In vitro evidence that bone formation may be coupled to resorption by release of mitogen(s) from resorbing bone

Abstract

Bone-derived proteins have been shown to stimulate the proliferation of bone-forming cells and to increase the rate of embryonic bone formation in vitro. The current studies were intended (1) to determine the tissue distribution of bone cell-active mitogen(s) in the embryonic chick, (2) to determine the cellular origin and the target cell specificity of the bone cell-active mitogen(s) in embryonic chick bone, (3) to determine whether the release of mitogenic activity from embryonic chick tibiae was proportional to bone resorption, and (4) to compare mitogenic activities prepared from different skeletal sources, with respect to M r, chemical stability, and mitogen activity kinetics. A bone cell-active mitogen(s) was identified in extracts of bone and cartilage but not in extracts of muscle, liver, intestine, or brain. (Mitogenic activity was determined as increased incorporation of 3[H]-thymidine into DNA in serum-free, calvarial cell cultures.) Together, the following three observations indicate an osteoblastic origin for the bone cell-active mitogen(s) in chick bone. First, the mitogen content of embryonic chick tibiae increased 4.5-fold, during eight days of serum-free in vitro growth ( P < .005). Second, conditioned medium (CM) from serum-free monolayer cultures of calvarial cells contained bone cell-active mitogen(s), but CM from parallel cultures of skin, liver, and intestinal cells did not. And, finally, the amount of bone cell-active mitogen(s) in calvarial cell CM was correlated with the amount of alkaline phosphatase (ALP) activity per cell, ie, an index of osteoblastic differentiation ( r = .92, P < .005). Treatment of calvarial cells with cycloheximide caused parallel decreases in protein synthesis and mitogen release ( r = .99, P < .01), indicating that the mitogen was not stored in calvarial cells. Mitogenic activities prepared from calvarial cell CM, tibial CM, and EDTA-extracts of tibiae increased 3[H]-thymidine incorporation in calvarial cell cultures, but not in parallel cultures of skin, liver or intestinal cells. Furthermore, the mitogenic response of calvarial cells to these activities was inversely proportional to ALP activity/cell ( r = .97, P < .001), suggesting a relative specificity for osteoprogenitor cells. In the presence of skeletal effectors (PTH and Cl 2MDP) the release of bone cell-active mitogen(s) from embryonic chick tibiae was proportional to bone resorption ( r = .915, P < .005) and inversely proportional to bone formation ( r = −.80, P < .05). When cycloheximide was added to this system we found that mitogen release was still correlated with resorption ( r = .996, P < .001), but not with protein synthesis ( r = .129), suggesting that the mitogen in CM did not reflect newly synthesized protein. Bone cell-active mitogen(s), prepared from calvarial cell CM, tibial CM, and EDTA-extracts of chick bone, could not be distinguished from each other on the basis of M r, chemical stability, or mitogen activity kinetics. Together, these data indicate that a bone cell-active mitogen(s) is produced by osteoblasts, deposited in bone, released in proportion to resorption, and relatively specific for osteoblast progenitor cells, and these observations are consistent with the hypothesis that osteoblasts may produce a delayed paracrine effector of bone volume. Since we also observed that the amount of ALP activity in calvarial cell cultures was directly proportional to mitogen production, but inversely proportional to mitogen response, these data further indicate that this mitogenic activity may be an acute paracrine effector of calvarial cell proliferation in vitro.