CCAAT/Enhancer-binding Protein and Activator Protein-1 Transcription Factors Regulate the Expression of Interleukin-8 through the Mitogen-activated Protein Kinase Pathways in Response to Mechanical Stretch of Human Airway Smooth Muscle Cells*

Ashok Kumar, Alan J. Knox {ddagger} and Aladin M. Boriek §

From the Department of Medicine, Baylor College of Medicine, Houston, Texas 77030, {ddagger} Division of Respiratory Medicine, City Hospital, University of Nottingham, Nottingham NG7 2RD, United Kingdom

Received for publication, December 16, 2002 , and in revised form, January 24, 2003.
    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Here we investigated the mechanisms by which mechanical stretch regulates the production of IL-8 in primary human airway smooth muscle cells (HASMC). Bronchial HASMC were subjected to cyclic mechanical stretch (12%, 1 Hz) using the computer-controlled Flexcell Strain system. Mechanical stretch increased IL-8 mRNA expression and protein production. Cyclic stretch of HASMC also increased the kinase activities of ERK1/2, JNK1, p38, and the DNA binding activities of AP-1 and C/EBP transcription factors with little effect on NF-{kappa}B. The inhibition of AP-1 and C/EBP transcriptional activities blocked the production of IL-8 in culture supernatants. Furthermore, the inhibition of ERK1/2 and p38 but not JNK1 caused a significant down-regulation in the expression and production of IL-8 in response to cyclic stretch. Although protein tyrosine kinases were required for the activation of both ERK1/2 and p38 kinase, stretch-activated channels, small GTPase proteins, and extracellular Ca2+ influx were required only for the activation of p38 kinase whereas phosphoinositide 3-kinase was needed for ERK1/2 activation. In addition, the phosphorylation of ERK1/2 was essential for the activation of AP-1 whereas p38 MAP kinase was needed for the activation of C/EBP. Our data demonstrate that the cyclic stretch of HASMC causes the increased production of IL-8 by activating the AP-1 and C/EBP transcription factors through the activation of ERK1/2 and p38 kinase signaling pathways.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Mechanotransduction is the fundamental mechanism by which mechanical forces acting through a cell initiates intracellular signaling. Therefore, this process promotes cellular growth and survival, influences metabolic processes including gene expression, and governs tissue architecture in various cell types (1, 2, 3). Whereas all adhesion-dependent cells appear to be sensitive to mechanical forces (4), this is especially evident in mechanocytes or cells routinely subjected to mechanical forces that include vascular endothelial and smooth muscle cells (5, 6, 7, 8), airway smooth muscle cells (9), osteocytes (10), cardiomyocytes (11), and skeletal muscle cells (12). Smooth muscle cells of airways are particularly responsive to mechanical stress as evident by the fact that in vitro, airway smooth muscle cells respond to cyclic mechanical strain with increased proliferation, cell reorientation, protein production, reorientation of stress fibers, and additional recruitment of focal adhesions (13, 14). Recent reports suggest that human airway smooth muscle cells (HASMC)1 has a further role, namely to act as an important source of pro-inflammatory factors such as interleukin-8 (IL-8), a chemokine that causes the activation and recruitment of neutrophils at the site of inflammation (15, 16, 17). However, the effect of mechanical stress on the regulation of IL-8 expression in HASMC remains unknown.

IL-8 gene expression is tightly controlled at several levels (18, 19). Many studies have revealed that a sequence spanning –1 to –133 nucleotides within the 5'-flanking region of the IL-8 gene is essential and sufficient for transcriptional regulation of the gene (18, 20, 21). Mutational and deletional analysis demonstrated that the promoter region of IL-8 is regulated in a highly cell type-specific manner (22, 23). Beside the usual "CCAAT" and "TATA" box-like structures, the IL-8 promoter also contains binding sites for NF-{kappa}B, AP-1, and C/EBP transcription factors (17, 18). Although, NF-{kappa}B was required for increased expression of IL-8 in all cell types studied, the AP-1 and C/EBP were dispensable for transcriptional activation in some cells but contribute to activation in other cells (22, 24, 25, 26, 27, 28).

Despite the rapid progress in identifying stress-induced signaling pathways and the identification of structural elements important in transcriptional activation, there is little information on how different signaling pathways interact with each other in order to mediate a particular biological response, such as expression of IL-8. Mitogen-activated protein (MAP) kinase pathways are among the best studied signaling pathways that are activated in response to various stimuli including mechanical stress (12, 29, 30). In mammalian cells, three parallel MAP kinase pathways have been described, i.e. extracellular signal-related kinase (ERK1/2), protein kinase 38 (p38), and c-Jun-N-terminal kinases (JNKs). These kinases act via the regulation of the activity of several transcription factors including AP-1, Elk1, and CCAAT/enhancer-binding protein (C/EBP) and therefore play an important role in initiating the expression of a variety of immediate and delayed response genes (31, 32, 33). Some earlier reports suggest that in response to inflammatory cytokines such as IL-1{beta} and TNF-{alpha}, the expression of IL-8 is regulated via MAP kinase pathways (34, 35, 36, 37). However, the molecular mechanism(s) by which mechanical forces are converted into the intracellular signals coupled with the nuclear expression of IL-8 in HASMC remains obscure.

In this study, we have investigated the signaling mechanisms that are involved in the transcriptional activation of the IL-8 gene in HASMC in response to cyclic mechanical stretch. Our data demonstrate that the expression of IL-8 in HASMC in response to mechanical stretch requires the activation of C/EBP and AP-1 but not the NF-{kappa}B transcription factor. Although mechanical stretching of HASMC causes the activation of all the three MAP kinases only ERK1/2 and p38 kinase are required for the stretch-induced expression of IL-8. Furthermore, our data demonstrate that different upstream signaling events are involved that regulate the activation of ERK1/2 and p38 MAP kinase in response to mechanical stretch.


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Wortmannin, LY290042 and H-7 were obtained from Sigma Chemical Co., Genistein, TMB-8, GF109203X, U0126, toxin A, and SP600125 were from CalBiochem (San Diego, CA). SB203580 was obtained from BIOMOL Research Laboratories (Plymouth Meeting, PA). Anti-JNK1 antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Phosphospecific rabbit polyclonal anti-p44/42 (Thr-202/Tyr-204), anti-p38 (Thr-180/Tyr-182), and anti p90RSK (Ser-380) were obtained from Cell Signaling Technology (Beverly, MA). NF-{kappa}B and AP-1 consensus oligonucleotides were obtained from Promega (Madison, WI). C/EBP consensus oligonucleotides were obtained from Santa Cruz Biotechnology. The human IL-8 ELISA kit was obtained from BD Biosciences PharMingen (San Diego, CA). Poly(dI·dC) was from Amersham Biosciences (Arlington Heights, IL). [{gamma}-32P]ATP (specific activity, 3000 (111 TBq) Ci/mmol) was obtained from PerkinElmer Life Sciences (Boston, MA). TRIzol reagent, oligo(dT)12–18 primer, reverse transcriptase, TaqDNA polymerase, Dulbecco's modified Eagle's medium cell culture and fetal bovine serum were from Invitrogen.

Cell Culture—Primary cultures of HASMC were prepared from explants as reported previously (17). Human tracheas were obtained from four postmortem individuals (two males aged 44 and 70 years and two females aged 52 and 70 years, respectively) within 12 h of death. The donors had no history of respiratory diseases and no evidence of airway abnormalities as determined by histological and pathological examination of the trachea and lungs. Cells at passages 7–8 were used for all experiments. It has been previously shown that the cells grown in this manner depict the immunohistochemical and light microscopic characteristics of typical HASMC (31).

Mechanical Stimulation using Cyclic Stretch—HASMC were plated onto type I collagen-coated flexible-bottom wells (Flex I plates, FlexCell International, McKeesport, PA) at a density of 2 x 105 cells/1 ml medium per well. The cells were incubated at 37 °C, 5% CO2. After the cells had been allowed to adhere for 48 h (>95% confluence), the cells were subjected to cyclic strain at 1 Hz (0.5 s of deformation alternating with 0.5 s of relaxation) for different time intervals, using a computer-controlled vacuum strain apparatus (Flexercell Strain Unit, FlexCell International) with a vacuum pressure that is sufficient to generate 12% strain. Replicate samples were maintained under static conditions, with no applied cyclic stretch.

Assay of ERK1/2, p90RSK, and p38 MAP Kinase—Using the Flexcell system the HASMC were subjected to cyclic stretching for different time intervals, and the activation of ERK1/2, p90RSK, and p38 MAP kinase was measured by immunoblotting with phosphospecific antibodies as described previously (12).

c-Jun Kinase Assay—The c-Jun kinase activity in stretched HASMC was determined by a method described earlier (12).

Electrophoretic Mobility Shift Assays—To determine the DNA binding activity of AP-1, C/EBP, and NF-{kappa}B, electrophoretic mobility shift assays (EMSA) were carried out as described previously (12, 39).

RNA Isolation and Reverse Transcription-Polymerase Chain Reaction (RT-PCR)—RNA isolation and RT-PCR analysis was performed using the procedure as described previously (40). Briefly, total RNA was extracted from 2 x 106 HASMC with TRIzol reagent (Invitrogen) following the manufacturer's suggestions. Total RNA samples (2 µg) was reverse-transcribed (RT) in a 20-µl reaction volume using oligo(dT)12–18 primers and SuperScriptTM II RNase H reverse transcriptase (Invitrogen) following the manufacturer's instructions. Ten percent of the RT reaction volume (2 µl, corresponding to 200 ng of original input RNA) was used for each subsequent 50-µl PCR reaction. Primer sequences that were used to amplify human TNF-{alpha}, IL-1{beta}, IL-8, and GAPDH are shown in Sequence I.


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SEQUENCE I
 

Hot start PCR reactions were conducted as 94 °C x 3'; (94 °C x 1', 55 °C x 30', 72 °C x 1') x 35 cycles; 72 °C x 5'. RT-PCR was also performed for GADPH as a control to check the amount and integrity of total RNA samples. The specificity of the amplified products was confirmed by the size of the templates and also by performing PCR reactions in the absence of RT products.

Decoy Oligonucleotide Technique—To understand the role of various transcription factors such as AP-1, C/EBP, and NF-{kappa}B in mechanical stretch-induced IL-8 production, we applied the "decoy" strategy to inhibit the activity of these transcription factors. HASMC plated in 6-well Bioflex plates were transfected with 10 µM phosphorothioate-modified double-stranded oligonucleotides (ODN) containing consensus sequences for either AP-1, C/EBP, or NF-{kappa}B transcription factors using LipofectAMINE Plus reagent (Invitrogen). The controls were transfected with corresponding scrambled oligonucleotide. The sequences of single-stranded ODNs are shown in Sequence II, where underlined nucleotides indicate the orthothionate-modified sequence.


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SEQUENCE II
 

The ODNs were annealed for 2 h with a steady temperature descent from 94 to 25 °C using a thermal cycler. The stability and hybridization of the oligonucleotide were confirmed in a 2.5% agarose gel.

Statistical Analysis—All experiments were repeated at least three times unless otherwise indicated. Results are expressed as mean ± S.D. Statistical analysis used Student's t test or analysis of variance to compare quantitative data populations with normal distribution and equal variance. A value of p < 0.05 was considered statistically significant unless specified.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
In this study we have investigated the effects of cyclic mechanical stretch on the expression and production of IL-8 in HASMC. We have also investigated the role of proinflammatory transcription factors and dissected the signaling pathways that led to the enhanced expression of IL-8 and protein production in response to mechanical stretch.

Cyclic Mechanical Stretch Causes an Augmented Expression of IL-8 mRNA in HASMC—HASMC were subjected to 15 min of 12% cyclic stretch at a frequency of 1 Hz. Eight hours later, total RNA was isolated, and RT-PCR was performed using specific primers for IL-1{beta}, IL-8, TNF-{alpha}, and GADPH. As shown in Fig. 1A, a significant up-regulation in mRNA level of IL-8 was observed whereas there was no effect on the level of IL-1{beta} and TNF-{alpha} expression. Furthermore, there was no difference in the mRNA level of a constitutively expressed GADPH gene. These data demonstrate clearly that cyclic stretching of HASMC leads to increased expression of IL-8. We also measured the IL-8 production in culture supernatants of HASMC after the application of mechanical stretch. As shown in Fig. 1B, a significant increase in the IL-8 level was observed in mechanically stretched cells. These data strongly suggest that cyclic stretch leads to an increased production of IL-8 protein in HASMC.



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FIG. 1.
Effect of cyclic mechanical stretch on the expression and production of IL-8 in HASMC. A, confluent cultures of HASMC were exposed to 12% cyclic mechanical stretch for 60 min. A, 8 h later, total RNA was isolated and subjected to RT-PCR analysis of IL-1{beta}, IL-8, TNF-{alpha}, and GADPH. A representative agarose gel shows a dramatic increase in the expression of IL-8 mRNA. The data also show no effect on the expression of IL-1{beta}, TNF-{alpha}, and GADPH. B, amount of IL-8 produced in culture supernatants assayed by ELISA of 1-h mechanically stretched HASMC. The assay was conducted 12 h after the completion of mechanical stretch protocol.

 

Cyclic Mechanical Stretch Activates AP-1 and C/EBP Transcription Factors—The IL-8 promoter is found between –1481 and +44 bp of the transcriptional start site and contains binding sites for AP-1, NF-{kappa}B, and C/EBP transcription factors (18, 19, 21, 27). We investigated the effects of cyclic mechanical stretch on the DNA binding activity of AP-1, NF-{kappa}B, and C/EBP transcription factors. As shown in Fig. 2, application of cyclic mechanical stretch of time intervals ranging from 2 to 60 min causes only a marginal increase in the NF-{kappa}B/DNA binding activity in these cells. On the other hand, the DNA binding activities of AP-1 and C/EBP transcription factors were enhanced in a time-dependent fashion. These results suggest that AP-1 and C/EBP could be two of the potential transcription factors that are activated by cyclic stretch and regulate the expression of IL-8 gene.



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FIG. 2.
Effect of mechanical stretching on the DNA binding activity of NF-{kappa}B, AP-1, and C/EBP transcription factors. HASMC were exposed to 12% mechanical stretching for indicated time intervals, and the DNA binding activities of NF-{kappa}B, AP-1, and C/EBP were determined as described under "Experimental Procedures." Representative EMSA gels show that the DNA binding activities of AP-1 and C/EBP transcription factors are significantly increased whereas there was only a minimal affect of NF-{kappa}B activity on mechanical stretching. Similar results were obtained in two other independent experiments.

 

Decoy Oligonucleotide of Either AP-1 or C/EBP Inhibits IL-8 Production in HASMC—To investigate whether any of the above transcription factors are involved in the increased production of IL-8 in HASMC in response to mechanical stretching, we employed the decoy oligonucleotide technique that is very effective in inhibition of the activity of transcription factors (41). HASMC were transfected for 4 h with the phosphorothiorate-modified double-stranded ODNs containing wild-type or mutated consensus sequences for AP-1, C/EBP, or NF-{kappa}B transcription factors followed by removal of transfection medium and replacement with fresh medium. HASMC were then subjected to 12% cyclic mechanical stretch at a frequency of 1 Hz for 60 min. The cells were again incubated at 37 °C, 5% CO2 for another 12 h. At the end of this incubation period the amount of IL-8 in the culture supernatants was measured using the IL-8 ELISA kit (BD Biosciences). As shown in Fig. 3, pretreatment of HASMC with wild-type AP-1 or C/EBP but not NF-{kappa}B oligonucleotides led to a significant inhibition in the stretch-induced production of IL-8. On the other hand scrambled oligonucleotide did not have any effect on IL-8 production in response to mechanical stretch (negative data not shown). These data strongly suggest that mechanical stretch-induced production of IL-8 in HASMC involves AP-1 and C/EBP transcription factors.



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FIG. 3.
Effect of decoy oligonucleotides on the production of IL-8 in HASMC in response to mechanical stretching. HASMC grown up to 80–90% confluence were transfected with 10 µM double-stranded NF-{kappa}B, AP-1, or C/EBP decoy oligonucleotides for 4 h followed by removal of the transfection medium and replacement with fresh medium. The cells were then subjected to 12% mechanical stretching for 1 h. Culture supernatants were collected after 12 h, and the amount of IL-8 was determined using ELISA. The data presented here show that decoy C/EBP and AP-1 oligonucleotides significantly inhibit the production of IL-8 by HASMC in response to cyclic mechanical stretching.

 

Cyclic Mechanical Stretch Activates ERK1/2, p90RSK, JNK1, and p38 MAP Kinases in HASMC—MAP kinase pathways are known to be activated in response to a number of stimuli and modulate the activity of many transcription factors as well the expression of a wide spectrum of genes (29). Data in Fig. 4A demonstrate that the application of cyclic mechanical stretch to HASMC activated ERK1/2 as early as 5 min, peaked at 15 min, and a slight decrease was observed thereafter. Cyclic stretching also caused a significant increase in the phosphorylation level of p90RSK, a physiological substrate of ERK1/2 (Fig. 4B), and p38 MAP kinase (Fig. 4D). Similarly, the activity of JNK1 was increased in response to mechanical stretch (Fig. 4C). The total cellular levels of ERK1/2, p90RSK, JNK1, and p38 were not affected by mechanical stretch (data not shown). These data thus suggest that cyclic mechanical stretching causes activation of MAP kinases as well as their downstream proteins such as p90RSK, which are directly phosphorylated by these kinases.



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FIG. 4.
Effect of cyclic mechanical stretching on the activation of ERK1/2, p90RSK, JNK1, and p38 kinase. HASMC were subjected to 12% cyclic mechanical stretching for the indicated amount of time and the activities of ERK1/2 (A), p90RSK (B), JNK1 (C), and p38 kinase (D) were determined by the immunoblotting and immunoprecipitation methods described under "Experimental Procedures." The representative data show that the cyclic mechanical stretch of HASMC activate ERK1/2, p90RSK, JNK1, and p38 kinase in a time-dependent manner.

 

Inhibition of ERK1/2 and p38 MAP Kinase Inhibits the Expression and Production of IL-8—Using specific pharmacological inhibitors we investigated the role of ERK1/2, JNK1, and p38 MAP kinases on the production of IL-8 from HASMC in response to cyclic mechanical stretching. Pretreatment of cells with 50 µM U0126 (a MEK1/2 inhibitor) or 20 µM SB203580 (a p38 kinase inhibitor) for 30 min caused a significant inhibition in the stretch-induced expression of IL-8 mRNA and its production in culture supernatants (Fig. 5). Although, the inhibition of both ERK1/2 and p38 kinase resulted in the decreased production of IL-8, p38 MAP kinase inhibition was more potent than ERK1/2 in down-regulating IL-8 production (Fig. 5). On the other hand, pretreatment of cells with 20 µM SP600125 (a JNK inhibitor) did not affect the expression or production of IL-8. These treatments did not have any significant affect on the viability of HASMC as measured by MTT assay (negative data not shown). These data thus suggest that ERK1/2 and p38 kinases are involved in the pathways that lead to the enhanced expression and augmented production of IL-8 in response to cyclic mechanical stretching of HASMC.



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FIG. 5.
Effect of inhibition of ERK1/2, JNK1, and p38 MAP kinase on the expression and production of IL-8 by HASMC in response to cyclic mechanical stretching. HASMC were pretreated with either 50 µM U0126 (ERK1/2 inhibitor) or 20 µM SP600125 (JNK inhibitor) or 20 µM SB203580 (p38 MAP kinase inhibitor) for 30 min followed by cyclic mechanical stretching for 1 h. A, 8 h later the expression of IL-8 in mechanically stretched HASMC was measured using RT-PCR and B, the production of IL-8 in culture supernatants was measured after 12 h following the 1-h stretch protocol. The data clearly show that both the expression and the production of IL-8 were significantly inhibited by pretreatment of HASMC with ERK1/2 and p38 kinase inhibitors whereas no effect was observed by inhibition of JNK.

 

Involvement of ERK1/2 and p38 Kinase in the Activation of AP-1 and C/EBP Transcription Factors in HASMC in Response to Cyclic Mechanical Stretch—Activation of MAP kinase signaling pathways is generally associated with the activation of nuclear transcription factors (42). Since the inhibition of ERK1/2 and p38 MAP kinase inhibited the expression and production of IL-8 in HASMC, we next investigated whether these kinases are involved in the pathways leading to the activation of AP-1 and C/EBP transcription factors. HASMC were pretreated with either 50 µM U0126 (a MEK1/2 inhibitor) or 20 µM SB203580 (p38 kinase inhibitor) for 30 min followed by mechanical stretching of HASMC for 1 h. Interestingly, pretreatment of HASMC with U0126 decreased the DNA binding activity of the AP-1 transcription factor without affecting the C/EBP transcription factor (Fig. 6). Contrary to this, SB203526 reduced the DNA binding activity of C/EBP transcription factor without having any affect on AP-1 (Fig. 6). These data strongly suggest that cyclic stretch regulate the activities of AP-1 and C/EBP transcription factor through the activation of ERK1/2 and p38 MAP kinase, respectively.



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FIG. 6.
Effect of inhibition of ERK1/2 and p38 kinase on the DNA binding activity of AP-1 and C/EBP transcription factors. HASMC were pretreated with 50 µM of U0126 (a MEK1/2 inhibitor) or 20 µM SB203580 (a p38 kinase inhibitor) for 30 min followed by the application of 12% cyclic mechanical stretching for 1 h followed by EMSA. Representative data show that inhibiting the activation of ERK1/2 inhibits the DNA binding activity of AP-1 whereas p38 kinase inhibitor decreases the activity of the C/EBP transcription factor.

 

Involvement of Protein Tyrosine Kinase (PTK), Phosphoinositide 3-Kinase (PI3K), Protein Kinase C (PKC), Protein Kinase A (PKA), and MEK1/2 in Mechanical Stretch-induced ERK1/2 and p38 Kinase in HASMC—Since activation of ERK1/2 and p38 MAP kinases is involved in the enhanced expression of IL-8 mRNA primarily through the activation of C/EBP and AP-1 transcription factors, we next investigated the upstream signaling mechanism(s) that leads to the activation of ERK1/2 and p38 MAP kinase in response to mechanical stretch. Protein tyrosine phosphorylation is one of the most important events that lead to the transduction of extracellular signals to the nucleus. Several receptor and non-receptor-associated PTK have been implicated in cell signaling (43). To understand whether PTK plays any role in the activation of ERK1/2 and p38 kinase, HASMC were preincubated with 50 µM genistein (a PTK inhibitor) for 30 min followed by cyclic stretching for 30 min. As shown in Fig. 7A, pretreatment of HASMC with genistein completely blocked the stretch-induced activation of both ERK1/2 and p38 MAP kinase. We also investigated the role of protein kinase A, protein kinase C, and MEK1/2 in cyclic stretch-induced activation of ERK1/2 and p38 in HASMC. As demonstrated in Fig. 7A, pretreatment of cells with H-7 (an inhibitor for PKA and PKC) only marginally affected the stretch-induced activation of ERK1/2. However, such treatment did not have any affect on the activation of p38 kinase. Additionally, our data show that ERK1/2 is the phosphorylated target of MEK1/2 as in the case of growth factor-induced ERK1/2 signaling. This is because the inhibition of MEK1/2 activity using U0126 completely inhibited the phosphorylation of ERK1/2 (Fig. 7A).



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FIG. 7.
Role of protein kinases, small GTPase proteins, SA channels, and extracellular Ca2+ ions in mechanical stretch-induced activation of ERK1/2 and p38 MAP kinase. A, HASMC grown to confluence were pretreated with indicated amounts of Genistein (a PTK inhibitor), H-7 (a PKA and PKC inhibitor), and U0126 (a MEK1/2 inhibitor) for 30 min followed by mechanical stretching for 30 min. A representative blot shows that PTK inhibitor genistein completely inhibits the activation of ERK1/2 and p38 kinase whereas the MEK1/2 inhibitor U0126 only inhibits the phosphorylation of ERK1/2. The activity of ERK1/2 and p38 kinase was not significantly affected by H-7. B, HASMC were pretreated with 50 µM LY290042 (a PI3K inhibitor) for 30 min followed by application of cyclic mechanical stretch for another 30 min, and ERK1/2 and p38 kinase activity was determined. The data show that PI3K inhibitor LY290042 inhibits the activity of ERK1/2 without having any effect on the activity of p38 kinase. C, HASMC were incubated with indicated amounts of toxin A (small GTPase proteins inhibitor), Gd3+ ions (SA channel inhibitor), and EGTA (Ca2+ ions chelator) for 18 h followed by mechanical stretching of these cells for 30 min. The data presented here show that the activity of p38 kinase is significantly inhibited by toxin A, Gd3+ ions, or EGTA treatment whereas no effect of these treatments was observed on the activity of ERK1/2. Similar data were obtained in another independent experiments.

 

PI3K plays a pivotal role in many signal transduction pathways and in the modulation of the activities of several transcription factors (44). To investigate whether PI3K is involved in the activation of ERK1/2 and p38 kinase, the HASMC were preincubated with 50 µM LY290042 (a PI3K inhibitor) for 30 min followed by application of cyclic mechanical stretch for 15 min. Interestingly, LY290042 only inhibited the activation of ERK1/2 without affecting the activation of p38 kinase (Fig. 7B). Similar results were obtained with wortmannin, another specific inhibitor of PI3K (data not shown).

Role of Stretch-activated (SA) Channels, Small GTPase Proteins, and Extracellular Ca2+ in Mechanical Stretch-induced Activation of ERK1/2 and p38 Kinase in HASMC—Activation of SA channels, which allows the influx of Na+, K+, and Ca2+ ions inside the cells, is considered one such early event that causes the activation of several signaling pathways in response to mechanical forces (45, 46). Smooth muscle cells were pretreated with 20 µM gadolinium (III) chloride (an inhibitor of SA channels) for 18 h followed by the application of cyclic mechanical stretch for 30 min. Interestingly, inhibition of SA channels with Gd3+ ions led to only marginal inhibition in the mechanical stretch-induced activation of ERK1/2 (Fig. 7C). On the other hand, the activity of p38 MAP kinase was completely blocked by treatment of cells with Gd3+ ions (Fig. 7C). Influx of Ca2+ ions from extracellular sources also plays the important role of second messenger in cell signaling in the activation of MAP kinase pathways in response to different stimuli, including mechanical stretch (47). To understand the role of Ca2+ in stretch-induced activation of ERK1/2 and p38 kinase in response to cyclic mechanical stretching, the HASMC were pretreated with a Ca2+ ion chelator EGTA before stretching. Pretreatment of cells with EGTA did not affect the activation of ERK1/2 (Fig. 7C, upper panel); however, the activity of p38 MAP kinase was significantly reduced by EGTA (Fig. 7C, lower panel). These data suggest that stretch-induced activation of p38 MAP kinase requires Ca2+ influx from an extracellular source.

We further sought to dissect the signaling pathways by studying the involvement of GTPase proteins. Small GTPases such as Rac, Rho, and cdc42 have been identified as important intermediates to the activation of the MAP kinase cascade (48, 49). The activity of these proteins is specifically inhibited by enterotoxin A from Clostridium difficile (50). Preincubation of HASMC with 100 ng/ml of toxin A caused a strong inhibition of mechanical stretch-induced activation of p38 kinase indicating the involvement of Rac and cdc42 in p38 kinase activation (Fig. 7C, lower panel). Virtually, no effect was observed on the activity of ERK1/2 by treatment of HASMC with toxin A (Fig. 7C, upper panel) suggesting that mechanical stretch-induced signals leading to the activation of ERK1/2 are not transduced through Rho-related GTPases.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
In this study we have investigated the effects of cyclic mechanical stretch on the expression and production of IL-8 in smooth muscle cells of human airways. We preferred to employ cyclic mechanical stretch rather than static constant stretch because the former closely approximates the in vivo mechanical loading experienced by HASMC. Our data demonstrate that cyclic mechanical stretching of HASMC induced the expression and the production of IL-8 in the culture supernatants. Cyclic stretch-induced IL-8 expression in HASMC was associated with a concomitant increase in the DNA binding activity of AP-1 and C/EBP transcription factors. In addition, cyclic stretch of HASMC increased the phosphorylation of ERK1/2, JNK1, and p38 MAP kinases. Therefore, the increased expression of IL-8 in response to cyclic stretch is attributed at least in part because of the increased activation of AP-1 and C/EBP transcription factors mainly through the activation of ERK1/2 and p38 MAP kinase signaling pathways. Additionally, the stretch-induced activation of ERK1/2 involves the activation of PI3K and PTK. In contrast, the activation of p38 kinase requires the extracellular Ca2+ influx, small GTPase proteins as well as the activation of SA channels and PTKs. A schematic representation of the signaling pathways that are involved in the enhanced expression and increased production of IL-8 in response to cyclic mechanical stretch of HASMC is depicted in Fig. 8.



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FIG. 8.
A putative mechanism of activation of different signaling pathways leading to enhanced expression and increased production of IL-8 in response to cyclic mechanical stretching of HASMC. The schematic representation of the signaling events clearly shows that the mechanical stretching of HASMC causes the activation of ERK1/2 and p38 kinase by two signaling pathways, which then modulate the activity of AP-1 and C/EBP transcription factors. The activation of AP-1 and C/EBP transcription factors leads to the expression of IL-8 mRNA followed by its translation into protein and subsequent release in the culture supernatants. SAC, stretch-activated channels.

 

Role of Mechanosensitive Transcription Factors and MAP Kinases in Stretch-induced IL-8 Expression in HASMC—Accumulating evidence suggests that the alteration in gene expression in response to mechanical forces is controlled mainly at the transcriptional level (12, 51). Such control is achieved through the specific mechanosensitive transcription factors that interact with regulatory sequences present in the promoter and enhancer regions of the target genes (1, 2, 3, 51, 52, 53). The sequence analysis has shown that the IL-8 promoter contains multiple potential regulatory transcription factor binding sites, including AP-1, C/EBP, NF-IL6, NF-{kappa}B, and octamer-binding protein motifs (18, 19, 21, 27). The induced expression of IL-8 in response to inflammatory mediators such as TNF, IL-1{beta}, or endotoxin in several epithelial and endothelial cells is mediated mainly through the activation of NF-{kappa}B and to some extent by AP-1 transcription factors (54, 55, 56). In contrast to the cytokine-induced activation of NF-{kappa}B, we observed a marginal increase in the activity of the NF-{kappa}B transcription factor in HASMC in response to cyclic mechanical stretch. On the other hand, a strong time-dependent activation of AP-1 and C/EBP transcription factors were observed in these cells in response to cyclic stretch (Fig. 2). Our data also suggest that the increase in the DNA binding activities of AP-1 and C/EBP transcription factors is essential for the expression and the production of IL-8. This is because the inhibition of the activity of either of these transcription factors by double-stranded decoy oligonucleotides significantly reduced the production of IL-8 by HASMC in response to cyclic stretch (Fig. 3). Our data also suggest that mechanical stretch-induced regulatory mechanism for IL-8 expression is different from that involved in either cytokine or growth factor-induced production of IL-8. Furthermore, NF-{kappa}B activation is dispensable in stretch-induced IL-8 expression. It has been shown that TNF requires only a minimal region of –130 bp relative to the transcriptional start site that contains NF-{kappa}B and AP-1 binding sequences to fully stimulate the transcription of IL-8 (18). Because the activity of the C/EBP transcription factor, which binds further upstream in the IL-8 promoter (57, 58) is required for the complete activation of IL-8, it further supports the contention that the regulatory mechanism of IL-8 by mechanical stretch is quite different from that occurring in the case of proinflammatory cytokines.

We also observed that mechanical stretching of HASMC increased the activity of ERK1/2, JNK1 p38, MAP kinase, and p90RSK, a downstream phosphorylation target of ERK1/2. Our data are consistent with other published reports demonstrating that mechanical stretching of smooth muscles leads to the activation of MAP kinase signaling pathways (Ref. 59, and references therein). The role of various MAP kinases in the expression and production of IL-8 is not well understood. In particular, it is not known among ERK1/2, JNK1, and p38 MAP kinase, which one is required for the induced expression of IL-8 in different cells. Furuichi et al. (60) have shown that production of IL-8 in hyperosmolarity-stimulated human bronchial epithelial cells requires the activation of JNK1 and p38 MAP kinase. Similarly it has been shown that sodium fluoride induces the production of IL-8 in lung epithelial cells through a p38-dependent mechanism (61). On the other hand other reports suggest that the activation of ERK1/2 pathway is indispensable for the expression of IL-8 in response to growth factors and other stimuli (62, 63). There is also evidence that suggest that the production of IL-8 does not require the activation of either ERK1/2 or p38 MAP kinase (64). Our data show that ERK1/2 and p38 MAP kinase are required for full transcriptional activation of IL-8 (Fig. 5). Interestingly, a recent report also implicates the role of ERK1/2 and p38 MAP kinases in stretch-induced IL-8 production in epithelial cell line (65).

We further investigated whether there is any link in the ERK1/2 and p38 kinase signaling with the downstream transcription factors that influence the expression of IL-8 in response to mechanical stretch. Interestingly, our results showed that the inhibition of ERK1/2 activity using U0126 significantly reduced the DNA binding activity of the AP-1 transcription factor (Fig. 6). Additionally, we observed that the DNA binding activity of C/EBP transcription factor was decreased in the presence of the p38 kinase inhibitor SB20358 (Fig. 6). Although, it has been established that MAP kinase pathways control the activity of AP-1 (32, 67, 68), the role of these pathways in regulation of C/EBP transcriptional activity is not very well understood. There is evidence that suggests that p38 MAP kinase can directly phosphorylate subunits of C/EBP transcription factor (69, 70). Consistent with these reports our present data show that p38 MAP kinase is involved in the activation of C/EBP transcription factor and the expression of IL-8 in the response of mechanical stretch in HASMC.

Mechanism of Activation of ERK1/2 and p38 Kinase in HASMC in Response to Cyclic Stretch—In the past decade, a significant amount of research has been conducted to understand the process of mechanical signal transduction. However, in the absence of specific cell surface receptors, it is still not clear how mechanical forces are converted into intracellular signals that lead to altered gene expression. Some investigators have proposed that transduction of mechanical signals to biological signals occur via the extracellular matrix/integrin/cytoskeletal axis (1). Mechanical stress causes deformation of the sarcolemma, which may directly or indirectly cause conformational changes in protein (and subsequent activation of them) that are anchored to the inner surface of cell membranes or in transmembrane proteins. Several effector enzymes such as protein tyrosine kinases, phospholipases, protein kinase C isoenzymes, and ion channels, such as the SA channels, are examples of sarcolemmal proteins that might be affected by mechanical stresses (1, 71).

To understand the upstream events that lead to the activation of ERK1/2 and p38 MAP kinase and hence the enhanced expression of IL-8, in response to mechanical stretching of HASMC, we investigated the role of different effector molecules and second messengers. We found that activation of both ERK1/2 and p38 kinase was completely abolished in the presence of genistein, a PTK inhibitor, suggesting that the activation of PTK is essential for the stretch-induced ERK1/2 and p38 kinase activation in HASMC (Fig. 7A). On the other hand, the activation of ERK1/2 and p38 kinase was not significantly altered in the presence of H-7, a PKA and PKC inhibitor (Fig. 7A) or in the presence of GF109203X (a PKC inhibitor, data not shown). The exact nature of the PTK that are involved in the mechanical stretch-induced ERK1/2 and p38 kinase is not known; however, it has been shown that focal adhesion kinase (FAK), a non-receptor tyrosine kinase localized at focal adhesion is involved in mechanosensing in fibroblast and endothelial cells (72, 73). Cyclic deformation of airway smooth muscle has been shown to increase the tyrosine phosphorylation of pp125FAK and paxillin adhesion proteins (13). Similarly the members of the Src family of protein tyrosine kinases such as c-Src have been suggested to constitute a part of mechanotransduction in endothelial cells in response to shear stress (Ref. 53, and references therein). Furthermore, there are additional reports suggesting that the production of IL-8 in myocytes and epithelial cells in response to infectious agents and proinflammatory cytokines require PTK (36, 64).

Small GTPase protein such as Rho, Rac, and cdc42 and a lipid kinase complex PI3K are important regulatory proteins that are involved in the initiation of different signaling pathways and regulating the major functions of the cell (76, 77, 78, 79). Our data with toxin A, an antagonist of GTPase proteins clearly showed that these proteins are involved in the stretch-induced activation of p38 kinase and not ERK1/2 in HASMC (Fig. 7C). On the other hand, the activation of ERK1/2 but not p38 required the activation of PI3K as the inhibition of PI3K by LY294002 only blocked the activation of ERK1/2 (Fig. 7B). Although, it has been established that both small GTPase proteins as well PI3K plays an essential role in G-protein-coupled receptor-mediated activation of the MAP kinase cascade, their role in stretch-induced cellular response is still enigmatic. Ikeda et al. (80) have shown that in endothelial cells, the activation of ERK1/2 in response to cyclic mechanical stretch involves PI3K and p21ras. Consistent with this study, our data demonstrate a similar role of PI3K in cyclic stretch-induced activation of ERK1/2. Our results also indicate that small GTPase proteins are essential in the mechanical stretch-induced p38 kinase activation but not ERK1/2 (Fig. 8). Since the activation of ERK1/2 and p38 required the participation of PI3K and small GTPase proteins respectively, it indicates that the upstream events that lead to the activation of ERK1/2 and p38 kinase in response to mechanical stretch are quite distinct, and therefore the expression of IL-8 is controlled by multiple signal transduction pathways.

Activation of mechanosensitive ion channels has been proposed as the transduction mechanisms between mechanical stress and various cellular responses (66). Recently, Inoh et al. (75) showed that the direct Ca2+ influx through SA channels is required for induced cyclic stretch-activation of NF-{kappa}B in cultured human fibroblast cells. The level of intracellular Ca2+ in skeletal muscles has been reported to increase in response to mechanical forces and this response is inhibited in the presence of Gd3+ ion or in the Ca2+-depleted solution (74). Similar to these studies we found that the activation of p38 kinase in HASMC is completely inhibited by pretreatment of the cells with Gd3+ ions or EGTA, a Ca2+ ion chelator suggesting that SA channels and Ca2+ influx from extracellular source is required for the activation of p38 kinase (Fig. 7C, lower panel). Interestingly, the activity of ERK1/2 was not affected by these treatments (Fig. 7C, upper panel). This would indicate that SA channels and Ca2+ do not play a role in the stretch-induced activation of ERK1/2 in HASMC. Furthermore, we ruled out the possibility of involvement of intracellular Ca2+ mobilization in stretch-induced activation of ERK1/2 or p38 kinase. This is because pretreatment of HASMC with TMB-8 (an inhibitor of intracellular Ca2+ ion) had no effect on the activation of either ERK1/2 or p38 MAP kinase (data not shown). Because SA channels and extracellular Ca2+ influx is required for the activation of p38 kinase and not for ERK1/2, our data further suggest that different effector molecules are involved in the signaling pathways that lead to the activation of ERK1/2 and p38 kinase in response to cyclic mechanical stretching. Based on the findings in this report, a putative sequence of signaling events that is initiated in response to cyclic stretch and causes secretion of IL-8 from HASMC is shown in Fig. 8.

In summary, our results provide fundamental information on the regulatory signaling mechanisms that lead to the enhanced expression and production of IL-8 from HASMC in response to cyclic mechanical stretch. Our data provide strong evidence that cyclic stretch-induced expression of IL-8 in HASMC is regulated by AP-1 and C/EBP transcription factors mainly through the activation of ERK1/2 and p38 MAP kinase signaling pathways, respectively.


    FOOTNOTES
 
* This work was supported in part by National Institutes of Health Grant HL-63134. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ To whom correspondence should be addressed: Pulmonary and Critical Care, Suite 520B, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030. Fax: 713-798-3619; E-mail: boriek{at}bcm.tmc.edu.

1 The abbreviations used are: HASMC, human airway smooth muscle cells; ERK1/2, extracellular regulated kinase-1 and 2; JNK1, c-Jun N-terminal kinase-1; p90RSK, p90 ribosomal S6 kinase; MAP, mitogen-activated protein; AP-1, activator protein-1; C/EBP, CCAAT/enhancer-binding protein; NF-{kappa}B, nuclear factor-{kappa}B; PI3K, phosphoinositide 3-kinase; PTK, protein tyrosine kinase; PKC, protein kinase C; PKA, cAMP-dependent protein kinase; SA, stretch-activated; FAK, focal adhesion kinase; EMSA, electrophoretic mobility shift assay; ODN, oligode-oxynucleotide; IL, interleukin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ELISA, enzyme-linked immunosorbent assay. Back



    REFERENCES
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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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