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1 Supplementary Material for Parathyroid Hormone Signaling through Low-density-lipoprotein-related Protein 6 Mei Wan, Chaozhe Yang, Jun Li, Xiangwei Wu, Hongling Yuan, Hairong Ma, Xi He, Shuyi Nie, Chenbei Chang, Xu Cao To whom correspondence should be addressed. mwan@uab.edu; and Cao@uab.edu This PDF file includes Supplementary Materials and Methods Supplemental figures 1-7 1

2 Supplementary Materials and Methods In vitro kinase assays. GST, GST-LRP5C and GST-LRP6C were purified from bacterial lysates by absorption to glutathione-agarose beads. GST-LRP5C and GST-LRP6C beads were washed with phosphorylation buffer (25 mm Tris-HCl, ph 7.5, 1 mm MgCl 2, 2 mm MnCl 2,.4 mm EDTA, 1 mm dithiothreitol, 2 mm orthovanadate, 1 mm NaF, 5 mm β-glycerophosphate, and 1 μm ATP) containing a protease inhibitor mixture (1 mm phenylmethylsulfonyl fluoride and 1 μg/ml antipain, chymostatin, leupeptin, and pepstatin A). [γ 32 P]ATP was then added to the mixture and incubated for 3 min at 3 C with PKA catalytic subunit. Phosphorylation status was analyzed on an 8.5% SDS-PAGE gel and autoradiography. Animals. In the intermittent injection model, PTH (1-34) (4µg/kg per day) or vehicle (equivalent volume of 1mM acetic acid in sterile PBS) in a final volume of 1µl was given daily by subcutaneous injection for 6 weeks to two-month-old male C57BL/J6 mice (6 per group). In the continuous infusion model, ALZET Osmotic Pumps (Model 24, DURECT Corp., Cupertino, CA, USA) were implanted subcutaneously into the backs of mice under anesthesia. Continuous infusion of PTH (1-34) or vehicle (equivalent volume of 1mM acetic acid in sterile PBS) was conducted to release 4µg/kg per day at the rate of.25µl/h for 6 weeks. To ensure continuous administration of active PTH (1-34), the original pump was removed and replaced by a new one in a different subcutaneous site every 2 weeks. Analysis of bone phenotype and histomorphometric analysis. Bone radiographs of excised femora and tibia were obtained using a soft X-ray apparatus (FAXITRON, wheeling, Illinois, USA). For histological analyses, the proximal tibia were resected and fixed in 1% buffered formalin for 48 h, decalcified in 1% ethylenediamine tetraacetic acid (EDTA) (ph 7.) for 2 days and embedded in paraffin. 5-μm thick longitudinally oriented sections of bone including metaphysis and diaphysis were processed for hematoxylin and eosin (H&E) staining. For double labeling, mice were given subcutaneous injections of demeclocycline (Sigma, St. Louis, MO, USA) at a dose of 2 mg/kg (in bacteriostatic water, ph 7.3) and calcein (Sigma, St. Louis, MO, USA) at dose of 1 mg/kg (in 2% sodium bicarbonate solution) 1 days and 3 days, respectively, before sacrifice. 1-μm thick nondecalcified sections of bone were processed for the dynamic analysis. 2

3 Sections were subjected to microphotography as the basis for histomorphometric measurements and the quantitative histomorphometric analyses were conducted in a blinded fashion with OsteoMeasure Software (OsteoMetrics Inc., Decatur, GA, USA). Two/three-dimensional parameters of trabecular bone of femur were measured in a 2-mm square, 1 mm distal to the lowest point of the growth plate in the secondary spongiosa. Bone volume (BV/TV, %) and Bone surface referent bone formation rate (BFR/BS, μm 2 /μm/day) were measured in four randomly selected visual fields per specimen, in a total of six specimens in each group. 3

4 Supplementary Figures A PTH (1-84) min β-catenin α-tubulin B Ratio of β-catenin/α-tubulin C β-catenin α-tubulin D Ratio of β-catenin/α-tubulin Con (M) Figure 1. Activation of β-catenin signaling by PTH in HEK293 cells. (A) Stabilization of β-catenin in HEK293 cells treated with 1-8 M PTH (1-84). Cells were transfected with PTH1R, serum deprived and treated with PTH for various times. Cytosolic fractions were prepared for detection of β-catenin by western blot. (B) Densitometric quantification of β-catenin protein as shown in (A). (C) Dose-dependent effect of PTH on β-catenin stabilization. Cells were transfected with PTH1R, serum deprived and treated with various doses of PTH (1-84) for 1h. Cytosolic fractions were prepared for detection of β-catenin levels by western blot. (D) Densitometric quantification of β-catenin protein as shown in (C). 4

5 A B C Osteoblasts containing β-catenin (%) ** ** * * Control.5h 2h 8h PTH 24h Figure 2. PTH single dose injection elevated β-catenin level in osteoblasts in mice. Immunohistochemical analysis of β-catenin level in tibia sections from 2-month-old male mice at the indicated time points after PTH (1-34) (2 μg/kg) injection. Representative of tissue sections obtained at different times after PTH injection and immunohistochemically stained for β-catenin and counterstained with hematoxylin. β-catenin-positive osteoblasts were stained in brown (A). β-catenin-positive osteoblasts were counted in a blinded fashion from three random high power fields per specimens in a 2-mm square, 1 mm distal to the lowest point of the growth plate in the secondary spongiosa, and a total of six specimens in each group were used. The quantification of β-catenin-positive osteoblasts is presented as percentage of total osteoblasts (B). *:p<.5; **:P<.1 (in comparison with control), n=6. 5

6 VSVG-LRP PTH (1-84) IP: Axin1 Blot: VSVG IP: VSVG Blot: VSVG Slerostin CM DKK1 CM Figure 3. Soluble Sclerostin and DKK1 inhibits PTH-induced axin-lrp6 binding. HEK 293 cells were transfected with PTH1R and VSVG-tagged LRP6 and treated with Sclerostin CM or DKK1 CM followed by treatment of 1-8 M PTH (1-84) for 3 min. The axinassociated LRP6 was determined by western blotting of the anti-axin1 immunoprecipitates. 6

7 sigfp silrp PTH (1-34) p-erk1/2 Total Erk1/2 p-creb Total CREB LRP6 Figure 4. Inhibitory effect of silrp6 on PTH-induced ERK1/2 and CREB phosphorylation. C2C12 cells expressing sigfp (control) or silrp6 together with PTH1R were treated with or without PTH (1-34) for 15 min, and lysates were analyzed by immunoblotting using antibodies specifically recognizing perk1/2, total ERK1/2, pcreb (Ser 133 ), total CREB and LRP6. 7

8 A B b 4 2. ** BV/TV (%) ** BFR/BS (μm 2 /μm/d) Vehicle PTH Vehicle PTH Intermittent Injection BV/TV (%) Vehicle PTH BFR/BS (μm 2 /μm/d) Vehicle PTH Continuous Infusion C 8

9 Figure 5. PTH intermittent injection increases, whereas PTH continuous infusion decreases the levels of β- catenin and phosphorylated LRP6 in osteoblasts in the mouse femurs. (A) PTH intermittent injection increases, whereas PTH continuous infusion decreases osteoblast formation and bone density. (i) Bone densities of distal femurs of mice by X-ray imaging after PTH (1-34) (4μg/kg) daily injection or continuous infusion for 6 weeks. (ii) Histological analyses of bone formation with HE-stained tibia longitudinal sections. (iii) Mineral apposition analyses by demeclocycline and calcein double labeling of trabecular bone. (B) Bone and histomorphometric indices were measured at the trabecular of the distal femur after PTH (1-34) daily injection or continuous infusion for 6 weeks. Total bone volume per tissue volume (BV/TV, %) and bone formation rate relative to bone surface (BFR/BS) were calculated. *:p<.5; **:P<.1 (in comparison with control), n=6. (C) Immunohistochemical staining of β-catenin (upper panel) and phosphorylated LRP6 (lower panel) counterstained with hematoxylin of consecutive sections from metaphyseal area of distal femurs of mice after treatment with vehicle control or PTH (1-34) through either daily injection (4µg/kg/day) or continuous infusion (4µg/kg/day) for 6 weeks. 9

10 A GST GST-LRP5C GST-LRP6C B PKA KD 116KD 93KD 53KD 37KD 29KD Non-specific GST-LRP5C GST-LRP6C GST Figure 6. Phosphorylation of LRP6 intracellular domain by PKA in vitro. (A) GST (Lane 1 and 2), GST-LRP5 (Lane 3 and 4), and GST-LRP6 (Lane 6 and 7) were incubated with [γ 32 P]ATP and PKA catalytic subunit for 3 min at 3 C. Phosphorylation status was analyzed on an 8.5% SDS-PAGE gel and autoradiography. Lane 5, molecular weight marker. (B) Purified GST (Lane 2), GST-LRP5 (Lane 3), and GST-LRP6 (Lane 4) showing on Commassie Blue gel. Lane 1, molecular weight marker. 1

11 HA- LRP5 VSVG- LRP6 Vehicle Forskolin (5µM) IP: axin1 Blot: HA WCL Blot: HA IP: axin1 Blot: VSVG WCL Blot: VSVG Figure 7. Effect of forskolin on LRP5-axin and LRP6-axin binding. HEK293 cells were transfected with PTH1R and HA-tagged LRP5 or VSVG-tagged LRP6. Cells were then treated with indicated vehicle or forskolin for 3min. The LRP5- or LRP6- associated axin was determined by Western blotting of the anti-axin1 immunoprecipitates. 11