A genetic basis for the “Adonis” phenotype of low adiposity and strong bones

Abstract

SPECIFIC AIMS The objective of this study was to determine whether mammalian Toll-like receptor 4, the homologue of Toll, could play a role in establishing and/or maintaining the body plan. PRINCIPAL FINDINGS 1. As C3H/HeJ mice with mutant TLR4 age they develop bones with higher mineral content, size, and density than wild-type mice To test whether TLR4 affects bone development, we compared the bone mineral content of mice, which have a loss-of-function (Pro⁷¹²His) mutation in TLR4 (C3H/HeJ), with strain-specific control mice. To assess the mineral content of bones we analyzed whole mice with dual-energy X-ray absorptiometry (DEXA) (Fig. 1 ⤻ ). TLR4 mutant mice had greater bone mineral content (PPPPPPPP Figure 1. Imaging of Toll-like receptor 4 mutant and WT mice. DEXA images of a sample pairing of anesthetized 24-wk-old female C3H mice: one C3H/HeJ mouse harboring a mutation in TLR4 (mutant) and a C3H/HeSnJ mouse (WT). Lines drawn by PIXIMUS software circumscribe calcified tissue (yellow) or all tissue measured (blue). Skulls were excluded for greater accuracy. Download figure Download PowerPoint Figure 2. TLR4 affects the body plan. Drosophila Toll responds to exogenous agonists by inducing host defenses and to strictly endogenous stimuli when influencing the body plan. Mammalian Toll-like receptors respond to exogenous and endogenous agonists by inducing host defenses. Toll-like receptor 4 affects the body plan, most likely through the actions of endogenous agonists. Download figure Download PowerPoint 2. As C57Bl/10ScNCr mice with a deletion of TLR4 age they develop bones with higher mineral content, size, and density than WT mice To determine whether differences in bone in C3H/HeJ mice result from loss of TLR4 function or a novel signaling pathway specific to TLR4 with a Pro⁷¹²His mutation, we asked whether C57Bl/10ScNCr mice, which have a deletion of the entire TLR4 gene, have the same phenotype. TLR4-negative mice had larger bones (PPPPPTLR4^lpsd mice, which have the Pro⁷¹²His mutation but a Balb/c genetic background. C.C3H-TLR4^lpsd mice had larger bones (PP=0.16) (Fig. 2)⤻ . These data in three strains of mice with two different mutations confirm that mice with loss of TLR4 function have larger bones, with more mineral content. 4. All mice continue to gain weight and body fat as they age; TLR4 mutant mice gain less body fat than WT mice Application of stress to bones, such as carrying increased body mass, leads to larger, denser, and stronger bones; thus, obesity is associated with denser bones and reduced risk of fracture in postmenopausal woman. To determine whether mice with mutations in TLR4 have an increased body mass as one explanation for larger bones with more mineral content, we compared the body weight of various mice. All mutant and control strains of mice tested continued to gain weight as they aged, but at varied rates. Mice with a TLR4 mutation in the C3H background were smaller than controls at all ages tested, as measured by DEXA (Fig. 2)⤻ . To determine whether mice with increased bone mineral content in association with TLR4 mutations have increased (or decreased) body fat, we analyzed fat mass and fat-free mass by DEXA and calculated % body fat. Surprisingly, as TLR4 mutant mice in all three strains grew, they gained up to 70% less body fat than controls (PPTLR4^lpsd mice were not studied over time). These data show that differences in body mass do not explain the changes observed in bone. 5. The phenotype of CD14 knockout mice mimicks the bone and fat changes seen in TLR4 mutant mice CD14 is required for signaling of free agonists via TLR4 unless the dose of agonist is very high, and CD14 is not thought to interact directly with TLR4 except perhaps when TLR4 is actively signaling. Although CD14 is required for responses to free agonists of TLR4, CD14 is not required for the activation of TLR4 by whole bacteria, which contain LPS on their surface. We studied B6.129S-Cd14^tm1Frm mice, which have a targeted deletion of CD14 and were backcrossed 20 times onto the C57Bl/6J strain. Although CD14 is a coreceptor for TLR2 and is not required for TLR4 to respond to bacterial infections, the phenotype of...