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- Looman, Camilla, et al.
(författare)
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An activating mutation in the PDGF receptor-beta causes abnormal morphology in the mouse placenta
- 2007
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Ingår i: International Journal of Developmental Biology. - : UPV/EHU Press. - 0214-6282 .- 1696-3547. ; 51:5, s. 361-370
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Tidskriftsartikel (refereegranskat)abstract
- An oncogenic D842V mutation in the platelet-derived growth factor (PDGF) alpha-receptor (Pdgfra) has recently been described in patients with gastrointestinal stromal tumors. In order to test if the same mutation would confer oncogenic properties to the homologous PDGF beta-receptor (Pdgfrb), the corresponding aspartic acid residue at position 849 of Pdgfrb was changed into valine (D849V) using a knock-in strategy. This mutation turned out to be dominantly lethal and caused death even in chimeras (from 345 transferred chimeric blastocysts, no living coat chimeras were detected). Experiments employing mouse embryonic fibroblasts (MEFs) indicated hyperactivity of the mutant receptor. The mutant receptor was phosphorylated in a ligand-independent manner and, in contrast to wild-type MEFs, mutant cells proliferated even in the absence of ligand. Knockout experiments have previously indicated a role for Pdgfrb in placental development. We therefore analyzed wild-type and Pdgfrb D849V chimeric placentas from different gestational stages. No differences were detected at embryonic days 11.5 and 13.5 (n=4). At embryonic day 17.5, however, chimeric placentas (n=3/4) displayed abnormalities both in the labyrinth and in the chorionic plate. The changes included hyper-proliferation of alpha-smooth muscle actin and platelet/endothelial cell adhesion molecule-1 positive cells in the labyrinth and cells in the chorionic plate. In addition, the fetal blood vessel compartment of the labyrinth was completely disorganized.
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- Looman, Camilla, 1977-
(författare)
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The ABC of KRAB zinc finger proteins
- 2003
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- All living organisms consist of cells and the identity of a cell is defined by the genes it expresses. To assure proper function, a cell receives continuous information on which genes to turn on and off. This information is, to a large extent, provided by transcription factors. Krüppel-related zinc finger proteins probably constitute the largest family of transcription factors in mammals and many of these proteins carry a potent repressor domain called Krüppel-associated box (KRAB). The human genome alone encodes more than 200 KRAB zinc finger proteins but still very little is known about their biological functions. The Krüppel-related zinc finger genes appear to have been involved in a massive expansion throughout evolution. To unravel some of the secrets underlying this evolutionary success, we studied the molecular evolution of KRAB zinc finger genes. We show that the frequently occurring duplications of these genes are accompanied by a low sequence constraint in their zinc finger region. In addition, we show that the number of zinc finger motifs carried within these proteins is far from fixed. New zinc finger motifs are frequently added while others are inactivated or even discarded from the coding region. The structurally independent Krüppel zinc finger motif has, through these mechanisms, served as a highly adaptive building block for the generation of new transcriptional regulators. The mouse, rat and human genomes carry four different variants of the KRAB domain – KRAB(AB), KRAB(Ab), KRAB(AC) and KRAB(A). This thesis presents the identification of a novel KRAB domain, KRAB C, as well as a functional analysis of the different KRAB domains. We conclude that all different KRAB domains share a common co-repressor, TIFβ, and effectively repress transcription. These functions are mainly mediated by the KRAB A box but are clearly influenced by the presence of a KRAB B, b or C box. Furthermore, we show that all KRAB zinc finger gene subfamilies originate from the KRAB(AB) zinc finger genes.In addition, this thesis includes a structural and functional analysis of four novel mouse and human KRAB zinc finger genes; MZF6D, HKr18, HKr19 and HZF12. Whereas HKr18 and HZF12 seem to be ubiquitously expressed, MZF6D and HKr19 show a more restricted expression pattern. Northern blot and in situ hybridisation analyses of MZF6D showed that the expression of this gene is restricted to meiotic germ cells. MZF6D might thus be involved in the formation of male gametes. The expression of HKr19, on the other hand, seems to be restricted to lymphoid cells, indicating a possible role for this KRAB zinc finger gene in the regulation of lineage commitment.
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- Magnusson, Peetra U., et al.
(författare)
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Platelet-derived growth factor receptor-beta constitutive activity promotes angiogenesis in vivo and in vitro
- 2007
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Ingår i: Arteriosclerosis, Thrombosis and Vascular Biology. - 1079-5642 .- 1524-4636. ; 27:10, s. 2142-2149
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Tidskriftsartikel (refereegranskat)abstract
- OBJECTIVE - Knockout studies have demonstrated crucial roles for the platelet-derived growth factor-B and its cognate receptor, platelet-derived growth factor receptor-β (PDGFR-β), in blood vessel maturation, that is, the coverage of newly formed vessels with mural cells/pericytes. This study describes the consequences of a constitutively activating mutation of the PDGFR-β (Pdgfrb) introduced into embryonic stem cells with respect to vasculogenesis/angiogenesis in vitro and in vivo. METHODS AND RESULTS - Embryonic stem cells were induced to either form teratomas in vivo or embryoid bodies, an in vitro model for mouse embryogenesis. Western blotting studies on embryoid bodies showed that expression of a single allele of the mutant Pdgfrb led to increased levels of PDGFR-β tyrosine phosphorylation and augmented downstream signal transduction. This was accompanied by enhanced vascular development, followed by exaggerated angiogenic sprouting with abundant pericyte coating as shown by immunohistochemistry/immunofluorescence. Pdgfrb embryoid bodies were characterized by increased expression of vascular endothelial growth factor (VEGF)-A and VEGF receptor-2; neutralizing antibodies against VEGF-A/VEGF receptor-2 blocked vasculogenesis and angiogenesis in mutant embryoid bodies. Moreover, Pdgfrb embryonic stem cell-derived teratomas in nude mice were more densely vascularized than wild-type teratomas. CONCLUSION - Increased PDGFR-β kinase activity is associated with elevated expression of VEGF-A and VEGF receptor-2, acting directly on endothelial cells and resulting in increased vessel formation.
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