Stimulation of creatine kinase activity in rat skeletal tissue in vivo and in vitro by protease-resistant variants of parathyroid hormone fragments.
We have reported that mid-region fragments of human parathyroid hormone (hPTH), exemplified by hPTH-(28-48), stimulated [3H]thymidine incorporation into DNA and increased the specific activity of the brain-type isoenzyme of creatine kinase (CK) in both skeletal-derived cell cultures (ROS 17/2.8 cells) and immature rat epiphyseal cartilage and diaphyseal bone, without stimulating cyclic AMP synthesis which is a prerequisite for bone resorption. In the present study, substitution of amino acids in hPTH-(28-48), which resulted in increased resistance to proteolysis, produced variants that stimulated skeletal systems at two orders of magnitude lower concentration than the wild-type fragment. We modified hPTH-(28-48) at Leu-37 by replacement with Met, Thr or Val. Under conditions in which 20% of the native hPTH-(28-48) resisted proteolysis by cathepsin D for 6 h, approx. 40% of the L37V mutant and 70% of the L37T mutant remained intact. Substitution of Met for Phe-34 in addition to Thr for Leu-37, or the substitution of Met for Phe-34 alone, produced 100%-resistant fragments. These variants at residue 34 caused maximal stimulation of CK in ROS 17/2.8 cells at 0.24 nM compared with 24 nM for hPTH-(28-48). The double mutant stimulated CK activity significantly in immature rats, at a minimum dose of 12.5 ng/rat, and caused maximal stimulation at 125 ng/rat, a 10-fold lower dose than for hPTH-(28-48). The effect of the double mutant lasted up to 24 h which differs from the stimulation by hPTH-(28-48) in which CK specific activity returns to the control level at 24 h. This same dose also significantly stimulated CK activity in gonadectomized rats. These results show the advantage of using protease-resistant mid-region variants of hPTH-(28-48) to stimulate bone cells, in terms of lower doses and longer duration of effectiveness, both in vitro and in vivo.
Sömjen
D
D
Tel Aviv Medical Center, Sackler Faculty of Medicine, Tel Aviv University, Israel.
Vargas
V
V
Waisman
A
A
Wingender
E
E
Tegge
W
W
Kaye
A M
AM
D000595:Amino Acid Sequence; D000818:Animals; D001842:Bone and Bones / Q000201:enzymology*; D002460:Cell Line; D003402:Creatine Kinase / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D010450:Endopeptidases / Q000378:metabolism*; D004789:Enzyme Activation; D005260:Female; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D010024:Osteoporosis / Q000628:therapy; D010281:Parathyroid Hormone / Q000737:chemistry / Q000378:metabolism / Q000502:physiology* / Q000627:therapeutic use; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D051381:Rats; D017208:Rats, Wistar
D010281:Parathyroid Hormone; D010446:Peptide Fragments; C069678:parathyroid hormone (28-48); D003402:Creatine Kinase; D010450:Endopeptidases