Regulatory factors on osteoclast formation and function.

It is increasingly recognised that exosomes can contribute to the pathogenesis of bone disease. This collaborative research project will investigate how exosomes regulate bone remodelling under normal and stress conditions. As such the proposal fits well with the BBSRC research priority 3, Bioscience for Health, and in particular the areas of "healthy ageing across the life course" and "the 3Rs in research using animals".

Bone homeostasis reflects the tightly regulated actions of osteocytes (OCY), osteoblasts (OB) and osteoclasts (OC); dysregulation of these systems can lead to bone disease. OB are the bone-forming cells which, when incorporated into bone matrix, can terminally differentiate into OCY. OCY, the most abundant cells in bone, form an extensive communication network that plays a central role in regulating bone turnover. O2 tension and pH have a profound effect on bone cell function with hypoxia and acidosis being implicated in the development of diseases such as osteoporosis. We have shown that hypoxia and acidosis stimulate OC formation and resorptive activity whilst inhibiting bone mineralisation. Moreover, acute hypoxia stimulates vesicular ATP release from OB.

Exosomes are small membrane-bound particles released from cells. They contain a diverse array of signalling molecules (e.g. proteins, miRNAs) and are increasingly viewed as key mechanism by which cells regulate local extracellular signalling. Evidence from different cell types suggests that environmental stress factors like hypoxia have a profound effect on exosome composition and release.This studentship will investigate the hypothesis that "environmental stress affects bone cell function by altering exosome release and composition". The research objectives are:

1. Defining the role of OB and OCY-derived exosomes in the regulation of bone cell function
The first objective of this studentship will be to fully characterise the composition of OB and OCY-derived exosomes under normal conditions. The ability of isolated exosomes to regulate bone cell differentiation, function (i.e. bone formation/resorption) and gene expression will also be examined.

This in vitro project will use primary OB and OC along with IDG-SW3 cells (an OB-to-late-OCY cell line). Experimental approaches that will be used to address the hypothesis include:
- Transmission electron, confocal and real-time fluorescence microscopy to examine exosome release and uptake
- FACS characterisation of isolated exosomes
- RNAseq and bioinformatics analysis of vesicle contents
- Real-time PCR, western blotting and/or ELISA for studying genes of interest
- Established assays for measuring bone formation and resorption

Bone cell co-cultures will also be used to establish if the actions on cell, differentiation and function recapitulate what is seen with isolated exosomes. Using these co-culture systems places the project at the cutting edge of in vitro bone biology techniques. Moreover, they allow bone formation and resorption to be studied without the need for in vivo work.

2. Understanding the effects of hypoxia and acidosis on OB and OCY-derived exosomes
The effect of long-term and acute exposure to hypoxia (2% O2) or acidosis (pH6.9) on exosome-mediated signalling in bone will be studied using the experimental approaches described in objective 1. This work will give valuable insights into how OB and OCY-derived signals are dysregulated under stress conditions.