Hormonal stimulation of bone cell proliferation

The recent demonstration of estrogen receptors in bone derived cells has stimulated the study of direct effects of sex steroids on bone. We have shown direct stimulation of proliferation by 17 beta-estradiol (E2) of ROS 17/2.8 rat osteogenic osteosarcoma cells, and other bone-derived cells in culture, as well as sex-specific stimulation of diaphyseal bone in vivo by estrogen and testosterone, using [3H]thymidine incorporation into DNA and stimulation of the specific activity of creatine kinase as markers. ROS 17/2.8 cells were used as models of osteoblast-like cells to study the reciprocal modulation of stimulation of bone cell proliferation by sequential treatment by sex steroid and calciotrophic hormones. Pretreatment with 1,25(OH)2D3 and PTH augmented stimulation by E2, while pretreatment with PGE2 followed by E2 resulted in no additional stimulation. Reciprocally, pretreatment with E2 significantly reduced the response to PGE2 while showing an insignificant effect on the response to the other hormones. Gonadectomized Wistar-derived rats provided a useful model system for study of postmenopausal osteoporosis. In diaphyseal bone, [3H]thymidine incorporation and creatine kinase activity decreased 4 weeks after gonadectomy. At that time, a single i.p. injection of E2 in females, and testosterone in males, resulted in a highly significant increase in both these parameters within 24 h.     

Hormonal regulation of [Ca(2+)](i) in periosteal-derived osteoblasts: effects of parathyroid hormone, 1,25(OH)(2)D(3) and prostaglandin E(2)

The effects of hormonal modulators of osteoblast function, parathyroid hormone, 1,25(OH)(2)D(3) and prostaglandins on [Ca(2+)](i) in periosteal-derived osteoblasts from rat femurs have been investigated. Our results show that application of parathyroid hormone PTH (10(-5) M) and prostaglandin E(2) (PGE(2)) (4 microM) result in a rapid heterogeneous elevation in [Ca(2+)](i) that, in the case of PTH, is dependent on both extracellular and intracellular sources of calcium. Variable responses to treatments have been found within populations of cells. The PGE(2) response is dose dependent. Treatment with 1,25(OH)(2)D(3) (10(-8) M) induces a brief (60-90 sec) elevation in [Ca(2+)](i) that is almost totally abolished in EGTA-buffered Ca(2+)-free medium. Interactive effects of multiple hormone treatments have been observed. Pretreatment with 1,25(OH)(2)D(3) results in near-total inhibition of the PTH and PGE(2) responses. In conclusion, modulation of [Ca(2+)](i) appears to play a role not only in the direct effects of osteotropic hormones on osteoblasts but also in the synergistic and antagonistic effects between circulating hormones.     

Differential stimulation by PGE(2) and calcemic hormones of IL-6 in stromal/osteoblastic cells

Formation of osteoclast-like cells in mouse bone marrow cultures induced by either 1,25-dihydroxyvitamin D(3) (1,25-(OH)(2)D(3)), parathyroid hormone (PTH) or prostaglandin E(2) (PGE(2)), respectively, shows partial dependence on interleukin-6 receptor (IL-6R) activation. This suggests that locally produced IL-6 could be relevant for osteoclast formation. Therefore, we evaluated the effects of 1,25-(OH)(2)D(3), PTH, and PGE(2) on IL-6 production in stromal/osteoblastic cell lines. It appeared that these bone resorptive factors differed widely in their ability to modulate IL-6 mRNA expression and, consequently, protein synthesis in each of the cell lines studied. While 1,25-(OH)(2)D(3) was marginally effective only in ST2 cells, and PTH caused a 2- to 20-fold increase in IL-6 levels MC3T3-E1 and UMR-106 cells, PGE(2) enhanced IL-6 production in the ST2 and MC3T3-E1 cell line by two to three orders of magnitude, respectively, and also induced IL-6 in fibroblastic L929 cells. PGE(2)-stimulated IL-6 release from mesenchymal cells seems to be important for autocrine/paracrine control of osteoclast formation in health and disease.     

Stimulation of creatine kinase BB activity by parathyroid hormone and by prostaglandin E2 in cultured bone cells.

Bone cells in culture responded to parathyroid hormone (PTH) and prostaglandin E2 (PGE2) by a 2-fold increase in creatine kinase (CK) activity. Combined treatment resulted in a higher response than with PTH alone. Calcitonin (CT) failed to stimulate CK activity, did not affect the response of CK to PTH, but inhibited slightly the increase in CK activity by PGE2. Bone-cell cultures grown in low [Ca2+] (0.125 mM), enriched in PTH-responsive osteoblast-like cells, responded to PTH, but not to PGE2 or CT, by increased CK activity. In both normal and low-[Ca2+] cultures, 8-bromo cyclic AMP did not affect CK activity, nor did it change the response of the cells to PTH, PGE2 or CT. The increase in CK activity was time- and dose-dependent and inhibited both by cycloheximide and by actinomycin D. The isoenzyme of CK stimulated was the CKBB form, the isoenzyme induced by other hormones. This appears to be the first report of the stimulation of CK activity by a polypeptide hormone or a prostaglandin. We suggest that stimulation of CKBB can serve as a marker for the action of a variety of hormones and growth promoters.     

Rat epiphyseal cells in culture: Responsiveness to bone-seeking hormones

Summary Cell cultures derived from young rat epiphyseal cartilage were grown for approximately 2 wk in BGJ _b medium supplemented with 10% fetal bovine serum to reach confluence. These cells were identified as chondrocytes as checked by morphology, the presence of alkaline phosphatase, and a positive type II collagen antibody reaction. The cells also responded to different hormonal treatment. Parathyroid hormone (PTH) increased cyclic AMP production by 50% within 15 min of treatment, whereas prostaglandin E₂ (PGE₂) caused an increase of 160%. Calcitonin (CT) did not affect cAMP production in these cells. DNA synthesis 24 h after hormonal treatment was increased by PTH (2.5-fold) and PGE₂ (2-fold), but not by CT. Among the vitamin D metabolites, 24,25(OH)₂D₃ increased significantly the [³H]thymidine incorporation into DNA, whereas 1,25(OH)₂D₃ effect was minimal. These results provide evidence for the use of these cell cultures as a model for cartilage in vitro when studying biological and hormonal responsiveness.     

1alpha,25(OH)2D3 and parathyroid hormone (PTH) signaling in rat intestinal cells: activation of cytosolic PLA2

In the current study, we have probed the role of cytosolic phospholipase A2 (cPLA2) activity in the cellular response to the calciotropic hormones, 1alpha,25,dihydroxy-vitamin D(3) [1alpha,25(OH)(2)D(3)] and PTH. Stimulation of rat enterocytes with either hormone, increased release of arachidonic acid (AA) 3H-AA] one-two fold in a concentration and time-dependent manner. The effect of either hormone on enterocytes was totally reduced by preincubation with the intracellular Ca(2+) chelator BAPTA-AM (5 microM), suggesting that the release of AA following cell exposure to the calciotropic hormones occurs mainly through a Ca(2+)-dependent mechanism involving activation of Ca(2+)-dependent cPLA2. Calciotropic homone stimulation of rat intestinal cells increases cPLA2 phosphorylation (three to four fold). This effect was decreased by PD 98059 (20 microM), a MAP kinase inhibitor, indicating that this action is, in part, mediated through activation of the MAP kinases ERK 1 and ERK2. Enterocytes exposure to 1alpha,25(OH)(2)D(3) (1nM) or PTH (10 nM) also resulted in P-cPLA2 translocation from cytosol to nuclei and membrane fractions, where phospholipase subtrates reside. Collectively, these data suggest that PTH and 1alpha,25(OH)(2)D(3) activate in duodenal cells, a Ca(2+)-dependent cytosolic PLA2 and attendant arachidonic acid release and that this activation requieres prior stimulation of intracellular ERK1/2. 1alpha,25(OH)(2)D(3) and PTH modulation of cPLA2 activity may change membrane fluidity and permeability and thereby affecting intestinal cell membrane function.     

Tamoxifen elicits its anti-estrogen effects in growth plate chondrocytes by inhibiting protein kinase C

17 beta-Estradiol (E(2)) regulates growth plate cartilage cells via classical nuclear receptor mechanisms, as well as by direct effects on the chondrocyte membrane. These direct effects are stereospecific, causing a rapid increase in protein kinase C (PKC) specific activity, are only found in cells from female rats and are mimicked by E(2)-bovine serum albumin (BSA), which cannot penetrate the cell membrane. E(2) and E(2)-BSA stimulate alkaline phosphatase specific activity and proteoglycan sulfation in female rat costochondral cartilage cell cultures, but traditional nuclear receptors do not appear to be involved. This study examined the effect of the anti-estrogen tamoxifen on these markers of chondrocyte differentiation; the gender-specificity of tamoxifen's effect on PKC, if tamoxifen has an effect on vitamin D metabolite-stimulated PKC, which is mediated via specific membrane receptors (1,25-mVDR; 24,25-mVDR) and whether the effect of tamoxifen is mediated by nuclear estrogen receptors. Tamoxifen dose-dependently inhibited the effect of E(2)-BSA on PKC, alkaline phosphatase and proteoglycan sulfation in confluent cultures of female resting zone (RC) cells and growth zone (GC) (prehypertrophic/upper hypertrophic zones) cells, suggesting that its action is at the membrane and not cell maturation-dependent. Neither the estrogen receptor (ER) antagonist ICI 182780 nor the ER agonist diethylstilbesterol affected E(2) or E(2)-BSA-stimulated PKC in female chondrocytes. Tamoxifen also inhibited the increase in PKC activity due to 1 alpha,25-(OH)(2)D(3) or 24R,25-(OH)(2)D(3) in growth plate cells derived from either female or male rats. Inhibition of PKC by tamoxifen may be a general property of membrane receptors involved in rapid responses to hormones.     

Metabolism of 25-hydroxyvitamin D3 and its regulation in rats during hypokinesis

The influence of short-(7 days) and long-term (28 days) hypokinesia on 25-hydroxyvitamin D3 metabolism was investigated in rats fed on a normal calcium (0.6%), normal phosphorus (0.6%), vitamin D-supplemented diet. The animals were given a single intraperitoneal dose of tritiated [26,27-3H]25(OH)D3 (200 pmol) eighteen hours before sacrifice. [3H]Labelled vitamin D3 metabolites were separated by high performance liquid chromatographic procedure, and their radioactivity levels in serum, kidney, intestinal mucosa and femoral bone were measured. Long-term hypokinesia resulted in decreased levels of [3H]1.25(OH)2D3 and increased levels of [3H]24.25(OH)2D3 in serum and kidney (3.15 +/- 0.62 vs. 4.33 +/- 0.41% and 5.34 +/- 0.69 vs. 3.76 +/- 0.29% for [3H]1.25(OH)2D3 and [3H]24.25(OH)2D3 in serum; 7.52 +/- 0.69 vs. 11.6 +/- 0.79% and 9.33 +/- 0.55 vs. 5.94 +/- 0.24% for those in kidney). The levels of [3H]1.25(OH)2D3 as well as of [3H] 24.25(OH)2D3 were decreased in intestinal mucosa and bone (21.5 +/- 1.46 vs. 30.1 +/- 3.04% and 7.30 +/- 0.58 vs. 9.18 +/- 0.78% for [3H]1.25(OH)2D3 and [3H]24.25(OH)2D3 in intestinal mucosa; 6.39 +/- 06.5 vs. 11.5 +/- 1.64% and 7.78 +/- 0.71 vs. 13.9 +/- 1.28% for those in bone). The data obtained suggest a suppressed synthesis of 1.25(OH)2D3 and enhanced production of 24.25(OH)2D3 in kidney as well as a diminished binding of 24.25(OH)2D3 in intestinal mucosa and bone in hypothetic rats. Possible causes of variations in biosynthesis of vitamin D3 active metabolites, and role of these variations in the disorders of calcium metabolism and bone state during hypokinesia are discussed.     

Involvement of prostaglandin E2 in the inhibition of osteocalcin synthesis by human osteoblast-like cells in response to cytokines and systemic hormones

The stimulation of the production of osteocalcin by human osteoblast-like cells in response to 1,25(OH)2D3 is antagonized by several agents that induce the synthesis of prostaglandin E2 (PGE2) including interleukin 1 (IL-1), tumour necrosis factor (TNF) and parathyroid hormone (PTH). The mechanism whereby these agents inhibit the synthesis of osteocalcin is not known. In this report we show that exogenous PGE2 inhibits this stimulatory action of 1,25(OH)2D3 on human osteoblast-like cells in a dose-dependent manner, suggesting that PGE2 may contribute to the inhibition of osteocalcin synthesis in response to these agents. Assessment of the inhibitory role of endogenous PGE2 synthesis in the action of rhIL-1 alpha, rhIL-1 beta and rhTNF alpha on the production of osteocalcin demonstrated that the inhibition by these agents could be partially overcome by the addition of indomethacin, an inhibitor of PGE2 synthesis. In contrast, the inhibitory action observed with bPTH (1-84) was unaffected by indomethacin. These observations indicate that endogenous PGE2 synthesis mediates, in part, some of the inhibitory actions of the cytokines on the induction of osteocalcin synthesis in response to 1,25(OH)2D3, but not of PTH. Since the antagonism of the synthesis of osteocalcin by rhIL-1 alpha, rhIL-1 beta and rhTNF alpha was not completely abolished following the inhibition of PGE2 synthesis this would indicate that additional PGE2-independent mechanisms also account for the action of these cytokines on osteocalcin production. The nature of these mechanisms is currently not known.     

Responses of rachitic cartilage cells to metabolites of vitamin D3

Responses of cultured cartilage cells to metabolites of vitamin D3 were studied. Cells were obtained from the epiphyseal growth plate of rachitic chicks and were exposed to physiological and pharmacological concentrations of three metabolites of vitamin D3, 25 hydroxyvitamin D3 (25(OH)D3), 24,25-dihydroxyvitamin D3 (24,25(OH)2D3) and 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). 1,25(OH)2D3 was found to reduce L-[U-14C]leucine incorporation into proteins and Na2 35SO4 incorporation into proteoglycans. The synthesis of 24,25(OH)2D3 from 25(OH)D3 was stimulated upon addition of 1,25(OH)2D3 to the cultures. Physiological concentrations of 24,25(OH)2D3 stimulated protein and proteoglycan synthesis. These findings support the notion that vitamin D3, through its active dihydroxylated metabolites, is directly involved in cartilage cells metabolism and healing of rickets.     

Prostaglandins enhance parathyroid hormone-evoked increase in free cytosolic calcium concentration in osteoblast-like cells.

Prostaglandins (PGs) are autocrine or paracrine hormones that may interact with circulating hormones such as parathyroid hormone (PTH) in bone. We examined the interaction of the PGs, PGF2 alpha, PGE2, and 6-keto-PGF1 alpha with PTH to enhance the rapid, initial transient rise in free cytosolic calcium ([Ca2+]i) and cAMP levels stimulated by PTH. Pretreatment of UMR-106, MC3T3-E1, and neonatal rat calvarial osteoblast-like cells by PGs resulted in an enhancement of the early transient rise in [Ca2+]i stimulated by PTH. PGF2 alpha was approximately 100 times more potent than PGE2. PGE2 itself was more potent than 6-keto-PGF1 alpha in enhancing PTH-stimulated rise in [Ca2+]i. Near-maximal augmentation was achieved at PGF2 alpha doses of 10 nM and PGE2 of 1 microM. The degree of augmentation in [Ca2+]i by PGF2 alpha was independent of preincubation time. PGF2 alpha pretreatment did not alter the EC50 for the PTH-induced [Ca2+]i increase but only the extent of rise in [Ca2+]i at each dose of PTH. The augmented increase in [Ca2+]i was mostly due to enhanced PTH-mediated release of Ca2+ from intracellular stores. PGF2 alpha did not stimulate an increase in PTH receptor number as assessed by [125I]-PTH-related peptide binding. PG pretreatment partially reversed PTH inhibition of cell proliferation, suggesting that an increase in [Ca2+]i may play a role in tempering the anti-proliferative effect of PTH mediated by cAMP. These studies suggest a new mode by which PGs can affect cellular activity.     

Localization of vitamin D3-responsive alkaline phosphatase in cultured chondrocytes

Alkaline phosphatase activity appears to be altered when chondrocyte cultures are incubated with 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3). This study examined whether the hormone-responsive enzyme activity is associated with alkaline phosphatase-enriched extracellular membrane organelles called matrix vesicles. Confluent, third passage cultures of rat costochondral growth cartilage (GC) or resting zone chondrocytes (RC) were incubated with 1,25-(OH)2D3 or 24,25-dihydroxyvitamin D3 (24,25-(OH)2D3) and enzyme specific activity was assayed in the cell layer or in isolated matrix vesicle and plasma membrane fractions. Alkaline phosphatase-specific activity in the matrix vesicles was enriched at least 2-fold over that of the plasma membrane and 10-fold over that of the cell layer. Matrix vesicle alkaline phosphatase was stimulated by 1,25-(OH)2D3 in GC cultures and by 24,25-(OH)2D3 in RC cultures. The cell layer failed to reveal these subtle differences. 1,25-(OH)2D3 increased GC enzyme activity but the effect was one-half that observed in the matrix vesicles alone. No effect of 1,25-(OH)2D3 on enzyme activity of the RC cell layer or of 24,25-(OH)2D3 on either GC or RC cell layers was detected. Thus, response to the metabolites is dependent on chondrocytic differentiation and is site specific: the matrix vesicle fraction is targeted and not the cells per se.     

1,25-Dihydroxyvitamin D(3) and prostaglandin E(2) act directly on circulating human osteoclast precursors.

1,25-Dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) and prostaglandin E(2) (PGE(2)) are known to influence osteoclast formation indirectly through their effects on osteoblasts. To determine whether 1, 25(OH)(2)D(3) and PGE(2) also have a direct effect on circulating osteoclast precursors, these factors were added to long-term cultures of human peripheral blood mononuclear cells (PBMCs) in the presence of osteoprotegerin ligand and macrophage colony-stimulating factor (M-CSF) (+/-dexamethasone). The number of TRAP(+) and VNR(+) multinucleated cells and the area of lacunar resorption were decreased when 1,25(OH)(2)D(3) alone was added. A marked increase in resorption pit formation was noted when the combination of 1, 25(OH)(2)D(3) and dexamethasone was added to PBMC cultures. Dose-dependent inhibition of osteoclast formation and lacunar resorption was seen when PGE(2) was added to PBMC cultures in both the presence and the absence of dexamethasone. Thus, 1,25(OH)(2)D(3) and PGE(2) not only influence osteoclast formation in the presence of bone stromal cells but also act directly on circulating osteoclast precursors to influence osteoclast differentiation.     

Localization of 1,25-(OH)2D3-responsive alkaline phosphatase in osteoblast-like cells (ROS 17/2.8, MG 63, and MC 3T3) and growth cartilage cells in culture

Previous studies have shown 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3)-responsive alkaline phosphatase in cultured growth zone cartilage chondrocytes is localized in extracellular matrix vesicles (MV). Since osteoblast-like cells also have 1,25-(OH)2D3-responsive alkaline phosphatase, this study determined whether the 1,25-(OH)2D3-responsive enzyme activity is localized to MV produced by these cells as well. Osteoblast-like cells from rat (ROS 17/2.8), mouse (MC 3T3), human (MG 63), and rat growth zone cartilage were cultured in Dulbecco's modified Eagle's medium containing 10(-7)-10(-12) M 1,25-(OH)2D3. Alkaline phosphatase total activity and specific activity were measured in the cell layer, MV, and plasma membrane (PM) fractions. MV and PM purity were verified by electron microscopy and MV alkaline phosphatase specific activity compared to PM (MV versus PM: ROS 17/2.8 6 x; MG 63, 5.5 x; MC 3T3, 33 x; GC, 2 x). There was a dose-dependent stimulation of MV alkaline phosphatase (5- to 15-fold increase at 10(-7)-10(-9) M) in all cell types in response to the 1,25-(OH)2D3. The PM enzyme was stimulated in a parallel fashion in the osteoblast cultures. No effect of 1,25-(OH)2D3 was observed in growth cartilage PM. Although MV accounted for less than 20% of the total activity they contributed 50% of the increase in alkaline phosphatase activity in the cell layer in response to 1,25-(OH)2D3 and MV specific activity was enriched 10 times over that of the cell layer. These are common features of MV produced by cells which calcify their matrix and suggest that hormonal regulation of MV enzymes may be important in primary calcification.     

Establishment of an osseous cell line from fetal rat calvaria using an immunocytolytic method of cell selection: characterization of the cell line and of derived clones

Using selective media and complement-mediated lysis of primary cultures of a fetal rat calvarial cell population, we have developed a cell line (OBCK6) that exhibits osteoblastic characteristics. OBCK6 cells demonstrated enhanced parathyroid hormone (PTH)-stimulated adenylate cyclase activity relative to the primary calvarial population, production of alkaline phosphatase activity and type 1 collagen, and the capacity to form mineralized nodules in unsupplemented medium after prolonged (22-26 day) culture. Two sublines, CFK1 and CFK2, which were isolated by dilution cloning, differed morphologically and with respect to growth rate. CFK1 cells demonstrated high PTH and prostaglandin E2-stimulated adenylate cyclase activity, whereas only low PTH-stimulated activity was observed in CFK2 cells. Retinoic acid and 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] each reduced PTH-stimulated adenylate cyclase activity in both the cell types. Retinoic acid and dexamethasone reduced and 1,25(OH)2D3 enhanced alkaline phosphatase activity in these cells. PTH significantly augmented alkaline phosphatase activity to a much greater extent in CFK1 than in CFK2 cells. Both CFK1 and CFK2 cells expressed type I but type III collagen, and neither expressed osteocalcin. Strong Alcian blue staining of CFK2 cells was suggestive of a cartilaginous phenotype. These three cell lines, therefore, demonstrated discrete characteristics of skeletal cell function and should provide important models for evaluation of mechanisms of mineralization and for control of skeletal cell growth and mesenchymal differentiation in vitro.     

The effect of 1,25-dihydroxyvitamin D3 on human osteoblast-like osteosarcoma cell: modification of response to PTH

The influence of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] on adenylate cyclase responsiveness in cultured osteoblastic cells was studied using a human osteosarcoma cell line SaOS-2. 1,25(OH)2D3 treatment had no effect on cell growth, cell protein and alkaline phosphatase activity. 1,25(OH)2D3 did not alter the basal production of cyclic AMP (cAMP) in intact cells, but the cAMP formation in response to parathyroid hormone (PTH), isoproterenol (ISO) and cholera toxin was attenuated by 1,25(OH)2D3. The response to forskolin, however, was unaffected by 1,25(OH)2D3 treatment. Islet activating protein failed to modify these 1,25(OH)2D3 effect. In cell free experiments, 1,25(OH)2D3 showed similar effect--that is, PTH and ISO-stimulated adenylate cyclase activity were attenuated, but forskolin-stimulated adenylate cyclase was unaffected. 1,25(OH)2D3 treatment had no effect on the kinetics of PTH binding to PTH receptor and on the ADP ribosylation of GTP stimulatory binding protein (Gs) in SaOS-2 cells. According to these results, 1,25(OH)2D3 appeared to change the coupling of Gs with adenylate cyclase, but does not affect receptor, Gs and adenylate cyclase themselves, nor GTP inhibitory binding protein.     

1 alpha, 25-Dihydroxyvitamin D3 augments clonal growth of macrophages from rat bone marrow progenitor cells and modulates their function

1 alpha, 25-Dihydroxyvitamin D3 (1,25(OH)2D3) was shown to enhance (approximately 2 fold) the colony-stimulating factor-dependent clonal growth of macrophage colonies and clusters from rat bone marrow progenitor cells. The proliferative capacity of macrophage progenitors in liquid cultures was likewise augmented (2-3 fold). Mononuclear phagocytes (macrophages, for simplicity) developing in the presence of 1,25(OH)2D3 showed a reduced capacity of migration. 1,25(OH)2D3 administered at bone marrow culture initiation led to augmentation of the phagocytic capability of macrophages in four-day cultures and to its suppression in macrophages in seven-day cultures. The observed patterns of modulation of differentiation and function by 1,25(OH)2D3 differ from the patterns we found for mouse bone marrow cells. The results suggest that the differential response to hormones observed in different species may include responses to 1,25(OH)2D3.     

PGE2 and dibutyryl-cAMP enhance the response of rat granulosa cells to FSH

Granulosa cells isolated from immature Sprague-Dawley rat ovaries produce progesterone (31.7 pg/micrograms cell protein) in response to an acute FSH stimulus (5 micrograms/ml NIH-FSH-S11, 2 H). After culture for 48 h in the absence of hormones (control culture), progesterone production by the granulosa cells in response to FSH is significantly reduced (2.9 pg/micrograms cell protein). Cells cultured with prostaglandin E2 (PGE2, 1 microgram/ml) or dibutyryl-cAMP (dbcAMP, 1 mM) exhibited a discernibly greater steroidogenic response to FSH (12.5 and 53.4 pg/microgram cell protein, respectively) than that of control cultures. Therefore the presence of PGE2 or dbcAMP in the culture medium helps to maintain the steroidogenic capacity of granulosa cells in culture. It is probable that this capacity is maintained at a locus distal to the production of cAMP by FSH. Paradoxically, granulosa cells cultured with PGE2 produce less cAMP in response to FSH stimulation than cells in control cultures (15.9 vs. 250.3 fm/micrograms cell protein). This may be due to a suppressive effect of prior exposure to PGE2 on the subsequent activity of adenylate cyclase when the FSH is introduced and a concomitant elevation of phosphodiesterase activity.     

The adenosine 3':5'-cyclic monophosphate response to prostaglandin E2 is altered in U937 cells in association with maturational events induced by activated T lymphocytes and 1,25-dihydroxyvitamin D3

Previous studies have shown that maturational changes can be induced in a human monocyte line, U937, by treatment with cellfree medium from lectin-stimulated cloned human T lymphocytes or the vitamin D metabolite, 1,25-dihydroxyvitamin D3 (1,25-[OH]2D3). Many of the maturational effects are accompanied by modification, new synthesis, or reorganization of cell surface membrane constituents, including proteins that function as receptors for ligands that modulate monocyte function. In the present studies, we examined the effects of monocyte differentiation on responsiveness to prostaglandin E2 (PGE)2. We found that the maturational effects of the lymphokine or 1,25[OH]2D3 were accompanied by a marked reduction in the PGE2-induced increase in cellular content of adenosine 3':5'-cyclic monophosphate (cAMP) and a shift in the dose-response curve consistent with a decrease in PGE2 receptor number or binding affinity. Incubation of cells with IFN-alpha or IFN-gamma produced by recombinant DNA technology did not influence PGE2 responsiveness, suggesting that the effects of the lymphokine were not mediated by these products. The absence of an effect on sodium fluoride- or forskolin-induced adenylate cyclase response in membranes prepared from treated cells suggests that the treatment conditions affect PGE2 receptor function rather than the adenylate cyclase enzyme complex. Changes in cell surface receptors for hormones such as PGE2 provide a potential mechanism for modulating the biologic activity of monocyte-macrophages during the process of cellular differentiation.