Neurogastroenterology & Motility Neurogastroenterol Motil (2015) 27, 135–146

doi: 10.1111/nmo.12491

ERK and p38MAPK pathways regulate myosin light chain phosphatase and contribute to Ca2+ sensitization of intestinal smooth muscle contraction E. IHARA ,*,† Q. YU ,* M. CHAPPELLAZ *

& J. A. MACDONALD *

*Smooth Muscle Research Group at the Libin Cardiovascular Institute of Alberta, Department of Biochemistry & Molecular Biology, University of Calgary, Calgary, AB, Canada †Department of Medicine and Bioregulatory Science, Kyushu University, Higashi-ku, Fukuoka, Japan

Key Messages Mitogen-activated protein kinases (MAPKs) may regulate gastrointestinal smooth muscle myosin phosphatase (MLCP) activity to influence Ca2+ sensitization and contractile force. Intestinal smooth muscle was dissected, and contractile force was measured in the presence of MAPK inhibitors. Differences in MLCP activity were observed during sustained GI smooth muscle contractions in the presence of MAPK inhibitors; however, prototypical MYPT1 and CPI-17 phosphorylations were not affected by MAPK inhibition.

Abstract Background Mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated protein kinase (ERK) and p38MAPK, are known regulators of smooth muscle contractility. The contraction of smooth muscle is mainly regulated by the phosphorylation of regulatory light chains of myosin II (LC20), which is driven by the balance between myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP). We hypothesized that one possible mechanism for MAPK-dependent modulation of intestinal smooth muscle contractility is via the regulation of MLCP activity. Methods Contractile responses to carbachol (CCh) and effects of MAPK inhibitors on CCh-induced contractions were assessed with isolated rat ileal longitudinal smooth muscle strips. Biochemical assessments of MLCP activity and myosin phosphatse targeting subunit (MYPT1) and CPI-17 phosphorylations were completed. Key Results Treatment of ileal smooth muscle with PD98059 (10 lM;

MEK inhibitor) or SB203580 (10 lM; p38MAPK inhibitor) significantly inhibited CCh-induced contractile force. Decreased MLCP activity was observed during sustained contractions induced by CCh; the MLCP activity was recovered by treatment with PD98059 and SB203580. However, MYPT1 (Thr697 and Thr855) and CPI-17 (Thr38) phosphorylations were not affected. Application of ML-7 (MLCK inhibitor) during CCh-induced sustained contraction elicited an MLCPdependent relaxation, the rate of which was accelerated by application of PD98059 and SB203580 with proportional changes in LC20 phosphorylation levels but not MYPT1 phosphorylation (Thr697 or Thr855). Conclusions & Inferences ERK and p38MAPK contribute to CCh-induced sustained contraction in a LC20 phosphorylation dependent manner. Moreover, both kinases inhibit MLCP activity possibly by a novel mechanism. Keywords calcium sensitization, ERK, ileum, intestine, mitogen-activated protein kinase, myosin phosphatase, p38MAPK, smooth muscle.

Address for Correspondence J. A. MacDonald, Department of Biochemistry & Molecular Biology, University of Calgary, HRIC GAA20, 3280 Hospital Drive NW, Calgary, AB, Canada T2N 4Z6. Tel: 403-210-8433; e-mail: [email protected] Received: 19 June 2014 Accepted for publication: 20 November 2014

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The coordinated regulation of gastrointestinal smooth muscle contractility contributes to gastrointestinal motility and the maintenance of general health and

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wellness.1–4 Alterations in motility and resultant changes in transit can contribute to abdominal pain, intestinal cramping, constipation, and/or diarrhea, which in turn can lead to systemic disease. Gastrointestinal smooth muscle possesses distinct properties that distinguish it from other types of visceral and vascular smooth muscle.5–8 Smooth muscle of the distal stomach, small intestine, and colon exhibits variable (phasic) tone on which are superimposed rhythmic contractions. Slow-waves of membrane depolarization and repolarization originate in pacemaker cells and are transmitted to the smooth muscle cells. The depolarization of SMCs primarily reflects activation of voltage-gated Ca2+ channels, resulting in Ca2+ entry and contraction. Concurrent stimulation of rhythmic smooth muscle by excitatory neurotransmitters elicits further depolarization and Ca2+ entry and activates intracellular signaling cascades that result in transient increases in intracellular Ca2+ concentration ([Ca2+]i). The contractile properties of smooth muscle are mainly regulated by the phosphorylation of regulatory light chains (LC20) of myosin II.9 To initiate contraction, the increase in [Ca2+]i activates myosin light chain kinase (MLCK), a Ca2+/calmodulin-dependent enzyme. MLCK phosphorylates LC20 on Ser-19, resulting in contraction of smooth muscle through increases in myosin ATPase activity and cross-bridge cycling. Myosin light chain phosphatase (MLCP) is responsible for the dephosphorylation of LC20 resulting in relaxation of smooth muscle.10,11 Although [Ca2+]i is the primary determinant of smooth muscle contraction, it is the balance between MLCK and MLCP activities that ultimately dictates the contractile tone of smooth muscle. Inhibition of MLCP that occurs independent from any alteration in [Ca2+]i leads to an increase in both LC20 phosphorylation and contractile force development. The activity of MLCP is central to the phenomenon of Ca2+ sensitization and can be regulated by G protein-coupled signaling pathways.8,12 Ca2+ sensitization can be mediated directly by phosphorylation of the myosin phosphatase targeting subunit of MLCP (MYPT1) and/or indirectly by phosphorylation of a protein kinase C (PKC)-potentiated phosphatase inhibitor protein17 kDa (CPI-17). Although several possible phosphorylation sites on MYPT1 have been reported, the Thr697 and Thr855 sites (numbering of the rat isoform) are the most extensively studied and have been confirmed to be important for the inhibition of MLCP activity.10,11 Likewise, phosphorylation of CPI-17 at Thr38 is the most extensively studied and can potentiate its inhibition of MLCP.13

Accumulated evidence has shown that mitogenactivated protein kinase (MAPK) pathways contribute to smooth muscle contraction.5,14–21 We have demonstrated that ERK1/2 and p38MAPK are important mediators not only of Ca2+ sensitization in normal intestinal smooth muscle,22 but also of hypercontractile responses observed in inflamed intestinal smooth muscle.23 Nevertheless, it has yet to be determined how MAPK signaling pathways contribute to smooth muscle contraction. The phosphorylation of caldesmon is thought to be one mechanism.20,24 Alternatively, p38MAPK can phosphorylate heat shock protein (HSP) 27 and contribute to smooth muscle contraction.6,25 Another possible mechanism by which MAPKs contribute to smooth muscle contraction is through the direct control of MLCP activity. In the present study, we examined whether MAPK signaling pathways are involved in the regulation of MLCP in rat intestinal smooth muscle.

MATERIALS AND METHODS Materials All chemicals were reagent grade unless otherwise indicated. PD98059, SB203580, GF109203x, carbachol, methoctramine, 4diphenylacetoxy-N-methylpiperidine-methiodide (4-DAMP), and ML-7 were obtained from Sigma (St. Louis, MO, USA). Polyclonal MYPT1 antibody generated against the N-terminal fragment of rat MYPT1 was a gift from Dr. Timothy Haystead (Duke University, Durham, NC, USA). Polyclonal antibodies specific for MYPT1 phosphorylated either at Thr697 (anti-[phospho-Thr697]-MYPT1; rat numbering) or Thr855 (anti-[phospho-Thr855]-MYPT1; rat numbering) and for CPI-17 were purchased from Upstate (Charlottesville, VA, USA).

Force measurement of rat ileal longitudinal smooth muscle strips Ileum was removed from male Sprague–Dawley rats anesthetized and euthanized according to protocols approved by the University of Calgary Animal Care and Use Committee. Ileal smooth muscle sheets were dissected and cut into longitudinal smooth muscle strips (250 lm 9 2 mm), and distal colonic smooth muscle sheets were cut into circular smooth muscle strips. For force measurements, muscle strips were tied with silk monofilaments to the tips of two fine wires, one of which was connected to a force transducer (SensoNor, AE801). Strips were stretched in the longitudinal axis until the resting force reached to 0.1 mN. Then, the strips were equilibrated for 30 min in normal extracellular solution (NES) containing 150 mM NaCl, 4 mM KCl, 2 mM calcium methanesulfonate (CaMS2), 1 mM magnesium methanesulfonate (MgMS2), 5.5 mM glucose, and 5 mM 4-(2hydroxyethyl) piperazine-1-ethanesulfonic acid, pH 7.3. Contraction in response to a 118 mM K+ extracellular solution (KES) was used to assess muscle quality. Force levels observed with NES and carbachol (CCh)-induced sustained contractions were designated as 0% and 100% respectively. All contractile mea-

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surements were carried out at room temperature (23 °C) with a computerized data acquisition system (PowerLab/8SP data recording unit and Chart software; ADInstruments, Colorado Springs, CO, USA).

lated proteins. The stoichiometries of LC20 and CPI-17 phosphorylation were calculated from the following equation: mol of Pi/mol protein = (y + 2z)/(x + y + z), where x, y, and z are the signal intensities of un-, mono-, and diphosphorylated protein bands respectively.

Measurement of MLCP activity

Data analysis

Samples of rat ileal smooth muscle strips were flash-frozen in liquid nitrogen and lyophilized overnight. MLCP activity in extracts of intact smooth muscle was assayed as described previously with some modifications.26 A lyophilized strip was homogenized in ice-cold homogenizing buffer (200 lL/strip of 20 mM Tris-HCl, pH 7.5 with 50 mM NaCl and protease inhibitors). Phosphatase assays were initiated by addition of 32Plabeled myosin light chain and terminated with ice-cold 25% (w/ v) trichloroacetic acid. BSA was added as a carrier protein, and the reactions were centrifuged to precipitate the proteins. After the pellets were washed extensively, 32P-phosphate release was measured by scintillation counting. Preliminary assays were completed to ensure linearity with respect to time. MLCP activity was expressed as 32P-phosphate released per min/mg protein and normalized to the activity measured in the absence of MAPK inhibitors.

All data are expressed as the mean  SEM. The Student’s t-test (two-tailed) was used to determine statistical significance between two groups with p < 0.05 considered to be significant.

RESULTS ERK1/2 and p38MAPK contribute to CChinduced contraction of intestinal smooth muscles Application of CCh to ileal smooth muscle strips induced a phasic contraction. The maximal force levels were obtained approximately 1 min after CCh stimulation and gradually declined to reach a sustained level within 15 min (Fig. 1A). The contraction induced by a second application of CCh was not significantly different from the first contraction, such that the first CChinduced contraction could be used as a reference response. The force level of the second CCh-induced sustained contraction at 15 min was 100.9  1.5% (n = 5), assigning the first CCh-induced sustained contraction to be 100%. It is possible that error exists as a result of “run-down” in contractile efficiency with the second contractile stimulus. In our experience, the contractile force generated with the first and second application of CCh under control conditions is highly reproducible and generally varies by less than 5%; however, the KCl responses exibit more variability among ileal smooth muscle strips. Interestingly, Y27632 (10 lM), a Rho-associated kinase (ROK) inhibitor, did not significantly inhibit CCh-induced contraction of rat ileal smooth muscle (Fig. 1B). Pretreatment with either PD98059 or SB203580 significantly inhibited CCh-induced contractile force (Fig. 1C, D, and H). The forces of the second CCh-induced sustained contraction at 15 min in the presence of 10 lM PD98059 and 10 lM SB203580 were 48.7  2.1% (n = 5) and 68.8  4.5% (n = 5) respectively. Furthermore, pretreatment with 4-DAMP (100 nM), but not methoctramine (100 nM), abolished CCh-induced contraction (Fig. 1E, F, and H), indicating that CCh-induced contraction of rat ileal smooth muscle was mediated by an M3 muscarinic receptor. Finally, GF109203x (10 lM), a broad-specificity PKC inhibitor, also significantly suppressed the CCh-induced sustained contraction (Fig. 1G and H; 72.3  2.5%, n = 5). Similar experi-

Western blot analysis of MYPT1 phosphorylation Ileal smooth muscle strips were flash frozen at indicated conditions by immersion in a dry-ice/acetone solution containing 10% (w/v) trichloroacetic acid and 10 mM DTT. The muscle strips were washed with an acetone solution containing 10 mM DTT and lyophilized overnight. Muscle proteins were extracted in a buffer containing 1% (w/v) SDS, 30 mM TrisHCl, pH 6.8, 12.5% (v/v) glycerol and APMSF with a glass-glass hand homogenizer. Proteins were resolved on 10% SDS-PAGE gels and then transferred to polyvinylidenedifluoride (PVDF) membranes. The blots were blocked with 5% (w/v) nonfat dry milk and then incubated with primary antibody (1 : 1000 dilution) in 25 mM Tris-HCl, 150 mM NaCl and 0.05% (v/v) Tween-20 (TBST) with 1% (w/v) nonfat dry milk. Blots were developed with Supersignal WestFemto-enhanced chemiluminescence (Pierce Chemical, Rockford, IL, USA). The bands were quantified by densitometry, and the relative phosphorylation levels were expressed as a function of the density of the total MYPT1 protein.

Measurement of LC20 and CPI-17 phosphorylation The phosphorylation status of LC20 and CPI-17 was examined with PhosTag SDS-PAGE gels as described previously.27 Lyophilized ileal smooth muscle strips were prepared as described for MYPT1 western blot analyses. Muscle proteins were extracted with a ground glass hand homogenizer in a buffer containing 1% (w/v) SDS, 30 mM Tris-HCl, pH 6.8, 12.5% (v/v) glycerol and APMSF. PhosTag ligand (final concentration, 30 lM) and MnCl2 (final concentration, 60 lM) were added to the separating-gel (10% polyacrylamide) before polymerization.28 After electrophoresis, gels were soaked in respective transfer buffer without methanol (25 mM Tris-HCl, 192 mM glycine, 2 mM EDTA, pH 8.3 for LC20, and 10 mM cyclohexylaminopropane sulfonic acid, 2 mM EDTA, pH 11 for CPI-17) and transferred to PVDF membranes. Western blotting was carried out with a polyclonal antibody that detected both phosphorylated and unphosphory-

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Figure 1 Effects of protein kinase inhibitors and selective muscarinic receptor antagonists on carbachol (CCh)-induced contraction of rat ileal longitudinal smooth muscle. Representative recordings of carbachol (10 lM)-induced contraction in the absence (A) or presence of 10 lM Y27632 (B), 10 lM PD98059 (C), 10 lM SB203580 (D), 100 nM methoctramine (E), 100 nM 4-DAMP (F), or 10 lM GF109203x (G) are shown. These inhibitors were applied to the strips 10 min before and during the second CCh-induced contraction. In (H), cumulative results are representative of n = 5 independent experiments. The force level of the first CCh-induced sustained contraction was set to be 100%. *- significantly different from vehicle control with Student’s t-test, p < 0.05; n.s.- not significantly different. Error bars indicate SEM.

(15 min) phases of CCh-induced contractions and then measured MLCP activities. As shown in Fig. 2, the activity of MLCP was not altered at 1 min after application of CCh (maximal contraction) but was decreased at 15 min (sustained contraction). In the latter condition, MLCP activity was decreased to 60.0  8.0% (n = 5) of the MLCP activity at rest. Interestingly, MLCP activity was recovered when MAPK inhibitors, PD98059 (10 lM) or SB203580 (10 lM), were applied to the strips. MLCP activities were 82  8% (n = 5) at 1 min and 177  39% (n = 5) at 15 min after application of CCh in the presence of PD98059 and 198  54% (n = 5) at 15 min in the presence of SB203580. These findings indicate that

ments were also carried out with colonic circular smooth muscle strips. In this case, the contribution of protein kinase and muscarinic receptor signaling to muscle contractility were comparable to those obtained for ileal smooth muscle (Fig. S1).

MLCP activity during CCh-induced sustained contraction is attenuated by MAPK inhibitors As described previously, the objective of this study was to determine whether MAPK pathways contributed to Ca2+ sensitization of intestinal smooth muscle via MLCP regulation. We quickly flash-froze ileal smooth muscle strips at basal, peak (1 min), and sustained

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was not mediated by MYPT1 phosphorylation at either of the two canonical inhibitory sites.

ERK1/2 and p38MAPK inhibition influences CPI17 phosphorylation at peak but not sustained CCh-induced contraction We next examined whether the phosphorylation of CPI-17 was affected by ERK1/2 and p38MAPK during CCh-induced contractions. The results of PhosTag SDS-PAGE gels revealed an increase in CPI-17 phosphorylation during CCh-induced contractions (Fig. 4A); two bands were seen for basal conditions, and three bands were observed for CCh-induced contractions. The two upper bands collapsed into a single lower band upon removal of Mn2+ from the PhosTag gel, indicating that the upper and middle bands correspond to diphosphorylated and mono-phosohorylated forms of CPI-17 respectively. As the phosphorylation of Thr38 is considered to play the most important role in regulating CPI-17 activity,13 we confirmed that the upper, but not middle, band was associated with phosphorylation of CPI-17 at Thr38 by re-blotting the stripped PVDF membrane with an anti[phospho-Thr38]-CPI-17 antibody (data not shown). The stoichiometry of CPI-17 phosphorylation at rest (0.16  0.04, n = 3) was significantly increased at 1 min (0.56  0.01, n = 3) and 15 min (0.46  0.07, n = 3), indicating that CPI-17 was activated during CCh-induced contraction in rat ileal smooth muscle. Interestingly, treatment with PD98059 or SB203580 increased phosphorylation levels of CPI-17 when compared to control (no inhibitors) at the peak (1 min) of CCh-induced contraction. However, neither PD98059 nor SB203580 influenced the phosphorylation of CPI17 at the sustained phase (15 min) of CCh-induced contraction (Fig. 4B and C). These results suggest that ERK1/2 and p38MAPK pathways did not influence CPI-17 inhibitory potential during sustained contractions of ileal smooth muscle with CCh.

Figure 2 Effects of MAPK inhibitors on MLCP activity during carbachol (CCh)-induced contraction of rat ileal smooth muscle. Smooth muscle strips were flash frozen and collected at rest, peak (1 min) or sustained phase (15 min) of CCh (10 lM)-induced contraction in presence of 10 lM PD98059 or 10 lM SB203580 and in the absence of these inhibitors (control). The collected strips were then used for measurement of MLCP activity as described in methods. The MLCP activity at rest with no inhibitors was set to be 1. *significantly different from vehicle control at rest; #- significantly different between indicated two groups; n.s.- no significant difference between groups. Student’s t-test, p > 0.05. Error bars indicate SEM. The cumulative results are representative of five independent experiments for measurement of MLCP activity.

ERK1/2 and p38MAPK pathways were involved in MLCP inhibition and Ca2+ sensitization of ileal smooth muscle.

ERK1/2 and p38MAPK effects on MLCP activity were not associated with changes in MYPT1 phosphorylation of Thr697 or Thr855 inhibitory sites As MLCP activity is commonly regulated directly by phosphorylation of MYPT1 or indirectly via phosphorylation of CPI-17, we next examined whether the phosphorylation of MYPT1 and/or CPI-17 during CChinduced contractions were affected by ERK1/2 or p38MAPK. Basal phosphorylation of MYPT1 at Thr697 was observed, but this phosphorylation was not significantly altered at 1 min or 15 min after CCh application (Fig. 3A and B). Furthermore, treatment with neither PD98059 (10 lM) nor SB203580 (10 lM) had any effect on Thr697 phosphorylation. Alternatively, MYPT1 phosphorylation at Thr855 was significantly increased during CCh stimulation (Fig. 3A and C) with the levels reaching 225  18% (n = 4) at 1 min and 222  24% (n = 4) at 15 min after CCh application. Treatment with PD98059 or SB203580 also had no effect on the phosphorylation of MYPT1 at Thr855 (Fig. 3A and C). Taken together, the phosphorylation of Thr855, but not Thr697, was associated with Ca2+ sensitization during CCh-induced contraction of rat ileal smooth muscle. However, the ERK1/2- and p38MAPK-dependent modulation of MLCP activity

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ML-7-induced relaxations of carbachol-induced sustained contractions were accelerated by addition of MAPK inhibitors To further confirm ERK1/2 and p38MAPK signaling pathways regulate the Ca2+ sensitization of MLCP activity during CCh-induced sustained contractions, we applied ML-7, an MLCK inhibitor, to ileal strips once sustained contractile force was developed (i.e., 15 min after CCh application). In this case, the contractile force gradually decreased as MLCK-dependent LC20 phosphorylation was blocked (Fig. 5A). The

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Figure 3 Effects of ERK and p38MAPK on the phosphorylation levels of MYPT1 during carbachol (CCh)-induced contraction of rat ileal smooth muscle. Smooth muscle strips were flash frozen and collected at rest, peak (1 min), or sustained phase (15 min) of CCh (10 lM)-induced contraction in presence of 10 lM PD98059 or 10 lM SB203580 and in the absence of the inhibitors (control). The collected strips were then subjected to a western blot analysis for MYPT1. In (A), a representative blot of MYPT1 phosphorylation levels at Thr697 and Thr855 is provided. The cumulative results are representative of four independent experiments for effects of PD98059 or SB203580 on MYPT1 phosphorylation levels at Thr697 (B) or Thr855 (C). The bands were quantified by densitometry, and the relative phosphorylation levels were expressed as a function of the total MYPT1 protein. The MYPT1 phosphorylation level at rest with no inhibitors (control) was set to be 1. *- significantly different from control at rest; n.s.- no significant difference between the indicated groups. Student’s t-test, p > 0.05. Error bars indicate SEM.

MYPT1 phosphorylation at Thr697 or Thr855 (Fig. 5F), although the phosphorylation of Thr855 was reduced at 30 min after CCh stimulation when compared to the levels found with CCh stimulation. The MYPT1 phosphorylation levels at Thr855 at 45 min in the presence of ML-7, PD98059 plus ML-7 and SB203580 plus ML-7 were 0.69  0.08 (n = 5), 0.58  0.07 (n = 5), and 0.83  0.10 (n = 5) respectively.

ML-7-induced relaxation was attributed to MLCP activity, which was revealed by administration of ML-7. Interestingly, the ML-7-induced relaxation was significantly accelerated by addition of either PD98059 or SB203580 (Fig. 5B–D). The contractile forces measured 30 min after application of ML-7 in the presence of PD98059 ( 2.9  3.7%, n = 4) or SB203580 (7.5  1.7%, n = 4) were significantly lower than those observed in the absence of MAPK inhibitors (17.1  1.3%, n = 4). The accelerated relaxations observed in the presence of both PD98059 and SB203580 were associated with proportional decreases in LC20 phosphorylation (Fig. 5E). Namely, the stoichiometry of LC20 phosphorylation at 15 min after CCh application (0.32  0.02, n = 6; just before the application of ML-7) was significantly decreased (0.19  0.02%, n = 6) at 45 min after CCh application in the presence of ML-7 (after treatment with ML-7 for 30 min). This value (0.19  0.02%, n = 6) was significantly higher than those with PD98059 (0.066  0.02%, n = 6) or SB203580 (0.12  0.09%, n = 6) (Fig. 5E). Furthermore, the phosphorylation of MYPT1 at both inhibitory sites was measured. The phosphorylation of Thr697 was not affected by the addition of MAPK inhibitors with ML-7 (Fig. 5F). The inclusion of PD98059 or SB203580 along with ML-7 did not have any additional effects on

DISCUSSION While it is known that MAPKs can influence smooth muscle contractility by altering thin filament dynamics and the ATPase activity of myosin II via caldesmon21,29 and/or HSP27,17,25 the comprehensive mechanism by which these MAPKs regulate contraction of the phasic smooth muscles (i.e., intestine) is not understood. Several reports have provided evidence for the regulation of tonic smooth muscle contractility by ERK and p38MAPK pathways. The ERK pathway was involved in phenylephrine-induced contraction of uterine artery18 and aorta,30 and in the angiotensin IIinduced contraction of lower esophageal sphincter.16,17 The p38MAPK pathway has been shown to contribute to angiotensin II- and endothelin-induced contraction of aortic smooth muscle cells14,19 and noradrenaline-

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Now, our findings also support a novel role for ERK and p38MAPK in the Ca2+ sensitization of intestinal smooth muscle through actions on MLCP activity. It is widely accepted that the Ca2+ sensitization of smooth muscle contractile force is dependent upon MLCP activity, which in turn is regulated by phosphorylation of MYPT1.10,11 There are two well-characterized inhibitory phosphorylation sites on MYPT1 (Thr697 and Thr855) that are known to suppress MLCP activity, and recent evidence supports differential roles for both.33–37 The phosphorylation of Thr697 appears to provide selective autoinhibition of MLCP activity33 whereas a variety of reports suggest that Thr855 phosphorylation influences the intracellular location and the stability of the MLCP-myosin complex.34–37 Neither ERK nor p38MAPK have been reported to directly phosphorylate MYPT1, and the sequences surrounding the Thr697 and Thr855 residues do not possess the necessary Ser/Thr-Pro consensus motif that is integral to the MAPK substrate-recognition mechanism. However, Matsumura et al. demonstrated that MYPT1 was phosphorylated at multiple sites (Ser432, Ser473, & Ser601) during mitotic events by the proline-directed cdc2 kinase,38,39 and these authors also suggested that MAPKs could phosphorylate these cdc2 sites. We have observed MAPK-dependent changes on the CCh-induced, sustained contractions of rat ileal smooth muscle which were associated with changes in MLCP activity and LC20 phosphorylation. These effects were not associated with any observable effect on the phosphorylation of MYPT1 at either the Thr697 or Thr855 inhibitory site. So, it will be important to establish whether MAPKs can directly phosphorylate MYPT1 and how this putative event might impact upon MLCP activity. Currently, the data do not support any action of MAPKs on the unique functionalities of the two inhibitory sites of MYPT1. In this regard, MAPKs did not appear to influence the spontaneous phosphorylation of MYPT1 at Thr697 under basal conditions, and hence the autoinhibitory capacity of phosphorylated MYPT1 appears to be unaffected. Moreover, the sensitivity of Thr855 phosphorylation during agonist stimulus of isolated ileal smooth muscle was also unaffected by application of MAPK inhibitors. The inhibition of MLCP activity by MAPKs was identified within the sustained phase of CCh-induced contractions. Contractile force was also reduced in the early phasic contractile response (i.e., CCh, 1 min) following application of SB203580 and PD98059; however, no significant MAPK-dependent change in MLCP activity was measured in this case. While beyond the scope of this study, MAPK pathways

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Figure 4 Analysis of ERK and p38MAPK effects on CPI-17 phosphorylation during carbacol (CCh)-induced contraction of rat ileal smooth muscle. Smooth muscle strips were flash frozen and collected at rest (NES), peak (1 min), or sustained phase (15 min) of CCh (10 lM)-induced contraction. The collected strips were then subjected to a western blot analysis for CPI-17 following separation by PhosTag SDS-PAGE (A). Only a single lower band was present upon removal of Mn2+, indicating that the middle and upper bands corresponded to mono- and di-phosphorylated forms of CPI-17 respectively. In (B), a representative blot of CPI-17 phosphorylation levels by Phos-Tag SDS-PAGE is provided for tissues samples collected at rest (NES), peak (1 min), or sustained phase (15 min) of CCh (10 mM)-induced contraction in the absence (control) or presence of 10 lM PD98059 and 10 lM SB203580. In (C), cumulative results are provided for three independent analyses of CPI-17 phosphorylation. Different exposure times were used for quantification to ensure that signals lay within the linear range of detection. Phosphorylation status of CPI-17 was assessed by stoichiometry, which was calculated from the following equation: mol of Pi/mol protein = (y + 2z)/(x + y + z), where x, y, and z are the signal intensities of un-, mono-, and diphosphorylated protein bands respectively. *- significantly different from control in the same condition. #- significantly different between the indicated groups; n.s.no significant difference from control in the same condition. Student’s t-test, p < 0.05. Error bars indicate SEM.

induced contraction of mesenteric artery.15 While these reports have revealed a role for MAPKs in the contractile function of tonic (i.e., vascular and sphincter) smooth muscles, recent data have indicated that MAPKs are also important contributors to phasic intestinal smooth muscle contractility.21–23,31,32

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Figure 5 Effects of ERK and p38MAPK inhibition on Ca2+ sensitization of rat ileal smooth muscle during ML-7-dependent relaxation from a carbachol (CCh)-induced contraction. Representative recordings are provided for the relaxant effects of the MLCK inhibitor (ML-7, 10 lM) on ileal longitudinal smooth muscle strips. ML-7 was applied to strips that were precontracted with CCh (10 lM, 15 min). Muscle strips were incubated in the absence (A) or presence of SB203580, 10 lM (B) and PD98059, 10 lM (C). Cumulative results for n = 4 independent experiments are provided in (D). *- significantly different from vehicle control (without PD98059 or SB203580) at the same time point; Student’s t-test, p < 0.05. In (E), a representative blot and the cumulative results of LC20 phosphorylation analysis by PhosTag SDS-PAGE are provided. Different exposure times were used to ensure that signals lay within the linear range of densitometry. LC20 phosphorylation stoichiometry was calculated as mol of Pi/mol protein = y/(x + y), where x and y are the signal intensities of un- and mono- protein bands respectively. No LC20 diphosphorylation was detected. In (F), a representative blot and the cumulative results are provided for the MYPT1 phosphorylation levels at Thr697 or Thr855. The bands were quantified by densitometry, and the relative phosphorylation levels were expressed as a function of the density of the total MYPT1 signal. The MYPT1 phosphorylation status at a CCh-induced sustained contraction before the addition of ML-7 was set to be 1. Error bars indicate SEM, n = 5. #- significantly different from carbachol-treated muscle; $- significantly different from ML-7-treated muscle; Student’s t-test, p < 0.05.

may also contribute to a Ca2+-dependent MLCK pathway. In other tissue types, it was shown that ERK1/2 could potentiate L-type Ca2+ conductances.40 Furthermore, it was also reported that neuronal Ntype Ca(v)2.2 calcium channels have ERK consensus phosphorylation sites, and the modulation of neuronal N-type voltage-dependent calcium channel activity might involve phosphorylation of Ca(v)2.2alpha1 by ERK.41

The phosphorylation of MYPT1 by MAPKs might not directly influence activity but could modulate the association of regulatory/scaffold proteins with the MLCP complex. This situation is thought to account for the antagonism observed between MYPT1 phosphorylation by cdc2 kinase at Ser473 and the signalling of polo-like kinase (PLK1) during cell division.39 The phosphorylation of MYPT1 by cdc2 kinase creates a binding motif that mediates MLCP-PLK1 complex

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tial of phospho-Thr38 or provide a signal to influence the intracellular targeting of CPI-17. Intriguingly, the MAPK-dependent elevations in P2-CPI-17 phosphorylation was associated with decreased force development (Fig. 1) yet no apparent change in MYPT1 inhibitory phosphorylation (Fig. 3) or MLCP activity (Fig. 2). Thus, we might speculate that MAPKs have dual effects on contractile force development that are temporally distinct; in the early phasic response via CPI-17 and in the sustained tonic response via MYPT1/ MLCP. Unfortunately, we could not determine how MAPK signaling pathways regulate MLCP activity in this study, but this unknown mechanism might work in both phasic and tonic responses via direct and indirect pathways. While we show minimal effects of ROK inhibition by the application of Y27632 on rat ileal force development here and previously,22 other studies have demonstrated a decrease in gastrointestinal smooth muscle force following application of Y27632, including a study of rat colon.48 One significant distinction between this earlier study and our own, is the use of native vs a-toxin permeabilized smooth muscle strips. Additionally, the particular strain of mouse may influence the degree to which ROK contributes to CCh-induced contraction of gastrointestinal tissues; for example, we have found significant reductions in force development in the longitudinal ileal smooth muscle of the 129 strain (data not presented) whereas minimal involvement of ROK in Ca2+ sensitization of ileal circular muscle from the Balb/c mouse was observed.23 Our additional investigations with other inhibitors (e.g., H-1152, data not shown) also suggest minimal involvement of ROK during CCh-induced contractions of rat ileal smooth muscle. A variety of kinase modules are known to provide inhibition of MLCP to increase both LC20 phosphorylation and contractile force development in smooth muscle, including integrin-linked kinase (ILK)49 and ZIPK.26 Recently, a novel small molecule inhibitor of ZIPK (i.e., HS38) was shown to reduce the contractile force and LC20 phosphorylation during CCh-dependent stimulation of a-toxin permeabilized rabbit ileal smooth muscle.50 As the application of HS38 provided a 30% reduction in the sustained contractile force, these results suggest ZIPK may also be as significant contributor to intestinal smooth muscle Ca2+ sensitization. G-protein-coupled receptors (GPCRs) can modulate ERK activity through the activation of nonreceptor type tyrosine kinases (e.g., Src, Fyn, Pyk2) or through the transactivation of growth factor receptor tyrosine kinases.51,52 However, the mechanisms by which

formation and prohibits downstream signaling of PLK1. The MAPK-dependent phosphorylation of MYPT1 has the potential to influence at least three scaffolds that are implicated in the coordinated management of MLCP activity: (i) the prostate apoptosis response (Par)-4; (ii) the myosin phosphatase Rhointeracting protein (M-RIP); and (iii) 14-3-3b. In the case of Par-4, its binding to MYPT1 was proposed to block access of kinases (such as zipper-interacting protein kinase [ZIPK]) to the inhibitory Thr697 site. Inhibitory phosphorylation of MYPT1 then requires ‘unlocking’ by phosphorylation (at Thr155 of Par-4) and displacement of Par-4 from the MLCP complex.42,43 Other groups have demonstrated a role for M-RIP in the regulation of MYPT1 phosphorylation in smooth muscle. In this case, M-RIP provides a scaffolding role to direct ROK-dependent phosphorylation of Thr855.44–46 In addition, the binding of 14-3-3b to MYPT1 may dissociate MLCP from myosin II and attenuate MLCP activity.47 MYPT1 phosphorylation at Ser472 was critical for the binding to 14-3-3 with epidermal growth factor (EGF) stimulation increasing both Ser472 phosphorylation and the binding of 14-3-3 to MYPT1. Finally, the phosphorylation of MYPT1 by MAPKs may promote the association of additional phosphatase activities to prevent accumulation of Thr697 and Thr855 phosphorylation or alternatively to influence MLCP localization. For example, changes in the localization of MYPT1 were associated with the inhibition of protein phosphatase-type 2A in HepG2 cells.35 These mechanisms, while still speculative, do offer attractive possibilities for novel MAPK-dependent regulation of MLCP and Ca2+ sensitization in the intestinal smooth muscle beds. Myosin light chain phosphatase activity can also be regulated indirectly by phosphorylation of the inhibitor protein, CPI-17.13 Our results show a discrepancy between total MLCP activity and CPI-17 phosphorylation state in the presence of MAPK inhibitors; this was most apparent at 1 min following stimulation with CCh. The phosphorylation of CPI-17 in the presence of MAPK inhibitors at 1 min was significantly higher than that in the absence (control); however, such a difference was not seen at 15 min after CCh stimulation (Fig. 4). It is important to note that the increase in CPI-17 phosphorylation at 1 min was generally associated with an increase in the P2-CPI-17 species. We are unable to define what additional CPI-17 phosphorylation event occurred coincident to the phosphorylation of Thr38 and what effect this dual phosphorylation might have on the biological function of CPI-17. Indeed, it is possible that the second phosphorylation may attenuate the inhibitory poten-

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appear to operate together to provide important regulation of intestinal smooth muscle Ca2+ sensitization and contractility.

GPCRs transactivate tyrosine kinases are heterogeneous and depend upon the particular complement of signaling molecules expressed within a given cell type. The muscarinic M3 receptor (a GPCR) is known to mediate carbachol-directed contraction of the ileum and other smooth muscle tissues,53 and we have demonstrated herein that ERK-dependent contraction of rat ileum and colon was ablated with M3 blockade. Previous reports54,55 indicate that the M3 receptor can be coupled to ERK via an EGF-receptor transactivation process. Alternatively, M3 receptors can increase [Ca2+]i and activate PKC through the Gq/a-PLCb pathway.51 Given that we found PKC and MAPKs to operate together22 in the regulation of intestinal smooth muscle contractility, ERK-induced contractile effects may not occur via EGF-receptor transactivation. The signaling of M3-receptor to ERK could also involve PKC-dependent pathways.54,55 For example, PKCd can activate CPI-17 through direct phosphorylation of Thr38, and PKCe can activate Ca2+-independent kinases, such as ZIPK and ILK,6 which in turn can modulate MLCP activity by phosphorylation of canonical inhibitory sites. Data from our lab have shown that conventional protein kinase C isoforms (PKCa, b, and c) can regulate MLCP in intestinal smooth muscle.27 Taken together, PKC, ERK, and p38MAPK pathways

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ACKNOWLEDGEMENTS E.I. was recipient of a Uehara Memorial Foundation Fellowship (Japan) and Canadian Association of Gastroenterology/Canadian Institutes of Health Research/AstraZeneca Fellowship. J.A.M. is recipient of a Canada Research Chair (Tier II) in Smooth Muscle Pathophysiology and a Senior Scholarship from Alberta Innovates – Health Solutions.

FUNDING This work was supported by grants from the Canadian Institutes of Health Research and the Crohn’s and Colitis Foundation of Canada.

DISCLOSURE No competing interest declared.

AUTHOR CONTRIBUTION EI, MC, and QY performed the research; EI and JAM designed the research study, analysed the data and wrote the article.

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SUPPORTING INFORMATION Additional supporting information may be found in the online version of this article at the publisher’s web site: Figure S1. Effects of protein kinase inhibitors and selective muscarinic receptor antagonists on carbachol (CCh)induced contraction of rat colonic circular smooth muscle.

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