Targeting the novel CD248-MMRN2-CLEC14A pathway in age-related bone loss

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Neag, Georgiana ORCID: https://orcid.org/0000-0002-3087-867X (2022). Targeting the novel CD248-MMRN2-CLEC14A pathway in age-related bone loss. University of Birmingham. Ph.D.

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Abstract

Resolving the mechanisms underlying bone formation would represent an important advancement to our fundamental understanding of bone homeostasis. We have recently identified a novel interaction between three proteins all with established angiomodulatory proprieties. Namely, we showed that the extracellular matrix protein MMRN2 is a shared ligand by the C-type lectin domain 14 family members CLEC14A and CD248 (1). Here, we explore the role of this interaction in regulation of trabecular bone formation during bone growth. H-type capillaries are a newly identified bone vessel subtype localised in the metaphysis, an anatomically distinct region of the long bone metaphysis where trabecular bone is formed (2). These vessels regulate osteoprogenitor migration to the metaphysis and their subsequent maturation by acting as an architectural and nourishing vascular meshwork. Unfortunately, in spite of the importance of H-type EC-osteoblast cross talk for the migration and maturation of osteoblasts, our understanding of the molecular constituents driving this bidirectional interaction are not yet fully understood.
Using high-resolution confocal microscopy, we imaged murine tibia sections labelled with immunofluorescent antibodies against endothelial cells and immature osteoblasts. Image analysis showed that that genetic deletion CLEC14A results in no alteration of H-type capillaries density or morphology, however, it does lead to shortening of the H-type vascular front in juvenile mice, indicating skeletal maturation-dependent plateauing of H-type vasculature development. In addition, imaging of CLEC14A-/- tibia sections uncovered changes in osteoblast angiotropism, marked by variation in the anatomical distribution of osteoblasts and type I collagen in the murine tibia. Micro–computed tomography and bone length analysis of CLEC14A-/- tibiae revealed increased tibia lengths and elevated trabecular bone parameters and an elongation of the trabecular bone front in CLEC14A-/- mice in a skeleton maturity stage-dependent fashion. Bulk RNA sequencing of the CLEC14A-/- neonatal pup calvaria revealed enrichment of genes in gene sets related to endochondral ossification and osteoblast maturation in addition to downregulation of mRNA expression of tight junction, extracellular matrix receptor interaction and cell adhesion molecules. In vitro culturing of CLEC14A-/- isolated osteoblasts confirmed a change in the status of osteoblast activity in response to induction of mineralisation stimulation. CLEC14A-/- osteoblasts produced more alkaline phosphatase and a higher density of bone mineral nodules in response to osteoinductive treatment. Blockade of the CLEC14A-MMRN2 interactions during bone development or in a disease model of ovariectomy-induced osteoporosis, did not phenocopy the genetic deletion of CLEC14A, indicating that perhaps the role of CLEC14A in bone formation is mediated by interactions with binding partners other than MMRN2. Data presented here provides evidence that CLEC14A plays an important role in regulating signalling in the bone during early postnatal bone development by supporting osteoblast maturation and mineralisation activity. Further research into the molecular mechanisms regulated by CLEC14A and its potential therapeutic translation is therefore warranted.

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

Supervisor(s):