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T K Gray

Publications and source records attributed to T K Gray.

At least 19 recordsLinked to original sources
PubMed1992

Estrogens modulate the responsiveness of osteoblast-like cells (ROS 17/2.8) stably transfected with estrogen receptor.

Recent studies have demonstrated the presence of estrogen receptor (ER) in both normal human osteoblast-like and osteoblast-like osteosarcoma cells. The number of ER in cultured osteoblastic cells is very low (200-500 sites/cell). This has complicated characterization of the biological role of estrogens in bone cells. To study the responsiveness of bone cells to estrogens, we established osteoblast-like cell lines expressing higher ER levels. ROS 17/2.8, an osteoblastic cell line, was stably transfected with the cDNA encoding for the mouse ER. After a selection period, positive clones were isolated and evaluated for the presence of ER by both Northern blot analysis and ligand binding assays. Using these techniques, we detected a significant increase in the level of both ER transcript and binding compared to that in wild-type cells. The levels of expressed ER protein were similar to those reported in normal human osteoblast-like cells in primary culture (approximately 2000 sites/cell). To test whether the exogenously inserted ER was responsive, both wild-type and ER stably transfected cells were transiently transfected with a reporter construct containing an estrogen-responsive element linked to a truncated thymidine kinase promoter and a chloramphenicol acetyltransferase (CAT) reporter gene. Exposure of the cells to increased concentrations of estradiol induced a slight increase in CAT activity in wild-type cells (approximately 1.5-fold) at maximal stimulation; however, it provoked a clear concentration-dependent increase in CAT activity in the ER stably transfected cells, with a maximal stimulation of approximately 10-fold. This event was receptor mediated, since ICI 164,384, an ER antagonist, blocked the enhancement of estradiol-induced CAT activity, and it was specific, since other steroid hormones did not stimulate CAT activity. Finally, we evaluated the ability of ER to modulate an endogenous estrogen-responsive gene by measuring the activity of the enzyme alkaline phosphatase. In addition, diethylstilbestrol, a synthetic estrogen agonist, increased the activity of both the CAT reporter gene and the endogenous alkaline phosphatase enzyme. In summary, we have established osteoblast-like cells expressing high levels of an exogenously inserted ER, which has characteristics similar to those of the endogenous ER in terms of its Kd. Finally, the exogenous ER regulates both exogenously inserted construct (VITERECAT) and endogenous properties of the cells (enzymatic activity and proliferation).

Alkaline Phosphatase↗
PubMed1989

Estradiol stimulates in vitro the secretion of insulin-like growth factors by the clonal osteoblastic cell line, UMR106.

UMR106 cells, a rat osteosarcoma derived clonal line, secreted insulin-like growth factors (IGF) in vitro. The IGF-II levels corrected for the cell numbers were 7-8 times higher than the IGF-I levels in the medium. Both growth factors were higher by 4-5 fold in medium conditioned by rapidly growing cells than in medium conditioned by confluent cells. The addition of 17-beta-estradiol (E) to the culture medium was associated with a statistically significant increase in the IGF concentrations. This increment was metabolite specific, not occurring with 17-alpha-E, the inactive epimer of E. 1,25(OH)2D3 also increased the IGF-I concentration but prior treatment with E blocked the response to 1,25(OH)2D3, demonstrating antagonistic actions of these two hormones on IGF secretion by osteoblast-like cells.

Animals↗
PubMed1989

Estrogens and the skeleton: cellular and molecular mechanisms.

Postmenopausal women lose bone mineral density and this loss can be prevented by estrogen administration. Although the skeletal effects of estrogens have been regarded previously as indirect, estrogen receptors have been discovered in cultured human osteoblasts and related cell lines. The UMR106 cell line derived from a rat osteogenic osteosarcoma is such an osteoblast model. We have shown direct effects of estradiol (E) on these cells in vitro, inhibiting growth and stimulating alkaline phosphatase activity (AP) corrected for cell number. This response was maximal at E conc. of 10(-10) M in serum and Phenol Red free medium, was metabolite specific and cell cycle-dependent. These cells contain high affinity binding sites with a Kd of 0.5 nM. Estrogen receptors were detected by the monoclonal antibody H-222 on Western blot after initial immunoprecipitation to concentrate the proteins. E treatment increased several enzymes including creatine kinase and LDH isoenzymes along with increments in intracellular transferrin. Transforming growth factor-beta is secreted by these cells. Secretion of this peptide was stimulated by E. TGF-beta mediated the transient growth inhibition associated with E treatment. Insulin like growth factors (IGF) are also secreted by these cells with IGF-II concentrations in the culture medium being eight times higher than IGF-I levels. E treatment increased the concentrations of both IGFs in the culture medium after a 3 day incubation. Exposure of E treated cells manifested a mitogenic response and reduced AP, indicating that E induced receptors for IGFs. These findings establish direct effects of E on osteoblastic cells in vitro and demonstrate responses to E at many levels. These osteoblast responses in vitro suggest an important role for sex steroids in the development and function of the osteoblast lineage.

Alkaline Phosphatase↗
PubMed1989

Biochemical effects of 17 beta-estradiol on UMR106 cells.

The effect of 17 beta-estradiol (E) on an osteoblast-like cell line, UMR106, was studied in vitro. The concentrations of transferrin and seven enzymes (gamma glutamyl transferase, alkaline phosphatase, acid phosphatase, lactate dehydrogenase, creatine kinase, alanine aminotransferase and aspartate aminotransferase) were measured in these cells after incubation in culture medium containing either E or the vehicle. E treatment increased five of the seven enzymes and increased the transferrin concentration in the UMR106 cells while simultaneously reducing the proliferation rates. 4-Hydroxytamoxifen, an estrogen antagonist, produced a mild estrogen agonist action on growth rates and enzyme concentrations in the UMR106 cells. When E was present simultaneously, the agonist properties of 4-hydroxytamoxifen were enhanced. These studies show that E enhanced activity of five enzymes and the transferrin content of UMR106 cells after a 2-day incubation. 4-Hydroxytamoxifen enhanced the E effect, illustrating that estrogen antagonists may manifest agonist or antagonist properties depending on the model. These results extend our previous observations showing a direct effect of E in vitro on osteoblast-like cells.

Acid Phosphatase↗
PubMed1989

Transforming growth factor beta mediates the estrogen induced inhibition of UMR106 cell growth.

A mitogenic response to transforming growth factor beta (TGF) occurred in the UMR106 cells cultured in serum-free medium and exposed serially to estradiol and TGF. This mitogenic response was lost when insulin was removed from the medium. TGF inhibited growth and increased the alkaline phosphatase content in the UMR106 cells cultured in medium lacking insulin. Prior exposure of the cells to estradiol enhanced this response. Monoclonal antibodies against TGF blocked the estradiol induced inhibition of growth after a two day incubation in medium devoid of insulin.

Alkaline Phosphatase↗
PubMed1988

Ring hydroxylation of 25-hydroxycholecalciferol by rat renal microsomes.

Two metabolites have been isolated from rat renal microsomes incubated with 25-hydroxycholecalciferol. Postmitochondrial supernatant fractions from kidneys of thyroidectomized and parathyroidectomized rats were incubated with magnesium acetate, potassium acetate, an NADPH generating system, and 25-hydroxycholecalciferol at a level of 20 micrograms/ml postmitochondrial supernatant for 60 min at 30 degrees C. Lipid extracts of the incubation mixtures were purified by silica gel TLC and HPLC. Two peaks were obtained. Metabolite chi 2 eluted at 18 min and metabolite chi 1 at 23 min when chromatographed on a silica column developed with hexane-isopropanol. Metabolites chi 1 and chi 2 were found to have maximal absorbance at 265 nm. Both metabolites were periodate sensitive, indicating vicinal hydroxyl groups. Mass spectral analysis of metabolite chi 2, which was isolated in greater quantity than metabolite chi 1, indicates that metabolite chi 2 had resulted from hydroxylation of the A ring. Results indicate that 25-hydroxycholecalciferol is hydroxylated on carbon 2 or carbon 4 by renal microsomes. Metabolites chi 1 and chi 2, because of similarity in chromatographic migration and periodate sensitivity, are, perhaps, isomers or 2- and 4-hydroxylated metabolites.

Animals↗
PubMed1987

Biological activity of 19-nor, 10-keto, 25-hydroxyvitamin D3.

19 nor, 10 keto, 25-hydroxyvitamin D3 (19/10-25OHD3) is a metabolite of 25-OHD3 produced in vitro by various phagocytes including normal human blood monocytes and transformed cell lines, U937 and HL-60. We recently reported that 19/10-25OHD3, induced differentiation of U937 cells. In these studies, 19/10-25OHD3 alone produced no detectable effect on the growth rates, surface adherence, and oxidative metabolism of U937 and HL-60 cells. When combined with lymphocyte-conditioned medium (LCM), 19/10-25OHD3 reduced proliferation, increased surface adherence and stimulated luminol-dependent luminescence (LDL) of the U937 cells. In contrast, the combination of 19/10-25OHD3 and LCM had no effect on the growth of HL-60 cells but did increase the surface adherence and the expression of a complement receptor component. 19/10-25OHD3 competed for tritium-labeled 1,25(OH)2D3 binding to receptors extracted from cultured human skin fibroblasts. This displacement capacity was 600 times weaker than that of unlabeled 1,25(OH)2D3. Incubation of human skin fibroblasts for 24 hr with 19/10-25OHD3 induced 25OHD3-24-hydroxylase activity in the fibroblasts. The inductive potency of 19/10-25OHD3 was 1/50 that of 1,25(OH)2D3. These results demonstrate bioactivity of 19/10-25OHD3 in several systems. At least one of these responses, the induction of 25OHD3-24-hydroxylase, is a receptor-mediated event. Some of the other responses may be independent of the cellular receptor for 1,25(OH)2D3. Interestingly, the potency of 19/10-25OHD3 was highest in the receptor-mediated response (1:50) and lower in the other parameters, ranging from 1:100 to 1:600 compared to 1,25(OH)2D3. This range of bioactivity in phagocytes and fibroblasts is presently explained.

Calcifediol↗
PubMed1987

17 beta-estradiol acts directly on the clonal osteoblastic cell line UMR106.

We studied the effect of 17 beta-estradiol (E) on the proliferation and alkaline phosphatase activity of cultured UMR106 cells, a clonal osteoblastic cell line. Growth rates were reduced and alkaline phosphatase activity was increased in cells incubated for 2 days in medium containing E (10(-8) M). In contrast, E had no effect on the growth rates or alkaline phosphatase of a human fibroblastic cell line, S90E. The effect of E was not observed with low cell density or at confluence. 1,25-Dihydroxyvitamin D3 antagonized the response to E. Preincubation of the cells with dexamethasone, a potent inducer of differentiation, reversed the effect of E or 1,25-dihydroxyvitamin D3. These results indicate that cellular and/or extracellular factors such as cell density, the phase of the cell cycle, the state of differentiation, and the presence or absence of other steroids influenced the response of UMR106 cells to E. Serum was removed from the culture medium to minimize the effect of the steroids, growth factors, and nutrients present in serum. A striking stimulation of alkaline phosphatase by E occurred with serum-free conditions. This stimulation was biphasic over an E concentration from 10(-12) to 10(-8) M, with the peak response at 10(-10) M. The action of E on UMR106 cells was metabolite-specific, since the isomer 17 alpha-estradiol produced no effect on proliferation rates or alkaline phosphatase activity. The cyclic AMP response to parathyroid hormone (residues 1-34) was not altered by E treatment of these cells. In contrast, dexamethasone exposure did increase the cyclic AMP response to parathyroid hormone. These results demonstrate a direct effect of E on an osteoblastic cell line. They also raise the possibility that similar or identical actions of E occur in cultured normal osteoblasts.

Alkaline Phosphatase↗
PubMed1987

17 Beta-estradiol inhibits the oxidative metabolism of U937 cells indirectly via lymphocytes.

The effect of 17 beta-estradiol (E) on the oxidative metabolism of U937 cells was studied. E had no direct effect on the proliferation, surface adherence, or luminol-enhanced luminescence (LEL) of the U937 cells. Exposure of U937 cells to lymphocyte-conditioned medium (LCM) and 1,25-dihydroxyvitamin D3 allowed a maximal LEL response by cells stimulated with phorbol myristate acetate. In contrast, LCM from lymphocytes exposed to E (LCM-E) did not stimulate LEL to the same extent as did an equal volume of LCM. Increasing the E concentration in the lymphocyte medium was associated with a dose-dependent reduction in the LEL response of the U937 cells. Mixing equal quantities of LCM and LCM-E significantly decreased LEL levels. LEL stimulated by LCM, gamma-interferon, or differentiation-inducing factor was reduced by the presence of LCM-E. The inhibitory action of LCM-E was reversible and metabolite specific. 17 alpha-E produced an effect that was only one tenth the magnitude of the E effect. These findings indicate that E can modulate the differentiation of phagocytes indirectly by altering the synthesis and/or secretion of lymphokines.

Calcitriol↗
PubMed1987

1,25-Dihydroxyvitamin D3 enhances phorbol ester-stimulated differentiation and protein kinase C-dependent substrate phosphorylation activity in the U937 human monoblastoid cell.

In the U937 human monoblastoid cell line, 1,25-dihydroxyvitamin D3[1,25(OH)2D3] through a specific interaction with the 1,25(OH)2D3 receptor promotes differentiation toward a more mature phenotype. In addition to this direct effect, 1,25(OH)2D3 also potentiates differentiation in response to lymphokines and (Bu)2cAMP. We examined the effect of 1,25(OH)2D3 on phorbol ester-stimulated differentiation. Either preincubation with or simultaneous exposure to 1,25(OH)2D3 enhanced phorbol ester-stimulated differentiation. Over a 72-h period, the increase in phorbol ester responsiveness was dependent on the duration of 1,25(OH)2D3 exposure. Enhancement of phorbol ester-induced differentiation was observed with 1,25(OH)2D3 concentrations ranging from 0.1-10 nM. The 1,25(OH)2D3 vitamin D metabolite was more potent than the 24,25-dihydroxyvitamin D3 and 25-hydroxyvitamin D3 metabolites in potentiating phorbol ester-induced differentiation. Phorbol esters can exert at least a portion of their effects on cellular function by activating protein kinase C. Thus, one mechanism by which 1,25(OH)2D3 could amplify signal transduction leading to potentiation of phorbol ester-stimulated differentiation would be by enhancing phorbol ester-stimulated phosphorylation. To examine this possibility, we measured protein kinase C-dependent substrate phosphorylation in extracts derived from cells pretreated with 1,25(OH)2D3. In extracts derived from cells treated with 1,25(OH)2D3, the protein kinase C-dependent phosphorylation of endogenous U937 substrates stimulated by calcium, phosphatidyl serine, and diolein was increased compared to that observed in vehicle-treated cells. The conditions required for 1,25(OH)2D3 to increase protein kinase C-dependent phosphorylation of endogenous substrates (concentration, duration of exposure, and metabolite specificity) were similar to those required to enhance phorbol ester-stimulated differentiation. Possibly mediating this enhanced phosphorylation was an increase in protein kinase C activity observed in extracts derived from 1,25(OH)2D3-treated cells.

Calcitriol↗
PubMed1987

The metabolite 1 alpha,25-dihydroxyvitamin D3 enhances lymphokine-mediated activation of the oxidative metabolism of the U937 cell line and phosphorylation of a 48-kD respiratory burst-associated protein.

U937 cells respond to a variety of stimuli with increased differentiation as manifested by reduced growth, increased adherence, increased expression of several surface receptors, and increased capacity for phagocytosis and formation of reactive oxygen intermediates. In the present study the effects of lymphocyte conditioned media, recombinant interferon-gamma (IFN-gamma), and 1 alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3) on the ability to form reactive oxygen intermediates by U937 cells were measured by using the luminol-dependent luminescence (LDL) assay. Neither 1,25(OH)2D3 alone nor IFN-gamma alone enhanced competence for phorbol myristate acetate-stimulated LDL. Cells were capable of moderate LDL after exposure to lymphocyte conditioned media, and this was enhanced by 1,25(OH)2D3 (10(-8) mol/L) and other vitamin D metabolites at higher concentrations. This effect was not secondary to accelerated production of myeloperoxidase, which is important in the LDL assay. Enhanced phorbol myristate acetate-stimulated phosphorylation of a 48-kd substrate was observed in 32P-labeled intact cells treated with 1,25(OH)2D3 alone or in combination with IFN-gamma. Treatment of cells with IFN-gamma or lymphocyte conditioned media did not alter phosphorylation. These results support the concept that 1,25(OH)2D3 plays a role in phagocyte differentiation and activation beyond the effects of lymphokines. Protein kinase C-mediated phosphorylation reactions may be necessary for the ability of U937 cells to reduce O2 and required for maximal activity under some conditions of incubation.

Calcitriol↗
PubMed1986

1,25-Dihydroxyvitamin D3 activates secretion of hydrogen peroxide by human monocytes.

Human blood monocytes cultured in the presence of 1,25(OH)2D3 developed enhanced competence for secretion of H2O2 relative to cells suspended in media. This effect was maximal at a concentration of 10(-8) M 1,25(OH)2D3. After 3 days of incubation, monocyte-derived macrophages (MDM) exposed to 1,25(OH)2D3 demonstrated competence for secretion of H2O2 equivalent to cells exposed to recombinant IFN-gamma. Both IFN-gamma and 1,25(OH)2D3 offset decay of this function among cells in culture after 7 days. Simultaneous exposure of cells to 1,25(OH)2D3 and IFN-gamma did not activate competence for H2O2 secretion more than either agent alone. 24,25(OH)2D3 and 25(OH)2D3 activated MDM but at higher concentration than required for 1,25(OH)2D3. Progesterone did not affect H2O2 production. Incubation of MDM with a monoclonal antibody directed against IFN-gamma inhibited activation induced by lymphokine, and to a lesser extent by cells activated with IFN-gamma; this antibody had an insignificant effect on cells treated with 1,25(OH)2D3. These results suggest that 1,25(OH)2D3 exerts a receptor-mediated effect on monocyte function that results in cellular activation as manifested by enhanced competence for secretion of H2O2. It is possible that smaller concentrations of 1,25(OH)2D3 present in serum are permissive for macrophage activation, or that monocytic phagocytes are exposed to high concentrations of vitamin D metabolites under some clinical circumstances.

24,25-Dihydroxyvitamin D 3↗
PubMed1986

Development of vitamin D3 25-hydroxylase activity in rat liver microsomes.

The ontogeny of vitamin D3 25-hydroxylase activity has been determined in liver microsomes of rat fetuses and neonates. Production of 25-hydroxyvitamin D3 was low (0.11 pmol/g liver/h) 3 days prior to birth. Production rates were 1.2, 2.2, 1.8, and 2.8 pmol/g liver/h on Day 0, Day 2, Day 7, and Day 15, respectively. 25-Hydroxyvitamin D3 production in neonates increased sixfold from Day 15 to Day 22 to a value twice that of the mothers (17.6 pmol/g liver/h compared with 7.3 pmol/g liver/h). Activity in the maternal microsomes was constant (0.22 to 0.30 pmol/mg protein/h) except for the day of parturition (0.54 pmol/mg protein/h) and Day 22 postpartum (0.44 pmol/mg protein/h). A cytosolic factor, present as early as 3 days prior to birth, was required for vitamin D3 25-hydroxylase activity in the fetuses and stimulated the 25-hydroxylase reaction (up to 2.5-fold) in neonates and mothers. The ability of cytosol to prevent degradation of vitamin D3 was also present in the fetal stage. These data suggest that microsomal vitamin D3 25-hydroxylase activity in rat liver microsomes develops slowly and reaches full activity near the weaning stage. Since the cytosolic factor(s) is/are present in the fetal stage, the limiting component in the maturation of vitamin D3 25-hydroxylase activity in liver microsomes is the development of the cytochrome P-450 vitamin D3 25-hydroxylase.

Aging↗
PubMed1986

Retinoids induce U937 cells to express macrophage phenotype.

The effects of two retinoids, all-trans-retinoic acid (t-RA) and 13-cis-retinoic acid (c-RA) were studied in a model of osteoclast precursors. The model employs the U937 cell line induced to differentiate when incubated with 1 alpha,25-dihydroxyvitamin D3 and conditioned medium from stimulated human lymphocytes. t-RA and c-RA (10(-8) to 10(-6) M) inhibited cellular growth rates and increased surface adherence. However, t-RA and c-RA both partially blocked the differentiation induced by 1,25(OH)2D3 and lymphokines. Thus, retinoids alone promoted the differentiation of U937 cells but partially blocked the differentiation induced by a vitamin D metabolite and lymphokines. These results suggest that vitamins A and D may exert antagonistic effects on the recruitment of osteoclast precursors.

Antigens, Surface↗
PubMed1986

Calcitriol levels in hypercalcemic patients with adult T-cell lymphoma.

Hypercalcemia is a frequent complication in patients with adult T-cell lymphoma. We measured serum calcitriol (1,25-dihydroxyvitamin D3) levels in five hypercalcemic patients with adult T-cell lymphoma and compared the values with those of five patients with mycosis fungoides, a T-cell neoplasm not associated with hypercalcemia. All five patients with adult T-cell lymphoma had calcitriol levels in or below the normal range. These data show that elevated calcitriol levels are not uniformly elevated in this disorder and may not be the usual cause of hypercalcemia in this subgroup of patients with lymphoma.

Calcitriol↗
PubMed1986

Mononuclear phagocytes secrete a protein that directly resorbs devitalized bone particles.

The radiolabelled bone particle assay was used as an in vitro model of bone resorption. Normal human peripheral blood monocytes and the monoblastic cell line, U937, increased 45Ca release from the devitalized bone particles. In contrast, normal human peripheral blood neutrophils, resting or stimulated, did not increase 45Ca release. Exposure of monocytes to gamma interferon (INF-gamma) stimulated secretion of hydrogen peroxide and inhibited 45Ca release from bone particles. U937 cells incubated with 1,25(OH)2D3 and lymphokines had increased secretion of oxygen reduction products and increased 45Ca release. Medium conditioned by incubation with U937 cells stimulated 45Ca release to the same extent as the U937 cells. The 45Ca releasing activity in the medium was resistant to extremes of temperature and acidification. This activity is not dependent on the presence of disulfide bonds and was not inhibited by collagenase or trypsin inhibitors. Exposure to a proteolytic agent reduced the activity by over 50%. These findings are consistent with the concept that mononuclear phagocytes secrete a substance, presumably a protein, which acts directly on the bone particles. The isolation and identification of this substance may increase our understanding of the mechanisms of bone loss associated with inflammatory processes.

Bone Resorption↗
PubMed1986

Acid phosphatase activity in mononuclear phagocytes and the U937 cell line: monocyte-derived macrophages express tartrate-resistant acid phosphatase.

Tartrate-resistant acid phosphatase (TRAcP) is used as a marker for osteoclasts, which are believed to be derived from phagocytic cells or phagocyte stem cell precursors. To further investigate the relationship between monocytic phagocytes and osteoclasts, acid phosphatase (AcP) activity was measured by three different techniques in human peripheral blood monocytes, monocyte-derived macrophages, and the U937 cell line. We found that cytochemistry and gel electrophoresis led to similar results, but that the colorimetric assay was inconsistent. Normal human peripheral monocytes expressed both tartrate-sensitive and -resistant AcP. In culture these cells formed polykaryons and expressed TRAcP activity that was further identified as an isoenzyme associated with bone tissue. In contrast, the U937 cells did not express TRAcP activity as measured by gel electrophoresis. Both U937 cells and monocytes possess material that interferes with interpretation of the colorimetric assay of AcP. The presence of TRAcP in monocyte-derived macrophages further supports the relationship between phagocytic cells and bone osteoclasts.

Acid Phosphatase↗