Loss-of-function mutations in the MEN1 tumor-suppressor gene cause the multiple endocrine neoplasia type 1 syndrome. Menin, the MEN1 gene product, is expressed in many tissues, including bone, where its function remains elusive. We conditionally inactivated menin in mesenchymal stem cells (MSCs) using paired-related homeobox 1 (Prx1)-Cre and compared resultant skeletal phenotypes of Prx1-Cre;Men1 f/f menin-knockout mice (KO) and wild-type controls using in vivo and in vitro experimental approaches and mechanics simulation. Dual-energy X-ray absorptiometry demonstrated significantly reduced bone mineral density, and 3-dimensional micro-CT imaging revealed a decrease in trabecular bone volume, altered trabecular structure, and an increase in trabecular separation in KO mice at 6 and 9 months of age. Numbers of osteoblasts were unaltered, and dynamic histomorphometry demonstrated unaltered bone formation; however, osteoclast number and activity and receptor activator of NF-κB ligand/osteoprotegerin (RANKL/OPG) mRNA profiles were increased, supporting increased osteoclastogenesis and bone resorption. In vitro, proliferative capabilities of bone marrow stem cells and differentiation of osteoblasts and mineralization were unaltered; however, osteoclast generation was increased. Gross femur geometrical alterations observed included significant reductions in length and in mid-metaphyseal cross-sectional area. Atomic force microscopy demonstrated significant decreases in elasticity of both cortical and trabecular bone at the nanoscale, whereas three-point bending tests demonstrated a 30% reduction in bone stiffness; finite element analysis showed morphological changes of the femur microgeometry and a significantly diminished femur flexural rigidity. The biomechanical results demonstrated the detrimental outcome of the accelerated osteoclastic bone resorption. Our studies have a twofold implication; first, MEN1 deletion from MSCs can negatively regulate bone mass and bone biomechanics, and second, the experimental and computational biomechanical analyses employed in the present study should be applicable for improved phenotypic characterization of murine bone. Furthermore, our findings of critical menin function in bone may underpin the more severe skeletal phenotype found in hyperparathyroidism associated with loss-of-function of the MEN1 gene.

Bone has a complex microenvironment formed by an extracellular matrix (ECM) composed mainly of mineralized type I collagen fibres. Bone ECM regulates signaling pathways important in the differentiation of osteoblast-lineage cells, necessary for bone mineralization and in preserving tissue architecture. Compared to conventional 2D cell cultures, 3D in vitro models may better mimic bone ECM and provide an environment to support osteoblastic differentiation. In this study, a biomimetic 3D osteoid-like dense collagen gel model was used to investigate the role of the nuclear protein menin plays in osteoblastic differentiation and matrix mineralization. Previous in vitro and in vivo studies have shown that when expressed at later stages of osteoblastic differentiation, menin modulates osteoblastogenesis and regulates bone mass in adult mice. To investigate the role of menin when expressed at earlier stages of the osteoblastic lineage, conditional knockout mice in which the Men1 gene is specifically deleted early (i.e., at the level of the pluripotent mesenchymal stem cell lineage), where generated and primary calvarial osteoblasts were cultured in plastically compressed dense collagen gels for 21 days. The proliferation, morphology and differentiation of isolated seeded primary calvarial osteoblasts from knockout (Prx1-Cre; Men1f/f) mice were compared to those isolated from wild-type (Men1f/f) mice. Primary calvarial osteoblasts from knockout and wild-type mice did not show differences in terms of proliferation. However, in comparison to wild-type cells, primary osteoblast cells derived from knockout mice demonstrated deficient mineralization capabilities and an altered gene expression profile when cultured in 3D dense collagen gels. In summary, these findings indicate that when expressed at earlier stages of osteoblast differentiation, menin is important in maintaining matrix mineralization in 3D dense collagen gel matrices, in vitro.

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