Taken together, a8 integrin-deficient MCs differed from wild type MCs with regard to morphology, cytoskeletal architecture and proliferative capacity, while a8 integrin-deficient VSMCs did not differ from wild type VSMCs. This is in keeping with our previous in vivo findings suggesting changes in the glomerular mesangium but not in the media of renal arteries in a8 integrin-deficient mice , although in normal mice both structures contain mesenchymal cells expressing a8 integrin. a8 integrin-deficient MCs downregulated expression of a-smooth muscle actin and desmin, but not vimentin, while expression of these mesenchymal proteins was not altered in a8 integrin-deficient VSMCs. a8 integrin-deficient MCs had higher expression levels of integrin chains a1, a2 and a6 compared to wild type MCs. Similar differences were not detected between a8 integrin-deficient and wild type VSMCs. Moreover, increased proliferation rates due to a lack of a8 integrin were only detected in MCs, not in VSMCs.
Several studies show that integrins can contribute to cell differentiation and to the maintenance of the phenotype of the cell via outside-in signaling from the surrounding matrix to the cytoskeleton and small adapter molecules inside the cell [4, 28]. Many integrins use a signaling pathway involving the β1 integrin chain and integrin linked kinase to regulate the cytoskeletal architecture of the cell . Moreover, integrins can alter the organization of the actin cytoskeleton via proteins of the rho family, which also regulate CTGF . CTGF, besides having profibrotic function, can act as a mediator of growth arrest . In MCs, disassembly of actin stress fibers with an inhibitor of rho family proteins resulted in inhibition of CTGF expression . We could show that MCs lacking a8 integrin rearrange their actin cytoskeleton and downregulate CTGF.
Changes in the cytoskeletal architecture can alter cell adhesion and motility . In a previous study, we showed that compared to wild type MCs, a8 integrin-deficient MCs adhered weaker to fibronectin and vitronectin, two ligands for a8 integrin, but adhered more easily on collagens, which are not ligands for a8 integrin . On the other hand, a8 integrin-deficient MCs migrated more easily on fibronectin or vitronectin than wild type cells . These results support the notion that α8 integrin could serve as an anti-migratory integrin, keeping MCs resting at their native location. Firm adhesion, as mediated by α8 integrin, inhibits migration in many cell types . Thus the decreased ability of α8 integrin-deficient MCs to adhere to fibronectin or vitronectin could contribute to the increased ability of these cells to migrate. Given these differences in migratory abilities, we hypothesized in the present study that wild type and α8 integrin-deficient MCs also differ in their cytoskeletal architecture and general morphology.
Downregulation of a-smooth muscle actin expression in a8 integrin-deficient MCs leads to a reduction in a-smooth muscle actin containing stress fibers and consequently to a reduction in firm adhesion. This in turn seems to lead to increased cell motility of a8 integrin-deficient MCs. Similar observations were made in VSMCs after siRNA knockdown of a8 integrin expression : Treatment with a8 integrin siRNA reduced expression of a-smooth muscle actin and increased cell migration, which is in contrast to our findings in a8 integrin-deficient VSMCs, where both the a8 integrin-deficient and the wild type genotype expressed a-smooth muscle actin in comparable amounts. The reasons for the discrepancy of the results of the studies in VSMCs after blockade of a8 integrin expression with siRNA and in a8 integrin-deficient VSMCs are unclear at present. Our results regarding a-smooth muscle actin expression in a8 integrin-deficient MCs are reminiscent of the findings of Zaghram et al.  after siRNA blockade of a8 integrin in VSMCs. We therefore wanted to investigate the differences in a8 integrin-deficient MCs and a8 integrin-deficient VSMCs: A compensatory increase of integrin chains a1, a2 and a6 was detected in a8 integrin-deficient MCs. A similar increase of integrin expression was not found in a8 integrin-deficient VSMCs. Thus, it seems possible that changes in the cytoskeletal architecture and a-smooth muscle actin expression in a8 integrin-deficient MCs is not a direct consequence of the lack of a8 integrin, but more likely due to the induction of other integrin chains. a6 integrin is usually not expressed in MCs, but is an integrin characteristic of epithelial cells, while a8 integrin is a typical mesenchymal integrin [35, 36]. During kidney development, downregulation of a8 integrin, possibly by WT-1 , results in epithelialization of mesenchymal cells and in the formation of tubular structures . For this reason, we tested if a8 integrin-deficient MCs exhibit reduced expression of other mesenchymal markers or increased expression of a typical epithelial marker, widely used in the detection of epithelial-mesenchymal transition [38, 39]. A reduction of desmin expression was readily detected, but vimentin expression was not reduced and E-cadherin expression was very low in a8 integrin-deficient MCs. These findings argue against the hypothesis that lack of a8 integrin, along with increased expression of a1, a2 and a6 integrins, leads to an epithelialization of MCs, but more likely might result in dedifferentiation of MCs. Why a8 integrin-deficient MCs undergo these changes in integrin expression and cytoskeletal architecture, while a8 integrin-deficient VSMCs do not, remains unclear. Discrepancies in the differentiation status might influence the ability of cells to dedifferentiate more easily than others. MCs and VSMCs might also use distinct transcriptional mechanisms, like it was described for smooth muscle cell and myofibroblast a-smooth muscle actin expression . Moreover, no explanation exists to date as to why VSMCs after blockade of a8 integrin with siRNA behave differently from a8 integrin-deficient VSMCs regarding a-smooth muscle actin expression and cytoskeletal rearrangements. As shown by Zargham et al. , blockade of a8 integrin with siRNA results in a dysregulation of the expression of other integrins, like an increased expression of the a2, a5 and av chains, or reduced expression of the a1 chain. In our isolations of a8 integrin-deficient VSMCs we did not observe significant increases in the expression of the a2, a5 and av chains, while the expression of the a1 chain indeed was reduced. One has to be aware that acute blockade of a8 integrin with siRNA in VSMCs might not be consistent with a genetic knockdown of a8, which is more comparable to a chronic deficiency from the time of VSMC differentiation on. As a consequence, many regulatory pathways might differ in the two cell types. Moreover, the findings with blockade of a8 integrin with siRNA was obtained in rat VSMCs , while our data are derived from mouse VSMCs. Species differences might exist with regard to VSMC biology.
Finally, differences in the properties of MCs and VSMCc lacking a8 integrin were detected regarding cell growth. While a8 integrin-deficient MCs had significantly increased proliferation rates on ligands for a8 integrin compared to wild type MCs , wild type and α8 integrin-deficient VSMCs showed a comparable growth response after stimulation. Thus it is conceivable that the cytoskeletal and matrix receptor changes in α8 integrin-deficient MCs may result in changes in proliferative capacities of these cells. Both α2 and α6 integrin chains, which are upregulated in α8 integrin-deficient MCs, can promote cell proliferation [41, 42]. On the other hand, increased proliferation rates in MCs lacking a8 integrin might be a consequence of rho-mediated disruption of actin stress fibers, leading to increased expression of CTGF, which was described to act anti-proliferative .