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In vivo analysis of the Pappalysins in relation to cilia and generation of transgenic models

Research output: Book/anthology/dissertation/reportPh.D. thesisResearch

The insulin-like growth factor (IGF) system comprises the ligands insulin, IGF-I and IGF-II and the associated tyrosine kinase receptors IGF-I receptor (IGFIR), insulin receptor (IR) and the hybrid receptor IGFIR/IR. IGF-II is expressed throughout human lifespan but has not been possible to study much due to experimental difficulties. IGF binding proteins (IGFBP) inhibit IGF-I and -II from associating with their receptors. The pappalysins pregnancy-associated plasma protein-A and -A2 (PAPP-A and PAPP-A2) are capable of liberating IGF from the IGFBPs, thus facilitating IGF signaling. However, stanniocalcin-1 and -2 (STC1 and STC2) inhibit PAPP-A and PAPP-A2 proteolytic activity decreasing local
IGF bioavailability. IGF signaling through the cellular appendage cilia is important in both cell mitogenesis and differentiation. Dysregulation of cilia development can result in the development of ciliopathies such as polycystic kidney disease (PKD). Knockout of PAPPA in PKD model mice has a protective effect reducing number and size of cysts, however the effect of PAPP-A on cilia function has never been studied.
The overall aim of this PhD project was to investigate if PAPP-A and PAPP-A2 influence cilia development and to aid in future IGF system research by the generation of transgenic models. For this purpose, the dissertation is divided into three studies.
Study I aimed to generate a papp-ab knockout zebrafish line. Zebrafish have two
orthologs of PAPP-A and one ortholog of PAPP-A2; Papp-aa, Papp-ab and Papp-a2. Both papp-aa and papp-a2 knockout zebrafish are commercially available but not papp-ab knockout zebrafish. A premature stop codon was introduced in exon 1 of papp-ab by CRISPR/Cas9. The papp-ab knockout zebrafish line was used in study II.
The aim of study II was to investigate the influence of PAPP-A and PAPP-A2 on the development of cilia. Cilia length was reduced in the embryonic kidney of papp-ab and papp-a2, but not papp-aa knockout zebrafish embryos. The reduced cilia length could be rescued by increasing IGF-I or papp-a2, indicating that the regulation of cilia length depends on IGF signaling. Overexpressing Stc1, but not Stc2 reduced pronephric cilia length to the same extent as papp-ab and papp-a2 knockout. This indicates that Stc-1 is capable of regulating cilia length, likely through inhibition of Papp-ab and/or Papp-a2. Overexpression of the pappalysins resulted in the development cysts a symptom of PKD.
The aim of study III was to generate stable IR-A and IR-B cell lines which would aid in the development of a new and sensitive IGF-II kinase receptor activation assay (KIRA) capable of detecting bioavailable IGF-II. The cell lines were established and have been given to our collaborator, Professor Jan Frystyk, who will develop the IGF-II KIRA.
Original languageEnglish
Place of publicationAarhus
PublisherAarhus Universitet
Number of pages158
Publication statusPublished - Aug 2020

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