SOX17, EGR-1 and KLF4 are supposed to bind to each other in human

All previous results we had so far led us to the hypothesis that all three factors might interact and therefore bind to each other in human vascular smooth muscle cells. To prove this, immuno- and co-immunoprecipitations were performed. Therefore cultured and proliferating coronary artery smooth muscle cells were used. The cells were scraped, washed twice with 1X PBS and afterwards resuspended in lysis buffer, containing 1% NP-40. After incubation on ice (20 min), the cells were sonicated and the resulting cell fragments were pelleted by centrifugation. The clear supernatant was incubated overnight at 4°C with the primary antibody and A-Sepharose. Next day, the lysates were washed five times with lysis buffer, the pellet was resuspended in loading dye (containing 0.1M DTT) and boiled at 95°C for 10 min. At least Western-Blots were done.

All three proteins could be immunoprecipitated from the human CASMCs, as well as c-myc, which was chosen as control for a non-binding protein. As isotype control, 3F10, an antibody, directed against a Hemaglutinin tag, was used. All samples were checked on one gel for SOX17 expression using a SOX17 specific primary antibody from SantaCruz. As one can see in figure 4.18, SOX17 protein is detectable in the SOX17, EGR-1 and KLF4 precipitated samples. In contrast to this the controls are negative for SOX17.

Figure 4.18 SOX17 is detectable in EGR-1 and KLF4 precipitated samples of human coronary artery smooth muscle cells in vivo. _{The lysates of the immunoprecipitations of EGR-1, KLF4,} SOX17, 3F10 and c-myc, as control for a non-SOX17-binding protein, were loaded on a 12.5 % SDS gel. The blot was analysed with a SOX17 specific primary antibody (goat, polyclonal; SantaCruz). Except of the two controls (IgG and c-myc), SOX17 is detectable in all three samples (lane 1 - 3).

For the precipitation of the proteins, the same concentration of antibody was used (1 µg/ml). Therefore it seems surprising that the amount of SOX17 detected in the EGR-1 precipitated sample is comparable to the amount of protein in the SOX17 precipitated sample. As it is unreasonable that the whole pool of SOX17 protein in the cell is bound specifically to EGR-1, this observation might be due to different precipitation capacities of the antibodies. At least there might also be differences in the amount of whole protein for the reason of a loss of protein during the precipitation protocol.

Nevertheless, the observations made in this co-immunoprecipitation lead us to the conclusion that all three transcription factors might specifically bind to each other in human CASMCs, building a stable protein complex. This complex could act as activator of proliferation associated genes and repress the expression of smooth muscle differentiation marker genes, as it was already shown for KLF4 (Dandre et al., 2004; Kawai-Kowase and Owens, 2006; Yoshida et al., 2006; Holycross et al., 1992). Of course these results would still have to be confirmed in further analysis, using different controls, checking for all antibody subtypes, as the used antibodies for EGR- 1, KLF4 and SOX17 are polyclonal. Moreover, one could perform the co- immunoprecipitations also the other way round, meaning a western-blot of EGR-1 on SOX17 and KLF4 precipitated lysates and a western-blot of KLF4 on SOX17 and EGR-1 precipitated samples. If these Western-Blots would show the same result, one could at least conclude that all three factors bind specifcally to each other in vascular SMCs, and as this complex is precipitated under proliferative conditions, indicate an involvement of the three interacting factors in promoting angiogenic processes, e.g. in pathological conditions, like atherosclerosis.

4.2. The FunGenES project

FunGenES (Functional Genomics in Engineered ES-cells) is a consortium whose goal is the identification and characterization of organospecific markers during embryonic development. Another objective is the development of new cellular and molecular tools to characterize gene function and to develop new ES-cell derived methods for high throughput screening of the toxicity of small molecules that are candidates for therapeutic interventions. The results could enable future therapeutic strategies for repair or regeneration of damaged or diseased organs, which might be

an alternative to organ transplantations. Moreover, the use of ES-cell derived screens could replace the use of animals in drug screening.

As endothelial cells and vascular smooth muscle cells derive mainly from the mesoderm, our goal was the identification and characterization of transcription factors involved in the differentiation of this lineage.

4.2.1. Differentiation of CGR8 ES-cells

The cultured mouse embryonic stem (ES) cells originate from the inner cell mass of the blastocyst of a day 3.5 old embryo. These cells are pluripotent, being able to differentiate in all three germ layers, comprising endoderm, ectoderm and mesoderm. As we are interested in vascular cells, like endothelial cells and vascular smooth muscle cells, we concentrated on the mesodermal lineage and the factors which are specific for mesoderm development, like e.g. Flk-1 (Shalaby et al., 1995). The differentiation of ES-cells mimics the embryonic development and it was shown that the in vivo expression of different cell lineage markers follows a comparable pattern to the in vitro differentiation. Thereby the ES-cell differentiation assay is a good model to examine the factors that are responsible for the cell lineage specification.

Figure 4.19 shows an overview of the differentiation protocol that was used in this study. The trypsinized cells were centrifuged and resuspended in differentiation medium, containing 20% FCS (Foetal Calf Serum), to a final concentration of 2.4 x 104. Afterwards the cells were placed in a drop-wise manner on the inner side of the lid of the plate (A). One drop of 20 µl contained around 500 cells. The cells stay like this for two days, accumulating at the bottom of the drop and starting differentiation (B). Afterwards the resulting embryoid bodies (EBs) were spilled in 10 cm bacteriological plates, containing 10 ml differentiation medium (C). The differentiating EBs stayed in suspension and grew for two more days. For further assays, in which a calculated number of cells/EBs is necessary, the EBs were then plated on e.g. 24- well plates (D). Here, the cells differentiated for 7 - 10 days and were analyzed afterwards, e.g. by immunofluorescence stainings or RT-PCR.

Figure 4.19 Differentiation of ES-cells (CGR8) using the hanging drop method. (A) Undifferentiated CGR8 embryonic stem cells (of a 3.5 day old embryo) were, up to a confluence of 70 - 80%, cultured on gelatin-coated plates. The cells were trypsinized, washed with 1X PBS and resuspended in 20% FCS containing differentiation medium to a final concentration of 2.4 x 104. The cells were placed in drops on the top of a 10 cm plate, each drop containing around 500 cells. Two days later the resulting embryoid bodies (B) were transferred in 10 cm bacteriological plates, containing 10 ml differentiation medium, where the EBs grow for around 5 - 7 days. The EBs were then plated in 24 well plates, growing for a few more days (3 - 6 days). The generated EBs differentiated in all three germ layers, mesoderm, endoderm and ectoderm.

Figure 4.20 (A) shows undifferentiated ES-cells, growing on a gelatin-coated plate. For the differentiation, the cells have to reach a confluence of around 70 - 80%. Figure 4.20 (B) shows 4 day old EBs cultured in differentiation medium. (C) and (D) are immunofluorescence stainings of Pecam-1 (C) and smooth muscle actin (D).

Figure 4.20 Differentiation of ES-cells (CGR8) by the hanging drop method. (A) Undifferentiated CGR8 embryonic stem cells with a confluence of 60 - 70%, cultured on gelatin-coated plates. (B) 4 day old EBs, generated by cell-suspensions, cultured in 10 cm bacteriological plates, containing 10 ml differentiation medium. At day 14 of embryonic development, the EBs, plated in 24 well plates, were stained by immunofluorescence with Pecam-1, detecting endothelial cells (C) and with smooth muscle actin, as cell-specific marker for vascular SMCs (D).

In this differentiation protocol, no cell lineage specific supplements were used so all various cell types can grow. At least it could be proven that the generation of mesoderm-deriving cell-lineages, in this case meaning endothelial and vascular smooth mucle cells, using the “hanging-drop” method, is possible. Using specific growth factors for the cultivation of the differentiating ES-cells, like VEGF or FGF, one could further on determine the differentiation of the ES-cells into endothelial specific lineages.