Bone-derived Cell Lines Research Articles

Identification of Sparus aurata bone morphogenetic protein 2: Molecular cloning, gene expression and in silico analysis of protein conserved features in vertebrates

Bone morphogenetic protein 2 (BMP-2) is a secreted signaling molecule that acts as an inducer of bone formation and a regulator of embryonic development. The objectives of this work were as follows: (1) to clone the full-length cDNA of BMP-2 in a marine fish model, (2) analyze its gene expression during development, in adult tissues and in cell lines, and (3) identify protein conserved features of vertebrate BMP-2. Using a combination of RT- and 5′-RACE-PCR, a 1653-bp fragment corresponding to Sparus aurata BMP-2 cDNA (SaBMP-2) was amplified. Levels of SaBMP-2 gene expression were estimated using quantitative real-time PCR and shown to be strongly increased (150-fold induction) at gastrulation, thus suggesting a key role for BMP-2 in fish development. Tissue distribution of SaBMP-2 mRNA revealed highest levels in the calcified tissues bone, caudal fin and scales and in liver. BMP-2 was also found to be highly expressed in S. aurata bone-derived cell lines VSa13 and VSa16 and to be up-regulated (more than 10-fold induction) in mineralized VSa13 chondrocyte-like cells. Using bioinformatic tools and all vertebrate protein sequences available, conserved features of BMP-2 were characterized. The mature protein was shown to be highly conserved across 20 species indicating that BMP-2 function has been conserved throughout evolution, a finding that is in agreement with the widely accepted view of the important role played by BMPs in vertebrate development.

Effects of Granulocyte Macrophage Colony-Stimulating Factor (GM-CSF) on the Interleukin-6 Expression in the Prostate Cancer Cell Line PC-3

Purpose: Interleukin-6 (IL-6) can stimulate a variety of tumors including prostatic carcinoma. Research has recently shown that IL-6 may act to stimulate the progression of prostatic cancer. IL-6 is elevated in the sera of patients with metastatic prostatic cancer and it has been shown to be a candidate marker of disease activity. To date, little work has been performed to characterize the nature of granulocyte macrophage colonystimulating factor (GM-CSF) and the expression of IL-6. The aim of this study is to evaluate the effects of GM-CSF on the expression of IL-6 in PC-3 cells. Materials and Methods: The bone-derived PC-3 cell line was used in this study. Reverse transcription polymerase chain reaction (RT-PCR) was performed to detect the GM-CSF and also the IL-6 mRNA expression. The IL-6 protein was measured by enzyme-linked immunosorbent assay (ELISA) after treatments with the hGM-CSF. Results: hGM-CSF was expressed in the PC-3 cell line. Our data indicated that the IL-6 mRNA expression was not increased at 4, 8 and 12 hours by the hGM-CSF in comparison to the control group, but it was slightly increased at 24 and 48 hours. The expression of IL-6 protein was increased at 4, 8, 12, 24 and 48 hours after hGM-CSF treatment, in comparison with the control group. Conclusions: The IL-6 mRNA expression was slightly increased by hGMCSF at 24 and 48 hours in comparison to the control group. Yet the IL-6 protein expression increased before the IL-6 mRNA expression. Therefore, hGM-CSF may modulate the post-transcription pathway of the IL-6 expression in prostate carcinoma cells. Our data suggest that GM-CSF may have a possible IL-6 mediated pathophysiologic role in prostate cancer. (Korean J Urol 2006;47:786-790)

Enhanced DNA Transfer Into Fish Bone Cells Using Polyethylenimine

The use of in vitro cell culture systems to assess gene function largely depends on the successful transfer of DNA into target cells. Well developed in mammals, transfection methods are still to be optimized for non-mammalian cell culture systems, like fish. Here we describe a rapid, cost-efficient, and successful method to transfer DNA into a fish bone-derived cell line using polyethylenimine (PEI) as the DNA carrier. Using this method, DNA transfer was remarkably enhanced in comparison with commercially available reagents, as demonstrated by the increased activity of both luciferase and green fluorescent protein observed in the transfected cells. Its efficiency in transferring DNA into a wide range of cell types, including non-mammalian and hard-to-transfect cells, in addition to a low cost, show that PEI is a reagent of choice for nonviral vector transfection.

Isolation of C15: A novel antibody generated by phage display against mesenchymal stem cell-enriched fractions of adult human marrow

Adult bone marrow stroma contains a source of mesenchymal stem cells (MSC) that have the capacity to self-renew and differentiate into multiple stromal lineages. These rare cells can be visualised indirectly by the formation of heterogeneous colonies, containing stem cells and their differentiated progeny in long-term culture. If MSC and their associated progenitor and precursor populations are to reach their full therapeutic potential, markers will be required to identify and characterize specific bone marrow stromal subsets. We sought to use phage display to generate antibodies against bone marrow mononuclear cells (BMMNC) enriched for colony forming cells. Initially, we identified our target cell population by comparing the colony forming efficiency (CFE) of CD49a-positive, STRO-1-positive and CD45-negative BMMNC subpopulations with unseparated BMMNC. Selection with anti-CD49a gave the greatest enrichment (19-fold) of colony forming cells and in light of these findings, we generated phage antibodies against CD49a-positive BMMNC by simultaneous positive/negative selection. A dominant clone (C15), generated after 3 rounds of selection, has been isolated and sequenced, then characterized for cell and tissue specificity. Sequence analysis showed that the V H and V L gene segments of C15 aligned most closely to the VH26/DP-47 and IGLV3S1/DPL16 germline V segments found in the synthetic repertoire. C15 bound to 4% of freshly isolated BMMNC and localized to osteoblastic cells and proximal marrow cells in areas of active bone formation in sections of osteophyte. C15 binding was upregulated in cultured bone marrow stromal cells (BMSC) and was also detected on bone-derived cell lines. This report demonstrates that phage display is a powerful tool for the isolation of antibodies against rare cell populations, and provides a platform for the future application of this technology in the search for antigens on MSC and other rare cell populations.

Characterization of Sparus aurata osteonectin cDNA and in silico analysis of protein conserved features: Evidence for more than one osteonectin in Salmonidae

Osteonectin is a matricellular protein involved in various cellular mechanisms but its exact function remains unclear despite numerous studies. We present here the cloning of Sparus aurata partial osteonectin cDNA and the reconstruction of 15 other sequences from both vertebrates and invertebrates, almost doubling the set of available sequences (a total of 35 sequences is now available). Taking advantage of the resulting large amount of data, we have created multiple sequence alignments and identified osteonectin putative conserved features (intra- and inter-disulfide bonds, collagen- and calcium-binding domains and phosphorylation sites) likely to be important for protein structure and function. This work also provides the first evidence for the presence of more than one osteonectin in some species. Finally, S. aurata osteonectin gene expression has been shown to initiate during larval development shortly after gastrulation, and to be high in bone-derived cell lines while down-regulated during extracellular matrix mineralization, further emphasizing the important role of osteonectin in skeletal development and bone formation.

Transient Versus Sustained Phosphorylation and Nuclear Accumulation of ERKs Underlie Anti-Versus Pro-apoptotic Effects of Estrogens

Sex steroids exert anti-apoptotic effects on osteoblasts/osteocytes but exert pro-apoptotic effects on osteoclasts, in both cases requiring activation of the extracellular signal-regulated kinases (ERKs). To explain the mechanistic basis of this divergence, we searched for differences in the kinetics of phosphorylation and/or in the subcellular localization of ERKs in response to 17beta-estradiol in the two cell types. In contrast to its transient effect on ERK phosphorylation in osteocytic cells (return to base line by 30 min), 17beta-estradiol-induced ERK phosphorylation in osteoclasts was sustained for at least 24 h following exposure to the hormone. Conversion of sustained ERK phosphorylation to transient, by means of cholera toxin-induced activation of the adenylate cyclase/cAMP/protein kinase A pathway, abrogated the pro-apoptotic effect of 17beta-estradiol on osteoclasts. Conversely, prolongation of ERK activation in osteocytes, by means of leptomycin B-induced inhibition of ERK export from the nucleus or overexpression of a green fluorescent protein-ERK2 mutant that resides permanently in the nucleus, converted the anti-apoptotic effect of 17beta-estradiol to a pro-apoptotic one. These findings indicate that the kinetics of ERK phosphorylation and the length of time that phospho-ERKs are retained in the nucleus are responsible for pro-versus anti-apoptotic effects of estrogen on different cell types of bone and perhaps their many other target tissues.

Molecular cloning, structure, expression, and chromosomal localization of the human Osterix (SP7) gene

We report the isolation of the human orthologue of the mouse Osterix (Osx/Sp7) gene, a C2H2 zinc finger transcription factor of the SP gene family and putative “master” regulator of bone cell differentiation. The human SP7 cDNA encodes a putative 431 amino acid protein that contains three consecutive C2H2 zinc finger repeats. The SP7 protein is highly conserved between mice and humans with an overall sequence identity of 95%. The expression of a SP7 mRNA transcript of approximately 3.2 kb is restricted to bone-derived cell lines in vitro but undetectable in any adult tissues including mandibular bone by Northern blot hybridization. The specific expression of SP7 mRNA in osteoblasts in vivo was further confirmed by in situ hybridization on human embryonic tissues. The highly restricted expression pattern and the divergence of the sequence outside of the zinc finger region distinguish SP7 as a unique member of the SP family. The SP7 gene consists of two exons, with exon 2 containing most of the protein coding sequence. The gene locus was mapped to chromosome 12q13.13 by fluorescent in situ hybridization (FISH). The identification and initial characterization of the SP7 gene will facilitate the study of the molecular regulation of osteoblast differentiation in humans.

Development of two bone-derived cell lines from the marine teleost Sparus aurata; evidence for extracellular matrix mineralization and cell-type-specific expression of matrix Gla protein and osteocalcin.

A growing interest in the understanding of the ontogeny and mineralization of fish skeleton has emerged from the recent implementation of fish as a vertebrate model, particularly for skeletal development. Whereas several in vivo studies dealing with the regulation of bone formation in fish have been published, in vitro studies have been hampered because of a complete lack of fish-bone-derived cell systems. We describe here the development and the characterization of two new cell lines, designated VSa13 and VSa16, derived from the vertebra of the gilthead sea bream. Both cell types exhibit a spindle-like phenotype and slow growth when cultured in Leibovitz's L-15 medium and a polygonal phenotype and rapid growth in Dulbecco's modified Eagle medium (D-MEM). Scanning electron microscopy and von Kossa staining have revealed that the VSa13 and VSa16 cells can only mineralize their extracellular matrix when cultured in D-MEM under mineralizing conditions, forming calcium-phosphate crystals similar to hydroxyapatite. We have also demonstrated the involvement of alkaline phosphatase, a marker of bone formation in vivo, and Gla proteins (osteocalcin and matrix Gla protein, MGP) in the process of mineralization. Finally, we have shown that VSa13 and VSa16 cell lines express osteocalcin and MGP in a mutually exclusive manner. Thus, both cell lines are capable of mineralizing in vitro and of expressing genes found in chondrocyte and osteoblast cell lineages, emphasizing the suitability of these new cell lines as valuable tools for analyzing the expression and regulation of cartilage- and bone-specific genes.

Telomerase activity and telomere length in primary and metastatic tumors from pediatric bone cancer patients.

The presence of telomerase activity has been analyzed in almost all tumor types and tumor-derived cell lines. However, there are very few studies that focus on the presence of telomerase activity in bone tumors, and most of them report analysis on very few samples or bone-derived cell lines. The objective of this study was to analyze the telomere length and telomerase activity in primary tumors and metastatic lesions from pediatric osteosarcoma and Ewing's sarcoma patients. The presence of telomerase activity was analyzed by the telomeric repeat amplification protocol assay, and the telomere length was measured by Southern blot. Results were related to survival and clinical outcome. Telomerase activity was detected in 85% of the bone tumor metastases (100% Ewing's sarcomas and 75% osteosarcomas) but only in 12% of the primary tumors (11.1% osteosarcomas and 12.5% Ewing's sarcomas). Bone tumor tissues with telomerase activity had mean telomere lengths 3 kb shorter than those with no detectable telomerase activity (p = 0.041). The presence of telomerase activity was associated with survival (p = 0.009), and longer event-free survival periods were found in patients who lacked telomerase activity compared with those who had detectable telomerase activity levels in their tumor tissues (p = 0.037). The presence of longer telomeres in primary pediatric bone tumors than in metastases could be indicative of alternative mechanisms of lengthening of telomeres for their telomere maintenance rather than telomerase activity. Nevertheless, the activation of telomerase seems to be a crucial step in the malignant progression and acquisition of invasive capability of bone tumors.

Biological evaluation of the effects of the surface roughness and composition of titanium discs on an immortalized human osteoblastic cell line.

In the search for methods to improve the biocompatibility of prosthetic materials, attention has recently been di- rected towards the usefulness of different surface topography and chemical modification and how they influence cellular be- havior. This in vitro study investigates the effect of surface roughness and composition of commercial grade 3 titanium (Ti) discs on a well-characterized human bone-derived cell line (Hobit). Cells were cultured on machined Ti discs (TOR), polished with ceramic particles (BUR), alumina-sand-blasted (S-Al), glass-blasted (S-V), hydroxyapatite-blasted (S-HA), hydrofluoric acid etched (HF), titanium plasma-sprayed (Ti-PS) and hydroxyapatite plasma-sprayed (HA-PS). Cell attachment, prolifera- tion, morphology and expression of alkaline phosphatase (AP), osteocalcin (OC) and type I collagen were studied. Cells at- tach to all surfaces, but proliferation differs among the variously treated materials: it is higher in TOR, BUR and HF than in S-Al, S-V, S-HA and Ti-PS, but much lower in HA-PS. Cells present the typical polygonal-morphology of the osteogenic cells and maintain the osteoblastic phenotype expressing AP activity and OC. (Journal of Applied Biomaterials & Biomechanics 2003; 1: 117-24)

Stromal factors involved in prostate carcinoma metastasis to bone.

Prostate carcinoma (PC) frequently metastasizes to bone, where it causes significant morbidity and mortality. Stromal elements in the primary and metastatic target organs are important mediators of tumor cell intravasation, chemoattraction, adhesion to target organ microvascular endothelium, extravasation, and growth at the metastatic site. The role of stromal factors in bone metastasis was determined with a cyclic DNA microarray comparison of a bone-derived cell PC cell line with a soft tissue-derived cell PC cell line and by evaluating the effects of selected stromal components on PC cell chemotaxis, cell adhesion to human bone marrow endothelium (HBME), and PC cell growth. The authors demonstrate that PC cells express protease-activated receptor 1 (PAR1; thrombin receptor), and its expression is up-regulated in PC compared with normal prostate tissue. In addition, this overexpression was very pronounced in bone-derived PC cell lines (VCaP and PC-3) compared with soft tissue PC cell lines (DUCaP, DU145, and LNCaP). The authors report that bone stromal factors, including stromal cell-derived factor 1 (SDF-1) and collagen Type I peptides, are chemoattractants for PC cells, and they demonstrate that some of these factors (e.g., extracellular matrix components, transforming growth factor beta, bone morphogenic proteins [BMPs], and SDF-1) significantly alter PC-HBME interaction in vitro. Finally, stromal factors, such as BMPs, can regulate the proliferation of PC cells in vitro. Soluble and insoluble elements of the stroma are involved in multiple steps of PC metastasis to bone. The authors hypothesize that PAR1 may play a central role in prostate tumorigenesis.

A functional thrombin receptor (PAR1) is expressed on bone-derived prostate cancer cell lines

Objectives To identify genes important in prostate cancer metastatic to bone. Bone-specific metastasis is a common feature of prostate cancer and a significant cause of morbidity. Methods To identify factors involved in organ-specific metastasis, we used cDNA microarray analysis to compare a bone-derived cell line, VCaP, with a soft tissue-derived cell line, DuCaP. Both cell lines were derived from the same patient and spontaneously passaged. Results Forty-five genes were differentially expressed, and only seven of these also had increased expression in VCaP compared with normal prostatic tissue. Of these, protease-activated receptor 1 (PAR1) was verified as having increased expression by reverse transcriptase-polymerase chain reaction and Northern blot analysis, as well as by immunohistochemistry. PAR1 expression in a panel of prostate cancer cell lines demonstrated increased expression in those cell lines derived from bone metastases. Alpha-thrombin stimulation of the VCaP cells produced a dose-dependent mobilization of intracellular calcium compared with DuCaP, suggesting that PAR1 expressed on the VCaP prostate cancer cell line is functional. Conclusions These data indicate that a functional PAR1 is expressed on prostate cancer cell lines. The prostate cancer cell lines expressing PAR1 appear to have an association with increased bone metastases.

Interleukin‐1α induces cyclooxygenase‐2 expression in bone‐derived endothelial cells

Histological studies have suggested that vascular endothelial cells in bone are members of a complex network that regulates bone development and remodeling by producing soluble factors or by mediating cell-cell adhesion. To clarify the role of bone-derived endothelial cell lines (BDECs) in bone remodeling, we established several clones of BDECs from the femurs of BALB/c mice after transformation with the SV40 virus. Then we examined the response of these clones to interleukin-1α (IL-1α). IL-1α is known to induce bone resorption in part by increasing the expression of cyclooxygenase-2 (COX-2) that is associated with the production of PGE2 in osteoblast-lineage cells. Treating the primary and established BDECs with IL-1α induced COX-2 mRNA expression. A transcriptional activation assay revealed that the treatment with IL-1α increased COX-2 promoter activity in a dose-dependent manner, and IL-1α promoted COX-2 protein expression in BDECs. Treatment with IL-1α promoted PGE2 production from BDECs in a dose-dependent manner. These results indicate that IL-1α stimulates PGE2 synthesis largely by inducing BDECs to express COX-2. Because PGE2 stimulates bone resorption, these vascular endothelial cells, as well as osteoblast cells, play important roles in bone remodeling. J. Cell. Physiol. 179:226–232, 1999. © 1999 Wiley-Liss, Inc.

Interleukin-1 alpha induced cyclooxygenase-2 expression in bone-derived endothelial cells.

Histological studies have suggested that vascular endothelial cells in bone are members of a complex network that regulates bone development and remodeling by producing soluble factors or by mediating cell-cell adhesion. To clarify the role of bone-derived endothelial cell lines (BDECs) in bone remodeling, we established several clones of BDECs from the femurs of BALB/c mice after transformation with the SV40 virus. Then we examined the response of these clones to interleukin-1alpha (IL-1alpha). IL-1alpha is known to induce bone resorption in part by increasing the expression of cyclooxygenase-2 (COX-2) that is associated with the production of PGE2 in osteoblast-lineage cells. Treating the primary and established BDECs with IL-1alpha induced COX-2 mRNA expression. A transcriptional activation assay revealed that the treatment with IL-1alpha increased COX-2 promoter activity in a dose-dependent manner, and IL-1alpha promoted COX-2 protein expression in BDECs. Treatment with IL-1alpha promoted PGE2 production from BDECs in a dose-dependent manner. These results indicate that IL-1alpha stimulates PGE2 synthesis largely by inducing BDECs to express COX-2. Because PGE2 stimulates bone resorption, these vascular endothelial cells, as well as osteoblast cells, play important roles in bone remodeling.

Cloning of a Novel cDNA Expressed during the Early Stages of Fracture Healing

Using differential mRNA display (DD-PCR), a novel cDNA, FxC1 (Fracture Callus 1) was isolated from the early stages of a healing fractured femur. Utilizing 5′ RACE PCR, a 598-bp full-length cDNA was obtained for FxC1 that contains an open reading frame (ORF) of 243 bp, encoding for an 80 amino acid protein. Within this ORF, a leucine zipper motif was present.In vitrotranscription/translation of the full-length cDNA generated the expected 9-kDa protein. Northern analysis reveals that this gene is expressed in calluses harvested from post-fracture day 5, 7 and 10, as well as in several other tissues and bone-derived cell lines. During the differentiation of MC3T3 cells along the osteoblast lineage, FxC1 expression increases 3- to 4-fold during the production and deposition of matrix proteins, suggesting a possible role for this protein in cell differentiation.

Calcium and Insulin-like Growth Factor I Stimulation of Sodium-Dependent Phosphate Transport and Proliferation of Cultured Rat Osteoblasts

Calcium (Ca) stimulates proliferation of osteoblasts in vitro, an effect proposed to be mediated by IGF I. Addition of 1 mM Ca or of 1 nM IGF I to the medium (0.3 mM Ca) of a rat bone-derived cell line, PyMS, stimulated not only DNA synthesis but also sodium-dependent (Nad) phosphate (Pi) uptake, the latter, within 2 h. These cells barely express and produce IGF I. IGF binding protein-3 which inhibits IGF action decreased neither basal nor Ca-stimulated but IGF I-stimulated NadPi transport and DNA synthesis, indicating that Ca stimulated NadPi transport and DNA synthesis independently of IGF I. The effects of Ca and IGF I on DNA synthesis were additive. 1 μM nifedipine blocked IGF I- and Ca-stimulated DNA synthesis but not NadPi transport, suggesting that Ca influx is not mediating the NadPi transport-enhancing IGF I signal but is required for IGF I-induced osteoblast proliferation.

Prostaglandin E2 stimulates sodium-dependent phosphate transport in osteoblastic cells via a protein kinase C-mediated pathway.

Eicosanoids are released by bone cells in response to physical, hormonal, and cytokine stimulation, and they can both inhibit and stimulate bone formation. We investigated the effect of PGE2 on sodium-dependent phosphate (Na(d)Pi) transport in a rat bone-derived cell line, PyMS. PGE2 and 12-O-tetradecanoyl phorbol-13-acetate (TPA), an activator of protein kinase C, increased Na(d)Pi uptake in a dose- and time-dependent manner. There was no change in Na-dependent alanine transport, and the effects of PGE2 and TPA were not associated with corresponding changes in cell number or protein content. Their effects on Na(d)Pi uptake were not additive. Calphostin C, an inhibitor of protein kinase C (10(-8) M) completely blocked TPA- and PGE2-stimulated Na(d)Pi uptake. The results are consistent with a crucial role of protein kinase C activation in the short term stimulation of Na(d)Pi transport by PGE2 in PyMS cells.

1α,25-Dihydroxyvitamin D 3 stimulates sodium-dependent phosphate transport in osteoblast-like cells

1α,25-Dihydroxyvitamin D 3 [1,25(OH) 2D 3] stimulate calcium and phosphate absorption in the duodenum. Because osteoblastic cells appear to be responsible for Pi deposition in bone, we analyzed the effect of 1,25(OH) 2D 3 on sodium-dependent phosphate (Na dPi) transport in newborn rat calvaria-derived osteoblasts and in a rat bone-derived cell line, PyMS. 1,25(OH) 2D 3 at 10 −9 mol/L stimulated Na dPi uptake 1.6-fold in PyMS and 1.8-fold in calvaria cells after 24 h, but not after 2 h and 6 h. Insulin-like growth factor (IGF)-I stimulated Na dPi transport 1.8-fold in both cell types within 2 h. There was no change in Na-dependent alanine transport or in Na-independent uptake of Pi and alanine, and the effects of 1,25(OH) 2D 3 on Na dPi transport were not associated with corresponding changes in cell number, protein content, or alkaline phosphatase activity. Actinomycin D, an inhibitor of transcription, prevented the stimulatory effect of 1,25(OH) 2D 3 (but not that of IGF-I) on Na dPi transport; stimulation of Na dPi transport by 1,25(OH) 2D 3 may depend on an increased de novo synthesis of transporters. Our studies confirm that IGF-I stimulates Na dPi uptake and show, for the first time, a specific stimulatory effect of 1,25(OH) 2D 3 on Na dPi transport in osteoblasts in vitro.

Human immunodeficiency virus infection of human bone derived cells.

Human immunodeficiency virus infection of a human bone derived cell line was initiated by either cell free virus or with a cell to cell transmission method. The human bone derived cells were examined for 8 weeks, and virus infection was not detected when assessed by microscopy, immunofluorescence, reverse transcriptase activity, or infection of cocultivated human T lymphoid cells susceptible to human immunodeficiency virus. Polymerase chain reaction analysis of human bone derived cells inoculated with the cell to cell infection format showed less than 0.1% infected cells. It is possible that the infected cells detected by polymerase chain reaction were lymphocytes used in the cell to cell infection format. Alternatively, latent infection may have been established in the bone derived cells with no apparent expression of the proviral genome. A large proportion of bone is represented by human bone derived cells, and it is unlikely that bone will contribute to a significant human immunodeficiency virus reservoir in vivo. The blood of bone allograft donors is likely to have a greater virus bioburden than is bone. Methods to sterilize bone should be assessed by their efficacy to inactivate the virus in blood contaminating the graft, and methods to detect human immunodeficiency virus deoxyribonucleic acid in a bone graft may be less sensitive than examining the donor's blood.

Stimulation of creatine kinase specific activity in human osteoblast and endometrial cells by estrogens and anti-estrogens and its modulation by calciotropic hormones.

We have previously demonstrated sex-specific stimulation of creatine kinase specific activity (CK) in bone cells both in vivo and in vitro, in primary culture cells derived from rat and human bone and in established human bone-derived cell lines. We found that the female-derived cell line, SaOS-2, responded to 17 beta-estradiol (E2) by increased CK specific activity. The effects of E2 on the CK activity in SaOS-2 cells was inhibited by 100-fold excess of 4-hydroxytamoxifen (Tam) as well as by the other antiestrogen, ICI 164,384. Tam by itself had some stimulatory effect whereas ICI 164,384 showed no estrogenic activity. We also demonstrated the estrogenic-like effect of another anti-estrogen, raloxifene (Ral), which is agonist only in the SaOS-2 osteoblast-like cells but not in the human endometrial, Ishikawa cell line. Ishikawa cells respond to E2 and to Tam by increased CK activity. In both osteoblasts and endometrial cell lines, Ral and Tam were inhibitory in the presence of E2. The effects of E2 on SaOS-2 cells are at least partially mediated by the estrogen receptor (ER) at the level of transcription as demonstrated by transient transfection experiments using the human creatine kinase promoter chloramphenicol acetyltransferase in these cells. Pretreatment of SaOS-2 with calcitropic hormones, either 1,25 dihydroxyvitamin D3 (1,25(OH)2D3) or human parathyroid hormone (1-34) (hPTH(1-34)) increased the stimulation of CK by E2 by 40-60% relative to E2 alone and significantly increased the sensitivity of the cells to E2 by lowering the effective hormonal dose needed for stimulation of CK by E2 by 100-fold. This stimulatory effect of pretreatment of the cells with 1,25(OH)2D3 was due to a 2.5-fold increase in the level of ER expression as measured directly by enzyme immunoassay in the SaOS-2/1 subline. The increase in the responsiveness to E2 by hPTH(1-34) was not due to an increase in ER level in the cells. We can conclude that in cell cultures as in vivo, Ral shows different effects depending on the cell type, namely estrogenic-like activity in skeletal cells but not in uterine cells. We can also conclude that as with rat-derived cells, in bone cells derived from human bone 1,25(OH)2D3 increased the sensitivity to E2 due to an increase in the number of ER in the cells, whereas PTH(1-34) augmented the response to E2 without increasing ER, by another, as yet unknown, mechanism. These studies suggest that the treatment of pathological bone disorders may be improved by combined hormone therapy.