Role of the short isoform of light chain kinase in the contraction of cultured cells as examined by its down-regulation

Jianjun Bao*†, Kazuhiko Oishi†, Tomohisa Yamada†, Liqun Liu*, Akio Nakamura*, Masaatsu K. Uchida†, and Kazuhiro Kohama*‡

*Department of Pharmacology, Gunma University School of Medicine, Maebashi, Gunma 371-8511, Japan; and †Department of Pharmacology, Meiji Pharmaceutical University, Kiyose, Tokyo 204-8588, Japan

Communicated by Setsuro Ebashi, National Institute for Physiological Sciences, Okazaki, Japan, May 17, 2002 (received for review April 12, 2002) GbaSM-4 cells, smooth muscle cells derived from brain basilar muscle without any signs of MLC20 phosphorylation (12). artery, which express both 210-kDa long and 130-kDa short iso- Obviously, an alternative regulation system must play an active forms of myosin light chain kinase (MLCK), were infected with an role. In the search for this system, we were interested in the actin- adenovirus vector carrying a 1.4-kb catalytic portion of MLCK– and myosin-binding properties of MLCK and expressed the cDNA in an antisense orientation. Western blot analysis showed N-terminal (2) and C-terminal (13) portions of MLCK as that the expression of short MLCK was depressed without affect- recombinant . They were tested for a regulatory role in ing long MLCK expression. The contraction of the down-regulated terms of the ability to modify the actin–myosin interaction in cells was measured by the cell-populated collagen-fiber method. vitro. We obtained positive answers (reviewed in ref. 14), even The tension development after stimulation with norepinephrine or though they are devoid of the catalytic domain. A23187 was depressed. The additional infection of the down- As the first step to examine whether our observations in vitro regulated cells with the adenovirus construct containing the same are related to a physiological role in regulating actual contraction insert in a sense direction rescued not only the short MLCK of smooth muscle, we tried to obtain smooth muscle cells that are expression but also contraction, confirming the physiological role devoid of MLCK expression by introducing into them an anti- of short MLCK in the contraction. To examine the role of long MLCK sense cDNA of MLCK (15). The effect of down-regulation of in the residual contraction persisting in the short MLCK-deficient MLCK was tested by chemotaxis of smooth muscle cells, an assay cells, long MLCK was further down-regulated by increasing the that is based on cellular motility. multiplicity of infection of the antisense construct. The additional In the present study, collagen gels populated by smooth muscle down-regulation of long MLCK expression, however, did not alter cells in culture were used to detect the isometric contraction on the residual contraction, ruling out the involvement of long MLCK stimulation with agonists (16). We observed a depressed con- in the contractile activity. Further, in the cells where short MLCK traction in the cells in which short MLCK was selectively was down-regulated specifically, the extent of phosphorylation of down-regulated. However, the depression was not associated 20-kDa myosin light chain (MLC20) after the agonist stimulation with changes in MLC20 phosphorylation—i.e., MLC20 was was not affected. This finding suggests that there are additional phosphorylated as well as MLC20 in control cells. factors to MLC20 phosphorylation that contribute to regulate smooth muscle contraction. Materials and Methods Cell Isolation and Culture. Smooth muscle cells were isolated from yosin light chain kinase (MLCK) phosphorylates the 20- the basilar artery of guinea pigs as described for guinea pig MkDa light chain of smooth muscle myosin (MLC20) in the stomach (16). The smooth muscle cells were grown on the ϩ presence of Ca2 and (reviewed in ref. 1). The kinase surface of plastic dishes in DMEM of high glucose containing 50 activity is exerted through the catalytic domain located in the units/ml penicillin and 50 ␮g/ml streptomycin supplemented central part of MLCK. The N-terminal portion of MLCK acts as with 10% FBS. One of the smooth muscle cells cultured in a low an actin-binding domain, where the amino acids responsible for density was isolated by using cloning rings was named GbaSM-4 the binding have been sequenced (2, 3). The C terminus of and used for the experiments. MLCK consists of a domain called telokin, which is expressed in smooth muscle cells as an independent gene product (4, 5). Adenovirus Construction and Purification. The 1,366-bp MLCK– Because telokin binds myosin (6), the C terminus of MLCK is cDNA corresponding to bp 1666–3031 of cDNA encoding rabbit considered to be a myosin-binding domain (7). Isoforms of the smooth muscle MLCK (17) was isolated from pBst͞SM3-FL by enzyme are high molecular weight (long MLCK) and low PCR as described (15). This fragment was then inserted into a molecular weight (short MLCK) kinases with molecular masses cosmid vector pAxCAwt (Takara Shuzo, Kyoto, Japan) derived Ϸ Ϸ of 210 and 130 kDa, respectively. The short MLCK is best from adenovirus stereotype 5 with the E1 and E3 regions deleted known as the conventional smooth muscle MLCK. However, the at the SwaI site (Fig. 1). The sense and antisense orientation of long MLCK, which is additionally furnished with 922–934 resi- the insert were confirmed by the nucleotide sequencing with dues at the N terminus of the short MLCK (8), is poorly Model 371 DNA sequencer (Applied Biosystems). Recombinant characterized (reviewed in ref. 9). Smooth muscle myosin phosphorylated by the catalytic do- main of MLCK is in an active form and interacts with actin Abbreviations: MLCK, myosin light chain kinase; AxCA–MLCK͞antisense, adenovirus bear- filaments. This mode of regulation is widely accepted as the ing MLCK-cDNA in the antisense orientation; AxCA–MLCK͞sense, adenovirus bearing MLCK-cDNA in the sense orientation; AxCA–NLacZ, adenovirus recombinant containing intracellular path for the induction of smooth muscle contraction bacterial ␤-galactosidase; AxCA-stuffer, adenovirus recombinant containing nonsense (reviewed in ref. 10). However, several observations of smooth cDNA; MLC20, the 20-kDa light chain of smooth muscle myosin; moi, multiplicity of muscle contraction cannot be explained by the mode of phos- infection; NE, norepinephrine. phorylation (reviewed in ref. 11). For example, when uterine Data deposition: The sequence reported in this paper has been deposited in the GenBank ϩ smooth muscle was subjected to prolonged incubation in Ca2 - database (accession no. AB070227). free medium, oxytocin was able to induce contraction of the ‡To whom reprint requests should be addressed. E-mail: [email protected].

9556–9561 ͉ PNAS ͉ July 9, 2002 ͉ vol. 99 ͉ no. 14 www.pnas.org͞cgi͞doi͞10.1073͞pnas.142298599 Downloaded by guest on September 28, 2021 trough. After 7 days of incubation, a string-shaped fiber was formed and used for the tension measurement.

Western Blot Analysis. SDS͞PAGE and immunoblotting with a poly(vinylidene difluoride) membrane (Immobilion PVDF Transfer Membrane; Millipore) were performed as described (15, 16). The membranes were treated with as follows. A monoclonal against chicken gizzard MLCK (Sigma, clone K36) was used to detect both 130- and 210-kDa MLCKs. Rho-kinase was detected with a polyclonal antibody (K-18, Santa Fig. 1. Schematic representation of construction of the recombinant ade- Cruz Biotechnology). Telokin was detected with the polyclonal novirus vectors. We obtained adenoviral recombinants bearing a rabbit anti-telokin antibody (15, 19). The immunoreactive proteins smooth muscle MLCK–cDNA fragment coding 1666–3031 bp in a sense or were visualized by using enhanced chemiluminescence Western antisense orientation by COS͞TPC method (18) and named then AxCA–MLCK͞ ͞ Ј ͞ blotting detection system (Amersham Pharmacia) and or 3,3 - antisense and AxCA–MLCK sense, respectively. diaminobenzidine (Sigma).

Measurement of Isometric Tension. adenoviruses containing the inserts were constructed by the Smooth muscle cell fiber of 20 COS͞TPC (terminal complex) method (18) using an mm in length was mounted vertically in a 10-ml organ bath adenovirus expression vector kit (Takara Shuzo, Kyoto). The containing Leibovitz’s L-15 medium at 37°C and equilibrated for at least1hataresting tension of 2 mN. The tension development obtained viruses bearing the sense MLCK–cDNA and the was recorded isometrically with a force displacement transducer antisense MLCK–cDNA are reffered to as AxCA–MLCK͞sense (TB-612T, Nihon Kohden, Tokyo). These procedures are essen- and AxCA–MLCK͞antisense, respectively. As controls, AxCA- tially the same as those reported (16). stuffer, an adenoviral recombinant containing 450 bp of non- sense cDNA, and AxCA–NLacZ, a vector expressing bacterial MLC20 Phosphorylation Assay. The MLC20 phosphorylation assay ␤-galactosidase tagged with a nuclear localization signal, were using glycerol-PAGE coupled with Western blotting was per- also used. formed as described (15, 20). Briefly, smooth muscle fibers were stimulated with 10Ϫ7 M norepinephrine (NE) or 5 ϫ 10Ϫ6 M Partial Sequence of MLCK from GbaSM-4 Cells. Total RNA from A23187 for a specified time, then flash-frozen. The phosphor- GbaSM-4 cells was prepared by using RNeasy Max kit (Qiagen, ylated MLC20 was detected by Western blotting (see above) Chatsworth, CA), and poly(A)-rich RNA was purified with after glycerol-PAGE with the antibodies as follows. The mono- QuickPrep mRNA purification kit (Amersham Pharmacia). The clonal MY-21 antibody to MLC20 (Sigma) was used to recognize first-strand cDNA was synthesized by priming with oligo(dT) both phosphorylated and unphosphorylated MLC20s. The phos- using Thermoscript reverse transcription (RT)-PCR system (In- phorylated MLC20s were confirmed with antibodies donated by vitorogen), and the MLCK–cDNA fragment was amplified with Y. Sasaki (20), which recognize the MLC20 monophosphory- Expand High Fidelity PCR system (Roche). Two degenerated, lated at Ser-19 and the MLC20 diphosphorylated at Thr-18 and oppositely oriented oligonucleotides were designed as PCR Ser-19 (15, 20). Western blots were quantified by densitometry primers by deducing their nucleotide sequences from the amino using the NIH IMAGE program (Version 1.62). acid sequences in two highly conserved regions of a variety of MLCKs. The PCR product was subcloned into pGEM-T Easy Data Analysis. All data are presented as means Ϯ SE. ANOVA (Promega), and its sequence was determined with BigDye and Student’s t test were used to assess differences. P Ͻ 0.05 was Terminator cycle sequencing FS kit (Applied Biosystems). The considered statistically significant. identity of the nucleotide sequence of the cDNA introduced into the cells was 87% as compared with the rabbit smooth muscle Results MLCK gene. Such a high identity might enable the down- Down-Regulated Expression of Short MLCK in Cells Harboring Antisense regulation despite the species difference. The nucleotide se- MLCK. To examine to what extent the adenoviral vector was quence was deposited in the GenBank nucleotide sequence introduced into GbaSM-4 cells, we allowed the ␤-galactosidase database under the accession number AB070227. vector of AxCA–NLacZ to infect the cells. The staining of the infected cells by X-Gal (5-bromo-4-chloro-3-indolyl Transduction of Adenoviral Vectors into Cells. Cultured GbaSM-4 ␤-D-galactoside) showed a Ն90% efficiency of transduction Ϸ cells at 70% confluence were exposed to respective adenoviral (Fig. 2A). vectors in 100 multiplicity of infection (moi) for 1 h unless Cell-populated collagen fibers were formed from the otherwise stated. The viruses were then removed, and the cells GbaSM-4 cells, those infected with AxCA-stuffer, and those were incubated in fresh DMEM supplemented with 1% FBS infected with AxCA–MLCK͞antisense. Equal quantities of total overnight for preparation of reconstituted smooth muscle fibers protein extracts from these fibers were subjected to Western (see below). For the rescue experiments, AxCA–MLCK͞sense blotting (Fig. 2B), which showed that the long MLCK isoform of or AxCA-stuffer was transduced at a titer of 160 moi into the Ϸ210 kDa and the short MLCK isoform of 130 kDa were cells that had been transduced by AxCA–MLCK͞antisense. expressed to a similar extent in untreated fibers and fibers infected with AxCA-stuffer. However, the expression of short Preparation of Cell-Populated Collagen Fibers. String-shaped, re- MLCK was markedly decreased in AxCA–MLCK͞antisense- constituted tissue fibers were prepared as described (16). Briefly, transduced fibers; the signals from the short MLCK of AxCA- dispersed cells were suspended in an ice-cold collagen solution stuffer-transduced fibers and AxCA–MLCK͞antisense- containing 3 ϫ 106 cells/ml cultured cells, 2.2 mg/ml collagen transduced fibers were 94.2% Ϯ 6.2% (n ϭ 4) and 7.6% Ϯ 2.3% type I-A, and 0.24 mg/ml collagen type IV in DMEM as (n ϭ 4), respectively, of the signals from untreated fibers. In described (16). Two milliliters of the suspension was poured into contrast, the signals of the long MLCK from the AxCA-stuffer- ͞ a trough and placed in a CO2 incubator (humidified 5% CO2 transduced fibers and the AxCA–MLCK͞antisense-transduced 95% air atmosphere) at 37°C for 2 h, then 15 ml of fresh DMEM fibers were 96.1% Ϯ 5.5% (n ϭ 4) and 80.3% Ϯ 3.7% (n ϭ 4), PHYSIOLOGY containing 10% FBS was added to each Petri dish containing the respectively, of the signals from untreated fibers. These results

Bao et al. PNAS ͉ July 9, 2002 ͉ vol. 99 ͉ no. 14 ͉ 9557 Downloaded by guest on September 28, 2021 developed maximum tensions of 0.96 Ϯ 0.15 mN (n ϭ 4) and 0.81 Ϯ 0.09 mN (n ϭ 4), respectively, on NE (10Ϫ7 M) stimu- lation. However, the maximum tension induced by NE of the fibers infected with AxCA–MLCK͞antisense was depressed to 0.23 Ϯ 0.04 mN (n ϭ 4). These contractile responses could be repeatedly evoked by NE stimulation (data not shown). When the fibers were stimulated with A23187 (5 ϫ 10Ϫ6 M), the maximum tensions of fibers of the untreated cells and AxCA-stuffer-treated cells were 0.45 Ϯ 0.05 mN (n ϭ 4) and 0.36 Ϯ 0.07 (n ϭ 4), respectively. However, fibers of AxCA͞ antisense-treated cells contracted with maximum tensions of 0.12 Ϯ 0.03 mN (n ϭ 4) on A23187 stimulation. We used A23187 as a calcium ionophor that allows Ca2ϩ to enter into cells. Unlike the stimulation by NE, contraction evoked by A23187 smaller (Fig. 3B) was not reversible (data not shown); the reason for this Fig. 2. (A) Infection of GbaSM-4 with the adenovirus vectors. GbaSM-4 cells remains to be determined. cultured in monolayers were infected with AxCA–NLacZ and incubated for 72 h. The transduction was verified by ␤-galactosidase staining. Original magnification, ϫ100. (B) Comparison of MLCK, telokin, and Rho-kinase ex- Rescue Experiments. To see whether the depressed contractile pression in fibers reconstituted from GbaSM-4 cells by Western blot analyses. activities of the cells infected with antisense MLCK vector of Each lane was loaded with 5 ␮g of protein and subjected to immunoblotting AxCA–MLCK͞antisense is attributable to the targeting of the with monoclonal antibody against MLCK and polyclonal antibodies to Rho- endogenous mRNA of short MLCK, GbaSM-4 cells harboring kinase and telokin. Lane 1, Untreated GbaSM-4 cells; lane 2, GbaSM-4 cells AxCA–MLCK͞antisense were further infected with the sense infected with the control vector of AxCA-stuffer; lane 3, GbaSM-4 cells in- MLCK vector AxCA–MLCK͞sense. We then reconstituted fi- fected with the antisense vector of AxCA–MLCK͞antisense; MLCK, MLCK bers from these cells to see whether the down-regulation of short expression; telokin, telokin expression; Rho-kinase, Rho-kinase expression. MLCK was rescued. As a control, AxCA-stuffer was introduced into the AxCA–MLCK͞antisense-treated cells. When the cells ͞ ͞ harboring AxCA–MLCK antisense were further infected with indicate that transduction of AxCA–MLCK antisense produced AxCA–MLCK͞sense, the short MLCK expression increased to the short MLCK-deficient fibers. On the other hand, the ex- 94.1% Ϯ 5.3% (n ϭ 3). However, no such increase was observed pression of telokin and Rho-kinase remained unaffected as when AxCA–MLCK͞antisense-transduced cells were further shown in Fig. 2B. treated with AxCA-stuffer; the short MLCK isoform expression remained at 11.2% Ϯ 3.8% (n ϭ 3). These data indicate that the Inhibition of Isometric Contraction by the Down-Regulation of Short down-regulation of short MLCK expression was rescued by the MLCK. The contraction of the fibers reconstituted from GbaSM-4 additional transduction of AxCA–MLCK͞sense. No obvious cells was monitored isometrically. As shown in Fig. 3, fibers of difference in the long MLCK expression was observed when the the untreated cells and the cells infected with AxCA-stuffer fibers with AxCA–MLCK͞antisense were further treated with AxCA–MLCK͞sense, because the signals of Ϸ210 kDa were 87.6% Ϯ 8.3% (n ϭ 3) for the AxCA–MLCK͞sense-treated cells, and 79.1% Ϯ 7.8% (n ϭ 3) for the AxCA-stuffer-treated cells. Concerning the rescue of tension development, the maximum tension on NE stimulation of the AxCA–MLCK͞antisense- treated fiber was increased from 0.23 Ϯ 0.04 mN (n ϭ 4) (Fig. 3) to 0.57 Ϯ 0.07 mN (n ϭ 4) by the additional transduction of AxCA–MLCK͞sense. Similar recovery was detected on the stimulation with A23187, from 0.12 Ϯ 0.03 mN (n ϭ 4) (Fig. 3) to 0.28 Ϯ 0.04 mN (n ϭ 3), after the additional transduction. However, no recovery was observed after the additional trans- duction of AxCA-stuffer (data not shown). It is notable that the rescue of contraction by the transduction of AxCA–MLCK͞sense was incomplete, although the expression of short MLCK was rescued almost completely. We speculate that the incomplete rescue in tension development is attributable to the nonspecific effect of the repeated infection with the adenoviral vectors.

Effects of the Treatment with Antisense MLCK on MLC20 Phosphory- lation. To examine whether phosphorylation of MLC20 is af- fected by down-regulation of the short MLCK isoform, the fibers reconstituted from the GbaSM-4 cells treated with AxCA– MLCK͞antisense or AxCA-stuffer were stimulated with 10Ϫ7 M NE or 5 ϫ 10Ϫ6 M A23187, and then subjected to Western blot analysis with a monoclonal antibody against MLC20. In AxCA- stuffer-transduced fibers, NE stimulation for 5 min caused a Fig. 3. (A) Typical record of contraction of cell-populated fibers. Fibers ͞ significant increase in MLC20 phosphorylation as expressed by infected with AxCA-stuffer (traces a and c) or AxCA–MLCK antisense (traces b ϩ and d) were contracted isometrically by stimulation with 10Ϫ7 M NE (traces a (monophosphorylated MLC20 diphosphorylated MLC20) per and b), or 5 ϫ 10Ϫ6 M A23187 (traces c and d). (B) Means Ϯ SE (n ϭ 4) in mN total MLC20 from the basal level of 25.2% Ϯ 2.7% (n ϭ 4) to Ϯ ϭ Ϯ of maximum tension are expressed by bars and error bars. ***, Significant at 58.9% 5.0% (n 4). The phosphorylation was 37.3% 2.3% P Ͻ 0.001 level compared with AxCA-stuffer-transduced fibers. (n ϭ 4) after 10 min of stimulation, and 25.7% Ϯ 2.3% (n ϭ 4)

9558 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.142298599 Bao et al. Downloaded by guest on September 28, 2021 In Fig. 4A Top, we assigned MLC-P and MLC-2P as mono- phosphorylated MLC20 and diphosphorylated MLC20, respec- tively. The phosphorylation of MLC20 at Ser-19 or at Thr-18͞ Ser-19 activates myosin functions (21). However, other amino acids such as Ser-1 or Ser-2 were also phosphorylatable (22). Thus, MLC-P and MLC-2P bands might contain MLC20 that is not related to the myosin activation. To exclude such a possi- bility, we carried out Western blot analysis using an antibody that recognizes MLC20 monophosphorylated at Ser-19 and another that recognizes MLC20 diphosphorylated at Thr-18 and Ser-19 (15, 20). We failed to detect significant differences in the phosphorylation levels between the fibers treated with AxCA- stuffer and those treated with AxCA–MLCK͞antisense (Fig. 4A Middle and Bottom). These findings are compatible with the above conclusion that MLC20 phosphorylation was not affected, even though short MLCK was down-regulated.

Effect of ML-9 and Y-27632 on the Tension Development of the Fiber Deficient in the Short MLCK Expression. Treatment of GbaSM-4 cells with the antisense vector of AxCA–MLCK͞antisense hardly down-regulated the expression of long MLCK and telokin (Fig. 2B), though they are the products of the same gene (23). Telokin assembles myosin into thick filaments (6), but to our knowledge, a regulatory role of telokin in smooth muscle contraction has not been reported. On the other hand, Rho-kinase, though not a product of the MLCK gene, regulates smooth muscle contraction (reviewed in ref. 25). Therefore, we investigated whether long MLCK and Rho-kinase exert regulatory roles in the contraction of the fibers reconstituted from Gba-SM4 cells, using ML-9 as an inhibitor for the former (26) and Y-27632 as an inhibitor for the latter (27). As shown in Fig. 5A, trace a, and Fig. 5B, the fibers developed a small tension of 0.25 Ϯ 0.07 mN (n ϭ 4) on 10Ϫ7 MNE stimulation. After pretreatment of the fibers with 3 ϫ 10Ϫ5 M ML-9 for 20 min, the same stimulation developed a tension of Fig. 4. Myosin light chain (MLC20) phosphorylation in reconstituted fibers. 0.23 Ϯ 0.05 mN (n ϭ 4) (Fig. 5B), indicating that the contraction (A) AxCA-stuffer-transduced (lanes 1, 2, and 3) or AxCA–MLCK͞antisense- was not affected (compare trace a with trace b in Fig. 5A). transduced (lanes 4, 5, and 6) fibers were stimulated with NE 10Ϫ7 M (lanes 3 However, the level of MLC20 phosphorylation 5 min after NE Ϫ and 6) for 5 min or 5 ϫ 10 6 M A23187 (lanes 2 and 4) for 2 min, then subjected stimulation was reduced by pretreatment with ML-9 from to glycerol-PAGE and transferred to a nitrocellulose membrane. Unphosphor- 52.3% Ϯ 2.4% (n ϭ 4) to 39.3% Ϯ 6.2% (n ϭ 4) (Fig. 5C). This ylated MLC20 (MLC), monophosphorylated MLC20 (MLC-P), and diphospho- reduction is attributable to the inhibition of kinase activity of rylated MLC20 (MLC-2P) were simultaneously detected with monoclonal anti- MLC20 antiboby as shown in the Top. The blots were also reacted with long MLCK, which was not associated with any changes in the anti-MLC-P antibody (Middle) and anti-MLC-2P antibody (Bottom) to detect contractile activity. Ser-19-monophosphorylated MLC20 and Thr-18͞Ser-19-diphosphorylated Y-27632 greatly decreased the unstimulated, resting levels of MLC20 (15, 20). (B and C) Time course of MLC20 phosphorylation in fibers tension of the fibers, as shown in Fig. 5A, trace c. Unlike the stimulated with 10Ϫ7 MNE(B)or5ϫ 10Ϫ6 M A23187 (C). Values are means Ϯ pretreatment with ML-9, however, NE stimulation 20 min after SE (n ϭ 4). pretreatment with Y-27632 failed to develop tension of the fiber (Fig. 5A, trace c). This failure is associated with the reduction in Ϯ ϭ ͞ MLC20 phosphorylation to 27.4% 5.1% (n 4) (Fig. 5C). after 15 min of stimulation. The phosphorylation of the AxCA Unlike the inhibitory effect of ML-9 on MLC20 phosphoryla- antisense-treated fibers was 22.7% Ϯ 1.9% (n ϭ 4) before Ϯ ϭ tion, the inhibition of MLC20 phosphorylation by Y-27632 was stimulation, 52.3% 4.2% (n 4) 5 min after NE stimulation, associated with that of tension development. Ϯ ϭ and 35.6% 3.6% (n 4) 10 min after NE stimulation (Fig. 4B). The total abolition of contraction induced by NE stimulation These results were unexpected, because the contraction of the in the presence of Y-27632 is crucially important in precluding fibers in which short MLCK was down-regulated was markedly the role of short MLCK remaining after AxCA–MLCK͞ affected (Fig. 3B). antisense treatment. As shown in Fig. 2B, about 7% of short MLC20 phosphorylation on A23187 stimulation is shown in MLCK remained in the fiber, and this may be the cause of the Fig. 4C. The basal phosphorylation of the fibers with AxCA- residual contraction of trace a in Fig. 5A. However, this is not the stuffer and AxCA–MLCK͞antisense was 23.1% Ϯ 1.6% (n ϭ 4) case, because the effect of Y-27632 could not be related to the and 20.4% Ϯ 2.8% (n ϭ 4), respectively. After stimulation for 2 conventional, short MLCK (see Discussion). min, 5 min, and 10 min, the phosphorylation of the AxCA- The other finding with Y-27632 is that it caused relaxation stuffer-treated fibers was 62.7% Ϯ 5.3% (n ϭ 4), 46.8% Ϯ 4.8% without any relaxants, indicating that the fibers are furnished (n ϭ 4), and 32.7% Ϯ 3.6% (n ϭ 4), respectively; and that of the with an intrinsic tension exerts an active role. AxCA–MLCK͞antisense-treated fibers was 55.6% Ϯ 6.1% (n ϭ 4), 43.7% Ϯ 5.1% (n ϭ 4), and 30.2% Ϯ 4.2% (n ϭ 4), Contractile Activities of Fibers in Which both the Short and Long MLCK respectively. Again, no differences in MLC20 phosphorylation Isoforms Were Down-Regulated to Various Extents. The ratio of virus were detectable, regardless of whether or not short MLCK was vector titer to cell number—i.e., moi—was fixed at 100 moi, PHYSIOLOGY down-regulated. because this moi allows down-regulation of short MLCK with

Bao et al. PNAS ͉ July 9, 2002 ͉ vol. 99 ͉ no. 14 ͉ 9559 Downloaded by guest on September 28, 2021 Fig. 5. Effects of ML-9 and Y-27632 on the contraction and on the MLC20 phosphorylation of smooth muscle fibers in which short MLCK was down-regulated. (A) AxCA–MLCK͞antisense-transduced fibers were first contracted with 10Ϫ7 M NE (trace a). The fibers were washed for 60 min and incubated with 3 ϫ 10Ϫ5 M ML-9 (trace b) or 1 ϫ 10Ϫ6 M Y-27632 (trace c) for 20 min, then a second contraction was induced by the addition of 10Ϫ7 M NE. After another washing for 60 min, the third contraction was induced (trace d). (B) Means of the maximal contraction induced by 10Ϫ7 MNE(n ϭ 4) was expressed by bars and error bars as Ϫ means Ϯ SE. ***, Significant at P Ͻ 0.01 level. (C) The extent of the MLC20 phosphorylation (%) was determined 5 min after the 10 7 M NE stimulation as described in the legend to Fig. 4. The bars and error bars express mean Ϯ SE (n ϭ 4). The double arrowheads and the arrowhead are the MLC20 phosphorylation before Ϫ stimulation (see Fig. 4B) and after 10 7 M NE stimulation (see Fig. 4B), respectively. **, Significant at P Ͻ 0.01; ***, significant at P Ͻ 0.001.

little effect on the long MLCK expression (Fig. 2B). In this effectively down-regulated long MLCK. Thus, the expression of section, we altered the expression of the short and long MLCK long MLCK does not parallel the tension development. These isoforms to various extents by changing the moi of the AxCA– data are in conformity with those obtained with ML-9 in the MLCK͞antisense vector to examine how the alteration modified previous section, precluding the involvement of long MLCK in the tension development (Table 1). The transduction of AxCA– tension development. MLCK͞antisense at 50 moi reduced the expression of short MLCK to 50.5% Ϯ 6.2% (n ϭ 4). The tension developed by NE Discussion Ϫ Ϫ (10 7 M) and A23187 (5 ϫ 10 6 M) was also reduced by about GbaSM-4, a cell line derived from vascular smooth muscle cells, half—i.e., to 65.5% Ϯ 11.1% (n ϭ 4) and 51.1% Ϯ 4.8 (n ϭ 4), expresses both the short and long isoforms of MLCK. In the respectively. At 100 moi, the expression of short MLCK was present study, we down-regulated almost specifically the expres- reduced to 8.1% Ϯ 2.4% (n ϭ 4), and the tension development sion of the short MLCK isoform in the vascular smooth muscle to 28.4% Ϯ 4.9% (n ϭ 4) on NE stimulation and to 33.3% Ϯ cells, resulting in the depressed contraction. The decrease in the 8.3% (n ϭ 4) on A23187 stimulation. The increase of moi to 200 contraction caused by the down-regulation of short MLCK was failed to further down-regulate short MLCK; short MLCK not associated with a decrease in the MLC20 phosphorylation. Ϯ ϭ expression remained at 7.0% 2.1% (n 6). Further depression The time course of the phosphorylation on stimulation by of tension development was also not observed; the fibers at 200 agonists was hardly altered by whether or not MLCK was Ϯ ϭ moi developed tension of 25.2% 7.5% (n 6) on NE down-regulated (Fig. 4), indicating that tension generation can Ϯ ϭ stimulation and 31.1% 6.3% (n 6) on A23187 stimulation. be inhibited without reducing MLC20 phosphorylation. How- Thus, the down-regulation of the expression of short MLCK ever, this issue is not sufficient to exclude the likelihood that the closely paralleled the reduction of the tension development. MLC20 phosphorylation is necessary for this contraction. The expression of long MLCK in the fibers infected at 50, 100, Does the residual kinase activity to phosphorylate MLC20 in Ϯ ϭ Ϯ ϭ and 200 moi was 95.3% 9.7% (n 4), 83.6% 3.9% (n 4), the cells in which short MLCK was down-regulated explain the Ϯ ϭ and 56.1% 4.9% (n 6), respectively. As described above, residual contraction of the fiber reconstituted from them? tension development in response to both NE and A23187 Western blots (Fig. 2B) showed that the long MLCK was hardly stimulations was depressed when moi was raised from 50 to 100. down-regulated and can thus be expected to take part in the However, the down-regulation of long MLCK was slight. On the activity. The other candidate for the activity is Rho-kinase (Fig. other hand, the increase in moi from 100 to 200 did not cause 2B), which might phosphorylate MLC20 directly and͞or by further decrease in tension development, but the increase in moi phosphorylating myosin phosphatase to reduce its activity (28). We examined this question by the use of kinase inhibitors (Fig. Table 1. MLCK isoform expression and contractility 5A) as follows. After 5 min of stimulation by NE, MLC20 of the down-regulated cells was phosphorylated to 52.3% Ϯ 4.2% (n ϭ Maximal contraction, % 4) as shown in Fig. 5C. When the Rho-kinase activity was moi Short MLCK, % Long MLCK, % NE A23187 inhibited by Y-27632, MLC20 phosphorylation was reduced to 27.4% Ϯ 5.1% (n ϭ 4). In this fiber, contraction by the same 50 50.5 Ϯ 6.2 95.3 Ϯ 9.7 65.5 Ϯ 11.1 51.1 Ϯ 4.8 stimulation was totally abolished (trace c in Fig. 5A). Therefore, Ϯ Ϯ Ϯ Ϯ 100 8.1 2.4 83.6 3.9 28.4 4.9 33.3 8.3 the abolition was associated with the reduction of MLC20 Ϯ Ϯ Ϯ Ϯ 200 7.0 2.1 56.1 4.9 25.2 7.5 31.1 6.3 phosphorylation by 24.9% (Fig. 5C), a figure that explains the The maximum tension of the AxCA–MLCK͞antisense-transduced fibers was abolition in terms of MLC20 phosphorylation. ML-9 also re- expressed relative (%) to that of the AxCA-stuffer-transduced fibers. Means Ϯ duced MLC20 phosphorylation to 39.3% Ϯ 6.2% (n ϭ 4) (Fig. SE (n ϭ 6). 5C). According to Birukov et al. (26), the reduction by 13.0% is

9560 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.142298599 Bao et al. Downloaded by guest on September 28, 2021 attributable to the inhibition of the activity of the long MLCK. The remaining concern is whether or not GbaSM-4 cells are However, this reduction was not associated with any changes in smooth muscle cells. GbaSM-4 cells were stained with antibodies contractile activity (traces a and b in Fig. 5A). ML-9 plus Y-27632 against differentiated smooth muscle marker proteins—i.e., inhibited MLC20 phosphorylation to the resting, unstimulated ␣ Ϯ ϭ SM-2 myosin heavy chain (31) and smooth muscle -actin (24) level of MLC20 phosphorylation—i.e., 16.1% 2.9% (n 4) (data not shown). The presence of long MLCK indicated that (Fig. 5C)—indicating the involvement of additional kinase(s) in they were furnished with embryonic properties (9). MLC20 phosphorylation. Thus, the kinase activity to phosphor- ylate MLC20 in the short MLCK-deficient cells is composed of In the previous study (15), the down-regulation of MLCK was the long MLCK, Rho-kinase and unidentified kinase(s). One of performed with a plasmid vector carrying the same antisense those exerting a regulatory role through MLC20 phosphoryla- cDNA, a method that requires screening of mutants with tion is Rho-kinase. Long MLCK appears not to be involved in antisense MLCK. Therefore, these mutants might acquire a the regulation of contraction. salvage pathway during the repeated subculture. However, sub- What mechanism(s) is involved in the contraction unassoci- culture was not required in the present method using the ated with MLC20 phosphorylation? We have reported that adenovirus vector, ruling out such a possibility. Together with caldesmon (29) and calponin (30) exert a stimulatory effect on the rescue experiments, this finding demonstrates that the the myosin ATPase activity of smooth muscle through myosin- depression in contraction of the present study is solely attribut- binding activity. Similarly, we recently observed that the myosin- able to the down-regulation of the short MLCK isoform. binding fragment of MLCK, which is devoid of kinase activity, stimulated the myosin ATPase activity (13). These observations were made only in vitro. Therefore, these proteins are candidates This work was supported by grants from the Naito Foundation, the for the regulatory proteins that activate smooth muscle contrac- Smoking Research Foundation, and by Grants-in-Aid for Scientific tion independently of MLC20 phosphorylation through the Research and the Special Coordination Funds of the Ministry of myosin-binding activity (reviewed in ref. 14). Education, Culture, Sports, Science, and Technology of Japan.

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