC inhibitors (3, 54). ACh-induced contraction of LES involves activation of M3 muscarinic receptors, which are coupled to phosphatidylinositol phospholipase C (PI-PLC) via Gq/11. PI-PLC hydrolyzes phosphatidylinositol 4,5-bisphosphate to generate inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). IP3 triggers Ca2+ release from the sarcoplasmic reticulum, activation of CaM-dependent MLCK, MLC phosphorylation, and contraction (55). In sharp contrast, ACh-induced MLC phosphorylation and contraction in ESO are inhibited by PKC inhibitors but not by CaM or MLCK inhibitors (54). In this case, ACh-induced contraction involves activation of M2 muscarinic receptors, which are coupled to both phosphatidylcholine-specific phospholipase C (PC-PLC) and phospholipase D (PLD) via Gi3 (55). Both phospholipases generate DAG, PC-PLC directly via hydrolysis of phosphatidylcholine (PC) and PLD indirectly via hydrolysis of PC to phosphatidic acid (PA) and hydrolysis of PA to DAG via phosphatidate phosphohydrolase. DAG activates PKC, a Ca2+-independent (novel) PKC isoform (57). The link between PKC
activation and the observed increase in MLC phosphorylation in ESO in response to ACh has not been elucidated. These results led to the unexpected conclusion that in ESO, unlike LES, MLCK is not the major kinase responsible for MLC phosphorylation and contraction.
In this study, we used a variety of molecular tools (phosphatase and kinase inhibitors and antibodies to signaling enzymes) in combination with intact and saponin-permeabilized isolated ESO and LES smooth muscle cells to compare the mechanisms of excitation-contraction coupling in these two smooth muscle types. Specifically, we examined the signaling pathways activated by phosphatase inhibition. LES contraction induced by phosphatase inhibition was reduced by MLCK inhibitors, supporting a central role of MLCK in this tissue. In contrast, MLCK plays no role in phosphatase inhibitor-induced contraction of ESO. Rather, contraction of ESO by phosphatase inhibitors is mediated by activation of a PKC-dependent pathway, which also involves mitogen-activated protein (MAP) kinase kinase (MEK), the MAP kinases ERK1 and ERK2, and activation of ILK. Thus, in ESO, phosphatase inhibitors do not evoke contraction by potentiating MLCK but likely inhibit MLCP and phosphatases that oppose the effects of activation of PKC
. Activation of PKC
then results in activation of a number of kinases, which include ERK1/2 and ILK.
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METHODS |
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Dispersion of smooth muscle cells. Tissue squares were digested in N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-buffered physiological solution containing 150 U/ml collagenase type II to dissociate smooth muscle cells as described previously (5, 35). The HEPES solution contained (in mM) 112.5 NaCl, 5.5 KCl, 2.0 KH2PO4, 10.8 glucose, 24.0 HEPES, 1.9 CaCl2, and 0.6 MgCl2, as well as 0.3 mg/ml basal medium Eagle (BME) amino acid supplement and 0.08 mg/ml soybean trypsin inhibitor. The solution was oxygenated (100% O2) at 31°C, and pH was adjusted to 7.4. During the digestion period, the gas flow rate was kept low to avoid agitating the tissue. At the end of the digestion period, the tissue was poured over a 500-µm Nitex mesh (Tetko, Elmsford, NY), rinsed in collagenase-free HEPES buffer to remove any trace of collagenase, and incubated in this solution at 31°C, gassed with 100% O2. The cells were allowed to dissociate freely for 1020 min. Care was taken not to agitate the fluid to avoid cell contraction in response to mechanical stress. All glassware used in this procedure was siliconized with a 0.05% silicon solution (Sigma, St. Louis, MO) to prevent the cells from adhering to the glass.
The isolated cells were relaxed, as their unstimulated length did not change after prolonged incubation in Ca2+-free medium. For instance, in intact LES cells, the initial cell length was 6772 µm in normal buffer and 6872 µm in Ca2+-free medium.
Preparation of permeable smooth muscle cells.
Cells were permeabilized to allow the use of microcystin-LR and various antibodies, which do not cross the intact cell membrane. After completion of the enzymatic phase of the digestion process, the partly digested muscle tissue was washed with an enzyme-free cytosolic buffer (buffer A) composed of (in mM) 20 NaCl, 100 KCl, 5.0 MgSO4, 0.96 NaH2PO4, 25 NaHCO3, 0.61 CaCl2, and 1 EGTA, as well as 2% bovine serum albumin, yielding 0.36 µM free Ca2+, calculated according to the method of Fabiato and Fabiato (19a). Investigators at our laboratory (3) previously showed that the maximal contractile response of permeabilized ESO smooth muscle cells to ACh requires 0.36 µM Ca2+ in buffer A. Buffer A was equilibrated with 95% O2-5% CO2 to maintain pH 7.2 at 31°C. Smooth muscle cells dispersed spontaneously in this medium. After dispersion, the cells were permeabilized by incubation for 3 min in buffer A containing saponin (75 µg/ml). After exposure to saponin, the cell suspension was centrifuged at 500 g, and the resulting pellet was resuspended in saponin-free buffer A containing 10 µM antimycin A, 1.5 mM ATP, and an ATP-regenerating system consisting of 5 mM creatine phosphate and 10 U/ml creatine phosphokinase (6, 55). After the cells were washed free of saponin, they were resuspended in the same buffer. Permeabilization did not affect the unstimulated cell length. For instance, the length of permeable unstimulated ESO cells was 70 µm and was the same as the length of unstimulated intact cells (7075 µm).
To measure phosphatase inhibitor-induced contractions, ESO and LES circular smooth muscle cells were exposed to okadaic acid for 2 min, to microcystin-LR for 1 min, or to antibodies against the catalytic subunit of type 1 protein phosphatase for 1 min. Cells were preincubated with appropriate concentrations of kinase inhibitors (ML-7, ML-9, chelerythrine, or SM-1) for 1 min before the addition of phosphatase inhibitor. Cells were incubated with appropriate concentrations of the MEK antagonist PD-98059 (66) for 1 h before the addition of phosphatase inhibitor. When PKC or MLCK antibodies were used, the permeabilized cells were incubated in antiserum for 1 h at 31°C before the addition of phosphatase inhibitor (7, 55). After exposure to phosphatase inhibitors, the cells were fixed in 1% acrolein. A drop of the cell-containing medium was placed on a glass slide and covered by a coverslip, and the edges were sealed with clear nail enamel to prevent evaporation.
Cell measurements. Thirty consecutive cells from each slide were observed through a phase-contrast microscope (Carl Zeiss, Oberkochen, Germany) and a closed-circuit television camera (model WV-CD51; Panasonic, Secaucus, NJ) connected to a Macintosh computer (Apple, Cupertino, CA). The Image software program (National Institutes of Health, Bethesda, MD) was used to measure cell length and for data accumulation. The average length of 30 cells, measured in the absence of agonists, was taken as a "control" length and compared with the length after the addition of test agents. The initial length of isolated ESO and LES cells was 65 ± 2 µm (mean ± SE). "Shortening" was defined as the percentage decrease in average length after agonist treatment compared with control length.
The cell shortening model used in this study has been used extensively by different investigators in our laboratory and elsewhere. Over the years, data obtained in isolated ESO and LES cells have been consistent and reproducible. Nevertheless, the degree of isolated cell shortening is less than the shortening capacity of other smooth muscles in intact tissues, and growing appreciation of the importance of cell matrix attachment and cytoskeletal structure in smooth muscle mechanics may suggest caution in extrapolating conclusions obtained from untethered cell preparations to in vivo muscle behavior.
Measurement of PKC activity. PKC activity in immunoprecipitates from ESO was measured with the Pierce colorimetric PKC assay (no. 29510; Rockford, IL). Briefly, a peptide substrate that was labeled with brightly colored fluorescent dye was incubated with the kinase-containing sample. The reaction mixture was applied to an affinity column that binds phosphorylated peptides. To wash nonphosphorylated peptide from the membranes, the column was washed twice with phosphopeptide binding buffer. To elute the phosphorylated peptide from the affinity membranes, the membranes were washed twice with phosphopeptide elution buffer. The phosphorylated product was quantified by measurement of its absorbance at 570 nm.
Measurement of ILK activity. ESO circular smooth muscle was homogenized (3- to 20-s burst, Tissue Tearer; Biospec Products, Bartlesville, OK) in a 3- to 20-s burst in 50 mM HEPES buffer (pH 7.5) containing 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 10 µg/ml leupeptin, 2.5 µg/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride, 5 mM sodium fluoride, and 1 mM sodium orthovanadate. The sample was centrifuged with a Fisher microfuge (15,000 g, 10 min, 4°C; Pittsburgh, PA), and the supernatant was collected. An ILK antibody (Upstate Biotechnology, Lake Placid, NY) was purified by dialysis with Slide-A-Lyzer cassettes (Pierce).
ILK was immunoprecipitated from the ESO sample with the Seize primary immunoprecipitation kit (no. 45335; Pierce). Briefly, purified ILK antibody was coupled to an aminolink coupling gel in a Handee spin cup column (Pierce). The ESO sample was applied to the ILK antibody-coupled spin cup column. ILK was eluted with immunopure elution buffer, and the eluent was neutralized with 1 M Tris, pH 9.5.
ILK activity was measured by adding 50 µl of ESO sample and 50 µl of kinase reaction buffer (in mM: 50 HEPES, pH 7.0, 10 MnCl2, 10 MgCl2, 2 NaF, and 1 Na3VO4) containing 5 µCi of [-32P]ATP (BLU502A; NEN Life Sciences, Boston, MA) to two plates coated with myelin basic protein (SMP106; NEN Life Sciences). The plates were incubated for 20 min at room temperature, and the reaction was stopped by washing the plates five times with ice-cold reaction buffer. One plate was washed after 0-min incubation, and the other plate was washed after 20-min incubation. The radioactivity of the plates was measured, and the difference between the 0- and 20-min plates in counts per minute was determined.
Protein determination. Protein content of ESO tissue was determined by colorimetric assay (Bio-Rad, Melville, NY) according to the method of Bradford (8a). Briefly, samples were diluted 1,000 times in distilled water and mixed with Bio-Rad protein assay dye reagent (1:4, vol/vol) before quantification of the color change of Coomassie brilliant blue G-250 dye measured at 595 nm.
Drugs and chemicals.
We purchased microcystin-LR and PD-98059 from Calbiochem (San Diego, CA), collagenase type II and soybean trypsin inhibitor from Worthington Biochemicals (Freehold, NJ), chelerythrine from LC (Boston, MA), PKC and PKC
II antibodies from GIBCO-BRL (Gaithersburg, MD), and ERK1, ERK2, and MLCK antibodies from Santa Cruz Biotechnology (Santa Cruz, CA). [
-32P]ATP was purchased from Amersham Pharmacia Biotech (Piscataway, NJ). Okadaic acid, ML-7, ML-9, saponin, BME amino acid supplement, HEPES, creatine phosphate, creatine kinase, ATP, antimycin A, CaM, MLC, and other reagents were purchased from Sigma. SM-1 (AKKLSKDRMKKYMARRKWQKTG) was synthesized in the Peptide Synthesis Core Facility at the University of Calgary (Calgary, AB, Canada). The purity of the peptide (>95%) was confirmed by analytical high-performance liquid chromatography and amino acid analysis.
Statistical analysis. Data are expressed as means ± SE. Statistical differences between multiple groups were tested by analysis of variance (ANOVA) for repeated measures and checked for significance by performing Scheffé's F-test.
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RESULTS |
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Because ML-7 and ML-9 may not be entirely selective for MLCK (64), we used a selective anti-MLCK antibody to inhibit okadaic acid-induced contraction of permeabilized LES and ESO cells. The antibody inhibited LES cell contraction (P < 0.001, ANOVA) but had no effect on contraction of ESO cells (Fig. 3). Similarly, the effect of an MLCK inhibitor peptide (SM-1) derived from the autoinhibitory domain of MLCK was tested on contraction of permeabilized cells induced by anti-PP1c. SM-1 inhibited LES contraction in a concentration-dependent manner (P < 0.001, ANOVA) but did not affect contraction of ESO (Fig. 4). These data clearly demonstrate that contraction induced by phosphatase inhibition is MLCK dependent in LES but not in ESO circular smooth muscle cells.
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MAP kinases mediate okadaic acid- and microcystin-LR-induced contraction of ESO but not of LES. Antibodies to ERK1 and ERK2 inhibited okadaic acid (109 M)- and microcystin-LR (109 M)-induced contractions of ESO in a concentration-dependent manner (P < 0.001, ANOVA) but had no significant effect on LES contractions (Figs. 10 and 11). Similarly, the MEK antagonist PD-98059 inhibited okadaic acid- and microcystin-LR-induced contractions of ESO in a concentration-dependent manner (P < 0.001, ANOVA) but had no effect on LES contractions (Fig. 12). These data suggest that ERK1 and ERK2 and the upstream kinase MEK are involved in okadaic acid- and microcystin-LR-induced contraction of ESO but not of LES.
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ILK antibodies inhibited okadaic acid-induced contraction of ESO permeable cells (Fig. 13), and ILK activity increased in a concentration-dependent manner in response to okadaic acid (Fig. 14). Finally, the MEK inhibitor PD-98059 inhibited both okadaic acid-induced contraction (as shown in Fig. 12) and ILK activity (Fig. 15).
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DISCUSSION |
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Several kinases in addition to MLCK have been shown to be capable of phosphorylating MLC at the activating site (Ser19); however, in most instances, phosphorylation has been demonstrated with the isolated light chain, but not intact myosin, as the substrate. Nevertheless, several kinases are capable of phosphorylating intact myosin at Ser19 of MLC: CaM-dependent PKII (16), MAP kinase-activated protein kinase-2 (39), ROK (2), ILK (13), and ZIP kinase (49). Of these candidates, ILK has been shown to associate with the contractile machinery of smooth muscle and therefore is likely to be a kinase responsible for Ca2+-independent myosin phosphorylation and contraction of rat tail arterial smooth muscle in response to the phosphatase inhibitor microcystin-LR (13, 65). However, we cannot rule out the involvement of other candidate kinases.
Because MLCK activation depends on activation of CaM, which requires a high Ca2+ concentration, phosphatase inhibition has been invoked to explain contraction at low cytosolic Ca2+ levels. The link between activation of the Ca2+-independent PKC and MLC phosphorylation in the ESO, however, has not been elucidated.
Inhibitors of protein serine/threonine phosphatases are useful tools for studying signaling pathways involved in the regulation of smooth muscle contraction. Okadaic acid is a widely used phosphatase inhibitor that is more potent toward type 2A than toward type 1 phosphatase (60), while microcystin-LR is less discriminating between types 1 and 2A phosphatases (61). In the present study, we found that okadaic acid and microcystin-LR caused a similar concentration-dependent contraction of ESO and LES, consistent with the involvement of a type 2A phosphatase in the contractile responses (Fig. 1). Concentration-dependent contraction of ESO and LES was also observed in response to anti-PP1c, implicating type 1 phosphatase in the contractile responses (Fig. 2). We conclude that both types 1 and 2A phosphatases are involved in the contractile responses of ESO and LES to phosphatase inhibitors.
Phosphatase inhibitor-induced contraction of LES was mediated by activation of MLCK, since the contraction was inhibited by the selective MLCK inhibitors ML-7 and ML-9 or an antibody to MLCK (Fig. 3) in a concentration-dependent manner. These data indicate that phosphatase (MLCP) inhibition in LES induces contraction through the classic CaM-MLCK pathway. In ESO, however, contraction induced by phosphatase inhibitors was not mediated by activation of MLCK, since it was unaffected by MLCK inhibitors or anti-MLCK.
To ensure the specificity of MLCK inhibition, we used SM-1, a synthetic peptide corresponding to the autoinhibitory sequence of MLCK (residues 783804 of chicken gizzard MLCK) (65), which is the most selective inhibitor of Ca2+/CaM-dependent MLCK (21, 30, 38). As observed with the other phosphatase inhibitors, contraction induced by anti-PP1c was inhibited by SM-1 in LES but not in ESO smooth muscle cells (Figs. 4 and 8). Taken together, these data strongly support the importance of MLCK in the initial contraction of LES smooth muscle and indicate that contraction of ESO smooth muscle is MLCK independent. In the case of ESO, phosphatase inhibitors must enhance the effect of kinases other than MLCK (e.g., ILK).
Because PKC has been implicated in ESO contraction (57), we examined the effect of the PKC inhibitor chelerythrine on okadaic acid- and microcystin-LR-induced contraction (Fig. 5). Chelerythrine inhibited ESO contraction in a concentration-dependent manner but had no effect on LES contraction in response to these phosphatase inhibitors, suggesting that in ESO, the phosphatase inhibitors inhibit a phosphatase that opposes the action of PKC. We verified that the PKC isoenzyme involved was PKC
, since anti-PKC
inhibited okadaic acid- and microcystin-LR-induced contraction in ESO but not in LES (Fig. 6), whereas anti-PKC
II had no effect on phosphatase inhibitor-induced contraction in either LES or ESO (Fig. 7). To resolve possible concerns regarding the specificity of these inhibitors, we also used anti-PP1c as a contractile agent, antibodies against MLCK, PKC
, and PKC
II (as a negative control), and the highly selective MLCK peptide inhibitor SM-1 (Fig. 8). ESO contraction induced by the PP1c antibody was not affected by SM-1, anti-MLCK, or anti-PKC
II but was inhibited by anti-PKC
. LES contraction, however, was inhibited by anti-MLCK and SM-1 but was unaffected by anti-PKC
or anti-PKC
II. These data confirm that anti-PP1c-induced phosphatase inhibition contracts LES through an MLCK-dependent pathway but contracts ESO through an MLCK-independent pathway. In addition, we found that phosphatase inhibition by okadaic acid stimulates PKC
activity in ESO, as demonstrated by translocation of the enzyme activity from the cytosol to the particulate fraction (Fig. 9).
Several possible mechanisms could be responsible for PKC activation and ESO contraction in response to phosphatase inhibition. Phosphatases may reverse phosphorylation-dependent activation of PKC, PKC-dependent phosphorylation of key substrates, or both. Phosphorylation of PKC regulates the activity and subcellular localization of the enzyme (48). The kinase, which is synthesized as a soluble unphosphorylated protein (8), must be phosphorylated before it is competent to respond to second messengers (17). The first phosphorylation of PKC (conventional, novel, and atypical PKC isoforms) occurs in the activation loop and is catalyzed by the phosphoinositide-dependent protein kinase PDK-1 (15). This phosphorylation is necessary for the catalytic activity of PKC (48). Phosphorylation by PDK-1 is followed by two autophosphorylations near the COOH terminus of PKC that stabilize the structure of the active site and the regulatory domain, allowing tighter binding of substrates and cofactors (17) and increasing the phosphatase resistance of PKC (8). The mature, phosphorylated species is half-phosphorylated at the activation loop and quantitatively phosphorylated at the two COOH-terminal sites, suggesting that dephosphorylation and/or phosphorylation at the active site may regulate kinase activity in response to stimuli (48). The phosphorylations act cooperatively to maintain the active (latent) conformation. The phosphorylated species is found in the cytosol or, on activation, is associated with membranes (26). PKC signaling is desensitized through dephosphorylation and proteolysis. After activation, PKC is dephosphorylated by a membrane-bound type 2A phosphatase. Okadaic acid has been shown to inhibit phorbol ester-induced dephosphorylation of PKC in intact COS cells (25). It is possible, therefore, that contraction induced by phosphatase inhibition may result from inactivation of membrane-bound type 2A phosphatase, leading to increased phosphorylation of PKC.
Alternatively, contraction induced by phosphatase inhibition may result from an increased pool of phosphorylated PKC substrates. Phosphatases (mostly type 2A but also type 1) have been shown to dephosphorylate several PKC substrates in a variety of cell preparations (19, 22, 62). For example, in intact Swiss 3T3 cells, okadaic acid prevented the dephosphorylation of the PKC substrate MARCKS that occurs after PKC activation by bombesin (11). In SH-SY5Y cells, a protein phosphatase (possibly type 2A) with high basal activity counteracts PKC-induced phosphorylation of myelin basic protein and other PKC substrates (41). A potential PKC substrate that has been implicated in the regulation of smooth muscle contraction is CPI-17, a heat-stable protein that potently inhibits MLCP activity when phosphorylated at Thr38. Thiophosphorylated CPI-17 causes Ca2+-independent contraction in -escin-permeabilized and Triton X-100-demembranated arterial smooth muscle (14, 37, 42). Thus it is possible that phosphatase inhibitors may facilitate phosphorylation of CPI-17 by PKC by preventing its dephosphorylation.
A connection between MAP kinase activation and PKC-mediated smooth muscle contraction has been proposed for some time (33, 34). In the ferret aorta, ERK1, ERK2, and possibly p38 MAP kinase are activated in response to phenylephrine (45). In colonic smooth muscle, MAP kinase is activated during PKC-dependent contraction and cotranslocated with heat shock protein 27 (HSP27) (68). Investigators at our laboratory (10) recently showed that PKC-dependent contraction, exemplified by ACh-induced contraction of ESO, may be associated with activation of MAP kinases. Contraction in response to ACh depends on ERK phosphorylation and/or phosphorylation of the HSP27-linked p38 MAP kinase. In this study, we observed that ERK1 and ERK2 antibodies nearly abolished okadaic acid- and microcystin-LR-induced contraction of permeabilized ESO, but not LES, cells (Figs. 10 and 11). In addition, the MEK antagonist PD-98059 inhibited, in a concentration-dependent manner, okadaic acid- or microcystin-LR-induced contraction in ESO but not in LES (Fig. 12). These data suggest that phosphatase inhibitor-induced contraction of ESO, but not LES, is associated with activation of ERKs, which are likely to participate in PKC-dependent contractile pathways.
One of the intermediate proteins in the PKC-MAP kinase contractile pathway may be ILK (13). The Ca2+ independence of ILK activity makes it eligible as a candidate intermediate protein in the Ca2+-independent PKC
-mediated contraction. ILK was identified and cloned on the basis of its interaction with the
1-integrin cytoplasmic domain (24), and its catalytic domain is homologous to other protein kinase catalytic domains. ILK interacts with various proteins, several of which are connected to the actin cytoskeleton and anchor it to the extracellular matrix. Our data show a link between the PKC
-MAP kinase contractile pathway and activation of ILK, because okadaic acid-induced contraction of ESO muscle cells is inhibited by ILK antibodies, and okadaic acid causes increased ILK activity, which is inhibited by the MEK inhibitor PD-98059 (Figs. 1315). Whether activation of ILK is a direct result of phosphorylation by ERK1/2 or whether other intermediate events may be involved remains to be demonstrated.
Other PKC substrates that have been implicated in the regulation of smooth muscle contraction are calponin (67) and caldesmon (63). Phosphorylation of these proteins alleviates their inhibition of the actin-activated MgATPase activity of phosphorylated smooth muscle myosin. Investigators at our laboratory (10) previously showed that PKC-dependent contraction, exemplified by ACh-induced contraction of ESO, is mediated by activation of two distinct pathways, one dependent on activation of ERK1/2 MAP kinases, as illustrated in the present study, and a distinct one dependent on phosphorylation of the HSP27-linked p38 MAP kinase, which has been shown to involve caldesmon and calponin (29, 50). ILK may be the kinase responsible for MLC phosphorylation in the ERK1/2 pathway. The kinase responsible for MLC phosphorylation in the p38-HSP27 pathway has not yet been defined. These possible contractile signal transduction pathways are illustrated in Fig. 16.
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GRANTS |
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FOOTNOTES |
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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.
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