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Mesangial cells organize the glomerular capillaries by adhering to the G domain of laminin alpha5 in the glomerular basement membrane.

Kikkawa Y, Virtanen I, Miner JH - J. Cell Biol. (2003)

Bottom Line: In addition, podocytes exhibited their typical arrangement in a single cell layer epithelium adjacent to the GBM, but convolution of glomerular capillaries did not occur.Finally, in vitro studies showed that integrin alpha3beta1 and the Lutheran glycoprotein mediate adhesion of mesangial cells to laminin alpha5.Our results elucidate a mechanism whereby mesangial cells organize the glomerular capillaries by adhering to the G domain of laminin alpha5 in the GBM.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine, Washington University School of Medicine, St Louis, MO 63110, USA.

ABSTRACT
In developing glomeruli, laminin alpha5 replaces laminin alpha1 in the glomerular basement membrane (GBM) at the capillary loop stage, a transition required for glomerulogenesis. To investigate domain-specific functions of laminin alpha5 during glomerulogenesis, we produced transgenic mice that express a chimeric laminin composed of laminin alpha5 domains VI through I fused to the human laminin alpha1 globular (G) domain, designated Mr51. Transgene-derived protein accumulated in many basement membranes, including the developing GBM. When bred onto the Lama5 -/- background, Mr51 supported GBM formation, preventing the breakdown that normally occurs in Lama5 -/- glomeruli. In addition, podocytes exhibited their typical arrangement in a single cell layer epithelium adjacent to the GBM, but convolution of glomerular capillaries did not occur. Instead, capillaries were distended and exhibited a ballooned appearance, a phenotype similar to that observed in the total absence of mesangial cells. However, here the phenotype could be attributed to the lack of mesangial cell adhesion to the GBM, suggesting that the G domain of laminin alpha5 is essential for this adhesion. Analysis of an additional chimeric transgene allowed us to narrow the region of the alpha5 G domain essential for mesangial cell adhesion to alpha5LG3-5. Finally, in vitro studies showed that integrin alpha3beta1 and the Lutheran glycoprotein mediate adhesion of mesangial cells to laminin alpha5. Our results elucidate a mechanism whereby mesangial cells organize the glomerular capillaries by adhering to the G domain of laminin alpha5 in the GBM.

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Analysis of mesangial cell adhesion to laminins in vitro. 96-well plates were coated with increasing concentrations of laminin-1 (closed squares) or laminin-10/11 (open circles) and incubated with mesangial cells at 37°C for 1 h. Human (A) and rat (B) mesangial cells readily attached to laminin-10/11, but were significantly less adherent to laminin-1. (C) Identification of receptors mediating adhesion of mesangial cells to laminin-10/11. Human mesangial cells preincubated with function-blocking antibodies against the indicated integrin subunits were added to laminin-10/11–coated wells. Values are expressed as percentages of the number of cells adhering in the absence of monoclonal antibodies. Each column represents the mean of triplicate assays. Error bars show the standard deviation. Anti–integrin α3 and β1 antibodies partially inhibited the adhesion of human mesangial cells to laminin-10/11, indicating that integrin α3β1 is a primary receptor. (D) The effect of sol-Lu on adhesion of human mesangial cells to laminin-10/11. Sol-Lu had little inhibitory effect on its own, but cooperated with anti–integrin β1 antibodies to inhibit adhesion by ∼90%. This indicates that Lu cooperates with integrin α3β1 to mediate mesangial cell adhesion to laminin α5 in the GBM.
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fig8: Analysis of mesangial cell adhesion to laminins in vitro. 96-well plates were coated with increasing concentrations of laminin-1 (closed squares) or laminin-10/11 (open circles) and incubated with mesangial cells at 37°C for 1 h. Human (A) and rat (B) mesangial cells readily attached to laminin-10/11, but were significantly less adherent to laminin-1. (C) Identification of receptors mediating adhesion of mesangial cells to laminin-10/11. Human mesangial cells preincubated with function-blocking antibodies against the indicated integrin subunits were added to laminin-10/11–coated wells. Values are expressed as percentages of the number of cells adhering in the absence of monoclonal antibodies. Each column represents the mean of triplicate assays. Error bars show the standard deviation. Anti–integrin α3 and β1 antibodies partially inhibited the adhesion of human mesangial cells to laminin-10/11, indicating that integrin α3β1 is a primary receptor. (D) The effect of sol-Lu on adhesion of human mesangial cells to laminin-10/11. Sol-Lu had little inhibitory effect on its own, but cooperated with anti–integrin β1 antibodies to inhibit adhesion by ∼90%. This indicates that Lu cooperates with integrin α3β1 to mediate mesangial cell adhesion to laminin α5 in the GBM.

Mentions: To further investigate interactions between mesangial cells and the laminin α5 G domain, we turned to in vitro adhesion assays that used primary human and rat mesangial cells and purified laminin preparations. We have not yet been able to isolate laminin trimers containing the chimeric α chains, and G domain preparations were not available, so we chose to use commercially available laminins. First, we compared the abilities of laminin-10/11 (α5β1/2γ1) and laminin-1 (α1β1γ1) to promote adhesion of mesangial cells. Quantitative analysis of both human and rat mesangial cell adhesion to surfaces coated with increasing concentrations of these proteins showed that laminin-10/11 had higher cell adhesion activity than laminin-1, especially at the lower protein concentrations (Fig. 8, A and B) . In addition, the cells spread less well on laminin-1 than they did on laminin-10/11 (unpublished data). These data provide an explanation as to why mesangial cells adhere poorly to GBM containing the α1 G domain (Fig. 5, F and J) but adhere well to normal GBM containing the α5 G domain (Fig. 5, D and I). We believe that these in vitro data using laminin trimers justify our conclusions concerning adhesiveness of G domains because in vivo mesangial cells adhere to wild-type α5 but not to Mr51, which contains α5 domains VI through I. Thus, adhesion to laminin-10/11 trimers is likely mediated primarily by the α5 G domain.


Mesangial cells organize the glomerular capillaries by adhering to the G domain of laminin alpha5 in the glomerular basement membrane.

Kikkawa Y, Virtanen I, Miner JH - J. Cell Biol. (2003)

Analysis of mesangial cell adhesion to laminins in vitro. 96-well plates were coated with increasing concentrations of laminin-1 (closed squares) or laminin-10/11 (open circles) and incubated with mesangial cells at 37°C for 1 h. Human (A) and rat (B) mesangial cells readily attached to laminin-10/11, but were significantly less adherent to laminin-1. (C) Identification of receptors mediating adhesion of mesangial cells to laminin-10/11. Human mesangial cells preincubated with function-blocking antibodies against the indicated integrin subunits were added to laminin-10/11–coated wells. Values are expressed as percentages of the number of cells adhering in the absence of monoclonal antibodies. Each column represents the mean of triplicate assays. Error bars show the standard deviation. Anti–integrin α3 and β1 antibodies partially inhibited the adhesion of human mesangial cells to laminin-10/11, indicating that integrin α3β1 is a primary receptor. (D) The effect of sol-Lu on adhesion of human mesangial cells to laminin-10/11. Sol-Lu had little inhibitory effect on its own, but cooperated with anti–integrin β1 antibodies to inhibit adhesion by ∼90%. This indicates that Lu cooperates with integrin α3β1 to mediate mesangial cell adhesion to laminin α5 in the GBM.
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Related In: Results  -  Collection

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fig8: Analysis of mesangial cell adhesion to laminins in vitro. 96-well plates were coated with increasing concentrations of laminin-1 (closed squares) or laminin-10/11 (open circles) and incubated with mesangial cells at 37°C for 1 h. Human (A) and rat (B) mesangial cells readily attached to laminin-10/11, but were significantly less adherent to laminin-1. (C) Identification of receptors mediating adhesion of mesangial cells to laminin-10/11. Human mesangial cells preincubated with function-blocking antibodies against the indicated integrin subunits were added to laminin-10/11–coated wells. Values are expressed as percentages of the number of cells adhering in the absence of monoclonal antibodies. Each column represents the mean of triplicate assays. Error bars show the standard deviation. Anti–integrin α3 and β1 antibodies partially inhibited the adhesion of human mesangial cells to laminin-10/11, indicating that integrin α3β1 is a primary receptor. (D) The effect of sol-Lu on adhesion of human mesangial cells to laminin-10/11. Sol-Lu had little inhibitory effect on its own, but cooperated with anti–integrin β1 antibodies to inhibit adhesion by ∼90%. This indicates that Lu cooperates with integrin α3β1 to mediate mesangial cell adhesion to laminin α5 in the GBM.
Mentions: To further investigate interactions between mesangial cells and the laminin α5 G domain, we turned to in vitro adhesion assays that used primary human and rat mesangial cells and purified laminin preparations. We have not yet been able to isolate laminin trimers containing the chimeric α chains, and G domain preparations were not available, so we chose to use commercially available laminins. First, we compared the abilities of laminin-10/11 (α5β1/2γ1) and laminin-1 (α1β1γ1) to promote adhesion of mesangial cells. Quantitative analysis of both human and rat mesangial cell adhesion to surfaces coated with increasing concentrations of these proteins showed that laminin-10/11 had higher cell adhesion activity than laminin-1, especially at the lower protein concentrations (Fig. 8, A and B) . In addition, the cells spread less well on laminin-1 than they did on laminin-10/11 (unpublished data). These data provide an explanation as to why mesangial cells adhere poorly to GBM containing the α1 G domain (Fig. 5, F and J) but adhere well to normal GBM containing the α5 G domain (Fig. 5, D and I). We believe that these in vitro data using laminin trimers justify our conclusions concerning adhesiveness of G domains because in vivo mesangial cells adhere to wild-type α5 but not to Mr51, which contains α5 domains VI through I. Thus, adhesion to laminin-10/11 trimers is likely mediated primarily by the α5 G domain.

Bottom Line: In addition, podocytes exhibited their typical arrangement in a single cell layer epithelium adjacent to the GBM, but convolution of glomerular capillaries did not occur.Finally, in vitro studies showed that integrin alpha3beta1 and the Lutheran glycoprotein mediate adhesion of mesangial cells to laminin alpha5.Our results elucidate a mechanism whereby mesangial cells organize the glomerular capillaries by adhering to the G domain of laminin alpha5 in the GBM.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine, Washington University School of Medicine, St Louis, MO 63110, USA.

ABSTRACT
In developing glomeruli, laminin alpha5 replaces laminin alpha1 in the glomerular basement membrane (GBM) at the capillary loop stage, a transition required for glomerulogenesis. To investigate domain-specific functions of laminin alpha5 during glomerulogenesis, we produced transgenic mice that express a chimeric laminin composed of laminin alpha5 domains VI through I fused to the human laminin alpha1 globular (G) domain, designated Mr51. Transgene-derived protein accumulated in many basement membranes, including the developing GBM. When bred onto the Lama5 -/- background, Mr51 supported GBM formation, preventing the breakdown that normally occurs in Lama5 -/- glomeruli. In addition, podocytes exhibited their typical arrangement in a single cell layer epithelium adjacent to the GBM, but convolution of glomerular capillaries did not occur. Instead, capillaries were distended and exhibited a ballooned appearance, a phenotype similar to that observed in the total absence of mesangial cells. However, here the phenotype could be attributed to the lack of mesangial cell adhesion to the GBM, suggesting that the G domain of laminin alpha5 is essential for this adhesion. Analysis of an additional chimeric transgene allowed us to narrow the region of the alpha5 G domain essential for mesangial cell adhesion to alpha5LG3-5. Finally, in vitro studies showed that integrin alpha3beta1 and the Lutheran glycoprotein mediate adhesion of mesangial cells to laminin alpha5. Our results elucidate a mechanism whereby mesangial cells organize the glomerular capillaries by adhering to the G domain of laminin alpha5 in the GBM.

Show MeSH
Related in: MedlinePlus