PROJECT SUMMARY/ABSTRACT Roughly 1/3 of bone mass accrual during life is realized during adolescence. Disruption of this critical window of skeletal maturation has lifelong implications for bone health and fracture risk. Systemic tetracyclines (i.e., minocycline, doxycycline) are commonly used to treat acne in adolescents, but the impact on bone is unclear. Preliminary studies were performed treating C57BL/6 mice with a clinically relevant dose of doxycycline or minocycline during pubertal/postpubertal development. Administering doxycycline or minocycline to specific- pathogen-free (SPF) mice caused dysbiotic shifts in the gut bacteriome and impaired skeletal maturation. Administering minocycline to mice reared under germ-free (GF) conditions did not affect the skeletal phenotype, which supports that tetracyclines’ effects on the maturing skeleton depend on the gut microbiota. Bile acids were identified as a novel candidate regulator contributing to gut microbiota effects on bone metabolism. Bile acids are synthesized in the liver and excreted into the intestine, where bacteria metabolize them. The intestinal FXR-FGF15 axis is a gut-liver endocrine axis that supports bile acid homeostasis. Bile acid activation of enterocyte-FXR induces the production of FGF15, which signals at hepatocyte-FGFR4 to inhibit CYP7A1- mediated bile acid synthesis. Bacteria have unique bile salt hydrolases (BSHs) that differentially deconjugate bile acids. Conjugation status affects bile acids’ potential to activate FXR. Shifts in intestinal bacteria composition alter BSHs' deconjugation of bile acids, which can disrupt the intestinal FXR-FGF15 axis. Preliminary studies showed that minocycline treatment blunted ileal FGF15 and enhanced hepatic Cyp7a1, which implies tetracyclines disrupt the intestinal FXR-FGF15 axis. Minocycline increased serum bile acids that are FXR antagonists, and this altered bile acid profile attenuated osteogenesis in cultured primary osteoblasts. Two aims will test the overall hypothesis: Tetracycline-induced gut dysbiosis disrupts the intestinal FXR- FGF15 axis, which impairs skeletal maturation through dysregulated serum bile acids that attenuate osteoblast- FXR signaling or reduced serum FGF15 activation of osteoblast-FGFR4 signaling. Aim 1 will utilize metagenomic approaches and fecal microbiota transfer from SPF to GF mice. Studies will delineate how minocycline- and doxycycline-induced changes in gut bacteria alter the transformation of intestinal bile acids to affect the FXR- FGF15 axis. Aim 2 relies on administering an intestinal-specific FXR agonist to define the role of the FXR-FGF15 axis in minocycline effects on the skeleton. Tamoxifen inducible osteoblast null mice will be used to delineate the role of osteoblast-FXR / osteoblast-FGFR4 in minocycline’s actions suppressing osteogenesis. The proposed work will define the relationship between tetracycline-induced gut dysbiosis, the FXR-FGF15 axis, osteoblast- FXR/FGFR4 signaling, a...