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


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.
Hildreth
Blake Eason
BE
Department of Veterinary Clinical Sciences, College of Veterinary Medicine, The Ohio State University, Columbus, Ohio 43210-1089, USA.
Werbeck
Jillian L
JL
Thudi
Nandu K
NK
Deng
Xiyun
X
Rosol
Thomas J
TJ
Toribio
Ramiro E
RE
D016475:3T3 Cells / Q000166:cytology / Q000187:drug effects / Q000502:physiology; D000469:Alkaline Phosphatase / Q000187:drug effects / Q000378:metabolism; D000818:Animals; D001846:Bone Development / Q000187:drug effects*; D002113:Calcification, Physiologic / Q000187:drug effects; D002294:Carcinoma, Squamous Cell / Q000235:genetics; D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D004355:Drug Stability; D006801:Humans; D051379:Mice; D010006:Osteoblasts / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D015675:Osteocalcin / Q000187:drug effects / Q000378:metabolism; D010281:Parathyroid Hormone / Q000627:therapeutic use*; D044162:Parathyroid Hormone-Related Protein / Q000235:genetics / Q000627:therapeutic use*; D010446:Peptide Fragments / Q000235:genetics / Q000627:therapeutic use*; D012333:RNA, Messenger / Q000235:genetics
D010281:Parathyroid Hormone; D044162:Parathyroid Hormone-Related Protein; D010446:Peptide Fragments; D012333:RNA, Messenger; C068871:parathyroid hormone-related protein (1-86); D015675:Osteocalcin; C066078:parathyroid hormone-related protein (1-141); D000469:Alkaline Phosphatase