8 integrin in mesangial cell adhesion, migration, and proliferationReceptors of the integrin family mediate cell-cell or cell-matrix interactions. Integrins are heterodimers consisting of an 
and a 
subunit. At least 18 - and 8 -chains are known to date, which combine to form 24 integrin receptors. Most receptors recognize more than one ligand, and each ligand is capable of binding several integrins, which leads to a wide variety of possible interactions. Many 1 and 3 integrins are receptors for extracellular matrix molecules mediating signals, which regulate cell adhesion, migration, and proliferation. Inhibition of integrin activity generally leads to detachment of adhesion-dependent cells. In these cells, attachment to extracellular matrix molecules is a prerequisite for migration and proliferation1. The contribution of integrins to cell migration and proliferation has been the subject of many studies in the past few years. Integrins involved in the migration of different cell types include 11, 21, 51 or v32,3,4. Some integrins, such as 51 and v3, are known to promote adhesion-dependent cell proliferation via the mitogen-activated protein kinase (MAPK) pathway, while others do not5.
81 integrin is a receptor for fibronectin, vitronectin, tenascin-C fragments, osteopontin, and nephronectin, but not for collagens6,7,8,9. It was shown to promote attachment of K562 cells6 on fibronectin. However, its contribution to cell migration or proliferation is presently unknown. In the kidney, the 8 integrin chain is expressed on vascular smooth muscle cells and on mesangial cells of the glomerulus10. During the DOCA-salt model of glomerular hypertension imposing mechanical stress on the glomerulus, mesangial 8 was up-regulated. Lack of 8 led to more severe glomerular lesions in this experimental disease model, suggesting that 8 is important in maintaining the integrity of the glomerular capillary tuft11. The way that 8 preserves tissue integrity is not yet understood. Therefore, we investigated the role of 8 for mesangial cell attachment, migration, and proliferation.
METHODS
Cultivation of mouse mesangial cells
Mesangial cells were isolated from kidneys of wild-type or 8-deficient mice (obtained from U. Müller, Basel, Switzerland) by the sieving method, as described previously12, using 63, 75, and 38 
m grid sieves. Renal development in mice with a homozygous deletion of the 8 gene is disturbed, leading to unilateral or bilateral agenesis. As a consequence, a considerable number of newborn 8-deficient mice die perinatally13. Most surviving 8-deficient mice have a kidney mass reduction of about 50%, without showing any gross alterations in glomerular structure. Glomeruli of these mice could be isolated with the same sieving protocol as wild-type mice. In the first two passages many 8-deficient mesangial cells were lost because they had a reduced ability to adhere. From the third passage onward, this was not the case, and cultivation of these cells became comparable to the cultivation of wild-type mesangial cells. Cultured mesangial cells showed a typical vascular smooth muscle–like morphology, positive immunostaining for smooth muscle -actin, and negative staining for markers of other cell types. Wild-type mesangial cells were also positive for 8 integrin, while mesangial cells from 8-deficient mice were negative for 8. By electron microscopy, phagocytotic inclusion bodies were detected in the cytoplasm of mesangial cells. Mesangial cells were grown in Dulbecco's modified Eagle's medium (DMEM; PAA Laboratories GmbH, Linz, Austria) containing 10% fetal calf serum (FCS), 5 g/mL insulin, 5 g/mL plasmocin (TEBU, Frankfurt, Germany), and 2 m L-glutamine (Sigma, Deisenhofen, Germany) in a 95% air-5% CO2 humified atmosphere at 37°C. Mesangial cells were used for experiments in passages 5–15. Before seeding of mesangial cells for experiments, test plates were coated with the matrix proteins in concentrations from 0.2 to 40 g/mL at 4°C overnight and blocked with 2% bovine serum albumin (BSA) at 37°C for 1 hour.
FACS analyses
A single cell suspension was prepared by trypsination. An aliquot was used for trypan blue staining to identify the fraction of dead cells. In all aliquots tested the fraction of dead cells was below 5% and did not differ between 8-deficient and wild-type cells. After centrifugation, cells were resuspended to 2 
106 cells/mL with staining buffer [5% FCS, 0.02% azide in phosphate-buffered saline (PBS)]. Cell suspension was aliquoted in 500 L per sample. The samples were incubated with 1 to 2 g/mL of antibody against v (Santa Cruz Biotechnology, Heidelberg, Germany) or 5 integrin (Quartett, Berlin, Germany) for 30 minutes on ice. Mesangial cells were then washed twice in staining buffer, incubated with 2 g/mL fluroscein isothiocyanate (FITC)-conjugated secondary antibody for 30 minutes on ice. After washing, labeled cells were analyzed with a Beckman Coulter Epics XL cytometer and System II software (Beckman Coulter, Krefeld, Germany).
Adhesion assays
Two different types of adhesion assays were applied: (1) a conventional attachment assay based on the determination of the number of adherent cells by measuring the activity of the lysosomal enzyme hexosaminidase, as described by Gauer et al14; and (2) the centrifugal assay for fluorescence-based cell adhesion (CAFCA), which is based on two mechanisms, synchronized cell matrix contact by the first step, and removal of non- or weakly adhered cells by the second reverse centrifugal step. The assay was performed as described15,16,17. In brief, trypsinized mesangial cells were washed and labeled with vital fluorochrome calcein AM (Molecular Probes, Inc., Eugene, OR, USA) for 5 to 10 minutes at 37°C. Labeled cells were washed again with FCS-free medium and seeded into the bottom unit of CAFCA miniplates (Tecan Group, Grodig, Austria) at a density of 1 106 cells/mL in medium containing 2% ink (Pelikan; Milano, Italy) to quench fluorescence. The miniplates were then placed in plastic holders (CAFCA; Tecan Group) and centrifuged at 142 g for 5 minutes. After a 20-minute incubation time and allowing cells to develop cell-matrix interactions, a top unit with an adhesive surface, filled with medium containing 2% ink, is fixed to the bottom unit. After a second reverse centrifugation step at 46 g for 5 minutes, the relation of bound (cells remaining in the bottom unit) to unbound cells (cells located in the top unit) were measured by top/bottom fluorescence detection in a computer-interfaced SPECTRAFluor microplate fluorometer (Tecan Group) and calculated with CAFCA software (Tecan Group).
Migration assays
Two types of migration assays were performed: (1) A transmigration assay was applied as recently described18. FluoroBlok Inserts (Falcon HTS; Becton Dickinson, Heidelberg, Germany), containing a proprietary light-opaque polyethylene terephthalate (PET) membrane to absorb visible light within 490 and 700 nm range with 8 m pores were coated with different matrix proteins, including fibronectin (20 g/mL), vitronectin (10 g/mL), collagen type I (20 g/mL), and collagen type III (20 g/mL), and saturated in FCS-free medium containing 1% BSA. Trypsinized mesangial cells were washed twice, labeled with 50 g/mL DiI (Molecular Probes, Leiden, The Netherlands), a vital lipophilic carbocyanine, for 10 minutes at 37°C, and put into inserts with a volume of 150 L at a density of 1 106 cells/mL. The inserts were then incubated in 24 Multiwell plates (Becton Dickinson, Heidelberg, Germany), each well filled with 700 L medium containing 0.1% BSA, for several hours. Measurements were performed within 0 (starting point) and 48 hours of incubation to observe transmigration. Transmigrated mesangial cells were detected with SPECTRAFluor fluorometer (Tecan); and (2) a chemotaxis assay was applied to investigate the chemotactic behavior of mesangial cells using a 48-well microwell modified Boyden chamber. The test wells were seeded with 0.5 Mio cells/mL. The method was performed as described by Li et al19.
Determination of cell proliferation
Cell proliferation was estimated according to two different protocols: (1) For measurement of [3H] thymidine uptake, mesangial cells were serum-starved for 72 hours in medium containing 0.1% FCS, seeded into matrix-coated 96-well plates, and stimulated with 2% FCS for 48 hours. [3H] thymidine uptake was determined as described before20; and (2) a 5-bromo-2'-deoxy-uridine (BrdU) incorporation assay into cellular DNA was performed using a BrdU Labeling and Detection Kit (#1299964; Roche, Mannheim, Germany). Mesangial cells were washed two times with PBS and serum-starved for 72 hours in medium containing 0.1% FCS. After trypsinating and washing they were seeded into culture slides (Falcon) which had been coated with matrix proteins and blocked with 2% BSA. After a 3-hour resting period for mesangial cells to attach to the matrix, mesangial cells were incubated with medium containing 2% FCS and BrdU for 48 hours at 37°C. Mesangial cells were then fixed with 70% ethanol (50 m glycine buffer; pH 2.0) and processed following the manufacturer's instructions. Incorporated BrdU was detected by an alkaline phosphatase–conjugated secondary antibody reacting with an NBT/X-phosphate substrate. Nuclei with a positive staining for BrdU were counted in a Leitz Aristoplan microscope (Leica Instruments, Nu
loch, Germany).
Western blot analysis and real-time PCR
Protein concentration of cell lysates was determined using a protein assay kit (Pierce, Rockford, IL). Protein samples containing 30 g total protein were denatured by boiling for five minutes and separated on an 8% denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel. After electrophoresis, the gels were electroblotted onto polyvinylidine difluoride (PVDF) membranes (Pall Filtron, Karlstein, Germany), blocked with 5% horse serum/Tris-buffered saline (TBS)/0.1% Tween 20 overnight, and incubated with the 2 integrin antibody (BD Biosciences, Heidelberg, Germany) for 2 hours. Immunoreactivity was visualized with a secondary horseradish peroxidase–conjugated anti-armenian hamster immunoglobulin G (IgG) antibody, using the enhanced chemiluminescence (ECL) system according to the manufacturer's instructions (Amersham, Braunschweig, Germany). To evaluate mRNA expression levels of 2, RNA was isolated from wild-type and 8-deficient mesangial cells as described20. mRNA levels of 2 were detected by real-time polymerase chain reaction (RT-PCR) using the TaqMan PCR system (Applied Biosystems, Weiterstadt, Germany) and using 18S RNA as a reference and cyber green as staining reagent. Primers were selected for mouse 2: forward primer: 5' TGA-CCA-GGT-TCT-GCA-GGA-TAG-A 3' and reverse primer 5' AGT-AGA-AAT-TGC-AGC-CAC-AGA-GTA-AC 3'. Real-time PCR was carried out and relative mRNA levels were calculated according to the supplier's protocols.
Expression of 
8 integrin cDNA in 293 human embryonic kidney (HEK) cells
Full-length 8 cDNA was cloned into the expression vector pcDNA3.1+/Neo. To obtain a deletion in the cytoplasmic domain of the 8 integrin chain, a 65 base pair (bp) fragment at the 3' end of the 8 cDNA was removed by an ExoIII/Mung Bean Deletion Kit (Stratagene, Amsterdam, The Netherlands) according to the suppliers' protocol. The resulting fragment was sequenced, 293 HEK cells were transfected with Lipotaxi (Stratagene), and neomycin-resistant pools were subcloned. Western blot and FACS analyses were performed to confirm comparable expression of the full-length and truncated 8 integrin chains.
RESULTS
Wild-type and 8-deficient mouse mesangial cells were tested for cell surface expression of 5 and v integrins to rule out any compensatory induction of these integrins. 51 is the main fibronectin receptor, while v integrins are vitronectin receptors. Furthermore, 5 and v are known to promote cell proliferation. Different expression levels of 5 or v in wild-type and 8-deficient mesangial cells could, therefore, conceal 8-dependent effects. As assessed by FACS analysis, both cell types express 5 and v integrins on their cell surface, the degree of expression being comparable between wild-type and 8-deficient mesangial cells Figure 1.
Figure 1.
FACS analyses of 5 and v integrin cell surface expression in wild-type and 8-deficient mesangial cells. Fluorescence intensity of 5 or v is not different in wild-type and 8-deficient cells (8-/-), arguing for comparable expression levels of these integrins in both cell types. Staining with preimmune rabbit immunoglobulin G (IgG) served as the control.
Using the conventional attachment assay, differences in adhesion of wild-type and 8-deficient mesangial cells were detected. Significantly less 8-deficient than wild-type mesangial cells adhered to fibronectin or vitronectin in coating concentrations between 0.5 to 2 g/mL Figure 2 a and b. In contrast, even more 8-deficient than wild-type mesangial cells adhered to collagens I or III, which are not ligands for 8 Figure 2 c and d. These findings could be confirmed by the CAFCA assay Figure 3. Fewer 8-deficient mesangial cells adhered to fibronectin or vitronectin (coating concentration 0.6 g/mL each), but more to collagen III (10 g/mL) as compared to wild-type cells. In addition, adhesion of 8-deficient and wild-type mesangial cells on osteopontin and thrombospondin was compared. Adhesion of 8-deficient mesangial cells to osteopontin (12.5 g/mL), a ligand for 8, was also reduced. Neither 8-deficient nor wild-type mesagial cells adhered to thrombospondin (10 g/mL), which is not a ligand for 8 Figure 3.
Figure 2.
Comparison of wild-type (WT) and 8-deficient (8-/-) mesangial cell attachment on different coating concentrations. (A) Fibronectin. (B) Vitronectin. (C) Collagen type I. (D) Collagen type III. A conventional attachment assay using hexosaminidase reagent was performed. Fewer 8-deficient than wild-type mesangial cells adhered to fibronectin or vitronectin on coating concentrations between 0.5 and 2.5 g/mL. In contrast, more 8-deficient than wild-type mesangial cells adhered to collagens.
Figure 3.
Comparison of wild-type (WT) and 8-deficient (8-/-) mesangial cell attachment on fibronectin, vitronectin, collagen type III, osteopontin, and thrombospondin by CAFCA assay. Bovine serum albumin was used as control. Adhesion of 8-deficient mesangial cells was weaker on the 8 ligands fibronectin, vitronectin, and osteopontin, but stronger on collagen III compared to wild-type mesangial cells. None of the cell types adhered to thrombospondin. *P < 0.001 8-deficient vs. wild-type mesangial cells.
To investigate the cause for the increased adhesive properties of 8-deficient mesangial cells on collagens, we compared expression of collagen receptors in wild-type and 8-deficient mesangial cells. In 8-deficient mesangial cells mRNA and protein expression of the 2 integrin chain was increased as compared to wild-type mesangial cells Figure 4, while differences in 1 expression were not detected.
Figure 4.
Detection of the 2 integrin chain in wild-type and 8-deficient (8-/-) mesangial cells by real-time polymerase chain reaction (PCR) (A) and Western blot analysis (B). (A) mRNA expression of 2 was significantly higher in 8-deficient mesangial cells compared to wild-type mesangial cells. *P < 0.05. (B) Protein expression of 2 was higher in 8-deficient mesangial cells compared to wild-type cells.
The ability of 8-deficient and wild-type mesangial cells to migrate was compared via a fluorescence-based transmigration assay (FATIMA). On collagens I and III neither 8-deficient nor wild-type mesangial cells migrated considerably during the time period investigated Figure 5. On fibronectin or vitronectin, migration of wild-type and 8-deficient mesangial cells was induced after 4 hours, reaching statistical significance at the 18-hour time point. Significantly more 8-deficient than wild-type mesangial cells had transmigrated after 18 hours Figure 5. Similar findings were obtained in a Boyden chamber chemotaxis assay. No migration was observed after 5 hours in response to collagens I and III, while cells migrated to fibronectin or vitronectin Figure 6. Again, significantly more 8-deficient than wild-type mesangial cells migrated to these matrices Figure 6.
Figure 5.
Comparison of wild-type (WT) and 8-deficient (8-/-) mesangial cell migration by FATIMA transmigration assay. (A) Fibronectin. (B) Vitronectin. (C) Collagen type I. (D) Collagen type III. On fibronectin and vitronectin, more 8-deficient mesangial cells transmigrated than wild-type mesangial cells, while on collagens, neither wild-type nor 8-deficient mesangial cells migrated.
Figure 6.
Comparison of wild-type (WT) and 8-deficient (8-/-) mesangial cell chemotaxis on fibronectin, vitronectin, collagen type I, and collagen type III by a modified Boyden chamber chemotaxis assay. More 8-deficient mesangial cells migrated toward fibronectin and vitronectin than wild-type mesangial cells, while collagens did not induce migration of wild-type or 8-deficient mesangial cells. *P < 0.001 8-deficient vs. wild type mesangial cells.
For [3H] thymidine incorporation assays, fibronectin and vitronectin coating concentrations of 10 g/mL were used, as attachment assays had revealed comparable adhesion efficacy of 8-deficient and wild-type mesangial cells at this coating concentration Figure 2 a and b. Collagens I and III were used for coating at 30 g/mL. The basal proliferation of synchronized 8-deficient and wild-type mesangial cells (FCS 0.01%) was comparable between the two cell types. The proliferative response of 8-deficient mesangial cells, however, was much more pronounced than the response of wild-type cells on fibronectin or vitronectin Figure 7 a and b. On collagens, in contrast, the proliferative response of wild-type and 8-deficient mesangial cells was not different Figure 7c. These findings were confirmed by BrdU incorporation assays. After induction of proliferation by 2% FCS, significantly more 8-deficient than wild-type mesangial cells were BrdU-positive when seeded on fibronectin or vitronectin Figure 8. On collagen III, however, the amount of BrdU-positive cells did not differ in wild-type and 8-deficient mesangial cells Figure 8. Differences in cell proliferation could be due to different expression levels of integrins other than 8. Possible candidates are 5 and v, which are both implicated in cell cycle regulatory pathways. However, our FACS analyses showed a comparable cell surface expression of 5 and v in wild-type and 8-deficient mesangial cells Figure 1, indicating that the observed differences in the proliferative response are indeed due to differences in 8 expression. To investigate the role of 8 integrin not only in cells underexpressing 8 but also in cells overexpressing 8, we transfected 293 HEK cells with a full-length 8 expression vector. As a control, other 293 HEK cells were transfected with an 8 construct with a deletion in its cytoplasmic domain conserving the regulatory GFFKR site. Untransfected 293 HEK cells do not express 8 integrin. Cells expressing the full-length 8 displayed a much smaller FCS-induced proliferative response on fibronectin than untransfected cells Figure 9. Expression of the truncated 8, which is unable to form a cytoplasmic integrin signaling complex, resulted in a nearly threefold stronger proliferative response compared to cells expressing full-length 8 Figure 9. Adhesion of the cells was not different on fibonectin in the coating concentration we used (10 g/mL).
Figure 7.
Cell proliferation of wild-type and 8-deficient mesangial cells, as assessed by [3H] thymidine incorporation assays. Upon stimulation, 8-deficient mesangial cells showed a significantly stronger proliferative response on fibronectin (A) or vitronectin (B) than wild-type cells. (C) On collagens, no differences in proliferation were observed between stimulated wild-type and 8-deficient mesangial cells *P < 0.001 8-deficient vs. wild-type mesangial cells.
Figure 8.
Cell proliferation of wild type and 8-deficient mesangial cells, as assessed by BrdU incorporation assays. Proliferation of stimulated 8-deficient mesangial cells was more pronounced on fibronectin or vitronectin than the proliferation of stimulated wild-type cells. On collagen III, no differences in proliferation were observed between stimulated wild-type and 8-deficient mesangial cells. *P < 0.001 8-deficient vs. wild-type mesangial cells.
Figure 9.
Cell proliferation of untransfected 293 HEK cells, 293 HEK cells expressing full-length 8 (HEK/FL), and 293 HEK cells expressing 8 with a cytoplasmic deletion (HEK/TR). Proliferation of HEK/FL was low compared to untransfected 293 HEK cells. In contrast, HEK/TR proliferated significantly more in response to fetal calf serum (FCS) than HEK/FL. *P < 0.001 HEK/TR vs. HEK/FL.
DISCUSSION
The results of the present study support the hypothesis that 8 is involved in the regulation of mesangial cell adhesion, migration, and proliferation. 8-deficient mesangial cells adhered weaker to fibronectin, which is abundant in the normal mesangial matrix, or to vitronectin, which is commonly present in diseased glomeruli. In contrast, 8-deficient mesangial cells migrated more easily on fibronectin or vitronectin than wild-type cells. Further, 8-deficient mesangial cells displayed a more pronounced proliferative response on fibronectin or vitronectin compared to wild-type mesangial cells. Taken together, 8 could serve to facilitate attachment of mesangial cells on fibronectin and vitronectin but inhibit mesangial cell migration on these matrices. Proliferation of mesangial cells on fibronectin and vitronectin seems to be negatively regulated by 8.
In the glomerular mesangium, several integrins can mediate cell adhesion. Most abundant receptors for collagens and laminin on mesangial cells are 11, 21, and 31. The mesangial integrin receptors for fibronectin are 51 and for vitronectin are v3 or v521,22. Our data argues for a contribution of 81 to mesangial cell adhesion to fibronectin and vitronectin. The decreased ability of 8-deficient mesangial cells to adhere to fibronectin or vitronectin was not due to an altered expression pattern of the principal receptors for fibronectin or vitronectin, the integrin chains 5 or v, respectively. Adhesion to collagen I and III, in contrast, was even increased in 8-deficient mesangial cells compared to wild-type cells, demonstrating matrix-specific effects on adhesion in our cell isolates. Increased adhesion of 8-deficient mesangial cells to collagens seems to be mediated by the collagen receptor integrin chain 2, which was found to be up-regulated in comparison to wild-type mesangial cells. This could be a compensating mechanism for the lack of 8 in these cells. According to our results, 81 may well be a mediator of firm adhesion of mesangial cells to the fibronectin in its surrounding matrix in vivo and thus serve as a structural support of glomerular tissue integrity.
Adhesion of cells to extracellular matrix via integrins is necessary to enable migration. Migration of mesangial cells is a typical feature of remodeling processes during glomerular injury23,24. Until today, only a few integrins have been assayed regarding their ability to influence mesangial cell migration. Kagami et al3 showed that 11 integrin could promote mesangial cell migration, as antibodies to 11 inhibited their motility. In general, most integrins investigated revealed promigratory properties. For example, v3 mediated endothelial cell motility during angiogenesis25, 51 was involved in shear stress-induced endothelial cell migration2, and 11 and 21 supported haptotaxis of endothelial cells toward collagen I26. In our studies, we found increased motility of 8-deficient mesangial cells compared to wild-type mesangial cells by transmigration and chemotaxis assays. These results support the notion that 81 could serve as an antimigratory integrin, keeping mesangial cells at rest at their native location. Firm adhesion, as mediated by 8, inhibits migration in many cell types4. Thus, the decreased ability of 8-deficient mesangial cells to adhere to fibronectin or vitronectin could contribute to the increased ability of these cells to migrate. Interestingly, a potent migratory response to fibronectin was described for mesangial cells27, but the integrin receptor involved in this process was not identified. A promising candidate may be the main fibronectin receptor, 51, as the findings of this study argue against a contribution of 81 to the migratory response of mesangial cells on fibronectin. In contrast to our data obtained in mesangial cells, 8 contributed to migration of neural crest cells as shown by inhibition studies28. The action of 8 on cell behavior thus seems to be dependent on the cell type.
Proliferation of mesangial cells is a hallmark of many glomerular diseases and has been implicated in the progression of glomerulosclerosis leading to the loss of glomerular function29. Several studies have supported a role for integrins in the regulation of cell proliferation30,31. Some chains, such as v, 5, or 1 associate with Shc and caveolin and thereby link integrin signaling to the extracellular signal-regulated protein kinase (ERK) pathway, which leads to cell proliferation5,32. In accordance with these data, v integrins were found to promote proliferation of endothelial cells during angiogenesis33. The integrins 51 and 64 regulated keratinocyte cell cycling in an ex vivo culture or in primaray keratinocytes adhering to the respective ligands34,35. In embryonic fibroblasts, proliferation seemed to be promoted by 11, as 1-deficient fibroblasts revealed a markedly reduced proliferation rate on collagens in vitro and 1-deficient mice had a hypocellular dermis36. In contrast, however, overexpression of 11 integrin in mesangial cells significantly inhibited cell cycle progression37, arguing for a cell type–specific role for integrins in the regulation of cell growth. Our data suggest that the 8 integrin chain exerts antiproliferative effects on mesangial cells. The findings obtained from 293 HEK cells support the concept of 8 as an antiproliferative integrin; overexpression of 8 leads to a reduction of the proliferative response of 293 HEK cells, while overexpression of a truncated 8, which lacks a large part of the cytoplasmic domain, restores part of the proliferative response in untransfected 293 HEK cells. The cytoplasmic domain of 5 and v is known to be crucial for integrin signaling, leading to regulation of growth and survival30. For example, deletion of the cytoplasmic domain of 5 impaired the signaling pathway leading to bcl-2 induction38. Why 293 HEK cells with a truncated 8 chain do not reach the proliferative response of untransfected 293 HEK cells is not clear, but overexpression of the 8 chains possibly leads to a shortage of 1 integrins to combine with other chains, like the pro-proliferative v or 5 chains. Signal transduction pathways of 8 are largely unknown, which leaves the possible intracellular signaling events leading to inhibition of proliferation by 8 to mere speculation. Recruitment of 8 to focal adhesions has been studied by some authors, but the findings are controversial, depending on the cell type investigated. 8 was localized to focal adhesions in cultured fibroblasts6 and mesangial cells10, but not in neuronal crest cells28. Strict colocalization of 8 integrin with focal adhesion kinase was observed in vivo in hair cells of the inner ear39, suggesting a signaling pathway of 81 via focal adhesion kinase. However, this assumption has not yet been tested functionally. Future studies will be required to elucidate the intracellular changes involved in the 8-mediated regulation of mesangial cell migration or proliferation. The inhibitory effect of 11 on mesangial cell proliferation seems to be due to the action of the cyclin-dependent-kinase inhibitor p27Kip137, because 11 overexpressing mesangial cells revealed an increased expression of p27Kip1 compared to control mesangial cells. Whether changes in the expression patterns of cell cycle inhibitors are mediated by the 8 chain will have to be elucidated in the future.
CONCLUSION
Our findings support the hypothesis that the 8 chain is involved in the regulation of the mesangial cell phenotype. We suggest that 8 helps to keep mesangial cells quiescent in a resting state, and promotes their tight adhesion to the surrounding matrix within the glomerulus.
References
| 1. | van der Flier A & Sonnenberg A. Function and interactions of integrins. Cell Tissue Res 2001; 305: 285–298. | Article | PubMed | ISI | ChemPort | |
| 2. | Urbich C, Dernbach E & Reissner A. et al Shear stress-induced endothelial cell migration involves integrin signaling via the fibronectin receptor subunits alpha(5) and beta(1). Arterioscler Thromb Vasc Biol 2002; 22: 69–75. | PubMed | ISI | ChemPort | |
| 3. | Kagami S, Kondo S & Loster KT. et al Alpha1beta1 integrin-mediated collagen matrix remodeling by rat mesangial cells is differentially regulated by transforming growth factor-beta and platelet-derived growth factor-BB. J Am Soc Nephrol 1999; 10: 779–789. | PubMed | ISI | ChemPort | |
| 4. | Holly SP, Larson MK & Parise LV. Multiple roles of integrins in cell motility. Exp Cell Res 2000; 261: 69–74. | Article | PubMed | ISI | ChemPort | |
| 5. | Wary KK, Mainiero F & Isakoff SJ. et al The adaptor protein Shc couples a class of integrins to the control of cell cycle progression. Cell 1996; 87: 733–743. | Article | PubMed | ISI | ChemPort | |
| 6. | Muller U, Bossy B & Venstrom K. et al Integrin alpha 8 beta 1 promotes attachment, cell spreading, and neurite outgrowth on fibronectin. Mol Biol Cell 1995; 6: 433–448. | PubMed | ISI | ChemPort | |
| 7. | Denda S, Reichardt LF & Muller U. Identification of osteopontin as a novel ligand for the integrin alpha8 beta1 and potential roles for this integrin-ligand interaction in kidney morphogenesis. Mol Biol Cell 1998; 9: 1425–1435. | PubMed | ISI | ChemPort | |
| 8. | Denda S, Muller U & Crossin KL. et al Utilization of a soluble integrin-alkaline phosphatase chimera to characterize integrin alpha 8 beta 1 receptor interactions with tenascin: Murine alpha 8 beta 1 binds to the RGD site in tenascin-C fragments, but not to native tenascin-C. Biochemistry 1998; 37: 5464–5474. | Article | PubMed | ISI | ChemPort | |
| 9. | Brandenberger R, Schmidt A & Linton J. et al Identification and characterization of a novel extracellular matrix protein nephronectin that is associated with integrin alpha8beta1 in the embryonic kidney. J Cell Biol 2001; 154: 447–458. | Article | PubMed | ISI | ChemPort | |
| 10. | Hartner A, Schocklmann H & Prols F. et al Alpha8 integrin in glomerular mesangial cells and in experimental glomerulonephritis. Kidney Int 1999; 56: 1468–1480. | Article | PubMed | ISI | ChemPort | |
| 11. | Hartner A, Cordasic N & Klanke B. et al The alpha8 integrin chain affords mechanical stability to the glomerular capillary tuft in hypertensive glomerular disease. Am J Pathol 2002; 160: 861–867. | PubMed | ISI | ChemPort | |
| 12. | Lovett DH, Sterzel RB & Kashgarian M. et al Neutral proteinase activity produced in vitro by cells of the glomerular mesangium. Kidney Int 1983; 23: 342–349. | PubMed | ISI | ChemPort | |
| 13. | Muller U, Wang D & Denda S. et al Integrin alpha8beta1 is critically important for epithelial-mesenchymal interactions during kidney morphogenesis. Cell 1997; 88: 603–613. | Article | PubMed | ISI | ChemPort | |
| 14. | Gauer S, Schulze-Lohoff E & Scheicher E. et al Glomerular basement membrane-derived perlecan inhibits mesangial cell adhesion to fibronectin. Eur J Cell Biol 1996; 70: 233–242. | PubMed | ISI | ChemPort | |
| 15. | Perri RT, Vercellotti G & McCarthy J. et al Laminin selectively enhances monocyte-macrophage-mediated tumoricidal activity. J Lab Clin Med 1985; 105: 30–35. | PubMed | ISI | ChemPort | |
| 16. | Spessotto P, Yin Z & Magro G. et al Laminin isoforms 8 and 10 are primary components of the subendothelial basement membrane promoting interaction with neoplastic lymphocytes. Cancer Res 2001; 61: 339–347. | PubMed | ISI | ChemPort | |
| 17. | Giacomello E, Neumayer J & Colombatti A. et al Centrifugal assay for fluorescence-based cell adhesion adapted to the analysis of ex vivo cells and capable of determining relative binding strengths. Biotechniques 1999; 26: 758–762. | PubMed | ISI | ChemPort | |
| 18. | Spessotto P, Giacomello E & Perri R. Improving fluorescence-based assays for the in vitro analysis of cell adhesion and migration. Mol Biotechnol 2002; 20: 285–304. | Article | PubMed | ISI | ChemPort | |
| 19. | Li Z, Calzada MJ & Sipes JM. et al Interactions of thrombospondins with alpha4beta1 integrin and CD47 differentially modulate T cell behavior. J Cell Biol 2002; 157: 509–519. | Article | PubMed | ISI | ChemPort | |
| 20. | Hartner A, Sterzel RB & Reindl N. et al Cytokine-induced expression of leukemia inhibitory factor in renal mesangial cells. Kidney Int 1994; 45: 1562–1571. | PubMed | ISI | ChemPort | |
| 21. | Adler S & Brady HR. Cell adhesion molecules and the glomerulopathies. Am J Med 1999; 107: 371–386. | Article | PubMed | ISI | ChemPort | |
| 22. | Gauer S, Yao J & Schoecklmann HO. et al Adhesion molecules in the glomerular mesangium. Kidney Int 1997; 51: 1447–1453. | PubMed | ISI | ChemPort | |
| 23. | Barnes JL, Hevey KA & Hastings RR. et al Mesangial cell migration precedes proliferation in Habu snake venom-induced glomerular injury. Lab Invest 1994; 70: 460–467. | PubMed | ISI | ChemPort | |
| 24. | Haseley LA, Hugo C & Reidy MA. et al Dissociation of mesangial cell migration and proliferation in experimental glomerulonephritis. Kidney Int 1999; 56: 964–972. | Article | PubMed | ISI | ChemPort | |
| 25. | Brooks PC, Clark RA & Cheresh DA. Requirement of vascular integrin alpha v beta 3 for angiogenesis. Science 1994; 264: 569–571. | PubMed | ISI | ChemPort | |
| 26. | Senger DR, Perruzzi CA & Streit M. et al The alpha(1)beta(1) and alpha(2)beta(1) integrins provide critical support for vascular endothelial growth factor signaling, endothelial cell migration, and tumor angiogenesis. Am J Pathol 2002; 160: 195–204. | PubMed | ISI | ChemPort | |
| 27. | Barnes JL & Hevey KA. Glomerular mesangial cell migration. Response to platelet secretory products. Am J Pathol 1991; 138: 859–866. | PubMed | ISI | ChemPort | |
| 28. | Testaz S, Delannet M & Duband J. Adhesion and migration of avian neural crest cells on fibronectin require the cooperating activities of multiple integrins of the (beta)1 and (beta)3 families. J Cell Sci 1999; 112: 4715–4728. | PubMed | ISI | ChemPort | |
| 29. | Schocklmann HO, Lang S & Sterzel RB. Regulation of mesangial cell proliferation. Kidney Int 1999; 56: 1199–1207. | Article | PubMed | ISI | ChemPort | |
| 30. | Giancotti FG. Complexity and specificity of integrin signalling. Nat Cell Biol 2000; 2: E13–E14. | Article | PubMed | ISI | ChemPort | |
| 31. | Giancotti FG & Ruoslahti E. Integrin signaling. Science 1999; 285: 1028–1032. | Article | PubMed | ISI | ChemPort | |
| 32. | Wary KK, Mariotti A & Zurzolo C. et al A requirement for caveolin-1 and associated kinase Fyn in integrin signaling and anchorage-dependent cell growth. Cell 1998; 94: 625–634. | Article | PubMed | ISI | ChemPort | |
| 33. | Varner JA & Cheresh DA. Tumor angiogenesis and the role of vascular cell integrin alphavbeta3. Important Adv Oncol 1996; 87: 69–87. |
| 34. | Bata-Csorgo Z, Cooper KD & Ting KM. et al Fibronectin and alpha5 integrin regulate keratinocyte cell cycling. A mechanism for increased fibronectin potentiation of T cell lymphokine-driven keratinocyte hyperproliferation in psoriasis. J Clin Invest 1998; 101: 1509–1518. | PubMed | ISI | ChemPort | |
| 35. | Mainiero F, Murgia C & Wary KK. et al The coupling of alpha6beta4 integrin to Ras-MAP kinase pathways mediated by Shc controls keratinocyte proliferation. EMBO J 1997; 16: 2365–2375. | Article | PubMed | ISI | ChemPort | |
| 36. | Pozzi A, Wary KK & Giancotti FG. et al Integrin alpha1beta1 mediates a unique collagen-dependent proliferation pathway in vivo. J Cell Biol 1998; 142: 587–594. | Article | PubMed | ISI | ChemPort | |
| 37. | Kagami S, Kondo S & Urushihara M. et al Overexpression of alpha1beta1 integrin directly affects rat mesangial cell behavior. Kidney Int 2000; 58: 1088–1097. | Article | PubMed | ISI | ChemPort | |
| 38. | Zhang Z, Vuori K & Reed JC. et al The alpha 5 beta 1 integrin supports survival of cells on fibronectin and up-regulates Bcl-2 expression. Proc Natl Acad Sci USA 1995; 92: 6161–6165. | PubMed | ChemPort | |
| 39. | Littlewood EA & Muller U. Stereocilia defects in the sensory hair cells of the inner ear in mice deficient in integrin alpha8beta1. Nat Genet 2000; 24: 424–428. | PubMed | |
Acknowledgments
The expert technical assistance of Hans Fees is gratefully acknowledged. We thank Dr. Karl Hilgers and Dr. Harald Schocklmann for critical discussion and reading of the manuscript. We thank Dr. Ulrich Muller for kindly providing us with 8-deficient mice and the 8 cDNA. This study was supported by a grant of the Deutsche Forschungsgemeinschaft, Bonn, Sonderforschungsbereich 423, TP A2, and a Vigoni grant from the DAAD.
