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EXTRACELLULAR MATRIX, CELL INTERACTIONS
1Department of Medicine, School of Medicine, University of Washington; and 2Veterans Affairs Puget Sound Health Care System, Seattle, Washington
Submitted 13 December 2005 ; accepted in final form 25 April 2006
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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4 and LN
2 chains, but not LN1, blocked IGFBP-5-induced migration. Anti-sense morpholino oligonucleotide inhibition of expression of LN4 substantially reduced expression of LN-8/9 (41
1/421, 411/421) and prevented IGFBP-5-induced migration. Anti-sense inhibition of lamb2 reduced expression of LN-9. Absence of LN-9 prevented IGFBP-5-induced migration, which was not preserved by continued expression of LN-8. The requirement for LN-9 was further supported by studies of T98G cells, which express predominantly LN-8. IGFBP-5 had little effect on migration in these cells, but increased migration when T98G cells were plated on LN-8/9. IGFBP-5-mediated mesangial cell migration was inhibited by antibodies that block attachment to 61-integrins but was unaffected by antibodies and disintegrins that block binding to other integrins. Furthermore, in cells with anti-sense inhibited expression of LN-9, integrin 61 was no longer detected on the cell surface. These studies suggest the specificity of mechanisms of migration induced by specific stimuli and for the first time demonstrate a unique function for LN-9 in mediating IGFBP-5-induced migration. migration; integrins; extracellular matrix
332, LN-332) (6) is one of the best-studied examples (41). Prevention of matrix binding reduces the response to PDGF. Insulin-like growth factor (IGF)-1 modulates surface expression of integrins and binding to different 1-integrin isoforms, which in turn influences attachment to LN. In Chinese hamster ovary and PC3 cells, IGF-1 increases adhesion to LN via integrin 1C, which can inhibit cell proliferation. In contrast, binding of integrin 1A to other matrix proteins enhances migration and proliferation (24). The mechanisms whereby IGF-1 induces migration differ in different cell types. In vascular smooth muscle cells (34), and the related glomerular mesangial cells (7), IGF-1 induces migration via v3 binding to fibronectin. Insulin-like growth factor binding protein (IGFBP)-5 is one of the IGFBPs with the capacity to modify cellular responses to IGF-1, but it also has direct effects on cells that are independent of IGF-1 and are influenced by binding to LN (54, 59). Changes in expression of IGFBP-5 occur with cancer and have been postulated to influence the risk for progression; however, the mechanism by which IGFBP-5 influences tumor invasion remains unknown (11, 23). Expression of IGFBP-5 temporally correlates with mesangial cell (MC) development during nephrogenesis and alterations in IGFBP-5 expression have been postulated to contribute to progression of diabetic nephropathy through increased matrix accumulation (9, 53). We have previously shown that IGFBP-5 stimulates MC migration and that this response differs from that induced by IGF-1, as the v3-integrin is not required. The present studies were undertaken to better define the requirements for IGFBP-5-induced migration.
The glomerular MC is a smooth muscle-like pericyte that provides structural support for endothelial cells and podocytes that make up the glomerular tuft. MC migration is critical during nephrogenesis when mesangial progenitors migrate into the vascular cleft of the developing glomerulus (5). In models of mesangiolysis, the mesangium is repopulated and repaired by MCs that migrate in from the extraglomerular mesangium (32). In other glomerular diseases, MCs migrate into the subendothelial space causing mesangial interposition (44). Little is known about the mechanisms that regulate MC migration in each of these circumstances. We have previously shown that MCs express predominantly LN-8/9 (411/421, 411/421) in vitro and in vivo (28, 29), and that MC migration on specific matrix substrates is influenced by the migratory stimulus (1, 7, 29). IGFBP-5 stimulates MC migration using a unique phenotype that is mimicked by plating MC on LN-8/9 (7). The present studies were conducted to further explore the role of LN isoforms in IGFBP-5-mediated migration.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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1-integrin (Sigma, St. Louis, MO), mouse anti-61-integrin (Chemicon, Temecula, CA), mouse anti-human 6-integrin with blocking activity (Abcam, Cambridge, UK), a nonblocking mouse anti-human 6-integrin, mouse anti-rat 3-integrin and goat anti-human LN4 (V20) (Santa Cruz Biotechnology, Santa Cruz, CA), mouse anti-collagen IV (Becton Dickinson, Bedford, MA), peroxidase-conjugated goat anti-rabbit IgG (Pierce, Rockford, IL) and Alexa Fluor secondary antibodies to mouse and rabbit IgG (Molecular Probes, Eugene, OR). Polyclonal rabbit antibodies to fibronectin, LN-1, LN4, LN1, and LN2 were generated as previously described (2, 28, 29). Mouse anti-rabbit -dystroglycan clone 11H6 was a gift from Dr. K. Campbell (Howard Hughes Medical Institute, University of Iowa, Iowa City, IA). Cell culture. Rat glomerular MC were prepared by modification (3, 4) of routine methods (39). In brief, minced rat kidney cortex was sieved. Isolated glomeruli were plated in medium containing a 1:1 mix of 20% FCS-RPMI 1640 and previously collected glomerular conditioned medium. Insulin routinely added to supplement MC cultures was omitted. MC outgrowths were harvested and passed in this medium for an additional week, after which the conditioned medium was omitted. MC were cloned and studied at passages 8–12. TG98 cells, a human glioblastoma cell line, was obtained from ATCC (Manassas, VA) and grown as described previously (20). These cells synthesize predominantly LN-8 with little LN-9 (20).
Cell migration. MC migration was measured with and without IGFBP-5201–218 (30 µg/ml) in a wounding assay as described previously (8). In brief, MCs were plated in 60-mm tissue culture dishes, grown to near confluence, growth arrested in 2% FCS-RPMI, and scraped with a sterile razor blade to create a linear wound across the dish. IGFBP-5201–218 peptide was added to the cultures and migration was examined 48 h later. Controls were maintained in 2% FCS-RPMI medium. Cultures were stained with toluidine blue and migrating cells along 1-mm length of the wound edge were counted in 0.1-mm increments. Five areas were examined per sample. Experiments were repeated three times. Data were analyzed as the total number of migrating cells and were expressed as a percentage of control. Individual control samples are also divided by the average for all controls and expressed as a percentage of control. A standard deviation for these results is calculated and shown in the graph to indicate the variability of repeat control experiments, as well as the experimental conditions. To test the role of specific LN isoforms, MCs previously treated with morpholino oligonucleotides as described below were used. To test the requirements for specific cell-matrix attachments, blocking antibodies were added at a 1:200 dilution with or without IGFBP-5. Normal serum or antibodies preabsorbed with specific substrate served as controls. The inhibitor kistrin was used at 10 nM.
To examine the role of LN isoforms in migration, T98G cells or MC were plated and grown to confluence. Cells were lysed with 20 mM NH4OH and washed away, leaving the matrix previously secreted by the cells. In separate experiments this matrix was solubilized and analyzed by Western blotting (40), confirming that the plate was coated with LN-8 by T98G cells or LN-8/9 by MC. T98G cells were then plated on these substrates at confluence and assayed for migration with and without IGFBP-5 as described above.
Morpholino anti-sense inhibition of protein expression.
Both lissamine-labeled and unlabeled anti-sense morpholino oligonucleotides for LN4 (A4AS), LN2 (B2AS), and several sense control oligonucleotides (StdC, A4S, B1S) were prepared by the manufacturer's instructions (Gene Tools, Philomath, OR) (36). The sequences used were as follows: LAMA4AS: 5'-AGCACCAGGCTCTGTTCCAAGCCAT; LAMB2AS: 5'-CACTCCATCCGTGTGGGTACTGGGC, StdC: 5'-CCTCTTACCTCAGTTACAATTTATA, LAMA4S: 5'-ATGGCTTGGAACACAGCCTGGTGCT, and LAMB1S: 5'-TGGGAGCGGCAGGAAATGGAAAGGC. The LN2 was derived from the published rat sequence (GenBank accession no. X16563). Rat sequences for LN4 and 1 were generated by RT-PCR from rat MC RNA and primers derived from mouse sequences as follows: LN4 (U69176
[GenBank]
) sense 5' GGGAGACCGGAGACAAAGTGAG, anti-sense 5' ACGCTGCGTTGGAGCAGACA and LN1 (M15525
[GenBank]
) sense 5'CGTGGGAGCGGCAGGAAAT, antisense 5' AAGGAGGACGCAGCAGGCAAG. The resulting products were sequenced by Dyedeoxy sequencing (Applied Biosystems, Foster City, CA). These sequences were used to design the anti-sense oligonucleotides. MC were plated in growth media at 200,000 cells per well in 24-well plates. Anti-sense and sense control morpholino oligonucelotides were administered using ethoxylated polyethylenimine according to the manufacturer's instructions. To test the effects of morpholino anti-sense inhibition, cell counts were performed in triplicate 24, 48, and 72 h after treatment. Total RNA was extracted 72 h after treatment for use in RT-PCR to examine LN chain mRNA expression. Protein expression was assessed by immunofluorescence microscopy and Western blot analysis. For use in immunofluorescence studies, cells were treated with lissamine-labeled oligonucleotides, trypsinized after treatment, plated in growth medium overnight, and then maintained in 2% FCS-RPMI 1640 medium for 48 h before fixation with 2% paraformaldehyde. For use in Western blots, cell layers were extracted at 72 h after treatment in PBS containing 0.1% Triton X-100, 0.025 M EDTA, 0.1 M N-ethylmalimide, 2 mM PMSF, and 0.02 mg/ml pepstatin A. For migration studies, cells were growth arrested for 24 h in 2% FCS-RPMI 1640 before treatment with morpholino oligonucleotides.
Immunofluorescence and light microscopy.
Slides were stained with antibodies to LN4, LN1, LN2, the 1-integrin chain, or the 61-integrin, followed by Alexa Fluor 488-labeled secondary antibodies as described previously (2). Slides were viewed using a Leitz microscope equipped for epi-illumination, and photographed using a digital RT-Color Spot camera. Migrating MC were fixed in 2% paraformaldehyde, stained with toluidine blue, and photographed with a Polaroid Land camera on a Leitz inverted microscope.
Western blot analysis.
Extracts of morpholino oligonucleotide-treated cell layers were normalized by cell number and cleared by centrifugation. Equal volume aliquots were immunoprecipitated with anti-LN-1 antibodies. The resulting immune complex was washed with 3% polyethylene glycol (MW 8,000) in borate buffer. The LN immune complexes were separated by SDS-PAGE and transferred to nitrocellulose. The membranes were incubated with anti-LN4 and anti-LN2 antibodies, followed by peroxidase-conjugated anti-rabbit IgG antibodies. Blots were developed with SuperSignal West Pico chemiluminescence substrate (Pierce) and exposed onto X-Omat film. The expression level of LN was quantified using an EDAS camera system and 1D software (Eastman Kodak, Rochester, NY). Proteins were extracted from secreted matrix of MC and T98G cells and analyzed by Western blot analysis using antibodies to LN subunits as described above.
RT-PCR.
Total RNA (0.5 µg) was prepared using RNace Total Pure kit (Bioline, Kenilworth, NJ) was used per RT-PCR reaction. An aliquot of the resulting cDNA generated for each condition was used for amplification of LN4, LN5, LN1, LN2, and GAPDH. The primers were as follows: LN4 (mouse sequence, GenBank accession no. U69176) sense 5'-TGGTCAGGTGACTCGCTTTG, anti-sense 5'-GCTCTTAACGTGCCGTCTGT; LN5 (mouse sequence, GenBank accession no. U37501) sense 5'-GCTGCGTACACTGCCCTCAAGT, anti-sense 5'-GATGCTGACGGCTGCAAACTGC; LN1 (rat sequence generated in our laboratory) sense 5'TTCCAGTCGCACCAGTTCTT, anti-sense 5'-GTCAGCAGATGCCAGGAGGA; LN2 (rat sequence, GenBank accession no. X16563) sense 5'-GTGGCAGTCGGAGAACGGTGTT, anti-sense 5'-CAGCGGGATTCCTGGGAAGTCA, and GAPDH sense 5'-ACCACAGTCCATGCCATCAC, anti-sense 5'-TCCACCACCCTGTTGCTGTA (Clontech, Palo Alto, CA). Amplification was preformed using HotStarTaq (Qiagen, Valencia, CA), and the amplification cycles were limited to ensure that the resulting products remained within the linear detection range. The expression level of LN and GAPDH mRNA was quantified with the use of a Kodak EDAS camera system and 1D software. The identity of the products was confirmed by Dyedeoxy sequencing (Applied Biosystems). To demonstrate that IGFBP-5 induced a change in LN mRNA expression, real-time PCR was performed on a multiplex quantitative PCR system (model Mx4000, Stratagene, La Jolla, CA) using the following primers: LN4 (AAGAAACCTTAGGAGTTGGTTATGGA, ATAAAACTTTGCCCGTTGAAATATG and 6FAM-CCAGAGGACTCCCTGATCTCTCGCAG-BHQ1), LN2 (TaqMan Gene Expression Assay; Applied Biosystems), LN1 (ATGACATCTCCTCGACCTTTCAG, GCCTCCGAGCCATCTCTCT, and 6FAM-CACACGCCACCCGTCCTCATCA-BHQ1), GAPDH (CGGCCTCGTCTCATAGACAAG, ACCAGGCGGCCAATACG and HEX-AAATCCGTTGACACCGACCTTCACCA-BHQ1).
Statistical analysis. Group means were compared using one-way ANOVA with subgroup testing by contrasts. A value of P < 0.05 was considered significant.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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4, LN2, and LN1.
Rat glomerular MC synthesize collagen IV, fibronectin, thrombospondin, LN-8 (411, 411) and LN-9 (421, 421), and small amounts of LN-2 (211, 211) and LN-10/11 (511/521, 511/521) (3, 4, 28). Total RNA was isolated from untreated or IGFBP-5-treated MC and subjected to real-time PCR analysis of mRNA expression for LN4, LN2, and LN1. As shown in Fig. 1, IGFBP-5 induced a twofold increase in expression of mRNA for subunits that compose LN-9. Because prior studies had shown no effect on other LN subunits (LN2, LN5, and LN1, data not shown), the effect appears to be specific for LN-9. Although this change in mRNA expression does not establish that newly synthesized LN-9 is required for IGFBP-5-induced migration, it suggested that this LN isoform might mediate migration in these cells.
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v3-integrins (7). Because data described above show that IGFBP-5 induced increased mRNA expression for LN-9, we examined the role of LN in mediating migration induced by IGFBP-5. Migration induced by IGFBP-5 was partially retarded when polyclonal antibody to LN-1 was added (Fig. 2). Cultured rat glomerular MC do not synthesize the LN1 chain (28); however, inhibition of migration by antibodies to LN-1 (111, 111) that bind to 1 and 1 chains suggests that LN isoforms synthesized by MC might be important. This was substantiated by antibodies to the globular domain of the LN4 chain, which significantly inhibited IGFBP-5-mediated migration. Preimmune serum or antibodies to the globular domains of LN2 and LN5 had no effect (data not shown). These data suggest that 4-containing isoforms support a migratory phenotype and that IGFBP-5-induced MC migration requires attachment to the 4 chain of LN-8/9. Because this antibody is specific to the LG4–5 domain, it suggests that the unprocessed carboxy-terminus of the LN4 subunit interacts with the cell. Disruption of a single type of cell-matrix attachment, in this case mediated by LN4, is not sufficient to detach cells, thus reduced migration does not merely represent detachment form the substrate. Furthermore, antibodies that inhibit IGFBP-5-mediated migration have no impact on IGF-1-dependent migration (data not shown), indicting that the cells are still able to migrate using other matrix attachment sites. These data indicate that migration induced by IGFBP-5 is similar to that induced by PDGF (29), and further characterizes the ways in which it differs from IGF-1 (9).
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2 to migrate.
The role of LN-8 and LN-9 in cell migration has received considerable attention recently because of their contribution to the invasiveness of cancer cells (42) and angiogenesis (15). Although regions in the globular domain of LN 4-chain that bind to integrins, dystroglycan, and heparin have been defined (52, 64, 65), little is known about the functional differences between LN isoforms with substituted -chains. Endothelial cells migrate on LN-8 using 31-, 61-, and v3-integrins (26); yet, specific integrin interactions with LN-9 have not been reported previously. In prior studies, we showed that MCs treated with IGFBP-5 develop a phenotype that mimics MC plated on an LN2 peptide (28); thus we postulated that IGFBP-5-mediated migration might require LN-9. The following experiments were performed to distinguish between these roles of LN-8 and LN-9. As shown in Fig. 3, antibodies to LN1 enhance basal MC migration after wounding and do not block migration induced by IGFBP-5. Conversely, antibodies to the LRE domain of LN2 (which supports MC attachment; see Ref. 28) inhibit migration induced by IGFBP-5. These observations suggest that attachment to LN-9 is required for IGFBP-5-mediated migration. Also, the finding that inhibition of attachment to LN-8 facilitates basal migration suggests that attachments to LN-8 may be severed as attachments to LN-9 are formed during IGFBP-5-mediated migration.
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4 or LN2 fail to migrate in response to IGFBP-5.
To further define the role of LN in IGFBP-5-mediated migration, LN4 and LN2 expression in MC were specifically inhibited by the use of anti-sense morpholino oligonucleotides (36). Inhibition of expression of the LN4 chain was expected to reduce synthesis of both LN-8 and LN-9. Inhibition of LN2 was expected to reduce expression of LN-9 while preserving expression of LN-8. As shown in Fig. 4A, MC treated with anti-sense morpholino oligonucleotides had cell counts that were slightly reduced compared with sense-treated controls; yet all cells continued to proliferate, which suggests that there was no significant toxicity from treatment with the morpholino oligonucleotides at doses used in these experiments. To confirm that anti-sense treatment knocked down expression of LN chains, matrix proteins were extracted and subjected to Western blot analysis. As shown in Fig. 4B, treatment with anti-sense morpholino oligonucleotides for LN4 decreased LN4 expression, indicating a loss of synthesis of both LN-8 and LN-9. The anti-sense morpholino for LN2 reduced expression of LN2, consistent with loss of LN-9, as well as other LN2-containing isoforms. To further evaluate the affect of anti-sense inhibition on LN chain expression, total RNA was isolated and mRNA levels were analyzed by RT-PCR. As shown in Fig. 4C, mRNA expression was unaffected for the chain targeted by the anti-sense treatment, as morpholinos reduce translation, but had no effect on transcription (36). There were no compensatory changes in expression of LN4, LN5, LN1, or LN2 mRNA with any of the anti-sense treatments; thus it is unlikely that alterations in other LN isoforms influenced the effects on MC migration (see below). Immunofluorescence microscopy (Fig. 4D) shows loss of expression of the appropriate LN chain in cells treated with anti-sense morpholino oligonucleotides. In those with suppression of LN2, the detection of abundant LN1 and LN4 indicates continued expression of LN-8 in these cells. This specificity of suppression of LN-9 allowed us to test the effects of loss of LN-9 with continued expression of LN-8. Suppression of LN4 results in a reduction in total laminin (Figs. 4D and 5). This was not surprising because LN4 is the predominant LN chain expressed by these cells and reduction in chain expression results in the loss of the heterotrimer. This reduction in total laminin is further illustrated in Fig. 5. Anti-sense treatment results in a significant reduction in laminin by both immunofluorescence microscopy and Western blot as detected by anti-LN-1 antibodies. These cultures were also stained with antibodies to collagen IV (data not shown) and fibronectin (Fig. 5). A fibrillar ECM containing these proteins was detected in anti-sense-treated cells, which was identical in appearance to normal cultures. This suggests that loss of LN-8/9 or LN-9 did not disrupt expression and assembly of other ECM proteins.
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4 and LN2 could specifically reduce expression of LN-8/9 and LN-9, respectively, we tested sense control and anti-sense-treated MC for their ability to migrate in response to IGFBP-5. By 48 h after creating a wound edge, sense control-treated MC migrated into and nearly filled the wound (Fig. 6A). In contrast, neither LN4 nor LN2 anti-sense-treated MCs, filled in the wound area after treatment with IGFBP-5. When migrating cells were counted as described above, it confirmed that migration of cells that failed to express LN-9 was significantly reduced in response to IGFBP-5 (Fig. 6B). Because the LN2 anti-sense cells continued to express LN-8, it implies that this LN isoform could not substitute for LN-9 in the migratory response to IGFBP-5. These cells were still able to migrate in response to IGF-1, a finding that was expected based on our previous studies (data not shown) (7, 9).
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v3 bound to fibronectin and that this integrin-matrix pair was not involved in migration induced by IGFBP-5 (9). To define the integrin required for IGFBP-5-induced migration, attachments to known matrix receptors were examined including integrins 1-, 61-, v3-, 31-, and -dystroglycan. As shown in Fig. 8, blocking antibodies to 1-integrins completely blocked IGFBP-5-mediated migration, whereas preimmune serum had no affect. Antibodies to the 6-integrin also blocked IGFBP-5-induced migration. To confirm the specificity of this result, a nonblocking antibody to the 6-integrin chain and a blocking antibody to the 3-integrin, were examined, and neither antibody affected migration. In keeping with previously published data (9), kistrin, a disintegrin that blocks the v3-integrin, had no effect on IGFBP-5-induced MC migration. In addition to integrins, LN binds to nonintegrin receptors including -dystroglycan (61). Blocking antibodies to -dystroglycan had no affect on IGFBP-5-induced migration. These data suggest that the integrin that mediates IGFBP-5-induced migration is 61. As the primary ligand for 61 is LN, these studies are consistent with those described above showing the requirement for LN-9.
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61-integrins.
As shown in Fig. 9, anti-sense inhibition of LN4 or LN2 was associated with absence of 61 expression on the surface of those cells that contained the oligonucleotides. These observations suggest that LN-9 is the only LN that binds to this integrin in MC and further supports the role of integrin 61 in IGFBP-5-mediated MC migration using LN-9. It is of note that staining only for the 1-integrin chain is still detected, which indicates that 1-integrins with other 6-chains are still expressed and engaged by other ligands. Several studies (30, 47) have shown that integrin expression, in cooperation with dystroglycan, is required for LN assembly on cell surfaces, yet Miner et al. (47) reported that basal expression of specific integrins was absent when their LN ligands were absent. Nothing is known about the role of LN-9 in mediating insertion of 61 into the cell membrane. Because this integrin is normally expressed by MC, it suggests that LN-9 may be required for its expression.
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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-actin-rich leading lamella that is associated with activation of Rac, and migration on fibronectin using v3-integrins (9, 34, 49). PDGF induces an elongated, bipolar phenotype and migration using a MC-derived LN containing the LN4 chain and a 1-integrin (29). In contrast, IGFBP-5-treated MC take on a spider-like morphology, a phenotype (7) that can be reproduced by plating on LN-8/9 (29) or the LRE peptide of the LN2 subunit (28). IGFBP-5-mediated MC migration is characterized by filopodia formation and is associated with activation of cdc42 (7). Intact IGFBP-5 can modify IGF-1 actions; however, the carboxy-terminal fragment of IGFBP-5 (including the AA201–218 peptide) has effects that are independent of IGF-1 (1). Furthermore, when added to IGF-1, IGFBP-5201–218 augments MC migration (1). A similar phenomenon in vascular smooth muscle cells requires binding of IGFBP-5 to cell-surface proteoglycan (31). These observations suggest that different migratory stimuli utilize specific cell-matrix interactions and signal transduction pathways. In this study, we demonstrate that IGFBP-5-mediated migration in MC specifically utilizes LN-9 bound to 61-integrins. Maneuvers that disrupt attachments to LN-8 enhance spontaneous migration after wounding and do not inhibit IGFBP-5-mediated migration. Because quiescent MCs are bound to both LN-8 and LN-9, these data suggest that cellular attachments to LN-8 are severed as new attachments to LN-9 are formed during IGFBP-5-mediated migration. Because IGFBP-5 induces an increase in expression of LN-9, new synthesis of this isoform might be required. Anti-sense inhibition of expression of LN-9 is associated with loss of integrin 61 from the cell surface, which suggests that these processes might be linked. Future studies are required this confirm relationship.
The 4 chain of LN-8 and -9 has received considerable attention recently because of studies showing that it is important to angiogenesis and microvessel integrity (26, 62), as well as being responsible for proper alignment of axons and the motor end plate in the neuromuscular junction (55). Although blood vessels appear to form normally in the LN4 null mutant mouse, LN4 is required for maintenance of vessel integrity and protection from vessel injury (62). In part, this is due to impaired recruitment of pericytes, a process known to require PDGF (10). Our observations showing that both PDGF (29) and IGFBP-5, two migratory stimuli for the pericyte-like MC require LN4, may explain the reduced number of pericytes in the LN4-null mice (62). The potential importance of LN4 to other vascular smooth muscle cells or pericytes has not been described previously.
Specific studies have attempted to define the relative roles of LN-8 and LN-9 in angiogenesis and tumor invasion. Glial tumors overexpress LN-8, particularly in blood vessels (42). This is in contrast to normal blood vessels that express predominantly LN-9. This difference was postulated to play a role in neovascularization and early tumor recurrence (42). Khazenzon et al. (36) have shown that in contrast to normal glial cells, glioblastoma cells stimulate endothelial cells to switch from expression of LN-9 to LN-8, and with that change in isoform, it promotes migration and glioblastoma invasion. When LN-8 expression is suppressed, there is less tumor invasion (36). Proliferating vessels and tumor invasion in vivo are associated with increased expression of -dystroglycan, a receptor for LN-8. The factor responsible for a shift in expression from LN-9 to LN-8 in glioblastomas in not known, but modulation of IGFBP-5 expression, which can have tumor-promoting or inhibitory effects, may play a role (11). To our knowledge, our studies are the first showing a specific role for LN-9, which differs from LN-8-dependent migration characterized in endothelial cells (19), monocytes (56), and lymphocytes (22). As LN-9 inhibits migration of lymphocytes (22), regulated expression of this isoform within the mesangium may retard infiltration of inflammatory cells. Conversely, stimuli other than IGFBP-5, which enhance expression of LN-8, might be anticipated in various forms of glomerulonephritis.
LN exerts effects on cells by binding to integrins and other matrix receptors. In endothelial cells LN-8/9 codistributes with v3-, 61-, and 31-integrins, and inhibition of binding of the 4LG1–4 domain of LN-8 blocks angiogenesis (20, 26). Subsequent studies (38, 50) with purified proteins and reconstituted cells have shown that LN4 binds only to 61-integrin; yet subsequent cooperation with v3 induces branching morphogenesis (25). In our studies blocking antibodies to the 6 and 1-integrin chains also blocked IGFBP-5-induced migration, which argues that the LN4 chain is binding to this integrin. The absence of an effect with kistrin indicates that cooperation with v3 was not necessary in our system. Although other LN subunits expressed by MC can also bind to 61-integrins, including LN2 and LN5, it is unlikely that they contribute to IGFBP-5-mediated MC migration. Attachment of MC to LN-2 and LN-10/11 induce a different phenotype than LN-9 (29) and antibodies to the globular domain of the LN2 chain had no affect on IGFBP-5-mediated migration (data not shown). Furthermore, these LN isoforms continue to be expressed in the cells with anti-sense-inhibited LN4 expression, yet they were unable to support IGFBP5-induced migration. Considerable data document interaction between the LN-subunits and integrins, but these binding studies have not elucidated the mechanisms that distinguish the biological response to LN isoforms that contain the same chain, but have substituted -chains. Our finding that MC lacking LN-9 did not express 61 on their surface indicates shifts in LN isoform expression may in turn affect the integrins that are expressed and the consequent biological events that follow. Functional differences have been attributed to isoforms of 6 and 1-integrin chains (13, 17, 24), yet it is unknown whether they have different binding specificities for LN-8 and LN-9. Additional studies are needed to further understand the molecular interactions between the LN-9 and 61-integrin, as well as the isoform of 61 that is involved.
In addition to binding to integrins, LN also binds to nonintegrin receptors, including dystroglycan (33). The extracellular domain of -dystroglycan binds to LN- chains, including 1, 2, 4, and 5; however, the affinity of binding for 4 is quite low (65). MCs express dystroglycan (16) (and unpublished data from our laboratory), yet the blocking antibody to dystroglycan had no affect on IGFBP-5-mediated migration. This suggests that even if LN-8/9 is bound to dystroglycan in quiescent MC, this attachment is not required for migration on LN-9. Recent studies have shown that LN4 binds to syndecan-4 (14, 45, 52) and that IGFBP-5-mediated migration that is independent of IGF-1 requires attachment of IGFBP-5 to a cell-surface proteoglycan (31). Additional studies are needed to explore the role of syndecan-4 in mediating MC migration and attachments to LN. Differences in engagement of matrix receptors in addition to 61-integrin may eventually explain the different biological functions of LN-8 and LN-9.
The glomerulus is a unique capillary structure in which the vascular smooth muscle, pericyte-like MC directly interfaces with both the glomerular endothelial cell and the podocytes. In each of the interfacing sites extracellular matrix with a specific composition is expressed (12, 27, 60). Some of this specificity is conveyed by the isoforms of LN. Evolution of expression of different LN isoforms corresponds to critical stages during glomerulogenesis (46) and becomes disrupted or altered during disease (2, 35). During glomerulogenesis, the mesangium first contains the 1 chain, which later is replaced by 4, the predominant -chain in the adult mesangium (29, 57). As the glomerulus matures, smaller amounts of 2 and 5 become detectable in the mesangial zone (35, 46). While the critical importance of the 5 chain has been demonstrated in elegant studies by Miner and colleagues (37, 48), the role of other LN chains is less clear. Null mutations of the 2 chain produce nephrotic syndrome in mice (51) and proteinuria and mesangial sclerosis in humans (66). Null mutations in the 4 chain are associated with microvascular disease that leads to progressive organ dysfunction later in life (63). Kidney architecture has not been reported in these animals, but is likely that they have abnormalities in renal microvessels as well. In culture, MCs synthesize predominantly LN-8/9, and small amounts of LN 2 and 5 chains (27, 28). Although key cellular properties have been mapped to peptides within specific LN chains, the role of LN1 compared with LN2 is less clear. As discussed above, a switch between LN-8 and LN-9 may influence blood vessel growth, quiescence with differentiation, responses to injury, and infiltration by inflammatory and malignant cells. Each of these effects may rely on a different set of growth factors, cells, and matrix proteins. These differences have been exploited in studies of retinal ischemia (21), where specific peptide inhibitors have shown the capacity to control abnormal vessel proliferation, while not impeding restoration of perfusion to ischemic retina. It is hoped that detailed characterization of similar interactions in normal and diseased glomeruli might provide insights with therapeutic implications for chronic kidney disease.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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