ARTICLE |
Correspondence to: Christine K. Abrass, (111A), Veterans Affairs Medical Center, 1660 S. Columbian Way, Seattle, WA 98108.
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Summary |
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Resident glomerular mesangial cells (MCs) have complex cytoskeletal organizations that maintain functional and structural integrity. The ability of cells to replicate, coordinate movement, change shape, and interact with contiguous cells or extracellular matrix depends on cytoskeletal organization. MCs synthesize insulin-like growth factor (IGF-I), express IGF-I receptors, and respond to IGF-I with increased proliferation. We noted that IGF-I treatment of mesangial cells was associated with a change in morphology. Therefore, these studies were undertaken to define specific IGF-I-mediated changes in cytoskeletal protein organization. Rat MCs were propagated from birth in culture without supplemental insulin. Quiescent, subconfluent cultures were treated with IGF-I (100 nM) for 1 hr. Rearrangements in f-actin, -smooth muscle actin, ß-actin, vimentin, and vinculin were seen by fluorescence microscopy. As the cytoskeleton rearranged,
-smooth muscle actin dissociated from the f-actin bundles and ß-actin became polymerized under the leading lamellar edge. Ultrastructural changes were consistent with increased membrane turnover and metabolic activity. The normally sessile mesangial cell was induced by IGF-I to express a wound-healing phenotype characterized by movement and increased pinocytosis. These changes are different from those induced by insulin and have important implications for mesangial cell function. (J Histochem Cytochem 45:583-593, 1997)
Key Words: IGF-I, cytoskeleton, glomerulus, mesangial cell, cell shape, migration
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Introduction |
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The cytoskeleton plays a critical role in determining cell shape and in trafficking stimuli that alter both gene expression and cell behavior. By virtue of its critical position between the extracellular matrix (ECM) and the nuclear matrix, the cytoskeleton transduces signals from outside the cell by integrins that attach to the ECM or with receptors for soluble ligands including hormones, cytokines, and growth factors (
Insulin-like growth factor I (IGF-I) plays crucial roles in somatic growth, cell proliferation, metabolism, and cell differentiation (
MCs in culture are routinely propagated in insulin (-smooth muscle actin (
SMA) and polymerization of ß-actin into the leading lamellar edge. The unique effects of insulin and IGF-I on cytoskeletal organization imply distinct signal transduction pathways and may be partially responsible for the specific effects of these agents on ECM synthesis.
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Materials and Methods |
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Materials
The following reagents were purchased from the designated suppliers: recombinant human IGF I (Collaborative Research; Waltham, MA), bodipy phalloidin 503/512 (Molecular Probes; Eugene, OR), monoclonal V9 mouse anti-swine vimentin (DAKO; Glostrup, Denmark), monoclonal mouse anti-human smooth muscle actin (SMA), monoclonal mouse anti-ß-actin, monoclonal mouse anti-vinculin (Sigma Biochemical; St Louis, MO), fluorescein (FITC)- and rhodamine (TRITC)-conjugated secondary antibodies (Jackson Labs; West Grove, PA), four-chamber tissue culture glass slides (Nunc; Naperville, IL), and Medcast (epon) (Ted Pella; Redding, CA).
Cell Culture
Rat glomerular MCs were prepared by modification (
Differential Interference Contrast Microscopy
Chamber slides were fixed for 15 min. at room temperature (RT) in 2% paraformaldehye in PBS, pH 7.6, and examined using a Leitz microscope equipped with differential interference contrast microscopy (DICM). Nomarski images were recorded on Kodak 100 black-and-white film.
Fluorescence Microscopy
Chamber slides were fixed as described above. After rinsing in PBS, cells were permeabilized with PBS containing 0.05% Triton X-100 for 3 min, then rinsed three times in PBS. Immunofluorescence was used to visualize vimentin, vinculin, SMA, or ß-actin. Slides were incubated with antibody for 20 min at RT, washed three times, and incubated with the appropriate fluorescein-conjugated secondary antibody for an additional 20 min. Controls included nonimmune IgG. Two different antigens in a cell were simultaneously visualized by double staining, using separate rhodamine- and fluorescein-conjugated secondary antibodies. To visualize actin, slides were stained with 12.5 µg/ml of bodipy phalloidin for 20 min at RT. All slides were mounted in Fluoromount and observed with a Leitz microscope equipped with epi-illumination. The fluorescent images were recorded on Kodak DX 400 black-and-white film.
Electron Microscopy
MCs from each experimental condition were rinsed with PBS and fixed in 2% glutaraldehyde in cacodylate buffer for 2 hr at 4C. Cells were washed with cacodylate buffer three times for 10 min each at RT, osmicated with 1% osmium tetroxide for 30 min at RT, and washed three times with cacodylate buffer. Cells were scraped with a rubber policeman, placed in microfuge tubes, briefly centrifuged at 1000 rpm, and sequentially dehydrated through ascending concentrations of alcohol (35-100%), through propylene oxide to Medcast, and polymerized (
Experimental Design
Short-term and chronic effects of IGF-I on the MC cytoskeleton were examined in triplicate on four separate occasions. For short-term studies, MCs (2 x 104) were plated in chamber slides and grown for 24 hr in 20% FCS-RPMI. MCs were rendered quiescent in 2% FCS-RPMI medium for 48 hr. Then fresh medium, with or without IGF-I, at the highest concentration (100 nM) that does not activate the insulin receptor was added. One hour later, cells were fixed and examined by differential interference contrast microscopy (DICM), immunofluorescence, and electron microscopy as described above. For long-term studies, MCs were propagated for 7 days in 20% FCS-RPMI or in medium containing 100 nM IGF-I, and were processed in the same way as in the short-term studies.
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Results |
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IGF-I induces rapid and specific effects on the MC cytoskeleton that correspond to a change in phenotypic morphology. Differential interference contrast (DIC) micrographs show that untreated MCs (Figure 1a) are phenotypically similar to smooth muscle cells in that they are oval, smooth, and polygonal to spindle-shaped. MCs treated with IGF-I for 1 hr display a different morphology (Figure 1b), with a triangular, spreading central body and a long filopodial extension. With prolonged exposure to IGF-I this change in morphology becomes more pronounced and is displayed by all of the cells in culture. After 7 days of exposure to IGF-I, MCs (Figure 1c) are elongated bipolar cells. The phenotype of untreated MCs is stable in prolonged culture and is identical to that shown in Figure 1a. Using DIC, membrane changes can also be seen. The MC membrane (Figure 1a) extends smoothly over the nucleus to the cell periphery. One hour after adding IGF-I (Figure 1b), blebs, rings, waves, and ruffles are seen in the large lamellar cell body, and knots or blebs are apparent in the tail. After exposure to IGF-I for a week, (Figure 1c), it is evident that filapodial terminals are flattened and splayed, with some distinct finger-like projections. These alterations in the membrane, as well as the gross morphological changes, are associated with reorganization of cytoskeletal elements.
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Staining of actin filaments (f-actin) by phalloidin (Figure 2) reveals a cytoskeletal arrangement that corresponds to the cell morphology exhibited in Figure 1. In untreated MCs (Figure 2a), stress fibers (bundled f-actin) smoothly traverse the cell in all directions and terminate in multiple focal adhesions that are uniformly distributed about the periphery of the cell. One hour after adding IGF-I (Figure 2b), f-actin fibers extend across the large lamella of the kite-like shapes. Sparse stress fibers are strung between focal adhesions and extend into the tails. In some cells (inset, Figure 2b), f-actin arcs are seen in the lamellapodia. Progressive elongation of the cells occurs after chronic exposure to IGF-I (Figure 2c). Roots of the bipolar cell terminate either in brightly condensed aggregates of f-actin or in a diffuse spray of thin fibers.
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In addition to the changes in f-actin, IGF-I rapidly modifies the noncontractile intermediate filaments (
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Staining for vinculin, a microfilament-associated protein that is an integral part of focal adhesions (
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Double staining with phalloidin [stains f-actin, usually composed of - and
-actin (
SMA, and subsequent staining of ß-actin, enabled us to examine bundling into actin-myosin "stress fibers" and the distribution of two actin isoforms (
SMA (Figure 5b) stains with low intensity, is monochromatically distributed throughout the cytoplasm, and extends to the cell periphery. Because the same slides were stained for f-actin and
SMA, it was possible to determine that
SMA was bundled into f-actin. In contrast, ß-actin (Figure 5c) is mottled and unevenly dispersed throughout the cell. After a 1-hr exposure to IGF-I (Figure 5d), the intensity of staining for f-actin appears diminished, whereas the staining intensity of
SMA (Figure 5e) is visibly increased. In kite-shaped cells,
SMA is perinuclear and extends into the tail, but it is absent from the cytoplasm of large lamellapodia and it is visibly dissociated from f-actin strands. Similarly, ß-actin (Figure 5f) appears increased, but it changes from a diffuse pattern into an organized structure identified by bright staining of condensates and long ruffles along specific borders of the cell periphery.
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Electron microscopy of cell cultures with and without IGF-I reveals ultrastructural changes that include the cytoskeleton, plasma membrane, and organelles. In untreated MCs (Figure 6a), the cells are evenly spread, with few filopodia, smooth oval nuclei, and moderate numbers of mitochondria, vacuoles, and rough endoplasmic reticulum. Under higher magnification (Figure 6b), organized cords of intermediate filaments are seen around the smooth extended nucleus, with little or no condensed perinuclear chromatin. There are few liposomes, lysosomes, or myelin bodies. After IGF-I is added (Figure 6c), MCs have many lamellapodia and microfilopodial extensions. The nuclei are invaginated, with perinuclear condensation of chromatin. Many dark bodies are seen in the cytoplasm. A section of a polarized, elongated cell reveals a large patch of stretched, parallel microfilaments, typical of anchoring bundles of actin fibers. To demonstrate the the differences in organelle structure in opposite ends of single cells, we show a higher magnification of adjacent cells (Figure 6d). One appears actively endocytic, with an extensive intermediate filament network supporting lysosomes, many mitochondria, and rough endoplasmic reticulum. The activity of the membrane is revealed by the presence of many coated pits, extensive multivesicular bodies, and fine networks of microfilaments in the plasma membrane surrounding the endosomes and coated pits. In the other MC, which appears to be more exocytotic, intermediate filaments are sparse, with resulting nuclear invaginations and regional reorganization of ultrastructural elements. The Golgi appears close to the nucleus. On the trans-Golgi side, there are many lysosomes full of lamellar bodies, endosomes, and debris. Parallel arrays of microfilaments under the plasma membrane and microfilapodia indicate adhesive activity. On further examination of MCs chronically exposed to IGF-I (Figure 6e), the majority of the cells appear reminiscent of macrophagic foam cells. They are characterized by elongated and invaginated nuclei, large foamy cytoplasm, and abundant extracellular matrix. With higher magnification (not shown), extensive Golgi, areas of extended rough endoplasmic reticulum, microfilapodia, and coated pits, vacuoles, and dark mitochondria with lipid-like droplets were seen.
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Discussion |
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The MC normally is a sessile, contractile cell in the hub of the glomerulus, where it maintains the dimensions and structural integrity of the capillary wall, while synthesizing and remodeling mesangial matrix. During glomerulogenesis and in a variety of glomerular diseases, MCs proliferate, express markers of an activated cell, and synthesize increased amounts of ECM. IGF-I appears to be one of the factors that can initiate these changes in MC function. These effects may in part be mediated by direct reorganization of the cytoskeleton. We recently described the effects of physiological concentrations of insulin on the MC cytoskeleton (
Cell shape and the cytoskeleton influence gene regulation (
The effects of IGF-I on the actin isoform SMA are unique and have not been described previously. Increased or neoexpression of
SMA is considered a marker of cell proliferation and differentiation (
SMA occurs in various renal diseases (
SMA in MCs propagated in insulin (
SMA co-localize as they are bundled into stress fibers. When these are induced to rapidly rearrange, the intensity of staining of stress fibers may increase whether or not the actual amount of
SMA has been modified (
SMA mRNA and protein synthesis (
SMA commonly reported in cultured MCs is due in part to the presence of insulin in the culture medium (
SMA in MCs. However, in IGF-I-treated MCs
SMA does not associate with stress fibers and
SMA does not visibly extend into lamellapodia along with the f-actin fibers. This dissociation of
SMA from other actin isoforms in f-actin fibers (
In cells, actin isoforms are located in distinct functional domains, suggesting that specific contractile protein isoforms support unique cellular functions. ß-Actin is preferentially distributed with the membrane-associated cytoskeleton, in a subset of stress fibers, in membrane spikes, and with motile cytoplasm (
Rearrangement of specific cytoskeletal proteins, such as ß-actin concentration, f-actin arcs, filamentous tails, and discrete coalescent focal adhesions, suggests that MCs can be activated to migrate, and to proliferate in response to IGF-I (
With the addition of IGF-I, MC endocytosis greatly increased. Early pathways of fluid-phase and receptor-mediated endocytosis are identical, but at a point they diverge into multivesicular bodies for recycling or to lysosomes for degradation (
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Acknowledgments |
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Supported by the Medical Research Service of the Department of Veterans Affairs.
Received for publication June 18, 1996; accepted November 13, 1996.
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