Individual and combined effects of intact PTH, amino-terminal, and a series of truncated carboxyl-terminal PTH fragments on alkaline phosphatase activity in dexamethasone-treated rat osteoblastic osteosarcoma cells, ROS 17/2.8

Abstract

The individual and combined effects of intact PTH, amino-terminal, and a series of truncated carboxyl \x=req-\ terminal PTH fragments on alkaline phosphatase activity were examined in dexamethasone-treated rat osteoblastic osteosarcoma cells ROS 17/2.8. Dexamethasone-induced alkaline phosphatase activity was inhibited not only by hPTH(1\p=n-\84) and amino-terminal PTH fragment hPTH(1\p=n-\34), but also by carboxyl-terminal PTH fragment hPTH(69\p=n-\84) in a dose-related fashion. At 10-7 mol/1, hPTH(1\p=n-\84) completely abolished dexamethasone-induced alkaline phosphatase activity, while hPTH(1\p=n-\34) and hPTH(69\p=n-\84) reduced alkaline phosphatase activity to 0.16\m=+-\0.02and 0.80\m=+-\0.03fold, respectively, of the control value obtained in the absence of PTH peptides. The combination of hPTH(1\p=n-\34) and hPTH(69\p=m-\84) resulted in reduction of alkaline phosphatase activity to the level obtained by hPTH(1\x=req-\ 84). The shorter carboxyl-terminal PTH fragment hPTH(71\p=n-\84) did not affect alkaline phosphatase activity or modulate the action of hPTH(1\p=n-\34). The longer carboxyl-terminal PTH fragment hPTH(53\x=req-\ 84) stimulated alkaline phosphatase activity up to 1.23 \ m=+-\ 0.03 fold and partially blunted the inhibitory effect of hPTH(1\p=n-\34) on alkaline phosphatase activity. These findings suggest that carboxyl-terminal PTH fragments could exert diverse effects on the target cells, depending on the length of deletion of amino-terminal amino acids of PTH molecule, and interact with amino-terminal PTH fragment. The two amino-terminal amino acids of hPTH(69\p=n-\84)and the 53\p=n-\68portion of hPTH(53\p=n-\84)might be responsible for the respective inhibitory and stimulatory effects of the peptides on alkaline phosphatase activity.

The [53–63] amino acid portion of the parathyroid hormone molecule is essential for the stimulatory effect of carboxyl-terminal PTH fragments on alkaline phosphatase activity in dexamethasone-treated rat osteoblastic osteosarcoma cells, ROS 17/2.8

“…On the other hand, it was shown that the carboxyl-terminal PTH fragment hPTH (53-84), stimulated dexamethasone-induced ALP activity in the cells [25,26]. In 1993, we confirmed the fact and subsequently demonstrated that intact PTH [hPTH (1-84)] inhibited dexamethasone-induced ALP activity more potently than aminoterminal PTH fragment hPTH (1-34), and that another carboxyl-terminal PTH fragment hPTH (69-84} inhibited, but not stimulated, dexamethasone-induced ALP activity in a dose-related fashion in the cells [27]. In that study, the combination of this carboxyl-terminal PTH fragment hPTH (69-84) and amino-terminal PTH fragment hPTH (1-34) inhibited ALP activity as potently as intact PTH on an equimolar basis, suggesting activity.…”

“…3]. The 69th and 70th amino acid of the PTH molecule was thought crucial for the inhibitory effect of carboxyl-terminal PTH fragments on ALP a c t i v i t y as previously reported [27]. It has been recently reported t h a t the amino-terminal portion of the hPTH (53-84) molecule structurally assumes an initial tight series of turns over which the rest of the molecule folds, and that the carboxylterminal portion of the hPTH (53-84) molecule is anchored to the compact amino-terminal portion of the hPTH (53-84) molecule in two regions through interactions of residues in the region 58-64 with residue 71 and residues 80-84 [28].…”

hPTH‐fragments (53–84) and (28–48) antagonize the stimulation of calcium release and repression of alkaline phosphatase activity by hPTH‐(1–34) in vitro

“…The C-terminal fragment PTH-(53-84) induces also ALP activity in bone cells and regulates calcium influx in chondrocytes [10]. Simultaneous exposure of cells and tissues to these PTH-fragments has been shown to modulate the changes in proliferation and differentiation induced by PTH-(1-34) [11][12][13]. From these studies it is apparent that the N-, C-and mid-regional PTH-fragments are biologically active on bone cells and are involved in the regulation of bone metabolism.…”

Type-1 Parathyroid Hormone (PTH)/PTH-Related Peptide (PTHrP) Receptors Activate Phospholipase C in Response to Carboxyl-Truncated Analogs of PTH(1–34)1

“…Because PKC may be activated independently of PLC, it is possible that the stimulation of PKC previously observed in ROS 17/2 cells was unrelated to activation of PLC via the PTH1R. On the other hand, it is quite possible [perhaps even likely (27)(28)(29)(30)] that ROS 17/2 cells and spleen cells express alternate species of receptors for PTH that could have inhibited the response(s) to hPTH in these cells or that might have supported a PLC-independent activation of PKC by hPTH(1-34) but not by hPTH . The ROS 17/2 cells almost certainly express the same rat PTH1R that we have studied here, because they were the source of the ROS 17/2.8 subclone from which the rat PTH1R cDNA used in our studies originally was cloned (2,5,21).…”