Colony-stimulating factor 1 regulates CTP: phosphocholine cytidylyltransferase mRNA levels.

Growth factor regulation of phosphatidylcholine (PtdCho) metabolism during the G1 stage of the cell cycle was investigated in the colony-stimulating factor 1 (CSF-1)-dependent murine macrophage cell-line BAC1.2F5. The transient removal of CSF-1 arrested the cells in G1. Incorporation of [3H]choline into PtdCho was stimulated significantly 1 h after growth factor addition to quiescent cells. Metabolic labeling experiments pointed to CTP:phosphocholine cytidylyltransferase (CT) as the rate-controlling enzyme for PtdCho biosynthesis in BAC1.2F5 cells. The amount of CT mRNA increased 4-fold within 15 min of CSF-1 addition and remained elevated for 2 h. The rise in CT mRNA levels was accompanied by a 50% increase in total CT specific activity in cell extracts within 4 h after the addition of CSF-1. CSF-1-dependent elevation of CT mRNA content was neither attenuated nor superinduced by the inhibition of protein synthesis with cycloheximide. The rate of CT mRNA turnover decreased in the presence of CSF-1 indicating that message stabilization was a key factor in determining the levels of CT mRNA. These data point to increased CT mRNA abundance as a component in growth factor-stimulated PtdCho synthesis.     

Involvement of CSF-1 in generating a stroma-independent hematopoietic stem cell line

The hematopoietic stem cell line, Myl-D7, is maintained by a self-renewing stem cell population that spontaneously generates myeloid, lymphoid, and erythroid progeny. MS-5 stromal cells are necessary for the growth of Myl-D7 cells. One component of the Myl-D7 cells proliferation activity released by MS-5 stromal cells was enriched by Q sepharose fractionation and shown to be colony stimulating factor-1 (CSF-1) by Western blotting, BAC1.2F5 cell bioassay and inhibition of Myl-D7 proliferation by CSF-1 antibody. The requirement of Myl-D7 cells for CSF-1 was also demonstrated independently by selecting for rare, stroma-independent Myl-D7 mutant clones able to grow without stroma and additional factors. Eighty-nine stroma-independent mutant clones were obtained and belonged to two classes. The majority of mutants did not secrete any growth promoting activity. The second, rarer class of mutants releases a factor that stimulates proliferation/survival for up to several months and approximately half of the secretors express high levels of CSF-1 mRNA. Wild type Myl-D7 grown with supernatants from the secretor cells retained the stem cell phenotype. These data suggest that CSF-1 may act as a key factor in stroma-regulated hematopoiesis and cell-cell interaction.     

Mouse mesangial cells produce colony-stimulating factor-1 (CSF-1) and express the CSF-1 receptor

CSF-1 stimulates the survival, proliferation, and differentiation of mononuclear phagocytes and may also play a role in placental development. The expression of CSF-1 and the CSF-1 receptor (CSF-1R) and their regulation were examined in cultures of mouse mesangial cells (MC). The concentration of CSF-1 in the medium of cultured MC increased linearly with time over 24 h. IFN-gamma stimulated and dibutyryl cyclic AMP inhibited CSF-1 production in a dose-dependent manner. MC expression of CSF-1 mRNA was shown by Northern blot analysis, and CSF-1 mRNA levels were increased within 4 h of IFN-gamma addition and inhibited within 4 h of dibutyryl cyclic AMP addition. Indirect immunofluorescence indicated that 90% of the untreated cultured MC expressed CSF-1. In addition, CSF-1R expression by MC was demonstrated by immunofluorescence with anti-receptor antibody, specific binding of [125I] CSF-1, and expression of the CSF-1R mRNA by Northern blot analysis. Thus, mouse MC, specialized pericytes of non-bone marrow origin, not only produce CSF-1 but also express receptors for CSF-1. The effects of CSF-1 on MC may be important in the control of immune function in the glomerulus.     

CSF-1-induced gene expression in macrophages: dissociation from the mitogenic response

Early gene expression associated with the mitogenic response to colony stimulating factor-1 (CSF-1) has been examined in BAC1.2F5, a CSF-1-dependent murine macrophage cell line. Stimulation of arrested cells by CSF-1 resulted in acute, transient elevation in c-fos and subsequently in c-myc mRNA levels. Dramatic, sustained elevations were observed for JE and KC mRNAs, which are induced by platelet-derived growth factor (PDGF) in 3T3 cells. The kinetics of expression of all four messages were similar to those reported in PDGF-stimulated fibroblasts, implying a program of gene expression common to these two mitogens. Granulocyte-macrophage CSF (GM-CSF) can replace CSF-1 in stimulating the growth of 2F5 cells. It induced mRNAs for c-fos, c-myc and JE but not KC. Therefore KC expression, although correlated with mitogenesis, is not required for proliferation. The effects of CSF-1 were also examined in cells cycling continuously in its absence: 2F5 cells incubated in GM-CSF and an autonomous variant subclone of 2F5. In either case, the only detected growth effect of CSF-1 was a reduction in doubling-time. Nevertheless, all four of the mRNAs induced by CSF-1 in arrested cultures of 2F5 were strongly induced with the same kinetics in these cycling cells. Thus it would appear that the functions mediated by this early-gene program are not restricted to the mitogenic stimulation of arrested cells.     

PGE1-independent MDCK cells have elevated intracellular cyclic AMP but retain the growth stimulatory effects of glucagon and epidermal growth factor in serum-free medium

Prostaglandin E1 (PGE1), a component in the hormone-supplemented, serum-free medium for the Madin Darby canine kidney (MDCK) cell line, has been proposed to increase MDCK cell growth by increasing intracellular cyclic AMP levels. The association between increased intracellular cyclic AMP and the growth stimulatory effect of PGE1 has been examined in normal MDCK cells and in PGE1-independent variants of MDCK. These variant cells have lost the PGE1 requirement for long term growth in defined medium. Normal MDCK cells had almost twofold higher intracellular cyclic AMP levels during growth in Medium K-1 (9.0 pmol/mg protein) than in Medium K-1 minus PGE1. Furthermore, PGE1-independent clone 1 had higher intracellular cyclic AMP levels in Medium K-1 minus PGE1 than normal MDCK cells in Medium K-1. This latter observation suggests that the PGE1 requirement for MDCK cell growth is associated with the low intracellular cyclic AMP levels of this cell line. An involvement of cyclic AMP in the growth response to PGE1 is supported by these observations, as well as by the growth stimulatory effects of other agents that affect cyclic AMP metabolism in MDCK cells. These agents include glucagon, isobutyl methylxanthine (IBMX), and dibutyryl cyclic AMP. The growth of PGE1-independent clone 1 was inhibited rather than stimulated by PGE1. Similarly, PGE1-independent cell growth was inhibited by IBMX and dibutyryl cyclic AMP. However, the growth response to one agent which increases cyclic AMP (glucagon) was retained in PGE1-independent clone 1. This result suggests that the effect of glucagon is not associated with increases in intracellular cyclic AMP. The growth stimulatory effect of epidermal growth factor (EGF) on normal MDCK cells was also studied. Although EGF does not act via a cyclic AMP-mediated mechanism, EGF increased normal MDCK cell growth and substituted for PGE1 in Medium K-1. Thus, EGF and PGE1 could possibly affect similar growth-related functions in MDCK cells, although by different pathways. This possibility was examined further, using PGE1-independent clone 1. EGF, like glucagon, was still growth stimulatory to the PGE1-independent cells. Consequently, the biochemical pathways by which EGF and PGE1 increase MDCK cell growth probably do not converge.     

ERK5/BMK1 is indispensable for optimal colony-stimulating factor 1 (CSF-1)-induced proliferation in macrophages in a Src-dependent fashion

CSF-1, by binding to its high-affinity receptor CSF-1R, sustains the survival and proliferation of monocyte/macrophages, which are central cells of innate immunity and inflammation. The MAPK ERK5 (also known as big MAPK-1, BMK1, or MAPK7) is a 98-kDa molecule sharing high homology with ERK1/2. ERK5 is activated by oxidative stress or growth factor stimulation. This study was undertaken to characterize ERK5 involvement in macrophage signaling that is elicited by CSF-1. Exposure to the CSF-1 of primary human macrophages or murine macrophage cell lines, as well as murine fibroblasts expressing ectopic CSF-1R, resulted in a rapid and sustained increase of ERK5 phosphorylation on activation-specific residues. In the BAC1.2F5 macrophage cell line, ERK5 was also activated by another mitogen, GM-CSF, while macrophage activators such as LPS or IFN-gamma and a number of nonproliferative cytokines failed. Src family kinases were found to link the activation of CSF-1R to that of ERK5, whereas protein kinase C or the serine phosphatases PP1 and PP2A seem not to be involved in the process. Treatment of macrophages with ERK5-specific small interfering RNA markedly reduced CSF-1-induced DNA synthesis and total c-Jun phosphorylation and expression, while increasing the expression of the cyclin-dependent kinase inhibitor p27. Following CSF-1 treatment, the active form of ERK5 rapidly translocated from cytosol to nucleus. Taken together, the results reported in this study show that ERK5 is indispensable for optimal CSF-1-induced proliferation and indicate a novel target for its control.     

Prostaglandin biosynthesis and stimulation of cyclic AMP in primary monolayer cultures of epithelial cells from mouse mammary gland.

Cyclic AMP levels in primary monolayer cultures of epithelial cells prepared from mid-pregnant mice are stimulated by prostaglandin E1 and E2. Prostaglandin F1alpha and F2alpha have only a slight effect upon cyclic AMP levels. In the absence of phosphodiesterase inhibitors the rise in cyclic AMP produced by PGE1 is only transient and the levels return to normal within 30 minutes. High concentrations (16 mM) of theophylline are needed to prevent this decline, suggesting that the phosphodiesterase activity of epithelial cells in culture is high. However, theophylline alone produced only a small increase in basal cyclic AMP levels even over a 2-hour period indicating that basal cyclic AMP is turned over more slowly than cyclic AMP produced in response to stimulation with PGE1. Both PGE and PGF synthesis were monitored using radioimmunoassay procedures previously reported. The observed levels were found to decrease as cell density increased and were sensitive to the addition of agents such as collagen and naproxen.     

Isolation and characterization of a cloned growth factor dependent macrophage cell line, BAC1.2F5

The SV40 transformed murine macrophage cell line, BAC1, proliferates in response to the colony stimulating factor, CSF-1 (Schwarzbaum et al., J. Immunol., 132:1158, 1984). In order to obtain a cell line suitable for biochemical and genetic studies of CSF-1 signal transduction, clones of BAC1 were established. Clones ranged from being completely autonomous to being completely dependent on CSF-1 for growth. Cells of one clone (2F5), which proliferated in response to either CSF-1 or granulocyte-macrophage CSF (GM-CSF) were characterized in detail. The kinetics of receptor-mediated internalization and intracellular destruction of CSF-1 were comparable to the kinetics observed with peritoneal exudate macrophages. CSF-1 was shown to regulate cell spreading, cell survival, protein degradation, and the duration of the G1 and S phases of the cell cycle. The 2F5 clone therefore exhibits a number of CSF-1 stimulated responses and is being used for genetic and biochemical studies of CSF-1 action.     

Two Possibly Distinct Prostaglandin E1 Receptors in N1E-115 Clone: One Mediating Inositol Trisphosphate Formation, Cyclic GMP Formation, and Intracellular Calcium Mobilization and the Other Mediating Cyclic AMP Formation

Prostaglandin E1 (PGE1)-mediated transmembrane signal control systems were investigated in intact murine neuroblastoma cells (clone N1E-115). PGE1 increased intracellular levels of total inositol phosphates (IP), cyclic GMP, cyclic AMP, and calcium ([Ca2+]i). PGE1 transiently increased inositol 1,4,5-trisphosphate formation, peaking at 20 s. There was more than a 10-fold difference between the ED50 for PGE1 at cyclic AMP formation (70 nM) and its ED50 values at IP accumulation (1 microM), cyclic GMP formation (2 microM), and [Ca2+]i increase (5 microM). PGE1-mediated IP accumulation, cyclic GMP formation, and [Ca2+]i increase depended on both the concentration of PGE1 and extracellular calcium ions. PGE1 had more potent intrinsic activity in cyclic AMP formation, IP accumulation, and cyclic GMP formation than did PGE2, PGF2 alpha, or PGD2. A protein kinase C activator, 4 beta-phorbol 12 beta-myristate 13 alpha-acetate, had opposite effects on PGE1-mediated IP release and cyclic GMP formation (inhibitory) and cyclic AMP formation (stimulatory). These data suggest that there may be subtypes of the PGE1 receptor in this clone: a high-affinity receptor mediating cyclic AMP formation, and a low-affinity receptor mediating IP accumulation, cyclic GMP formation, and intracellular calcium mobilization.     

The effects of hormones on cyclic adenosine 3':5'-monophosphate accumulation in transitional epithelium of the urinary bladder.

Transitional epithelium lining rabbit urinary bladders was isolated and studied in vitro. The homogeneity of the isolated epithelium was demonstrated by light and electron microscopical monitoring as well as cell culture studies. Transitional epithelium responded to epinephrine and prostaglandin E1 (PGE1) in the presence of 2mM 1-methyl, 3-isobutylxanthine (MIX) with increases in intracellular levels of cyclic adenosine 3':5'-monophosphate (cyclic AMP). Corticotropin, aldosterone, insulin, parathyroid hormone and vasopressin were slightly but significantly stimulatory under similar conditions. Glucagon and oxytocin were not stimulatory at the concentrations tested. The effects of epinephrine and PGE1 were potentiated by 2mM MIX 20-fold or greater. The cells were slightly more sensitive to PGE1 then to epinephrine. The prostaglandin produced a noticeable response at about 10nM, while effects of epinephrine were discernible at 0.1muM. Maximal responses to both effectors were seen at about 10muM. The action of 10muM epinephrine, but not 10muM PGE1, was completely abolished by 0.1mM propranolol. Responses to combinations of epinephrine and PGE1 were additive. Cyclic AMP accumulated in the incubation medium of transitional epithelial cells exposed to epinephrine, PGE1, MIX, or combinations of the agonists. The appearance of cyclic AMP in the medium was slow compared to the rate of intracellular accumulation, but reached significant levels following prolonged stimulation.     

Prostaglandin E2 and F2 alpha inhibit growth of human gastric carcinoma cell line KATO III with simultaneous stimulation of cyclic AMP production

The effects of prostaglandins (PGs) on the growth of human gastric carcinoma cell line KATO III were investigated. PGE2 as well as PGF2 alpha significantly and dose-dependently inhibited the growth of this gastric carcinoma cell line (PGE2 greater than PGF2 alpha). This inhibition of cell growth by the PGs was associated with the increase in cyclic AMP production (PGE2 greater than PGF2 alpha), whereas inositol-phospholipid turnover was not affected by either PGE2 or PGF2 alpha as assessed by the formation of 3H-inositol phosphates. Furthermore, the proliferation of these gastric carcinoma cells was also suppressed by the administration of forskolin as well as of dibutyryl cyclic AMP. These results suggest that PGE2 and PGF2 alpha inhibit the growth of cultured human gastric carcinoma cells KATO III via stimulation of cyclic AMP production.     

Hormonal activation of adenylate cyclase in mouse melanoma metastatic variants.

The ability of melanocyte stimulating hormone (MSH), adrenocorticotropic hormone (ACTH), and prostaglandin E1 (PGE1) to stimulate the accumulation of cyclic AMP was examined in intact mouse melanoma cells of varying metastatic potential. F1 cells (low metastatic potential) had significantly greater cyclic AMP levels in response to all three hormones than F5 (intermediate metastatic potential) and F10 (high metastatic potential) cells. The ranking of the response was as follows: MSH, F1 greater than F5 greater than F10, ACTH, F1 greater than F5 greater F10, PGE, F1 greater than F10 greater F5. In contrast to the above, the degree of hormonal stimulation of adenylate cyclase in broken cell preparations was virtually identical in all three melanoma cell lines. Control enzyme activity was depressed in both F5 and F10 relative to F1. The conflicting results between studies of intact vs. broken cell preparations could not be explained by increased cyclic AMP phosphodiesterase activity in F5 and F10 cells. We conclude that as the melanoma cells increase in metastatic potential, there is a significant loss in the ability of their cyclic AMP system to respond appropriately to hormonal stimuli.     

Forskolin inhibits the Gs-stimulated adenylate cyclase in rat ascites hepatoma AH66F cells

Forskolin increased intracellular cyclic AMP and augmented cyclic AMP formation by prostaglandin E1 (PGE1) in normal rat hepatocytes and ascites hepatoma AH66 cells. However, in AH66F cells which were derived from the AH66 cell line, the diterpene only slightly increased the cyclic AMP level, and dose-dependently inhibited the accumulation caused by PGE1. Forskolin dose-dependently activated adenylate cyclase in these membranes, and the magnitude of activation by forskolin was largest in the following order: hepatocytes, AH66 cells, and AH66F cells. This difference may be based on the number of forskolin-binding sites. The binding affinity of forskolin for each cell membrane was similar. The number and affinity of forskolin-binding sites in these cells were not influenced by 5'-guanylylimidodiphosphate [Gpp(NH)p]. In hepatocytes and AH66 cells, forskolin and other adenylate cyclase activators such as PGE1, GTP, Gpp(NH)p, F-, and Mn2+ synergistically increased the enzyme activity. In AH66F cells, the forskolin-stimulated activity was hardly influenced by the GTP analog, and forskolin diminished the activities induced by the GTP analog in a manner similar to that of diterpene alone. Forskolin (10 microM) also significantly inhibited the activities induced by PGE1, GTP, and F-. The effect of forskolin with Mn2+ was additive in AH66F cells. The data suggest that forskolin promotes the interaction between the stimulatory guanine nucleotide-binding protein and the catalytic unit in the membrane of normal hepatocytes and AH66 cells, but it interferes with the coupling in AH66F cells.     

PGE1-mediated cyclic AMP refractoriness: effects of cycloheximide and indomethacin.

Human synoviocytes in culture respond to prostaglandin E1 (PGE1) by increasing their intracellular concentration of cyclic AMP. Readdition of PGE1 to cells previously treated with PGE1 elicits no change in the intracellular concentration of cyclic AMP. This refractory state is partially prevented by the inhibitors of protein synthesis, puromycin (PM) and cycloheximide (CH). Indomethacin (IM), which reduces angiotensin tachyphylaxis, does not prevent the occurrence of refractoriness to PGE1 with respect to accumulation of cyclic AMP. This agent does alter the release of cyclic AMP from human synovial cells. We postulate that other factors, independent of new protein synthesis, are necessary for the development of the complete PGE1 refractory state in these cells.     

Stimulation of Cloudman melanoma tyrosinase activity occurs predominantly in G2 phase of the cell cycle

A widely accepted notion is that an increasing cellular cyclic AMP (cAMP) concentration is prerequisite for increasing tyrosinase activity and melanin synthesis and for regulating proliferation of pigment cells. alpha-Melanocyte stimulating hormone (alpha-MSH) increases cAMP and tyrosinase activity in Cloudman melanoma cells. Prostaglandins (PGs) E1 and E2 increase melanoma cell tyrosinase activity and inhibit proliferation. Both PGs, but not alpha-MSH, block the progression of Cloudman melanoma cells from G2 phase of the cell cycle into M or G1. Only PGE1 and not PGE2 causes an elevation of cellular cAMP concentrations. The adenylate cyclase inhibitor 2',5'-dideoxyadenosine (DDA) at 5 x 10(-4) M effectively blocks the increased cAMP synthesis by cells treated with 10 micrograms/ml PGE1. The addition of DDA, however, enhances the melanogenic response of melanoma cells to 10 micrograms/ml PGE1 or PGE2, 10(-7) M alpha-MSH, 10(-4) M isobutylmethylxanthine, 10(-4) M dibutyryl cyclic AMP. DDA also augments the effects of PGE1 or PGE2 on the melanoma cell cycle. Moreover, when DDA is added concomitantly with alpha-MSH, more cells are recruited into G2 than observed in untreated controls. Neither alpha-MSH nor DDA alone has any effect on the cell cycle. These findings undermine the role of cAMP in the melanogenic process and suggest that blocking melanoma cells in G2 may be required for the remarkable stimulation of tyrosinase activity observed with PGE1 or PGE2 alone or in combination with DDA. The observed block in G2 may be essential for the synthesis of sufficient mRNA, which is required for stimulation of tyrosinase activity.     

Differential effects of prostaglandin F2 alpha and of prostaglandins E1 and E2 on cyclic 3',5'-monophosphate production and intracellular calcium mobilization in avian uterine smooth muscle cells

The effects of prostaglandins (PGs) E1 (PGE1), E2 (PGE2) and F2 alpha (PGF2 alpha) on cyclic 3',5'-adenosine monophosphate (cAMP) production and intracellular Ca mobilization were examined in smooth muscle cells of chicken uterus grown in primary culture. At subnanomolar concentrations, both PGE1 and PGE2 significantly suppressed cAMP levels. However, at higher concentrations (0.1-100 microM), both agonists caused a dose-related increase in cAMP production. PGF2 alpha, on the other hand, had no effect on cAMP production. Forskolin (1-100 microM), which also stimulated cAMP production in a dose-dependent fashion, potentiated the effects of both PGE1 and PGE2. In digitonin-permeabilized uterine cells preloaded with 45Ca2+, the addition of PGF2 alpha caused a biphasic 45Ca2+ efflux. There was a small but significant 45Ca2+ release (10.0 +/- 1.5%) within 30 s (rapid phase), followed by a larger one (32.0 +/- 2.0%) within 5 min (slow phase). PGE2, at doses above 1 nM (which significantly increased cAMP accumulation), promoted 45Ca2+ sequestration. This action of PGE2 was observed as early as 1 min and was complete by 5 min. In addition, 0.001 nM PGE2 (a dose that was ineffective on 45Ca2+ mobilization) enhanced PGF2 alpha-induced 45Ca2+ mobilization from 22.5 +/- 5% to 57.0 +/- 3.5%. These results show that PGs of the E series have distinctly different effects on cAMP production and intracellular Ca mobilization. PGF2 alpha action may be linked directly to intracellular Ca mobilization, whereas the effects of PGE may be exerted at multiple sites depending on its local concentration. At low concentrations, its action may be mediated by the suppression of cAMP levels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of thioacetamide on cytochrome P-450 synthesis in rat liver.

Both calcitonin and prostaglandin E2 (PGE2) stimulate adenylate cyclase activity in the human breast cancer cell line (T 47D). The maximum cyclic AMP response to calcitonin exceeds that of PGE2. When maximal concentrations of the two hormones were added simultaneously to the cells, the amount of cyclic AMP generated was less than that seen with calcitonin alone. When cells were treated with the protein toxin of Bordetella pertussis (islet-activating protein; IAP) which inactivates the inhibitory regulatory component (Ni) of adenylate cyclase, there was no change in basal or calcitonin-responsive adenylate cyclase in intact cells. However, the PGE2 response was augmented at all dose levels, and this effect was dependent on the concentration of IAP. Moreover, in cells pretreated with IAP, simultaneous addition of PGE2 and calcitonin resulted in additivity rather than in inhibition of cyclic AMP production. The additivity of the response to calcitonin and PGE2 after IAP treatment implies activation of separate pools of adenylate cyclase catalytic subunit by the two hormones. These data are consistent with a model in which calcitonin acts on adenylate cyclase in T 47D cells through stimulatory regulatory components alone, whereas PGE2 acts on the same cells through both stimulatory and inhibitory components. The Ni input can limit the maximum effect of PGE2 and is capable of limiting calcitonin effects when the two agonists are used simultaneously.