A mouse tumor-derived osteolytic factor stimulates bone resorption by a mechanism involving local protaglandins production in bone

Culture medium which was conditioned by tissue of a CE mouse breast tumor in vitro containes dose-dependent osteolytic activity. The osteolytic activity was not soluble in dichloromethane and ethylacetate, indicating that it was not attributable to vitamine D metabolites or prostaglandins. HOwever, breast tumor-conditioned medium stimulated production and release of prostaglandin E₂ from mouse calvaria in vitro, and the stimulation of bone resorption in vitro by breast tumor-conditioned medium was blocked by a dose of indomethacin that prevented stimulation of mouse calvarial prostaglandin E₂ production and release. The resorptive activity of parathyroid hormone(PTH) was not affected by the same dose of indomethacin, suggesting that the osteolytic factor was not PTH. This was further supported by observation that mouse kidney cell cAMP production was stimulated by PTH, but not only by the aqueous phase of ethylacetate-extracted breast tumor-conditioned medium. In addition to osteolytic activity, breast tumor-conditioned medium contained a dose-dependent bone cell mitogenic activity, demonstrated by the stimulation of [³H]thymidine incorporation into trichloroacetic acid-insoluble macromolecules and a corresponding increase in bone cell number in monolayer cultures of bone cells. Breast tumor-conditioned medium also contained a dose-dependent transforming growth factor-(TGF)-like activity as defined by its ability to transform anchorage-dependent growth of nontransformed cells to anchorage-independent growth. The TGF in breast tumor-conditioned medium did not compete with epidermal growth factor (EGF) for EGF receptor binding, but its transforming activity was greatly enhanced by EGF, indicating that it was a β-type TGF. Both the osteolytic and mitogenic activities were nondialyzable, sensitive to reducing agent, and not removable by dichloromethane and ethylacetate extractions. Furthermore, the TGF activity was not removed by the ethylacetate extraction. Thus, the possibility that these activities in breast tumor-conditioned medium might be mediated by the same molecule must be considered. In summary, our data suggest that the CE mouse mammary carcinoma cells produce and secret into the culture medium an osteolytic factor which is neither PTH nor prostaglandin and which stimulates local synthesis in bone of prostaglandin E₂ which in turn increased bone resorption in vitro.     

Prostaglandin-related bone resorption in cultured neonatal mouse calvaria: evaluation of biopotency of nonsteroidal anti-inflammatory drugs

The objective of this study was the development of an assay based on suppression of endogenous prostaglandin synthesis in cultured neonatal mouse calvaria for evaluation of the biopotency of nonsteroidal anti-inflammatory drugs in bone. In preliminary trials, osteolytic activity due to spontaneous prostaglandin production over a 72 h culture period was found highly variable, and could not be stabilized by addition of the common precursor arachidonic acid to the culture medium. Eventually, continuous exposure of mouse calvaria to moderate concentrations of thrombin (greater than or equal to 14 U/ml medium) proved to be satisfactory to achieve stable rates of bone resorption through continuous stimulation of prostaglandin synthesis from endogenous sources. Notably, the extent of net calcium release into the medium was highly reproducible in different experiments. As an example for possible applications of the bioassay, the ability of acemetacin to interfere with prostaglandin synthesis in bone, which had not been assessed before, was evaluated in a comparative assay with indomethacin and acetylsalicylic acid. While 1 X 10(-8) M acemetacin appeared to augment thrombin-induced bone resorption, as did 5 X 10(-6) M acetylsalicylic acid, a dose-dependent inhibition of calcium release was observed between 10(-7)-10(-5) M acemetacin. In this respect, the biopotency of indomethacin was 50 times higher than that of acemetacin and exceeded that of acetylsalicylic acid by a factor of more than 2000. These data could be useful for the appraisal of multiple effects of the investigated drugs on prostaglandin-related bone turnover.     

Androgens increase osteoblast-stimulating activity of human breast cancer cells in vitro

Bone metastases of breast cancers produce not only osteolytic but also osteosclerotic lesions. The latter are often observed after androgenic treatment of the tumor. Potential production of osteoblast stimulating activity (ObSA) in breast cancer cell lines, and possible androgen control of this activity have been investigated. Conditioned media (CM) collected from 4 breast cancer cell lines (MCF-7, ZR75, MDA-MB 231, BT20) was tested in vitro on ROS 17/2,8 osteoblast-like cells and on osteoblasts derived from human bone biopsies. The parameters monitored in osteoblasts were [3H]thymidine incorporation, alkaline phosphatase activity, and osteocalcin secretion. Serum-free media conditioned during 24 h by MCF-7 cells presented the highest ObSA. CM decreased thymidine incorporation in DNA and increased alkaline phosphatase activity in a dose-dependent manner. Bone GLA protein (osteocalcin) secretion by human osteoblasts was not increased however in the presence of CM. MCF-7 cells were cultured in the presence of dihydrotestosterone (DHT) [1-100 nM] for 5 days. Serum-free, DHT-free CM collected after an additional 24 h, contained alkaline-phosphatase stimulating activity which was DHT dose-dependent. Estradiol and 1,25(OH)2D3 failed to elicit a comparable increase of the ObSA in the CM. In conclusion, MCF-7 cells product factor(s) that interfere with bone remodeling. The DHT modulation of ObSA parallels the estradiol control of MCF-7 cells osteolytic lesions in relation with Prostaglandin E secretion. Sex hormones at physiological and pharmacological levels might thus control both osteosclerotic and osteolytic lesions observed in bone deposits of hormone dependent cancers.     

Mouse osteoblasts synthesize collagenase in response to bone resorbing agents

Bone cells isolated from mouse calvariae by a sequential digestion procedure have many osteoblast characteristics: they respond to PTH and prostaglandin E2 by activation of adenylate cyclase but not to calcitonin, they stain for alkaline phosphatase and they make only type I collagen. In confluent monolayer culture, they do not secrete collagenase in appreciable quantities, unless stimulated with resorptive substances such as PTH, prostaglandin E2, 1,25(OH)2 vitamin D-3 and monocyte-conditioned medium. This suggests they play a direct role in bone resorption.     

骨髓基质干细胞的分离纯化及培养

目的 建立骨髓基质干细胞(MSCs)良好的分离纯化和培养方法。方法 将小鼠骨髓基质干细胞自殷骨中分离,应用贴壁选择法结合细胞克隆挑选法进行分离纯化,应用细胞生长因子(EGF和PDGF-BB)刺激法进行MSCs的体外培养和传代,倒置显微镜下观察分离培养的细胞并照像记录。结果,培养获得了纯化的呈梭形成纤雏样细胞的骨髓基质干细胞。在生长因子EGF和PDGF-BB的共同作用下,传代MSCs生长旺盛,形态均一。结论 该方法是简便高效的骨髓基质干细胞的分离纯化和培养方法。     

Epidermal growth factor stimulates prostaglandin production and bone resorption in cultured mouse calvaria.

Murine epidermal growth factor (EGF) stimulated the production of prostaglandin E₂ (PGE₂) and bone resorption in neonatal mouse calvaria in organ culture. The effect of EGF on bone resorption occurred at low concentrations of the polypeptide (half-max stimulation = 0.4 ng/ml, 6.6 × 10^?11 M). All concentrations of EGF which stimulated resorption also stimulated the production of PGE₂ by bone; concentrations of EGF which did not stimulate resorption did not enhance PGE₂ production. EGF-induced formation of PGE₂ and bone resorption were inhibited completely by indomethacin (200 ng/ml) and hydrocortisone (3 × 10^?6 M). Indomethacin did not inhibit the bone resorption-stimulating activity of exogenous PGE₂. The time courses of action of EGF, parathyroid hormone and exogenous PGE₂ on bone resorption were similar. Brief exposure (15 or 60 min) to EGF (10 ng/ml) did not cause bone resorption or an increase in PGE₂ accumulation in a subsequent 48-h incubation in the absence of EGF. High concentrations (30 to 100 ng/ml) of bovine fibroblast growth factor (FGF) also stimulated the production of PGE₂ and bone resorption. We conclude that concentrations of EGF equal to or less than those present in mouse plasma stimulate the resorption of mouse bone in organ culture by a mechanism that involves the enhanced local production of PGE₂.     

Consequences of daily administered parathyroid hormone on myeloma growth, bone disease, and molecular profiling of whole myelomatous bone

Background

Induction of osteolytic bone lesions in multiple myeloma is caused by an uncoupling of osteoclastic bone resorption and osteoblastic bone formation. Current management of myeloma bone disease is limited to the use of antiresorptive agents such as bisphosphonates.

Methodology/Principal Findings

We tested the effects of daily administered parathyroid hormone (PTH) on bone disease and myeloma growth, and we investigated molecular mechanisms by analyzing gene expression profiles of unique myeloma cell lines and primary myeloma cells engrafted in SCID-rab and SCID-hu mouse models. PTH resulted in increased bone mineral density of myelomatous bones and reduced tumor burden, which reflected the dependence of primary myeloma cells on the bone marrow microenvironment. Treatment with PTH also increased bone mineral density of uninvolved murine bones in myelomatous hosts and bone mineral density of implanted human bones in nonmyelomatous hosts. In myelomatous bone, PTH markedly increased the number of osteoblasts and bone-formation parameters, and the number of osteoclasts was unaffected or moderately reduced. Pretreatment with PTH before injecting myeloma cells increased bone mineral density of the implanted bone and delayed tumor progression. Human global gene expression profiling of myelomatous bones from SCID-hu mice treated with PTH or saline revealed activation of multiple distinct pathways involved in bone formation and coupling; involvement of Wnt signaling was prominent. Treatment with PTH also downregulated markers typically expressed by osteoclasts and myeloma cells, and altered expression of genes that control oxidative stress and inflammation. PTH receptors were not expressed by myeloma cells, and PTH had no effect on myeloma cell growth in vitro.

Conclusions/Significance

We conclude that PTH-induced bone formation in myelomatous bones is mediated by activation of multiple signaling pathways involved in osteoblastogenesis and attenuated bone resorption and myeloma growth; mechanisms involve increased osteoblast production of anti-myeloma factors and minimized myeloma induction of inflammatory conditions.     

EGF-like ligands stimulate osteoclastogenesis by regulating expression of osteoclast regulatory factors by osteoblasts: implications for osteolytic bone metastases

Epidermal growth factor (EGF)-like ligands and their receptors constitute one of the most important signaling networks functioning in normal tissue development and cancer biology. Recent in vivo mouse models suggest this signaling network plays an essential role in bone metabolism. Using a coculture system containing bone marrow macrophage and osteoblastic cells, here we report that EGF-like ligands stimulate osteoclastogenesis by acting on osteoblastic cells. This stimulation is not a direct effect because osteoclasts do not express functional EGF receptors (EGFRs). Further studies reveal that EGF-like ligands strongly regulate the expression of two secreted osteoclast regulatory factors in osteoblasts by decreasing osteoprotegerin (OPG) expression and increasing monocyte chemoattractant protein 1 (MCP1) expression in an EGFR-dependent manner and consequently stimulate TRAP-positive osteoclast formation. Addition of exogenous OPG completely inhibited osteoclast formation stimulated by EGF-like ligands, while addition of a neutralizing antibody against MCP-1 exhibited partial inhibition. Coculture with bone metastatic breast cancer MDA-MB-231 cells had similar effects on the expression of OPG and MCP1 in the osteoblastic cells, and those effects could be partially abolished by the EGFR inhibitor PD153035. Because a high percentage of human carcinomas express EGF-like ligands, our findings suggest a novel mechanism for osteolytic lesions caused by cancer cells metastasizing to bone.     

Stimulation of creatine kinase BB activity by parathyroid hormone and by prostaglandin E2 in cultured bone cells.

Bone cells in culture responded to parathyroid hormone (PTH) and prostaglandin E2 (PGE2) by a 2-fold increase in creatine kinase (CK) activity. Combined treatment resulted in a higher response than with PTH alone. Calcitonin (CT) failed to stimulate CK activity, did not affect the response of CK to PTH, but inhibited slightly the increase in CK activity by PGE2. Bone-cell cultures grown in low [Ca2+] (0.125 mM), enriched in PTH-responsive osteoblast-like cells, responded to PTH, but not to PGE2 or CT, by increased CK activity. In both normal and low-[Ca2+] cultures, 8-bromo cyclic AMP did not affect CK activity, nor did it change the response of the cells to PTH, PGE2 or CT. The increase in CK activity was time- and dose-dependent and inhibited both by cycloheximide and by actinomycin D. The isoenzyme of CK stimulated was the CKBB form, the isoenzyme induced by other hormones. This appears to be the first report of the stimulation of CK activity by a polypeptide hormone or a prostaglandin. We suggest that stimulation of CKBB can serve as a marker for the action of a variety of hormones and growth promoters.     

Osteoprotegrin and the bone homing and colonization potential of breast cancer cells

Breast cancer cells preferentially metastasize to bone, leading to the formation of primarily osteolytic lesions. Osteoprotegerin (OPG) plays multifactorial roles in the development of osteolytic bone metastases. An increase in the ratio of receptor activator of nuclear factor kappaB ligand (RANKL) to OPG increases osteoclastogenesis within the bone microenvironment. OPG also acts as a survival factor for cancer cells by protecting them from tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) mediated apoptosis. This study compares OPG production in vitro in a number of breast cancer cell lines exhibiting both differences in metastatic capacity and in preferential metastasis to bone. Our studies demonstrated that OPG expression by MDA-231, MDA-MET, and MDA-231/K cancer cells was directly correlated with bone specific homing and colonization potential but not with metastasis of cancer cells to other organs; both in IL-1 beta stimulated and control cells. We also demonstrated expression of other bone-related markers including type I collagen, osteocalcin, osteopontin, and Runx2 in these cells. However, the generally lower expression of these markers in the bone selective cell line MDA-MET suggested that increased OPG expression in the bone specific variant was not merely a consequence of enhanced osteomimicry by these cells but that it has a significant role in the metastatic process. Co-culture of breast cancer cells with osteoblastic cells (hFOB 1.19) led to an overall downregulation in OPG production, which was not affected by the bone homing and colonization potential of the cell lines, suggesting that OPG alone is not indicative of osteolytic bone activity by breast cancer cells.     

Inhibition by 17 beta-estradiol of PTH stimulated resorption and prostaglandin production in cultured neonatal mouse calvariae

Previous attempts to show a direct effect of physiological concentrations of 17 beta-estradiol (beta E2) on bone in vitro have been unsuccessful. We describe a culture system using neonatal mouse calvariae in which beta E2 in the range 1 pM to 1 nM inhibited parathyroid hormone (PTH) stimulated prostaglandin E2 (PGE2) release by 50 to 70% in the presence and absence of cortisol. In addition, beta E2 reduced medium calcium concentration and release of previously incorporated 45Ca by 10 and 20%, respectively, in PTH stimulated cultures. Indomethacin did not block beta E2 effects on resorption. 17 alpha-Estradiol (alpha E2) reduced PTH stimulated 45Ca release but not PGE2 release. Thus, beta E2 has direct effects on bone consistent with its known effects to decrease bone resorption in vivo.     

Targeted overexpression of BSP in osteoclasts promotes bone metastasis of breast cancer cells

Bone is one of the most common sites of breast cancer metastasis while bone sialoprotein (BSP) is thought to play an important role in bone metastasis of malignant tumors. The objective of this study is to determine the role of BSP overexpression in osteolytic metastasis using two homozygous transgenic mouse lines in which BSP expression is elevated either in all the tissues (CMV-BSP mice) or only in the osteoclasts (CtpsK-BSP mice). The results showed that skeletal as well as systemic metastases of 4T1 murine breast cancer cells were dramatically increased in CMV-BSP mice. In CtpsK-BSP mice, it was found that targeted BSP overexpression in osteoclasts promoted in vitro osteoclastogenesis and activated osteoclastic differentiation markers such as Cathepsin K, TRAP and NFAT2. MicroCT scan demonstrated that CtpsK/BSP mice had reduced trabecular bone volume and bone mineral density (BMD). The real-time IVIS Imaging System showed that targeted BSP overexpression in osteoclasts promoted bone metastasis of breast cancer cells. The osteolytic lesion area was significantly larger in CtpsK/BSP mice than in the controls as demonstrated by both radiographic and histomorphometric analyses. TRAP staining demonstrated a twofold increase in the number of osteoclasts in the bone lesion area from CtpsK/BSP mice compared with that from wild type mice. We conclude that host tissue-derived BSP also plays important roles in breast cancer metastasis through inducing tumor cell seeding into the remote host tissues. Furthermore, osteoclast-derived BSP promotes osteoclast differentiation in an autocrine manner and consequently promotes osteolytic bone metastasis of breast cancer.     

Heparin-like polysaccharides reduce osteolytic bone destruction and tumor growth in a mouse model of breast cancer bone metastasis

TGF-β regulates several steps in cancer metastasis, including the establishment of bone metastatic lesions. TGF-β is released from bone during osteoclastic bone resorption and it stimulates breast cancer cells to produce osteolytic factors such as interleukin 11 (IL-11). We conducted a cell-based siRNA screen and identified heparan sulfate 6-O-sulfotransferase 2 (HS6ST2) as a critical gene for TGF-β-induced IL-11 production in highly bone metastatic MDA-MB-231(SA) breast cancer cells. HS6ST2 attaches sulfate groups to glucosamine residues in heparan sulfate glycosaminoglycans. We subsequently showed how heparin and a high-molecular-weight Escherichia coli K5-derived heparin-like polysaccharide (K5-NSOS) inhibited TGF-β-induced IL-11 production in MDA-MB-231(SA) cells. In addition, K5-NSOS inhibited bone resorption activity of human osteoclasts in vitro. We evaluated the therapeutic potential of K5-NSOS and fragmin in a mouse model of breast cancer bone metastasis. MDA-MB-231(SA) cells were inoculated into the left cardiac ventricle of athymic nude mice which were treated with fragmin, K5-NSOS, or vehicle once a day for four weeks. Both heparin-like glycosaminoglycans inhibited weight reduction, decreased osteolytic lesion area, and reduced tumor burden in bone. In conclusion, our data imply novel mechanisms involved in TGF-β induction and support the critical role of heparan sulfate glycosaminoglycans in cancer metastasis as well as indicate that K5-NSOS is a potential antimetastatic and antiresorptive agent for cancer therapy. This study illustrates the potential to translate in vitro siRNA screening results toward in vivo therapeutic concepts.     

Paracrine interactions in bone-secreted products of osteoblasts permit osteoclasts to respond to parathyroid hormone

During bone remodeling, activation of resorption is followed by a cycle of formation and this ordered sequence of events has long suggested that local interactions between osteoclasts and osteoblasts are an important regulatory mechanism in bone metabolism. To study this phenomenon, we have prepared bone cells containing primarily osteoclasts by brief digestion of mice calvariae in collagenase, overnight attachment to polystyrene tissue culture flasks in serumless medium supplemented with OB (osteoblast) cell conditioned medium and subsequent growth in low serum. These OC (osteoclast) cells were found to be highly enriched in acid phosphatase activity and expressed cAMP responses to PTH (parathyroid hormone) and prostaglandin E2 but exhibited no PTH-stimulated hyaluronate synthesis in contrast to prostaglandin E2. PTH effects on hyaluronate, however, could be restored upon coculture of OC cells with OB cells (noncontact) or with OB cell conditioned medium, thereby suggesting that OB cells regulate OC cell PTH responsiveness and/or differentiation by soluble cell products secreted into the medium.     

Blood coagulation and bone metabolism: some characteristics of the bone resorptive effect of thrombin in mouse calvarial bones in vitro

Chronic inflammatory processes are often associated with bone resorption. Stimulated by the current great interest in the role of coagulation factors in inflammation and immune injury, we have studied the effect of thrombin on mouse calvarial bones in vitro. Thrombin caused a dose-dependent (0.1-7 U/ml) stimulation of 45Ca release from neonatal mouse calvarial bones. Thrombin also stimulated the mobilization of stable calcium and inorganic phosphate, the release of 3H from [3H]proline-labelled calvaria, the production of lactate and the release of the lysosomal enzymes, beta-glucuronidase and beta-N-acetylglucosaminidase. Thrombin also enhanced 45Ca release from fetal rat long bones, although this bone resorption assay was less sensitive to thrombin than the mouse calvarial system. The bone resorption stimulatory activity of thrombin in mouse calvaria could be inhibited by calcitonin and an increased concentration of phosphate in the culture medium. Thrombin-induced 45Ca release in mouse calvaria was sensitive to inhibition by hydrocortisone and dexamethasone. By contrast, 45Ca release response to parathyroid hormone was insensitive to corticosteroids. The prostaglandin synthetase inhibitors indomethacin, meclofenamic acid and naproxen and 5,8,11,14-eicosatetraynoic acid reduced 45Ca release from thrombin-stimulated calvaria. However, significant stimulation by thrombin could be achieved also in bones treated with inhibitors of arachidonate metabolism. The results obtained suggest that thrombin can stimulate cell-mediated bone resorption by an osteoclast-dependent mechanism. The mechanism of action may involve both prostaglandin-dependent and prostaglandin-independent pathways. Our findings indicate that thrombin may contribute to the bone resorptive processes seen in periodontal disease and rheumatoid arthritis.     

Gamma interferon inhibits basal and interleukin 1-induced prostaglandin production and bone resorption in neonatal mouse calvaria

Production of the osteolytic arachidonic acid metabolites, prostaglandin (PG) E2, PGI2 and PGF2 alpha, by neonatal mouse calvariae was quantitated by gas chromatography/mass spectrometry. Mouse recombinant interleukin 1 (rIL-1) raised medium levels of PGE2 and PGI2 (measured as 6-keto-PGF1 alpha) in the dose range tested (1.0-10.0 U/ml culture medium), while an effect on PGF2 was only observed at 10 U/ml. Bone resorption in response to rIL-1 reached a plateau at 3.0 U/ml. Mouse recombinant gamma-interferon (rIFN-gamma) between 100-500 U/ml suppressed basal PG synthesis and spontaneous resorption of cultured bone. In addition, IFN-gamma at 100 U/ml prevented stimulation of PG synthesis by 3.0 U/ml rIL-1 and thereby reduced the bone resorbing activity of the cytokine by at least 60%. 5 X 10(-7) M indomethacin was equally effective in suppression of PG synthesis and bone resorption. The present study provides evidence that IFN-gamma inhibits PG synthesis and consequently resorption of cultured bone.     

NF-kappaB in breast cancer cells promotes osteolytic bone metastasis by inducing osteoclastogenesis via GM-CSF

Advanced breast cancers frequently metastasize to bone, resulting in osteolytic lesions, yet the underlying mechanisms are poorly understood. Here we report that nuclear factor-kappaB (NF-kappaB) plays a crucial role in the osteolytic bone metastasis of breast cancer by stimulating osteoclastogenesis. Using an in vivo bone metastasis model, we found that constitutive NF-kappaB activity in breast cancer cells is crucial for the bone resorption characteristic of osteolytic bone metastasis. We identified the gene encoding granulocyte macrophage-colony stimulating factor (GM-CSF) as a key target of NF-kappaB and found that it mediates osteolytic bone metastasis of breast cancer by stimulating osteoclast development. Moreover, we observed that the expression of GM-CSF correlated with NF-kappaB activation in bone-metastatic tumor tissues from individuals with breast cancer. These results uncover a new and specific role of NF-kappaB in osteolytic bone metastasis through GM-CSF induction, suggesting that NF-kappaB is a potential target for the treatment of breast cancer and the prevention of skeletal metastasis.     

Structural changes induced in capillary endothelial cells by growth factors: Altered distribution of cytoskeletal elements and organelles in cells spreading in the presence of tumor conditioned medium and hypothalamus derived growth factor

Using high-voltage and conventional electron microscopy of cell whole mounts, we have investigated the effects of tumor-conditioned medium and hypothalmus-derived growth factor on the structure of capillary endothelial cells during their attachment and spreading in tissue culture. Cells were cultured in A, Dulbecco's Modified Eagle's Medium (DMEM) and 10% calf serum; B, equal parts of A and 48 hr mouse sarcoma conditioned medium; and C, A containing 10 units of hypothalamus-derived growth factor. Cells cultured in all three media were fully spread, and to the same extent, by 4 hr after plating. While spreading, cells cultured in DMEM alone developed prominent stress fibers and contained numerous bundles of microtubules which formed radical tracts along which mitochondria and other organelles rapidly moved to the cell periphery. Stress fibers were thinner and microtubule tracts fewer in number in cells cultured in tumor-conditioned medium. In 4 hr, organelles moved only part of the distance to the cell margin. Stress fibers were rudimentary and microtubules randomly orientated in cells exposed to hypothalamus-derived growth factor. Most organelles remained near the cell nucleus. The dramatic decrease in stress fibers and microtubule tracts in cells grown in tumor-conditioned medium and hypothalamus-derived growth factor and the subsequent decreased capacity of the cells to move organelles toward their periphery could have some functional significance relative to the growth-promoting activity of these substances.     

Effect of protein and steroidal osteotropic agents on differentiation and epidermal growth factor-mediated growth of the CFK1 osseous cell line.

The effects of factors known to influence bone metabolism were examined using the osseous cell line CFK1. Parathyroid hormone (PTH) and dexamethasone (DEX) appeared to enhance the formation of cell foci of CFK1 cells in culture whereas retinoic acid (RA) caused a marked alteration in individual cell morphology. Bone morphogenetic protein (BMP-2) and PTH increased alkaline phosphatase activity, however, this index of differentiation was suppressed by epidermal growth factor (EGF), DEX, and RA. BMP-2 and EGF each stimulated DNA synthesis in a dose-dependent manner and enhanced cell numbers, but, no synergistic response of EGF and BMP-2 was observed. PTH and DEX failed to significantly alter cell number or EGF-stimulated DNA synthesis or cell proliferation. Although RA treatment of CFK1 cells resulted in a reduction in cell number compared to control, pretreatment with RA enhanced EGF-stimulated DNA synthesis and proliferative effects. At least part of this effect was by increasing the EGF receptor binding capacity of the cells. Furthermore, using cell cycle analysis, addition of EGF stimulated the progression of RA-treated cells into the DNA synthesis (S) phase with a reduced lag time. EGF and BMP-2, therefore, appear to exert a role in the expansion dynamics of the CFK1 population although BMP-2 may also enhance differentiation. PTH and DEX may act primarily to modulate the differentiated function of the CFK1 cells. RA inhibited cell proliferation and may mediate differentiation towards a less established cell population with upregulation of EGF receptors. The CFK1 cell model may, therefore, provide insight into microenvironmental control of growth and differentiation of precursor osseous cells.