Abstract
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, α-smooth muscle actin, β-actin, vimentin, and vinculin were seen by fluorescence microscopy. As the cytoskeleton rearranged, α-smooth muscle actin dissociated from the f-actin bundles and β-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.
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 (Ben-Ze'ev 1991; Watson 1991; Ingber and Folkman 1989). When ligands engage their respective receptors, components of the cytoskeleton rapidly rearrange. Grossly, this can be reflected by a change in cell shape. Ultrastructurally, it is associated with reorganization of cytoplasmic organelles. Moreover, as attachments between the cytoskeleton and the nuclear matrix reorganize, certain regions of the chromatin are peripheralized, which facilitates transcription of particular genes (Ben-Ze'ev 1991). Current studies are just beginning to define the specific effects of individual ligands on cytoskeletal reorganization and the functional changes that accompany this restructuring.
Insulin-like growth factor I (IGF-I) plays crucial roles in somatic growth, cell proliferation, metabolism, and cell differentiation (Humbel 1990; Sara and Hall 1990). These general activities are relevant to the kidney because IGF-I promotes nephrogenesis (Hammerman 1995), improves recovery from ischemic renal injury (Miller et al. 1994), induces mesangial cell (MC) proliferation (Abrass et al. 1988; Conti et al. 1988a,b) and alters glomerular matrix synthesis (Feld et al. 1995). In excess, IGF-I has been proposed to contribute to accumulation of mesangial matrix and progressive glomerulosclerosis (Bell and Madri 1990; Striker et al. 1989). MCs synthesize (Aron et al. 1989; Conti et al. 1988a) and express receptors for IGF-I (Abrass et al. 1988; Arnqvist et al. 1988; Conti et al. 1988b). Therefore, they can be stimulated by endocrine and autocrine pathways (Camussi et al. 1993; Catanese et al. 1993). Autocrine and paracrine IGF-I may be particularly important during tissue repair and localized growth (Bang and Hall, 1992; Sara and Hall 1990). Although ligand occupancy of the IGF-I receptor initiates activation of various signal transduction pathways (Humbel 1990), little is known about the specific signals that induce growth, differentiation, or cytoskeletal organization.
MCs in culture are routinely propagated in insulin (Kreisberg and Karnovsky 1983), and in that condition they express IGF-I receptors and few insulin receptors (Abrass et al. 1988). To specifically define the actions of insulin and IGF-I, we propagated rat MCs from birth in culture without supplemental insulin. These MCs express both insulin and IGF-I receptors, which are downregulated by the addition of insulin to the culture medium (Abrass et al. 1995). We have shown that both insulin and IGF-I stimulate MC proliferation (Abrass et al. 1988). However, the effects of these ligands on cell shape and ECM composition differ (Abrass et al. 1994, 1995). Recently, we showed that the effects of insulin on MC shape and cytoskeletal organization were direct and were not the consequence of secondary alterations in ECM composition (Berfield et al. 1996). Here we report that IGF-I initiates rapid cytoskeletal rearrangement in MCs that differs from that induced by insulin. IGF-I-induced cytoskeletal organization mimics that of cells that move or those with macrophagic properties (Warn et al. 1993; Camussi et al. 1990). We report discordant organization of f-actin and β-smooth muscle actin (αSMA) and polymerization of β-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.
Materials and Methods
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 (αSMA), monoclonal mouse anti-β-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 (Abrass et al. 1994, 1995) of routine methods (Kreisberg and Karnovsky, 1983). In brief, glomeruli were isolated by sieving minced rat kidney cortex and plated in medium containing a 1:1 mix of 20% FCS-RPMI 1640 and previously collected glomerular conditioned medium (Abrass et al. 1995). Unlike most MC cultures (Kreisberg and Karnovsky 1983) to which insulin (1 μM) is added to facilitate growth, no supplemental insulin was added. MC outgrowths were harvested and passaged in the same medium for an additional week, after which conditioned medium was omitted. MCs were cloned and studied at passages 8–12. These cells display characteristics of MCs in vivo, and express insulin and IGF-I receptors. Their growth characteristics, phenotype, and ECM composition have been compared to those of MCs routinely propagated in the presence of supplemental insulin as reported previously (Abrass et al. 1994, 1995). Before our reports of these cells, MCs were routinely propagated with supplemental insulin (1 μM) from birth in culture to the time of study (Kreisberg and Karnovsky 1983). Insulin was then transiently omitted from the culture medium at the time of plating for particular experiments when other additives are examined. We have found that after the fourth passage such cells retain the phenotype we described for insulin-treated MCs (Berfield et al. 1996; Abrass et al. 1994, 1995). The studies reported herein were all performed with MCs propagated in culture without supplemental insulin.
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, αSMA, or β-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 Med-cast, and polymerized (Sjostrand 1968). A Sorvall MT6000 ultramicrotome was used to prepare sections (70 nm) and specimens were examined with a JEOL S100 electron microscope.
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 × 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.
Results
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.
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.
Differential interference contrast microscopy shows the effects of IGF-I on the shape of rat MCs. Untreated MCs (a) show a smooth muscle-like morphology with tails (arrow). MCs exposed to 100 nM IGF-I for 1 hr (b) demonstrate a kite-like morphology. MCs chronically cultured (1 week) with 100 nM IGF-I (c) develop an elongated fusiform shape. Bar = 25 μm.
Phalloidin staining of f-actin and stress fibers. Untreated MCs (a) have thin stress fibers that traverse the cell in all directions. In MCs exposed to IGF-I for 1 hr (b), stress fibers detach from peripheral focal adhesions and rearrange into elongated actin fibers that stretch along the long axis of the cell (arrows). Actin arcs (arrow in inset) in the lamellipodia are seen in some cells. In MCs chronically incubated with 100 nM IGF-I (c), the long thin, actin fibers are drawn out into spindly forms, with small condensates at the end (arrows). Bar = 25 μm.
In addition to the changes in f-actin, IGF-I rapidly modifies the noncontractile intermediate filaments (Steinert and Roop 1988) of the cytoskeleton. In untreated MCs (Figure 3a), vimentin forms a perinuclear ring and radiates out to the cell periphery, consistent with extension from the nucleus to the actin network under the plasma membrane (Carmo-Fonseca and David-Ferreira 1990; Georgatos and Blobel 1987). This network is reorganized by IGF-I treatment (Figure 3b). In the kite-like cells, vimentin radiates outward from the perinuclear area to the lamellapodia. Long corded strings of vimentin are seen in the tail. In other cells the filamentous network is gone, leaving a diffuse cytoplasm with bright condensed spots of vimentin. Similar to f-actin (Figure 2c), the cells chronically exposed to IGF-I have thin vimentin strands stretched from the perinuclear area to the bipolar ends of the cells (not shown).
Immunofluorescent staining for vimentin. In untreated MCs (a), vimentin forms a beaded perinuclear ring (arrows) that evenly extends outward to the cell periphery. (b) A variety of vimentin forms are seen in MCs exposed to IGF-I. Some cells demonstrate condensed vimentin around the nucleus, with a definitive tail (arrow), and others have distinct fibers of vimentin that extend from the perinuclear ring outward to the periphery and terminate in areas of loose, untethered cytoplasmic bands (arrows). In a few cells, vimentin is diffuse and spotty (arrowhead). Bar = 25 μm.
Staining for vinculin, a microfilament-associated protein that is an integral part of focal adhesions (Miyamoto et al. 1995; Bendori et al. 1989), illustrates how the membrane of the MC is tacked down onto the extracellular matrix or directly to the membrane of adjacent cells. In Figure 4a, vinculin is regularly dispersed around the periphery of the cell in cell-cell and cell-matrix adhesions. In MCs treated with IGF-I (Figure 4b), vinculin is still abundantly distributed, albeit unevenly, around the cell membrane in lamellapodial areas. Vinculin is also found at sites of extracellular focal adhesions, but these are reduced in number and are clustered in polarized and trailing foot areas. Because elements of the actin network are linked to the plasma membrane through vinculin-rich focal adhesions and to the nucleus through intermediate filaments (Miyamoto et al. 1995; Dunlevy and Couchman 1993; McHugh and Crawford 1991; Bendori et al. 1989; Geiger 1987), the corresponding patterns of actin (Figure 2), vimentin (Figure 3) and vinculin (Figure 4) support the morphological changes seen in the DIC photographs (Figure 1). In aggregate, these changes imply that some focal adhesions have been released and new ones have formed.
Immunofluorescence micrographs of vinculin. In untreated MCs (a), vinculin is evenly dispersed around the cell periphery (arrows). After addition of IGF-I (b), vinculin congregates into variable patterns (arrows) around the main cell body and into coalesced adhesive edges along tails, or clustered claws at the ends of polarized extensions (arrowheads). Bar = 25 μm.
Double staining with phalloidin [stains f-actin, usually composed of α- and γ-actin (McHugh and Crawford 1991)] and antibody to αSMA, and subsequent staining of β-actin, enabled us to examine bundling into actin-myosin “stress fibers” and the distribution of two actin isoforms (McHugh and Crawford 1991).
Untreated MCs (Figure 5a) have thin stress fibers that are diffusely distributed across the cell. In these cells, αSMA (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 αSMA, it was possible to determine that αSMA was bundled into f-actin. In contrast, β-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 αSMA (Figure 5e) is visibly increased. In kite-shaped cells, αSMA 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, β-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.
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.
Actin staining of untreated MCs (a-c) and MC + IGF-I (d-f) for f-actin (a,d), αSMA (b,e) and β-actin (c,f). a and d and b and e are from the same double-stained field. Untreated MCs have f-actin bands and sparse stress fibers (a) and αSMA (b) is similar to f-actin distribution (arrows). IGF I-induced actin rearrangement (d) shows loosened f-actin bundles (arrowhead) and long stress fibers (arrows) in polarized cells. (e) αSMA is condensed and increased in intensity but does not extend out to lamellapodial areas (arrows). In untreated MCs, β-actin (c) is unevenly distributed. In some cells, the β-actin arrangement is similar to αSMA, as it is dispersed throughout the cell body. However, even in these cells β-actin does not extend all the way to the periphery of the cell. In other cells, β-actin is patchy around the nucleus and in cytoplasmic rings. After exposure to IGF-I (f), β-actin is increased in intensity and concentrated around the nucleus. In addition, many cells have large areas of concentrated β-actin and peripheral ribbons of β-actin under the plasma membrane of ruffled edges (arrows and higher magnification inset). Bar = 25 μm.
Electron micrographs. Untreated MCs (a) have few microfilopodia and a smooth, extended nucleus. Higher magnification (b) reveals organized perinuclear intermediate filaments (arrows), moderate endoplasmic reticulum, few lipid droplets, and lysosomes (small arrows). There are few coated pits. In cells treated with IGF-I (c), elongated MCs have anchoring bands of parallel arrays of microfilaments (arrows). The nuclei have invaginations, and DNA is condensed peripherally (arrowhead). There are abundant dark lysosomes, mitochondria, multivesicular bodies, and filopodia. At higher magnification (d) in the upper cell (a), on the trans side of the Golgi (arrowheads) are many lysosomes which are filled with “myelin” or recycled plasma membrane and endosomes. Dense parallel arrays of actin are seen along the peripheral edges of the cell (arrows). At the bottom of the picture (c), multiple coated pits and multivesicular body (arrows) are seen in the plasma membrane. MCs chronically exposed to IGF-I (e) have invaginated nuclei with condensed peripheral DNA. The cells have extended frothy cell bodies with moderate numbers of pseudopodia and abundant ECM. Bars = 0.4 μm.
Discussion
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 (Berfield et al. 1996). Both insulin and IGF-I induce rapid cytoskeletal reorganization. Insulin causes initial rounding of MCs, followed by cell extension. In the present studies we demonstrate that IGF-I alone induced rapid, specific effects on the cytoskeleton of the MC, resulting in alterations of cell shape that have not been described previously. IGF-I induces a tree-like or kite-shaped morphology, followed by formation of an extremely elongated bipolar cell.
Cell shape and the cytoskeleton influence gene regulation (Ben-Ze'ev 1991; Watson 1991). The cytoskeleton, via a interfilamentous network of vimentin, connects molecules at the cell surface to lamin-B on the outer surface of the nuclear envelope (Ingber 1993; Carmo-Fonseca and David-Ferreira, 1990; Steinert and Roop 1988; Georgatos and Blobel 1987). In turn, this interacts with the nuclear matrix, forming a continuous fibrous network that ultimately ends up at specific sites on chromosomes (Puck and Krystosek 1992; Geiger 1987). Condensation or deformation of the cytoskeleton exposes sets of genes at the nuclear periphery that are more susceptible to activation by transcription factors (Ingber 1993; Puck and Krystosek 1992; Geiger 1987). In the present study, ultra-structural changes indicated that IGF-I induced a release in tensegrity of the cytoskeletal network of MCs that was accompanied by nuclear invagination and peripheral DNA condensation (Ingber 1993; Puck and Krystosek 1992; Krystodek and Puck 1990; Geiger 1987). Therefore, IGF-I-associated changes in MC protein synthesis (Feld et al. 1995) may in part be mediated through its effects on the cytoskeleton. Although this association between IGF-I, cytoskeletal rearrangement, peripheral nuclear condensation of DNA, and activation of transcription of specific genes has not been confirmed in MCs, it has been shown that IGF-I-mediated gene regulation in fibroblasts, mesenchymal, and granulosa cells is affected by cytochalasins that disrupt the integrity of the intermediate filament network (Puck and Krystosek 1992; Ben-Ze'ev 1991; Unemori and Werb 1986; Werb et al. 1986).
The effects of IGF-I on the actin isoform αSMA are unique and have not been described previously. Increased or neoexpression of αSMA is considered a marker of cell proliferation and differentiation (Carey et al. 1992), and increased MC expression of αSMA occurs in various renal diseases (Berfield et al. 1996; Alpers et al. 1992; Johnson et al. 1991). It has been postulated that increased αSMA in MCs propagated in insulin (Sappino et al. 1990), treated with endotoxin (Bursten et al. 1991) or cytokines (Camussi et al. 1993), and in diseased mesangia in vivo (Johnson et al. 1991; Alpers et al. 1992) represents an “activated” phenotype. Normally, f-actin (filament actin can form from various actin isoforms), myosin, and αSMA colocalize 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 αSMA has been modified (Berfield et al. 1996). In some circumstances, the ligand that induces cytoskeletal rearrangement may also increase αSMA mRNA and protein synthesis (Foo et al. 1992). We previously demonstrated that the bright staining of αSMA commonly reported in cultured MCs is due in part to the presence of insulin in the culture medium (Berfield et al. 1996). We now show that IGF-I also increases the intensity of staining for αSMA in MCs. However, in IGF-I-treated MCs αSMA does not associate with stress fibers and αSMA does not visibly extend into lamellapodia along with the f-actin fibers. This dissociation of αSMA from other actin isoforms in f-actin fibers (McHugh and Crawford 1991) may be intimately involved with cell activation and movement.
In cells, actin isoforms are located in distinct functional domains, suggesting that specific contractile protein isoforms support unique cellular functions. β-Actin is preferentially distributed with the membrane-associated cytoskeleton, in a subset of stress fibers, in membrane spikes, and with motile cytoplasm (Janmey and Chaponnier 1995; Hill et al. 1994; Hill and Gunning 1993; Hoock et al. 1991). β-Actin mRNA has also been found to be recruited to the periphery of wounded endothelial cells, correlating with an increase of β-actin in the motile regions (Hoock et al. 1991). In untreated MCs, β-actin is distributed diffusely throughout the cytoplasm, but within an hour of adding IGF-I, β-actin becomes condensed in patchy areas and brightly concentrated in distinct peripheral areas of the lamella. These morphological changes in IGF-I-treated MC indicate induction of a motile phenotype (Warn et al. 1993; Camussi et al. 1990).
Rearrangement of specific cytoskeletal proteins, such as β-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 (Abrass et al. 1988). IGF-I receptors interact with ras GTPase-activating protein in the plasma membrane (Yamamoto et al. 1992), which in turn activates rac and rho and initiates filopodia formation (Ridley and Hall 1992). IGF-I may initiate MC motility in this manner. In addition, cytoskeletal rearrangement induces integrins to activate and release procollagenase, prostromelysin, and meprin (Walker et al. 1994; Werb et al. 1986). These enzymes degrade ECM and aid in cellular movement. MCs have been found to move in response to TNF (Camussi et al. 1993) and PDGF (Barnes and Hevey 1990). IGF-I has been reported to facilitate migration of endothelial cells, and fibroblasts during injury (Mueller et al. 1991), but induction of a motile configuration of cytoskeleton in MCs in response to IGF-I has not been previously reported. Because Jones et al. (1993) have shown that IGF-I stimulates migration of vascular smooth muscle cells, it is conceivable that IGF-I has a similar effect on MCs. We believe that these observations are important because motile responses to growth factors are necessary during glomerulogenesis (Hammerman 1995; Liu et al. 1993) and in glomerular diseases typified by mesangial interposition. IGF-I may be particularly important in renal growth (Miller et al. 1990), wound healing (Taylor and Alexander 1993), and ischemic renal injury (Miller et al. 1994). Circulating IGF-I, as well as locally synthesized IGF-I can influence the kidney (Conti et al. 1988a). Based on the presence of significant amounts of IGF-I in all human and animal wounds (Mueller et al. 1991), clinical use of IGF-I for healing chronic wounds, especially in burn patients and diabetics, has recently increased (Bondy et al. 1994; Kolaczynski and Caro 1994). Wounded endothelial cells (Taylor and Alexander 1993), and MCs from diabetic mice (Elliot et al. 1993) secrete increased amounts of IGF-I. Therefore, these particular observations may be relevant to IGF-I effects on MCs in a variety of clinical circumstances.
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 (Bretscher 1984). IGF-I appears to increase both pathways. Motile cells randomly endocytose membrane, which is then returned to the leading edge of the cell. When motility factors or IGF-I are added to MDCK cells, ruffling, which is associated with endocytosis, occurs (Kirkeeide et al. 1993; Dowrick and Warn 1991). Similar patterns have been seen in fibroblasts (Dunlevy and Couchman 1993) and in bovine arterial smooth muscle cells (Davies and Ross 1980; Haigler et al. 1979) after cytokine exposure. This is the first report of similar patterns in MCs. The increased endocytosis, lysosomes, and multivesicular bodies seen by electron microscopy in cells exposed to IGF-I, and the “foam cell” appearance of MCs chronically exposed to IGF-I, indicate that both fluid-phase and receptor-mediated endocytosis have been stimulated. This change appears specific to IGF-I, because insulin treatment of MCs does not induce foam cell formation (Berfield et al. 1996). Therefore, we hypothesize that IGF-I induces MCs to express an activated, motile, macrophagic, wound-healing phenotype (Alpers et al. 1992; Carey et al. 1992; Johnson et al. 1991; Sappino et al. 1990).
Footnotes
Acknowledgements
Supported by the Medical Research Service of the Department of Veterans Affairs.