Inhibition of the p38 MAP kinase pathway destabilizes smooth muscle ...

Am J Physiol Lung Cell Mol Physiol 282: L1117–L1121, 2002; 10.1152/ajplung.00230.2000.

Inhibition of the p38 MAP kinase pathway destabilizes smooth muscle length during physiological loading OREN J. LAKSER,1,2 ROBERT P. LINDEMAN,1,2 AND JEFFREY J. FREDBERG1 1 Physiology Program, Harvard School of Public Health; and 2 Boston Children’s Hospital, Boston, Massachusetts 02115 Received 19 July 2000; accepted in final form 2 January 2002

Lakser, Oren J., Robert P. Lindeman, and Jeffrey J. Fredberg. Inhibition of the p38 MAP kinase pathway destabilizes smooth muscle length during physiological loading. Am J Physiol Lung Cell Mol Physiol 282: L1117–L1121, 2002; 10.1152/ajplung.00230.2000.—We tested the hypothesis that mechanical plasticity of airway smooth muscle may be mediated in part by the p38 mitogen-activated protein (MAP) kinase pathway. Bovine tracheal smooth muscle (TSM) strips were mounted in a muscle bath and set to their optimal length, where the active force was maximal (Fo). Each strip was then contracted isotonically (at 0.32 Fo) with ACh (maintained at 10⫺4 M) and allowed to shorten for 180 min, by which time shortening was completed and the static equilibrium length was established. To simulate the action of breathing, we then superimposed on this steady distending force a sinusoidal force fluctuation with zero mean, at a frequency of 0.2 Hz, and measured incremental changes in muscle length. We found that TSM strips incubated in 10 ␮M SB-203580-HCl, an inhibitor of the p38 MAP kinase pathway, demonstrated a greater degree of fluctuation-driven lengthening than did control strips, and upon removal of the force fluctuations they remained at a greater length. We also found that the force fluctuations themselves activated the p38 MAP kinase pathway. These findings are consistent with the hypothesis that inhibition of the p38 MAP kinase pathway destabilizes muscle length during physiological loading.

namics can explain much of the force and stiffness inhibition (4), it is thought that important changes in the cytoskeleton are also induced by changes of the muscle load in time (8, 21). Here we have investigated the hypothesis that activation of p38 mitogen-activated protein (MAP) kinase may modulate muscle mechanical responses to imposed load fluctuations during contractile stimulation. We show that the p38 MAP kinase inhibitor SB-203580-HCl increased the degree of smooth muscle lengthening induced by load fluctuations. As such, these data suggest that activation of p38 MAP kinase stabilizes airway smooth muscle subjected to dynamic loading conditions that approximate those that prevail in vivo. Moreover, these data suggest that associated mechanical stresses activate the p38 MAP kinase pathway. METHODS

by reversible airway obstruction, airway inflammation, and airway hyperresponsiveness to nonspecific agonists. The end result is excessive airway narrowing. Airway narrowing is driven by the action of airway smooth muscle and its actomyosin contractile machinery, but myosin exerts its mechanical effects within a cytoskeletal scaffolding that is extensible and in a continuous state of remodeling (6, 8, 21, 23, 24). In this connection, load fluctuations are imposed continuously on airway smooth muscle by the tidal action of breathing. These fluctuations are known to inhibit the development of active force and stiffness (7, 9, 22, 27) and result in smooth muscle lengthening (4). Although the direct effects of tidal stretch on actomyosin bridge dy-

Muscle preparations. Bovine tracheas obtained from a local slaughterhouse were sectioned into 4–5-ring segments and stored for no longer than 24 h in cold phosphate-buffered saline. Muscle strips, measuring 2 ⫻ 1 ⫻ 10 mm, were dissected out by removing the inner and outer layers of connective tissue and adjoining cartilage. Each end of the strip was glued to small brass clips. The strips were then suspended in a glass tissue bath using a fine steel rod. The top of the rod was attached to a servo-controlled lever arm via a miniature force transducer. The lower clip was latched onto a glass hook fused to the bottom of the bath. We established previously that the servo-lever and mounting system add no appreciable compliance compared with the muscle itself (4). Finally, the bath was perfused with Krebs-Henseleit solution, maintained at a volume of 50 ml, aerated with 95% O2-5% CO2, and maintained at 37°C. Each bovine tracheal smooth muscle (TSM) strip was mounted in the muscle bath (as described above) and allowed to equilibrate for 1 h. The strip was then set to its optimal length (Lo) using electric field stimulation adjusted for optimal response. Once at Lo, the strip was maximally stimulated with 10⫺4 M ACh establishing the optimal force Fo. After Lo and Fo had been established, the ACh was flushed out of the bath. Protocol 1: force fluctuations. SB-203580-HCl (10 ␮M, treatment) or an equivalent volume (0.05 ml) of distilled H2O (control) was added to the 50-ml muscle bath, and the smooth

Address for reprint requests and other correspondence: J. J. Fredberg, Physiology Program, Harvard School of Public Health, 665 Huntington Ave., Boston, MA 02115 (E-mail: jfredber@hsph. harvard.edu).

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mitogen-activated protein; contraction; plasticity; perturbed myosin binding

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muscle strips were allowed to incubate for 45 min. Each strip was then contracted isotonically (at 0.32 Fo) with 10⫺4 M ACh and allowed to shorten for 180 min, by which time its static equilibrium length (LSE) was established. Then, to simulate the effects of tidal breathing, sinusoidal force (load) fluctuations of amplitude (␦F) (4, 8, 16, 24, 32, and back to 8% Fo) with zero mean, at a frequency of 0.2 Hz, were superimposed upon the steady distending force (Fig. 1). We then measured the incremental changes in muscle length that accumulated progressively over the course of many tidal cycles. We also measured resulting changes of muscle length that occurred within each tidal event and that were phasic with tidal changes in the muscle load. From the tidal force and length fluctuations we computed muscle stiffness, a measure of the slope of the force-length loop. Protocol 2: p38 MAP kinase activity. At this time there is no commercially available product that recognizes bovine heat shock protein (HSP) 27. Therefore, we measured activation of a kinase upstream of HSP27, namely, p38 MAP kinase. Muscle strips were set to Lo, where Fo was maximal. Each strip was then isotonically contracted (at 0.32 Fo) with 10⫺4 M ACh and allowed to shorten for 180 min, by which time its LSE was established. Sinusoidal ␦F (0, 16, or 32% Fo) with zero mean, at a frequency of 0.2 Hz, were superimposed upon the steady distending force. After 15 min, each strip was quickly removed from the apparatus, flash-frozen in liquid nitrogen, and stored at ⫺80°C. The tissue was homogenized using a probe homogenizer at 4°C in a buffer contain-

ing 20 mM Tris (pH 7.5), 5 mM EGTA, 1 mM Na3VO4, 20 mM ␤-glycerophosphate, 10 mM NaF, 1 mM dithiothreitol, 1 ␮g/ml aprotinin, and 0.1 mM phenylmethylsulfonyl fluoride. The homogenate was spun on a table-top microfuge for 30 s, and 200 ␮l of supernatant was removed and boiled in Laemmli buffer containing 5% 2-mercaptoethanol. Protein concentration was determined according to the Bradford method. Samples were resolved on 12% SDS polyacrylamide minigels, transferred to polyvinylidene difluoride membrane, and probed with antibodies against tyrosine-phosphorylated p38 or total p38 (commercially available kit from New England Biolabs, Beverly, MA) according to the manufacturer’s instructions. Protein loading was identical for all samples. Specific bands were detected by electrochemiluminescence. p38 MAP kinase phosphorylation was then measured by densitometry. The ratio of phosphorylated to total p38 MAP kinase was determined to control for any variation in protein loading. Statistical analysis. Changes in length (L/Lo) with increasing ␦F were expressed as means ⫾ SE. This relationship was represented by the equation L/Lo ⫽ a ⫹ b(␦F)2 where a represents the statically equilibrated length (i.e., when ␦F ⫽ 0), and b represents a measure of the sensitivity of the dynamically equilibrated length to the ␦F. Thus a is a measure of isotonic shortening, and b is a measure of fluctuationdriven lengthening. Values of a and b for control strips and strips pretreated with the p38 MAP kinase inhibitor were compared and analyzed by the t-test. All other data were also expressed as means ⫾ SE and compared via t-tests. P values ⬍0.05 were considered significant. RESULTS

Fig. 1. Evolution of change in length (L) over time, from its optimal length (Lo) for representative muscle strips during contraction with 10⫺4 M ACh against a steady-state force component of 0.32 Fo. Force fluctuations of graded amplitudes (␦F) are superimposed upon the isotonic load. Change in L is compared for a control strip (top) and a strip pretreated with 10 ␮M SB-203580-HCl (bottom). Fo, maximal active force. AJP-Lung Cell Mol Physiol • VOL

In a representative control strip (Fig. 1, top), stimulation with 10⫺4 M ACh caused the muscle to shorten until it reached a steady-state length that was statically equilibrated (LSE). Whereas muscle that is contracted isometrically reaches a steady-state tension within minutes, muscle that is contracted isotonically continues to shorten for a much longer time before becoming statically equilibrated. When small force fluctuations (4 and 8% Fo) were subsequently superimposed on the steady distending force (0.32 Fo), there was little change in muscle length. However, when larger-amplitude force fluctuations were superimposed, the strip lengthened and became dynamically equilibrated at a length that substantially exceeded the LSE. When ␦F was dropped back to 8% Fo, the strip did not reshorten to the same length as with the initial 8% force fluctuations. The dynamically equilibrated lengths for all control strips showed similar findings (Fig. 2). The strip pretreated with SB-203580-HCl (Fig. 1, bottom) also shortened when activated by ACh. The treated strips tended to a steady-state length that was shorter than the control strips (0.49 vs. 0.59 Lo, Fig. 1), but this difference did not achieve statistical significance (Fig. 2, P ⫽ 0.219, comparing a ⫽ 0.562 ⫾ 0.037 for control strips vs. a ⫽ 0.499 ⫾ 0.030 for treated strips). There was little change in length with imposition of small force fluctuations (4 and 8% Fo) in the treated strips (Fig. 2), but when larger force fluctuations (ⱖ16%) were superimposed on the steady distending load, the muscle strips lengthened. The representative strip pretreated with the inhibitor 282 • MAY 2002 •

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Fig. 2. Fluctuation-driven muscle lengthening: pooled data for control strips (gray triangles) and strips pretreated with 10 ␮m SB203580-HCl (circles) are compared. The dynamically equilibrated lengths are plotted as change in L from Lo on the y-axis, for given ␦F on the x-axis. Solid lines represent the curves predicted by the algorithm y ⫽ a ⫹ bx2 for control strips (gray line) and strips exposed to the inhibitor (darker line). a is a measure of isotonic shortening, and b is a measure of fluctuation-driven lengthening. Also shown is the change in L for control (broken gray line and triangles) and treated strips (broken darker line and circles) when the ␦F was reduced back down to 8% Fo.

lengthened to a greater extent than did the control strip (1.15 vs. 0.75 Lo, at ␦F ⫽ 32% in Fig. 1). Overall, treated strips demonstrated significantly greater lengthening than control strips (Fig. 2, P ⫽ 0.013, comparing b ⫽ 0.00020 ⫾ 0.00002 for control vs. b ⫽ 0.00037 ⫾ 0.00005 for treated strips) despite being activated by the same dose of ACh and being loaded in an identical manner. When the ␦F was dropped back to 8% Fo, the treated strips did not reshorten to the length observed at the original 8% Fo, indicating an important role for loading history and resulting plasticity. All strips (treated and control) became less stiff with larger force fluctuations (Fig. 3). Fluctuation-driven lengthening is better appreciated when the same data are normalized by the LSE, rather than Lo (Fig. 4). There was a greater degree of smooth muscle lengthening, for a given ␦F, in strips exposed to the p38 MAP kinase inhibitor than for control strips. The force developed during the isometric contraction was the same in muscle strips treated with the inhibitor and control strips. Similarly, the force was maintained in both groups of muscle strips for the entire 180 min. After the tidal loading maneuvers, the isometric force generation capacity of the muscle was not compromised, retaining 85% or more of its initial capacity. Activation of airway smooth muscle with ACh results in increased phosphorylation of p38 MAP kinase (12). We found that imposition of load fluctuations on airway smooth muscle resulted in further activation (phosphorylation) of p38 MAP kinase (Fig. 5). AJP-Lung Cell Mol Physiol • VOL

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Fig. 3. Pooled observations for change in stiffness (E) from stiffness at static equilibrium length (LSE) (Eo) with increasing ␦F. Control strips (gray triangles) are compared with strips pretreated with SB-203580-HCl (circles). DISCUSSION

The principal findings of this report are as follows. We found little change in smooth muscle length with imposition of small load fluctuations but substantial lengthening when ␦F was increased to 16% or more of Fo. For any given ␦F, blocking p38 MAP kinase activity with the inhibitor SB-203580-HCl caused the muscle to lengthen to a greater extent. Last, the force fluctuations themselves induced activation of the p38 MAP kinase pathway. We have shown previously that fluctuation-driven muscle lengthening can be explained in large part by the effect of the load fluctuations on myosin binding (4). That is, when the load fluctuations are large enough

Fig. 4. Pooled data for all strips replotted as change in L from LSE (␦F ⫽ 0), instead of Lo, on the y-axis, for given ␦F on the x-axis. Control strips (gray bars) are compared with strips pretreated with 10 ␮M SB-203580-HCl (darker bars). †P ⬍ 0.05, compared with preceding ␦F (for control and treated strips); ‡P ⬍ 0.01, compared with preceding ␦F (for control and treated strips); *P ⬍ 0.05, control vs. treated strips. 282 • MAY 2002 •

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Fig. 5. Densitometry (in pixels) of the ratio of phosphorylated p38 mitogen-activated protein (MAP) kinase to total p38 MAP kinase in baseline (inactivated) smooth muscle strips and in strips activated with 10⫺4 M ACh and exposed to force fluctuations of 0, 16, and 32% of Fo. Data are normalized to strips activated with ACh alone, without imposition of force fluctuations (0%).

(ⱖ16% of Fo), actomyosin binding becomes perturbed, muscle stiffness decreases, and the muscle lengthens. Muscle length becomes dynamically equilibrated. However, bridge-based mechanisms alone cannot account for the incomplete reshortening of the muscle as ␦F is reduced. It appears that, during the force fluctuation protocol, the smooth muscle strips have undergone a plastic change. This conclusion is consistent with the observations of others, who have suggested that plasticity may play a major role in changes in the muscle contractile state length as a result of imposed stretch (8, 21, 24). Moreover, these findings are consistent with the hypothesis that activation of p38 MAP kinase stabilizes airway smooth muscle and limits the bronchodilating effects of deep inspirations. Our data demonstrate that the load fluctuations themselves acted as stressors that activate the p38 MAP kinase pathway (Fig. 5). These data suggest, therefore, that in addition to activation of the p38 MAP kinase pathway by thermal, osmotic, and biochemical stessors, the p38 pathway is also activated by mechanical stress. Together, these results suggest that although myosin is the primary effector molecule of the contractile response of smooth muscle, myosin exerts its mechanical effects within cytoskeletal scaffolding that is both deformable and in a continuous state of remodeling. The increased responsiveness of TSM to load fluctuations in the presence of the p38 MAP kinase inhibitor provides some insight into the type of plastic change that could be occurring. In the control situation, activation of the smooth muscle strips with ACh resulted in the activation of the p38 MAP kinase pathway, as demonstrated here and by others (12, 13), and we have shown here that a p38 MAP kinase inhibitor destabilized muscle mechanics. To explain the mechanical effects that were observed, HSP27 is a logical candiAJP-Lung Cell Mol Physiol • VOL

date molecule. Activation of p38 MAP kinase is known to result in the downstream activation of HSP27, which in turn stabilizes the actin cytoskeleton (13, 26, 28). Moreover, Gerthoffer and Gunst (6) have speculated recently that HSP27 may play a key role in cytoskeletal plasticity in airway smooth muscle, and the data in this report are consistent with that possibility. HSPs are a family of molecules induced by a variety of stressors including elevated temperature, osmotic stress, ultraviolet light, exposure to reactive oxygen species, and some inflammatory cytokines. The activation of these HSPs increases a cell’s capacity to survive these stressors. HSP27, one member of this family, is an actin binding protein that is constitutively expressed at high levels in smooth muscle. It is believed to modulate the polymerization of actin and to remodel the cytoskeleton by binding to and capping barbed ends of microfilaments and stabilizing them (13, 26, 28). Phosphorylation of HSP27 has been shown to be a necessary event for the migration of airway smooth muscle cells (12), and it has been proposed that HSP27 phosphorylation may be necessary for smooth muscle contraction (13). Other investigators have demonstrated that HSP27 is activated upon routine contraction of airway smooth muscle cells (12). HSP27 lies downstream of p38 MAP kinase, which phosphorylates MAP kinase-activated protein kinases-2/3 and ultimately leads to the phosphorylation (activation) of HSP27 (15, 28). We speculate, but could not demonstrate, that when muscle strips were incubated with the p38 MAP kinase inhibitor before activation with ACh, activation of HSP27 was blocked (12, 15), thereby limiting its stabilizing effect on the cytoskeleton. As such, force fluctuations generated more muscle lengthening. An alternative possibility is some nonspecific effect of the inhibitor on activation of the motor proteins, but muscle strips pretreated with SB-203580-HCl and then activated with ACh demonstrated no difference in phosphorylation of the 20-kDa myosin regulatory light chain compared with control strips activated with ACh. How does this all relate to airway narrowing in asthma? It has long been thought that spontaneous asthmatic obstruction behaves as if it were caused by an intrinsic impairment of the bronchodilating effect of a deep inspiration (3, 5, 18–20). This bronchodilating effect can be explained in large part by perturbed equilibria of actomyosin binding caused by imposed load fluctuations in the physiological range (4). Muscle stiffness decreases, and the muscle lengthens and becomes dynamically equilibrated. We have shown here that a p38 MAP kinase inhibitor modulates that process. In that connection, there is evidence in the literature that there are elevated levels of HSP70 in the airways of asthmatic individuals (1, 25). There is also a suggestion that there may be higher levels of HSP27, in particular in allergic asthmatic individuals (11). Our results demonstrate that activation of the p38 MAP kinase pathway limits the ability of force fluctuations 282 • MAY 2002 •

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to induce smooth muscle lengthening, and although we did not measure HSP27 directly, our data suggest that the limitation of fluctuation-driven smooth muscle lengthening may be due to the downstream activation of HSP27 and its cytoskeletal stabilizing effect. Our data suggest that if asthmatic individuals do have greater levels of activated HSP27 in their airways, then the resulting stabilization of the muscle may limit the fluctuation-driven lengthening and bronchodilation associated with deep inspirations. In summary, we have shown that activation of the p38 MAP kinase pathway may modulate muscle mechanical responses to imposed load fluctuations during contractile stimulation. The presence of SB-203580HCl increased the degree of smooth muscle lengthening induced by load fluctuations. As such, these data suggest the hypothesis that activation of p38 MAP kinase stabilizes airway smooth muscle and may limit the bronchodilating effect of deep inspirations. The authors thank Dr. William Gerthoffer and Dr. Stephanie Shore for advice and guidance. This work was supported by National Heart, Lung, and Blood Institute Grants HL-33009 and HL-59682. REFERENCES 1. Bertorelli G, Bocchino V, Zhuo X, Chetta A, Del Donno M, Foresi A, Testi R, and Olivieri D. Heat shock protein 70 upregulation is related to HLA-DR expression in bronchial asthma. Effects of inhaled glucocorticoids. Clin Exp Allergy 28: 551–560, 1998. 2. Cuenda A, Rouse J, Doza YN, Meier R, Cohen P, Gallagher TF, Young PR, and Lee JC. SB 203580 is a specific inhibitor, of a MAP kinase homologue which is stimulated by cellular stresses and interleukin-1. FEBS Lett 364: 229–233, 1995. 3. Fish JE, Ankin MG, Kelly JF, and Peterman VI. Regulation of bronchomotor tone by lung inflation in asthmatic and nonasthmatic subjects. J Appl Physiol Respir Environ Exercise Physiol 50: 1079–1086, 1981. 4. Fredberg JJ, Inouye DS, Mijailovich SM, and Butler JP. Perturbed equilibrium of myosin binding and its implications in bronchospasm. Am J Respir Crit Care Med 159: 959–967, 1999. 5. Gaynard P, Orehek J, Grimaud C, and Charpin J. Bronchoconstrictor effects of a deep inspiration in patients with asthma. Am Rev Respir Dis 111: 433–439, 1975. 6. Gerthoffer WT and Gunst SJ. Signal transduction in smooth muscle invited review: focal adhesion and small heat shock proteins in the regulation of actin remodeling and contractility in smooth muscle. J Appl Physiol 91: 963–972, 2001. 7. Gunst SJ. Contractile force of canine airway smooth muscle during cyclical length changes. J Appl Physiol 55: 759–769, 1983. 8. Gunst SJ, Meiss RA, Wu MF, and Rowe M. Mechanisms for the mechanical plasticity of tracheal smooth muscle. Am J Physiol Cell Physiol 268: C1267–C1276, 1995.

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