Figure 1.
Preparation of Recombinant hPTHrP1-34 and 1-84.
(a) Determination of the recombinants by PCR and double-enzyme (EcoR I+ BamH I) digestion. LaneM1, l-Hind III digest DNA marker; Lane1-3, double-enzyme digestion patterns of recombinant hPTHrP1-34, hPTHrP1-84 and pGEX-2TK; LaneM2, DL2000 DNA marker; Lane4-5, PCR band patterns of recombinant hPTHrP1-34 and hPTHrP1-84. SDS-PAGE analysis of the GST-hPTHrP1-34 (b) or GST-hPTHrP1-84 (d) fusion protein under inducing condition and stained with coomassie blue. LaneM, molecular weight marker; Lane1, total cell soluble protein of BL21, control; Lanes2–6, cells with pGEX-2TK/hPTHrP1-34 or pGEX-2TK/hPTHrP1-84 after inducing with IPTG 0 h, 1 h, 2 h, 3 h, 4 h respectively. SDS-PAGE analyses of hPTHrP1-34 (c) or hPTHrP1-84 (e) peptide expression and purification. LaneM, molecular weight marker; Lane1, IPTG induction of BL21 transformed with pGEX-2TK without insert (expression control); Lane2, BL21 transformed with pGEX-2TK/hPTHrP1-34 or pGEX-2TK/hPTHrP1-84 without IPTG induction (induction control); Lane3, IPTG induction of BL21 transformed with pGEX-2TK/hPTHrP1-34 or pGEX-2TK/hPTHrP1-84; Lane4, The proteins not bound to GSTrap FF column; Lane5, Purified hPTHrP1-34 or hPTHrP1-84 obtained after binding to GSTrap FF column and cleaved by thrombin; Lane6, GST-tag eluted from GSTrap FF column. (f) Western blot analysis of purified recombinant hPTHrP1-34 and hPTHrP1-84 with N-terminal hPTHrP antibody.
Figure 2.
Effects of recombinant hPTHrP1-34 and 1-84 on BMCs.
Cells from 18-day primary BMC cultures incubated in the absence (Control) or presence of 10−7 M hPTHrP1-34 (hPTHrP1-34) or presence of 10−7 M hPTHrP1-84 (hPTHrP1-84) on day 0 were stained cytochemically for ALP (a. CFU-f ALP) and with sirius red for total collagen(c. CFU-f Col) and with the von Kossa method for calcified colonies(e. CFU-f Ca) and with methylene blue to show total CFU-f (g. Total CFU-f) as described in Materials and Methods. The positive number of CFU-f ALP (b), CFU-f Col (d), CFU-f Ca (f) and Total CFU-f number (h) per dish are the mean±SEM of triplicate determinations from three replicate experiments, respectively. (i) Real-time RT-PCR of cells extracts from 14-day primary BMC cultures for the expression of Cbfa I, ALP, Col I and OCN. Messenger RNA expression assessed by real-time RT-PCR is calculated as a ratio to the GAPDH mRNA level and expressed relative to levels of Control group. Each value is the mean ± SEM of three trials. *, P<0.05; **, P<0.01; ***, P<0.001 compared with Control group; #, P<0.05 compared with hPTHrP1-34 group.
Figure 3.
Effects of recombinant hPTHrP1-34 and 1-84 on serum chemistry and on expression of calcium transporters in kidney.
(a) Serum calcium, (b) phosphorus and (c) ALP ratio were determined after 4-week administration in vehicle-treated sham-operated mice (Sham), vehicle-treated OVX mice (Control), hPTHrP1-34-treated OVX mice (hPTHrP1-34) and hPTHrP1-84-treated OVX mice (hPTHrP1-84). (d) Western blots of renal extracts for expression of CB28K, CB9K, NCX1 and TRPV5. β-tubulin was used as loading control for Western blots. (e) CB28K, CB9K, NCX1 and TRPV5 protein levels relative to β-tubulin protein level and expressed relative to levels of Sham group. Each value is the mean ± SEM of determinations in six mice of each group.*, P<0.05; ***, P<0.001 compared with Sham group; #, P<0.05 compared with Control group; △, p<0.05 compared with hPTHrP1-34 group.
Figure 4.
Effects of recombinant hPTHrP1-34 and 1-84 on bone volume.
(a) Representative contact radiographs of thoracic vertebrae. Representative (b) cross-sections and (c) longitudinal sections of 3D reconstructed thoracic vertebrae. Micrographs of decalcified paraffin sections of lumbar vertebrae stained with H&E (d) and total collagen (f). Scale bars represent 200 µm in d and f. Trabecular bone volume relative to the tissue volume [BV/TV (%), e] and the cortical thickness (g) of vertebrae were determined by histomorphometric analysis as described in Materials and Methods. Each value is the mean ± SEM of determinations in six mice of each group. *, P<0.05; **, P<0.01 compared with Sham group; #, P<0.05; ##, P<0.01 compared with Control group; △, P<0.05; △△, P<0.01 compared with hPTHrP1-34 group.
Figure 5.
Effects of recombinant hPTHrP1-34 and 1-84 on osteoblastic bone formation parameters.
(a) Representative micrographs of calcein double labeling and (c) sections stained with the von Kossa procedure in the trabeculae were imaged from ethanol fixed and undecalcified LR white resin embedded sections of vertebrae. (b) MAR of trabeculae was determined. (d) Osteoid volume was determined in undecalcified von Kossa-stained sections and is presented as a percent of bone volume (OV/BV, %) of trabeculae. Micrographs of decalcified paraffin sections of vertebrae stained with H&E (e) and histochemically for ALP (g). (f) Number of osteoblasts per mm bone parameter (N.Ob/B.Pm, #/mm) and (h) ALP positive area as a percent of the tissue area were determined in the vertebrae. Scale bars represent 25 µm in a and c and 50 µm in e and g. (i) Real-time RT-PCR of long bone extracts for the expression of Cbfa I, ALP, Col I and OCN. Messenger RNA expression assessed by real-time RT-PCR is calculated as a ratio to the GAPDH mRNA level and expressed relative to levels of Sham group. Each value is the mean ± SEM of determinations in six mice of each group. *, P<0.05; **, P<0.01 compared with Sham group; #, P<0.05; ##, P<0.01 compared with Control group; △, P<0.05 compared with hPTHrP1-34 group.
Figure 6.
Effects of recombinant hPTHrP1-34 and 1-84 on osteoclastic bone resorption parameters and half-life analyses of recombinant hPTHrP1-34 and 1-84 in vivo.
(a) Representative micrographs of paraffin embedded sections of vertebrae stained histochemically for TRAP and photographed at a magnification of 200. Scale bars in a represents 50 µm. (b) Number of TRAP positive osteoclasts per mm bone parameter (N.Oc/B.Pm, #/mm) and (c) the surface of osteoclasts relative to the bone surface (Oc.S/B.S, %) were determined in the trabeculae of TRAP-stained vertebrae. Each value is the mean ± SEM of determinations in 6 animals of each group. (d) Real-time RT-PCR was performed on long bone extracts for RANKL and OPG mRNA as described in Materials and Methods. Messenger RNA expression assessed by real-time RT-PCR analysis is calculated as a ratio to the GAPDH mRNA level and expressed relative to levels of Sham group. Ratio of RANKL/OPG relative mRNA levels was calculated and was presented as the mean ± SEM of determinations in six animals of each group. (e) Plasma immunoreactive PTHrP determined using ELISA after subcutaneous injection of vehicle or hPTHrP1-34 or hPTHrP1-84. Blood samples were drawn at the following time points after injection of study drugs: 15 and 30 minutes, then 1, 2, 4, 6 hours. Each value is the mean ± SEM of determinations in 6 animals of each group. *, P<0.05; **, P<0.01 compared with Sham group.