1Department of Molecular Pharmacology, Physiology, and Biotechnology, Brown University, Providence, Rhode Island 02912; and 2Department of Biological Sciences, Marquette University, Wisconsin 53233
Submitted 29 April 2004 ; accepted in final form 11 August 2004
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ABSTRACT |
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asthma; carbachol; deep inspiration; gene expression; histamine
Actin and myosin are major contractile proteins in airway smooth muscle cells. -Smooth muscle (
-SM) actin is the major actin isoform in differentiated smooth muscle cells in vivo (23). Smooth muscle myosin exists in multiple isoforms (11, 41). They include myosin heavy chain head isoforms (SMA, SMB), myosin heavy chain tail isoforms (SM1, SM2), and myosin light chain isoforms (LC17a, LC17b). Several laboratories have reported correlation between myosin heavy chain SMB isoform expression and myosin ATPase activity or shortening velocity in smooth muscle (3, 12, 22, 26, 40). Relative expression of the SM1 and SM2 myosin heavy chain isoforms has also been found to correlate with maximal shortening of single vascular smooth muscle cells (31). Similarly, several laboratories have reported correlation between LC17a and LC17b isoform expression and myosin ATPase activity or shortening velocity (29), but other laboratories did not observe such correlation (10). The regulation of
-SM actin and myosin mRNA transcription appears to involve complex combinations of multiple modules (23, 30). Such a complex system of gene transcriptional control may allow multiple stimulatory and inhibitory inputs into the regulation of
-SM actin and myosin mRNA transcription. In this study, we tested the hypothesis that sinusoidal length oscillation and receptor activation interactively regulate the abundance of mRNA encoding
-SM actin and myosin isoforms in intact bovine tracheal smooth muscle. ERK1/2 MAPK has been implicated in gene regulation and cell proliferation in airway smooth muscle (13, 33). U0126 inhibits MEK1/2, thereby inhibiting ERK1/2 phosphorylation and activation. Accordingly, we studied the effect of U0126 on receptor-mediated
-SM actin and myosin mRNA expression in bovine tracheal smooth muscle.
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MATERIALS AND METHODS |
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Sinusoidal length oscillation. The procedures for tissue equilibration and length manipulation were described previously (2, 18). Basically, a computer program controlled the sending of voltages to the length input port of the lever system at regular time intervals, thereby inducing sinusoidal oscillation of the lever arm and the muscle strip attached to the lever arm. For length oscillation experiments, muscle strips were first stretched to 12 g and then allowed to equilibrate for 1 h in PSS bubbled with air at 37°C. The muscle strips were then activated for 3 min with K-PSS, a solution similar to PSS in composition except that 104.95 mM NaCl was substituted by an equimolar concentration of KCl to check muscle viability. Muscle strips were then allowed to relax in PSS for 15 min and stretched to 12 g every 15 min during another hour of equilibration in PSS. Muscle strips were adjusted to optimal length (Lo) for contraction by releasing the muscle strips quickly to 2.5 g and then stimulated by K-PSS for 10 min. The active force developed in this contraction was recorded as Fo, which was used as an internal standard to normalize active force produced by carbachol and histamine in subsequent contractions. After the recording of Fo, muscle strips were allowed to relax in PSS for 15 min, and the muscle length was measured with a caliper (resolution = 0.1 mm). For slack length experiments, muscle strips were allowed to hang freely in a muscle chamber containing the solution to which the oscillating muscle strips were exposed.
Experimental protocols. In PSS experiments, muscle strips were unstimulated and equilibrated in PSS at slack length or at sinusoidal length oscillation. In histamine experiments, muscle strips were stimulated by 100 µM histamine at slack length or at sinusoidal length oscillation. In carbachol experiments, muscle strips were stimulated by 1 µM carbachol at slack length or at sinusoidal length oscillation. For each protocol, muscle strips were allowed to equilibrate in the solution for 15 min before sinusoidal length oscillation. Using a computer-controlled Dual Mode Lever System (model 300 B; Aurora Scientific) and DMC/DMA Version 3.1 software, we oscillated muscle strips at a frequency of 1 Hz and an amplitude of 10% Lo. We recorded peak force during length oscillation for 20 cycles at the beginning and every hour of length oscillation. Solutions in muscle chambers were changed every hour.
RNA extraction and reverse transcription. At the end of each experiment, muscle strips were weighed and then homogenized in 500 µl of lysis solution with 1% mercaptoethanol (GenElute Mammalian Total RNA Kit; Sigma). Tissue weights ranged from 30 to 50 mg. Tissue homogenate was then centrifuged at 16,000 rpm for 1 min at 20°C. The supernatant was collected for RNA extraction with the minicolumns according to the manufacturer's instructions. The reverse transcription (RT) procedure was similar to that described by Eddinger et al. (10). The extracted RNA was first mixed with 20 µg/ml oligo(dT) (Promega) and 500 µM dNTP (Promega) and heated at 68°C for 5 min. The mixture was rapidly cooled in wet ice for 20 s and then placed on a heating block set at 42°C. The following chemicals were then added to the tube for first-strand synthesis of cDNA: first-strand buffer (Invitrogen), 2 U/ml RNasin (Promega), 0.1 µg/µl acetylated BSA (Promega), 1.25 mM DTT (Promega), and 10 U/µl Superscript II RNase H-reverse transcriptase (Invitrogen). The RT reaction was carried out at the manufacturer's recommended temperature of 42°C for 2 h.
Polymerase chain reaction and DNA chip analysis.
Using aliquots of the RT product from each muscle sample, we set up four separate polymerase chain reaction (PCR) tubes to amplify the expression of GAPDH, myosin heavy chain isoforms SMA and SMB, myosin light chain isoforms LC17a and LC17b, and -SM actin. The primer sequences for GAPDH and
-SM actin were designed with GeneFisher software (15) based on the published mRNA sequences for bovine GAPDH and
-SM actin in the National Center for Biotechnology Information Entrez Nucleotides database (accession numbers U85042 and BM431010, respectively). The forward primer sequence (5' to 3') for GAPDH was CTG GGG TCT TCA CTA CCA, corresponding to nucleotide positions 259276 of the bovine GAPDH mRNA sequence. The reverse primer sequence (5' to 3') for GAPDH was TTG AGA GGG CCC TCT GA, corresponding to nucleotide positions 743759 of the bovine GAPDH mRNA sequence. The expected size of the PCR product was 500 bp. The forward primer sequence (5' to 3') for
-SM actin was AGC ATC CAA CCC TTC TCA, corresponding to nucleotide positions 84101 of the bovine
-SM actin mRNA sequence. The reverse primer sequence (5' to 3') for
-SM actin was TTC TCG AGG GAG GAG GA, corresponding to nucleotide positions 465482 of the bovine
-SM actin mRNA sequence. The expected size of the PCR product was 398 bp.
The primer sequences for myosin heavy chain SMA and SMB isoforms were the same as described by Eddinger and Meer (12) and corresponded to nucleotide positions 610629 and 806787 as published by Nagai et al. (32). These primers flank the 21-nucleotide exon insert that is present in SMB but absent in SMA isoforms. The forward primer sequence (5' to 3') for SMA/SMB was CAG TCC ATT CTC TGC ACA GG. The reverse primer sequence (5' to 3') for SMA/SMB was TCA TTC TTG ACC GTC TTG GC. The expected sizes of the PCR products for SMA and SMB were 197 and 218 bp, respectively. As reviewed by Babu et al. (4), alternative splicing of the smooth muscle myosin gene near the 5' end generates the myosin SMA and SMB isoforms. It is noteworthy that exon 5b for the seven-amino acid insert is separated from the flanking exon 5a and exon 6 by introns. Therefore, PCR products generated from amplification of the genomic DNA would have different sizes from the PCR products generated from RT-PCR of the mRNA. Because the primers for SMA/SMB were designed based on the sequence of rabbit smooth muscle myosin (32), we sequenced the SMA PCR product from bovine tracheal smooth muscle for comparison. DNA sequencing was done by automated DNA cycle sequencing at Brown University's DNA Sequencing Facility with the Big Dye Terminator kit 1.1 (Applied Biosystems, Foster City, CA). We have sequenced the SMA PCR product from both the forward and reverse directions with the appropriate primers. Results from the two directions of sequencing were consistent in producing the data in Table 1. We compared the bovine SMA PCR product sequence to the cDNA sequence for rabbit myosin heavy chain mRNA (GenBank accession no. M77812). As shown in Table 1, excluding the primer sequences, 146 of the 157 nucleotide bases (93%) of the bovine SMA PCR product sequence were identical to the rabbit SMA cDNA. The deduced amino acid sequences from the two sets of cDNA sequences indicated that 51 of the 52 amino acid residues (98%) were identical. We checked the bovine SMA PCR product sequence against the nucleotide sequences in GenBank with BLAST and found significant matches with smooth muscle myosin heavy chain sequences from human, mouse, and rat. The estimated probability that the matches happened by chance was extremely small (8 x 10421 x 1031). The sequence of the seven-amino acid insert in the myosin SMB isoform is highly conserved and identical in rat, rabbit, and mouse smooth muscles (4). The sequence data together with the BLAST results suggest that bovine tracheal smooth muscle expresses predominantly the myosin SMA isoform. It is noteworthy that human airway smooth muscle also expresses only the SMA isoform (28). We further confirmed our results by Western blotting with antibodies specific for smooth muscle myosin SMA and SMB isoforms (from T. J. Eddinger). We found that bovine tracheal smooth muscle expressed predominantly the SMA isoform, with the SMB signal indistinguishable from nonspecific binding (data not shown). Therefore, bovine and human airway smooth muscles are similar in expressing predominantly the SMA isoform (28).
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For the PCR reaction, an aliquot of RT product was added to a PCR mix containing Taq buffer (Promega), 1.5 mM MgCl2 (Promega), each dNTP at 0.2 mM, 1 µM forward primer, and 1 µM reverse primer. The PCR reaction tubes were placed in a thermocycler (Gene Amp PCR System 2400; Applied Biosystems), and the solution was heated to 80°C. The cycle was paused, and 1 U of Taq polymerase (Promega) was added to each tube. After 2 min at 94°C, the PCR temperature protocol consisted of 30 cycles of 94°C denaturation for 90 s, 55°C annealing for 2 min, and 72°C primer extension for 3 min. The PCR products were then allowed to cool gradually to 4°C and stored at 20°C until DNA chip analysis.
PCR products were analyzed by using the DNA500 chip and a bioanalyzer (2100 Bioanalyzer; Agilent Technologies). A DNA500 chip contains interconnected fluid wells and microchannels. A DNA500 chip was set up by filling the microchannels with a sieving polymer and fluorescence dye. An internal standard with known sizes and concentrations of DNA fragments was loaded into the chip. Samples of PCR products were loaded into the sample wells of the chip. The DNA500 chip was then placed inside the Agilent 2100 Bioanalyzer, which consists of a 16-pin electrode cartridge, a laser detector, and a computer. The computer regulated the voltage at the 16-pin electrode to inject individual samples into the separation microchannel to separate the PCR products by size. The separated PCR products were detected by their fluorescence at the laser detector. Using the information from running the internal standard before running the samples, the Agilent 2100 Bioanalyzer estimates the size and concentration of individual PCR products. DNA500 chips were set up and loaded with PCR products according to the manufacturer's instructions. Of the 12 sample wells available for analysis, the first and last wells were loaded with a 100-bp DNA ladder (Invitrogen) to detect any drift in the system during DNA chip analysis. The remaining 10 wells were used for the analysis of PCR products. The bioanalyzer analyzed PCR products by size and concentration. The bioanalyzer accurately estimated the sizes of the PCR products as follows: GAPDH, 500 bp; -SM actin, 398 bp; LC17a, 208 bp; LC17b, 253 bp; SMA, 197 bp; and SMB, 218 bp. Tissue wet weight of each muscle strip was measured before RNA extraction and used for standardization of PCR products.
Statistics. Data are shown as means ± SE; n represents the number of tracheal rings. Student's t-test was used for the comparison of two means (P < 0.05 considered significant).
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RESULTS |
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Figure 1C shows the peak forces developed by PSS-, histamine-, and carbachol-treated muscle strips after 4 h of sinusoidal length oscillation. During the 20 oscillatory cycles immediately after 4 h of length oscillation, peak forces developed by histamine- and carbachol-treated muscle strips both remained significantly higher than the peak force developed by PSS-treated muscle strips (P < 0.05). Furthermore, peak forces developed by histamine- and carbachol-stimulated muscle strips were also significantly different after 4 h of length oscillation.
Control experiments for RT-PCR and DNA chip analysis.
Figure 2 shows the typical patterns of DNA chip analysis: lane a shows the bands of the 100-bp DNA ladder; lanes b and c represent negative RT and PCR controls, which do not show any bands at the expected positions of the PCR products; lane d shows the PCR product of GAPDH at the expected 500-bp position; and lane e shows the PCR products of the myosin heavy chain SMA and SMB splice variants, at the expected 197- and 218-bp positions. We found that SMB was typically low or not detectable in bovine tracheal smooth muscle. Figure 2, lane f, shows the PCR products of the myosin light chain LC17a and LC17b splice variants at the expected 208- and 253-bp positions; lane g shows the PCR product of -SM actin at the expected 398-bp position. These results demonstrate the accuracy of PCR product analysis by the system of DNA chip analysis.
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Effect of sinusoidal length oscillation on mRNA expression in unstimulated, histamine-stimulated, and carbachol-stimulated tissues.
In these experiments, PSS-treated muscle strips were either held at slack length or placed at sinusoidal length oscillation for 2 or 4 h. As shown in Fig. 4, sinusoidal length oscillation significantly downregulated -SM actin expression by 44% in unstimulated muscle strips at 4 h (Fig. 4B) but showed no other significant changes. In contrast, when muscle strips were stimulated by 100 µM histamine and 1 µM carbachol, sinusoidal length oscillation did not significantly alter the abundance of mRNA encoding GAPDH, SMA, SMB, LC17a, and LC17b (Fig. 5). To determine the effect of muscle length per se on mRNA expression, we also compared unstimulated muscle strips held either at Lo or slack length in PSS but did not detect any significant differences in mRNA expression between muscle strips at the two muscle lengths (data not shown). Therefore, oscillatory strain appeared to be necessary to induce downregulation of
-SM actin expression in unstimulated muscle strips.
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DISCUSSION |
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Histamine- and carbachol-activated tissues did not respond to sinusoidal length oscillation with downregulation of -SM actin expression (Fig. 5), suggesting antagonistic interactions between mechanical stretch and receptor activation in regulating
-SM actin expression in intact airway smooth muscle. This pattern of antagonistic interaction between mechanical stretch and receptor activation was also observed in carbachol-induced mRNA expression experiments. Whereas carbachol significantly upregulated myosin heavy chain SMA expression in muscle strips held at slack length, carbachol did not significantly alter SMA expression in muscle strips with sinusoidal length oscillation (Fig. 7). Recently, Gosens et al. (17) investigated the effect of long-term (8 days) cholinergic receptor activation on contractile protein expression in organ-cultured bovine tracheal smooth muscle. They found that 1 µM methacholine induced a small (
20%) but statistically insignificant increase in myosin heavy chain protein expression, whereas 100 µM methacholine significantly downregulated myosin heavy chain protein expression. In this study, we observed a statistically significant 65% increase in myosin heavy chain SMA expression induced by 1 µM carbachol. These results together suggest that cholinergic receptor agonists may have a bimodal and concentration-dependent effect on myosin heavy chain expression in airway smooth muscle.
Carbachol also significantly upregulated GAPDH expression in bovine tracheal smooth muscle (Fig. 7). This effect was detectable in our study when we normalized PCR products by tissue wet weight. Recently, Glare et al. (16) reported variable GAPDH expression in bronchoalveolar lavage fluid cells and endobronchial biopsy tissues collected from control and asthmatic patients. Furthermore, recent findings suggest that GAPDH may be involved in the initiation of apoptosis in neurons and skeletal muscle cells (7, 20). These findings suggest that GAPDH is inadequate for the normalization of mRNA expression. The observed carbachol-induced upregulation of GAPDH expression in this study does not appear to represent a general increase in transcriptional activity, as the expression of -SM actin and myosin light chain LC17a and LC17b did not change significantly in carbachol-stimulated tissues. Furthermore, we found that GAPDH expression was upregulated by cholinergic receptor activation regardless of whether muscle strips were held at slack length or at sinusoidal length oscillation (Fig. 7). Therefore, unlike SMA expression, upregulation of GAPDH expression by cholinergic receptor activation appeared to be insensitive to the mechanical state of airway smooth muscle.
ERK1/2 MAPK has been implicated in gene regulation and cell proliferation in airway smooth muscle (13, 33). Cholinergic receptor activation by 1 µM carbachol has been found to stimulate ERK1/2 MAPK activation in airway smooth muscle (14). We previously found that 10 µM U0126 completely inhibited ERK1/2 MAPK activation induced by 1 µM carbachol (data not shown). In this study, we found that the upregulation of GAPDH and SMA expression by 1 µM carbachol was not significantly affected by 10 µM U0126 (Fig. 9). Therefore, cholinergic receptor-mediated upregulation of SMA and GAPDH expression appears to be mediated by mechanisms other than the ERK1/2 MAPK pathway in bovine tracheal smooth muscle. Actinomycin D is an inhibitor of RNA polymerase and has been used to inhibit gene transcription in bovine tracheal smooth muscle (21). However, actinomycin D (5 µg/ml) did not significantly change SMA and GAPDH mRNA abundance in carbachol-stimulated tissues (Fig. 10). This finding suggested that cholinergic receptor-mediated increases in SMA and GAPDH mRNA abundance were not due to increases in gene transcription. Recently, p38 MAPK-mediated mRNA stabilization has been implicated in cytokine-induced increase in cyclooxygenase-2 mRNA abundance in airway smooth muscle (35). It has been recognized that mRNAs having AU-rich elements in their 3'-untranslated regions are stabilized by p38 MAPK (8). Bakheet et al. (5) compiled a database of AU-rich element mRNAs and found a wide repertoire of functionally diverse proteins. A search on their ARED 2.0 website revealed myosin, tropomyosin, and NADH dehydrogenase mRNAs as having AU-rich elements. Cholinergic receptor stimulation has been found to stimulate p38 MAPK phosphorylation in bovine tracheal smooth muscle (24). Therefore, we speculate that p38 MAPK-mediated mRNA stabilization could be an important mechanism in carbachol-induced increases in SMA and GAPDH mRNA abundance in bovine tracheal smooth muscle.
Histamine did not significantly alter the expression of GAPDH, myosin heavy chain SMA and SMB, myosin light chain LC17a and LC17b, or -SM actin in bovine tracheal smooth muscle (Fig. 6). Panettieri et al. (34) found that histamine induced proliferation and c-fos expression in cultured airway smooth muscle cells but did not significantly alter the ratio of muscle-specific myosin heavy chain to total myosin heavy chain. Therefore, Panettieri et al. (34) concluded that histamine did not induce significant phenotypic modulations toward the production of more muscle-specific myosin heavy chains. Our observation that histamine did not significantly alter myosin heavy chain SMA expression confirms the findings of Panettieri et al. (34) in intact airway smooth muscle. We found that bovine tracheal smooth muscle expresses almost exclusively the myosin heavy chain SMA isoform, with near-zero SMB expression. Furthermore, we found that sinusoidal length oscillation did not significantly affect the relative ratios of myosin heavy chain SMA and SMB isoforms or LC17a and LC17b isoforms in bovine tracheal smooth muscle (Figs. 46). Interestingly, Ma et al. (28) also found no change in the expression of SMA in human asthmatic airway smooth muscle, together with the absence of SMB expression. These findings together suggest that phenotypic plasticity of myosin isoform expression may be limited in intact airway smooth muscle cells in vivo.
Results from this study are summarized in the model of interactive regulation of the abundance of mRNA encoding -SM actin and myosin heavy chain by sinusoidal length oscillation and receptor activation shown in Fig. 11. In this model, the downregulation of
-SM actin expression by sinusoidal length oscillation is opposed by histaminergic and cholinergic receptor activation. Similarly, the upregulation of myosin heavy chain SMA expression by cholinergic receptor activation is opposed by sinusoidal length oscillation. To our knowledge, this is the first report of interactive regulation of the abundance of mRNA encoding
-SM actin and myosin heavy chain by sinusoidal length oscillation and receptor activation in intact airway smooth muscle. However, the upregulation of GAPDH by cholinergic receptor activation is not sensitive to sinusoidal length oscillation, suggesting the coexistence of mechanoinsensitive pathways of gene regulation in airway smooth muscle cells.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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