Heavy Metal Ion Regulation of Gene Expression: MECHANISMS BY WHICH LEAD INHIBITS OSTEOBLASTIC BONE-FORMING ACTIVITY THROUGH MODULATION OF THE Wnt/β-CATENIN SIGNALING PATHWAY

Abstract

Exposure to lead (Pb) from environmental sources remains an overlooked and serious public health risk. Starting in childhood, Pb in the skeleton can disrupt epiphyseal plate function, constrain the growth of long bones, and prevent attainment of a high peak bone mass, all of which will increase susceptibility to osteoporosis later in life. We hypothesize that the effects of Pb on bone mass, in part, come from depression of Wnt/β-catenin signaling, a critical anabolic pathway for osteoblastic bone formation. In this study, we show that depression of Wnt signaling by Pb is due to increased sclerostin levels in vitro and in vivo. Downstream activation of the β-catenin pathway using a pharmacological inhibitor of GSK-3β ameliorates the Pb inhibition of Wnt signaling activity in the TOPGAL reporter mouse. The effect of Pb was determined to be dependent on sclerostin expression through use of the SOST gene knock-out mice, which are resistant to Pb-induced trabecular bone loss and maintain their mechanical bone strength. Moreover, isolated bone marrow cells from the sclerostin null mice show improved bone formation potential even after exposure to Pb. Also, our data suggest that the TGFβ canonical signaling pathway is the mechanism by which Pb controls sclerostin production. Taken together these results support our hypothesis that the osteoporotic-like phenotype observed after Pb exposure is, in part, regulated through modulation of the Wnt/β-catenin pathway.

Keywords: TGF-β signaling; Wnt signaling; bone; gene expression; lead toxicity; osteoblast; osteoporosis; sclerostin.

FIGURE 1.

Pb inhibits osteogenesis, which is partially restored by co-treatment with Wnt3a. Mouse calvarial osteoblasts were differentiated to form bone nodules and treated with 0 (control (Ctrl)), 0.5, 2.0, and 5.0 μm Pb and in the presence of 50 ng/ml Wnt3a. A and C, mineralization was assessed by alkaline phosphatase staining (A) on day 10 and alizarin red staining (C) on day 20. Scl, sclerostin. B and D, images are representative of one trial; quantification of alkaline phosphatase (B) and alizarin red (D) staining was performed with ImageJ. Data represent mean ± S.E. for three trials. *, p < 0.05 versus control, #, p < 0.05 versus 2.0 μm Pb.

FIGURE 2.

Pb inhibits Wnt signaling in osteoblasts. A, real-time PCR analysis on total RNA in calvarial osteoblasts following a 24-h exposure to increasing Pb. We examined the expression of two Wnt signaling antagonists (SOST and DKK1) and Wnt signaling agonists (Wnt3a and Wnt5a). Osteoblastic cells were treated with control or Pb-conditioned medium at the dose indicated and then stimulated with Wnt3a for 12 h. B, representative Western blots (above) of the active form of β-catenin (β-cat) and Wnt antagonists with quantification (below). C and D, data from luciferase (Luc) reporters of TOPFLASH (C) and 7-kb upstream SOST promoter activity (D) are shown when activated with Wnt3a and treated with Pb. Veh, vehicle. Data represent mean ± S.E. for three trials. *, p < 0.05 versus control, #, p < 0.05 versus activation + Pb.

FIGURE 3.

Pb depresses Wnt3a stimulated β-catenin signaling in TOPGAL cells. Bone marrow from TOPGAL transgenic mice was isolated and stimulated with osteogenic medium. Subsequently, cell cultures were given treatment of either Wnt or BIO in combination with Pb. A, images are representative of X-gal-stained cell cultures from each treatment group: BIO and Wnt treatment with and without Pb. B, β-catenin signaling was measured following activation from either Wnt3a or BIO in the context of Pb using a luminescent substrate for β-galactosidase. DMSO, dimethyl sulfoxide. C, sclerostin protein levels in medium following 72 h of Pb were measured with ELISA. Data represent mean ± S.E. for three trials. *, p < 0.05 versus control, #, p < 0.05 versus vehicle + Pb.

FIGURE 4.

Effect of increasing Pb on long bone length, bone turnover markers, and osteoblast precursors in sclerostin-deficient mice. A, gross measurements of total femur length were taken and are presented for each treatment group. B, MSC populations from Pb-treated 3-month-old WT and SOST-KO mice were isolated and sorted for their positive surface expression of Sca-1. The percentage and total number of Sca-1⁺ cells were quantified. C–F, serum bone formation marker P1NP (C), resorption markers CTX-1 (D) and TRAP5b (E), and corticosterone (F) were measured using standard ELISA methods. Data represent mean ± S.E. of 5 mice/group. *, p < 0.05, **, p < 0.005 versus 0 Pb WT, #, p < 0.05, ##, p < 0.005 versus 0 Pb KO.

FIGURE 5.

Effect of Pb exposure on bone volume and quality in vertebrae of SOST knock-out mice. Top, images are representative transverse sections from each control and Pb-exposed group in WT and SOST-KO mice. Bar: 200 μm. Bottom, trabecular bone was analyzed in the fourth lumbar vertebra in 6-month-old mice for the following bone parameters: BV/TV, Tb.N, and Tb.Sp. Pb decreased bone mass and bone properties in WT mice. Bone in KO mice was unaffected by Pb. Data represent mean ± S.E. of 5 mice/group. *, p < 0.05, **, p < 0.005 versus 0 Pb WT.

FIGURE 6.

Pb inhibits osteogenesis and is rescued, in part, in cells from SOST knock-out mice. Bone marrow was harvested from 3-month-old WT and SOST-KO mice treated with 0, 50, or 500 ppm of Pb in drinking water. A, mesenchymal cells were induced by osteogenic medium to form bone nodules. Left, representative alkaline phosphatase staining on day 14 of differentiation. Right, quantification. B, osteoclastogenesis was initiated in marrow cells for 7 days. Left, representative osteoclasts are presented; right, TRAP-positive stains were quantified. Data represent mean ± S.E. for three trials. *, p < 0.05 versus WT control, #, p < 0.05 versus KO control.

FIGURE 7.

The TGFβ/Smad3 signaling axis regulates sclerostin expression. A, mature primary osteoblasts express sclerostin mRNA in vitro. A TGFβ receptor inhibitor, TβRI-V#616456, strongly up-regulates transcription, whereas TGFβ itself significantly inhibits sclerostin mRNA production. Ctrl, control. B, immunofluorescent analysis at the femoral epiphysis demonstrates that in the absence of osteoblastic TGFβ receptor 2, there is more intense staining for sclerostin protein in the cells surrounding the growth plate. (left, representative images; right, quantification right). C, the number and intensity of sclerostin expression in osteocytes are elevated in mice that are devoid of Smad3 (left, representative images; right, quantification right). D, similarly, serum levels of sclerostin in 3-month-old Smad3^+/− and Smad3^−/− mice were increased. E, representative Western blot analysis of bone marrow cells isolated from the animals shown in panel C for sclerostin (Scl) and Smad3 expression. Data represent mean ± S.E. for three trials. *, p < 0.05 versus control.

FIGURE 8.

Pb blocks TGFβ signaling by inhibiting Smad3 phosphorylation. A, following TGFβ induction, increasing concentrations of Pb significantly diminished Smad3 reporter activity in osteoblastic cell cultures. B, a constitutively active Smad3 rescues p3TP-lux reporter activity in Pb-treated cells. Data represent mean ± S.E. for five trials; *, p < 0.05, **, p < 0.005 versus TGFβ alone, +, p < 0.005 versus control. C, Western blot analysis demonstrates that TGFβ can increase the level of phospho-Smad3 (pSMAD3 in the nucleus and that Pb can inhibit this effect. The total amount of nuclear Smad3 was unaffected. D, Pb inhibits phosphorylation of Smad3 in a cell-free system at 300–600 nm. Biotin-SA was used as a loading control in these experiments.