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.

Figure 1. Foxo1 haploinsufficiency rescues the low bone formation phenotype of Lrp5^+/– mice.

(A) Representative vertebral section images from 2-month-old mice. Mineralized bone matrix is stained in black by Von Kossa reagent. Original magnification, ×4. (B) Representative 3D images of transverse CT slices of proximal tibia obtained by high-resolution μCT analysis. (C) Serum serotonin levels (n = 4 mice/group). (D) Osteoblast proliferation as the number of BrdU-stained osteoblasts (Obs) per trabecular area (T.Ar.) or as the percentage of total osteoblasts per trabecular area (n = 6 mice/group) in sections of femurs. (C and D) *P < 0.05 versus WT, ^#P < 0.05 versus Lrp5^+/–Foxo1^+/– group. (E and F) Real-Time PCR analysis of indicated genes in bone (n = 6 mice/group). *P < 0.05 versus WT, ^#P < 0.05 versus Lrp5^+/–Foxo1^+/– group. (G) Serum serotonin levels (n = 4 mice/group). *P < 0.05 versus WT, ^#P < 0.05 versus Lrp5^–/–Foxo1^+/– group. (H) Serum serotonin levels in WT, Lrp5^+/–Foxo1^+/–, and LP533401-treated Lrp5^+/–Foxo1^+/– mice. The gut-derived serotonin synthesis inhibitor LP533401 was administered as indicated at 200 mg/kg/d by oral gavage for 4 weeks. Mice were 2 months old (n = 6 mice/group). *P < 0.05 versus WT, ^#P < 0.05 versus LP533401-treated Lrp5^+/–Foxo1^+/– group.

Figure 2. Serotonin suppresses osteoblast proliferation through FOXO1.

(A) SOD2 activity in osteoblasts treated with serotonin (5-HT; 100 μM) for 6 hours (n = 3). *P < 0.05 versus vehicle. (B) Calvaria cells cotransfected with Lrp5 and FOXO-Luc plasmids and treated with vehicle or 100 μM serotonin for 24 hours (n = 3). EV, empty vector. *P < 0.05 versus empty vector, ^#P < 0.05 versus FOXO-Luc vehicle. (C) Real-time PCR analysis of the expression of Ccnd1 and Ccnd2 in calvaria cells treated with serotonin (100 μM) for 6 hours (n = 3). *P < 0.05 versus vehicle. (D) Calvaria cells from WT and Foxo1_ob^–/– mice were treated with serotonin (100 μM) for 48 hours, and proliferation was assessed by Cell Titer One (left) or BrdU incorporation (right) (n = 3). *P < 0.05 versus vehicle. (E and F) Calvaria cells from WT and Foxo1_ob^–/– mice were treated with serotonin (100 μM) for 48 hours. Gene expression of (E) Ccnd1 and (F) Ccnd2 (n = 3). *P < 0.05 versus vehicle. (G) Western blot analysis showing phosphorylation of PKA in WT and Foxo1-deficient osteoblasts treated with serotonin (100 μM) for 30 minutes. (H) Serum serotonin levels (n = 4 mice/group). *P < 0.05 versus WT, ^#P < 0.05 versus Lrp5^+/–Foxo1_ob^–/– group.

Figure 3. Serotonin regulates FOXO1 transcriptional activity via its HTR1B receptor in osteoblasts.

(A) Representative vertebral section images from 2-month-old mice. Mineralized bone matrix is stained in black by Von Kossa reagent. Original magnification, ×4. (B) Representative 3D images of transverse CT slices of proximal tibia obtained by high-resolution μCT analysis. (C) In vivo osteoblast proliferation assessed in femoral sections expressed as the number of BrdU-stained osteoblasts per trabecular area or as the percentage of total osteoblasts per trabecular area (n = 3 mice/group). (D) Real-time PCR analysis of indicated genes in bones (n = 4 mice/group). (C and D) *P < 0.05 versus WT, ^#P < 0.05 versus Htr1b^+/–Foxo1_ob^+/– group. (E) Calvaria cells from WT or Htr1b^–/– mice transfected with FOXO-Luc and treated with vehicle or serotonin (100 μM) (n = 3). Mice were 2 months old. *P < 0.05 versus respective vehicle.

Figure 4. Serotonin regulates a FOXO1-CREB interaction.

(A and B) Phosphorylation status of (A) ATF4 and (B) CREB in OB-6 cells treated with serotonin (100 μM). (C) OB-6 cells transfected with expression plasmids for PKA, the active domain of CREB fused to GAL4, and a construct with GAL4-binding sites upstream of the luciferase gene and treated with 100 μM serotonin for 24 hours (n = 3). *P < 0.05 versus PKA plasmid vehicle. (D and E) Immunoprecipitation and immunoblotting in OB-6 cells treated with serotonin (100 μM) for 1 hour. Blots were representative of 3 experiments. ND, not detected. (F) Binding of GST-FOXO1 fragments and His-CREB for mapping the FOXO1 interaction domain. (G) Binding of GST-FOXO1 (aa 290–655) and His-CREB fragments for mapping the CREB interaction domain. (H) Calvaria cells transfected with indicated plasmids and treated with vehicle or serotonin (100 μM) for 24 hours (n = 3). *P < 0.05 versus EV vehicle; ^#P < 0.05 versus respective vehicle. (I) Serum serotonin levels, (J) SOD2 activity in tibial lysates, and (K and L) immunoprecipitation and immunoblotting in femoral lysates from WT and Lrp5^–/– mice (n = 3–4 mice/group). *P < 0.05 versus WT.

Figure 5. Differential regulation of FOXO1 interactions with ATF4 and CREB by serotonin.

(A) Serum serotonin levels, (B–D) real-time PCR analysis of indicated genes in bones, (E and F) immunoblotting in femoral lysates, and (G and H) immunoprecipitation and immunoblotting in femoral lysates of WT, Lrp5^–/–, and LP533401-treated Lrp5^–/– mice. The gut-derived serotonin synthesis inhibitor LP533401 was administered daily by gavage at 200 mg/kg/d for 7 days (n = 4 mice/group). Mice were 2 months old. *P < 0.05 versus WT, ^#P < 0.05 versus LP533401-treated Lrp5^–/– group.

Figure 6. PKA/JNK1-dependent signaling localizes FOXO1 to the nucleus.

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.

Figure 7. Gut-derived serotonin inhibits bone formation by regulating FOXO1-CREB interaction.

(A) Representative vertebral section images from 2-month-old mice. Mineralized bone matrix is stained in black by Von Kossa reagent. Original magnification, ×4. (B) Representative 3D images of transverse CT slices of proximal tibia obtained by high-resolution μCT analysis. (C) Osteoblast proliferation expressed as the number of BrdU-stained osteoblasts per trabecular area or as the percentage of total osteoblasts per trabecular area in sections of femurs (n = 6 mice/group). (D and E) Real-time PCR analysis of indicated genes in bones (n = 4 mice/group). Mice were 2 months old. *P < 0.05 versus WT, ^#P < 0.05 versus Creb_ob^+/–Foxo1_ob^+/– group by ANOVA.

Figure 8. Model illustrating the mechanism of serotonin/FOXO1 interaction in osteoblasts.

Under physiological conditions, FOXO1 interacts in the nucleus with both ATF4 and CREB to maintain normal osteoblast proliferation. The association of FOXO1 with ATF4 promotes, whereas the one with CREB suppresses, expression of FOXO1-regulated transcriptional targets. An increase in circulating serotonin levels disrupts the interaction of FOXO1 with CREB. This favors the formation of ATF4-FOXO1 heterodimers. These ATF4-dependent or -independent interactions lead to increased transcriptional activity of FOXO1 and suppression of cell-cycle progression genes. Additionally, following release from its complex with CREB, high levels of available FOXO1 may interact with other transcription factor(s).