Stimulation by defined parathyroid hormone fragments of cell proliferation in skeletal-derived cell cultures

Abstract

We have reported previously that parathyroid hormone (PTH) acts on cultured bone cells to stimulate creatine kinase (CK) activity and [3H]thymidine incorporation into DNA via phosphoinositide turnover, in addition to its other actions via increased cyclic AMP production. We also found that mid-region fragments of PTH stimulate [3H]thymidine incorporation into avian chondrocytes. In the present study of mammalian systems, we demonstrate differential effects of defined synthetic PTH fragments on CK activity and DNA synthesis, as compared with cyclic AMP production, in osteoblast-enriched embryonic rat calvaria cell cultures, in an osteoblast-like clone of rat osteosarcoma cells (ROS 17/2.8) and in chondroblasts from rat epiphysial cartilage cell cultures. Unlike full-length bovine (b)PTH-(1-84) or the fully effective shorter fragment human (h)PTH-(1-34), fragments lacking the N-terminal region of the hormone did not increase cyclic AMP formation, whereas they did stimulate increases in both DNA synthesis and CK activity. Moreover, the PTH fragment hPTH-(28-48) at 10 /M inhibited the increase in cyclic AMP caused by 10 nm-bPTH-(1-84). The increase of CK activity in ROS 17/2.8 cells caused by bPTH-(1-84) or hPTH-(28-48) was completely inhibited by either cycloheximide or actinomycin D, as was shown previously for rat calvaria cell cultures. These results indicated the presence of a functional domain of PTH in the central part of the molecule which exerts its mitogenic-related effects on osteoblast-and chondroblast-like cells in a cyclic AMP-independent manner. Since cyclic AMP formation by PTH leads to bone resorption, specific mid-region fragments of PTH might prove suitable for use in vivo to induce bone formation without concomitant resorption. INTRODUCTIONIn conjunction with its regulation of calcium homeostasis, parathyroid hormone (PTH) can stimulate both net bone formation and resorption, depending on the conditions of application. Increased levels of cyclic AMP [1] are considered to mediate the action of PTH in causing bone resorption [2] and stimulation of ornithine decarboxylase activity [3,4]. On the other hand, PTH has also been shown to exert proliferative and anabolic effects,

Stimulation of cell proliferation in skeletal tissues of the rat by defined parathyroid hormone fragments

“…The increases are dose-and time-dependent, as shown previously using in vitro models of chondrocytes [32] and both osteoblast-enriched calvaria cell cultures and ROS 17/2.8 osteoblast-like cells [33]. These results are in accord with data in vivo for stimulation of proliferation by bPTH-(1-84) and bPTH-(1-34) in ovariectomized rats [38] and intact rats [30,38,39], dogs [4] and humans [30].…”

“…To corroborate that under the circumstances of these experiments the increase in CK specific activity in bone is indeed a valid marker of cell proliferation, changes in DNA synthesis were measured by [3H]thymidine incorporation [33]. The earliest time at which PTH increased [3H]thymidine incorporation was determined by injecting rats with an optimal dose of 2,ug of PTH-(1-34)/rat, equivalent to 13 nm (Fig.…”

“…Biochemicals were obtained from Sigma (St. Louis, MO, U.S.A.). hPTH- (25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39) and hPTH-(34-47) were synthesized at the Gesellschaft fur Biotechnologische Forschung, Braunschweig, Germany.…”

“…2); namely, significant increases at all three times measured in the diaphysis and epiphysis, and at 1 and 24 h in kidney. The mid-portion fragment hPTH-(28-48), which has mitogenic activity in vitro [32,33], also increased CK activity after injection of 1.25 ,ug/rat, equivalent to 14 nm (Fig. 3).…”

“…Moreover, increased bone formation in hyperparathyroid states in man [31] and animals [23,24] has also been demonstrated. Recently we have shown that the central portion of the PTH molecule increases cell proliferation in chondrocytes [32] and in osteoblasts [33] without affecting cyclic AMP production. Therefore it was important to determine whether these fragments from the mid region of the PTH molecule can induce proliferation of bone cells in vivo and thus have the potential to be used to increase bone formation in pathological conditions such as osteoporosis.…”