Analysis of cortactin- and HS1/cortactin-deficiency in mice

In general, the use of KO mice has important advantages compared to cellular studies.

On one hand, the deletion of a gene in the whole organism allows examining its role in embryogenesis, brain and organ formation, and behavior all of which require functional cytoskeleton (Erck et al., 2005; Lommel et al., 2001; Rust et al., 2010). On the other

hand, KO mice can be used as source for organs or primary cells, which can be applied to in vitro experiments.

Mice lacking cortactin were viable, fertile and did not show obvious phenotypes in SPF animal housing. This demonstrated that cortactin was not essential during embryogenesis, although contradictory results were obtained using a gene trap approach instead of a conditional KO (Yu et al., 2010). Yu et al. reported that heterozygous matings did not produce KO pups and examination of fertilized oocytes revealed that no KO embryos in the two-cell stage could be observed. Thus, Yu et al.

claimed that cortactin was essential for asymmetric division in oocytes. As cortactin KO mice were born mostly at Mendelian ratio (see below), a role for cortactin in embryonic cell division can be excluded, and the results from Yu et al. indicate a complication in the gene trap approach.

Heterozygous matings of two different mouse populations served as source to analyze the frequency of the different genotypes and sexes in the offspring. In both mouse lines, WT, heterozygous as well as homozygous pups from both sexes were born, but the frequencies differed from Mendelian ratios in some cases. Crossings of heterozygous mice harboring the deleted Neo allele produced more male than female offspring, but male KO mice were underrepresented (Figure 3-10). Interestingly, in heterozygous matings of mice carrying the deleted cortactin allele, more female pups were born, and especially heterozygous females were more frequent than calculated (Figure 3-11). These results demonstrate that although in principle all combinations of genotypes and sexes were generated, still the different allele composition of parental mice biased the frequency of certain combinations in the offspring. The reasons for these differences are unknown, but could be due to the different genetic background of the cell lines or the conditions in the animal husbandry.

In cortactin KO mice, the hematopoietic homolog of cortactin, HS1, was still present. In order to generate a knockout lacking both class II NPFs found in mammals, crossings between mice with the cortactin deleted Neo allele and HS1 KO mice were arranged by Frank Lai. The double KO was also fertile, viable and without apparent phenotype.

However, HS1 KO mice were previously shown to be impaired in antibody production, immunoreceptor-induced proliferation of B and T cells (Taniuchi et al., 1995) and blood coagulation (Kahner et al., 2007). These defects were certainly also present in the double KO mice, but neither was the immune system challenged with pathogens nor were mice wounded, so none of these phenotypes were observed in our animal husbandry. Mating statistics of crossings of mice homozygous for HS1 KO and heterozygous for cortactin KO revealed that these mice were born as expected from

In the literature, cortactin was described as essential component in the formation of podosomes in cell types as various as osteoclasts, smooth muscle cells and v-Src- transformed fibroblasts. Osteoclasts depleted for cortactin neither formed podosomes nor sealing rings, and were defective in bone resorption (Tehrani et al., 2006a).

Podosome formation was also reported to be abolished in smooth-muscle cells after cortactin knockdown (Zhou et al., 2006).

In the present study, peritoneal macrophages isolated from control mice and littermates deleted for cortactin or both cortactin and HS1, respectively, all displayed podosomes with unaltered morphology and typical protein composition (Figure 3-13 and Figure 3-16). The number of cells forming podosomes was also comparable in control and KO macrophages (Figure 3-14 and Figure 3-17). It could be excluded that HS1 was upregulated to compensate for the absence of cortactin, as western blot analysis detected equal amounts of HS1 in both control and cortactin KO macrophages (Figure 3-15). More importantly, also the double KO macrophages lacking both cortactin and HS1 were able to form podosomes. In conclusion, at least in macrophages, podosomes can be assembled in the absence of cortactin. Yet it remains to be answered whether macrophages are the only cell type forming podosomes independently of cortactin. To address this question, osteoclasts and smooth muscle cells from cortactin KO mice should be examined regarding the existence of podosomes, because the essential role for cortactin could be specific for these cell types. However, the impairment in podosome assembly obtained with RNAi-induced knockdown of cortactin could also have other reasons such as off-target effects, as this is one major disadvantage of silencing genes using RNAi (Jackson et al., 2003).

Jackson et al. could demonstrate that direct silencing of non-targeted genes required as few as eleven contiguous nucleotides of identity to the siRNA, so misinterpretation from RNAi experiments can easily occur. Thus, the potential role for cortactin in podosome formation in osteoclasts and smooth-muscle cells should be re-evaluated.

Although podosome formation and recruitment of typical proteins in cortactin and HS1/cortactin KO macrophages was normal, nothing is known about the functionality of podosomes lacking cortactin and/or HS1. In order to cross endothelial barriers, macrophages recruit metalloproteases to podosomes, where they are secreted into the extracellular lumen and decompose the ECM (Linder, 2007). Preliminary results from a collaboration with Christiane Wiesner and Stefan Linder (UKE Hamburg), where macrophages from cortactin KO mice were subjected to matrix degradation assays revealed that upon cortactin depletion macrophages displayed a severe impairment in gelatin degradation (unpublished data). These experiments have to be repeated in the

future to verify whether or not cortactin may indeed play a role in the degradation process of the ECM mediated by podosomes.