FOXO1 orchestrates the bone-suppressing function of gut-derived serotonin
Aruna Kode, Ioanna Mosialou, Barbara C. Silva, Marie-Therese Rached, Bin Zhou, Ji Wang, Tim M. Townes, Rene Hen, Ronald A. DePinho, X. Edward Guo, Stavroula Kousteni
Aruna Kode, Ioanna Mosialou, Barbara C. Silva, Marie-Therese Rached, Bin Zhou, Ji Wang, Tim M. Townes, Rene Hen, Ronald A. DePinho, X. Edward Guo, Stavroula Kousteni
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Research Article Bone biology

FOXO1 orchestrates the bone-suppressing function of gut-derived serotonin

Abstract

Serotonin is a critical regulator of bone mass, fulfilling different functions depending on its site of synthesis. Brain-derived serotonin promotes osteoblast proliferation, whereas duodenal-derived serotonin suppresses it. To understand the molecular mechanisms of duodenal-derived serotonin action on osteoblasts, we explored its transcriptional mediation in mice. We found that the transcription factor FOXO1 is a crucial determinant of the effects of duodenum-derived serotonin on bone formation We identified two key FOXO1 complexes in osteoblasts, one with the transcription factor cAMP-responsive element–binding protein 1 (CREB) and another with activating transcription factor 4 (ATF4). Under normal levels of circulating serotonin, the proliferative activity of FOXO1 was promoted by a balance between its interaction with CREB and ATF4. However, high circulating serotonin levels prevented the association of FOXO1 with CREB, resulting in suppressed osteoblast proliferation. These observations identify FOXO1 as the molecular node of an intricate transcriptional machinery that confers the signal of duodenal-derived serotonin to inhibit bone formation.

Authors

Aruna Kode, Ioanna Mosialou, Barbara C. Silva, Marie-Therese Rached, Bin Zhou, Ji Wang, Tim M. Townes, Rene Hen, Ronald A. DePinho, X. Edward Guo, Stavroula Kousteni

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Figure 6

PKA/JNK1-dependent signaling localizes FOXO1 to the nucleus.

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PKA/JNK1-dependent signaling localizes FOXO1 to the nucleus. Immunoblot ...
Immunoblot showing (A) PKA, (B) JNK1, and (C) AKT phosphorylation in OB-6 cells treated with serotonin (100 μM). (D and E) Proliferation in primary osteoblasts transfected with (D) PKA siRNA oligos or control scrambled siRNA oligos or (E) JNK1 siRNA or control scrambled siRNA oligos, in the presence or absence of siRNA-resistant JNK1, followed by treatment with vehicle or serotonin (100 μM) for 48 hours (n = 3). (F) Proliferation in primary osteoblasts transfected with indicated siRNAs or control scrambled siRNA oligos, followed by treatment with vehicle or serotonin (100 μM) for 48 hours (n = 3). (D–F) *P < 0.05 versus vehicle. (G) Immunoblot of JNK1 phosphorylation in OB-6 cells transfected with PKA siRNA and control scrambled siRNA oligos. (H) Subcellular localization of Foxo1 in primary osteoblasts transfected with JNK1 siRNA oligos or control scrambled siRNA oligos, followed by cotransfection with FOXO1-GFP and nRFP plasmids, in the presence or absence of siRNA-resistant JNK1. Transfected cells were treated with serotonin (100 μM) for 6 hours. Subcellular localization of FOXO1 was expressed as the percentage cytoplasmic or nuclear localization (n = 3). Original magnification, ×4. *P < 0.05 versus vehicle. (I–K) Immunoprecipitation and immunoblotting of CREB and FOXO1 in OB-6 cells (I) treated with PKA inhibitor (H-89) or (J) transfected with JNK1 siRNA, with or without siRNA-resistant JNK1 or control scrambled siRNA oligos, or (K) treated with the JNK inhibitor SP600125 at 10 μM. (L) Immunoblotting for JNK1 and JNK2 protein levels in OB-6 cells. Blots were representative of 3 experiments.