PTH was studied for its effects on bone formation in cultured rat calvariae. 0.01-10 nM PTH stimulated [Hjthymidine incorporation into DNA by up to 4.8-fold. Although continuous treatment with PTH for 24-72 h inhibited [3Hlproline incorporation into collagen, transient (24 h) treatment enhanced 1H1-proline incorporation into collagen 24-48 h after the hormone was removed. The collagen stimulated by PTH was type I and the effect was observed in the periosteum-free bone and was not blocked by hydroxyurea. Furthermore, treatment with 1-100 nM PTH for 24 h increased insulin-like growth factor (IGF) I concentrations by two to fourfold, and an IGF I antibody prevented the PTH stimulation of collagen synthesis, but not its mitogenic effect. In conclusion, continuous treatment with PTH inhibits calvarial collagen, whereas transient treatment stimulates collagen synthesis, and the stimulatory effect is mediated by local production of IGF I.

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Basic fibroblast growth factor (bFGF) was studied for its effects on bone formation in cultured rat calvariae. bFGF at 0.1-100 ng/ml stimulated [3Hlthymidine incorporation into DNA by up to 4.4-fold. bFGF also increased the number of colcemid-induced metaphase arrested cells and the DNA content. Transient (24 h) treatment with bFGF enhanced 1H1-proline incorporation into collagen 24-48 h after the factor was removed; this effect was DNA synthesis dependent and blocked by hydroxyurea. The collagen stimulated by bFGF was type I, and this effect was observed primarily in the periosteum-free bone. In contrast, continuous treatment with bFGF for 24-96 h inhibited VHlproline incorporation into type I collagen. bFGF did not alter collagen degradation. In conclusion, bFGF stimulates calvarial DNA synthesis, which causes an increased number of collagen-synthesizing cells, but bFGF has a direct inhibitory effect on collagen synthesis.

Insulin-like growth factors I and II (IGF-I and -II) are polypeptides secreted by skeletal cells and are considered regulators of bone formation. IGF-I and -II were studied for their effects on collagen synthesis and degradation in cultures of intact fetal rat calvariae and on type I collagen transcript levels in osteoblast-enriched (Ob) cells from fetal rat parietal bone. IGF-I and -II increased [3H]proline incorporation into type I collagen independently of their effect on cell replication. IGF-I and -II also decreased collagen degradation in calvarial cultures. Both factors had similar actions, although IGF-I stimulated collagen synthesis at 10 nM, and IGF-II at 30 nM. In Ob cells, IGF-I and -II also increased [3H]proline incorporation into type I collagen, but the effect was seen at 100 nM, and neither factor decreased collagen degradation. Slot blot analysis of IGF-I- and IGF-II-treated cells, using a rat type I collagen cDNA probe, revealed an increase in type I collagen transcripts. In conclusion, IGF-I and -II increase bone collagen synthesis and decrease collagen degradation in cultures of intact calvariae; the effect on collagen synthesis correlates with an increase in transcript levels in Ob cells.

Transforming growth factor beta (TGF-beta), a potent regulator of bone formation, has bifunctional effects on osteoblast replication and biochemical activity that appear differentiation dependent. We now show that cell surface binding sites for TGF-beta vary markedly among fibroblasts, bone-derived cells, and highly differentiated osteosarcoma cultures from fetal rats. Expression of betaglycan and type II receptors decline relative to type I receptor expression in parallel with an increase in osteoblast-like activity, predicting that the ratio among various TGF-beta binding sites could influence how its signals are perceived. Bone morphogenetic protein 2 (BMP-2), which induces osteoblast function, does not alter TGF-beta binding or biochemical activity in fibroblasts and has only small effects in less differentiated bone cells. In contrast, BMP-2 rapidly reduces TGF-beta binding to betaglycan and type II receptors in osteoblast-enriched primary cell cultures and increases its relative binding to type I receptors in these cells and in ROS 17/2.8 cultures. Pretreatment with BMP-2 diminishes TGF-beta-induced DNA synthesis in osteoblast-enriched cultures but synergistically enhances its stimulatory effects on either collagen synthesis or alkaline phosphatase activity, depending on the present state of bone cell differentiation. Therefore, BMP-2 shifts the TGF-beta binding profile on bone cells in ways that are consistent with progressive expression of osteoblast phenotype, and these changes distinguish the biochemical effects mediated by each receptor. Our observations indicate specific stepwise actions by TGF-beta family members during osteoblast differentiation, developing in part from changes imprinted by BMP-2 on TGF-beta receptor stoichiometry.

PTH stimulates bone resorption and formation, but the mechanism of its anabolic effect is unknown. The effects of PTH on bone formation could be mediated by local regulators, either by altering their binding to receptors or by modulating their synthesis. Cell extracts from PTH-treated osteoblast-enriched cultures isolated from fetal rat parietal bones were examined by Northern blot analysis for changes in mRNAs encoding insulin-like growth factor I (IGF-I), transforming growth factor-beta, and beta 2-microglobulin. PTH did not influence transforming growth factor-beta or beta 2-microglobulin transcript levels. In contrast, PTH-(1-34) had a biphasic stimulatory effect on IGF-I transcript levels; 0.1-10 nM PTH increased IGF-I transcripts by 100-200% after a 6-h treatment, while 100 nM PTH induced a 100% increase. In addition, PTH at 0.01-10 nM increased immunoreactive IGF-I (iIGF-I) in culture medium by 40-200% at 24 h. Maximal increases in IGF-I transcripts occurred at 6 h, while iIGF-I accumulated throughout 24 h of culture. These results are compared to the effects of 0.5-50 nM GH, which increased IGF-I transcripts by 30% and iIGF-I by 50-100%. Therefore, PTH enhanced local IGF-I synthesis by increasing IGF-I transcripts, and this effect may in part mediate the anabolic actions of PTH on bone.

Prostaglandin E2 (PGE2) affects both bone resorption and formation, but its mechanism of action remains unclear. PGE2 is known to elevate intracellular cAMP levels in a variety of culture systems. Agents that increase cAMP in primary osteoblast-enriched (Ob) cultures enhance the synthesis of skeletal insulin-like growth factor I (IGF-I), a potent anabolic factor for bone. A 5 min exposure to PGE2 at 0.01-1 microM enhanced cAMP synthesis in Ob cultures by 8- to 54-fold, and within 6 h produced up to a 3- fold increase in steady state prepro-IGF-I transcripts. The stimulatory effect of PGE2 on IGF-I messenger RNA was first evident within 4 h of treatment and remained elevated for at least 24 h. Furthermore, 0.01-1 microM PGE2 increased immunoreactive IGF-I polypeptide accumulation by 1.9- to 4.7-fold. In contrast PGE2 did not elevate steady state IGF-II mRNA or polypeptide levels within this time frame. Although PTH increase cAMP, intracellular calcium, and PGE2 production by bone cells, our previous studies indicate that the stimulatory effect of PTH on IGF-I production is cAMP, but not calcium dependent. Inhibition of PGE2 synthesis by exposure to indomethacin did not alter basal or PTH-stimulated IGF-I levels, substantiating that the effect of PTH on IGF-I is not PGE2 dependent. These studies indicate that PGE2 production, a feature common to many agents that enhance bone resorption, could contribute to the coupling of bone resorption and new bone formation, by way of its ability to increase cAMP and, consequently, IGF-I synthesis by the osteoblast.