Sphingosine-1-phosphate promotes osteogenesis by stimulating osteoblast growth and neovascularization in a vascular endothelial growth factor-dependent manner.

Sphingosine-1-phosphate (S1P) plays multiple roles in bone metabolism and regeneration. Here, we have identified a novel S1P-regulated osteoanabolic mechanism functionally connecting osteoblasts (OBs) to the highly specialized bone vasculature. We demonstrate that S1P/S1PR3 signaling in OBs stimulates vascular endothelial growth factor a (VEGFa) expression and secretion to promote bone growth in an autocrine and boost osteogenic H-type differentiation of bone marrow endothelial cells in a paracrine manner. VEGFa-neutralizing antibodies and VEGF receptor inhibition by axitinib abrogated OB growth in vitro and bone formation in male C57BL/6J in vivo following S1P stimulation and S1P lyase inhibition, respectively. Pharmacological S1PR3 inhibition and genetic S1PR3 deficiency suppressed VEGFa production, OB growth in vitro, and inhibited H-type angiogenesis and bone growth in male mice in vivo. Together with previous work on the osteoanabolic functions of S1PR2 and S1PR3, our data suggest that S1P-dependent bone regeneration employs several nonredundant positive feedback loops between OBs and the bone vasculature. The identification of this yet unappreciated aspect of osteoanabolic S1P signaling may have implications for regular bone homeostasis as well as diseases where the bone microvasculature is affected such as age-related osteopenia and posttraumatic bone regeneration.

Sphingosine-1-phosphate (S1P) is a signaling lipid that regulates bone growth and regeneration. In the present study, a novel regenerative mechanism was connected to S1P signaling within the bone. Activation of its receptor S1PR3 in bone-forming osteoblasts led to secretion of vascular endothelial growth factor a (VEGFa), the most potent vessel-stimulating factor. This stimulated the development of specialized vessels of the bone marrow, the H-type vessels, that supported overall bone regeneration. These findings foster our understanding of regular bone metabolism and suggest that S1P-based drugs may help treat diseases such as age-related osteopenia and posttraumatic bone regeneration, conditions crucially dependent on functional bone microvasculature.

Functional Assessment of Coding and Regulatory Variants From the DKK1 Locus.

The DKK1 gene encodes an extracellular inhibitor of the Wnt pathway with an important role in bone tissue development, bone homeostasis, and different critical aspects of bone biology. Several BMD genome-wide association studies (GWASs) have consistently found association with SNPs in the DKK1 genomic region. For these reasons, it is important to assess the functionality of coding and regulatory variants in the gene. Here, we have studied the functionality of putative regulatory variants, previously found associated with BMD in different studies by others and ourselves, and also six missense variants present in the general population. Using a Wnt-pathway-specific luciferase reporter assay, we have determined that the variants p.Ala41Thr, p.Tyr74Phe, p.Arg120Leu, and p.Ser157Ile display a reduced DKK1 inhibitory capacity as compared with WT. This result agrees with the high-bone-mass (HBM) phenotype of two women from our cohort who carried mutations p.Tyr74Phe or p.Arg120Leu. On the other hand, by means of a circularized chromosome conformation capture- (4C-) sequencing experiment, we have detected that the region containing 24 BMD-GWA variants, located 350-kb downstream of DKK1, interacts both with DKK1 and the LNCAROD (LncRNA-activating regulator of DKK1, AKA LINC0148) in osteoblastic cells. In conclusion, we have shown that some rare coding variants are partial loss-of-function mutations that may lead to a HBM phenotype, whereas the common SNPs associated with BMD in GWASs belong to a putative long-range regulatory region, through a yet unknown mechanism involving LNCAROD. © 2020 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Short-Term Effect of Estrogen on Human Bone Marrow Fat.

Bone marrow fat, an unique component of the bone marrow cavity increases with aging and menopause and is inversely related to bone mass. Sex steroids may be involved in the regulation of bone marrow fat, because men have higher bone marrow fat than women and clinical observations have suggested that the variation in bone marrow fat fraction is greater in premenopausal compared to postmenopausal women and men. We hypothesized that the menstrual cycle and/or estrogen affects the bone marrow fat fraction. First, we measured vertebral bone marrow fat fraction with Dixon Quantitative Chemical Shift MRI (QCSI) twice a week during 1 month in 10 regularly ovulating women. The vertebral bone marrow fat fraction increased 0.02 (95% CI, 0.00 to 0.03) during the follicular phase (p = 0.033), and showed a nonsignificant decrease of 0.02 (95% CI, -0.01 to 0.04) during the luteal phase (p = 0.091). To determine the effect of estrogen on bone marrow fat, we measured vertebral bone marrow fat fraction every week for 6 consecutive weeks in 6 postmenopausal women before, during, and after 2 weeks of oral 17-ß estradiol treatment (2 mg/day). Bone marrow fat fraction decreased by 0.05 (95% CI, 0.01 to 0.09) from 0.48 (95% CI, 0.42 to 0.53) to 0.43 (95% CI, 0.34 to 0.51) during 17-ß estradiol administration (p < 0.001) and increased again after cessation. During 17-ß estradiol administration the bone formation marker procollagen type I N propeptide (P1NP) increased (p = 0.034) and the bone resorption marker C-terminal crosslinking telopeptides of collagen type I (CTx) decreased (p < 0.001). In conclusion, we described the variation in vertebral bone marrow fat fraction among ovulating premenopausal women. And among postmenopausal women, we demonstrated that 17-ß estradiol rapidly reduces the marrow fat fraction, suggesting that 17-ß estradiol regulates bone marrow fat independent of bone mass.