EXPERIMENTAL
CELL
RESEARCH
194,186189
(1991)
Density-Related Expression of Caldesmon and Vinculin in Cultured Rabbit Aortic Smooth Muscle Cells VLADIMIR P. SHIRINSKY, KONSTANTIN G. BIRUKOV, VICTOR E. KOTELIANSKY, MARINA A. GLUKHOVA, ELPIS SPANIDIS,* JANET D. ROGERS,* JULIE H. CAMPBELL,*” AND GORDON R. CAMPBELL? Cardiology Research Center of the USSR Academy of Medical Sciences, 121552 Moscow, USSR; *Baker Medical Research Institute, Melbourne; and TDepartment of Anatomy, University of Melbourne, Victoria, Australia
Quantitative immunoblotting techniques were used to study the effects of seeding density on the expression of caldesmon and vinculin variants, which are sensitive markers of vascular smooth muscle cell (SMC) phenotypic modulation in culture. Rabbit aortic SMC were seeded at different densities: 13 x 10’ cells/cma (high density), 3 X 10’ cells/cm’ (medium density), and 0.2 X 10’ cells/cma (low density) and cultured in the presence of 5% fetal calf serum. Irrespective of cell density and growth phase, caldesmon,,, was gradually and irreversibly substituted by caldesmon,, , but at high seeding density this substitution proceeded at a slower rate. The fraction of meta-vinculin (smooth muscle variant of vinculin) was reduced after seeding SMC in culture, but was reestablished when the cells reached confluency. Thus, high SMC seeding density is essential but not sufficient to keep vascular SMC cultured in the presence of serum in the contractile phenotype. o 1991 Academic
Press,
Inc.
INTRODUCTION Vascular smooth muscle cells (SMC) enzyme isolated and seeded in primary culture at a density below 5 X 10’ cells/ml (7 X lo4 cells/cm2) undergo a phenotypic modulation over the first few days then proliferate if serum mitogens are present [ 11. Ultrastructural examination shows that these SMC lose the bulk of their myofilaments and develop extensive endoplasmic reticulum [2, 31. This process is accompanied by a reduction in quantity of contractile proteins and substitution with nonmuscle counterparts [3-61. Following confluence the SMC may reexpress smooth muscle myosin and a-actin mRNA [3,4,7], but this is dependent on the initial SMC seeding density and thus the number of cell population doublings [3]. In the confluent state, visceral SMC from guinea pig vas deferens [8] and ureter [9] and the ’ To whom correspondence and reprint requests should dreesed at Baker Medical Research Institute, Commercial Prahran, Victoria 3181, Australia. Fax: 61-3-5211362. 0014~4327/91$3.00 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
chicken gizzard [lo] gain (or regain) the ability to contract; however, data on the contractile activity of confluent vascular SMC are limited [ 111. The aim of the present study was to investigate the effect of SMC seeding density on the expression of caldesmon and vinculin-cytoskeletal proteins reported to be sensitive markers of phenotypic modulation of vascular SMC in culture over 2 weeks in primary culture [5,6, 121. Caldesmon is the Ca2+-calmodulin-dependent regulator of actomyosin interaction in smooth muscle and nonmuscle cells [13-151. It is mainly expressed in mature rat and human aortic SMC as the 150-kDa isoform,2 and caldesmon,,, is substituted by caldesmon,, variant in the course of culture [5, 61. Vinculin and meta-vinculin are found in dense plaques where SMC actin filaments are anchored to the plasma membrane [20,21]. Meta-vinculin is a vinculin variant restricted to muscle cells [12,18-201. Meta-vinculin comprises about 40% of the total vinculin immunoreactivity in the mature human aortic SMC while in fetal SMC as well as in those proliferating in culture it is almost absent [13]. Our data demonstrate that the expression of caldesmon significantly differs from that of vinculin, and that high seeding density is important, but not sufficient, for the complete preservation of SMC contractile phenotype in culture. MATERIALS
AND METHODS
Elastase (type IV from bovine pancreas with 5 units/mg of tryptic activity), collagenase (type IV from Clostridium histolyticum) and soybean trypsin inhibitor were purchased from Sigma Chemical CO. (St. Louis, MO). Nitrocellulose (0.45 pm) was from Schleiaher and Schuell (FRG). Tissue culture plastic ware was purchased from Sterilin, Ltd. All other reagents were of tissue culture grade or analytical grade.
’ Molecular mass of heavy and light caldesmon variants was shown to be somewhat different from that determined by electrophoretic methods [16, 171. In this study, to distinguish between caldesmon variants we used the technique of immunoblotting, and therefore used the terms CaD,, for heavy caldesmon and CaDrr for light isoform.
be adRoad,
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12345 FIG. 1. Immunochemical detection of vinculin and caldesmon variants in cultured rabbit aortic SMC: (1) 7.5% SDS-PAGE of total SMC proteins; (2-5) total SMC protein transfer stained with antibodies to vinculin (2,3) and caldesmon (4,5) revealed by peroxidase-conjugated secondary antibodies (2,4) as well as 1261-labeled secondary antibodies and subsequent autoradiography (3,5). M, myosin, A, actin.
of Caldesmn
On the basis of densitometry of caldesmon bands (revealed on immunoblots by peroxidase reaction) and of radioactivity counting of the excised caldesmon bands detected with lz51-labeled secondary antibodies, the initial ratio of CaD,,/CaD,, was determined to be about 6 (Fig. 2). Hence, approximately 85% of caldesmon immunoreactivity is concentrated in caldesmon,, band in a freshly isolated suspension of rabbit aortic SMC. This ratio decreased progressively with time of culture indicating the shift to the expression of caldesmon,, isoform (Fig. 2, middle row). There was no increase in Cal&,/ CaD,, value either when SMC reached confluency and stopped to divide and incorporate thymidine (data not shown) or when FCS was withdrawn from the culture medium in low density culture (Fig. 2, upper row and middle row). However, the extent of the shift towards the expression of CaD,, depended on the cell seeding density. At higher densities more CaD,,, was expressed in the cells of the same culture age.
Expression of Vinculin SMC from media of the 9- to 12-week-old rabbit aorta were isolated enzymatically according to Charnley-Campbell et al. [22] and cultured in 90-mm plastic petri dishes (63 cm* growth area) in medium 199 (Commonwealth Serum Laboratories, Melbourne, Australia) supplemented with 5% fetal calf serum (FCS), 60 pg/ml penicillin, and 2 mM L-glutamine at 37’C in the atmosphere of 95% air, 5% CO,. SMC (10 ml) were seeded in 63-cm2 petri dishes at concentrations of 8 X 10s cell/ml (13 X 10’ cells/cm*, high density), 2 X l@ cells/ml (3 X 10” cells/cm’, medium density), and 10’ cells/ml (0.2 X 10’ cells/cm’, low density). SMC attached and spread within the first 2 days after seeding, and culture medium was replaced every third day. The seeding efficiency of the cells varied from 80-95% and was highest for the high seeding density. Samples of the total SMC proteins for SDS-PAGE were collected by rapid scraping of cells washed three times in ice-cold phosphatebuffered saline (PBS) in 100 ~1 of the sample buffer containing 0.2 M Tris-HCI, pH 6.8,32% (v/v) glycerol, 6.4% (w/v) SDS, 1.2 M 2-mercaptoethanol, 0.2% (w/v) bromophenol blue, and boiling for 3 min. Final volume of the sample did not exceed 350 pl. SDS-PAGE was conducted according to Laemmli [23]. Protein transfer to the nitrocellulose was done as described by Towbin et aL [24]. Nonspecific binding sites on the nitrocellulose were blocked by 1 h soaking of the paper in PBS with 0.5% dry milk powder and 0.1% Tween 20. Intermediate washes were accomplished by PBS-Tween 20 or PBS alone. Rabbit polyclonal affinity-purified antibodies to chicken gizzard caldesmon have been characterized previously [25]. To recognize vinculin variants mouse monoclonal antibodies raised against human uterus meta-vinculin (clone VIIFS) were used [26]. The antibodies recognized both protein variants equally. To reveal caldesmon and vinculin variants position on immunoblots, secondary sheep antirabbit and rabbit anti-mouse peroxidase-conjugated antibodies were used (Silenus Laboratories, Australia). 4Chloronapthol was utilized as the substrate for peroxidase reaction, and for quantitative immunoblotting, izWabeled antibodies instead of peroxidase-labeled antibodies were used (Fig. 1). Quantitation of radioactivity incorporated in caldesmon and vinculin bands was conducted as previously outlined [12]. The method was linear within the range of protein loadings used.
Variants
Variants
The measurement of meta-vinculin to vinculin ratio in freshly isolated rabbit aortic SMC provided the value of 0.5 (Fig. 2, bottom row). Thus, meta-vinculin incorporated approximately 33% of the total vinculin immunoreactivity in these cells. During the first 5-6 days metaVin/Vin ratio decreased more than twice in the medium density culture but returned to the original level a week later (Fig. 2B, bottom row). The drop in meta-Vin/Vin ratio in the low density culture during the first days of cultivation was more profound and irreversible (Fig. 2C, bottom row). Meta-vinculin was hardly detectable in these cells by our methods. The removal of FCS from the growth medium on Day 11 did not affect the expression of vinculin variants in low density culture within the following 7 days (Fig. 2C, bottom row). In SMC plated at high density the vinculin variant ratio remained unaltered throughout 2 weeks of culture and equalled the initial value of 0.5 (Fig. 2A, bottom row). DISCUSSION Several lines of evidence suggest that SMC which have undergone a change in phenotype in primary culture may regain their original phenotype after achieving a confluent monolayer. In the confluent state, rabbit aortic SMC regain the expression of smooth musclespecific myosin heavy chains and cu-actin mRNA depressed during modulation and proliferation [3,7]. Concomitantly the volume fraction of myofilaments increases in the confluent SMC returning to levels similar to those in cells prior to culturing [3]. It has thus been
188
SHIRINSKY HMH DENSITY CULTURE
ET
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(C)
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5c
-
--
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/
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FIG. 2. Changes of cell number, at high density (A), medium density ratio (bottom row) were determined growth medium. The data represent
CaD,,/CaD,, ratio and meta-VinNin (B), and low density (C). Cell number as a function of days in culture. Arrows the mean value of two experiments.
proposed that in these circumstances the reversibility of phenotypic modulation depends on the initial cell seeding density, which in turn determines the number of cell doublings the SMC undergo before achieving confluency in primary culture. In the present work we have extended our studies of rabbit aortic SMC modulation in culture and looked at the seeding density-related changes in expression of caldesmon and vinculin proteins associated with the smooth muscle cytoskeleton and contractile apparatus [ 13, 211. The dependence of vinculin variant expression on SMC seeding density was similar to that found for a-actin [3]. After an initial drop in meta-VinlVin ratio, which lasted from the start of the culture until the onset of intensive cell division, we observed a complete resto-
ratio in the course of rabbit aortic SMC culture. Cells were seeded (upper row), CaD,,/CaD,, ratio (middle row) and meta-VinlVin in panel C indicate the timepoint when FCS was removed from the
ration of the ratio back to 0.5 in the medium density culture following confluence and inhibition of SMC proliferation. SMC seeded at low density did not achieve confluent state during the time of experiment (18 days) and we observed irreversible loss of meta-vinculin from these cells. These results are in agreement with the previously reported loss of meta-vinculin from focal contacts of cultured human aortic SMC [12] and suggest that cell-to-cell contacts, rather than cell-to-substratum interactions, regulate meta-vinculin expression. This may explain why, in high density culture where contacts between cells are established just after spreading, there was no relative regression of meta-vinculin expression. Thus meta-vinculin, together with (Yactin and smooth muscle myosin heavy chains, maybe-
CALDESMON
AND
VINCULIN
IN
long to a class of SMC protein markers whose expression in cultured cells depends on intercellular interactions. Further experiments need to be performed to confirm this tenet. Caldesmon expression was, however, very different from that of vinculin. Irrespective of cell seeding density and growth phase we observed a gradual substitution of CaD,,, with CaD,, during cell cultivation which was reflected in the decrease of CaD,,/CaD,, ratio. Nevertheless, at higher cell density the rate of exchange of CaD,, for CaD,, was significantly decreased. As seen in Fig. 2 on Day 8 of culture, Cal&,, comprised 70% of caldesmon immunoreactivity in high density cultures whereas in medium density cultures and low density cultures this value was 38% and 0.05%, respectively. Our data are consistent with findings in cultured rat and human aortic SMC where the substitution of CaD,,, with CaD,, was also not found to be reversible [5,6]. At present, the B&H, cell line remains the only cell type known to reexpress CaD,,, after growth arrest [5]. Thus, the density of SMC is important for the expression of protein markers associated with SMC contractile phenotype, e.g., a-actin mRNA, smooth muscle myosin heavy chains, meta-vinculin, and caldesmon,,, . However, the lack of cell density-dependent reappearance of caldesmon150 indicates that the establishment of contact between SMCs is not sufficient for complete reversal of phenotypic modulation and the expression of certain proteins is controlled by different mechanisms.
CULTURED
Charnley-Campbell, J. H., Campbell, J. Cell Biol. 8, 379-383.
2.
Charnley, J. H., Campbell, G. R., McConnell, J. D., and Groschel-Stewart, U. (1977) Cell Tissue Res. 177, 503-522. Campbell, J. H., Kocher, O., Skalli, O., Gabbiani, G., and Campbell, G. R. (1989) Arteriosclerosis 9, 633-643. Rovner, A. S., Murphy, R. A., and Owens, G. K. (1986) J. Biol. Chem. 261, 14,740-14,745. Ueki, N., Sobue, K., Kanda, K., Hada, T., and Higashino, K. (1987) Proc. Natl. Acad. Sci. USA 84,9049-9053.
3. 4. 5.
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November 19,199O version received January
28, 1991
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Glukhova, M. A., Kabakov, A. E., Frid, M. G., Ornatsky, 0. I., Belkin, A. M., Mukhin, D. N., Orekhov, A. N., Koteliansky, V. E., and Smirnov, V. N. (1988) Proc. Natl. Acad. Sci. USA 85, 9542-9546.
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H., Fingerle, J., Rupp, Ch. C. (1988) Exp.
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J. H., Campbell, G. R., and Burnstock, G. (1974) J. Exp. Morphol. 32,297-323. J. H., and Campbell, G. R. (1975) Cytobiology 11,358-
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Campbell, G. R., Charnley, Anat. 117,295-321.
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Gunther, S., Alexander, R. W., Atkinson, M. A. (1982) J. Cell Biol. 92, 289-298.
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Belkin, A. M., Ornatsky, 0. L., Kabakov, A. E., Glukhova, M. A., and Koteliansky, V. E. (1988) J. Biol. Chem. 263,6631-6635.
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Sobue, K., Muramoto, Y., Fujita, M., and Kakiuchi, Proc. Natl. Acad. Sci. USA 78, 5652-5655.
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Sobue, K., Tanaka, T., Kanda, K., Ashino, N., and Kakiuchi, (1985) Proc. Natl. Acad. Sci. USA 82,5025-5029.
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Lash, J. A., Sellers, J. R., and Hathaway, Chem. 261,16,155-16,160.
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Bryan, J., Imai, M., W. G. (1989) J. Biol.
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Bryan,
J. H., and
Burnstock,
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W. J., and Gimbrone,
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Lee, R., Moore, P., Cook, Chem. 264, 13,873-13,879.
J., Lee, R., Chang,
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S. J., and Lin, W. G. (1989)
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Feramisco, J. R., Smart, J. E., Burridge, Thomas, G. P. (1982) J. Biol. Chem. 257,
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Sihciano, 533-535.
M.,
M.,
and Kojima,
J. D., and Craig,
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Nature
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Geiger, B., Dutton, A. H., Tokuyasu, K. T., and (1980) Proc. Natl. Acad. Sci. USA 77,4127-4131. Charnley-Campbell, Physiol. Reu. 59,
D., and
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J., Campbell, 1-61.
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Laemmli,
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Towbin, H., Staehlin, Sci. USA 76,4350-4354.
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Shirinsky, V. P., Birukov, N. B. (1984) FEBS Lett.
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Hoshino,
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U. K. (1970)
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T., and Gordon,
227, J. (1970)
K. G., Vorotnikov,
Singer,
Ross,
300, S. J.
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Glukhova, M. A., Frid, M. G., and Koteliansky, Biol. Chem. 265, 13,042-13,046.
V. E. (1990)
J.
CELL
RESEARCH
194,186189
(1991)
Density-Related Expression of Caldesmon and Vinculin in Cultured Rabbit Aortic Smooth Muscle Cells VLADIMIR P. SHIRINSKY, KONSTANTIN G. BIRUKOV, VICTOR E. KOTELIANSKY, MARINA A. GLUKHOVA, ELPIS SPANIDIS,* JANET D. ROGERS,* JULIE H. CAMPBELL,*” AND GORDON R. CAMPBELL? Cardiology Research Center of the USSR Academy of Medical Sciences, 121552 Moscow, USSR; *Baker Medical Research Institute, Melbourne; and TDepartment of Anatomy, University of Melbourne, Victoria, Australia
Quantitative immunoblotting techniques were used to study the effects of seeding density on the expression of caldesmon and vinculin variants, which are sensitive markers of vascular smooth muscle cell (SMC) phenotypic modulation in culture. Rabbit aortic SMC were seeded at different densities: 13 x 10’ cells/cma (high density), 3 X 10’ cells/cm’ (medium density), and 0.2 X 10’ cells/cma (low density) and cultured in the presence of 5% fetal calf serum. Irrespective of cell density and growth phase, caldesmon,,, was gradually and irreversibly substituted by caldesmon,, , but at high seeding density this substitution proceeded at a slower rate. The fraction of meta-vinculin (smooth muscle variant of vinculin) was reduced after seeding SMC in culture, but was reestablished when the cells reached confluency. Thus, high SMC seeding density is essential but not sufficient to keep vascular SMC cultured in the presence of serum in the contractile phenotype. o 1991 Academic
Press,
Inc.
INTRODUCTION Vascular smooth muscle cells (SMC) enzyme isolated and seeded in primary culture at a density below 5 X 10’ cells/ml (7 X lo4 cells/cm2) undergo a phenotypic modulation over the first few days then proliferate if serum mitogens are present [ 11. Ultrastructural examination shows that these SMC lose the bulk of their myofilaments and develop extensive endoplasmic reticulum [2, 31. This process is accompanied by a reduction in quantity of contractile proteins and substitution with nonmuscle counterparts [3-61. Following confluence the SMC may reexpress smooth muscle myosin and a-actin mRNA [3,4,7], but this is dependent on the initial SMC seeding density and thus the number of cell population doublings [3]. In the confluent state, visceral SMC from guinea pig vas deferens [8] and ureter [9] and the ’ To whom correspondence and reprint requests should dreesed at Baker Medical Research Institute, Commercial Prahran, Victoria 3181, Australia. Fax: 61-3-5211362. 0014~4327/91$3.00 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
chicken gizzard [lo] gain (or regain) the ability to contract; however, data on the contractile activity of confluent vascular SMC are limited [ 111. The aim of the present study was to investigate the effect of SMC seeding density on the expression of caldesmon and vinculin-cytoskeletal proteins reported to be sensitive markers of phenotypic modulation of vascular SMC in culture over 2 weeks in primary culture [5,6, 121. Caldesmon is the Ca2+-calmodulin-dependent regulator of actomyosin interaction in smooth muscle and nonmuscle cells [13-151. It is mainly expressed in mature rat and human aortic SMC as the 150-kDa isoform,2 and caldesmon,,, is substituted by caldesmon,, variant in the course of culture [5, 61. Vinculin and meta-vinculin are found in dense plaques where SMC actin filaments are anchored to the plasma membrane [20,21]. Meta-vinculin is a vinculin variant restricted to muscle cells [12,18-201. Meta-vinculin comprises about 40% of the total vinculin immunoreactivity in the mature human aortic SMC while in fetal SMC as well as in those proliferating in culture it is almost absent [13]. Our data demonstrate that the expression of caldesmon significantly differs from that of vinculin, and that high seeding density is important, but not sufficient, for the complete preservation of SMC contractile phenotype in culture. MATERIALS
AND METHODS
Elastase (type IV from bovine pancreas with 5 units/mg of tryptic activity), collagenase (type IV from Clostridium histolyticum) and soybean trypsin inhibitor were purchased from Sigma Chemical CO. (St. Louis, MO). Nitrocellulose (0.45 pm) was from Schleiaher and Schuell (FRG). Tissue culture plastic ware was purchased from Sterilin, Ltd. All other reagents were of tissue culture grade or analytical grade.
’ Molecular mass of heavy and light caldesmon variants was shown to be somewhat different from that determined by electrophoretic methods [16, 171. In this study, to distinguish between caldesmon variants we used the technique of immunoblotting, and therefore used the terms CaD,, for heavy caldesmon and CaDrr for light isoform.
be adRoad,
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Expression
12345 FIG. 1. Immunochemical detection of vinculin and caldesmon variants in cultured rabbit aortic SMC: (1) 7.5% SDS-PAGE of total SMC proteins; (2-5) total SMC protein transfer stained with antibodies to vinculin (2,3) and caldesmon (4,5) revealed by peroxidase-conjugated secondary antibodies (2,4) as well as 1261-labeled secondary antibodies and subsequent autoradiography (3,5). M, myosin, A, actin.
of Caldesmn
On the basis of densitometry of caldesmon bands (revealed on immunoblots by peroxidase reaction) and of radioactivity counting of the excised caldesmon bands detected with lz51-labeled secondary antibodies, the initial ratio of CaD,,/CaD,, was determined to be about 6 (Fig. 2). Hence, approximately 85% of caldesmon immunoreactivity is concentrated in caldesmon,, band in a freshly isolated suspension of rabbit aortic SMC. This ratio decreased progressively with time of culture indicating the shift to the expression of caldesmon,, isoform (Fig. 2, middle row). There was no increase in Cal&,/ CaD,, value either when SMC reached confluency and stopped to divide and incorporate thymidine (data not shown) or when FCS was withdrawn from the culture medium in low density culture (Fig. 2, upper row and middle row). However, the extent of the shift towards the expression of CaD,, depended on the cell seeding density. At higher densities more CaD,,, was expressed in the cells of the same culture age.
Expression of Vinculin SMC from media of the 9- to 12-week-old rabbit aorta were isolated enzymatically according to Charnley-Campbell et al. [22] and cultured in 90-mm plastic petri dishes (63 cm* growth area) in medium 199 (Commonwealth Serum Laboratories, Melbourne, Australia) supplemented with 5% fetal calf serum (FCS), 60 pg/ml penicillin, and 2 mM L-glutamine at 37’C in the atmosphere of 95% air, 5% CO,. SMC (10 ml) were seeded in 63-cm2 petri dishes at concentrations of 8 X 10s cell/ml (13 X 10’ cells/cm*, high density), 2 X l@ cells/ml (3 X 10” cells/cm’, medium density), and 10’ cells/ml (0.2 X 10’ cells/cm’, low density). SMC attached and spread within the first 2 days after seeding, and culture medium was replaced every third day. The seeding efficiency of the cells varied from 80-95% and was highest for the high seeding density. Samples of the total SMC proteins for SDS-PAGE were collected by rapid scraping of cells washed three times in ice-cold phosphatebuffered saline (PBS) in 100 ~1 of the sample buffer containing 0.2 M Tris-HCI, pH 6.8,32% (v/v) glycerol, 6.4% (w/v) SDS, 1.2 M 2-mercaptoethanol, 0.2% (w/v) bromophenol blue, and boiling for 3 min. Final volume of the sample did not exceed 350 pl. SDS-PAGE was conducted according to Laemmli [23]. Protein transfer to the nitrocellulose was done as described by Towbin et aL [24]. Nonspecific binding sites on the nitrocellulose were blocked by 1 h soaking of the paper in PBS with 0.5% dry milk powder and 0.1% Tween 20. Intermediate washes were accomplished by PBS-Tween 20 or PBS alone. Rabbit polyclonal affinity-purified antibodies to chicken gizzard caldesmon have been characterized previously [25]. To recognize vinculin variants mouse monoclonal antibodies raised against human uterus meta-vinculin (clone VIIFS) were used [26]. The antibodies recognized both protein variants equally. To reveal caldesmon and vinculin variants position on immunoblots, secondary sheep antirabbit and rabbit anti-mouse peroxidase-conjugated antibodies were used (Silenus Laboratories, Australia). 4Chloronapthol was utilized as the substrate for peroxidase reaction, and for quantitative immunoblotting, izWabeled antibodies instead of peroxidase-labeled antibodies were used (Fig. 1). Quantitation of radioactivity incorporated in caldesmon and vinculin bands was conducted as previously outlined [12]. The method was linear within the range of protein loadings used.
Variants
Variants
The measurement of meta-vinculin to vinculin ratio in freshly isolated rabbit aortic SMC provided the value of 0.5 (Fig. 2, bottom row). Thus, meta-vinculin incorporated approximately 33% of the total vinculin immunoreactivity in these cells. During the first 5-6 days metaVin/Vin ratio decreased more than twice in the medium density culture but returned to the original level a week later (Fig. 2B, bottom row). The drop in meta-Vin/Vin ratio in the low density culture during the first days of cultivation was more profound and irreversible (Fig. 2C, bottom row). Meta-vinculin was hardly detectable in these cells by our methods. The removal of FCS from the growth medium on Day 11 did not affect the expression of vinculin variants in low density culture within the following 7 days (Fig. 2C, bottom row). In SMC plated at high density the vinculin variant ratio remained unaltered throughout 2 weeks of culture and equalled the initial value of 0.5 (Fig. 2A, bottom row). DISCUSSION Several lines of evidence suggest that SMC which have undergone a change in phenotype in primary culture may regain their original phenotype after achieving a confluent monolayer. In the confluent state, rabbit aortic SMC regain the expression of smooth musclespecific myosin heavy chains and cu-actin mRNA depressed during modulation and proliferation [3,7]. Concomitantly the volume fraction of myofilaments increases in the confluent SMC returning to levels similar to those in cells prior to culturing [3]. It has thus been
188
SHIRINSKY HMH DENSITY CULTURE
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AL.
DENSITY CULNRE
(A)
LOW DENSITY
CULTURE
(B)
(C)
lot 0
5c
-
--
h
l
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/
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6
L L1
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0 6
aA
l
0:
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6
0
12
15
3
6
0
12
16
DAYS IN UILTURt?
FIG. 2. Changes of cell number, at high density (A), medium density ratio (bottom row) were determined growth medium. The data represent
CaD,,/CaD,, ratio and meta-VinNin (B), and low density (C). Cell number as a function of days in culture. Arrows the mean value of two experiments.
proposed that in these circumstances the reversibility of phenotypic modulation depends on the initial cell seeding density, which in turn determines the number of cell doublings the SMC undergo before achieving confluency in primary culture. In the present work we have extended our studies of rabbit aortic SMC modulation in culture and looked at the seeding density-related changes in expression of caldesmon and vinculin proteins associated with the smooth muscle cytoskeleton and contractile apparatus [ 13, 211. The dependence of vinculin variant expression on SMC seeding density was similar to that found for a-actin [3]. After an initial drop in meta-VinlVin ratio, which lasted from the start of the culture until the onset of intensive cell division, we observed a complete resto-
ratio in the course of rabbit aortic SMC culture. Cells were seeded (upper row), CaD,,/CaD,, ratio (middle row) and meta-VinlVin in panel C indicate the timepoint when FCS was removed from the
ration of the ratio back to 0.5 in the medium density culture following confluence and inhibition of SMC proliferation. SMC seeded at low density did not achieve confluent state during the time of experiment (18 days) and we observed irreversible loss of meta-vinculin from these cells. These results are in agreement with the previously reported loss of meta-vinculin from focal contacts of cultured human aortic SMC [12] and suggest that cell-to-cell contacts, rather than cell-to-substratum interactions, regulate meta-vinculin expression. This may explain why, in high density culture where contacts between cells are established just after spreading, there was no relative regression of meta-vinculin expression. Thus meta-vinculin, together with (Yactin and smooth muscle myosin heavy chains, maybe-
CALDESMON
AND
VINCULIN
IN
long to a class of SMC protein markers whose expression in cultured cells depends on intercellular interactions. Further experiments need to be performed to confirm this tenet. Caldesmon expression was, however, very different from that of vinculin. Irrespective of cell seeding density and growth phase we observed a gradual substitution of CaD,,, with CaD,, during cell cultivation which was reflected in the decrease of CaD,,/CaD,, ratio. Nevertheless, at higher cell density the rate of exchange of CaD,, for CaD,, was significantly decreased. As seen in Fig. 2 on Day 8 of culture, Cal&,, comprised 70% of caldesmon immunoreactivity in high density cultures whereas in medium density cultures and low density cultures this value was 38% and 0.05%, respectively. Our data are consistent with findings in cultured rat and human aortic SMC where the substitution of CaD,,, with CaD,, was also not found to be reversible [5,6]. At present, the B&H, cell line remains the only cell type known to reexpress CaD,,, after growth arrest [5]. Thus, the density of SMC is important for the expression of protein markers associated with SMC contractile phenotype, e.g., a-actin mRNA, smooth muscle myosin heavy chains, meta-vinculin, and caldesmon,,, . However, the lack of cell density-dependent reappearance of caldesmon150 indicates that the establishment of contact between SMCs is not sufficient for complete reversal of phenotypic modulation and the expression of certain proteins is controlled by different mechanisms.
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