Greater Bone Formation of Y2 Knockout Mice Is Associated with Increased Osteoprogenitor Numbers and Altered Y1 Receptor Expression

Pernilla Lundberg,Nathalie Brouard,Amanda Sainsbury,Paul Simmons,Meriem Lamghari,Herbert Herzog,Edith M. Gardiner,Susan J. Allison,Stephanie Rost,Paul A. Baldock,Nicola J. Lee,Ronaldo F. Enriquez,John A. Eisman

doi:10.1074/jbc.m609629200

Abstract

Germ line or hypothalamus-specific deletion of Y2 receptors in mice results in a doubling of trabecular bone volume. However, the specific mechanism by which deletion of Y2 receptors increases bone mass has not yet been identified. Here we show that cultured adherent bone marrow stromal cells from Y2(-/-) mice also demonstrate increased mineralization in vitro. Isolation of two populations of progenitor cell types, an immature mesenchymal stem cell population and a more highly differentiated population of progenitor cells, revealed a greater number of the progenitor cells within the bone of Y2(-/-) mice. Analysis of Y receptor transcripts in cultured stromal cells from wild-type mice revealed high levels of Y1 but not Y2, Y4, Y5, or y6 receptor mRNA. Interestingly, germ line Y2 receptor deletion causes Y1 receptor down-regulation in stromal cells and bone tissue possibly due to the lack of feedback inhibition of NPY release and subsequent overstimulation of Y1 receptors. Furthermore, deletion of Y1 receptors resulted in increased bone mineral density in mice. Together, these findings indicate that the greater number of mesenchymal progenitors and the altered Y1 receptor expression within bone cells in the absence of Y2 receptors are a likely mechanism for the greater bone mineralization in vivo and in vitro, opening up potential new treatment avenues for osteoporosis.

Highlights

The control of bone remodeling has been traditionally thought to be regulated primarily by endocrine systems and by locally acting factors such as cytokines and growth factors
The presence of vasoactive intestinal peptide receptors has been demonstrated on both these cell types and vasoactive intestinal peptide has been shown to regulate the activity of both osteoblasts and ostesorting; colony forming units (CFUs), colony forming unit; PBS, phosphate-buffered saline; MACS, magnetic activated cell sorting; Bone mineral content (BMC), bone mineral content; BMD, bone mineral density
Selection based on 5-flurouracil resistance and fluorescence-activated cell sorting (FACS) using wheat germ agglutinin and stem cell antigen-1 (Sca-1) was used for the identification of a candidate osteoprogenitor cell population in mouse bone marrow [28]

Summary

Introduction

The control of bone remodeling has been traditionally thought to be regulated primarily by endocrine systems and by locally acting factors such as cytokines and growth factors. Culturing and Colony Formation of Sorted MSC and Progenitor Cells—Sca-1ϩ and Sca-1ϪCD51ϩ cells from wild-type and Y2Ϫ/Ϫ mice were plated at a density of 1000 cells/well in 9.6cm2 plates (ϳ100 cells/cm2, BD Biosciences Labware) and cultured in control media (see section above) containing 20% fetal bovine serum, initially in 5% O2/10% CO2/85% N2 (Air Liquide, Melbourne, Australia) at 37 °C for 3 days, changed into regular culture conditions at 37 °C and 5% CO2.

Results

Conclusion