Role of the short isoform of myosin light chain kinase in the contraction of cultured smooth muscle 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 proteins. 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 calmodulin (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 antibodies as follows. A monoclonal antibody 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 protein 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).