Isoproterenol induces actin depolymerization in human airway smooth muscle cells via activation of an Src kinase and GS
Carol A. Hirshman,1
Defen Zhu,1
Thomas Pertel,1
Reynold A. Panettieri,2 and
Charles W. Emala1
1Department of Anesthesiology, College of Physicians and Surgeons of Columbia University, New York, New York; and 2Pulmonary and Critical Care Division, Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania
Submitted 13 December 2004
; accepted in final form 7 January 2005
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ABSTRACT
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In a previous study, we showed that isoproterenol induced actin depolymerization in human airway smooth muscle cells by both protein kinase A (PKA)-dependent and -independent signaling pathways. We now investigate the signaling pathway of PKA-independent actin depolymerization induced by isoproterenol in these cells. Cells were briefly exposed to isoproterenol or PGE1 in the presence and absence of specific inhibitors of Src-family tyrosine kinases, phosphatidylinositol-3-kinase (PI3 kinase), or MAP kinase, and actin depolymerization was measured by concomitant staining of filamentous actin with FITC-phalloidin and globular actin with Texas red DNase I. Isoproterenol, cholera toxin, and PGE1 induced actin depolymerization, indicated by a decrease in the intensity of filamentous/globular fluorescent staining. Pretreatment with the Src kinase inhibitors 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyriimidine (PP2) or geldanamycin or the PKA inhibitor Rp-cAMPS only partly inhibited isoproterenol- or PGE1-induced actin depolymerization. In contrast, PP2 and geldanamycin did not inhibit forskolin-induced actin depolymerization, and AG-213 (an EGF receptor tyrosine kinase inhibitor) did not inhibit isoproterenol- or PGE1-induced actin depolymerization. PI3 kinase or MAP kinase inhibition did not inhibit isoproterenol-induced actin depolymerization. Moreover, isoproterenol but not forskolin induced tyrosine phosphorylation of an Src family member at position 416. These results further confirm that both PKA-dependent and PKA-independent pathways mediate actin depolymerization in human airway smooth muscle cells and that the PKA-independent pathway by which isoproterenol induces actin depolymerization in human airway smooth muscle cells involves Src protein tyrosine kinases and the Gs protein.
protein kinase A; tyrosine 416; phosphatidylinositol-3-kinase; 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyriimidine; geldanamycin
THE ACTIN CYTOSKELETON IS a dynamic structure that undergoes rearrangement under the control of signaling molecules that are intimately involved in regulating the contractile status of cells. In cultured human airway smooth muscle cells, many agonists that contract airway smooth muscle induce actin polymerization (10, 39), whereas most agonists that relax airway smooth muscle induce actin depolymerization (12). Contractile agonists induce actin polymerization by pathways involving the heterotrimeric G proteins G
i-2 and G
q signaling to RhoA (10, 11). A study from this laboratory demonstrated that isoproterenol induced actin depolymerization in cultured human airway smooth muscle cells by two separate signaling pathways: a protein kinase A (PKA)-dependent and a PKA-independent pathway (12).
In the PKA-dependent pathway
-adrenergic receptors transduce signals from isoproterenol and other catecholamines to the heterotrimeric G protein, Gs, which in turn activates adenylyl cyclase to produce cAMP (25). cAMP in turn activates its effector, cAMP-dependent protein kinase (PKA). Because inhibition of RhoA induces actin depolymerization in these cells (39) and because PKA inactivates RhoA by phosphorylating serine 188 on RhoA (5), PKA most likely induces actin depolymerization by inhibiting RhoA.
The intermediates in the PKA-independent signaling pathway by which isoproterenol induces actin depolymerization are not known. Desensitized
-adrenergic receptors have been shown to signal to Gi proteins and phosphatidylinositol-3-kinase (PI3 kinase) (4). The protein tyrosine kinase inhibitors tyrphostin A23 and genistein reduced isoproterenol-induced actin depolymerization in human airway smooth muscle cells (12), suggesting that nonreceptor protein tyrosine kinases and perhaps Gi proteins are involved in this response. The nonreceptor tyrosine kinases are grouped into families based on sequence homology. One of these families, the Src family of protein tyrosine kinases are expressed in a wide variety of cell types (15) and have been shown to participate in a wide variety of cellular processes including cytoskeletal reorganization (38). Src family tyrosine kinases contain many phosphorylation sites, but autophosphorylation of Tyr416 at the activation loop is a critical step leading to full activation of the protein (37). Moreover, the Src family of protein tyrosine kinases have been shown to communicate with large numbers of different receptors, including G protein-coupled receptors (37), and the heterotrimeric G protein, Gs, has been shown to autophosphorylate Tyr416 (21).
The goals of the present study were to identify the heterotrimeric G protein by which isoproterenol induces actin depolymerization in human airway smooth muscle cells in the PKA-independent signaling pathway and to determine whether the protein tyrosine kinase intermediates involved in this signaling pathway belong to the Src family. To determine whether Src family tyrosine kinases were intermediates in the PKA-independent pathway, we therefore compared the amount of actin depolymerization induced by isoproterenol in untreated cultured human airway smooth muscle cells and in cells that were pretreated with two structurally unrelated Src-family protein tyrosine kinase inhibitors, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyriimidine (PP2) and geldanamycin; the EGF receptor tyrosine kinase inhibitor AG-213; pertussis toxin, which inhibits Gi; and wortmannin, which inhibits PI3 kinase. To determine whether Gs was an intermediate in this pathway, we also compared the amount of actin depolymerization induced by cholera toxin, which directly activates the Gs protein, and prostaglandin E1 (PGE1), which activates a receptor that couples to Gs distinct from that of isoproterenol. In addition, we determined whether Src kinase inhibition reversed the actin depolymerization induced by PGE1. To further confirm the involvement of Src tyrosine kinases and Gs in this pathway, we exposed human airway smooth muscle cells to isoproterenol and measured the amount of Src that is tyrosine phosphorylated at Tyr416.
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METHODS
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Cell culture.
In brief, primary cultures of human tracheal smooth muscle cells (30) were maintained in Ham's F-12 medium containing 10% fetal bovine serum at 37°C in an atmosphere of 5% CO2-95% air. Immunoblot analysis of these cells identified expression of
-actin, myosin heavy chain, and desmin, confirming the smooth muscle phenotype of the cells. For the fluorescence microscopy studies the cells were plated on eight-well microscope slides (Nunc Chambers, Naperville, IL) and grown until almost confluent. For the immunoblotting, cells were grown to confluence in T-75 flasks.
Fluorescence staining.
Fluorescence microscopy was performed by methods previously described in our laboratory with minor modifications (39). Because activation of the cytoskeleton by nonspecific stimuli is a major concern, physical manipulation of the slides was kept to a minimum. In brief, cells were fixed and agonist action terminated by the addition of 3.7% (final concentration) fresh paraformaldehyde in phosphate-buffered saline (PBS) for 15 min. After three washes with PBS, cells were permeabilized with 0.2% Triton X-100 in PBS for 5 min. Cells were next pretreated with blocking solution (1% BSA-0.1% Triton X-100 in PBS) for 15 min. Cells were simultaneously stained with FITC-labeled phalloidin (FITC-phalloidin, 1 µg/ml) and Texas red-labeled DNase I (Texas red-DNase I, 10 µg/ml) in 1% BSA in PBS to localize pools of filamentous (F)-actin and monomeric or globular (G)-actin, respectively (14). The cell staining was performed for 20 min in the dark at room temperature. The wells were washed twice with PBS, and a coverslip was mounted on the slide with the mounting medium Vectashield H-1000 (Vector, Burlingame, CA) to prevent rapid photobleaching. All fluorescence intensity measurements are routinely obtained from all images within 15 s of exposure to UV excitation, ensuring that photobleaching was not a significant factor in our results (12). Incubation, fixation, and staining were always performed in parallel for all wells on a slide.
Fluorescence.
Actin pools were visualized with a fluorescence microscope (Olympus IX70; Tokyo, Japan), and the images were captured and stored using Metamorph software (Universal Imaging, West Chester, PA) on a personal computer. The fluorescence intensities of FITC-phalloidin and Texas red-DNase I were calculated simultaneously from a view containing >15 cells. The excitation and emission wavelengths for FITC-phalloidin are 490 and 525 nm, respectively, whereas the excitation and emission wavelengths for Texas red-DNase I are 596 and 615 nm, respectively. To standardize the florescence intensity measurements among experiments, the time of image capturing, the image intensity gain, the image enhancement, and the image black level in both channels were optimized before each experiment and kept constant throughout each experiment. Representative images were taken in triplicate from each well and were digitized (640 x 484 pixels) with a color resolution from 0 (minimum)- to 255-bit (maximum) intensity. After measuring the total and background intensity of FITC-phalloidin and Texas red-DNase, we subtracted background fluorescent intensity from each image and calculated the filamentous-to-globular (F/G) ratios. To control for day-to-day variations in staining intensity, we always compared untreated cells with treated cells on the same microscope slide, as cells on the same slide undergo identical culture, fixation, permeabilization, staining, and microscopy conditions, allowing meaningful comparisons between samples.
Protocols for the fluorescence microscopy studies.
After serum deprivation for 24 h, cells were exposed to vehicle or the agonists isoproterenol (100 µM for 10 min), forskolin (10 µM for 10 min), PGE1 (10 µM for 10 min), or cholera toxin (10 µg/ml for 4 h), in some cases in the presence or absence of the PKA inhibitor the Rp diastereomer of adenosine 3',5'-cyclic monophosphorothioate (Rp-cAMPS, 100 µM for 30 min), the selective Src protein tyrosine kinase inhibitor PP2 (10 µM for 30 min), or PP2 and Rp-CAMPS in combination. In other studies cells were pretreated with the Src tyrosine kinase inhibitor geldanamycin (5 µM for 30 min) or the EGF receptor tyrosine kinase inhibitor AG-213 (10 µM for 30 min) before agonist treatment. In separate studies cells were pretreated with pertussis toxin (100 ng/ml for 4 h) to inhibit Gi and wortmannin (0.1 µM for 30 min) to inhibit PI3 kinase and exposed to the agonist isoproterenol. This concentration of pertussis toxin for 4 h has been shown to inhibit Gi in these cells (39), whereas wortmannin at this concentration is specific for PI3 kinase (19). Because both PI3 kinase and MAP kinase may both be required in this signaling pathway (27), studies involving PI3 kinase inhibition were repeated in the presence and absence of pretreatment with the MAP kinase inhibitor PD-98059 (50 µM for 30 min).
Isoproterenol, forskolin, Rp-cAMPS, pertussis toxin, and cholera toxin were dissolved in water. PGE1 was dissolved in 0.1% ethanol. PP2, geldanamycin, AG-213, wortmannin, and PD-98059 were dissolved in DMSO such that the final concentration of DMSO was 0.050.1%. Previous studies from our laboratory have shown that 0.1% DMSO has no effect on actin polymerization or depolymerization in these cells (18).
Western blotting.
Confluent cells in T-75 flasks were serum deprived for 24 h, washed twice with PBS at room temperature, and recovered from culture flasks with trypsin/EDTA. Cells were washed three times with PBS to remove the trypsin/EDTA and aliquotted into microcentrifuge tubes. Cells were treated with isoproterenol (100 µM for 3 min) or forskolin (10 µM for 5 min) at 37°C, after which Laemmli's gel loading buffer (62.5 mM Tris·HCl, pH 6.8, 2% SDS, 10% glycerol, 5% 2-mercaptoethanol, and 0.004% bromphenol blue) was added to stop the reaction. The amount of protein was confirmed by protein assay using BSA as a standard. Samples (50 µg) were electrophoresed through 12% SDS-polyacrylamide gels and transferred to polyvinylidene difluoride (PVDF) membranes. The PVDF membranes were blocked for 1 h at room temperature with 5% nonfat milk in Tris-buffered saline with 0.1% Tween 20 and were then probed with a rabbit polyclonal anti-p-cSrc (Y416) antibody (1:500) overnight at 4°C. After being washed three times, membranes were incubated for 1 h at room temperature with anti-rabbit antibody coupled to horseradish peroxidase. The signal from the immunoreactive bands was detected by enhanced chemiluminescence according to the manufacturer's recommendations (Amersham) and quantified on a scanner coupled to a personal computer.
Materials.
Isoproterenol, forskolin, PGE1, pertussis toxin, cholera toxin, and FITC-phalloidin were obtained from Sigma (St. Louis, MO). Texas red-DNase I was obtained from Molecular Probes (Eugene, OR). PP2, geldanamycin, PD-98059, wortmannin, and AG-213 were purchased from Calbiochem (La Jolla, CA). Rp-cAMPS was purchased from Biolog (Bremen, Germany). Vectashield H-1000 was obtained from Vector Laboratories (Burlingame, CA). p-c-Src (Tyr416)-R antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA).
Statistical analysis of data.
All data are presented as means ± SE. Immunoblot intensities were compared by paired t-test, whereas F/G actin ratios were compared by analysis of variance with repeated measures and Bonferroni posttest comparisons when appropriate with Instat software (Graph Pad, San Diego, CA). P < 0.05 was considered significant.
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RESULTS
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Exposure of serum-deprived human airway smooth muscle cells to isoproterenol (100 µM) (Fig. 1) or to forskolin (10 µM) for 10 min decreased the FITC-phalloidin staining intensity of F-actin and increased the Texas red DNase I staining of G-actin. The F/G-actin fluorescence ratio decreased from 3.2 ± 0.11 in untreated cells to 2.2 ± 0.11 and 2.5 ± 0.14 in isoproterenol- and forskolin-treated cells, respectively (P < 0.001 for each agonist compared with the control group and P < 0.05 for isoproterenol compared with forskolin-treated cells, n = 6 experiments) (Fig. 2). These data confirm previous data (12) that isoproterenol and forskolin induced actin depolymerization in these cells.

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Fig. 1. Representative photomicrographs of cultured human airway smooth muscle cells stained with FITC-phalloidin and Texas red-DNase I to illustrate filamentous (F)-actin (left column) and globular (G)-actin (right column) fibers. Cells treated with isoproterenol alone (Iso, 100 µM for 10 min) showed decreased F-actin staining and increased G-actin staining compared with the untreated control cells indicating actin depolymerization. Pretreatment for 30 min with either 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyriimidine (PP2, 10 µM) or Rp diastereomer of adenosine 3',5'-cyclic monophosphorothioate (Rp-cAMPS, 100 µM) each partially reduced the actin depolymerization, whereas the combined pretreatment with both PP2 and Rp-cAMPS for 30 min completely blocked actin depolymerization induced by isoproterenol.
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Fig. 2. Fluorescence staining ratios of F- to G (F/G)-actin in human airway smooth muscle cells exposed to 100 µM Iso for 10 min in the absence or presence of either PP2 (10 µM) or Rp-cAMPS (Rp, 100 µM) or the combination of PP2 and Rp-cAMPS for 30 min or forskolin (Forsk, 10 µM) in the presence or absence of PP2. PP2 or Rp-cAMPS each partially inhibited the decrease in the F/G-actin ratio induced by Iso. The combined treatment with both PP2 and Rp-cAMPS completely blocked the Iso-induced decrease in the F/G-actin ratio. PP2 was without effect on the forskolin-induced decrease in the F/G-actin ratio. *P < 0.001 compared with control; #P < 0.05 compared with forskolin; $P < 0.01 compared with control; @P < 0.001 compared with Iso (n = 6).
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To investigate the role of Src protein tyrosine kinases in the PKA-independent pathway by which isoproterenol induced actin depolymerization, we pretreated cells with the specific Src protein tyrosine kinase inhibitor PP2 (10 µM), the specific competitive antagonist of the action of cAMP on PKA Rp-cAMPS (100 µM), or both PP2 and Rp-cAMPS 30 min before isoproterenol exposure. PP2 and Rp-cAMPS pretreatment each only partly inhibited the actin depolymerization induced by isoproterenol. However, the combined pretreatment with PP2 and Rp-cAMPS totally inhibited the actin depolymerization induced by isoproterenol (Figs. 1 and 2). In cells pretreated with PP2, isoproterenol decreased the F/G-actin ratio to only 2.9 ± 0.11 (P < 0.001 compared with isoproterenol alone). In cells pretreated with Rp-cAMPS, isoproterenol decreased the F/G-actin fluorescence ratio to only 2.7 ± 0.08 (P < 0.001 compared with control and to isoproterenol alone). In contrast, when cells were pretreated with both PP2 and Rp-cAMPS, the F/G-actin ratio in cells exposed to isoproterenol was 3.3 ± 0.16 (P > 0.05 compared with control and P < 0.001 compared with isoproterenol alone, n = 6 experiments; Fig. 2).
However, PP2 pretreatment did not inhibit forskolin-induced actin depolymerization (Fig. 2). The F/G-actin fluorescence ratio was 2.5 ± 0.17 in PP2-pretreated cells exposed to forskolin (P > 0.05 compared with forskolin alone, Fig. 2). PP2 in the absence of agonist did not induce actin polymerization or depolymerization. The F/G-actin fluorescence ratio was 3.2 ± 0.12 in PP2-treated cells (P > 0.05 compared with control, n = 6).
To evaluate the role of Gs protein in this pathway, we next compared the ability of PGE1 with isoproterenol to induce actin depolymerization in these cells and questioned whether PGE1-induced actin depolymerization was also partly inhibited by either PP2 or Rp-cAMPS. PGE1, like isoproterenol, induced actin depolymerization in these cells that was partly inhibited by either PP2 or Rp-cAMPS (Fig. 3). The F/G-actin fluorescence ratio decreased from 2.8 ± 0.16 in untreated cells to 1.6 ± 0.10 and 1.8 ± 0.12 in isoproterenol- and PGE1-treated cells, respectively (P < 0.001 for each agonist compared with the control group). In cells pretreated with PP2, isoproterenol decreased the F/G-actin ratio to only 2.3 ± 0.14 (P < 0.001 compared with isoproterenol alone), and PGE1 decreased the F/G-actin ratio to 2.3 ± 0.17 (P < 0.001 compared with PGE1 alone). In cells pretreated with Rp-cAMPS, isoproterenol decreased the F/G-actin fluorescence ratio to only 2.1 ± 0.15 (P < 0.001 compared with isoproterenol alone), and PGE1 decreased the F/G-actin ratio to only 2.2 ± 0.13 (P < 0.001 compared with PGE1 alone). Ethanol (0.1%, the vehicle for PGE1) had no effect on actin polymerization. The F/G-actin ratio was 2.6 ± 0.18 in ethanol-pretreated cells (P > 0.05 compared with control, n = 6).

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Fig. 3. Fluorescence staining ratios of F/G-actin in human airway smooth muscle cells exposed to 100 µM Iso or 10 µM prostaglandin E1 (PGE1) for 10 min in the absence or presence of either PP2 (10 µM) or Rp-cAMPS (100 µM). PP2 or Rp-cAMPS each partially inhibited the decrease in the F/G-actin ratio induced by either Iso or PGE1. Ethanol (EtOH, 0.1%), the vehicle for PGE1, was without effect. *P < 0.001 compared with control; #P < 0.001 compared with Iso; $P < 0.001 compared with PGE1 (n = 6).
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In a separate series of studies, cells were pretreated with geldanamycin (5 µM) (a selective Src protein tyrosine kinase inhibitor that is structurally unrelated to PP2) or Rp-cAMPS (100 µM) for 30 min before isoproterenol or PGE1 exposure. Geldanamycin in the absence of agonist had no effect on actin polymerization or depolymerization in these cells. Isoproterenol and PGE1 each induced actin depolymerization in these cells, and geldanamycin and Rp-cAMPS pretreatment each partly inhibited the actin depolymerization induced by isoproterenol (Fig. 4A) or PGE1 (Fig. 4B). The F/G-actin fluorescence ratio averaged 2.7 ± 0.16 and 3.1 ± 0.23 in untreated and geldanamycin-treated cells, respectively (P > 0.5). Isoproterenol significantly decreased the F/G-actin fluorescence ratio to 1.8 ± 0.09 (P < 0.001), whereas geldanamycin and Rp-cAMPS pretreatment reduced the isoproterenol-induced decrease in the F/G-actin fluorescence ratio to 2.5 ± 0.22 and 2.2 ± 0.11, respectively (P < 0.01 for geldanamycin and Rp-cAMPS compared with isoproterenol alone; n = 5 experiments) (Fig. 4A). In another series of studies, the F/G-actin fluorescence ratio averaged 2.7 ± 0.13 and 2.9 ± 0.20 in untreated and geldanamycin-treated cells, respectively (P > 0.5). PGE1 significantly decreased the F/G-actin fluorescence ratio to 1.9 ± 0.08 (P < 0.001), whereas geldanamycin and Rp-cAMPS pretreatment reduced the PGE1-induced decrease in the F/G-actin fluorescence ratio to 2.4 ± 0.13 and 2.4 ± 0.12, respectively (P < 0.01 for geldanamycin and Rp-cAMPS compared with PGE1 alone, n = 7 experiments; Fig. 4B).

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Fig. 4. A: fluorescence staining ratios of F/G-actin in human airway smooth muscle cells exposed to 100 µM Iso for 10 min with or without pretreatment with geldanamycin (geldana, 5 µM for 30 min) or Rp-cAMPS (100 µM for 30 min). Geldanamycin or Rp-cAMPS each partially reversed the Iso-induced decrease in F/G-actin staining ratios. (n = 5). B: fluorescence staining ratios of F/G-actin in human airway smooth muscle cells exposed to PGE1 (10 µM for 10 min) with or without pretreatment with geldanamycin (5 µM for 30 min) or Rp-cAMPS (100 µM for 30 min). Geldanamycin or Rp-cAMPS each partially reversed the PGE1-induced decrease in F/G-actin staining ratios. *P < 0.001 compared with control; #P < 0.01 compared with Iso or PGE1 (n = 7).
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In a separate series of studies, to determine whether a protein tyrosine kinase inhibitor that does not inhibit Src protein tyrosine kinases reduced isoproterenol- or PGE1-induced actin depolymerization in these cells, human airway smooth muscle cells were pretreated with the EGF receptor tyrosine kinase inhibitor AG-213 (10 µM) or with Rp-cAMPS (100 µm) for 30 min and exposed to either isoproterenol or PGE1 for 10 min. Isoproterenol and PGE1 decreased the F/G-actin fluorescence ratio from 2.5 ± 0.04 to 1.7 ± 0.03 and 1.9 ± 0.05, respectively (P < 0.001 compared with control). Both isoproterenol- and PGE1-induced actin depolymerization was reduced by Rp-cAMPS, but AG-213 pretreatment did not inhibit actin depolymerization in isoproterenol- or in PGE1-exposed cells. Rp-cAMPS pretreatment reduced the isoproterenol- and PGE1-induced decrease to 2.1 + 0.06 and 2.2 ± 0.07, respectively (P < 0.001 for isoproterenol and 0.01 for PGE1, Fig. 5). In AG-213-pretreated cells exposed to isoproterenol and PGE1 the F/G-actin fluorescence ratio averaged 1.8 ± 0.09 and 2.0 ± 0.14 (P > 0.05 compared with isoproterenol or PGE1 alone, Fig. 5). AG-213 in the absence of isoproterenol or PGE1 had a small effect on actin depolymerization. The F/G-actin fluorescence ratio averaged 2.3 ± 0.13 (P < 0.05, n = 5, Fig. 5).

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Fig. 5. Fluorescence staining ratios of F/G-actin in human airway smooth muscle cells exposed to 100 µM Iso or 10 µM PGE1 for 10 min in the absence or presence of AG-213 (10 µM for 30 min) or Rp-cAMPS (100 µM for 30 min). Rp-cAMPS but not AG-213 partially inhibited the decrease in the F/G-actin ratio induced by Iso or PGE1. *P < 0.050.001 compared with control; #P < 0.01 compared with PGE1; $P < 0.001 compared with Iso (n = 5).
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To further confirm that isoproterenol signaled to Gs rather than to the Gi protein and PI3 kinase, we exposed human cultured airway smooth muscle cells to vehicle, cholera toxin (10 µg/ml for 4 h), isoproterenol (100 µM for 10 min), pertussis toxin (100 ng/ml for 4 h), and wortmannin (0.1 µM for 30 min), or isoproterenol pretreated with either pertussis toxin or wortmannin. Both cholera toxin and isoproterenol decreased the F/G-actin ratio, but neither pertussis toxin nor wortmannin inhibited isoproterenol-induced actin depolymerization. Cholera toxin and isoproterenol decreased the F/G-actin ratio from 2.9 ± 0.48 to 2.1 ± 0.33 and 2.0 ± 0.39 (P < 0.001 compared with control), respectively (n = 5) (Fig. 6). In pertussis toxin- and wortmannin-pretreated cells exposed to isoproterenol, the F/G-actin ratio averaged 2.3 ± 0.33 and 2.0 ± 0.39, respectively (P > 0.05 compared with isoproterenol alone) (Fig. 6). Because both PI3 kinase and MAP kinase may both be required in this signaling pathway (27), studies involving PI3 kinase inhibition were repeated in the presence and absence of pretreatment PD-98059 (50 µM for 30 min). Pretreatment with both wortmannin and PD-98059 had no significant effect on actin reorganization in isoproterenol-exposed cells. Isoproterenol decreased the F/G-actin ratio from 4.2 ± 0.16 to 3.2 ± 0.15 in the absence of combined MAP kinase and PI3 kinase inhibition (P < 0.001 compared with control and to 3.2 ± 0.16) and presence of these inhibitors (P > 0.05 compared with isoproterenol, n = 6).

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Fig. 6. Fluorescence staining ratios of F/G-actin in human airway smooth muscle cells were decreased by exposure to Iso (100 µM for 10 min) or cholera toxin (10 µg/ml for 4 h). The Iso-induced decrease in the F/G-actin ratio was not blocked by pretreatment with wortmannin (0.1 µM for 30 min) or pertussis toxin (100 ng/ml for 4 h). *P < 0.010.001 compared with control (n = 5).
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To directly measure the activation and phosphorylation of Src family tyrosine kinases following isoproterenol treatment, we performed immunoblotting studies using an antibody specific for phosphorylation of Tyr416 of Src family tyrosine kinases. Isoproterenol (100 µM) for 3 min significantly increased Tyr416 phosphorylation (control, 10.5 ± 0.36; isoproterenol, 14.7 ± 0.44 band intensity units; n = 9; Fig. 7A). In contrast, forskolin (10 µM) for 5 min resulted in no significant change in phosphorylation of Y416 of tyrosine kinases (control, 11.3 ± 0.26; forskolin, 10.6 ± 0.26 band intensity units; n = 8; Fig. 7B).

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Fig. 7. Representative immunoblots of Iso-induced (100 µM, A) and forskolin-induced (10 µM, B) autophosphorylation of an Src tyrosine kinase at Tyr416. Exposure of cells to Iso for 3 min increased Tyr416 phosphorylation, whereas forskolin exposure for 5 min had no effect on Tyr416 phosphorylation.
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DISCUSSION
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This study extends previous observations from our laboratory showing that isoproterenol-induced actin polymerization in human airway smooth muscle cells occurs in part by a cAMP-independent pathway involving tyrosine kinases (12) by demonstrating that an Src family tyrosine kinase and Gs protein are intermediates in this pathway (Fig. 8).

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Fig. 8. PKA-dependent pathway and proposed PKA-independent pathway mediating actin depolymerization in cultured human airway smooth muscle cells.
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Two types of studies were used to examine the role of Src family tyrosine kinases in isoproterenol-induced actin depolymerization: selective inhibitor studies using fluorescence microscopy and antibody studies using immunoblotting. We used dual fluorescence labeling with FITC-phalloidin and Texas red-DNase I adapted from the method of Knowles and McCulloch (14) to image and quantify actin depolymerization in these studies. FITC-phalloidin specifically labels F-actin, whereas Texas red DNase I specifically labels G-actin. Dual labeling techniques with concurrent labeling of F- and G-actin pools (14, 32, 36) is widely used to correct for differences in cell size or number per image and to reduce their influence on fluorescence intensities. This technique has been used by many laboratories to quantify changes in stress fibers and reorganization of the actin cytoskeleton (7, 11, 23, 39). The avoidance of confounding factors, such as variations in staining of the FITC and Texas red or photobleaching that could have influenced our fluorescence intensity measurements or the measured F/G-actin ratios, have been discussed in our previous publications (12).
We first compared the effect of two selective inhibitors of Src family kinase members, PP2 (9, 33) and geldanamycin (45), to that of a non-Src tyrosine kinase inhibitor, AG-213, on actin depolymerization induced by isoproterenol or forskolin. Both PP2 and geldanamycin, at concentrations that are known to be selective for Src protein tyrosine kinases, partly inhibited isoproterenol-induced actin depolymerization but were without effect on forskolin-induced actin depolymerization. In addition, AG-213 was without effect on either isoproterenol- or forskolin-induced actin depolymerization, implicating Src family tyrosine kinases in this pathway.
In addition to transducing the signal from isoproterenol to the Gs protein, the
2-adrenergic receptor undergoes internalization after phosphorylation by G protein-coupled receptor kinases to activate Gi, PI3 kinase, an Src family kinase, and MAP kinase (41). Our data showing that inhibition of Gi, PI3 kinase, and MAP kinase had no effect on isoproterenol-induced actin depolymerization suggest that a signaling pathway linking internalized
-adrenergic receptors to actin reorganization is unlikely, although PI3 kinase and MAP kinase mediate cell proliferation in these cells (2).
Four receptors for PGE (EP1, -2, -3, and -4 receptors) have been identified and cloned (26). Although both EP2 and EP4 receptors couple to Gs protein (26), the human cultured airway smooth muscle cells used in this study express EP2 receptors predominantly (31). The similar results on actin depolymerization obtained with PGE1 and isoproterenol suggest that the PKA-independent pathway that mediates actin depolymerization signals though Gs. Studies with cholera toxin, which catalyzes ADP-ribosylation of the
-chain of Gs thereby stabilizing the protein in an active form (28), provide further confirmation.
Data showing an increase in tyrosine phosphorylation of an Src family kinase at amino acid residue 416 by isoproterenol but not forskolin further confirms the role of Src protein tyrosine kinases in a PKA-independent pathway mediating actin depolymerization by isoproterenol in human airway smooth muscle cells. This antibody that recognizes phosphorylation at residue 416 was selected for the following reasons. Autophosphorylation of Tyr416 at the activation loop is a critical step leading to full activation of Src family tyrosine kinases (37, 44). Although the mechanism by which other G proteins activate Src family tyrosine kinases is not known, Gs
and Gi
can directly interact with and activate Src (8, 21). Moreover, Gs
has been shown to tyrosine phosphorylate residue 416 (21).
Src family tyrosine kinases are a major group of cellular signal transducers, and the Src kinase inhibitors used in this study block the activity of many Src family members including Src, Yes, Lck, Fyn, and Hck (9, 24). The antibody used to identify isoproterenol-induced tyrosine phosphorylation in this study is an affinity-purified rabbit polyclonal antibody raised against a peptide that includes Tyr416 of c-Src of human origin that reacts with many Src family members. Thus this study does not identify the Src kinase family member that mediates actin depolymerization in human airway smooth muscle cells. There are at least two possible explanations for the small but significant increase in Tyr416 phosphorylation induced by isoproterenol. First, the antibody used recognizes the phosphorylated form of many Src family tyrosine kinases, only one of which is increased by isoproterenol. Second, isoproterenol, in addition, may induce tyrosine phosphorylation of sites in addition to Tyr416.
The Src kinases expressed in airway smooth muscle and the receptors to which they couple have not yet been identified. c-Src is expressed in vascular tissue (17, 29, 40), whereas Fyn, Lyn, Hck, and Fgr are found in human aortic smooth muscle cells (6), and Src, Fyn, and Yes have been identified in cultured canine colonic smooth muscle cells (34). In canine colonic smooth muscle cells, muscarinic stimulation via M2 muscarinic receptors couple to Src but not to Fyn (34), whereas in vascular smooth muscle angiotensin II activation of c-Src mediates contraction (29), not relaxation.
-Adrenergic agonists are known to mediate a number of biological effects that do not use the classic PKA pathway, and recent studies have identified Src protein tyrosine kinases as intermediates in these pathways. In S49 mouse lymphoma cells and in human T cell lymphoma cells (Jurkat cells),
-adrenergic receptor stimulation induces apoptosis through a pathway involving the Lck family of protein tyrosine kinases (8). In HEK-293 cells
-adrenergic receptor stimulation of MAP kinases ERK1 and ERK2 requires the activation of an Src tyrosine kinase (20). In 3T3-L1 cells Gs activation inhibits adipogenesis through the protein tyrosine kinase Syc (42).
Because agents that inhibit actin polymerization significantly alter the contractile properties of smooth muscle (3, 13, 22, 43), it is likely that the actin depolymerization induced by
-adrenergic receptor activation in this study plays a role in the regulation of airway smooth muscle tone. In guinea pig trachealis a PKA-independent pathway appears to mediate airway smooth muscle relaxation (35), and Gs in a PKA-independent manner opens the Ca2+-activated K channel in airway smooth muscle (16). One may speculate that Src protein tyrosine kinases may be involved in this process, since c-Src has been shown to tyrosine phosphorylate and close this channel in vascular smooth muscle (1).
It is unlikely that nonspecific effects by the inhibitors used in this study would alter our conclusions. The inhibitors at the concentrations used in this study are relatively selective for the targets referred to in Table 1. Rp-cAMPS is a specific competitive inhibitor of PKA I and II. Although both PP2 and geldanamycin at 4080 times the concentration used in this study inhibit EGF receptors, AG-213 (a selective EGF receptor antagonist) was without effect. Wortmannin, at concentrations higher than was used in this study, inhibits both myosin light chain kinase and PI3 kinase. However, wortmannin did not alter the F/G-actin ratio in our cells, nor did the MAP kinase inhibitor PD-98059. Pertussis toxin, which ADP-ribosylates Go as well as Gi protein, again had no effect in these cells.
In conclusion, this study demonstrates that isoproterenol induces actin depolymerization in human airway smooth muscle cells by two separate signaling pathways: a PKA-dependent pathway and a PKA-independent pathway involving an Src protein tyrosine kinase.
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GRANTS
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National Heart, Lung, and Blood Institute Grant HL-62340 supported this work.
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FOOTNOTES
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Address for reprint requests and other correspondence: C. A. Hirshman, Dept of Anesthesiology, College of Physicians and Surgeons of Columbia Univ., 630 W. 168th St., P&S Box 46, New York, New York 10032 (E-mail: cah63{at}columbia.edu)
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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REFERENCES
|
---|
- Alioua A, Mahajan A, Nishimaru K, Zarei MM, Stefani E, and Toro L. Coupling of c-Src to large conductance voltage- and Ca2+-activated K+ channels as a new mechanism of agonist-induced vasoconstriction. Proc Natl Acad Sci USA 99: 1456014565, 2002.[Abstract/Free Full Text]
- Ammit AJ and Panettieri RA Jr. Invited review: the circle of life: cell cycle regulation in airway smooth muscle. J Appl Physiol 91: 14311437, 2001.[Abstract/Free Full Text]
- Battistella-Patterson AS, Wang S, and Wright GL. Effect of disruption of the cytoskeleton on smooth muscle contraction. Can J Physiol Pharmacol 75: 12871299, 1997.[CrossRef][ISI][Medline]
- Daaka Y, Luttrell LM, and Lefkowitz RJ. Switching of the coupling of the beta2-adrenergic receptor to different G proteins by protein kinase A. Nature 390: 8891, 1997.[CrossRef][ISI][Medline]
- Dong JM, Leung T, Manser E, and Lim L. cAMP-induced morphological changes are counteracted by the activated RhoA small GTPase and the Rho kinase ROKalpha. J Biol Chem 273: 2255422562, 1998.[Abstract/Free Full Text]
- Dumler I, Weis A, Mayboroda OA, Maasch C, Jerke U, Haller H, and Gulba DC. The Jak/Stat pathway and urokinase receptor signaling in human aortic vascular smooth muscle cells. J Biol Chem 273: 315321, 1998.[Abstract/Free Full Text]
- Fan J, Mansfield SG, Redmond T, Gordon-Weeks PR, and Raper JA. The organization of F-actin and microtubules in growth cones exposed to a brain-derived collapsing factor. J Cell Biol 121: 867878, 1993.[Abstract]
- Gu C, Ma YC, Benjamin J, Littman D, Chao MV, and Huang XY. Apoptotic signaling through the
-adrenergic receptor. J Biol Chem 275: 2072620733, 2000.[Abstract/Free Full Text]
- Hanke JH, Gardner JP, Dow RL, Changelian PS, Brissette WH, Weringer EJ, Pollok BA, and Connelly PA. Discovery of a novel, potent, and Src family-selective tyrosine kinase inhibitor. Study of Lck- and FynT-dependent T cell activation. J Biol Chem 271: 695701, 1996.[Abstract/Free Full Text]
- Hirshman CA and Emala CW. Actin reorganization in airway smooth muscle cells involves Gq and Gi-2 activation of Rho. Am J Physiol Lung Cell Mol Physiol 277: L653L661, 1999.[Abstract/Free Full Text]
- Hirshman CA, Togashi H, Shao D, and Emala CW. G
i-2 is required for carbachol-induced stress fiber formation in human airway smooth muscle cells. Am J Physiol Lung Cell Mol Physiol 275: L911L916, 1998.[Abstract/Free Full Text]
- Hirshman CA, Zhu D, Panettieri RA, and Emala CW. Actin depolymerization via the
-adrenoceptor in airway smooth muscle cells. A novel PKA-independent pathway. Am J Physiol Cell Physiol 281: C1468C1476, 2001.[Abstract/Free Full Text]
- Jones KA, Perkins WJ, Lorenz RR, Prakash YS, Sieck GC, and Warner DO. F-actin stabilization increases tension cost during contraction of permeabilized airway smooth muscle in dogs. J Physiol 519: 527538, 1999.[Abstract/Free Full Text]
- Knowles GC and McCulloch CAG. Simultaneous localization and quantification of relative G and F actin content: optimization of fluorescence labeling methods. J Histochem Cytochem 40: 16051612, 1992.[Abstract/Free Full Text]
- Korade-Mirnics Z and Corey SJ. Src kinase-mediated signaling in leukocytes. J Leukoc Biol 68: 603613, 2000.[Abstract/Free Full Text]
- Kume H, Mikawa K, Takagi K, and Kotlikoff MI. Role of G proteins and KCa in the muscarinic and
-adrenergic regulation of airway smooth muscle. Am J Physiol Lung Cell Mol Physiol 268: L221L229, 1995.[Abstract/Free Full Text]
- Laniyonu A, Eto S, Wang JH, and Hollenberg MD. Detection of sarcoma virus family tyrosine kinase activity in coronary arterial tissue. Can J Physiol Pharmacol 73: 15521560, 1995.[ISI][Medline]
- Lesh RE, Emala CW, Lee HT, Zhu D, and Hirshman CA. Inhibition of geranylgeranylation blocks agonist-induced actin reorganization in human airway smooth muscle cells. Am J Physiol Lung Cell Mol Physiol 281: L824L831, 2001.[Abstract/Free Full Text]
- Lopez-Ilasaca M, Crespo P, Pellici PG, Gutkind JS, and Wetzker R. Linkage of G protein-coupled receptors to the MAPK signaling pathway through PI 3-kinase. Science 275: 394397, 1997.[Abstract/Free Full Text]
- Luttrell LM, Ferguson SS, Daaka Y, Miller WE, Maudsley S, Della Rocca GJ, Lin F, Kawakatsu H, Owada K, Luttrell DK, Caron MG, and Lefkowitz RJ. Beta-arrestin-dependent formation of beta2 adrenergic receptor-Src protein kinase complexes. Science 283: 655661, 1999.[Abstract/Free Full Text]
- Ma YC, Huang J, Ali S, Lowry W, and Huang XY. Src tyrosine kinase is a novel direct effector of G proteins. Cell 102: 635646, 2000.[CrossRef][ISI][Medline]
- Mehta D and Gunst SJ. Actin polymerization stimulated by contractile activation regulates force development in canine tracheal smooth muscle. J Physiol 519: 829840, 1999.[Abstract/Free Full Text]
- Mills JW, Falsig PS, Walmod PS, and Hoffmann EK. Effect of cytochalasins on F-actin and morphology of Ehrlich ascites tumor cells. Exp Cell Res 261: 209219, 2000.[CrossRef][ISI][Medline]
- Mimnaugh EG, Chavany C, and Neckers L. Polyubiquitination and proteasomal degradation of the p185c-erbB-2 receptor protein-tyrosine kinase induced by geldanamycin. J Biol Chem 271: 2279622801, 1996.[Abstract/Free Full Text]
- Morris AJ and Malbon CC. Physiological regulation of G protein-linked. Physiol Rev 79: 13731430, 1999.[Abstract/Free Full Text]
- Narumiya S, Sugimoto Y, and Ushikubi F. Prostanoid receptors: structures, properties, and functions. Physiol Rev 79: 11931226, 1999.[Abstract/Free Full Text]
- Nebl G, Fischer S, Penzel R, and Samstag Y. Dephosphorylation of cofilin is regulated through Ras and requires the combined activities of the Ras-effectors MEK and PI3K. Cell Signal 16: 235243, 2004.[CrossRef][ISI][Medline]
- Neer EJ and Clapham DE. Roles of G protein subunits in transmembrane signalling. Nature 333: 129134, 1988.[CrossRef][ISI][Medline]
- Oda Y, Renaux B, Bjorge J, Saifeddine M, Fujita DJ, and Hollenberg MD. cSrc is a major cytosolic tyrosine kinase in vascular tissue. Can J Physiol Pharmacol 77: 606617, 1999.[CrossRef][ISI][Medline]
- Panettieri RA, Murray RK, DePalo LR, Yadvish PA, and Kotlikoff MI. A human airway smooth muscle cell line that retains physiological responsiveness. Am J Physiol Cell Physiol 256: C329C335, 1989.[Abstract/Free Full Text]
- Penn RB, Pascual RM, Kim YM, Mundell SJ, Krymskaya VP, Panettieri RA Jr, and Benovic JL. Arrestin specificity for G protein-coupled receptors in human airway smooth muscle. J Biol Chem 276: 3264832656, 2001.[Abstract/Free Full Text]
- Rao JY, Bonner RB, Hurst RE, Liang YY, Reznikoff CA, and Hemstreet GP III. Quantitative changes in cytoskeletal and nuclear actins during cellular transformation. Int J Cancer 70: 423429, 1997.[CrossRef][ISI][Medline]
- Salazar EP and Rozengurt E. Src family kinases are required for integrin-mediated but not for G protein-coupled receptor stimulation of focal adhesion kinase autophosphorylation at Tyr-397. J Biol Chem 276: 1778817795, 2001.[Abstract/Free Full Text]
- Singer CA, Vang S, and Gerthoffer WT. Coupling of M2 muscarinic receptors to Src activation in cultured canine colonic smooth muscle cells. Am J Physiol Gastrointest Liver Physiol 282: G61G68, 2002.[Abstract/Free Full Text]
- Spicuzza L, Belvisi MG, Birrell MA, Barnes PJ, Hele DJ, and Giembycz MA. Evidence that the anti-spasmogenic effect of the beta-adrenoceptor agonist, isoprenaline, on guinea-pig trachealis is not mediated by cyclic AMP-dependent protein kinase. Br J Pharmacol 133: 12011212, 2001.[CrossRef][ISI][Medline]
- Symons MH and Mitchison TJ. Control of actin polymerization in live and permeabilized fibroblasts. J Cell Biol 114: 503513, 1991.[Abstract]
- Thomas SM and Brugge JS. Cellular functions regulated by Src family kinases. Annu Rev Cell Dev Biol 13: 513609, 1997.[CrossRef][ISI][Medline]
- Thomas SM, Soriano P, and Imamoto A. Specific and redundant roles of Src and Fyn in organizing the cytoskeleton. Nature 376: 267271, 1995.[CrossRef][ISI][Medline]
- Togashi H, Emala CW, Hall IP, and Hirshman CA. Carbachol-induced actin reorganization involves Gi activation of Rho in human airway smooth muscle cells. Am J Physiol Lung Cell Mol Physiol 274: L803L809, 1998.[Abstract/Free Full Text]
- Touyz RM, Wu XH, He G, Salomon S, and Schiffrin EL. Increased angiotensin II-mediated Src signaling via epidermal growth factor receptor transactivation is associated with decreased C-terminal Src kinase activity in vascular smooth muscle cells from spontaneously hypertensive rats. Hypertension 39: 479485, 2002.[Abstract/Free Full Text]
- Wan Y, Kurosaki T, and Huang XY. Tyrosine kinases in activation of the MAP kinase cascade by G-protein-coupled receptors. Nature 380: 541544, 1996.[CrossRef][ISI][Medline]
- Wang HY and Malbon CC. G(s)alpha repression of adipogenesis via Syk. J Biol Chem 274: 3215932166, 1999.[Abstract/Free Full Text]
- Wright G and Hurn E. Cytochalasin inhibition of slow tension increase in rat aortic rings. Am J Physiol Heart Circ Physiol 267: H1437H1446, 1994.[Abstract/Free Full Text]
- Xu W, Doshi A, Lei M, Eck MJ, and Harrison SC. Crystal structures of c-Src reveal features of its autoinhibitory mechanism. Mol Cell 3: 629638, 1999.[CrossRef][ISI][Medline]
- Yamaki H, Iguchi-Ariga SM, and Ariga H. Inhibition of c-myc gene expression in murine lymphoblastoma cells by geldanamycin and herbimycin, antibiotics of benzoquinoid ansamycin group. J Antibiot (Tokyo) 42: 604610, 1989.[ISI][Medline]
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