Rat Osteosarcoma Cells Research Articles

Expression and regulation of galectin 3 in rat osteoblastic cells.

Galectin 3 is an endogenous soluble beta-galactoside-specific lectin originally identified and termed epsilon BP or IgE-binding protein in rat basophilic leukemia cells, but its wide tissue distribution and the multiple contexts in which it has been isolated have suggested that its function may not be limited to IgE binding but may include a role in cell growth regulation and differentiation, neoplastic transformation, and cell adhesion (Liu, 1990, Crit. Rev. Immunol., 10:289-306; Barondes et al., 1994, J. Biol. Chem., 269:20807-20810). After immunoscreening of a lambda gt11 cDNA expression library made from bone-nodule forming cultures of fetal rat calvaria (RC) cells with an antibody raised against osteoblastic cells (Turksen et al., 1992, J. Histochem. Cytochem., 40:1339-1352), three cDNA clones were isolated and sequenced; the sequence matched that of rat galectin 3. Galectin 3 mRNA was detected in various fetal and adult rat tissues, including calvaria and cultured RC cells. In RC cells and the rat osteosarcoma cell line ROS 17/2.8, galectin 3 mRNA expression increased with time in culture, in contrast to its behavior in fetal rat skin fibroblasts (RSF) in which its expression decreased with time in culture. In a second rat osteosarcoma line, UMR 106.01, galectin 3 mRNA was almost nondetectable. The synthetic glucocorticoid dexamethasone (Dex) enhanced galectin 3 expression in RSF cell cultures, while 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) had no significant effect. In contrast, Dex downregulated and 1,25(OH)2D3 upregulated galectin 3 expression in RC and ROS 17/2.8 cells, especially at later time points in culture when expression of osteoblast-associated differentiation markers by these cell types is most marked. Immunolabeling with an antibody against rat galectin 3 to identify galectin 3 protein showed that cells labelled within both the ROS 17/2.8 and RC populations but with marked intercellular heterogeneity of intensity. Our data support the conclusion that galectin 3 is a previously unrecognized product of osteoblastic cells, that galectin 3 mRNA and protein expression increases with time in vitro concomitant with other markers of osteogenesis, including formation of bone nodules and expression of osteoblast-associated markers such as alkaline phosphatase, bone sialo-protein, and osteocalcin, and that its expression is regulated by hormones such as glucocorticoids and 1,25(OH)2D3 that modulate other aspects of the osteoblast phenotype.

Characteristics of tumor cell bioactivity in oncogenic osteomalacia

Oncogenic osteomalacia is a condition where renal phosphate wasting occurs causing defective mineralisation, in the presence of a tumor. Cultures of cells were established from a hemangiopericytoma resected from a patient with oncogenic osteomalacia. Conditioned media from the cells inhibited phosphate uptake in opossum kidney cells and stimulated production of cAMP in rat osteosarcoma cells, a standard parathyroid hormone (PTH)-like assay. This cAMP stimulation was suppressed by the PTH analogue, 3–34 bPTH and also by heat and trypsin treatment of the media. Tests of conditioned media for PTH and parathyroid hormone related protein (PTHrP) immunoreactivity were negative, however, and no hybridisation to probes for PTH, PTHrP or human stanniocalcin was detected in tumor cell RNA on Northern blot. These data support the hypothesis that tumors responsible for oncogenic osteomalacia produce a humoral substance that reduces renal phosphate reabsorption and provide evidence that the factor may act via PTH/PTHrP receptors.

Growth hormone induces tyrosine phosphorylation of annexin I in rat osteosarcoma cells

Growth hormone induces tyrosine phosphorylation of annexin I in rat osteosarcoma cells

Growth hormone induces tyrosine phosphorylation of annexin I in rat osteosarcoma cells.

GH induces phosphorylation of a number of cellular proteins, of which several have now been identified, such as mitogen-activated protein kinase, insulin receptor substrate-1, and members of the JAK kinase and STAT families of proteins. However, other phosphorylated proteins remain unidentified. Growth factors and cytokines, including epidermal growth factor, insulin, pp60v-scr, and angiotensin II, induce a rapid phosphorylation of annexin I, a 35-kDa member of the annexin family of Ca2+ and phospholipid-binding proteins. The osteoblast-like rat osteosarcoma cell-line UMR-106.01, in which GH acts as a mitogen via a high affinity GH receptor, was used as a model for GH-induced protein phosphorylation. It is demonstrated by immunoblotting and immunoprecipitation techniques that GH induces the phosphorylation of annexin I on tyrosine residues. This phosphorylation is dose and time dependent. Induction of annexin I phosphorylation is delayed compared with that of JAK2. These results identify annexin I as a protein that becomes tyrosine phosphorylated under the influence of GH and show that phosphorylation of annexin I is a general phenomenon that follows activation of a cell by hormones or cytokines.

Hydrostatic Pressure Has Different Effects on the Assembly of Tubulin, Actin, Myosin II, Vinculin, Talin, Vimentin, and Cytokeratin in Mammalian Tissue Cells

Hydrostatic pressures in the range of hundreds of atmospheres are known to disrupt cytoskeletal organization in tissue culture cells, with profound changes in cell shape. The molecular mechanisms of these effects are poorly understood. To determine the effect of pressure on the cytoskeleton, and thus to provide better indicators of the molecular mechanisms, we used fluorescent antibody staining to compare the organizations of seven different cytoskeletal proteins in HeLa cells and rat osteosarcoma cells (ROS-17/2.8) subjected to different pressures up to 400 atm. Pressures of 300 atm or more caused cells of both lines to “round up” and to withdraw their lamellar extensions. However, this response varied within a population of cells, with some cells remaining spread at pressures that caused their neighbors to round up. The most resistant to rounding were those cells touching other cells, and the occasional giant cells. As expected, the rounded cells showed disruption of actin stress fibers and of vinculin and talin at focal contacts. The unrounded cells showed less disruption in the organization of these same proteins. Microtubules and myosin II filaments appeared resistant to 400 atm pressure in both cell types, whether rounded or unrounded. However, in HeLa cells, the intermediate filaments, vimentin and cytokeratin, depolymerized and formed small vesicles when pressures exceeded 200 atm, and this occurred in rounded as well as unrounded cells. In osteosarcoma cells, which do not have cytokeratin, vimentin did not depolymerize. We discuss different mechanisms that might explain these responses to pressure, including direct effects on the equilibria of protein polymerization and less direct effects on regulatory mechanisms, such as phosphorylation pathways, that control cytoskeletal organization. The latter type of explanation seems more consistent with both the variability of response within cell populations and the difference in vimentin's response in one cell line compared with the other.

Serum stimulation of parathyroid hormone-related peptide gene expression in ROS 17/2.8 osteosarcoma cells through transcriptional and posttranscriptional mechanisms.

The gene encoding PTH-related peptide (PTHrP), a protein that plays a primary role in the development of humoral hypercalcemia of malignancy, is widely expressed in normal and neoplastic tissues. This study demonstrates that expression of the PTHrP gene has features of early response genes, including up-regulation after serum repletion of serum-starved ROS 17/2.8 (rat osteosarcoma) cells. The PTHrP messenger RNA (mRNA) levels were induced within 30 min and peaked at 4 h. Elevated mRNA levels were accompanied by an increase in secreted PTHrP. The serum effects on PTHrP mRNA levels were blocked by actinomycin D, suggesting a requirement for gene transcription. Nuclear run-on assays revealed a 3-fold increase in PTHrP gene transcription 4 h after exposure to serum. Deletions of the 5' flanking sequence of the rat PTHrP gene fused to the chloramphenicol acetyltransferase gene and transfected into ROS 17/2.8 cells showed that the serum-responsive region is located between -1.05 kb and -0.3 kb upstream of the transcription start site. PTHrP mRNA levels were also induced by cycloheximide, another feature common to early response genes. The PTHrP mRNA half-life in serum-starved cells was 56 min. Serum treatment prolonged the half-life 2.7-fold, suggesting serum-induced stabilization of the mRNA. Insulin and epidermal growth factor also induced PTHrP mRNA expression in a time-dependent manner analogous to serum, indicating that the effects of serum may be mediated, at least partially, through these agents. In summary, serum up-regulated PTHrP mRNA expression through both transcriptional and posttranscriptional mechanisms. This rapid stimulation by growth factors suggests that PTHrP may contribute to the early cellular response after growth factor stimulation.

FRAG1, a gene that potently activates fibroblast growth factor receptor by C-terminal fusion through chromosomal rearrangement.

A constitutively active form of fibroblast growth factor 2 (FGFR2) was identified in rat osteosarcoma (ROS) cells by an expression cloning strategy. Unlike other tyrosine kinase receptors activated by N-terminal truncation in tumors, this receptor, FGFR2-ROS, contains an altered C terminus generated from chromosomal rearrangement with a novel gene, designated FGFR activating gene 1 (FRAG1). While the removal of the C terminus slightly activates FGFR2, the presence of the FRAG1 sequence drastically stimulates the transforming activity and autophosphorylation of the receptor. FGFR2-ROS is expressed as a unusually large protein and is highly phosphorylated in NIH 3T3 transfectants. FRAG1 is ubiquitously expressed and encodes a predicted protein of 28 kDa lacking significant structural similarity to known proteins. Epitope-tagged FRAG1 protein showed a perinuclear localization by immunofluorescence staining. The highly activated state of FGFR2-ROS appears to be attributed to constitutive dimer formation and higher phosphorylation level as well as possibly altered subcellular localization. These results indicate a unique mechanism of receptor activation by a C terminus alteration through a chromosomal fusion with FRAG1.

Alteration of the hormonal bioactivity of parathyroid hormone-related protein (PTHrP) as a result of limited proteolysis by prostate-specific antigen

Objectives To discover whether the proteolytic activity of prostate-specific antigen (PSA) affects the structure and function of parathyroid hormone-related protein (PTHrP), as both are abundant components of human seminal plasma. Methods The ability of PTHrP to act as a substrate was studied by incubating a synthetic polypeptide, consisting of 34 amino acid residues of the amino-terminal domain of PTHrP, with purified PSA. The incubate was then analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, high-pressure liquid chromatography separation, amino-terminal peptide sequencing and mass spectrometry. The physiologic effect of the proteolytic activity of PSA on PTHrP was studied by measuring any alteration in PTHrP ( 1–34)-induced elevation of cyclic adenosine monophosphate (cAMP) production by UMR-106 rat osteosarcoma cells in culture. All cell culture experiments were performed with PSA and PTHrP (1–34) at physiologic concentrations. Results Our data show that PSA proteolytically cleaves PTHrP (1–34) after either residue 22 or 23, generating three peptide fragments. Both cleavages occur carboxy terminally of a phenylalanine residue. The cAMP production in rat osteosarcoma cells, induced by the amino-terminal portion of PTHrP (1–34), as a result of its structural similarity with parathyroid hormone (PTH), was abated by PSA in a dose- and timedependent fashion. In contrast, heat-inactivated PSA had no effect on cAMP production. Conclusions Our study demonstrates that PTHrP is a substrate for PSA. The cleavage of the amino-terminal portion of PTHrP completely disrupts its ability to interact with the PTH/PTHrP receptor and thus inhibits its PTH-like activity. The proteolytic processing of PTHrP by PSA may play an important role in the post-translational/post-secretional regulation of prostatic PTHrP activities, which are believed to include regulation of prostate growth and differentiation.

1α,25(OH)2Vitamin D3-Mediated Stimulation of Outward Anionic Currents in Osteoblast-like ROS 17/2.8 Cells

1α,25-dihydroxyvitamin-D3[1α,25(OH)2D3] is known to potentiate in osteoblast-like rat osteosarcoma cells (ROS 17/2.8) the influx of Ca2+through voltage-activated L-type Ca2+channels. In the present study, by means of the whole-cell patch-clamp technique, we found that physiological concentrations of 1α,25(OH)2D3(5–5000 × 10−11M) also stimulate, in a dose-dependent manner, outwardly rectifying anionic currents over the course of 1–5 minutes. These currents, recorded in the presence of 130 mM glutamate or ≈140 mM Cl−in the external solution, were blocked by 200 μM DIDS, a stilbene derivative selective for Cl−channels. We conclude that, in addition to the already known effects of 1α,25(OH)2D3on Ca2+channels, the hormone also enhances in ROS 17/2.8 cells an outward anion conductance. This is the first report concerning activation of anion channels by 1α,25(OH)2D3.

Conformational studies on analogs of recombinant parathyroid hormone and

Through the use of oligonucleotide-directed mutagenesis we have generated variants of a recombinant human parathyroid (PTH) hormone-(1-34)-homoserine (RPTH) in which a positively charged residue (Arg or Lys), a negatively charged residue (Glu), or a neutral residue (Gly) has been substituted at every position throughout the peptide. These 106 PTH analogs have been tested for their ability to stimulate cAMP production in the rat osteosarcoma cell line, UMR106. Analysis of these peptides led to the construction of several analogs containing multiple substitutions at sites of potential structural importance. Several of these analogs were shown to have 3-5-fold enhanced activity and receptor affinity. Circular dichroism (CD) and lipid binding studies were then performed on these analogs. Circular dichroism demonstrates enhanced helical content in the presence of lipid vesicles, particularly anionic lipid. The [Arg15,19,22,Lys29]RPTH (+6RPTH) analog requires higher concentrations of trifluoroethanol to attain enhanced helicity. The intrinsic tryptophan fluorescence of the peptides are blue shifted more in the presence of the anionic lipid dimyristoyl phosphatidylglycerol (DMPG) than with the zwitterionic lipid dimyristoyl phosphatidylcholine (DMPC). Effects of the peptides on the phase transition behavior of DMPC shows that +6RPTH has less effect on the lipid than does RPTH. This difference in lipid interaction is also exhibited with isothermal titration calorimetry, in which RPTH reacts exothermally with DMPG, while +6RPTH shows little or no heat change. The pH dependence of binding of the hydrophobic probe 1,1'-bis(4-anilino)-naphthalene-5,5'-trisulfonic acid, also shows a difference in exposure of hydrophobic sites between RPTH and +6RPTH. The +6RPTH has about a 5-fold greater affinity for receptor binding. We suggest that this enhanced activity is a consequence of the altered lipid interaction of +6RPTH, combined with increased conformational flexibility, particularly in the carboxyl-terminal region of the molecule.

AP-1 regulation of the rat bone sialoprotein gene transcription is mediated through a TPA response element within a glucocorticoid response unit in the gene promoter

Bone sialoprotein (BSP), a protein which has been implicated in the initial mineralization of newly-formed bone, provides an early phenotypic marker for differentiated osteoblasts. BSP expression is induced by glucocorticoids in association with osteoblast differentiation, and a glucocorticoid response element (GRE) overlapping a putative TRE (TPA, 12-O-tetradecanoyl-phorbol 13-acetate, response element) site has been identified in the rat BSP promoter (Ogata et al., 1995). Since AP-1 and the glucocorticoid receptor have a central role in regulating cell proliferation and differentiation, we have studied AP-1 activity, stimulated by 100 ng/ml TPA in normal fetal rat calvarial cells and in transformed rat osteosarcoma cells (ROS 17/2.8). A transient induction of both c-fos and c-jun mRNAs by TPA was observed in both cell populations, together with an associated suppression of BSP mRNA in the fetal rat calvarial cells. Rat BSP promoter constructs, transiently transfected into ROS 17/2.8 cells, were used to show that TPA suppressed transcription of a luciferase construct (-938/+60; pLUC6) that included the GRE/TRE, but not transcription of shorter contructs lacking this element. Notably, suppression of pLUC6 transcription by TPA was abrogated in the presence of the synthetic glucocorticoid, dexamethasone. Gel mobility shift analyses were performed using two double-stranded synthetic oligonucleotides. These encompassed the TRE and either the distal pair of GRE half-sites (-936/ -910; GRE3) or the proximal pair of GRE half-sites (-925/-899; GRE 4) that comprise the GRE/AP-1 element. The assay showed binding of both AP-1 complexes and recombinant c-Jun homodimers. Additionally, either the c-Jun or glucocorticoid receptor could displace its counterpart from the GRE/TRE but not from consensus GRE and TRE oligonucleotides, indicating that the abrogation of AP-1-mediated gene suppression by glucocorticoids could involve competitive binding. These studies, therefore, have identified a glucocorticoid response unit through which c-Fos and c-Jun can suppress the expression of BSP in proliferating pre-osteoblastic cells and through which glucocorticoids can ameliorate the effects of AP-1 and promote osteoblast differentiation and the associated expression of BSP.

Effect of endogenously produced parathyroid hormone-related peptide on growth of a human hepatoma cell line (Hep G2).

A well differentiated human hepatoma cell line (Hep G2) was used to explore potential roles for PTH-related peptide (PTHrP) as an autocrine/paracrine growth factor. Using Northern analysis or reverse transcription-PCR, Hep G2 cells were found to express messenger RNAs for both PTHrP and the cloned PTH/PTHrP receptor, and the cells exhibited specific binding for [125I]PTHrP(1-36). Hep G2 growth medium was found to contain relatively large amounts of immunoreactive PTHrP (30 vs. 1-2 pM in medium not exposed to cells), and the PTHrP in growth medium (conditioned medium) was shown to contain N-terminal PTHrP biological activity, as assessed by the ability of the medium to stimulate cAMP production in rat osteosarcoma cells (ROS 17/2.8). Conditioned medium produced a dose-dependent severalfold increase in ROS cell cAMP that could be blocked by the PTHrP receptor antagonist [Asn10,Leu11,DTrp12]PTHrP-(7-34). PTHrP in Hep G2 cells also was detected by immunocytochemistry using antiserum to either synthetic PTHrP-(1-34) or recombinant PTHrP-(-5 to 139). Furthermore, these antisera were found to inhibit the ability of PTHrP-(1-34) to stimulate ROS cell cAMP production. When either antiserum (1:800-1:100 dilution) was added to subconfluent Hep G2 cells in medium containing 5% FBS for 3 days, a dose-related 40-50% increase in cell number occurred that could be inhibited by concurrent addition of 10 microM synthetic PTHrP-(1-36). The results show that Hep G2 cells synthesize and secrete both immunoreactive and biologically active PTHrP. As neutralization of PTHrP secreted by these cells by the addition of antiserum to PTHrP resulted in increased cell growth, the findings suggest that PTHrP can function as an autocrine or paracrine growth factor to suppress the growth of these human hepatoma cells.

DNA sequences in the rat parathyroid hormone-related peptide gene responsible for 1,25-dihydroxyvitamin D3-mediated transcriptional repression.

Expression of the gene encoding PTH-related peptide (PTHrP), a protein that plays a primary role in the development of humoral hypercalcemia of malignancy, is down-regulated at the transcriptional level by 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. Deletions of the 5'-flanking region of the rat PTHrP gene, when fused to the chloramphenicol acetyl-transferase gene and transfected into ROS 17/2.8 (rat osteosarcoma) cells, showed that the 1,25-(OH)2D3 responsive region is located between -1.05 and -0.71 kb upstream of the transcription start site. Further mapping of this region revealed that a 123-bp fragment is able to confer 1,25-(OH)2D3 responsiveness to a heterologous (SV40) promoter. This region contains two potential vitamin D response elements (VDREs). One of these motifs resembles the negative VDRE (nVDRE) from the PTH gene, which is also down-regulated by vitamin D3. The other element resembles the canonical VDRE (two hexanucleotide motifs separated by three nucleotides), which has been characterized in a number of genes whose expression is modulated by vitamin D3. Electrophoretic mobility shift assays using nuclear extracts from ROS 17/2,8 cells and from vitamin D receptor. (VDR)-enriched COS 1 cells revealed that both elements interact with the VDR. This protein-DNA interaction is disrupted by an anti-VDR antibody. Therefore, modulation of PTHrP gene transcription by 1,25-(OH)2D3 is mediated by the VDR interacting with one or both of the identified motifs in the 5'-flanking sequence of the gene.

Bone-selective analogs of human PTH(1-34) increase bone formation in an ovariectomized rat model.

Intermittent parathyroid hormone (PTH) therapy increases bone mass. The purpose of this study was to determine if analogs of human PTH(1-34) (hPTH[1-34]), which differ from the native sequence in their receptor-activating properties, could promote bone formation in an ovariectomized (OVX) osteopenic rat model. We synthesized two hPTH(1-34) analogs with single substitutions for serine in the 3-position that in vitro are partial agonists in kidney. In the renal cell line OK, maximal cyclic adenosine monophosphate (cAMP) activation by [His(3)]hPTH(134) was 50%, and maximal cAMP activation by [Leu(3)]hPTH(1-34) was 20% of that produced by hPTH(1-34). Both analogs were full agonists in UMR-106 rat osteosarcoma cells and other bone-derived systems, but both had reduced potency compared with hPTh(1-34). Six-month-old retired breeder Sprague-Dawley rats were ovariectomized, and five animals underwent sham operation. On day 56 post-OVX, five sham-operated and five pre-PTH treatment OVX animals were sacrificed, and the remaining animals were randomized into 10 groups of six animals each. All other animals were injected with one of the hPTH analogs or hPTH(1-34) at 0, 4, 40, or 400 mu g/kg of body weight (BW)/day and were killed on day 84. Histomorphometry of the proximal tibia metaphysis and biochemical markers of bone turnover (osteocalcin and pyridinoline cross-links) were the primary endpoints. The cancellous bone volume was significantly lower at day 56 post-OVX (pretreatment) and at day 84 post-OVX (post-vehicle treatment) than at baseline. None of the compounds significantly increased the cancellous bone volume. Trabecular number declined after OVX and did not change with hPTH treatment. In contrast, the trabecular thickness declined after OVX but was higher after treatment with 40 mu g/kg of BW/day or 400 mu g/kg of BW/day of hPTH(1-34). In OVX rats, the mineralizing surface was higher than baseline at day 56 and fell toward control levels by day 84. All three peptides produced marked dose-related increases in the mineralizing surface and bone formation rates, but the two analogs were less potent than hPTH(1-34). Likewise, all peptides produced significant dose-related increases in the serum osteocalcin level. The osteoclast surface was not affected by OVX but was decreased with medium and high doses of hPTH(1-34). Pyridinoline cross-link excretion was not significantly affected by treatment with hPTH(1-34) but responded with a dose-dependent decrease to treatment with [His3]hPTH(1-34). These data suggest that bone selective analogs of hPTH(1-34) maintain the ability to induce bone formation but are less potent than hPTH(1-34).

Poly(L-lactic acid) foams with cell seeding and controlled-release capacity.

A synthetic porous three-dimensional structure that can mimic the architecture of actual tissues, provide sustained release of nutrients or growth factors, and serve as a template for cell seeding would be an ideal substrate for tissue engineering. Poly(l-lactic acid) (PLLA) foams were fabricated for this purpose, based on the principle of phase separation from homogeneous naphthalene solutions. Complex shapes could be readily fabricated, and resulting foams had relatively uniform, open cells throughout the matrix. Densities and total pore-surface areas were in the range of 0.05-0.1 g/cm3 and 0.8-1.3 m2/g, respectively. The loss tangent of these foams ranged from 0.07 to 0.128, as measured by thermomechanical analysis. Naphthalene residue in the resulting foams went below 0.2 wt% after extensive vacuum sublimation. Feasibility of incorporating drugs or nutrients into such a highly porous structure was demonstrated by the dispersion of two model compounds, bromothymol blue (BTB) and sulforhodamine B (SD), in the matrix. Sustained release of BTB from the foam with a porosity as high as 87% was observed for more than 2 months. Alkaline phosphatase, as a model protein to be incorporated, lost approximately 30% of its bioactivity during the fabrication. As a cell-culture substrate, the PLLA foams performed as well as the flat PLLA surface in supporting the growth of rat osteosarcoma cells (ROS 17/2.8) and in maintaining their functions such as alkaline phosphatase activity and osteocalcin synthesis. UMR-106 cells cultured in the foam also expressed a higher degree of mineralization than those cultured on the flat PLLA substrate.

CD44 Expression in Fetal Rat Bone:In Vivoandin VitroAnalysis

Knowledge of the expression pattern of the cell surface glycoprotein CD44 in the development and differentiation of bone is limited. We investigated CD44 expression (a) in bone sections of 21-day-old fetal rat calvaria (RC), metatarsals, and tibiae, (b) in primary cultures of RC cells undergoing differentiationin vitro,and (c) in three rat osteosarcoma cell lines: ROS 17/2.8, UMR 106.01, and UMR 106.06. By immunocytochemistry, Western, Northern, and reverse transcription polymerase chain reaction analyses, we found that osteoblastic cells express the “hematopoietic” or “standard” CD44 (CD44s) isoform. Osteoblastic cellsin vivoandin vitrostained at all detectable stages of differentiation, but intercellular heterogeneity of CD44s staining was evident, with lesser staining in preosteoblastic cells and greater staining in mature osteoblasts and osteocytes. As cells in RC cultures differentiated and formed bonein vitro,CD44s mRNA and protein levels as measured on immunoblots were invariant. All three osteosarcoma cell lines expressed CD44s mRNA and protein. The synthetic glucocorticoid dexamethasone, which stimulates osteogenesis in RC cellsin vitroand regulates a number of osteoblast-associated genes, had no apparent effect on either CD44s protein or mRNA levels. The widespread presence of CD44s in osteoblastic cells at various maturational stages suggests that further analyses will be required to determine what role CD44s may play in osteogenesis and in bone tissue organization.

Affinity Labeling of the 1α,25-Dihydroxyvitamin D3 Receptor

Genomic actions of the calciotropic hormone 1 alpha, 25-dihydroxyvitamin D3 (1,25(OH)2D3) involves a multistep process that is triggered by the highly specific binding of 1,25(OH)2D3 to 1 alpha, 25-dihydroxyvitamin D3 receptor, VDR. In order to study this key step in the cascade, we synthesized 1 alpha,25-dihydroxy[26(27)-3H]vitamin D3-3-deoxy-3 beta-bromoacetate (1,25(OH)2[3H]D3-BE) and 1 alpha,25-dihydroxyvitaminD3-3 beta-[1-14C]bromoacetate(1,25(OH)2D3-[14C]BE) binding-site directed analogs of 1,25(OH)2D3, and affinity-labeled baculovirus-expressed recombinant human VDR (with 1,25(OH)2[3H]D3-BE), and naturally occurring VDRs in cytosols from calf thymus homogenate and rat osteosarcoma (ROS 17/2.8) cells (with 1,25(OH)2D3-[14C]BE). In each case, specificity of labeling was demonstrated by the drastic reduction in labeling when the incubation was carried out in the presence of an excess of nonradioactive 1 alpha,25(OH)2D3. These results strongly suggested that 1,25(OH)2[3H]D3-BE and 1,25(OH)2D3-[14C]BE covalently modified the 1,25(OH)2D3-binding sites in baculovirus-expressed recombinant human VDR and naturally occurring calf thymus VDR and rat osteosarcoma VDR, respectively.

Expression of parathyroid hormone-related protein in cells of osteoblast lineage

The expression of parathyroid hormone-related protein (PTHrP) was studied in a range of cell cultures representative of the osteoblast lineage and in rat calvarial sections. Primary newborn rat calvarial cells, a rat preosteoblastic cell line (UMR 201), a mouse stromal cell line (ST 2), a mouse calvaria-derived osteoblastic cell line (KS 4), and rat osteosarcoma cell lines (UMR 106-01 and -06), all expressed PTHrP when examined by reverse transcription polymerase chain reaction (RT-PCR). Using a radioimmunoassay we also demonstrated PTHrP in the conditioned medium of the cultured cells, with the exception of UMR 106-01 and -06 cells. Treatment of UMR 201 cells with all-trans-retinoic acid which induces them to acquire a more differentiated phenotype, also led to a time-dependent decrease in PTHrP mRNA levels as determined by RT-PCR, Northern blot analysis, and in situ hybridization. Decreased PTHrP levels in the conditioned medium of the treated cells was also observed. These results suggested that PTHrP production might be greater in less mature osteoblasts. Examination of the populations obtained from newborn rat calvariae by sequential collagenase digestion revealed that the early digests exhibited low ALP activity, low expression of PTH/PTHrP receptor mRNA, and no adenylate cyclase response to PTHrP(1-34). These populations showed the highest level of mRNA and production of PTHrP. Cells from later digests, the "osteoblast-rich" populations, had reduced PTHrP expression. Immunohistochemistry and in situ hybridization in sections of newborn rat calvariae showed PTHrP expression in cuboidal osteoblasts located adjacent to bone and in spindle-shaped cells in the periosteal region. It is concluded that PTHrP is produced by cells of the osteoblast lineage, supporting the hypothesis that PTHrP may function physiologically as a paracrine factor in bone.

Comparison among the actions of 1,25(OH)2D3, interleukin 1α and interleukin 6 on cellular proliferation and diferentiation (alkaline phosphatase activity) in rat osteosarcoma cell cul tures (UMR-106)

Comparison among the actions of 1,25(OH)2D3, interleukin 1α and interleukin 6 on cellular proliferation and diferentiation (alkaline phosphatase activity) in rat osteosarcoma cell cul tures (UMR-106)

Expression and Regulation by Insulin of Glut 3 in UMR 106-01, a Clonal Rat Osteosarcoma Cell Line

Expression of the glucose transporter GLUT 3 is mainly restricted to neuronal tissues, with lower levels in testis and placenta. In addition, GLUT 3 has recently been reported in neonatal rat calvaria byin situhybridisation. We report the co-expression of GLUT 1 and GLUT 3 mRNA and protein in UMR 106-01, a clonal osteosarcoma cell line. By semi-quantitative analysis we show that GLUT 3 protein is expressed at levels comparable to GLUT 1. Insulin stimulates glucose uptake in UMR 106-01 cells. GLUT 3 mRNA and protein are increased by chronic (16 h) treatment with insulin, and the increase in GLUT 3 mRNA is not inhibited by cycloheximide. Regulation of GLUT 3 mRNA by insulin has not been previously shown. UMR 106-01 represents a useful model for investigating regulation of GLUT 3 expression.