We and others have previously shown that the laminin α5 chain is widely expressed in mice (Miner et al., 1995
; Sorokin et al., 1997b
). Based on knockout studies, laminin α5 has roles in several important developmental processes, including neural tube closure, digit septation, placentation, and lung and kidney development (Miner et al., 1998
; Miner and Li, 2000
; Nguyen et al., 2002
). We found that most of the developmental defects are associated with basement membrane breakdown or discontinuity resulting from the absence of α5. Here, we have begun to investigate the role of laminin α5 using a combined transgenic/knockout approach, which effectively substitutes either all or part of the α5 G domain with analogous segments of α1. The result is expression of full-length chimeric laminin α chains capable of trimerizing with β and γ chains and incorporating into basement membranes. A similar strategy was previously used in vitro to map a synaptic basement membrane localization domain on the laminin β2 chain (Martin et al., 1995
). This approach has allowed us to uncover a laminin α5 G domain–specific function in glomerulogenesis that might never have been found through traditional knockouts. It also demonstrates the feasibility of using widely expressed transgenes encoding altered basement membrane proteins to replace existing knocked out genes, effectively generating knockins without the use of further gene targeting in ES cells.
Expression of the Mr51, and presumably Mr5G2, chimeric α chains on the Lama5
−/− background was able to rescue the breakdown of the GBM () that normally occurs in Lama5
−/− glomeruli when laminin α1 is eliminated (Miner and Li, 2000
). The mechanism of α1 elimination is unknown; but if it is not purely transcriptional, then it must somehow be selective for α1 because α5 is not eliminated. It is likely that primary sequence differences or domain structural differences account for the selective elimination of α1. The G domain of α1, present in Mr51, could have carried a signal for elimination, but our results suggest this not to be the case because Mr51 was not eliminated. We are continuing to investigate this interesting issue using additional α chain transgenes.
The major defect in the Lama5
−/−; Mr51 and Lama5
−/−; Mr5G2 embryos was ballooning of the glomerular capillaries. This same defect was observed in mice lacking mesangial cells due to absence of PDGFB/PDGF receptor β signaling (Lindahl et al., 1998
). However, in our case, mesangial cells were clearly present (, and J), so we concluded that they must not be adhering properly to the GBM to maintain capillary looping. As the only known differences between normal and Lama5
−/−; Mr51/Mr5G2 GBMs are the G domain substitutions, and laminin α chain G domains have been shown to harbor recognition sites for numerous cell adhesion receptors (Colognato and Yurchenco, 2000
), we hypothesized that mesangial cells normally adhere to the α5 G domain but were unable to adhere tightly to either the complete α1 G domain or to α1 LG3–5. Our in vitro studies confirmed that both human and rat mesangial cells adhere better to α5-containing laminins than to α1-containing laminin ().
With regard to mechanisms for mesangial adhesion to the α5 G domain, mesangial cells express several β1 integrins, including α1
, and α8
(Gauer et al., 1997
; Sterk et al., 1998
; unpublished observations). Furthermore, immuno-EM studies have shown that β1-containing integrins are concentrated at the mesangial cell surface adjacent to the GBM and the mesangial matrix (Kerjaschki et al., 1989
). Antibody inhibition studies demonstrated that integrin α3
plays a major role in mesangial cell adhesion to laminin-10/11 (). In support of this, the glomerular capillaries of Itga3
−/− kidneys are dilated (Kreidberg et al., 1996
), suggesting a defect in mesangial adhesion to the GBM but the fact that the capillaries are not ballooned suggests that another receptor normally cooperates with α3
and is able to partially compensate in Itga3
−/− mesangial cells. We found that Lu is expressed on mouse mesangial cells and cooperates with β1 integrins to mediate adhesion in vitro (unpublished data; D). The fact that Lu was found to be involved is consistent with the fact that Mr5G2 does not support capillary loop formation in vivo, because we have shown that sol-Lu does not bind Mr5G2 (Kikkawa et al., 2002
mutant mice being generated in our laboratory will allow us to more directly address the function of Lu in glomerulogenesis.
An important issue to consider here is the relationship of mesangial cells with laminins in the mesangium, a non–basement membrane ECM, which mesangial cells secrete and in which they are embedded. Several different laminins are found in the mesangium, including substantial amounts of laminins-1 (α1
), -2 (α2
), and -10 (α5
), but others can be detected at lower levels (Miner, 1999
). It has not been possible to determine the relative levels of these laminins, but one would suspect that, based on our findings, decreased levels of laminin-10 or increased levels of laminin-1, as might occur in disease states, could correlate with reduced adhesion of mesangial cells to the mesangial matrix. On the other hand, the fact that mesangial cells are almost totally surrounded by their matrix may make this issue irrelevant, as weaker adhesion may be tolerated, both in disease and in normal states. This would be in contrast to the relationship of mesangial cells to the GBM, with which they make contact only at the bases of the capillary loops. A more robust adhesion to the GBM may be necessary in this setting of limited contact in order to counteract the force of blood pressure. Therefore, interaction with the G domain of α5, normally the only α chain in the GBM, would ensure a tight adhesion.
Two other laminin mutant mice with kidney defects have been described. In mice lacking laminin β2, the β1 chain compensates and allows an ultrastructurally normal basement membrane to form. However, the glomerular filter fails as a barrier to plasma proteins, and the mice die at 3 wk of age with massive proteinuria (Noakes et al., 1995
). No defects in capillary looping were observed, consistent with the fact that laminin α5, as part of laminin-10 (α5
), is present in the GBM (unpublished observations); thus, mesangial cells should still be capable of binding to the GBM and maintaining capillary looping. On the other hand, mice lacking the binding site for nidogen on laminin γ1 exhibit glomerular capillary aneurysms similar to the ballooned capillaries we have reported here. The aneurysms were associated with GBM discontinuities (Willem et al., 2002
), and we suggest that these GBM defects prevent mesangial cells from adhering and maintaining the integrity of the capillary loops.
is expressed basally on podocytes (Korhonen et al., 1990
; Kreidberg et al., 1996
), yet detachment of podocytes from the GBM in Lama5
−/− Mr51/Mr5G2 glomeruli, as occurred with mesangial cells, was not observed. There are two possibilities to explain this. First, integrin α3
on podocytes may serve primarily as a signal transducing receptor rather than as an anchoring one. Dystroglycan is also expressed on podocytes but not on mesangial cells (unpublished data), and together with Lu, this may be sufficient for adhesion of podocytes to the GBM. Second, podocytes and mesangial cells may be adhering only weakly to the chimeric laminins through integrin α3
. This may be sufficient for long-term adhesion of podocytes to the GBM but not for mesangial cells. Capillary looping was evident in immature Lama5
−/−; Mr51/Mr5G2 glomeruli, but mesangial cell adhesion to the GBM was apparently too weak to counteract the force of blood pressure, leading to de-adhesion and capillary ballooning. In support of this is our finding that adhesion activity of laminin-10/11 (α5
) was stronger than that of laminin-1 (α1
) for both human and rat mesangial cells ().
In conclusion, the laminin α5 chain plays a crucial role in maintaining glomerular capillary loop structure. We mapped the adhesive site in vivo to the LG3–5 modules of the G domain. Adhesion of mesangial cells to the laminin α5 G domain is mediated by integrin α3β1 and Lu. Mesangial cells express contractile proteins and are similar to smooth-muscle cells. Their frequency and extent of contraction in response to vasoactive substances are thought to determine the glomerular filtration rate. It is interesting to speculate that defective interactions between mesangial cells and laminin α5 in the GBM may be a feature of diverse glomerulopathies in the adult kidney.