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Published ahead of print on April 22, 2004, doi:10.1165/rcmb.2003-0272OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 31, pp. 266-275, 2004
© 2004 American Thoracic Society
DOI: 10.1165/rcmb.2003-0272OC

Phophatidylinositol-3 Kinase/Mammalian Target of Rapamycin/p70S6K Regulates Contractile Protein Accumulation in Airway Myocyte Differentiation

Andrew J. Halayko, Sreedharan Kartha, Gerald L. Stelmack, John McConville, John Tam, Blanca Camoretti-Mercado, Sean M. Forsythe, Marc B. Hershenson and Julian Solway

Departments of Medicine and Pediatrics, University of Chicago, Chicago, Illinois; Department of Physiology, and Section of Respiratory Diseases (Asthma/COPD Research Centre), University of Manitoba, and Biology of Breathing Group, Manitoba Institute of Child Health, Winnipeg, Manitoba, Canada; and Departments of Pediatrics and Communicable Diseases, Molecular and Integrative Physiology, University of Michigan, Ann Arbor, Michigan

Address correspondence to: Andrew J. Halayko, Ph.D., University of Manitoba, Section of Respiratory Diseases, RS321-810 Sherbrook Street, Winnipeg, MB, R3A 1R8 Canada. E-mail: ahalayk{at}cc.umanitoba.ca


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Increased airway smooth muscle in airway remodeling results from myocyte proliferation and hypertrophy. Skeletal and vascular smooth muscle hypertrophy is induced by phosphatidylinositide-3 kinase (PI(3) kinase) via mammalian target of rapamycin (mTOR) and p70S6 kinase (p70S6K). We tested the hypothesis that this pathway regulates contractile protein accumulation in cultured canine airway myocytes acquiring an elongated contractile phenotype in serum-free culture. In vitro assays revealed a sustained activation of PI(3) kinase and p70S6K during serum deprivation up to 12 d, with concomitant accumulation of SM22 and smooth muscle myosin heavy chain (smMHC) proteins. Immunocytochemistry revealed that activation of PI3K/mTOR/p70S6K occurred almost exclusively in myocytes that acquire the contractile phenotype. Inhibition of PI(3) kinase or mTOR with LY294002 or rapamycin blocked p70S6K activation, prevented formation of large elongated contractile phenotype myocytes, and blocked accumulation of SM22 and smMHC. Inhibition of MEK had no effect. Steady-state mRNA abundance for SM22 and smMHC was unaffected by blocking p70S6K activation. These studies provide primary evidence that PI(3) kinase and mTOR activate p70S6K in airway myocytes leading to the accumulation of contractile apparatus proteins, differentiation, and growth of large, elongated contractile phenotype airway smooth muscle cells.

Abbreviations: Dulbecco's modified Eagle's medium, DMEM • glycogen synthase kinase-3ß, GSK-3ß • insulin-like growth factor-1, IGF-1 • mammalian target of rapamycin, mTOR • 3-phosphoinositide–dependent protein kinase, PDK1 • phosphatidylinositol-3 kinase, PI(3) kinase • smooth muscle myosin heavy chain, smMHC


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
It is well established that the airways from humans with asthma undergo structural remodeling marked by chronic inflammation, subepithelial fibrosis, and excessive accumulation of contractile airway smooth muscle (1). Airway smooth muscle thickening results from myocyte hyperplasia and hypertrophy in central and small airways (2), and is a principal factor underlying excessive airways narrowing in asthma. Though there has been considerable investigation of molecular signals that regulate myocyte proliferation (3) and of transcription of genes encoding contractile apparatus-associated proteins (4–6), the signaling mechanisms that regulate synthesis and accumulation of contractile apparatus proteins during cellular hypertrophy and differentiation have not been studied in depth (7). Accumulation of smooth muscle proteins is an important component of the normal differentiation of the airways and vasculature, and is associated with smooth muscle cell hypertrophy and growth in the bladder and uterus, and during pathogenesis of atherosclerosis, and systemic and pulmonary hypertension.

Signaling cascades downstream from phosphatidylinositol-3 kinase (PI(3) kinase) play a principal role in mediating protein synthesis and hypertrophy of vascular smooth muscle and skeletal muscle cells (8–10). In addition, activation of PI(3) kinase by insulin-like growth factor-1 (IGF-1) inhibits the loss of contractile apparatus-associated proteins by contractile vascular and visceral smooth muscle cells in vitro (11, 12). Activation of PI(3) kinase, in response to ligands such as angiotensin II, epidermal growth factor, insulin, and IGF-1, generates membrane phospholipids that induce activity of 3-phosphoinositide–dependent protein kinase (PDK1) (13), which in turn activates the serine/threonine kinase Akt1 (13), leading to regulation of a large number of further downstream protein targets involved with cellular processes including protein synthesis, glucose metabolism, proliferation, migration, and apoptosis (14).

Downstream targets of PI(3) kinase that are associated with promoting protein synthesis and accumulation include glycogen synthase kinase-3ß (GSK-3ß), p70S6 kinase (p70S6K), and PHAS-1/4E-BP (14, 15). Akt1 phosphorylates and inhibits GSK-3ß resulting in downstream de-inhibition of the translation initiator eIF2 (16, 17). Akt1 can also phosphorylate, and in part activate, the rapamycin-sensitive threonine/serine kinase, mammalian target of rapamycin (mTOR), which has as downstream targets both p70S6K and the translation repressor PHAS-1/4E-BP1 (18–20). Previous work shows that p70S6K activation, which regulates efficiency of protein translation through phosphorylation of the 40S ribosomal protein S6 (21, 22), is required for PI(3) kinase-mediated differentiation and hypertrophy of skeletal myotubes (8, 15) and for angiotensin II–induced vascular smooth muscle hypertrophy (10). Though the principal site required for mTOR-dependent activation of p70S6K is Thr389, which resides in a region between catalytic and auto-inhibitory domains (23), full activation of p70S6K is achieved through hierarchical phosphorylation of seven Ser/Thr sites targeted by mTOR, PDK1, and other PI(3) kinase–dependent kinases (24).

Despite the knowledge that signaling downstream from PI(3) kinase and mTOR plays a critical role in IGF-1 and angiotensin II–induced vascular smooth muscle and skeletal muscle hypertrophy (8, 10, 15), little is known about how these pathways and the factors that induce them may contribute to airway smooth muscle hypertrophy and differentiation. We previously described a novel insulin-supplemented serum-free primary cultured canine airway smooth system in which a subset of large, elongated contractile myocytes develop with 5- to 7-fold increase in the abundance of contractile smooth muscle phenotype-specific proteins such as smooth muscle myosin heavy chain (smMHC), and the actin binding protein SM22 (25, 26). We used this culture model to test the hypothesis that PI(3) kinase and downstream signaling pathways are required for the regulated accumulation of contractile-phenotype proteins during myocyte elongation and differentiation that occurs during prolonged serum deprivation. Our studies indicate that PI(3) kinase and p70S6K exhibit sustained and significant activation concomitant with the accumulation of SM22 and smMHC. Moreover, accumulation of these contractile proteins and formation of large, elongated fully contractile myocytes was prevented by treatment with pharmacologic inhibitors of either PI(3) kinase or mTOR, was independent of ERK pathway activation, and occurred in the absence of increased steady-state abundance of mRNAs encoding SM22 and smMHC. Collectively these data provide evidence that PI(3) kinase and mTOR signaling are required for differentiation and growth of contractile airway smooth muscle cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Airway Smooth Muscle Culture
Canine airway smooth muscle cell primary cultures were established with myocytes enzymatically-dissociated from trachealis muscle from adult mongrel dogs as we have previously described (4, 27). For all experiments second passage cultures were used. To induce a functionally contractile phenotype in ~ 1/6 of cultured myocytes, confluent cultures were maintained in serum-free medium (25, 28). Briefly, myocyte cultures were grown to confluence on uncoated plastic culture plates or glass coverslips in Dulbecco's modified Eagle's medium (DMEM)/F-12 medium containing 10% fetal bovine serum, then the cells were maintained in serum-free DMEM/F-12 supplemented with ITS (insulin 5 µg/ml, transferrin 5 µg/ml, selenium 5 ng/ml) for up to 14 d. In some experiments, rapamycin (10 nM), LY290004 (20 µM), or PD98058 (20 µM) was added during serum deprivation.

Western Analysis
Culture protein lysates studied were prepared in Tris-SDS buffer, size fractionated by SDS-PAGE, then electroblotted to nitrocellulose (25, 27). Blots were immunolabeled using a biotin-conjugated streptavidin–horseradish peroxidase or horseradish peroxidase–conjugated secondary antibody strategy and developed on film using ECL Plus reagents (Amersham, Piscataway, NJ). Primary antibodies used included, mouse monoclonal anti–smooth muscle myosin heavy chain (hSMv) and mouse anti–ß-actin (Sigma, St. Louis, MO), rabbit anti-SM22 (29), rabbit-anti-phospho-Thr308-Akt1/PKB{alpha} (Upstate Biotech Inc., Lake Placid, NY), and rabbit anti-Akt1 (Cell Signaling Tech., Beverly, MA). Densitometry to measure relative band intensity was performed using a Hewlett Packard scanner and Scanplot Software as described (30).

Northern Analysis
Whole cell RNA was prepared using a single step phenol/guanidinium thiocyanate method using RNA STAT-60 (Tel-Test, Inc., Friendswood, TX). RNA (20 µg) was size fractionated by electrophoresis using a 1.2% agarose-formaldehyde gel then transferred to Hybond N+ membrane (Amersham). 32P-labeled probes were generated by random primer labeling of cDNAs for SM22 and smMHC mRNA. Prehybridization and hybridization were completed at 55°C in ExpressHyb (BD Biosciences Clontech, Mississauga, ON, Canada), then membranes were washed with 1X SSC/ 0.5% SDS and 0.1X SSC at 55°C and subjected to autoragiography. Relative band intensity on films was measured using a Hewlett Packard scanner and Scanplot Software (30).

Immunocytochemistry
Airway myocytes were seeded onto pre-cleaned, uncoated glass coverslips in culture dishes. Cells were grown to confluence and then maintained in serum-free medium for 7 d as described above, in the presence or absence of rapamycin (10 nM). Cells were then fixed, permeablized, and immunolabeled as described (4, 25), using mouse-anti-smMHC (clone hSMv) (Sigma) (dilution 1:100) alone, and in combination with rabbit anti-phosphorylated Akt1(Ser473) (dilution 1:100) or rabbit-anti-phosphorylated p70S6K(Thr421/Ser424) (dilution 1:100; Cell Signaling Tech.). FITC- and Cy3-conjugated secondary antibodies (dilution 1:100; Jackson ImmunoResearch, West Grove, PA) were used to detect primary antibody bound to labeled cells. Nuclei were labeled with Hoechst 33342 (10 µg/ml). Coverslips were mounted using anti-fade medium and digitally imaged (25).

S6 Kinase Activity
Phosphorylation of p70S6K at Thr 389 was measured by immunoblot assay (Upstate Biotechnology); Thr 389 is a site targeted for phosphorylation by mTOR, whose phosphorylation is required for full function of S6 kinase (20, 21, 31). Total p70S6K was assayed in parallel by Western Blot using rabbit-anti-p70S6K (Cell Signaling Tech.). Protein lysates were made from myocyte cultures using RIPA buffer (40 mM Tris, 1% deoxycholic acid, 150 mM NaCl, 1% NP-40, pH 8.0) containing protease inhibitors (1 mM PMSF, and 5 µg/ml each of pepstatin, leupetin, and aprotinin) and phosphatase inhibitors (1 mM Na3VO4, and 20 mM NaF). Total protein content was determined using the Bio-Rad Protein Assay Kit based on the Bradford method. Lysates were diluted 1:4 in 5x SDS-loading buffer, size fractionated by 10% SDS-PAGE, and subjected to immunoblotting using the anti–phospho-p70S6K antibody (diluted 1:200; 20 µg of total protein lysate were loaded for each gel lane).

The specific enzymatic activity of p70S6K was measured by an in vitro assay according manufacturer's instructions (Upstate Biotechnology). This assay monitors phosphorylation of a specific substrate (AKRRRLSSLRA) modeled after the major phosphorylation sites in S6 kinase. All steps were performed at 4°C unless noted otherwise. Cells from 100-mm dishes were extracted in 200 µl RIPA buffer then incubated overnight at with 1 µl anti-p70S6K antibody (Santa Cruz, Santa Cruz, CA). The lysates were then incubated with 30 µl of Protein A-sepharose beads for 2 h to immunoprecipitate immune complexes. Beads were washed three times with RIPA buffer and once with assay dilution buffer supplied by the manufacturer, before resuspending the beads in 20 µl assay dilution buffer. Thereafter, 10 µl of substrate, 10 µl 32P-ATP (10 µCi), and 10 µl inhibitor cocktail (20 µM PKC inhibitor peptide, 2 µM protein kinase A inhibitor peptide, 20 µM R24571 [calmodulin-dependent kinase inhibitor]) were added samples were incubated for 10 min (30°C). Immediately thereafter, 25 µl of sample mixture were spotted to P81 paper, which was then washed three times with 0.75% phosphoric acid, and then once with acetone. Each square was transferred to a scintillation vial and its radioactivity measured as CPM.

PI(3)-Kinase Activity Assay
Following established methods (32), myocyte cultures in 100-mm dishes were extracted at 4°C with 100 µl RIPA buffer then stored on ice. An aliquot (30 µl) of Protein G beads, which had been incubated with anti-phosphotyrosine (4G10) (Upstate Biotechnology) (100 µl beads with 20 µl antibody) overnight then washed in RIPA buffer, was added to 200 µl of cell extract and gently shaken for 2 h at 4°C. The beads were washed with RIPA buffer, three times with PI(3)-kinase buffer (10 mM Tris, pH 7.5, 0.2 mM EGTA, 100 mM NaCl, 2 mM MgCl2), and then resuspended in 50 µl of kinase buffer. Thereafter, freshly sonicated phosphatidyl inositol (250 µg), phosphatidyl inositol monophosphate (250 µg), and phosphatidyl serine (50 µg) were added, and 10 µCi of 32P-ATP to start the reaction. The mixture was incubated for 30 min (30°C), then the reaction was stopped by adding 100 µl chloroform:methanol:HCl (100:200:2), then after mixing well 100 µl of chloroform and then 100 µl distilled water were added. Lipids were twice extracted with chloroform:methanol:HCl (2:1:2), then the lipid layer was dried. For chromatographic separation, the lipids were dissolved in chloroform:methanol (95:5) and phosphatidylinositol-3-phosphate (PIP) was resolved by silica gel thin layer chromatography using 1-propanol:2M acetic acid (65:35, vol:vol) as solvent system. The dried plates were then subjected to autoradiography, and spots corresponding to PIPs were quantified by densitometry (30).

Statistical Analysis
Data are presented as mean ± SEM. Comparisons between groups were made as indicated either by unpaired Student's t test, or by Kruskal-Wallis one-way ANOVA with post hoc Dunn test. Statistical tests were performed using Instat Software 2.0 (GraphPad Software, San Diego, CA). P values < 0.05 were considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Time Course for Accumulation of Contractile Phenotype mRNA and Proteins
The abundance of mRNA for smMHC and SM22 was determined by Northern blot analysis (Figure 1). Tracheal myocytes were plated at ~ 30% confluence and grown to 100% confluence in the presence of 10% serum, and then maintained in ITS-supplemented serum-free medium for up to 12 d. Transcript abundance increased by more than 2-fold to a near-maximum plateau during the phase of rapid cell proliferation between initial myocyte seeding (Day –7) and confluence (Day 0). Thereafter high levels of mRNA for SM22 and smMHC were maintained for at least 12 d after serum withdrawal from confluent cultures. This pattern differs markedly from the temporal pattern of SM22 and smMHC protein accumulation we have shown previously (25). Whereas mRNA for SM22 and smMHC accumulate to highest levels near confluence in serum-fed conditions, at confluence the abundance of the proteins they encode is less than 15% of the highest levels reached in serum-free culture. In addition, though accumulation of SM22 and smMHC protein occurs in parallel with development of large, elongated functional contractile myocytes in serum-free conditions, the abundance of corresponding mRNA does not increase during the same time. The earlier increase in mRNA undoubtedly reflects the high transcriptional activities of the SM22 and smMHC promoters in subconfluent, serum-fed cells, which in turn falls by over 80% in 7-d serum-deprived cultures (4). Importantly, our observation of dissociation between the abundance of mRNA and protein for SM22 and smMHC in confluent myocytes before serum deprivation indicates that subsequent contractile protein accumulation is not solely controlled through regulation of mRNA levels; rather, pathways regulating post-transcriptional events in serum-free cultures are likely involved.



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  		Figure 1. Northern blot analysis reveals that mRNA for smMHC and SM22 accumulate in serum-fed canine airway myocyte primary cultures as they grow to confluence, and transcript levels remain high for at least 12 d thereafter in ITS-supplemented serum-free culture conditions. (A) Representative autoradiogram (from two independent experiments) showing mRNA for SM22 and smMHC using the culture conditions described in MATERIALS AND METHODS. Cells from a P0 primary culture were re-seeded at ~ 25% confluence in DMEM/F-12 + 10% fetal bovine serum media, and total RNA was collected beginning 24 h later (corresponding to Day –7), and thereafter when cultures reached ~ 70% confluence (Day –4), ~ 100% confluence (Day 0) (at which time media was changed to a serum-free formulation as indicated by the arrow), and thereafter on Day 4, Day 8, and Day 12 of culture in serum-free conditions. Ethidium bromide (EtBr) staining of the gel prior to membrane transfer is shown in the bottom panel to confirm equal loading of different samples. (B) Comparison of mRNA abundance by densitometry for the Northern blot shown in A. Transcript abundance is expressed as relative units normalized to the value measured for the corresponding mRNA at Day –7 (thus both SM22 and smMHC abundance is 1.0 at Day –7). Broken line with triangle symbols, SM22; Solid line with circle symbols, smMHC.

 
The PI(3) Kinase/mTOR/p70S6K Pathway Is Activated during Prolonged Serum Deprivation of Canine Tracheal Smooth Muscle Cells
As previous work has demonstrated a critical role for PI(3) kinase signaling during skeletal myotube formation and hypertrophy (15, 33) and in vascular smooth muscle cell differentiation (10), we investigated the possibility that this pathway might be involved with growth and differentiation of airway myocytes maintained in ITS-supplemented serum-free cultures. Total PI(3) kinase activity was measured by in vitro assay using anti-phosphotyrosine immunoprecipitates from prolonged serum deprived canine tracheal myocyte cultures (Figure 2A). In individual experiments a substantial (~ 20-fold) increase in PI(3) kinase activity developed at 2 or 5 d of serum-free culture, and elevated enzymatic activity was maintained for at least 9 d (Figure 2C). To further confirm the activation of PI(3) kinase signaling in serum-free culture, we used an immunoblot assay to measure PDK1-mediated phosphorylation of Akt1 at Thr308 in the catalytic domain (Figure 2B). Increased Akt1 phosphorylation mimicked the temporal pattern of PI(3) kinase activation, with no change in total Akt1 abundance. In additional studies, a specific pharmacologic inhibitor of PI(3) kinase (LY294002, 20 µM), was included during serum-free culture conditions. Addition of LY294002 completely abolished the increase in activity of PI(3) kinase that was otherwise induced during prolonged culture in ITS-containing serum-free media (Figure 2C).



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  		Figure 2. Serum deprivation leads to prolonged activation of PI(3) kinase and its downstream signaling effector Akt1 in canine airway myocyte primary cultures. (A) In vitro measurement of total PI(3) kinase activity in anti-phosphotyrosine immmunoprecipitates prepared from cell lysates of canine myocyte cultures in ITS-supplemented serum-free culture for 0–9 d. A typical thin layer chromatography autoradiogram is shown that demonstrates the generation of radiolabeled PIP by PI(3) kinase. (B) Immunoblot assay reveals the sustained phosphorylation of Akt1 at Thr308 (labeled as pAkt1 in the upper panel) for up to 12 d after confluent canine tracheal cultures are switched to serum-free conditions. Total Akt1 in the same samples is shown in the lower panel. Blots are representative of three independent experiments. A quantity of 20 µg of total protein lysate was loaded in each lane. Proteins were separated by 10% SDS-PAGE. (C) Quantification of in vitro PI(3) kinase activity by densitometry of PIP detected on autoradiograms. LY294002 (20 µM) blocked the sustained elevation in PI(3) kinase seen during prolonged serum deprivation. Shaded bars, control; solid bars, LY294002. For each experiment, data were normalized to the activity on Day 0. The values are means from three independent experiments ± SE. *P < 0.05 compared with untreated, time-matched controls using Kruskal-Wallis one way ANOVA and Dunn Test.

 
Because p70S6K is an essential downstream signaling intermediate for PI(3) kinase–mediated skeletal myotube differentiation and hypertrophy (8, 15), we postulated that it may also be involved with contractile protein accumulation during development of large contractile myocytes during prolonged serum deprivation of canine tracheal myocytes. Thus, we first used an immunoblot assay to assess the activation of p70S6K by mTOR, which is a signaling intermediate in the PI(3) kinase cascade (15). Figure 3A shows that phosphorylation of p70S6K at Thr389, a site in the regulatory domain targeted by mTOR, increased markedly by 2 d of serum withdrawal and persisted for at least 7 d; total p70S6K abundance was unchanged during the same period. We next quantified the enzymatic activity of p70S6K immunoprecipitated from lysates of cultured canine tracheal myocytes after 0, 2, 5, and 7 d of growth in serum-free media using an in vitro enzyme assay. In parallel with the immunoblot assay described above, p70S6K activity was relatively low in 100% confluent (Day 0) cells prior to serum deprivation, it increased significantly by 2 d after serum deprivation, and this elevated level was maintained for at least 7 d (Figure 3B). In additional studies, we included specific pharmacologic inhibitors of PI(3) kinase or mTOR, LY294002 (20 µM) and rapamycin (10 nM), respectively, in serum-free cultures and assessed their individual effects on p70S6K activation. Figure 3B shows that inhibition of PI(3) kinase and mTOR completely blocked p70S6K activation during ITS-supplemented serum-free culture, thus confirming their role as required upstream regulators of p70S6K activity in airway smooth muscle cells.



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  		Figure 3. Serum deprivation leads to prolonged activation of p70S6K in canine airway myocyte primary cultures. (A) Immunoblot assay (upper panel) revealing a sustained phosphorylation of Thr389 on p70S6K (labeled phospho-p70S6K) for at least 7 d after confluent cultures are switched to serum-free media. Total p70S6K in the same samples is shown in the lower panel. Blots are representative of three independent experiments. A quantity of 20 µg of total protein lysate was loaded in each lane. Proteins were separated by 10% SDS-PAGE. (B) In vitro p70S6K assay of cell lysates taken from confluent myocyte cultures and up to 7 d thereafter in serum-deprived conditions. The addition of LY294002 (20 µM) or rapamycin (10 nM) to inhibit PI(3) kinase or mTOR, respectively, abrogated p70S6K activity in serum-free conditions. Data from each experiment were normalized to activity on Day 0. Shaded bars, control; solid bars, LY294002; slashed bars, rapamycin. The values shown are means from three independent experiments ± SE. *P < 0.05 compared with untreated, time-matched controls using Kruskal-Wallis one-way ANOVA and Dunn Test.

 
The PI(3) Kinase/mTOR/p70S6K Signaling Cascade Is Activated Selectively in Airway Myocytes that Accumulate Contractile Proteins during Prolonged Serum Deprivation
As only a subpopulation of primary cultured airway myocytes acquire an elongate, contractile phenotype with prolonged serum deprivation (25), we employed immunocytochemistry to more definitively characterize the level of PI(3) kinase–mediated signaling in individual myocytes. PI3K/PDK1-induced activation of Akt1 was assessed by a double labeling strategy for smMHC and Ser473-phosphorylated Akt1 (pSer473-Akt1) in myocytes serum deprived form up to 10 d (Figure 4). Cytochemical analyses showed that whereas pSer473-Akt1 was homogeneously distributed in the cytoplasm of all myocytes in confluent cultures before serum deprivation, a marked increase in cytoplasmic pSer473-Akt1 became evident almost exclusively in the subset of myocytes that accumulated smMHC during serum deprivation. Furthermore, the intensity of staining for pSer473-Akt1 appeared to become reduced in the myocyte subpopulation that failed to acquire a contractile phenotype in serum-free culture. In similar experiments the activation of p70S6K was also investigated, employing a double labeling strategy for smMHC and phospho-Thr421/Ser424 p70S6K (Figure 5). Consistent with studies examining Akt1 activation (Figure 4), phospho-p70S6K was selectively increased in the cytoplasm of myocytes that became elongated and accumulated abundant smMHC over 7 d serum deprivation. In addition to cytoplasmic labeling, abundant nucleus-associated phosphorylated p70S6K that did not appear to change with serum deprivation, and that did not distinguish elongate and nonelongate myocytes, was readily evident. It is likely that nuclear S6 kinase labeling is largely due to the presence of p85S6K that can be detected with the antibody we used, and has been reported to have an exclusively nuclear localization pattern (34). Collectively our immunocytochemical analyses confirm that PI3K/mTOR/p70S6K signaling is significantly and selectively activated in the subpopulation of airway myocytes that become elongate and accumulate contractile apparatus–associated proteins during prolonged serum-free culture.



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  		Figure 4. PI(3) kinase–mediated signaling via Akt1 is selectively activated in airway myocytes concomitant with elongation, and accumulation of smMHC during prolonged serum deprivation. Images represent matched double fluorescent immunocytochemistry for smMHC (red) (A, C, E) and Ser473-phosphorylated Akt1 (pS473-Akt1) (B, D, F) at Day 0 (A, B), Day 5 (C, D), and Day 10 (E, F) in ITS-supplemented serum-free culture medium. Secondary antibodies used were Cy3-conjugated (smMHC) or FITC-conjugated (pSer473Akt1) and nuclei are labeled with Hoechst 33342 (blue). Arrows indicate myocytes that develop a smMHC-rich elongate phenotype. Bar = 30 µm.

 


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  		Figure 5. p70S6K is selectively activated in airway myocytes concomitant with elongation and accumulation of smMHC during prolonged serum deprivation. Images represent matched double fluorescent immunocytochemistry for smMHC (red) (A, C) and Thr421/Ser424-phosphorylated p70S6K (pThr421/pSer424-p70S6K) (B, D) at Day 0 (A, B) and Day 7 (C, D) in ITS-supplemented serum-free culture medium. Secondary antibodies used were Cy3-conjugated (smMHC) or FITC-conjugated (pThr421/pSer424-p70S6K). Arrows indicate myocytes that develop a smMHC-rich elongate phenotype. Bar = 30 µm.

 
PI(3) Kinase/mTOR Signaling Is Required for Contractile Protein Accumulation and Differentiation of Large Contractile Airway Smooth Muscle Cells
Having confirmed the concomitant induction of PI(3) kinase signaling through mTOR and p70S6K during prolonged serum deprivation, we next tested whether activation of these signaling effectors was necessary for differentiation of contractile phenotype airway myocytes. Our previous studies showed that a distinct subpopulation of ~ 20% of airway myocytes acquire a functionally contractile phenotype characterized by a large elongated morphology and an abundance of contractile apparatus proteins such as smMHC and SM22 during prolonged culture in serum-free conditions (25). Figures 6A and 6C demonstrate the concomitant increase in size and accumulation of the contractile protein smMHC that occurs in these myocytes during prolonged serum deprivation. Treatment of serum-deprived cultures with rapamycin (10 nM) for up to 7 d to prevent mTOR-mediated p70S6K activity appeared to completely block both the development of morphologically large elongate myocytes and the accumulation smMHC (Figure 6B and 6D). These effects of rapamycin appeared to be somewhat specific, as concomitant immunoblot analysis (Figures 7 and 8) showed that ß-actin protein abundance was not altered by rapamycin treatment. Also, additional studies revealed rapamycin had no influence on de novo synthesis of ß-galactosidase from plasmid expression vectors during transient transfection of serum-fed, subconfluent canine tracheal smooth muscle cells (data not shown). Consistent with previous reports using cultured skeletal muscle cells (8), prolonged treatment of serum-deprived airway myocytes with rapamycin did not lead to cell detachment or a significant loss of cell viability. Thus the qualitative results in Figure 6 strongly suggest that differentiation of contractile airway smooth muscle cells requires prolonged mTOR-dependent activation of p70S6K.



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  		Figure 6. Inhibition of mTOR and p70S6K prevents differentiation of primary cultured canine tracheal myocytes in prolonged serum-free culture. Cultures were grown to confluence then maintained in serum-free conditions in the absence or presence of rapamycin (10 nM) for 7 d. (A) Phase contrast micrograph showing bundles of differentiated elongate canine tracheal myocytes (indicated by arrows) that develop in prolonged serum-free cultures. Bar = 30 µm. (B) Phase contrast micrograph showing the effects rapamycin on prolonged serum-free cultures. Note the absence of the typical elongate, phase dark contractile myocytes seen in A. Bar = 30 µm. (C) Fluorescent immunocytochemistry for smMHC (green), which accumulates in elongate, contractile phenotype myocytes that develop during prolonged serum deprivation (indicated by arrows). Nuclei are labeled with Hoechst 33342 (blue). Bar = 10 µm. (D) Fluorescent immunocytochemistry for smMHC (green) in myocytes from 7-d serum-free cultures treated with rapamycin (10 nM). Note the absence of elongate smMHC rich myocytes compared with that seen in C. Nuclei are labeled with Hoechst 33342 (blue). Images are representative of those obtained in three independent experiments. Bar = 10 µm.

 


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  		Figure 7. Inhibition of mTOR or PI(3) kinase during prolonged serum deprivation of canine tracheal myocyte cultures blocks accumulation of SM22 associated with differentiation to a functionally contractile state. (A) Western blots showing SM22 (upper panel) and ß-actin (lower panel) abundance in whole cell lysates from confluent canine myocyte cultures maintained in serum-free conditions for 0 to 7 d, and in serum-free cultures that included either LY294002 (20 µM) or rapamycin (Rapa) (10 nM). Proteins were size fractionated by 12% SDS-PAGE, and 10 µg of total cell lysate was loaded in each lane. (B) Comparison of SM22 abundance by densitometry revealed that both LY294002 and rapamycin significantly inhibited accumulation during prolonged serum-free culture (solid circles, no drug; solid squares, LY294002; solid triangles, rapamycin). For each experiment data were normalized to Day 0. The values shown are means from three independent experiments ± SE. *P < 0.05 compared with control, time-matched controls using Kruskal-Wallis one-way ANOVA and Dunn Test.

 


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  		Figure 8. The accumulation of smMHC associated with differentiation to a functionally contractile state during prolonged serum deprivation of canine tracheal myocyte cultures is blocked by inhibitors of mTOR or PI(3) kinase, but is unaffected by an inhibitor of ERK. (A) Western blot for smMHC (upper panel) and ß-actin (lower panel) in whole cell lysates from confluent canine myocyte cultures in the absence and presence of LY294002 (20 µM) in serum-free conditions for 0–7 d. (B) Western Blot for smMHC (upper panel) and ß-actin (lower panel) in whole cell lysates from confluent canine myocyte cultures maintained in serum-free conditions, or in serum-free conditions containing either rapamycin (Rapa) (10 nM) or PD98058 (PD) (20 µM) for 0–9 d. Proteins were size fractionated by 6% SDS-PAGE, and 10 µg of total cell lysate was loaded in each lane. (B) Comparison of smMHC abundance by densitometry revealed that both LY294002 and rapamycin significantly inhibited accumulation that occurred over 7 d in serum-free culture (solid circles, no drug; solid squares, LY294002; solid triangles, rapamycin). For each experiment, data were normalized to Day 0. The values shown are means from three independent experiments ± SE. *P < 0.05 compared with control, time-matched controls using Kruskal-Wallis one-way ANOVA and Dunn Test.

 
As mTOR is known to be an important regulator of protein synthesis by promoting translation initiation and elongation via phosphorylation of p70S6K and PHAS-1/4E-BP1 (24), we further quantified the role of PI(3) kinase and downstream mTOR-mediated p70S6K activity in contractile protein accumulation in airway smooth muscle cells. We measured the effects of the pharmacologic inhibitors LY294002 and rapamycin on SM22 and smMHC accumulation using Western blot analysis. Inhibition of either PI(3) kinase or mTOR was highly effective in preventing accumulation of SM22 (Figure 7) and smMHC protein (Figure 8) seen in association with airway smooth muscle cell elongation and differentiation during prolonged serum-free culture. As noted above, neither rapamycin nor LY294002 treatment affected nonsarcomeric ß-actin abundance, suggesting a somewhat specific effect on accumulation of contractile apparatus–associated proteins. As a further control for the specificity of the pharmacologic inhibitors used, in some cultures we included PD98059, a specific inhibitor of MEK and the ERK pathway that is known to activate p90S6 kinase required for airway smooth muscle cell proliferation (35). Unlike experiments with inhibitors of PI(3) kinase or mTOR/p70S6K, inhibition of the ERK pathway had no effect on accumulation of smMHC and differentiation of a contractile phenotype by airway myocytes. These data reveal that activation of PI(3) kinase and a downstream signaling cascade that includes mTOR and p70S6K is required for airway smooth muscle cell contractile apparatus protein accumulation and acquisition of a large elongate morphology associated with myocyte differentiation. Of note, inhibition of mTOR or its upstream effector PI(3) kinase was equally effective at blocking the accumulation of SM22 and smMHC. This suggests that the principal pathways mediating accumulation of contractile proteins in airway myocytes are likely downstream of mTOR, rather than being associated with more upstream branches of PI(3) kinase regulated signaling pathways.

Inhibition of the PI(3) Kinase/mTOR/p70S6K Signaling Cascade Does Not Affect Steady-State mRNA Abundance for smMHC and SM22
To more confidently determine if the inhibition of PI(3) kinase or mTOR/p70S6K prevents differentiation of elongate contractile airway myocytes by inhibiting accumulation of smooth muscle contractile apparatus–associated genes, we further tested the effects of rapamycin and LY294002 on steady-state mRNA abundance for SM22 and smMHC during prolonged serum deprivation (Figure 9). Northern blot analysis revealed that the high SM22 and smMHC mRNA levels maintained after serum withdrawal are not decreased by inhibition of PI(3) kinase or mTOR/p70S6K. Indeed, in some but not all experiments rapamycin appeared to slightly increase steady state mRNA for both SM22 and smMHC. An example of such an effect is revealed in the sample blot shown in Figure 9. These studies provide strong evidence that pharmacologic blockade of PI(3) kinase/mTOR/p70S6K-dependent accumulation of contractile apparatus proteins does not depend upon decreases in the steady-state abundances of corresponding mRNAs.



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  		Figure 9. Northern blot analysis reveals that inhibition of PI(3) kinase or mTOR activity for up to 7 d in serum-deprived canine airway myocyte primary cultures had no effect on the content of mRNA for smMHC and SM22. Upper and middle panels show representative blots for smMHC and SM22 transcripts, respectively, and indicate that the transcripts are maintained for at least 7 d in serum-deprived conditions (Control). Neither the addition of LY294002 (20 µM) nor of rapamycin (Rapa) (10 nM) for up to 7 d in serum-free culture reduced mRNA abundances. The blot shown is representative of three independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our previous studies using canine tracheal myocytes in long-term serum-deprived culture revealed a striking reduction of over 80% in the transcription activity of promoters for SM22 and smMHC (4) at the same time that SM22 and smMHC protein accumulates by 5- to 7-fold (25). As these data suggested post-transcriptional mechanisms may be critical for differentiation of large, elongated contractile phenotype airway smooth muscle cells, the current study was undertaken to identify signaling pathways required for this process. Our results demonstrate that levels of mRNA for SM22 and smMHC are not increased during prolonged culture in serum-free, insulin-supplemented conditions that otherwise promoted the development of large, elongated contractile phenotype myocytes (Figure 1). During the same time we observed a significant and sustained activation of PI(3) kinase and downstream signaling targets including Akt1, mTOR, and p70S6K almost exclusively in myocytes that acquired an elongate contractile phenotype (Figures 2–6). Importantly, sustained induction of this pathway, which drives protein synthesis and is required for hypertrophy of vascular smooth muscle cells and skeletal myotubes (8, 10–12, 15), is required both for the selective accumulation of contractile apparatus–associated proteins and the differentiation of contractile airway smooth muscle cells (Figures 6–8). Conversely, inhibition of PI(3) kinase or mTOR did not diminish steady-state abundance of mRNA for SM22 and smMHC (Figure 9). These results reveal that disparate signaling mechanisms likely regulate transcription of genes encoding contractile apparatus–associated proteins and the synthesis and accumulation of the proteins themselves. Moreover, a principal role for PI(3) kinase, chiefly through downstream signal transduction branches involving mTOR and p70S6K, in the differentiation and growth of large contractile airway myocytes is strongly indicated.

There has been considerable investigation pointing to an important role for PI(3) kinase and downstream targets in signaling cascades that promote protein synthesis, differentiation, and hypertrophy of visceral and vascular smooth muscle and skeletal myotubes (8, 11, 12, 15, 33). PI(3) kinase signaling also appears to be important for promoting ERK-independent DNA synthesis, cyclin D1 expression, and cell cycle progression of cultured airway myocytes (32, 36–38). Though the precise mechanisms by which hormones and mitogens induce either differentiation or proliferation are not understood, it is clear that divergent signaling pathways mediate mitogenic and myogenic responses of smooth muscle and skeletal muscle cells. IGF-1 induces an initial mitogenic response in skeletal myotubes, including increases in cyclin D1 and c-fos mRNA, that requires MEK/ERK signaling (33). This is followed by a myogenic response, myotube hypertrophy, and the accumulation of contractile proteins that requires the PI(3) kinase/Akt1/mTOR and PI(3) kinase/Akt1/GSK3 signal transduction pathways (8, 15, 33). Using primary cultured embryonic chicken gizzard smooth muscle cells Hayashi and colleagues (11, 12) demonstrated that mitogen-mediated induction of ERK or p38 MAPK promotes myocyte de-differentiation and proliferation, whereas induction of PI(3) kinase by IGF-1 was required to prevent the spontaneous reduction in expression of contractile phenotype–associated proteins that occurs in primary culture. Our experiments differed significantly from these studies in that we investigated the requirement of PI(3) kinase, mTOR, p70S6K, and ERK in promoting the accumulation of smooth muscle–specific contractile apparatus proteins during hypertrophic growth and differentiation to a functionally contractile phenotype. We recently reported that PI(3) kinase is required expression of sm-{alpha}-actin and myosin light chain kinase in a transformed human bronchial smooth muscle cell line (39), and in the present study we significantly extend this observation by reporting that PI(3) kinase–mediated signal transduction is required for the differentiation and elongation of contractile airway myocytes. Conversely, signaling through MEK/ERK appears to play little or no role in this process, as blockade with PD98059 had no effect.

As the synthesis of contractile apparatus–associated proteins is a key component of myogenesis and hypertrophy of smooth muscle and skeletal muscle, our studies focused on the role of signaling pathways downstream from PI(3) kinase that affect the accumulation of SM22 and smMHC during development of contractile phenotype airway myocytes. Our studies revealed the sustained induction of both PI(3) kinase and p70S6K activity in a select subpopulation of airway myocytes during prolonged insulin-supplemented serum-free culture. Moreover, sustained activation of PI(3) kinase/mTOR/p70S6K signaling was required to drive both the accumulation of SM22 and smMHC, and for development of large, elongated, contractile myocytes. PI(3) kinase is a dual specificity kinase that targets the 3-position of phosphatidylinositol 4,5-bisphosphate and the serine/threonine residues on target proteins. The generation of phosphatidylinositol 3,4,5-trisphosphate leads to plasma membrane association and activation of PDK1 and Akt1. PDK1 mediates activation of Akt1 by phosphorylating Thr308 in the Akt1 catalytic domain (13, 40) and Ser473 in the regulatory domain (14). The activation of p70S6K through Akt1/mTOR and/or parallel pathways involving PDK1 appears to be essential for increased protein accumulation during angiotensin II–induced vascular smooth muscle hypertrophy (10, 41), insulin-mediated differentiation and hypertrophy of skeletal myotubes (15, 33), and in preventing skeletal muscle atrophy (8). Though the medium we used for prolonged serum deprivation was supplemented with insulin, which activates PI(3) kinase, the molecular mechanisms responsible for the activation of PI(3) kinase/mTOR/p70S6K in only a distinct subpopulation of airway myocytes are not clear. Our previous studies have shown airway smooth muscle cells to be phenotypically heterogeneous, and primary cultures are comprised of stable, functionally and phenotypically distinct sublineages (25, 28, 42, 43). Nonetheless, we have also reported the steady-state abundance of mRNA for muscle-specific markers such as SM22 and smMHC to be more-or-less equal in all airway myocytes in serum-free culture (4). Thus our current results suggest that phenotypic and functional heterogeneity of airway myocyte subpopulations may arise through the divergent activation of signal pathways controlling post-transcriptional pathways that regulate the synthesis and accumulation of contractile smooth muscle–specific proteins.

Multiple upstream pathways, including those initiated by PI(3) kinase, that control global and selective protein synthesis converge on p70S6K, which only becomes fully activated through phosphorylation of seven serine/threonine residues by effectors that include PKC{zeta}, PDK1, mTOR, and possibly Akt1 (19, 24, 44, 45). Our studies using primary cultured tracheal myocytes demonstrate that independent inhibition of either PI(3) kinase with LY29004, or mTOR with rapamycin, was equally effective in blocking activation of p70S6K and the accumulation of SM22 and smMHC protein during prolonged insulin-supplemented serum-free culture. Despite some reports that LY294002 may also inhibit mTOR-regulated cell reposnses indepndent of PI(3) kinase (46), our data clearly indicate that the principal signaling network for airway myocyte differentiation and cell growth in these conditions includes PI(3) kinase, mTOR, and p70S6K. Though it is likely that PI(3) kinase effects are mediated through PDK1, our studies did not discern the relative contribution of PDK1/Akt/mTOR, PDK1 alone, or PDK/PKC{zeta} in the activation of p70S6K. During prolonged serum-free culture a sustained increase in phosphorylation of Thr308 and Ser473 on Akt1 was observed, suggesting that it may be activated; however, future detailed studies will be required to determine a direct role, if any, of Akt1 in differentiation of myocyte subpopulations. Nonetheless, our observations that inhibition of mTOR or its upstream effector PI(3) kinase was equally effective in blocking myocyte differentiation and hypertrophy precludes a role for pathways upstream from mTOR that could regulate protein synthesis in parallel with PI(3) kinase/mTOR/p70S6K. Thus, it is unlikely that Akt1-mediated inhibition of GSK-3ß leading to de-inhibition of the translation initiator eIF2 (16, 17) or the direct activation of mTOR involving amino acid sufficiency (47, 48) are essential for the accumulation of SM22 and smMHC by airway myocytes in prolonged serum-free culture.

We did not investigate the involvement of PHAS-1/4E-BP phosphorylation by mTOR, which causes dissociation of PHAS-1 from the translation initiator eIF-4E and increased protein synthesis (18–20). mTOR, which associates with both p70S6K and the translation repressor PHAS-1 through the scaffold protein raptor (49), represents an important branch point for downstream pathways that regulate protein synthesis. PHAS-1 is phosphorylated during angiotensin II–induced vascular smooth muscle hypertrophy (10, 41), and during IGF-1–induced differentiation and hypertrophy of skeletal myotubes (15, 33). Due to its role in vascular smooth muscle cell hypertrophy, future investigation of the role of mTOR/PHAS-1 in airway myocyte differentiation may be warranted. Our experiments did not assess the possible role that reduced degradation rate of SM22 and smMHC might play in the accumulation of these proteins during prolonged serum-free culture; therefore, we cannot exclude the possibility that this process may, in part, also contribute to our findings.

Differentiation of airway myocytes is complex, requiring integration of multiple pathways that regulate transcriptional and post-transcriptional mechanisms. Our current studies reveal that though PI(3) kinase and mTOR are essential for regulating the accumulation of SM22 and smMHC protein in at least one subpopulation of airway myocytes, they do not appear to affect the abundance of the mRNAs for SM22 and smMHC. We reported that transcription activity of smMHC and SM22 promoters is selectively reduced by over 80% in airway myocytes in serum-free culture compared with serum-supplemented culture (4, 50). Several studies report that serum-fed myocytes use an essential signal transduction pathway involving the monomeric GTPase RhoA and its downstream target ROCK-1 to induce nuclear localization of serum response factor (SRF), a transcription factor required for transcription from promoters for smooth muscle contractile apparatus associated proteins such as SM22 and smMHC (4, 5, 51–53). Our current studies demonstrate that in addition to RhoA/ROCK-mediated transcription, synthesis and accumulation of contractile apparatus–associated proteins under the control of the PI(3) kinase/mTOR/p70S6K signaling cascade is a fundamental process during smooth muscle cell differentiation and hypertrophy. Collectively our current and prior observations using the unique culture system developed for our studies significantly extend our existing understanding, identifying for the first time distinctly different signaling pathways required for the segregated regulation of transcriptional and post-transcriptional events leading to expression and accumulation of proteins associated with the contractile apparatus. This insight greatly broadens awareness of mechanisms that could contribute to increased airway smooth muscle mass and changes in intrinsic contractile properties associated with asthma.


   Acknowledgments
 
This study was supported by NHLBI Grants HL56399, HL64095, HL07605, HL54685, and HL63314, Canadian Institutes of Health Research, Canada Foundation for Innovation, Manitoba Health Research Council, and Merck Frosst/Inspiraplex.

Received in original form July 21, 2003

Received in final form April 9, 2004


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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