Development of a Whole Organism Platform for Phenotype-Based Analysis of IGF1R-PI3K-Akt-Tor Action

Abstract

Aberrant regulation of the insulin-like growth factor (IGF)/insulin (IIS)-PI3K-AKT-TOR signaling pathway is linked to major human diseases, and key components of this pathway are targets for therapeutic intervention. Current assays are molecular target- or cell culture-based platforms. Due to the great in vivo complexities inherited in this pathway, there is an unmet need for whole organism based assays. Here we report the development of a zebrafish transgenic line, Tg(igfbp5a:GFP), which faithfully reports the mitotic action of IGF1R-PI3K-Akt-Tor signaling in epithelial cells in real-time. This platform is well suited for high-throughput assays and real-time cell cycle analysis. Using this platform, the dynamics of epithelial cell proliferation in response to low [Ca2+] stress and the distinct roles of Torc1 and Torc2 were elucidated. The availability of Tg(igfbp5a:GFP) line provides a whole organism platform for phenotype-based discovery of novel players and inhibitors in the IIS-PI3K-Akt-Tor signaling pathway.

The metalloproteinase Papp-aa functions as a molecular switch linking IGF signaling to adaptive epithelial growth

“…Since low [Ca 2+ ] stress treatment did not change papp-aa mRNA levels (this study) or igfbp5 mRNA levels (Liu et al, 2017) in wild-type fish, and because disruption of the Igfbp/IGF complex was sufficient to activate IGF signaling and induce NaR cell reactivation under normal [Ca 2+ ] conditions, we speculate that a [Ca 2+ ]-regulated post-transcriptional mechanism(s) exists that suppresses Papp-aa proteolytic activity under normal [Ca 2+ ] conditions. Human PAPP-A contains three Lin-12/Notch repeat (LNR) modules (Boldt et al, 2004).…”

“…Because NaR cells are located on the yolk sac skin, they are more sensitive to permeabilization by protease K treatment, a key step in the whole mount in situ hybridization procedure. Thus, to assess pappalysin expression, NaR cells were isolated by FACS from Tg(igfbp5a:GFP) fish, in which NaR cells are labeled by GFP expression (Liu et al, 2017) and analyzed. The mRNA levels of papp-aa were 2-fold higher than those of papp-ab and papp-a2 (Fig.…”

“…0.003% (w/v) Nphenylthiourea (PTU) was added to the media to inhibit pigmentation. Tg(igfbp5a:GFP) fish were generated in a previous study (Liu et al, 2017). The pappaa p170/+ fish were obtained from the Marc Wolman lab.…”

“…We have recently found that genetic deletion of the major Papp-a/Papp-a2 substrate IGF binding protein 5a (Igfbp5a) resulted in greatly reduced calcified bone mass even though igfbp5a is not expressed in skeletal tissues in zebrafish (Liu et al, 2018). Zebrafish igfbp5a is specifically expressed in a population Ca 2+ -transporting epithelial cells located in the yolk sac epidermis Liu et al, 2017). These cells, known as ionocytes or NaR cells, are functionally similar to human intestinal epithelial cells (Hwang, 2009;Lin and Hwang, 2016).…”

“…NaR cells take up Ca 2+ from the surrounding aquatic habitats to maintain body Ca 2+ homeostasis. These cells, normally non-dividing and quiescent, rapidly exit the quiescent state and proliferate in response to low [Ca 2+ ] stress Liu et al, 2017). Genetic deletion of igfbp5a blunted the low [Ca 2+ ] stress-induced IGF signaling and NaR cell proliferation (Liu et al, 2018).…”