ARTICLE |
Correspondence to: Marvin H. Stromer, Dept. of Animal Science, 3116 Molecular Biology Bldg., Iowa State U., Ames, IA 50011-3260.
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Summary |
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We studied the specific expression patterns and distributions of 1 and ß1 integrin subunits, the major cell adhesion receptors in smooth muscle, in developing smooth muscle cells from 16-, 18-, and 20-day embryonic gizzards and from 1- and 7-day post hatch chick gizzards by SDS-PAGE, immunoblotting, and immunoelectron microscopy. Antibodies raised against
1 and ß1 integrins isolated from avian gizzards were used as probes. Gels and blots showed that the amount of
1 and ß1 integrins increased as age increased, with major increases at 1 and 7 days post hatch. Image analysis of immunoelectron micrographs demonstrated that statistically significant labeling increases occurred between embryonic Days 16 and 18, between embryonic Day 20 and 1 day post hatch, and between 1 day and 7 days post hatch. Immunolabeling with both anti-
1 and anti-ß1 integrin was prominent at membrane-associated dense plaques (MADPs) and at filament anchoring regions at cell ends. This indicates that
1 and ß1 integrin expression coincides temporally with the intracellular proliferation and reorientation of myofilaments. The similarity in distribution patterns of
1 and ß1 integrins during development suggests that the two integrin subunits are synchronously expressed during development and do not appear sequentially. (J Histochem Cytochem 46:119-125, 1998)
Key Words: integrin, developing smooth muscle, immunoelectron microscopy, immunoblots
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Introduction |
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Integrins, a family of heterodimeric transmembrane receptors that mediate adhesion to the extracellular matrix (ECM), are also very important for cell migration, cell adhesion, embryogenesis and differentiation, and signal transduction (for reviews see - and 8 ß-integrin subunits have been identified (
- and ß-subunits give rise to the great diversity of integrin receptors and their versatility in ligand binding sites (
1ß1 dimer is 10 x 12 nm, and the arm domains, which consist of part of the extracellular domain, the transmembrane domain, and the intracellular domain, are 17-20 nm long (
-subunit (
-actinin, vinculin, and talin (
The predominant integrins in chick gizzard smooth muscle cells are the 165-kD 1 (
1-integrin subunit is both a primary receptor for collagen IV (
1-integrin in several types of developing avian embryonic tissues, including skeletal muscles, visceral, and vascular smooth muscles, have been investigated by using immunoblots and fluorescence microscopy (
1-integrin disappeared when the cells became contractile (
7-integrin appeared after the beginning of terminal differentiation (
1 was present early in differentiation and continued to be expressed in adult smooth muscle cells (
1-integrin in muscle was frequently correlated with expression of laminin and collagen IV. The ECM is evidently involved in the control of integrin expression. Vascular smooth muscle cells in the medial layer of normal arteries interact with collagen IV and express
1ß1 integrins, but cultured cells from medial explants express only
2ß1-integrins, which are needed for cell migration on collagen I substrates (
1-integrin gene could not spread or migrate on collagen IV substrates but could on collagen I substrates (
1-integrin.
During embryogenesis and neonatal development, smooth muscle cells undergo remarkable phenotypic changes, including reorganization of the ECM (-actinin, vinculin, talin, paxillin, tensin, zyxin, filamin, and tyrosine kinases, have been localized or have been proposed to be located at or associated with MADPs (for review see
The objectives of this study were to investigate the expression patterns of both 1- and ß1-integrins in embryonic and neonatal smooth muscle cells to determine if the
1- and ß1-subunits were sequentially or synchronously expressed, to determine when the amounts of
1- and ß1-integrins increased during development, and to localize both
1- and ß1-integrins in these cells by using high-resolution immunogold labeling to determine when the subunits could be localized in relation to other assembling components in these cells.
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Materials and Methods |
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Purification of Integrin 1- and ß1-subunits
Turkey gizzard 1- and ß1-integrins were prepared from stripped membrane fractions by the method of
Antibodies
Electroeluted 1-integrin (0.2-0.3 mg) was dialyzed against PBS (2.6 mM KCl, 137 mM NaCl, 1.5 mM KH2PO4, 15.2 mM Na2HPO4, pH 7.2) before emulsification 1:1 with Freund's complete adjuvant (initial injection) and with Freund's incomplete adjuvant (booster injections) for SC injections into a New Zealand White rabbit. Animal care and management in these studies were done in compliance with the guidelines of the Iowa State University Committee on Animal Care. The ß1-integrin (0.4-0.5 mg) without any adjuvant was injected directly into a sterile wiffle ball that had previously been implanted in the thoracic region of a New Zealand White rabbit (
1- and ß1-integrins were collected. The ß1-integrin polyclonal antibody was affinity-purified by using immunoblots as described in
1-integrin generously donated by Dr. M. Paulsson (University of Cologne, Germany) and a monoclonal anti-ß1-integrin from the Hybridoma Bank were only used to identify
1- and ß1-integrins in Western blots of column fractions. Neither of these antibodies was satisfactory for immunoelectron microscope labeling, thus requiring the production of
1- and ß1-integrin antibodies for localization.
SDS-PAGE and Immunoblotting
Smooth muscle samples were taken from gizzards of 16-, 18-, and 20-day embryos and 1- and 7-day post hatch chicks. The integrin-containing Triton X-100 extract was prepared by the same method used with adult gizzards, except that homogenization steps were three times for 10 sec each. Protein concentrations were determined by the Bio-Rad Protein Assay (Bio-Rad; Hercules, CA). To minimize protein degradation, gels were run the day after preparation of the extracts. Electrophoresis was done on 10% polyacrylamide gels (acrylamide: N,N'-methylenebisacrylamide = 77:1 w/w) by a combination of the methods of 1-integrin (1:10,000) and affinity-purified anti-ß1-integrin (1:200) was done by the method of
Tissue Preparation for Immunoelectron Microscopy
Smooth muscle samples for immunoelectron microscopy were taken from gizzards of 16-, 18-, and 20-day embryos and from 1- and 7-day post hatch chicks. Tissues were prepared by a modification of the method of
Immunoelectron Microscopy
The polyclonal antibody to avian gizzard 1-integrin was used at 1:50 dilution, and the affinity-purified polyclonal antibody to avian gizzard ß1-integrin was used undiluted. The protein A-12-nm gold complex was produced by the method of
Sample Evaluation
A minimum of 25 attachment plaques were measured and colloidal gold particles associated with each plaque were counted for each animal age. Plaques were identified in randomly selected micrographs, and the number of gold particles per µm2 of plaque area was determined for both embryonic and neonatal samples. To compare labeling intensities of plaques, where plaque area could readily be determined, with labeling associated with filament anchoring sites at the ends of cells, the area of which could not be determined, we counted the number of gold particles per µm of cell surface in each of these sites in neonatal samples. The t-test was used to determine statistical significance.
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Results |
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Purification of 1- and ß1-integrins
The 1-integrin-rich fractions (no. 60 to no. 110) from the DEAE-cellulose column were chromatographed on a hydroxylapatite column to remove some high and low molecular weight contaminating proteins. SDS-PAGE analysis showed that
1-integrin was prominent in hydroxylapatite column fractions no. 45 to no. 70. The
1-integrin band was cut out of the gel and the protein was recovered by electroelution. The ß1-integrin-rich fractions (no. 140 to no. 160) from the DEAE-cellulose column were chromatographed on a HiTrap SP column. Contaminating proteins were retained by the HiTrap SP column, and ß1-integrin was in the flow-through.
Characterization of 1- and ß1-integrin Antibodies
Immunoblotting with anti-1-integrin showed that only the
1-integrin subunit was recognized (Figure 1, Lane 2). Immunoblotting with anti-ß1-integrin heavily labeled the ß1-integrin subunit but also weakly labeled some minor components (Figure 1, Lane 3). To ensure the specificity of the ß1-integrin antibody, affinity-purification was done. The affinity-purified antibody recognized only the ß1-integrin band in blots (Figure 1, Lane 4). The
1- and ß1-integrin antibodies from outside sources identified
1- and ß1-integrins, respectively, in the column fractions (results not shown).
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Changes in 1- and ß1-integrin Expression
Results from SDS-PAGE (Figure 2A) and from immunoblots (Figure 2B) of stripped membrane fractions demonstrated that the 165-kD 1-integrin band is already present in relatively small amounts as a percentage of total membrane proteins at embryonic Day 16 and that amounts of
1-integrin increase rapidly as age increases. The increase in the 130-kD ß1-integrin band (Figure 2C) with increasing age shows a similar pattern. Both
1- and ß1-integrins increase markedly after hatching (cf. Lanes 4 and 5 vs Lanes 1, 2, and 3 in Figure 2B and Figure 2C). It is evident that, compared with the embryonic and neonatal samples in Lanes 1-5 in Figure 2, adult gizzard smooth muscle cells (Lane 6) contain more of both
1- and ß1-integrin.
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Immunoelectron Microscopic Labeling
Because both the density and the pattern of labeling with antibodies to 1- and ß1-integrins were very similar (see Table 1 and Table 2), we have chosen to include micrographs from each animal age that show typical labeling with anti-
1-integrin only. Representative micrographs from each sample are shown in Figure 3, and quantitation of the labeling obtained is shown in Table 1 and Table 2.
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Attachment plaques were consistently labeled at all developmental stages included in this study. Sixteen-day embryonic smooth muscle cells contain both large areas of cytoplasm that have no recognizable filaments and relatively small, somewhat diffuse attachment plaques (Figure 3A). The lowest density of labeling was associated with these immature plaques. At 18 days, embryonic cells contain more filaments that are associated with plaques (Figure 3B) and labeling intensities have increased significantly over that seen at 16 days (Table 1). Plaques increase further in both size and density by 20 days of embryonic development (Figure 3C) and, although labeling density has increased (Table 1), the increase is not statistically significant. By 22 days of development (1 day post hatch), labeling associated with plaques (Figure 3D) increased significantly by 1.8 to 2 times that observed 2 days earlier. Additional significant increases in label density occurred by 28 days of development (7 days post hatch) (Table 1; Figure 3G). The overall increase in plaque labeling from Day 16 to Day 28 was 4.6 times with anti-1-integrin and 4 times with anti-ß1-integrin.
As the size of the filament compartment increased during development, filaments became oriented more nearly parallel to the longitudinal cell axis and the intracellular patches of filaments expanded towards the ends of the developing smooth muscle cell. Cell ends were a second site that was consistently labeled by anti-1- and anti-ß1-integrins. By 22 days of development (1 day post hatch), the linear boundary of the cell end was adequately developed to permit reproducible measurements of the end boundary and to compare labeling at the cell ends with labeling at the attachment plaques. In 22-day samples (Figure 3E), the number of gold particles per µm of plaque and of attachment area at the cell end was virtually identical with both antibodies (Table 2). Labeling densities per µm of plaques and cell ends in 28-day (7 days post hatch) samples were greater than in 22-day samples (Table 2; Figure 3F). Plaque labeling increased approximately 30% with the
1-integrin antibody and 20% with the ß1-integrin antibody. In contrast, the cell end-labeling increased 50% with the
1-integrin antibody and 40% with the ß1-integrin antibody. The increases were statistically significant at cell ends with both antibodies and at plaques with anti-
1-integrin.
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Discussion |
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We have used SDS-PAGE, immunoblotting, immunoelectron microscopy, and image analysis to demonstrate that expression of both 1- and ß1-integrin increases synchronously as age of developing smooth muscle cells increases. Our SDS-PAGE and immunoblots both show a consistent increase in both
1- and ß1-integrin in gizzard smooth muscle cells from embryonic Day 16 through 7 days post hatch. This steady increase differs from the patterns observed by
1- and ß1-integrins were lower at 24 weeks of gestation than at 10 weeks, increased again after birth, and reached adult levels by 1.5 years after birth. Although the relative developmental stages in the human smooth muscle studied by
1- and ß1-integrin expression. We also show that the timing of increased synthesis of
1- and ß1-integrins coincides with the increase in intracellular filaments and the reorientation of these filaments in a direction more nearly parallel with the long axis of the cell. The low level of labeling we observed in 16-day embryonic cells coincides with the appearance of intracellular patches of myofilaments that occupy only a small central portion of the cell. Between 20 and 22 days (1 day post hatch) of development, a major increase in cell volume occupied by myofilaments is accompanied by the greatest increase in integrin content. The timing of the increase in myofilaments in these smooth muscle cells from the digestive tract is probably dictated by the need to crush and grind feed taken in by mouth after hatching. The myofilaments in smooth muscle cells consist of both contractile and cytoskeletal filaments (
The patterns of increase in labeling from 10 to 28 days of development were very similar for 1 and for ß1 antibodies (Table 1 and Table 2). Although it has previously been predicted that
1- and ß1-integrins should be synchronously expressed during development, we know of no published reports in which this has been demonstrated. The slightly lower labeling of attachment plaques (Table 1) and cell ends (Table 2) with anti-ß1-integrin is probably due to a slight loss in binding activity after affinity purification rather than a real difference in amount of
1- and ß1-integrins. This interpretation is supported by
1- and ß1-integrin are required. This suggests that
1- and ß1-integrin should be present during development in similar if not identical amounts, and is consistent with our immunoblots (Figure 2B and Figure 2C).
We also observed that, as increasing numbers of collagen fibrils became visible in the extracellular space, intensity of integrin labeling at attachment plaques and cell ends increased. Although collagen fibrils were not seen in association with all attachment plaques, we observed an increase in collagen fibrils near plaques from one to three at embryonic Days 16, 18, or 20 to six or seven collagen fibrils near plaques at 1 and 7 days post hatch. This increase coincided with the major increase in integrin labeling (Table 1). It has been demonstrated that 1ß1-integrin dimers are collagen IV receptors (
1ß1-integrin binding site is approximately 100 nm from the N-terminus of collagen IV (
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Acknowledgments |
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Supported in part by a grant from the American Heart Association, Iowa Affiliate.
We thank Dr M. Paulsson (University of Cologne, Germany) for providing the 1-integrin antibody. We also thank Mary Sue Mayes, Suzy Sernett, Jo Philips, and Lynn Newbold for excellent technical assistance.
This is Journal Paper J-17204 of the Iowa Agriculture and Home Economics Experiment Station, Ames, IA 50011, Projects 3349 and 2127.
Received for publication February 27, 1997; accepted July 10, 1997.
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