university of groningen central role of rho-kinase in the

University of Groningen

Central role of Rho-kinase in the pathophysiology of allergic asthmaSchaafsma, Dedmer

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Chapter 7

Insulin increases the expression of contractile phenotypic markers in airway smooth muscle

Dedmer Schaafsma, Karol D. McNeill, Gerald L. Stelmack, Reinoud Gosens, Hoeke A. Baarsma, Erin Frohwerk, Bart G. J. Dekkers, Jelte-Maarten

Penninks, Karen M. Ens, S. Adriaan Nelemans, Johan Zaagsma, Andrew J. Halayko, Herman Meurs

Submitted (2006)

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Abstract We have previously demonstrated that long-term exposure of bovine tracheal smooth muscle (BTSM) strips to insulin induces a functional hypercontractile phenotype. To elucidate molecular mechanisms by which insulin might induce maturation of contractile phenotype airway smooth muscle (ASM) cells, we investigated effects of insulin stimulation in serum-free primary BTSM cell cultures on protein accumulation of specific contractile phenotypic markers, and on the abundance and stability of mRNA encoding these markers. In addition, we used microscopy to assess insulin effects on ASM cell morphology, phenotype, and induction of PI-3-kinase signaling. It was demonstrated that protein and mRNA levels of smooth muscle specific contractile phenotypic markers, including sm-myosin, are significantly increased after stimulation of cultured BTSM cells with insulin (1 µM) for 8 days as compared to cells treated with serum-free media, while mRNA stability was unaffected. In addition, insulin-treatment promoted the formation of large, elongate ASM cells, characterized by dramatic accumulation of contractile phenotype marker proteins and phosphorylated p70S6K (downstream target of PI-3-kinase associated with ASM maturation). Insulin effects on protein accumulation and cell morphology were abrogated by combined pretreatment with the Rho-kinase inhibitor Y-27632 (1 µM) or the PI-3-kinase inhibitor LY-294002 (10 µM), indicating that insulin increases the expression of contractile phenotypic markers in BTSM in a Rho-kinase and PI-3-kinase dependent fashion. In conclusion, insulin increases transcription and protein expression of contractile phenotypic markers in ASM. This could have important implications for the use of recently approved aerosolized insulin formulations in diabetes mellitus. Introduction Chronically inflamed airways are subject to airway remodeling, which may contribute importantly to airway hyperresponsiveness (AHR) and progressive decline of lung function in chronic and severe asthma [1-3]. It is well recognized that mature airway smooth muscle (ASM) cells retain the ability for phenotype plasticity, enabling them to exert contractile, proliferative, migratory and synthetic functional responses that likely contribute to asthma pathogenesis [4,5]. Modulation of ASM phenotype is governed by growth factors, neurotransmitters and inflammatory mediators. In vitro, ASM cells and intact tissue strips switch to a less contractile, more proliferative phenotype when exposed to high concentrations of fetal bovine serum (FBS) or peptide growth factors [4,6]. Such a phenotype shift is accompanied by a decrease in contractile ability that occurs concomitantly with a dramatic decrease in expression of specific contractile proteins, such as smooth muscle myosin heavy chain (sm-MHC) [4]. On the other hand, induction of a (hyper)contractile phenotype in cultured myocytes occurs as a consequence of growth arrest by serum-deprivation, a process characterized by enhanced

Insulin-induced hypercontractile ASM phenotype

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expression of contractile proteins and contraction regulatory proteins, such as calponin and myosin light chain kinase (MLCK) [4,7]. In airways from asthmatic patients, excessive accumulation of contractile ASM has frequently been described [8], and likely results from both myocyte hyperplasia and hypertrophy in central and small airways [9-11]. ASM thickening is considered to be the pivotal component of airway remodeling that underpins excessive airway narrowing in asthma [12]. Prolonged serum-deprivation of ASM cells has been used successfully to study processes involved in the development of large, elongate contractile ASM cells. Maturation of ASM cells was accompanied by an increase in protein expression of specific contractile phenotypic markers such as sm-MHC [4,7] that occurred in the absence of any change in sm-MHC mRNA abundance [13], while transcriptional activity was reduced [14]. Myocyte elongation and sm-MHC protein accumulation is dependent on protein translational regulation by a pathway involving phosphatidylinositol-3-kinase (PI-3-kinase), mammalian Target of Rapamycin (mTOR) and p70S6K [13]. Transcription of genes encoding specific contractile proteins appears to mediated primarily by the Rho/Rho-kinase pathway [3,15,16]. This signaling pathway promotes nuclear translocation of the transcription factor serum response factor (SRF), which binds CArG box elements to activate promoter function [14,17,18]. Notably, studies describing the effects of prolonged serum-deprivation on ASM phenotype and function have been performed in the absence of serum, but also in the presence of insulin, which was added to serve as a survival/attachment promoting factor. To date, however, it has not been determined to what extent insulin may contribute directly to ASM maturation via the signaling pathways that are known to be critical in this process. Long-term exposure (8 days) of bovine tracheal smooth muscle (BTSM) strips to insulin has been shown to induce a functional hypercontractile ASM phenotype characterized by increased contractile response to methacholine and KCl, and decreased proliferative response to growth factor [19]. Beyond its biological importance, the effects of insulin on ASM function are of clinical relevance, as the use of aerosolized insulin formulations has recently been approved in Europe and the U.S.A. for the treatment of diabetes mellitus type 1 and 2 [20]. To elucidate molecular mechanisms by which insulin might underpin maturation of contractile phenotype ASM cells, we investigated the effects of insulin stimulation in serum-free primary cultures on protein accumulation of specific contractile phenotypic markers, and on the abundance and -stability of mRNA encoding contractile phenotype markers. In addition, we used microscopy to assess insulin effects on ASM cell morphology, phenotype, and induction of PI-3-kinase signaling. As Rho-kinase [3,15] and PI-3-kinase [13] are tightly associated with transcription and translation of contractile phenotypic markers, respectively, we also studied the effects of the selective Rho-kinase inhibitor Y-27632 and the selective PI-3-kinase inhibitor LY-294002 on insulin-induced protein expression and phenotype maturation.

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Methods Airway smooth muscle cell and organ culture Bovine tracheae were obtained from local slaughterhouses and rapidly transported to the laboratory in Krebs-Henseleit (KH) buffer of the following composition (mM): NaCl 117.5, KCl 5.60, MgSO4 1.18, CaCl2 2.50, NaH2PO4 1.28, NaHCO3 25.00 and glucose 5.50, pregassed with 5% CO2 and 95% O2; pH 7.4. After the removal of mucosa and connective tissue, tracheal smooth muscle was chopped using a McIlwain tissue chopper, three times at a setting of 500 �M and three times at a setting of 100 �M. Tissue particles were washed two times with sterile Dulbecco’s modification of Eagle’s medium (DMEM) supplemented with NaHCO3 (10 mM), HEPES (20 mM), sodium pyruvate (1 mM), nonessential amino acid mixture (1:100), gentamicin (45 �g/ml), penicillin (100 U/ml), streptomycin (100 �g/ml), amphotericin B (1.5 �g/ml) and 0.5 % fetal bovine serum (FBS). Enzymatic digestion was performed in the same medium, supplemented with collagenase P (0.75 mg/ml), papain (1 mg/ml) and soybean trypsin inhibitor (1 mg/ml). During digestion, the suspension was incubated in an incubator shaker at 37 ˚C, 55 rpm for 20 min, followed by a 10 min period of shaking at 70 rpm. After filtration of the obtained suspension over 50 �m gauze, bovine tracheal smooth muscle (BTSM) cells were washed three times in medium supplemented with 10% FBS (S+). Cells were grown to 70-80 % confluence (time-point 0) in 6-well plates, after which they were kept in DMEM (S0) or DMEM supplemented with insulin (1 �M) in the presence or absence of (+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl) cyclohexane carboxamide (Y-27632, 1�M) and/or 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY-294002, 10 �M) for 2, 4 or 8 days. Each condition was performed in triplicate for each experiment. For all protocols, cells in passage 1-3 were used. In separate experiments, organ culture of BTSM strips under serum-deprived or insulin-stimulated conditions was performed as described previously [19]. Western analysis To obtain whole cell lysates, cells were washed once with ice-cold phosphate-buffered saline (PBS, composition (mM): NaCl 140.0; KCl 2.6; KH2PO4 1.4; Na2HPO4.2H2O 8.1; pH 7.4) and then lysed in ice-cold RIPA buffer (composition: 40 mM Tris, 150 mM NaCl, 1 % Igepal, 1 % deoxycholic acid, 1 mM NaF, 1 mM Na3VO4, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 7 µg/ml pepstatin A, 1 mM PMSF, pH 8.0). Equal amounts of protein were subjected to electrophoresis and transferred to nitrocellulose or polyvinylidene difluoride membranes. Membranes were subsequently blocked in blocking buffer (composition: Tris-HCl 50.0 mM; NaCl 150.0 mM; Tween-20 0.1%, dried milk powder 5%) for 90 minutes at room temperature. Next, membranes were incubated overnight at 4 °C with primary antibodies (sm-myosin (diluted 1:200), calponin (diluted 1:400) and �-actin (diluted 1:2000); all dilutions in blocking buffer). After three washes with Tris Buffered Saline Tween 20 (0.1 % TBST, containing Tris-HCl 50.0 mM; NaCl 150.0 mM, Tween 20 0.1 %) of 10 min each, membranes were incubated with horseradish peroxidase-labelled secondary antibodies (dilution 1:3000 in blocking buffer) at room temperature for 90 min, followed by an additional three washes


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with 0.1 % TBST. Bands were subsequently visualized on film using enhanced chemiluminescence reagents (Amersham, Buckinghamshire, UK) and analyzed by densitometry (TotallabTM). All bands were normalized to �-actin expression. RNA extraction and reverse transcriptase PCR Total RNA was extracted using a RNeasy RNA Mini Kit (Qiagen, U.S.A) in accordance with the manufacturer’s instructions. Reverse transcription (first strand cDNA synthesis) was performed with 2 �g of total RNA (in x �l), 1 �l Oligo dT12-18 primer (500 mg/ml, Invitrogen) and 10-x �l ddH2O. After heating this mixture for 5 min at 65 °C, 9 �l of reaction mixture, consisting of 1 �l dNTP PCR mix (10 mM, Amersham, Canada), 4 �l 5x first-strand buffer, 2 �l DTT (0.1 M), 1 �l RNaseOUT (40 U) and 1 �l Moloney murine leukemia virus reverse transcriptase (M-MLV RT, 200 U, Invitrogen) was added. Subsequently, the samples were incubated at 42 °C for 120 minutes, after which the reaction was inactivated by heating the samples at 72 °C for 15 minutes and putting them on ice. cDNA was stored at -20 °C until further use. PCR amplification was performed in a total volume of 50 �l which included 1 �l RT reaction mixture, 0.5 �M of each forward and reverse oligonucleotide, 1x PCR buffer with 1.5 mM MgCl, 0.2 mM dNTP PCR mix and 1.25 U of Platinum Taq Polymerase (Invitrogen). The following primers were used: to amplify a 162-bp fragment of Bos Taurus (bovine) insulin receptor cDNA; 5’-AAA CGG ACG GAT TCT GAC TTT-3’ (forward) and 5’-GTG ATC TCT GAG CTC CGT TTG -3’ (reverse); to amplify a 150-bp fragment of bovine Insulin-Like Growth Factor Receptor (IGFR)-1 cDNA; 5’-CCG GGA GGT CTC CTC TAC TA-3’ (forward) and 5’-TTG TGT CCT GAG TGT CTG TCG-3’ (reverse); and, to amplify a 247-bp fragment of bovine IGFR-2 cDNA; 5’-CGT GTT TGA TCT GAA CCC ACT-3’ (forward) and 5’-CCC CGT GTA GTT CAG GGT TAT-3’ (reverse). PCR comprised of a denaturing step of 94°C for 5 minutes followed by amplification of 10 cycles at 94°C for 1 minute, 67°C for 1 minute decreasing 1°C per cycle, and 72°C for 1 minute, and then 20 cycles of 94°C for 1 minute, 57°C for 1 minute, and 72°C for 1 minutes, with a final extention at 72°C for 5 minutes. PCR products were separated on a 2% agarose gel. Real Time PCR cDNA was subjected to real time PCR, which was performed with a LightCycler (Roche Molecular Biochemicals) and LightCycler FastStart DNA Master SYBR Green I (SYBR-GR, Roche Molecular Biochemicals) in accordance with the manufacturer’s instructions. The following primer sets were used: to amplify a 158-bp fragment of bovine sm-MHC-I cDNA; 5’-ATG TTC CAG TCC ACA ATA GGA GA-3’ (forward) and 5’-TGT GTC AAT GGC AGA ATC AAT AG-3’ (reverse); and, to amplify a 136-bp fragment of G3PDH cDNA; 5’-AGC AAT GCC TCC TGC ACC ACC AAC-3’ (forward) and 5’-CCG-GAG GGG CCA TCC ACA GTC T (reverse). Changes in sm-MHC-I cDNA were calculated relative to G3PDH.

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Analysis of mRNA stability Cells were grown to 70-80 % confluence then were kept in S0 or medium containing insulin (1 �M) for 0, 6 or 24 hr in the presence of actinomycin D (1 �g/ml) to inhibit de novo RNA-synthesis. Total RNA was extracted and real time PCR was used to assess sm-MHC-I expression at each time point. G3PDH mRNA abundance, which was unaltered by actinomycin D over the time course of our experiments, was used to normalize sm-MHC-1 transcript abundance. Phase-contrast microscopy and immunocytochemistry For all imaging experiments, before plating cells in 6-well plates, a pre-cleaned, sterile glass coverslip was placed in each well. BTSM cells were grown to 70-80% confluence as described above, then were maintained in S0 or S0 supplemented with insulin (1�M) in the absence or presence of Y-27632 (1 �M) and/or LY-294002 (10 �M) for 2, 4 or 8 days. Coverslips were removed from each well to obtain phase contrast images using an Olympus AX70 microscope equipped with digital image capture system (ColorView Soft system with Olympus U CMAD2 lens). Using AnalySIS® image processing software, a scale bar representing 100 �m was added to each picture. Quantitative analysis of cell number and length was performed using Image Pro Plus® software. Immunocytochemistry was performed as we have previously described [13]. Briefly, cells were maintained in S0 or S0 supplemented with insulin (1�M ) for 8 days, were fixed with 4% paraformaldehyde and permeabilized using 0.1% Triton-X 100. Thereafter cells were incubated in blocking solution containing 10% normal donkey serum, and then stored overnight at 4°C in diluted primary antibody solutions that included monoclonal mouse anti-sm-MHC (clone hSMv, Sigma-Aldrich, St. Louis, MO), monoclonal mouse anti-calponin (clone hCP, Sigma-Aldrich, St.Louis, MO), and rabbit-anti-phosphorylated p70S6K(Thr421/Ser424) (Cell Signaling Tech., Beverly, MA). FITC- and Cy5-conjugated secondary antibodies (Jackson ImmunoResearch, West Grove, PA) were used to detect primary antibody bound to cells. Nuclei were labeled with Hoechst 33342 (10 µg/mL). Coverslips were mounted using ProLong Anti-fade Gold (Invitrogen Canada, Burlington, ON) and images were captured using an Olympus LX70 inverted microscope equipped with a high resolution Ultra Pix FSI CCD camera controlled by Ultraview 4.0 Software (Olympus Canada, Markham, ON). Data analysis All data represent means ± s.e.mean from n separate experiments. Statistical significance of differences was evaluated by the Student's t-test for paired observations or the multiple measurement ANOVA, where appropriate. Differences were considered to be statistically significant when P<0.05. Materials Dulbecco’s modification of Eagle’s medium (DMEM), fetal bovine serum, bovine serum albumin, sodium pyruvate solution (100 mM), non-essential amino acid mixture, gentamicin solution (10 mg/ml), penicillin/streptomycin solution (5000 U/ml; 5000


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�g/ml) and amphotericin B solution (Fungizone, 250 �g/ml) were obtained from Gibco BRL Life Technologies (Paisley, UK). Mouse monoclonal anti-�-actin, mouse monoclonal anti-sm-�-actin, human apo-transferrin, soybean trypsin inhibitor and insulin (from bovine pancreas) were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Mouse monoclonal anti-sm-myosin and mouse monoclonal anti-calponin were from Neomarkers (Fremont, CA, USA). Primary and secondary antibodies used for immunocytochemistry included monoclonal mouse anti-sm-MHC (clone hSMv, Sigma-Aldrich, St. Louis, MO), monoclonal mouse anti-calponin (clone hCP, Sigma-Aldrich, St.Louis, MO), and rabbit-anti-phosphorylated p70S6K(Thr421/Ser424) (Cell Signaling Tech., Beverly, MA). FITC- and Cy5-conjugated secondary antibodies were obtained from Jackson ImmunoResearch, West Grove, PA. Collagenase P and papain were from Boehringer (Mannheim, Germany). L(+)ascorbic acid was from Merck (Darmstadt, Germany). All other chemicals used were of analytical grade. Results Time-dependent effects of insulin on accumulation of contractile proteins The abundance of the contractile proteins sm-myosin and calponin was determined by Western analysis (Figure 1). To ensure that we were able to effectively study effects of insulin on ASM cell differentiation, the experiments were initiated using subconfluent cultures, as at 100% confluence, likely due to density-dependent mechanisms, ASM cells can start to differentiate spontaneously even in the presence of serum [4]. BTSM cells were grown to 70-80 % confluence in the presence of serum (S+)(day 0), after which they were maintained in serum-free conditions to enable assessment of the direct and exclusive effects of insulin exposure for up to 8 days. Insulin exposure induced a significant and continual accumulation of both sm-myosin (Figure 1A, D) and calponin (Figure 1B, D) over the 8 days of study. Indeed, protein abundance was increased by ~9-fold and ~4-fold for sm-myosin and calponin, respectively, compared to day 0. In contrast, in the absence of insulin contractile protein abundance increased by only ~1- (sm-myosin) and ~2-fold (calponin); which was not statistically significant for sm-myosin (P=0.2 for sm-myosin). Overall, 8 days after serum withdrawal insulin exposure induced at least 5-times more sm-myosin and 2-times more calponin than was observed for myocytes maintained in serum-free culture without added insulin. Importantly, no difference was detected in myocyte attachment or viability in serum-deprived cultures in the presence and absence of insulin (data not shown).

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Days of stimulation0 2 4 6 8

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Figure 1. Insulin induces contractile protein accumulation in BTSM cells in a time-dependent fashion. Western analysis was performed using whole cell lysates prepared from prolonged insulin stimulated (1 µM, closed symbols) or serum-deprived (S0, open symbols) BTSM cell cultures. (A) Comparison by densitometry showed that insulin significantly increases sm-myosin protein expression as compared to expression on day 0 and expression after 8 d. of serum-deprivation. No significant increase was observed under serum-deprived conditions. (B) Comparison by densitometry showed that insulin significantly augments calponin protein expression as compared to expression on day 0 and expression after 8 d. of serum-deprivation. In addition, calponin expression was also enhanced under serum-deprived conditions as compared to day 0. (C) As compared to expression levels in 8 d. serum-deprived BTSM strips, expression of sm-myosin, determined by densitometric analysis, is significantly augmented in homogenates prepared from 8 d. insulin-stimulated BTSM strips. (D) Representative Western blots for sm-myosin (upper panel), calponin (middle panel) and �-actin (lower panel) in whole cell lysates from BTSM cell cultures kept under insulin-stimulated or serum-deprived conditions for 0-8 d. For each experiment expression levels were normalized to that on day 8 under insulin stimulated conditions. Data shown represent means ± s.e.mean of 4 (A), 8 (B) and 3 (C) independent experiments. *P<0.05, **P<0.01, ***P<0.001 compared to 8 d. S0; #P<0.05, # #P<0.01 compared to day 0. To be sure that the insulin effects were not an artifact of using primary cultured myocytes, we also measured the effects of 8 day insulin exposure on sm-myosin abundance in organ cultured BTSM strips (Figure 1C). As observed for the primary


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cultured myocytes, prolonged insulin stimulation resulted in a significantly greater abundance of sm-myosin compared to BTSM strips cultured in serum-free media without insulin. This strongly suggests that insulin effects on contractile protein abundance are consistent between cultured cells and intact BTSM. Furthermore, the results with organ culture are fully in line with the previously demonstrated development of hypercontractile responses in BTSM strips exposed to insulin in prolonged organ culture [19].

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Figure 2. Insulin increases sm-MHC mRNA content, without affecting its stability, in BTSM cells in a time dependent fashion. Real time PCR was performed using total RNA extracted from whole cell lysates prepared from prolonged insulin stimulated (1 µM, closed symbols) or serum-deprived (S0, open symbols) BTSM cell cultures. (A) Analysis shows that insulin significantly increases sm-MHC mRNA content in a time-dependent fashion, similar to that observed for protein accumulation. Serum deprivation tends to increase sm-MHC mRNA content as well, although no significant differences were observed. (B) In the presence of actinomycin D (1 µg/ml), which inhibits de novo RNA synthesis, no differences between insulin stimulated and serum-deprived conditions were observed for up to 24 hr. Thus, the effects of insulin on sm-MHC mRNA abundance (as compared to serum-deprivation) are not due to an increased mRNA stability. Data shown represent means ± s.e.mean of 3 independent experiments. *P<0.05, compared to 8 d. S0; #P<0.05 compared to day 0. Time-dependent effects of insulin on accumulation of contractile phenotype mRNA sm-MHC is considered to be one of the most stringent markers for mature contractile ASM cells [4]. Therefore, we determined the effects of insulin exposure of cultured BTSM cells on the abundance of mRNA for sm-MHC using real time PCR (Figure 2A). After 8 days, insulin stimulation had induced a significantly greater increase in sm-MHC mRNA abundance compared to serum-deprived conditions (~1.5-fold). Moreover, sm-

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MHC mRNA abundance in insulin exposed cultures was almost 3-fold greater than that measured on day 0, and mimicked the time-dependent accumulation of sm-myosin protein (Figure 1). Though there appeared to be a trend for accumulation of sm-MHC mRNA in insulin-deficient cultures, after 8 days exposure the abundance of this transcript was not significantly different from day 0 (P=0.2). In a separate set of experiments we determined the effects of serum-deprivation and insulin stimulation on sm-MHC mRNA stability. Actinomycin D (1 µg/ml), an inhibitor of de novo RNA synthesis, was added to BTSM cells at day 0, when subconfluent cultures were switched to serum-free culture media. Thereafter, we monitored sm-MHC mRNA abundance for up to 24 hours in the presence or absence of insulin. As shown in Figure 2B, no effects of insulin on the rate of sm-MHC mRNA degradation were observed in comparison with the serum-deprived control. This suggests that the potentiating effects of insulin on sm-MHC mRNA accumulation in prolonged serum-free culture may occur at the level of transcription. Of note, no effects of actinomycin D were observed on G3PDH mRNA expression, which was used to normalize real time PCR data obtained for sm-MHC. Intracellular signaling underlying insulin-induced contractile protein accumulation In ASM, Rho-kinase has emerged to be critically involved in transcription of genes encoding for contractile proteins [3,15], while at the level of protein translation, a critical role for PI-3-kinase signaling has been identified in ASM cell differentiation [13]. To study the involvement of Rho-kinase and PI-3-kinase in insulin-stimulated expression of contractile phenotype marker proteins, we co-incubated BTSM cells with insulin and selective inhibitors of Rho kinase (Y-27632, 1 µM) and PI-3-kinase (LY-294002, 10 µM) [21-23][Chapters 6 & 8] for 8 days (Figure 3). Inhibition of Rho-kinase or PI-3-kinase significantly reduced accumulation of both sm-myosin and calponin in the presence of insulin. In the case of calponin, complete blockade was evident so accumulation in the presence of each of the inhibitors was similar to that seen in serum-free cultures in the absence of insulin. These data may suggest that the excessive accumulation of sm-myosin and calponin stimulated by insulin is dependent on both Rho-kinase and PI-3-kinase signaling. Remarkably, in the presence of insulin we observed an additive effect of the Rho-kinase and PI-3-kinase inhibitors in which accumulation of sm-myosin and calponin was virtually abolished. This observation suggests Rho-kinase and PI-3-kinase signaling may act in parallel, potentially through different mechanisms, during ASM maturation in the presence of insulin.


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Figure 3. Rho-kinase and PI-3-kinase are required for insulin-induced contractile protein accumulation. Western analysis was performed using whole cell lysates prepared from BTSM cell cultures that were maintained under insulin-stimulated (1 µM) or serum-deprived (S0) conditions in the absence or presence of Y-27632 (1 µM), LY-294002 (10 µM) or both for 8 d. (A) Comparison by densitometry showed that the increased sm-myosin protein expression in response to 8 d. insulin stimulation is strongly reduced after co-incubation with Y-27632 (1 µM) or LY-294002 (10 µM) and is even further reduced by combined pretreatment. In addition, LY-294002, but not Y-27632, affected sm-myosin expression under serum-deprived conditions as well. (B) Densitometric analysis revealed that the insulin-induced increase in calponin is normalized by Y-27632 (1 µM) or LY-294002 (10 µM) and is even further decreased by combined pretreatment with these inhibitors. As for sm-myosin, LY-294002, but not Y-27632, decreased calponin content under serum-deprived conditions. (C) Representative Western blots for sm-myosin (upper panel), calponin (middle panel) and �-actin (lower panel) in whole cell lysates from BTSM cell cultures kept under insulin-stimulated (1 µM) or serum-deprived (S0) conditions in the absence or presence of Y-27632, LY-294002 or both for 8 d. For each experiment expression levels were normalized to that on day 8 under insulin stimulated conditions. Data shown represent means ± s.e.mean of 4 (A) and 5 (B) independent experiments. **P<0.01, ***P<0.001 compared to insulin; #P<0.05, # #P<0.01, # # #P<0.01 compared to S0.

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Notably, in contrast to our observation using insulin-stimulated cultures, no effect of Rho-kinase inhibition was observed on contractile protein expression in insulin-free conditions after 8 days. However, the effects of PI-3-kinase inhibition were similar to that observed in insulin-stimulated cultures (Figure 3). Moreover, no additive effects of combined treatment with Y-27632 and LY-294002 in insulin-deficient conditions were measured. Collectively, these observations suggest that serum-deprivation-induced increases in protein abundance are dependent on PI-3-kinase, but not Rho-kinase, while both PI-3-kinase and Rho-kinase appear to be necessary for insulin-augmented accumulation of sm-myosin and calponin.

Figure 4. Maturation of a subset of BTSM cells into large, elongate contractile phenotype myocytes in insulin-supplemented cultures. Primary cultured BTSM cells on glass coverslips were grown in DMEM/10% FBS to 70%-80% confluence, then were maintained for 8 days in serum-free DMEM supplemented with 1�M insulin. Cells were fixed and labeled for calponin (green) or sm-MHC (red). Original images were captured using either a 10x objective (A & B) or 40x objective (C & D). Nuclei (blue) are stained with Hoechst 33342. Images are typical of those obtained for 3 different primary cultures.

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Effects of insulin on airway smooth muscle cell morphology and phenotype It has been previously established for canine ASM cells, that serum deprivation in the presence of insulin induces the maturation of a select subset of myocytes that are characteristically large and elongate and exhibit abundant accumulation of contractile proteins such as sm-MHC and calponin [7,24]. Thus, we assessed myocyte phenotype and morphology resulting from prolonged serum deprivation in the presence and absence of insulin. BTSM cells were grown to 70 % confluence on glass coverslips, after which they were serum-deprived or stimulated with insulin for up to 8 days. Consistent with previous reports, ~1/6 to 1/5 of BTSM cells exhibited dramatic accumulation of contractile phenotype marker proteins in insulin-supplemented, serum-free media (Figure 4). In the subconfluent state (70%), where both proliferating and nonproliferating cells were present, only low level labeling of sm-MHC and calponin that was without clear filamentous organization was evident ( not shown).


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Figure 5. Inhibition of Rho-kinase or PI-3-kinase prevents the formation of large, elongate contractile ASM cells in insulin-stimulated BTSM cell cultures. Shown in this Figure are phase contrast micrographs of (A) some differentiated elongate BTSM cells after 8 days of serum-deprivation and (B) bundles of differentiated elongate BTSM cells that develop after 8 days of insulin stimulation, a process which is prevented in the presence of Y-27632 (1 µM, C) or LY-294002 (10 µM, D). Bar=100 µM. (E) Quantitative analysis using Image Pro Plus® software shows a time-dependent increase in the number of elongate spindle-shaped BTSM cells in response to insulin stimulation (closed circles), which is significantly different from serum-deprived conditions in the absence of insulin (open circles), and which is strongly prevented in the presence of Y-27632 (triangles) or LY-294002 (squares). #P=0.07, *P<0.05, compared to insulin. We used phase contrast microscopy to quantify myocyte elongation during prolonged serum-deprivation (Figure 5). No significant morphological changes associated with cell

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elongation were observed for up to 4 days in insulin-deficient serum-free cultures; rather, a relative homogenous population of BTSM cells was maintained. After 8 days, however, a limited number of aggregations of elongate cells similar in shape to sm-MHC and calponin-rich myocytes observed by immunocytochemistry were evident (Figure 5A, E). In contrast, in insulin-supplemented cultures, the formation of large, elongate myocytes was clearly evident within 2 days of serum-withdrawal, and this number increased markedly, nearing a plateau in number by 4 days that was only ~20% higher after 8 days (Figure 5B, E). These data further reveal the key role of insulin in facilitating ASM maturation in primary culture. As Rho-kinase and PI-3-kinase were required for contractile protein accumulation in BTSM cells (Figure 3), we also studied the effects of Y-27632 and LY-294002 on cell morphology under serum-free conditions in the presence and absence of insulin. Consistent with our earlier immunoblot analyses (Figure 3) Rho-kinase inhibition had little effect on development of elongate myocytes in insulin-deficient cultures, whereas inhibition of PI-3-kinase appeared to completely prevent maturation of elongate contractile morphology myocytes in insulin-deficient cultures. Formation of large, elongate contractile ASM cells during 8-days culture in insulin-supplemented serum-free media was however, largely dependent on both Rho-kinase and PI-3-kinase (Figure 5C, D & E). These findings parallel our earlier results using immunoblotting that revealed dependence on both Rho-kinase and PI-3-kinase for insulin-augmented accumulation of sm-myosin and calponin. In some experiments Trypan Blue was added to the cells to establish cell viability in all conditions. Although LY-294002 induced ~1% loss in cell viability, it was clear that at the concentrations used, these inhibitors had no significant effect on cell attachment nor did they promote cell death. As our data suggest that PI-3-kinase is required for myocyte maturation in the presence and absence of insulin, we next used fluorescence microscopy to examine whether activation of the downstream signaling effector, p70S6K, was selectively associated with BTSM cell maturation (Figure 6). After 4 days serum-withdrawal in the presence or absence of insulin we observed that phospho- Thr421/Ser424-p70S6K was elevated in all myosin-enriched myocytes. Moreover, this pattern was maintained through 8 days of study (not shown). These data strongly suggest that activation of PI-3-kinase, in parallel with the dramatic augmentation in the number of myocytes induced to a contractile phenotype by insulin, is associated with the accumulation of sm-MHC and calponin during maturation of individual BTSM cells.


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Insulin receptor identification in airway smooth muscle In the present study, we report significant effects of insulin in augmented ASM phenotype maturation. However, to date, insulin receptor expression in ASM has not been described, though expression of the insulin receptor and the insulin-like growth factor (IGF) receptors are well established in skeletal muscle [25] and vascular smooth muscle [26]. We performed reverse transcriptase PCR, using primers for the bovine insulin receptor and the bovine IGF receptors 1 and 2, to determine the profile of receptors expressed by primary cultured BTSM cells. In accordance with previous findings in rabbit ASM [27], we demonstrate the presence of mRNA for IGF receptors 1 and 2. Moreover, we demonstrate for the first time the presence of mRNA for the insulin receptor in ASM (Figure 7). Discussion Our results demonstrate that prolonged insulin stimulation of ASM cells induces a time-dependent increase in the expression of contractile ASM phenotypic markers in a Rho-kinase and PI-3-kinase dependent fashion. Moreover, this is associated with the formation of large, elongate ASM cells that are selectively enriched in sm-MHC and calponin, and are highly reminiscent of previously described (hyper)contractile canine ASM cells [7]. To date, induction of a hypercontractile phenotype in ASM cells has been attributed to mechanisms associated with prolonged serum-deprivation. Our current studies, however, strongly indicate that insulin has a direct effect that significantly potentiates phenotype maturation to a far greater extent than can be attained in its absence in serum-free conditions. There has been considerable investigation on identifying signaling pathways underlying the development of large, elongate contractile phenotype ASM cells. An important role for PI-3-kinase and its downstream targets has emerged in signaling cascades that promote protein synthesis, differentiation and hypertrophy in ASM [13] and a variety of other muscle types [28-30]. Recently, it was found PI-3-kinase is required for the expression of sm-�-actin and the contraction regulatory protein MLCK in a transformed human bronchial smooth muscle cell line [31]. Moreover, it was demonstrated that under insulin-supplemented serum free conditions, PI-3-kinase mediated signaling involving Akt1, mTOR and p70S6K is critical for accumulation of contractile proteins such as SM22 and sm-MHC in canine ASM cells [13].

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Figure 6. The downstream effector of PI-3-kinase, p70S6K, is selectively activated in airway myocytes during maturation to a contractile phenotype. Images shown are from BTSM cells after 4 days in serum-free culture in the absence (A-C) and presence (D-F) of insulin (1�M). Myocytes were double labeled for both sm-MHC (red) and phospho- Thr421/Ser424-p70S6K (green). Nuclei (blue) are stained with Hoechst 33342. Merged images (C and F) of sm-MHC and phospho- Thr421/Ser424-p70S6K are also shown. Images are typical of those obtained from triplicate wells of three different primary cultures. Figure 7. mRNA abundance of insulin target receptors in BTSM cells. Reverse transcriptase PCR was performed using total RNA extracted from BTSM cell cultures. (A) Photograph showing mRNA abundance of the bovine insulin receptor in 4 different BTSM cell cultures. Bovine liver was used as a positive control. (B) Photograph showing mRNA abundance of the bovine insulin-like growth factor receptor 1 and 2 in BTSM cell cultures.

A B C

F E D


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Another downstream target involved in PI-3-kinase/mTOR signaling is PHAS-1/4E-BP. Temperature-shift-induced differentiation and hypertrophy of immortalized human ASM cells was found to be accompanied by an augmented PI-3-kinase/mTOR-induced phosphorylation of PHAS-1, causing dissociation of PHAS-1 from the translation initiator protein eukaryotic initiation factor (eIF)-4E and subsequent increased protein synthesis [32]. Moreover, a direct link between insulin and PI-3-kinase signaling has been reported in C2C12 myoblasts; it was demonstrated that PI-3-kinase and mTOR were required for insulin-induced differentiation of these cells [33]. Completely in agreement with these findings, we now demonstrate that PI-3-kinase is required for the accumulation of contractile apparatus proteins and the formation of elongate, contractile phenotype ASM cells, both under serum-free and insulin-stimulated culture conditions. Notably, our studies are the first to show directly that insulin potentiates airway myocyte maturation in serum-free culture. An essential role for RhoA/Rho-kinase signaling in driving the transcription of genes encoding for contractile proteins such as sm-MHC, sm-�-actin, calponin and SM22 in smooth muscle has become evident [3,14-16]. Activation of the promoters of these genes is under combinatorial control by a number of transcription factors [16], but virtually all harbour a pair of essential CArG box elements [CC(A/T)6GG] that bind dimers of the MADS transcription regulator family member, SRF [14,17,18]. Localization and activation of SRF in the nucleus and subsequent induction of smooth muscle specific genes are chiefly governed by RhoA / Rho-kinase signaling in ASM [15]. The RhoA/Rho-kinase pathway regulates actin filament dynamics that modulate translocation of SRF co-activators to further support transcription of contractile smooth muscle specific genes [18,34]. Our present findings reveal Rho-kinase is required for the induction of a hypercontractile ASM phenotype in response to insulin, however the pathway appears to play an inconsequential role in the absence of insulin. This suggests that insulin stimulation may activate transcription of genes for contractile proteins. Although insulin-induced gene transcription has been proposed to be Rho/Rho-kinase dependent [35,36], clearly the specific link to this pathway is complex and requires in depth study, which is beyond the scope of this manuscript. Previous studies using transient transfection of canine ASM cells have shown that under serum-free, insulin-supplemented conditions the activity of promoters for sm-MHC and SM22 is markedly reduced from that measured when cells are grown in the presence of serum [14]. However, to date there are no reports of studies using either luciferase reporter assays or assays of nuclear mRNA synthesis to assess the specific effects of insulin on transcription of genes that contribute directly or indirectly to the extent of (airway) smooth muscle specific gene expression. Though such experiments are beyond the scope of the present study, our current findings from studies using actinomycin D indicate insulin has no effect on the stability of mRNA for contractile apparatus associated proteins, but rather on the gene transcription of these proteins. As the use of aerosolized insulin formulations has recently been approved in the U.S.A. and Europe for the treatment of diabetes mellitus type 1 and 2 [20], effects of insulin on

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ASM function and phenotype might not only be of pharmacological interest but might also be of clinical relevance. We have previously demonstrated that prolonged insulin exposure of BTSM strips results in the induction of a functional hypercontractile phenotype [19]. Completely confirming those findings, we now demonstrate that insulin stimulation, as in BTSM cells, results in an increased contractile protein expression in these intact muscle strips. Several phase 2 and 3 trials for inhaled insulin formulations show efficacy and tolerability that is comparable to subcutaneous (s.c.) insulin use [37-39]. Importantly, due to a low biological availability of inhaled insulin, effective dosing for inhaled delivery is ~10-fold higher than that used for s.c. injections to achieve satisfactory glycemic control [40-43]. It has not been directly determined whether large quantities of inhaled insulin might induce some adverse effects on ASM function, including ASM hypercontractility. However, it has been reported that inhalation of insulin might be accompanied by an increased incidence of cough, dyspnoea, sinusitis and pharyngitis [20]. Accordingly, very recently we have demonstrated that insulin has acute contractile effects on guinea pig tracheal smooth muscle preparations (DS, unpublished observations)[Chapter 5]. Moreover, it was reported that insulin caused a decrease in FEV1, which was indicated to be of small and uncertain clinical significance [20]. Since due to a poor absorption by asthmatics compared to healthy subjects, diabetic patients with asthma may need to inhale even a higher dose of insulin as compared to nonasthmatic diabetics [41], and since patients suffering from asthma, as well as patients with COPD, are hyperresponsive to inhaled contractile stimuli [44-46], it could be envisaged that these effects on FEV1 might be more significant in these patients. Moreover, effects of insulin on AHR and airway inflammation have been reported in rat models of diabetes [47,48]. It was found that airway constriction in response to electrical field stimulation was decreased in diabetic rats due to an increased function of the neuronal inhibitory M2-muscarinic receptor in the lungs [47]. Upon allergen challenge neuronal M2-receptor function was preserved and eosinophilia did not occur around airway nerves in these animals [48]. Conversely, treatment of these diabetic animals with insulin induced M2-receptor dysfunction, AHR and eosinophilia after allergen challenge. Further investigations are clearly warranted to identify acute and chronic effects of insulin on human ASM and to establish the effects of insulin inhalation on ASM function and phenotype under specific pathophysiological conditions (e.g. in asthma and COPD models). In conclusion, we demonstrate prolonged insulin stimulation induces expression of contractile phenotypic markers in a time-dependent fashion, completely confirming previous findings on ASM contractility [19]. Moreover, Rho-kinase and PI-3-kinase are required for the insulin-induced effects, both for contractile protein accumulation and formation of large, elongate contractile ASM cells. These findings might be of both pharmacological as pharmacotherapeutical interest and might significantly contribute to the ongoing discussion on the use of aerosolized insulin formulations for the treatment of diabetes mellitus type 1 and 2.


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Acknowledgements These studies were financially supported by grants to DS from the Netherlands Asthma Foundation (NAF grant 01.83), the research school of Behavioural and Cognitive Neurosciences (BCN) and Stichting Astma Bestrijding (SAB). RG is the recipient of a Marie Curie Outgoing International Fellowship (008823) from the European Community. This work was supported, in part, by grants to AJH from the Sick Kids Foundation / Institute of Human Development, Child and Youth Health (#XG05-011), Canadian Institutes of Health Research (CIHR), the Manitoba Institute of Child Health, and the Canada Research Chairs Program. AJH holds a Canada Research Chair in Airway Cell and Molecular Biology.

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