Heparan sulfate proteoglycans in epithelial morphogenesis and physiology: knock out of Syndecan-1 using CRISPR-Cas9

Abstract

Prostate morphogenesis, as in other branched organs, such as kidney, salivary gland and lung, depends on well regulated epithelial-stromal interactions. The two compartments exchange molecular clues either by ECM remodeling, cell contact or soluble signaling molecules. Some of these molecules, such as growth factors, bind to heparan sulfate proteoglycans (HSPGs), which dictate the dynamics of the communication between the epithelium and stroma, concentrating these factors locally and thereby stimulating or impeding their functions. Prostate development begins by induction of the urogenital sinus epithelium. Testis testosterone down-regulates the expression of sulfatase-1 leading to a "accumulation" of tri-sulfated HS, and interference in this axis hampers prostate induction. In this project we hypothesized that heparanase-1 would participated in this axis given its ability to both to cleave enzymatically HS and then modulate its interaction with growth factors and their cognate receptors, and bind to HSPG to promote aggregation and/or endocytosis, then modulating the kinetics of extracellular signaling molecules. Our results so far showed that heparanase-1 knock down hampers epithelial outgrowth and lumen formation, while HS-sulfation is important for branching and lumen formation. In 3D cell culture of isolated cells from ventral prostate, heparanase-1 seems to be important for lumen formation. Using RWPE-1, a cell line derived from human prostate non-tumor epithelial cells, chlorate-induced sulfation-block severely compromised the formation of organized spheroids. Finally we observed that the expression of syndecan-1 is up-regulated when RWPE-1 is in contact with Matrigel. In order to achieve molecular specificity, we decided to approach this issue by targeting syndecan-1. In Esko's lab, they are interested in the role of HSPG in binding and clearing triglyceride-rich lipoprotein and is using the CRISPR/Cas9 system to inactivate syndecan-1 in hepatoma cells. We will perform experiments to silence syndecan-1 using CRISPR/Cas9 to determine how it is involved in prostate physiology. (AU)