E-Poster Presentation 30th Australian and New Zealand Bone and Mineral Society Annual Scientific Meeting 2020

Unexpected skeletal phenotype in a mesenchymal-targeted Calcitonin-like receptor (CLR) knockout model (#82)

Natalie K Wee 1 2 , Sanja Novak 2 , Ivo Kalajzic 2
  1. Bone Cell Biology and Disease, St Vincent's Institute of Medical Research, Fitzroy, Victoria, Australia
  2. Reconstructive Sciences, University of Connecticut Health Center, Farmington, Connecticut, USA

The calcitonin peptide family exerts a range of effects on bone cells. Calcitonin gene-related peptide (CGRP), adrenomedullin and intermedin promote osteoblast proliferation and differentiation through actions mediated by Calcitonin receptor-like receptor (CLR; gene: Calcrl) and Receptor Activity-Modifying proteins (RAMPs; genes: Ramp1,2,3). CGRP is a signal in pain transmission and is released from nerves at sites of trauma. While CGRP stimulates osteoblast proliferation and differentiation in vitro, there is limited evidence that endogenous CGRP directly regulates the skeleton. The periosteum, outer surface of bone, is richly innervated with CGRP+ nerves, therefore CGRP may regulate periosteal bone apposition. Our aims were to: 1) evaluate how periosteal cells respond to CGRP and 2) deplete CGRP-CLR signalling in mesenchymal cells.

We identified CGRP receptor components, Calcrl and Ramp1, were expressed in periosteal cells, with Ramp1 expression increased 7-fold during osteoblastic differentiation in vitro. CGRP treatment of periosteal cells led to cAMP activation, increased cell number by 20% and increased mineralisation by 40%. We also generated a mesenchymal-specific constitutive deletion model of CLR using Prxcre. Although CLR deletion was expected to reduce bone formation, we observed significantly 27% higher trabecular bone mass in 10-week-old male mice (Prxcre+,Calcrlfl/fl) compared to Cre- littermates. Cortical thickness and maximum moment of inertia were significantly greater (10% and 16% respectively) in these mice compared to controls, suggesting greater bone formation on the periosteal surface.

These findings in vivo using Prxcre-induced CLR deletion differ from known CGRP effects on mesenchymal cells and appear more in line with observations from the calcitonin/CGRP double knockout and calcitonin receptor knockout models that each have increased bone formation. Targeting CLR deletion using a constitutive promoter (Prxcre) that is broadly expressed in multiple lineages within bone makes the model difficult to interpret. Further investigation of CLR cell specificity and signalling may uncover novel mechanisms that increase bone mass.