Fetal Bone Cells Research Articles

Transforming Growth Factor-β Regulation of Collagenase, 72Kda-Progelatinase, Timp and Pai-1 Expression in Rat Bone Cell Populations and Human Fibroblasts

Quiescent cultures of normal fetal rat calvarial bone cell populations (RC I and RC IV) and human fibroblasts were incubated with 1.0 ng/ml TGF-beta and the conditioned culture media were processed individually to separate collagenase and 72 kDa-progelatinase from TIMP, the tissue inhibitor of matrix metalloendoproteinases, using mini-columns of heparin- and gelatin-Sepharose. Collagenase synthesis was decreased progressively by TGF-beta in fibroblasts despite a 1.6-fold increase in secreted protein levels and a approximately 1.8-fold increase in 72 kDa-progelatinase synthesis. The human fibroblasts and the osteoblast-enriched RC IV cells showed a greater TGF-beta-induced stimulation in 72 kDa-progelatinase levels over controls compared with the RC I cells. In contrast to RC IV cells, in which TIMP mRNA levels were increased 2.9-fold by TGF-beta, the constitutive level of TIMP transcripts in the RC I cells was greater than 20-fold over that of the RC IV cells, but was not elevated by TGF-beta. TGF-beta also increased TIMP expression in fibroblasts approximately 1.7-fold and PAI-1 levels approximately 5-fold in RC IV cells, and greater than 10-fold in fibroblasts.

Parathyroid hormone modulates transforming growth factor beta activity and binding in osteoblast-enriched cell cultures from fetal rat parietal bone.

Transforming growth factor beta (TGF-beta) is produced by bone cells, is abundant in bone matrix, and regulates bone cell biochemical processes. In osteoblast-enriched fetal rat parietal bone cell cultures, low TGF-beta doses increase DNA synthesis, whereas higher levels are less mitogenic, stimulate collagen production, and decrease alkaline phosphatase activity. Parathyroid hormone by itself has minimal effects on these processes, but it opposes the effects of TGF-beta and alters TGF-beta binding to its receptors in osteoblast-enriched cultures. Some functions ascribed to parathyroid hormone in bone may therefore result from alterations in TGF-beta activity, suggesting that the local effects of TGF-beta in bone are under systemic hormonal control.

Response of cultured bone cells to a capacitively coupled electric field: inhibition of cAMP response to parathyroid hormone.

Fetal rat calvarial bone cells grown in monolayers were exposed to a capacitively coupled electrical field of 2.62 mV/cm for 2.5-30 min. There was a 59% increase in cAMP concentration after electrical stimulation, compared with a 3615% increase after parathyroid hormone (PTH) administration. PTH administration after electrical stimulation resulted in less cAMP synthesis than following either electrical stimulation alone or PTH administration alone. When PTH administration was delayed for 30 min following cessation of the electrical stimulation, there was an inhibition in the cAMP response. This suggests that one possible mechanism of electrically induced osteogenesis is the desensitization of the bone cell to PTH. Since the effect of PTH is to increase bone resorption, desensitizing or blocking the effects of PTH at the bone cell level would result in a net increase in bone formation.

Differential effects of transforming growth factor-beta on the synthesis of extracellular matrix proteins by normal fetal rat calvarial bone cell populations.

To determine the effects of transforming growth factor-beta (TGF-beta) on the different cell types that exist in bone, cell populations (I-IV), progressively enriched in osteoblastic cells relative to fibroblastic cells, were prepared from fetal rat calvaria using timed collagenase digestions. TGF-beta did not induce anchorage-independent growth of these cells, nor was anchorage-dependent growth stimulated in most populations studied, despite a two- to threefold increase in the synthesis of cellular proteins. In all populations the synthesis of secreted proteins increased 2-3.5-fold. In particular, collagen, fibronectin, and plasminogen activator inhibitor synthesis was stimulated. However, different degrees of stimulation of individual proteins were observed both within and between cell populations. A marked preferential stimulation of plasminogen activator inhibitor was observed in each population, together with a slight preferential stimulation of collagen; the effect on collagen expression being directed primarily at type I collagen. In contrast, the synthesis of SPARC (secreted protein acidic rich in cysteine/osteonectin was stimulated approximately two-fold by TGF-beta, but only in fibroblastic populations. Collectively, these results demonstrate that TGF-beta stimulates matrix production by bone cells and, through differential effects on individual matrix components, may also influence the nature of the matrix formed by different bone cell populations. In the presence of TGF-beta, osteoblastic cells lost their polygonal morphology and alkaline phosphatase activity was decreased, reflecting a suppression of osteoblastic features. The differential effects of TGF-beta on bone cell populations are likely to be important in bone remodeling and fracture repair.

Mitogenesis in fetal rat bone cells simultaneously exposed to type beta transforming growth factor and other growth regulators.

Type beta transforming growth factor (TGF-beta) is found in large amounts in bone tissue, and is a potent mitogen for osteoblast-enriched cell cultures obtained from fetal rat parietal bone. Because other local and systemic factors may be presented to bone cells simultaneously with TGF-beta, it is important to understand the effects of this complex growth regulator in such circumstances. Unlike the effects observed in many tissue systems, TGF-beta does not invariably inhibit the mitogenic response of bone cells to other growth promoters. In contrast, other factors such as epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and type alpha tumor necrosis factor (TNF-alpha) limit the response of osteoblastic bone cells to TGF-beta. TGF-beta is a much weaker mitogen for fibroblastic cells obtained from fetal rat bone, whereas fetal bovine serum, EGF, bFGF, and TNF-alpha are more potent stimulators. In addition, TGF-beta does not significantly impair the response of the fibroblastic bone cells to the other tested agents. These findings reinforce a role of TGF-beta as an anabolic bone growth regulator, and suggest that its function may be modified by other local or systemic agents that can also affect bone cells.

Human bone cellsin vitro

Human bone cell cultures were established by maintaining collagenase-treated, bone fragments in low Ca++ medium. The resulting cell cultures exhibited a high level of alkaline phosphatase activity and produced a significant increase in intracellular cAMP when exposed to the 1-34 fragment of human parathyroid hormone. With continued culture, the cells formed a thick, extracellular matrix that mineralized when cultures were provided daily with normal levels of calcium, fresh ascorbic acid (50 μg/ml) and 10 mM β-glycerol phosphate. Biosynthetically, these cells produced type I collagen (without any type III collagen), and the bone-specific protein, osteonectin. In addition, the cells produced sulfated macromolecules electrophoretically identical to those positively identified as the bone proteoglycan in parallel cultures of fetal bovine bone cells. This technique provides a useful system for the study of osteoblast metabolismin vitro.

Identification and control of collagenase secreted by fetal rat bone cells in culture

Identification and control of collagenase secreted by fetal rat bone cells in culture

Characterization of insulin-like growth factor I receptors on cultured rat bone cells: regulation of receptor concentration by glucocorticoids.

A specific receptor for insulin-like growth factor I (IGF-I) has been demonstrated in cultured fetal rat osteoblast-like bone cells. Specific binding of [125I]IGF-I to bone cells incubated at 15 C reached a steady state by 5 h. Half-maximal inhibition of [125I]IGF-I binding by unlabeled IGF-I was observed at 7 ng/ml. Multiplication-stimulating activity, insulin, and proinsulin were less effective than unlabeled IGF-I in competing for receptor occupancy. Scatchard analysis showed a curvilinear plot, with a Ka similar to that observed in human fibroblasts. Incubation of cell monolayers with glucocorticoids resulted in a concentration-dependent increase in [125I]IGF-I binding. This increase in [125I]IGF-I binding was dependent on cell density. After a 2-day exposure to dexamethasone, no increase in binding was observed in sparsely plated cells; however, an increase in binding was observed after 3 days in culture (log phase) and was maximal by 5 days (peak log phase). These data indicate that rat bone cells possess a specific receptor for IGF-I with binding characteristics similar to those reported in human fibroblasts, and that IGF-I receptor concentrations are increased by exposure to glucocorticoids. A role for glucocorticoids and IGF-I in rat bone proliferation is suggested by these findings.

Fetal bovine bone cells synthesize bone-specific matrix proteins.

We isolated cells from both calvaria and the outer cortices of long bones from 3- to 5-mo bovine fetuses. The cells were identified as functional osteoblasts by indirect immunofluorescence using antibodies against three bone-specific, noncollagenous matrix proteins (osteonectin, the bone proteoglycan, and the bone sialoprotein) and against type 1 collagen. In separate experiments, confluent cultures of the cells were radiolabeled and shown to synthesize and secrete osteonectin, the bone proteoglycan and the bone sialoprotein by immunoprecipitation and fluorography of SDS polyacrylamide gels. Analysis of the radiolabeled collagens synthesized by the cultures showed that they produced predominantly (approximately 94%) type I collagen, with small amounts of types III and V collagens. In agreement with previous investigators who have employed the rodent bone cell system, we confirmed in bovine bone cells that (a) there was a typical cyclic AMP response to parathyroid hormone, (b) freshly isolated cells possessed high levels of alkaline phosphatase, which diminished during culture but returned to normal levels in mineralizing cultures, and (c) cells grown in the presence of ascorbic acid and beta-glycerophosphate rapidly produced and mineralized an extracellular matrix containing largely type I collagen. These results show that antibodies directed against bone-specific, noncollagenous proteins can be used to clearly identify bone cells in vitro.

Vitamin D and Pregnancy: The Maternal-Fetal Metabolism of Vitamin D

A model of the maternal-fetal metabolism of vitamin D3 is depicted in Fig. 2. 25-OHD3 of maternal origin is metabolized by the maternal kidneys to the potent metabolite, 1,25-(OH)2D3, which acts on the maternal intestine, kidneys, and skeleton. The maternal kidneys and other organs can produce 24,25-(OH)2D3, although this pathway may be suppressed near the end of gestation. The placenta has selective permeability to the vitamin D3 metabolites, with 25-OHD3 crossing from the mother to the fetus more readily than the dihydroxylated metabolites. The onset of the placental synthesis of 1,25-(OH)2D3 during gestation is unknown. Likewise the regulation of the placental 25-OHD3-1 alpha-hydroxylase is unknown. 1,25-(OH)2D3 of placental origin may enter the maternal or the fetal circulation or act locally on the placenta by inducing the synthesis of proteins involved in the cellular transport of Ca. Perhaps one placenta cell type synthesizes 1,25-(OH)2D3 and another cell type possessing a cytoplasmic receptor for 1,25-(OH)2D3 responds to this metabolite. The function of the 24,25-(OH)2D3 produced by the placenta is unknown. The concentration of free 25-OHD3 and free 1,25-(OH)2D3 in the fetal circulation exceeds the maternal levels due to the differences in the DBP concentrations of the two bloodstreams. The 1,25-(OH)2D3 in the fetal bloodstream may originate from either the placenta or the fetal kidneys. The latter site may not be active in utero due to the hypercalcemia and hyperphosphatemia relative to the maternal levels of these ions. 1,25-(OH)2D3 in the fetal bloodstream acts on those fetal tissues containing cytoplasmic receptors for this metabolite. The intestinal mucosa apparently lacks these receptors until sometime during neonatal life. In contrast, fetal bone cells possess receptors for the 1,25-(OH)2D3. The 24,25-(OH)2D3 in the fetal bloodstream may also be involved in the growth and differentiation of the fetal skeleton. However, the precise role of both metabolites in the fetus remains conjectural.

Isolation of specific bone cell types by free-flow electrophoresis.

The bone cells and fibroblasts from fetal rat calvaria can be isolated by collagenase digestion of the collagen matrix and separated into specific cell types by free-flow electrophoresis. The method involves injection of a specially prepared suspension of cells into a stream of buffer across which is maintained an electric field of 60 V/cm. The fetal bone cell types are differentially deflected toward the anode where they can be collected. Free-flow electrophoresis of this heterogenous cell preparation yields three distinguishable peaks which can be identified by morphologic, morphometric, and enzymatic characteristics. All three cell peaks have greater than 95% viability as judged by trypan blue exclusion and will grow to confluent monolayers in culture. The data indicate that these cell peaks may be comprised of osteoclasts and/or preosteoclasts, osteoblasts and/or preosteoblasts, and fibroblasts.

Dissociation of specific binding of 25-OH-D 3 and resorption in fetal rat bones

Specific binding of 25-OH-D 3 was measured in cytosol prepared from isolated fetal rat bone cells. Binding was significant after 2 h incubation at 0°C and appeared to be approaching a plateau at 4 h. Binding was half-maximal at 1.7 × 10 −10 M 25-OH-D 3. 24(R),25-(OH) 2D 3, which was approximately equipotent with 25-OH-D 3 in bone culture, had approximately the same binding activity. 1,25-(OH) 2D 3, which was 2–3 orders of magnitude more active on resorption, was a much weaker competitor for binding. Vitamin D 3, which was inactive in culture, was at least as effective a competitor as 1,25-(OH) 2D 3. The results suggest that the cytosol site which specifically binds 25-OH-D 3 is not the mediator of the bone-resorbing activity.

Responses of fetal rat bone cells and bone organ cultures to the ionophore, A23187

The ionophore A23187 produced a rapid transient increase in the rate of calcium uptake by isolated fetal rat bone cells. There was no effect on calcium efflux or total cellular calcium. The magnitude of the effect on influx was amplified when the cell were incubated at 4 degrees C. Cellular metabolic functions and resorption of cultured fetal rat bones (release of 45Ca from pre-labeled long bone) were affected by A23187 in a biphasic manner: cell cyclic AMP (cAMP) was increased by 0.1 and 0.3 mug/ml of the ionophore, whereas 10 mug/ml was either ineffective or lowered the cAMP levels. The high A23187 concentration abolished the stimulatory effects of parathyroid hormone and methylisobutylxanthine. Concentrations of 0.1 and 0.3 mug/ml A23187 stimulated bone resorption. The effect was abolished by calcitonin. Ionophore concentrations above 1 mug/ml produced less bone resorption. These higher concentrations antagonized the bone-resorbing effect of parathyroid hormone and 1,25-dihydroxyvitamin D3. A23187 at 5 and 10 mug/ml decreased bone cell lactate and ATP. Thus at low concentrations, A23187 produced effects on bone similar to those of parathyroid hormone, suggesting that calcium is the primary initiator of PTH-induced bone resorption. At the higher concentrations A23187 may have a general inhibitory effect on cell metabolism.