TGFbeta inducible early gene-1 directly binds to, and represses, the OPG promoter in osteoblasts.

TGFbeta inducible early gene-1 (TIEG) is a member of the Krüppel-like family of transcription factors (KLF10) that plays an important role in TGFbeta mediated Smad signaling. In order to better understand the role of TIEG in bone, we generated TIEG knockout (KO) mice. Calvarial osteoblasts (OBs) isolated from these mice exhibit a reduced ability to support osteoclastogenesis in vitro. Gene expression studies revealed decreased receptor activator of NF-kappaB ligand (RANKL) and increased osteoprotegerin (OPG) expression in TIEG KO OBs, suggesting a potential role for TIEG in regulating the expression of these genes. Since OPG and RANKL are two important regulators of osteoclast (OC) differentiation, we sought to determine if TIEG directly regulates their expression. Luciferase constructs, containing fragments of either the mouse OPG promoter (-1486 to +133 bp) or the RANKL promoter (-2000 to +1 bp) were each cloned into the pGL3 basic reporter vector and transiently transfected into TIEG KO calvarial OBs with and without a TIEG expression vector. No significant changes in the activity of the RANKL promoter were detected in the presence of TIEG. However, OPG promoter activity was inhibited in the presence of TIEG protein suggesting that TIEG directly represses the expression of OPG in OBs. In order to determine the region of this promoter through which TIEG acts, sequential 5'-deletion constructs were generated. Transient transfection of these constructs revealed that the TIEG regulatory element(s) reside within a 200 bp region of the OPG promoter. Transient ChIP analyses, using a TIEG-specific antibody, revealed that TIEG binds to this region of the OPG promoter. Since we have previously shown that TIEG regulates target gene expression through Sp-1 sites, we examined this region of the OPG promoter for potential TIEG binding elements and identified four potential Sp-1 binding sites. Site-directed mutagenesis was used to determine if TIEG utilizes these Sp-1 elements to regulate the activity of the OPG promoter. The data demonstrate that two Sp-1 sites are likely to be involved in TIEG's repression of the OPG promoter. Taken together, these results confirm that TIEG directly binds to and inhibits OPG promoter activity in OBs, partially explaining the inability of TIEG KO OBs to fully support osteoclast differentiation.

Comparison and characterization of nuclear isolation procedures as applied to chick oviduct.

A number of methods for the preparation of chick oviduct nuclei have been compared. Nuclei have been isolated in hypertonic sucrose and citric acid and the product has been characterized with respect to cleanliness, ultrastructure, RNA polymerase activity, RNA integrity, and chromatin composition. The study demonstrates that the choice of oviduct nuclear isolation procedure will depend markedly on the purpose for which the nuclei are required. Thus, nuclei prepared entirely in high-molarity sucrose retain the highest levels of RNA polymerase. Those prepared rapidly in the presence of citric acid retain nuclear RNA in an essentially undegraded state. Finally, a bulk preparation is described which, because of its adaptability and high yield of morphologically intact nuclei using large amounts of tissue, is ideal for use in preparing chromatin. Conditions are described by which isolated nuclei can be stored for up to 6 months and retain their morphology, chemical characteristics, and RNA polymerase activity.

Nonhistone chromosomal proteins and gene regulation.

Evidence from several model systems suggests that nonhistone chromosomal proteins may regulate gene expression in eukaryotic cells. The data indicate that the synthesis of new species of nonhistone chromosomal proteins as well as modifications of preexisting nonhistone chromosomal proteins are involved in the control of transcription. However, from the vast number of proteins included in this class, it is apparent that, in addition to regulating the transcription of defined genome loci, the nonhistone chromosomal proteins include enzymes that have a general function, proteins that are involved in determining the structure of chromatin, as well as proteins that serve as recognition sites for binding of regulatory macromolecules. The presence of a nucleoplasmic pool of nonhistone chromosomal proteins which may exchange with the chromatin has also been reported (89). While it is clear that the nonhistone chromosomal proteins play a key role in the regulation of gene expression, the exact manner in which they interact with the genome to initiate, modify, or augment the transcription of specific RNA molecules remains to be resolved.

Progesterone binding to hen oviduct genome: specific versus nonspecific binding.

Data are presented to explain discrepancies in the literature involving the in vitro binding of steroid receptor complexes to isolated nuclei and chromatin. The type of binding in vitro of the progesterone-receptor complex to nuclei, chromatin, or DNA of hen organs is largely determined by the ionic strength of the medium. Low ionic conditions (0.01 to 0.05 molar potassium chloride) result in a nonspecific, nonsaturable binding, while high ionic conditions (0.15 to 0.20 molar potassium chloride) create a tissue-specific, saturable binding. Pure DNA binds the steroid receptor complex extensively in low salt but very little in the higher salt conditions.

Evidence of estrogen receptors in normal human osteoblast-like cells.

In seven strains of cultured normal human osteoblast-like cells, a mean of 1615 molecules of tritium-labeled 17 beta-estradiol per cell nucleus could be bound to specific nuclear sites. The nuclear binding of the labeled steroid was temperature-dependent, steroid-specific, saturable, and cell type-specific. These are characteristics of biologically active estrogen receptors. Pretreatment with 10 nanomolar estradiol in vitro increased the specific nuclear binding of progesterone in four of six cell strains, indicating an induction of functional progesterone receptors. RNA blot analysis demonstrated the presence of messenger RNA for the human estrogen receptor. The data suggest that estrogen acts directly on human bone cells through a classical estrogen receptor-mediated mechanism.

Estrogen-TGFbeta cross-talk in bone and other cell types: role of TIEG, Runx2, and other transcription factors.

It is well established that E(2) and TGFbeta have major biological effects in multiple tissues, including bone. The signaling pathways through which these two factors elicit their effects are well documented. However, the interaction between these two pathways and the potential consequences of cross-talk between E(2) and TGFbeta continue to be elucidated. In this prospectus, we present known and potential roles of TIEG, Runx2, and other transcription factors as important mediators of signaling between these two pathways.

Steroid hormone regulation of nuclear proto-oncogenes.

The role of the nuclear proto-oncogenes as rapidly responding nuclear regulators in the cascade model for steroid hormone action is proposed. In this model, the nuclear proto-oncogenes respond within minutes to steroids and would code for regulatory proteins that in turn enter the nucleus to positively or negatively regulate "late" structural gene transcription and mRNA processing. The potential involvement of the nuclear matrix and one of its components, a receptor binding factor (RBF-1) for steroid receptors of these genes, is discussed. The nuclear proto-oncogenes, thus, may serve as important "early" regulatory genes and as excellent universal markers in all tissues in steroid hormone action. Proto-oncogenes are known to be regulatory genes since genetic alterations of amplification, mutations, chromosome translocation, and other rearrangements which result in overexpression, inactivation, and/or loss of regulatory control result in malignant transformation of the cell. It is now known that as many as six different proto-oncogenes must undergo alteration to induce the loss of cellular control of replication and/or for transformation to occur. The nuclear oncogenes, representing only one class of several of the known proto-oncogenes, code for transcription factors and enzymes involved in regulating these factors and other features of gene transcription. Possibly RNA processing is another function. Many of these genes and their responses to steroids are described in this chapter. The steroid receptors belong to a family which themselves represent transcription factors in that they reside in the nucleus and bind to specific DNA elements or other transcription factors to alter gene transcription and/or mRNA processing. The receptors for the various steroids are described as having a common structural motif and function using the above mentioned pathways. In the majority of cases cited, the steroid receptors mediate the rapid regulation of the nuclear proto-oncogene transcription. The role of palindromic SRE in or near these genes is to bind steroid hormone receptor dimers to regulate transcription. More powerful steroid response units (SRU), composed of two or more response elements for steroids or transcription factors, can act at great distances from the gene as enhancers for steroid- regulated transcription. What has become obvious from the studies of steroid effects on nuclear proto-oncogenes, is that, despite the tremendous scientific strides that have been made toward understanding gene regulation by steroids, we are still relatively naive.(ABSTRACT TRUNCATED AT 400 WORDS)

The skeletal response to estrogen is impaired in female but not in male steroid receptor coactivator (SRC)-1 knock out mice.

Estrogen (E) is critical for the maintenance of bone mass in both female and male mice and steroid receptor coactivator (SRC)-1 has been shown to be important for mediating E effects on bone, at least in female mice. In the present study, we defined the skeletal phenotype of male SRC-1 knock out (KO) mice and compared it with their female littermates. Further, to determine the role of SRC-1 in mediating effects of E on bone in male mice, we examined the skeletal effects of gonadectomy (gnx) with or without E replacement in male mice and placed these findings in the context of our previous studies in female SRC-1 KO mice. Analysis of a large group of male (WT, n=67; SRC-1 KO, n=56) and female (WT, n=66; SRC-1 KO, n=70) mice showed a significant decrease in trabecular volumetric bone mineral density (vBMD) in SRC-1 KO mice compared to their WT littermates in both genders (male SRC-1 KO, 275+/-3 vs. WT, 295+/-3 mg/cm(3), P<0.001; female SRC-1 KO, 210+/-2 vs. WT, 221+/-2 mg/cm(3), P<0.001). Following gnx and E replacement (10 microg/kg/day), we previously demonstrated that SRC-1 KO female mice have a defect in E action in trabecular, but not in cortical bone. In contrast, we now demonstrate that the same dose of E administered to gnx'd male SRC-1 KO mice was sufficient to prevent trabecular bone loss in these mice. For example, in WT female mice, gnx followed by E replacement maintained spine BMD (1.2+/-3.4% vs. baseline) as compared to gnx without E replacement (-12.7+/-2.6%, P<0.001 vs. sham); this effect of E was absent in SRC-1 KO female mice. By contrast, the identical dose of E was equally effective in maintaining spine BMD in E-treated gnx'd male WT (-5.2+/-5.1% vs. baseline) and male SRC-1 KO (-5.4+/-5.3%) mice, respectively, as compared to gnx'd mice without E treatment (WT, -17.6+/-2.5%, P=0.02; SRC-1 KO, -28.6+/-2.6%, P<0.001 vs. sham). E treatment was effective in suppressing cancellous bone turnover in both gnx'd WT and SRC-1 KO male mice as determined by significant reductions in osteoblast and osteoclast numbers; however, in female mice, E treatment only suppressed bone turnover in WT but not in SRC-1 KO mice. Collectively, these findings demonstrate that loss of SRC-1 results in trabecular osteopenia in male and female mice, but in contrast to female mice, this is not due to any detectable resistance to E action in trabecular bone in male SRC-1 KO mice.

TIEG-null mice display an osteopenic gender-specific phenotype.

TGFbeta inducible early gene-1 (TIEG) was originally cloned from human osteoblasts (OB) and has been shown to play an important role in TGFbeta/Smad signaling, regulation of gene expression and OB growth and differentiation. To better understand the biological role of TIEG in the skeleton, we have generated congenic TIEG-null (TIEG(-/-)) mice in a pure C57BL/6 background. Through the use of DXA and pQCT analysis, we have demonstrated that the femurs and tibias of two-month-old female TIEG(-/-) mice display significant decreases in total bone mineral content, density, and area relative to wild-type (WT) littermates. However, no differences were observed for any of these bone parameters in male mice. Further characterization of the bone phenotype of female TIEG(-/-) mice involved mechanical 3-point bending tests, micro-CT, and histomorphometric analyses of bone. The 3-point bending tests revealed that the femurs of female TIEG(-/-) mice have reduced strength with increased flexibility compared to WT littermates. Micro-CT analysis of femurs of two-month-old female TIEG(-/-) mice revealed significant decreases in cortical bone parameters compared to WT littermates. Histomorphometric evaluation of the distal femur revealed that female TIEG(-/-) mice also display a 31% decrease in cancellous bone area, which is primarily due to a decrease in trabecular number. At the cellular level, female TIEG(-/-) mice exhibit a 42% reduction in bone formation rate which is almost entirely due to a reduction in double labeled perimeter. Differences in mineral apposition rate were not detected between WT and TIEG(-/-) mice. Taken together, these findings suggest that female TIEG(-/-) mice are osteopenic mainly due to a decrease in the total number of functional/mature OBs.

Overexpression of the TGFbeta-regulated zinc finger encoding gene, TIEG, induces apoptosis in pancreatic epithelial cells.

Members of the TGFbeta family of peptides exert antiproliferative effects and induce apoptosis in epithelial cell populations. In the exocrine pancreas, these peptides not only regulate normal cell growth, but alterations in these pathways have been associated with neoplastic transformation. Therefore, the identification of molecules that regulate exocrine pancreatic cell proliferation and apoptotic cell death in response to TGFbeta peptides is necessary for a better understanding of normal morphogenesis as well as carcinogenesis of the pancreas. In this study, we have characterized the expression and function in exocrine pancreatic epithelial cells of the TGFbeta-inducible early gene (TIEG), a Krüppel-like zinc finger transcription factor encoding gene previously isolated from mesodermally derived osteoblastic cells. We demonstrate that this gene is expressed in both acinar and ductular epithelial cell populations from the exocrine pancreas. In addition, we show that the expression of TIEG is regulated by TGFbeta1 as an early response gene in pancreatic epithelial cell lines. Moreover, overexpression of TIEG in the TGFbeta-sensitive epithelial cell line PANC1 is sufficient to induce apoptosis. Together, these results support a role for TIEG in linking TGFbeta-mediated signaling cascades to the regulation of pancreatic epithelial cell growth.

Bone morphogenetic protein-6 production in human osteoblastic cell lines. Selective regulation by estrogen.

Bone morphogenetic proteins (BMPs) induce differentiation of osteoblast and chondroblast lineage cells from uncommitted mesenchymal precursors. Because estrogen has potent osteochondrogenic actions, we investigated its effect on BMP production in two estrogen-responsive, human immortalized cell lines (hFOB/ER3 and hFOB/ER9) that display the mature osteoblast phenotype. These cell lines were produced by stable transfection of the estrogen receptor (ER) gene into immortalized fetal osteoblasts at low ( approximately 800 ER/ nucleus) and at high ( approximately 3, 900 ER/nucleus) levels, respectively. As assessed by reverse transcriptase PCR, treatment with 17beta-estradiol (10(-)10 - 10(-)7 M) increased steady-state levels of BMP-6 mRNA dose dependently by twofold in the hFOB/ER3 cells and by over threefold in the hFOB/ER9 cells. Messenger RNA levels for transforming growth factors-beta1 and -beta2 and BMPs-1 through -5 and -7 levels were unchanged. The results were confirmed by sequence determination of the PCR product and by Northern blot analysis for total RNA. 17beta-estradiol increased BMP-6 protein production sixfold by Western analysis. Cotreatment with antiestrogens (ICI 182,780 or 4-hydroxytamoxifen) antagonized the effects of 17beta-estradiol. These data suggest that some of the skeletal effects of estrogen on bone and cartilage may be mediated by increased production of BMP-6 by osteoblasts.

Effects of stable transfection of human fetal osteoblast cells with estrogen receptor-alpha on regulation of gene expression by tibolone.

Tibolone is a synthetic steroid which undergoes tissue selective metabolism into several metabolites having estrogenic, progestogenic or androgenic activities. The effects of 3 alpha-hydroxy tibolone (Org 4094), 3 beta-hydroxy tibolone (Org 30126) and their sulfated metabolites were investigated on human fetal osteoblasts (hFOB). Tibolone had no effect on selected osteoblast marker proteins in estrogen-receptor negative hFOB cells. In contrast, 3 alpha-hydroxy and 3beta-hydroxy tibolone resulted in dose-dependent increases in alkaline phosphatase activity in estrogen receptor (ER) alpha-positive hFOB cells. The maximum increase for both metabolites was comparable to the effects of an optimal dose of 17beta-estradiol, and occurred at 10 muM. At 20 muM, both metabolites increased mRNA levels for alkaline phosphatase and type 1 collagen and protein levels for osteocalcin. Sulfated metabolites of tibolone also increased alkaline phosphatase activity. The estrogen receptor antagonist ICI 182, 780 inhibited stimulation of alkaline phosphatase activity by sulfated and non-sulfated tibolone metabolites, but was more potent on the former. Taken together, these results suggest that stable transfection of ER alpha into hFOB cells confers regulation by 3 alpha-hydroxy and 3beta-hydroxy tibolone metabolites of osteoblast metabolism.

Effect of TGF-beta inducible early gene deficiency on flexor tendon healing.

The role of transforming growth factor beta (TGF-beta) in tendon healing is still not clearly established. TGF-beta affects gene expression primarily through the activation of the Smad signaling pathway. The first step in the Smad pathway is the expression of TGF-beta inducible early gene (TIEG). Recently, a TIEG knockout mouse has been developed. The purpose of this study was to examine the healing potential of flexor tendons in mice lacking the TIEG gene, and to further examine what role the TIEG pathway plays in flexor tendon repair. Twenty-two mice, consisting of 11 normal wild-type mice and 11 TIEG knockout mice, were euthanized at 8 to 12 weeks of age. The second through fifth FDL tendons of both hind feet were transected and repaired in zone 2. The repaired tendons were removed from the mice and placed into tissue culture. Tendons were then examined at days 3, 7, 14, 21, and 42 after surgery. Hematoxylin and eosin (HE) staining and immunohistochemical staining for TGF-beta, collagen type I, and collagen type III were performed. Reverse transcriptase polymerase chain reaction (RT-PCR) was performed to examine expression of TGF-beta1, beta2, beta3, and collagen type I and III. At 42 days after surgery, HE staining showed coaptation of lacerated tendon ends in both groups. Both groups showed healing of the lacerated tendon, but the chronologic expression pattern of TGF-beta was different between the knockout and normal tendons. TIEG deficient tendons had delayed expression of TGF-beta when compared with control tendons. The collagen mRNA expression pattern was similar with both groups, but the expression level was different, with TIEG knockout tendons having a lower expression of collagen type I mRNA (p < 0.001). TGF-beta is thought to play a major role in tendon healing. Healing of tendons in the TIEG knockout mouse suggests the possibility of tendon healing in the absence of the Smad pathway. The knockout mouse model described in the present study provides a novel means for further understanding of the tendon healing process through isolated deletion of specific growth factors.

Effects of metabolism on the binding of polycyclic hydrocarbons to nuclear subfractions of cultured AKR mouse embryo cells.

A high-affinity localization of [2H]methylcholanthrene and/or its metabolites to a specific nuclear fraction (Fraction l) between 4 and 72 hr of exposure is described. Other carcinogenic hydrocarbons, such as benzo(a)pyrene and dibenz(a,h)anthracene, demonstrate a similar markded localization in Fraction l after 24 hr of incubation. The weak carcinogen dibenz(a,c)anthracene, as well as steroid hormones, sho little localization in this fraction. Two types of binding are measured:and organic solvent: extractable (non-covalent) binding and a nonextractable (covalent) binding. Maximal levels of the combined extractable and nonextractable binding per mass DNA are found at 24 hr of exposure, while at 48 and 72 hr of exposure the binding is reduced. The highest level of the nonextractable binding per mass DNA is also observed at the 24-hr exposure period. However, as the period of exposure increases, the proportion of the total nuclear-bound radioactivity representing the non-extractable type increases. Analysis by high-pressure liquid chromatography of the extractable radioactivity from the fractions indicates that the longer the period of exposure, the greater the extent of metabolism of 3-methylchol-anthrene. When 7,8-benzoflavone (a flavanoid hydroxylase inhibitor) is included in the incubations, practically all metabolic alterations of the parent compound are prevented. In addition, the time-dependent increase in nonextractable radioactivity from all nuclear subfractions is prevented. A metabolic-dependent "covalent" binding of carcinogenic polycyclic aromatic hydrocarbons to the barious nuclear subfractions of chromatin is suggested. This covalent binding is markedly localized in a specific fraction of the chromatin containing rapidly labeled nascent RNA.

Separation and characterization of transcriptionally active and inactive nuclear subfractions of AKR mouse embryo cells.

This study was initiated with the objective of separating and characterizing two or more nuclear subfractions, which could then be compared with respect to their relative propensity for binding carcinogenic polycyclic aromatic hydrocarbons. Nuclei were isolated from cloned AKR-2B mouse embryo cells, which are susceptible to transformation by chemical carcinogens. The nuclei were mechanically sheared and subfractions were separated by sedimentation through a 0.17 to 1.7 M sucrose gradient. When the cells were treated with [3H]uridine for 30 min, most of the label incorporated into RNA was recovered in the top region of the gradients, which represented Nuclear Subfraction I. The majority of the chromatin DNA, however was localized in the bottom region (Subfraction II) and the pellet (Subfraction III). Precipitation (with CaCl2) of the rapidly labeled RNA of Subfraction I along with the chromatin DNA suggested that the label was present in nascent RNA chains still attached to the chromatin. Thus, the transcripitionally active chromatin seemed to be localized in Nuclear Subfraction I. The chromatin of Subfraction I was also the best template for RNA synthesis in vitro with exogenous bacterial polymerase. The protein and RNA content of subfraction I was greater than that of the other two subfractions and whole chromatin. Electron microscopy revealed the presence of membrane material in Subfraction I and II, with little such material in Subfraction III. Subfraction I differed from Subfractions II and II and whole chromation with respect to thermal denaturation of the DNA and histone composition (as determined by gel electrophoresis). The acidic protein composition (as determined by gel electrophoresis) differed for the chromatin of all three nuclear subfractions.

Binding of [3H]benzo(a)pyrene metabolites to the AKR mouse embryo cell line nuclear proteins.

Nuclear proteins from AKR-2B mouse embryo cells incubated 24 hr with [3H]benzo(a)pyrene (1 microgram/ml; 10 microCi/ml) were purified to remove nucleic acids and characterized by isoelectric focusing and sodium dodecyl sulfate gel electrophoresis on polyacrylamide gels. Analysis of the nuclear proteins by isoelectric focusing revealed a distribution of protein in the pH gradient with two prominent bands of proteins, one focusing at pH 5 and the other at pH 11, both of which bound significant amounts of the carcinogen. The acid-soluble proteins from the nuclear preparation were analyzed by isoelectric focusing, and the results also revealed two peaks of protein and radioactivity at pH's of 5 and 11. Further analysis of the acid-soluble fraction on acid urea gels revealed that significant radioactivity was associated with proteins that migrated near but slower than histone H1 which may be the high-mobility group proteins 1 and 2. Radioactivity also comigrated with histones H3 and H2B. Low levels of radioactivity were associated with histone H1 and H4.

Binding of polycyclic aromatic hydrocarbons to transcriptionally active nuclear subfractions of AKR mouse embryo cells.

The objective of this study was to examine the binding of carcinogenic polycyclic aromatic hydrocarbons in well-characterized nuclear subfractions from transformable cells in culture. A cloned line of AKR mouse embryo cells was exposed to culture medium containing [3H]-3-methyl-cholanthrene (MC) (0.4 mug/ml) 670 Ci/mole). Cellular uptake and nuclear binding were determined after 4 hr of exposure. The addition of unlabeled MC up to 10 mug/ml did not cause reduction of [3H]MC cellular uptake or nuclear binding. From 2 to 5% of the total cellular MC was localized in the nuclei. All nuclear subfractions obtained from mechanically sheared nuclei and separated on sucrose gradients showed some MC binding; however, a high-affinity, high-specific-activity binding of MC was associated only with the slower-sedimenting component shown to represent that fraction of nuclear chromatin that is transcriptionally active. Conditions that caused the precipitation of this chromatin also resulted in the precipitation of the radioactive compound, thus suggesting that the MC was physically bound to the chromatin. Unlabeled MC (10 mug/ml) saturated this high-affinity MC binding to the transcripitionally active chromatin but did not saturate the binding to the other nuclear fractions. The binding of another potent carcinogen, [3H]-1,2,5,6-dibenzanthracene, and the "weak" carcinogen, [3H]-1,2,3,4-dibenzanthracene (3,4-DBA), to whole nuclei and nuclear subfractions was also determined. The concentration, specific activity, and time of treatment were identical with those used for MC. The level of binding of [3H]-1,2,5,6-dibenzanthracene was approximately 3-fold greater in whole nuclei on a per mass DNA basis than in those of either the MC or the 3,4-DBA. The binding of MC and 3,4-DBA to whole nuclei was approximately equal. As with MC, the [3H]-1,2,5,6-dibenzanthracene demonstrated a peak of high specific activity binding to the slower-sedimenting fraction of chromatin while the 3,4-DBA displayed considerably less binding to this fraction.

Identification of breast cancer cell line-derived paracrine factors that stimulate osteoclast activity.

Metastatic breast cancer causes destruction of significant amounts of bone, and, although bone is the most likely site of breast cancer metastasis, little is understood about interactions between tumor cells and bone-resorbing osteoclasts. We have investigated the paracrine factors produced by breast cancer cells that are involved in increasing osteoclast activity. We have determined by immunoassay that the human breast cancer cell line MDA MB 231 (231) cultured in serum-free medium secretes transforming growth factors type beta(TGF-beta) 1 and 2, macrophage colony-stimulating factor (M-CSF), granulocyte macrophage colony-stimulating factor (GM-CSF), interleukin (IL) -1 and -6, tumor necrosis factor alpha (TNF-alpha), insulin-like growth factor II (IGF II), and parathyroid hormone-related peptide. To determine which of these are involved in increased bone destruction, we have fractionated serum-free 231-conditioned media and measured these fractions for effects on osteoclast resorption activity using multiple activity assays. The pattern of responses was complex. Several fractions stimulated osteoclast resorption either by increasing the number of osteoclasts binding to the bone or by elevating the resorption activity of the individual osteoclasts. Other fractions inhibited osteoclast activity. Analysis of active fractions for the factors identified in the 231-conditioned medium revealed that the presence of TNF-alpha and IGF-II was restricted to separate fractions that stimulated osteoclast resorption activity. The fractions that inhibited osteoclast resorption activity contained M-CSF, IL-6, TGF-beta2, and GM-CSF. No TGF-beta1 or IL-1 was detected in any of the active fractions. Our data support the hypothesis that breast cancer cells modulate osteoclast activity using multiple regulatory factors that increase both the number of mature osteoclasts attached to the bone and the bone resorption activity of these individual osteoclasts. Once it is understood how metastatic breast cancer elevates osteoclast-mediated bone loss, effective therapies to slow the progression and/or prevent this bone loss will become possible.

Bisphosphonates directly regulate cell proliferation, differentiation, and gene expression in human osteoblasts.

Bisphosphonates are widely used clinically to treat bone diseases in which bone resorption is in excess. However, the mechanism of bisphosphonate action on bone is not fully understood. Studies of direct action of bisphosphonates on bone have been limited mainly to their effects on bone-resorbing osteoclast cells, with implications that some activity may be mediated indirectly through paracrine factors produced by the bone-forming osteoblast cells. Little is known about the direct effects of bisphosphonates on osteoblasts. In this report, the direct actions of several bisphosphonates on cell proliferation, gene expression, and bone formation by cultured human fetal osteoblasts were examined. Osteoblast cell proliferation was decreased, and cytodifferentiation was increased in a dose-dependent manner in cultures treated with the bisphosphonate pamidronate. In addition, pamidronate treatment increased total cellular protein, alkaline phosphatase activity, and type I collagen secretion in osteoblasts. Consistent with the above-mentioned findings, the rate of bone formation was also increased in osteoblasts cultured with pamidronate. The actions of two other bisphosphonates, the weak-acting etidronate and the potent new analogue zoledronate, were also compared with the action of pamidronate on proliferation of immortalized human fetal osteoblast (hFOB) cells and rate of bone formation. Pamidronate and zoledronate decreased hFOB cell proliferation with equal potency, whereas etidronate decreased proliferation only at much higher concentrations. Studies comparing EDTA and etidronate indicate that etidronate may act indirectly on the hFOB cells by reducing free divalent ion concentrations, whereas pamidronate and zoledronate appear to act on the hFOB cells by a direct action. Both pamidronate and zoledronate increase hFOB cell bone formation, whereas no increase is observed with etidronate and EDTA. Taken together, these observations strongly suggest that treatment with pamidronate or zoledronate enhances the differentiation and bone-forming activities of osteoblasts.

Effects of estrogen and progesterone on transcription, chromatin and ovalbumin gene expression in the chick oviduct.

The effects of estrogen, progesterone and estrogen + progesterone combined on nuclear transcriptional processes in oviducts of immature chicks, previously withdrawn from estrogen, are reported. The responses to the steroids of the endogenous nuclear RNA polymerase activities, both nucleolar (I) and nucleoplasmic (II), the chromatin compositions and template capacities, and the appearance of ovalbumin messenger RNA (mRNA) are compared. When immature chicks (previously treated at 14 days with estrogen) are withdrawn from estrogen treatment, there is a gradual reduction in both polymerase activities. Diurnal variations in polymerase II activties in the oviduct of withdrawn chicks required that subsequent experiments include time-matched controls. The hormones alter RNA polymerase II and II activities in vivo as assayed in isolated nuclei. Progesterone represses the polymerase I and II activities, while estrogen alone and estrogen + progesterone enhance both polymerase activities immediately after injection. Diethylstillbestrol, a synthetic estrogen, causes changes similar to those of estrogen. The effects of these steroids on the polymerases are detected within 15 min of hormone injection. Changes in the capacities of chromatins to serve as template for RNA synthesis in general correlated with changes in polymerase II activities. Interestingly, in the case of estrogen treatment, the acidic chromatin protein (but not histone) levels fluctuate positively with the template capacities of the chromatin. An antagonism between estrogen and progesterone is observed in the responses of both RNA polymerases I and II activities as well as in the chromatin template capacity. Levels of messenger RNA coding for ovalbumin, as detected by hybridization with labeled complementary DNA, increase in oviducts of withdrawn chicks within 2--3 of the injection of estrogen, progesterone or estrogen + progesterone. This rapid accumulation of ovalbumin mRNA is not accompanied in each case by a similar increase in polymerase II activity or chromatin template capacity.