This project had two main aims: to identify novel interactors of WWP2 and to determine their role in the process of chondrogenesis. The interactors were identified in the first year of the project. During the second (final) year we investigated the impact of WWP2 and its interactors on cartilage and skeletal development. The work was performed at the National Institute of Health in the USA and at Newcastle University in the UK, respectively.
A significant amount of time was allocated to generating tools necessary for the project to progress. Using these tools, we identified several potential WWP2 interactors/substrates. The proteins were identified by co-immunoprecipitations (Co-IPs) using the catalytically inactive WWP2 in combination with mass spectrometry. Further, several possible WWP2 substrates were identified in cells depleted of WWP2 (using siRNA) and in cells over-expressing tetracycline-inducible, active WWP2. The proteins were identified using quantitative mass spectrometry.
Five of the identified proteins were confirmed as WWP2 interactors in co-IPs and/or in-vitro binding assays. In-vitro ubiquitination assays indicate that four of the proteins are ubiquitinated by WWP2.
These five WWP2 interactors were investigated for their impact on the chondrogenesis in human mesenchymal stem cells (hMSCs). WWP2 and the interactors were depleted in hMSCs using siRNA prior to the induction of the chondrogenesis. The chondrogenesis process, resulting in cartilage pellets formation, takes 14 days to complete in our experimental conditions and most of the cartilage-specific genes peak in expression at day 7. Using real-time RT-PCR we determined the expression of the cartilage-specific genes (e.g. collagen2, aggrecan, collagen10, and Sox9). Deletion of WWP2 resulted in impairment of the chondrogenesis process. Moreover, deletion of one of the investigated interactors also had a negative impact, and deletion of another had a positive impact on the cartilage-specific gene expression and cartilage pellet formation.
In addition, we generated a novel transgenic mouse, Wwp2-C-KO, which lacks the first exon of the cartilage specific isoform of Wwp2 using CRISPR/Cas9. We performed RNA-sequencing in costal chondrocytes isolated from ribs of 7 days old mice. Deletion of the cartilage-specific isoform resulted in subtle changes at the level of gene expression.
We also evaluated the skeletal development in Wwp2-C-KO mice. We performed bone measurements, quantitative computed tomography (-QCT) scans of the skulls and histological staining on the legs; H&E to visualise tissue structure and cellular organization, and safranin ‘O’ to detect cartilage proteoglycans. Wwp2-C-KO mice showed differences in skull shape morphology along with delayed endochondral ossification.
Aspects of these results have been presented at two international meetings the 9th Cold Spring Harbor meeting on The Ubiquitin Family, Cold Spring Harbor, New York, USA; and at FASEB Science Research Conferences on Ubiquitin and Cellular Regulation, Snowmass, Colorado, USA.
Currently, we are drafting a manuscript for a scientific publication from the obtained data. In addition, we secured funding for 12 months, which will allow as to publish and apply for more funding in the future. In conclusion, our main goals were accomplished.
