PTHrP 1-141 and 1-86 increase in vitro bone formation

Abstract

Background: Parathyroid hormone-related protein (PTHrP) has anabolic effects in bone, which has led to the clinical use of N-terminal fragments of PTHrP and PTH. Since 10% to 20% of fractures demonstrate healing complications and osteoporosis continues to be a debilitating disease, the development of bone-forming agents is of utmost importance. Due to evidence that regions of PTHrP other than the N-terminus may have bone-forming effects, this study was designed to compare the effects of full-length PTHrP 1-141 to N-terminal PTHrP 1-86 on in vitro bone formation.

Materials and methods: MC3T3-E1 pre-osteoblasts were treated once every 6 d for 36 d with 5, 25, and 50 pM of PTHrP 1-141 or 1-86 for 1 or 24 h. Cells were also treated after blocking the N-terminus, the nuclear localization sequence (NLS), and the C-terminus of PTHrP, individually and in combination. Area of mineralization, alkaline phosphatase (ALP), and osteocalcin (OCN) were measured.

Results: PTHrP 1-141 and 1-86 increased mineralization after 24-h treatments, but not 1-h. PTHrP 1-141 was more potent than 1-86. Treatment with PTHrP 1-141 for 24-h, but not 1-86, resulted in a concentration-dependent increase in ALP, with no effect after 1-h. Exposure to both peptides for 1- or 24-h induced a concentration-dependent increase in OCN, with 24-h exceeding 1-h. Antibody blocking revealed that the NLS and C-terminus are anabolic.

Conclusions: Both PTHrP 1-141 and 1-86 increased in vitro bone formation; however, PTHrP 1-141 was more effective. The NLS and C-terminus have anabolic effects distinct from the N-terminus. This demonstrates the advantage of PTHrP 1-141 as a skeletal anabolic agent.

Fig. 1

Treatment protocol for MC3T3-E1 pre-osteoblast mineralization assays with OM or 1- and 24-hr of treatment with vector, PTHrP 1-86, or PTHrP 1-141. Schematic represents (1) 6-day treatment cycle. Grey areas represent exposure to vector, PTHrP 1-86, or PTHrP 1-141 and black areas represent exposure to OM. Black arrows represent when either OM, vector, PTHrP 1-86, or PTHrP 1-141 was aspirated and cells treated with OM. Numbers indicate the day during each 6-day cycle.

Fig. 2

Western blot of PTHrP 1-141 in whole-cell extracts produced by only the LCC15-PTHrP 1-141 cell line (22 kDa band, far right column). From left to right, LCC15-vector and LCC15-PTHrP 1-141 cell lines and each cell line after the addition of a Golgi blocker (GB, Golgi Plug™, BD Biosciences).

Fig. 3

Area of mineralization at day 36 after treatment with OM, vector, PTHrP 1-86, and PTHrP 1-141 for (A) 24-hrs or (B) 1-hr once every 6 days, where PTHrP 1-141 and 1-86 significantly increased mineralization after 24-hr treatments, with minimal effect using 1-hr. In addition, PTHrP 1-141 was more potent than 1-86. Bars represent mean ± 1 standard deviation of three individual wells. Along the x-axis, pM represents the concentration of PTHrP 1-141, PTHrP 1-86, and equivalent pM vector. Within each concentration, “*” indicate a statistically significant difference from OM, “†” from OM and vector, “#” from OM, vector, and PTHrP 1-86, and “Ω” from OM and PTHrP 1-141. Between concentrations, “a” indicates a statistically significant difference from 5 pM, “b” from 25 pM, and “c” from both 5 and 25 pM.

Fig. 4

ALP activity (U/g secreted protein) at day 18 after treatment with OM, vector, PTHrP 1-86, and PTHrP 1-141 for (A) 24-hrs or (B) 1-hr once every 6 days, where treatment with PTHrP 1-141 for 24-hr, but not 1-86, resulted in a concentration-dependent increase in ALP activity, which exceeded 1-86. No effect was observed after 1-hr. See Fig. 3 for legend.

Fig. 5

OCN concentrations (ng/mg secreted protein) at day 36 after treatment with OM, vector, PTHrP 1-86, and PTHrP 1-141 for (A) 24-hrs or (B) 1-hr once every 6 days, where treatment with both peptides for 1- or 24-hrs induced a concentration-dependent increase in OCN secretion; however, 24-hr consistently exceeded 1-hr. See Fig. 3 for legend.

Fig. 6

Area of mineralization (A), ALP activity (B), and OCN concentrations (C) after treatment with 50 pM of PTHrP 1-86 or 1-141 for 24-hr once every 6 days, both alone and after treating PTHrP 1-141 with each of the following individually or all possible combinations thereof: anti-N-terminal antibody, anti-NLS antibody, and anti-C-terminal antiserum. PTHrP 1-86 was treated with only anti-N-terminal antibody. Region-specific blockade reveals that the NLS and C-terminus are capable of eliciting anabolic effects distinct from, or complimentary to, the N-terminus of the peptide. Bars represent mean ± 1 standard deviation of four to six individual wells. Between treatment groups, statistically significant differences from the group in parenthesis are noted by: 1) “*” (PTHrP 1-141), 2) “†” (PTHrP 1-141 and PTHrP 1-141 + N-terminal blockade), 3) “#” (PTHrP 1-141 and all blocking combinations except N- and C-terminal blockade and complete blockade), 4) “Ω” (PTHrP 1-141 and all blocking combinations except NLS and C-terminal blockade), 5) “^” (PTHrP 1-141 and all blocking combinations except NLS and C-terminal blockade and complete blockade, 6) “@” (PTHrP 1-141 and all blocking combinations except complete blockade), and 7) “$” (PTHrP 1-141 and all blocking combinations). Specifically for (C), 1) “%” (PTHrP 1-141 and PTHrP 1-141 + N-terminal blockade, PTHrP 141 + NLS blockade, and PTHrP 1-141 + N-terminus and NLS blockade and 2) “N” (PTHrP 1-86).