Characterising the therapeutic potential of PEPITEMin age-related bone loss, skeletal remodelling and repair

Lewis, Jonathan (2022). Characterising the therapeutic potential of PEPITEMin age-related bone loss, skeletal remodelling and repair. University of Birmingham. Ph.D.

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Abstract

Bone is a constantly active organ, undergoing osteoclast-led bone resorption and osteoblast-induced bone formation. In homeostatic conditions, levels of resorption and formation are equivalent, ensuring there is no net change in bone mass. However, in osteoporosis there is an imbalance, leading to increased resorption compared to formation, ultimately increasing the risk of fracture. Currently, the main treatments include parathyroid hormone (targeting osteoblasts) and bisphosphonates (targeting osteoclasts), however there are issues with non-responders, patient compliance and side-effects. Therefore, there is a need for new therapeutics to boost bone growth, preventing initial bone loss and quickening fracture repair. We have previously identified a novel, endogenous peptide known as PEPITEM which, through the release of S1P, has anti-inflammatory actions.

In this thesis, we aimed to explore the response of bone to PEPITEM, looking at homeostatic bone, diseased bone and the direct action on osteoblasts and osteoclasts. We hypothesised that PEPITEM would act directly on bone, decreasing osteoclastogenesis and resorption, and enhancing osteogenesis and mineralisation.

We found increased tibiae and vertebrae trabecular parameters in murine bone following daily treatment with PEPITEM, indicating improved bone growth. The bioactivity of PEPITEM was maintained in disease: using ovariectomised mice as a model of rapid bone loss, PEPITEM therapy protected from bone loss. Overall, these data suggest a role for PEPITEM as pro-anabolic agent with therapeutic potential.

In vitro, we observed murine and human osteoblasts increased their anabolic potential in response to PEPITEM treatment, leading to enhanced alkaline phosphatase activity and mineral production. Whilst there was no direct effect of PEPITEM on cultured osteoclasts, the number of osteoclasts on the bone surface decreased in PEPITEM treated mice, and osteoclastogenesis was reduced in vitro by molecules released from osteoblasts treated with PEPITEM. This indicates the ability for PEPITEM to induce crosstalk between osteoblasts and osteoclasts, leading to dual actions of decreased resorption and increased formation on the bone.

The molecular mechanism responsible for eliciting the bioactivity of PEPITEM in osteoblasts was also investigated, revealing a possible role for sphingosine-1-phosphate (S1P). S1P treatment increased osteogenesis and decreased osteoclastogenesis in human cells in vitro, but conversely had the inverse effects on murine cells (decreased osteoblast mineralisation and increased osteoclast numbers). Additionally, we identified for the first-time that the expression of cadherin-15 protein in osteoblasts localised at sites of adherens junctions. Protein expression was maintained in calvarial osteoblasts following knockdown of gene expression using siRNA, revealing high levels of protein storage and recycling. By contrast, siRNA knockdown of cadherin-15 in human osteoblasts led to decreased osteogenic potential of osteoblasts, suggesting an essential role in osteogenesis.

In conclusion, we have identified a novel, endogenous peptide which has dual action on osteoblasts and osteoclasts in vivo and in vitro, leading to increased bone growth in health and disease. PEPITEM therefore has potential to be used as a therapeutic in musculoskeletal diseases involving bone loss to induce bone repair processes. However, further work is required to understand the mechanism that controls its bioactivity.

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

Supervisor(s):