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With the knowledge that estrogen replacement therapy can circumvent postmenopausal osteoporosis and with the discovery of estrogen receptors (ER) in cultures of normal osteoblast-like cells, extensive investigations have been directed toward understanding the role of the ER in normal bone homeostasis. ROS 17/2.8 and UMR-106-01, two established osteoblast-like cell lines derived from rat osteosarcomas, have been shown to have estrogen-regulated biologic responses. Only the ROS 17/2.8 cell line has been reported to contain ER. In this study, high-affinity, saturable binding sites characteristic of the ER were detected in UMR-106-01 cells by binding assays with the high-affinity ligand, [125I]17 beta-estradiol. An initial immunoconcentration step before western blot analysis also allowed detection of the full-length ER protein. In addition, northern blot analysis indicated that the entire ER transcript was expressed and that the half-life of the ER message was increased following cycloheximide treatment. Message levels were also regulated by removal of serum and treatment with estradiol. An estrogen-regulated reporter vector, ERET81CAT, was transfected into the UMR-106-01 cells to determine whether the detected level of ER was transcriptionally functional. Using this assay, estrogen responsiveness was evident; however, the response was inconsistent. Multiple factors, such as serum, estradiol, and cell density, influence the ER levels in these cells and probably cause fluctuations in the abundance of receptors available to induce the CAT response. When the cells are responsive, the ICI 164,384 antagonist could block the estrogen-induced activation of CAT.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

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.

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.

Metabolism of 3H-25OHD3 was studied in pregnant, D-deprived rats and their fetuses at 21 days’ gestation. Significant changes in the circulating levels of 3H-24,25(OH)2D3 and 3H-1,25(OH)2D3 in maternal and fetal plasma occurred from 4 to 12 h after the administration of 223 ng of 3H-250HD3. 3H-1,25(OH)2D3 appeared before 3H-24,25(OH)2D3 in both the mother and fetuses. Maternal plasma contained more 3H-1,25(OH)2D3 than fetal plasma and, contrariwise, fetal plasma contained more 3H-24,25(OH)2D3 than maternal plasma. Maternal nephrectomy studies showed that the in vivo synthesis of 3H-24,25(OH)2D3 at 6 h was dependent on circulating levels of 3H-1,25(OH)2D3. These results revealed aspects of maternal-fetal metabolism of 250HD3 similar to those in the non-pregnant state as well as others unique to pregnancy.

SummaryVitamin D metabolism was studied in pregnant, D-deficient rats and their fetuses. D-depleted, pregnant rats were supplemented with [3H]25OHD3 on the 19th day of pregnancy. The distribution and metabolism of radiolabeled D metabolites was different in maternal and fetal blood, kidneys, and small intestine. 24,25(OH)2D3 was the predominant dihydroxylated D metabolite in the fetus, whereas 1,25(OH)2D3 was the predominant dihydroxylated D metabolite in the mother. The ratio of 24,25(OH)2D3:1,25-(OH)2D3 was 12-fold greater in fetal plasma than maternal plasma. Maternal nephrectomy reduced the metabolism of [3H]25OHD3 to 24,25(OH)2D3 (43%) and 1,25(OH)2D3 (75%). However, plasma levels of these two metabolites were unchanged in the fetuses of these animals when compared with levels observed in fetuses from mothers with intact kidneys. These results suggest the possibility of independent control of 25OHD3 metabolism by the feto-placental unit and raise questions as to the possible role of 24,25(OH)2D3 in f...

The kidneys are thought to be the only organs capable of 1 alpha-hydroxylation of vitamin D and its metabolites. We have examined the in vivo conversion of 3H-(25,26)-25-hydroxyvitamin D3(25OHD3) to 3H-(25,26)-1 alpha,25-dihydroxyvitamin D3 [1 alpha,25(OH)2D3] in vitamin D-deficient, pregnant and nonpregnant rats. As expected, nephrectomy of nonpregnant, vitamin D-deficient rats prevented the conversion of 25OHD3 to 1 alpha,25(OH)2D3. In contrast, nephrectomy of pregnant, vitamin D-deficient rats reduced but did not abolish the formation of 1 alpha,25(OH)2D3 from its precursor. The identity of the radioactive metabolite formed from 3H-25OHD3 which circulated in nephrectomized, pregnant rats was established as 1 alpha,25(OH)2D3 by comigration with synthetic 1 alpha,25(OH)2D3 on high-pressure liquid chromatography. The simultaneous absence of 1 alpha,25(OH)2D3 in the fetal kidneys indicated that the site of 1 alpha-hydroxylation after nephrectomy of the pregnant rat was probably extra-renal in origin. Two sites of 1 alpha-hydroxylation of 25OHD3, one renal and the other extra-renal, either fetoplacental or maternal, may exist in the pregnant, vitamin D-deficient rat.

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.

A model of the maternal-fetal metabolism of vitamin D3 is depicted in Fig. 2. 25-OHD3 of maternal origin is metabolized by the maternal kidneys to the potent metabolite, 1,25-(OH)2D3, which acts on the maternal intestine, kidneys, and skeleton. The maternal kidneys and other organs can produce 24,25-(OH)2D3, although this pathway may be suppressed near the end of gestation. The placenta has selective permeability to the vitamin D3 metabolites, with 25-OHD3 crossing from the mother to the fetus more readily than the dihydroxylated metabolites. The onset of the placental synthesis of 1,25-(OH)2D3 during gestation is unknown. Likewise the regulation of the placental 25-OHD3-1 alpha-hydroxylase is unknown. 1,25-(OH)2D3 of placental origin may enter the maternal or the fetal circulation or act locally on the placenta by inducing the synthesis of proteins involved in the cellular transport of Ca. Perhaps one placenta cell type synthesizes 1,25-(OH)2D3 and another cell type possessing a cytoplasmic receptor for 1,25-(OH)2D3 responds to this metabolite. The function of the 24,25-(OH)2D3 produced by the placenta is unknown. The concentration of free 25-OHD3 and free 1,25-(OH)2D3 in the fetal circulation exceeds the maternal levels due to the differences in the DBP concentrations of the two bloodstreams. The 1,25-(OH)2D3 in the fetal bloodstream may originate from either the placenta or the fetal kidneys. The latter site may not be active in utero due to the hypercalcemia and hyperphosphatemia relative to the maternal levels of these ions. 1,25-(OH)2D3 in the fetal bloodstream acts on those fetal tissues containing cytoplasmic receptors for this metabolite. The intestinal mucosa apparently lacks these receptors until sometime during neonatal life. In contrast, fetal bone cells possess receptors for the 1,25-(OH)2D3. The 24,25-(OH)2D3 in the fetal bloodstream may also be involved in the growth and differentiation of the fetal skeleton. However, the precise role of both metabolites in the fetus remains conjectural.

The effect of 17β-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 aspettate 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 white 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.

SummaryThe administration of SCT, natural and synthetic, had no apparent effect on the ileal water and electrolyte transport in the rabbit. The failure of SCT to influence ileal transport of water and electrolytes in the rabbit, as it does in man, may be due to differences in the rabbit intestinal response to a foreign peptide hormone.The authors wish to acknowledge the assistance of Kathy Dodson and Janet Patterson in the typing of this manuscript.

The progress in knowledge about Vitamin D metabolism and mechanisms of its action in the last ten years has brought us to the formulation of several questions about hormonal factors involved in phosphate transport mechanisms, phosphate homeostasis and phosphate homeostasis hormonal regulation. Beside broadly described phosphaturic effect of parathyroid hormone also active metabolite of Vitamin D3, 1,25 (OH)2D3, in serum phosphate homeostasis recently has been suggested as an important factor /1/.

U937 is a human-derived lymphoma cell line that has monoblastic properties and high-affinity receptors for 1 alpha,-dihydroxyvitamin D3. Incubation of these cells with the vitamin D metabolite at 10 nM for 5 days produced marked stimulation in adherence and ingestion of Staphylococcus aureus (645% of control) and of C3b receptor (CR1) expression (292% of control) and a slight increase in hexose monophosphate shunt activity without changing cell growth rates or Fc fragment receptor expression. The changes in cellular association of S. aureus and the CR1 were detected as early as 48 hr of incubation and peaked between 3 and 5 days. Similar changes in the CR1 were induced by 25-hydroxy- and 24,25-dihydroxyvitamin D3 at micromolar concentrations. Dexamethasone, hydrocortisone, and progesterone had no effect on CR1 expression. U937 cells incubated in the presence of vitamin D metabolites exhibited a change in their phenotype. These results suggest that vitamin D metabolites may contribute to monocyte/macrophage differentiation.