Lrp5 controls bone formation by inhibiting serotonin synthesis in the duodenum

Abstract

Loss- and gain-of-function mutations in the broadly expressed gene Lrp5 affect bone formation, causing osteoporosis and high bone mass, respectively. Although Lrp5 is viewed as a Wnt coreceptor, osteoblast-specific disruption of beta-Catenin does not affect bone formation. Instead, we show here that Lrp5 inhibits expression of Tph1, the rate-limiting biosynthetic enzyme for serotonin in enterochromaffin cells of the duodenum. Accordingly, decreasing serotonin blood levels normalizes bone formation and bone mass in Lrp5-deficient mice, and gut- but not osteoblast-specific Lrp5 inactivation decreases bone formation in a beta-Catenin-independent manner. Moreover, gut-specific activation of Lrp5, or inactivation of Tph1, increases bone mass and prevents ovariectomy-induced bone loss. Serotonin acts on osteoblasts through the Htr1b receptor and CREB to inhibit their proliferation. By identifying duodenum-derived serotonin as a hormone inhibiting bone formation in an Lrp5-dependent manner, this study broadens our understanding of bone remodeling and suggests potential therapies to increase bone mass.

Figure 1. Increased Tph1 expression and serum serotonin levels in Lrp5−/− mice

(A) Real-time PCR analysis of gene expression in WT, Lrp5−/− and β-Cat(ex3)_osb−/− bones. (B) Proliferation analysis (BrdU labeling) in vivo and ex vivo of WT and Lrp5−/− osteoblasts. (C) Microarray analysis of WT and Lrp5−/− bones. (D) Increased Tph1 expression in osteoblasts (Osb) and bones of Lrp5−/− mice by real-time PCR analysis. (E–F) Real-time PCR analysis of Tph1 expression in tissues of WT and Lrp5−/− mice (E), and in primary osteoblasts (Osb) and gut of WT mice (F). (G–H) Real-time PCR analysis of Tph1 expression in gut versus bone (G), and serum serotonin levels (H) in WT, Lrp5+/− and Lrp5−/− mice at indicated ages. (I) Serum serotonin levels in two OPPG patients and age matched controls (n=6).

Figure 2. Serotonin inhibits osteoblast proliferation and decreasing serum serotonin levels corrects Lrp5 −/− mice bone phenotype

(A) Ex vivo proliferation rates of WT and Lrp5−/− osteoblasts treated with serotonin (50μM) for 24 hours. (B) Real-time PCR analysis of Cyclins and Col1a1 expression in WT osteoblasts treated with serotonin (50μM) for 24 hours. (C) TOPFLASH reporter activities in osteoblasts in response to serotonin (10–50 μM) or lithium chloride (LiCl, 10mM). (D–E) Serum serotonin levels (D) and histological analysis of vertebrae (E) of WT and Lrp5−/− mice fed a normal diet or a 75% tryptophan-less diet. Mineralized bone matrix is stained in black by von Kossa reagent. Histomorphometric parameters. BV/TV%, bone volume over trabecular volume; Nb.Ob/T.Ar., number of osteoblasts per trabecular area; BFR, bone formation rate; OcS/BS, osteoclast surface per bone surface. (F–H) Serum serotonin levels (F), real-time PCR analysis of Cyclin expression in bone (G) and histomorphometric analyses (H) in WT and Lrp5−/− mice treated with the serotonin synthesis inhibitor pCPA (100 mg/kg).

Figure 3. Lrp5 regulates serotonin synthesis through its expression in gut

(A–B) Real-time PCR analysis of Lrp5 (A) and Tph1 (B) expression in the gut and long bones (Bone) of Lrp5_gut−/− and Lrp5_osb−/− compared to +/+;Villin-Cre and +/+;α₁(I)Col-Cre mice, respectively. (C) Serum serotonin levels in +/+;Villin-Cre, Lrp5_gut−/−, +/+;α₁(I)Col-Cre and Lrp5_osb−/− mice. (D) Histomorphometric analysis of vertebrae of WT, +/+;Villin-Cre, Lrp5_gut−/−, Lrp5KI_gut+/−, +/+;α₁(I)Col-Cre, Lrp5_osb−/− and Lrp5KI_osb+/− mice. (E) Real-time PCR analysis of Cyclins and Col1a1 expression in long bones of WT, Lrp5_gut−/− and Lrp5_osb−/− mice. (F) In vivo osteoblast proliferation in WT, Lrp5_gut−/− and Lrp5_osb−/− mice. (G) Western blot analysis of Lrp5 high bone mass (G171V) cDNA expression (Flag) in bone and gut of Lrp5KI_osb+/− and Lrp5KI_gut+/− compared to +/+;α₁(I)Col-Cre and +/+;Villin-Cre mice respectively. (H–I) Real-time PCR analysis of Tph1 expression (H) in the gut and long bones (Bone) and serum serotonin levels (I) in Lrp5KI_osb+/− and Lrp5KI_gut+/− compared to +/+;Villin-Cre and +/+;α₁(I)Col-Cre mice, respectively. (J–K) Real-time PCR analysis of Cyclins and Col1a1 expression (J) and in vivo osteoblast proliferation (K) in long bones of WT, Lrp5KI_gut+/− and Lrp5KI_osb+/− mice. (L) Plasma serotonin levels in two high bone mass (HBM) patients and age matched controls (n=3).

Figure 4. Duodenal-derived serotonin regulates bone formation

(A–B) Real-time PCR analysis of Tph1 expression in gut and long bones (Bone) and Tph2 expression in the brainstem (BS) of Tph1_gut−/− and Tph1_osb−/−compared to +/+;Villin-Cre and +/+;α₁(I)Col-Cre mice respectively. (C–E) Serum serotonin levels (C) and bone histomorphometric analysis (vertebrae) (D–E) in WT, +/+;Villin-Cre, Tph1_gut−/−, +/+;α₁(I)Col-Cre and Tph1_osb−/− mice. (F) Real-time PCR analysis of Cyclins and Col1a1 expression in long bones of WT, Tph1_gut−/− and Tph1_osb−/− mice. (G) In vivo osteoblast proliferation in WT, Tph1_gut−/− and Tph1_osb−/− mice.

Figure 5. Serotonin inhibits osteoblasts proliferation through Htr1b

(A–B) Real-time PCR analysis of serotonin receptors expression in primary osteoblasts (A) and of Htr1b expression in different tissues of WT mice (B). (C–E) Histomorphometric analysis of vertebrae of 3 month-old WT, Htr2a−/− (C), Htr2b_osb−/− (D) and Htr1b−/− (E) mice. (F) Real-time PCR analysis of Cyclins, Runx2, Osx, and Atf4 expression in long bones of WT, Htr1b−/−, Htr2a−/− and Htr2b_osb−/− mice. (G) Real-time PCR analysis of CycD1 expression in primary osteoblasts of WT, Htr1b−/−, Htr2a−/− and Htr2b_osb−/− mice treated with serotonin (50μM). (H) In vivo osteoblast proliferation in WT, Htr1b−/−, Htr2a−/− and Htr2b_osb−/− mice. (I) Histomorphometric analysis of vertebrae of 6 week-old WT and Htr1b_osb+/− mice.

Figure 6. Lrp5 mediates its effect through CREB

(A) Histomorphometric analysis of vertebrae of WT, single (Lrp5+/−, Runx2+/−, Osx+/−, Atf4+/− or Creb_osb+/−) and double (Lrp5+/−;Runx2+/−, Lrp5+/−;Osx+/−, Lrp5+/−;Atf4+/− or Lrp5+/−;Creb_osb+/−) heterozygous mutant mice. (B–C) Western blot analysis of CREB phosphorylation (B) and chromatin-immunoprecipitation assay of CREB binding to the consensus cAMP response element (CRE) in CycD1 promoter (C) at 0, 3, 6, 12 and 24 hours upon serotonin (50μM) treatment. Coding sequence (CDS) PCR, IgG pulldown and ATF4 binding to the same site were used as negative controls. (D) Real-time PCR analysis of CycD1 expression in the long bones of WT, Lrp5+/−, Lrp5−/−, Creb_osb+/−, Osx+/− and Runx2+/− mice. (E) Real-time PCR analysis of Creb expression in the long bones of WT, Lrp5−/−, Lrp5_gut−/−, Lrp5_osb−/−, Lrp5KI_gut+/− and Lrp5KI_osb+/− mice.

Figure 7. Genetic interaction between Lrp5 and serotonin

(A) Bone histomorphometric analysis (vertebrae) of WT, Htr1b+/−, Tph1_gut+/−, Htr1b+/−;Tph1_gut+/−, Lrp5−/−, Lrp5−/−;Htr1b+/− and Lrp5−/−;Tph1_gut+/− mice. (B) Bone histomorphometric analysis (vertebrae) of WT, β-Cat_gut+/−, Tph1_gut+/−;β-Cat_gut+/−, Lrp5+/− and Lrp5+/−;β-Cat_gut+/− mice. (C) Serum serotonin levels and real-time PCR analysis of Tph1 expression in the gut of WT and β-Cat_gut+/− mice. (D) Bone histomorphometric analysis (vertebrae) of WT, WT(Ovx), Tph1_gut−/− and Tph1_gut−/−(Ovx) mice. (E) Model of the Lrp5-dependent regulation of bone formation. Lrp5 favors bone formation and bone mass accrual by inhibiting Tph1 expression and serotonin synthesis in enterochromaffin cells. Following its binding to Htr1b serotonin inhibits Creb expression and function, this results in a decrease in CycD1 expression and osteoblast proliferation.