Transforming growth factor-beta modulation of glycosaminoglycan production by mesenchymal cells of the developing murine secondary palate

Development of the mammalian secondary palate requires proper production of the extracellular matrix, particularly glycosaminoglycans (GAGs) and collagen. Endogenous factors that regulate the metabolism of these molecules are largely undefined. A candidate for a locally derived molecule would be transforming growth factor beta 1 (TGF beta 1) by virtue of its potency as a modulator of extracellular matrix metabolism by several cell lines. We have thus attempted to assign a regulatory role for TGF beta 1 in modulation of GAG production and degradation by mesenchymal cells of the murine embryonic palate (MEPM). Treatment with TGF beta 1 or TGF beta 2, but not IGF-II, resulted in a stimulation of total GAG synthesis. Furthermore, cells treated with both TGF beta 1 and TGF alpha showed a synergistic increase in GAG synthesis if pretreated with TGF beta 1 but not TGF alpha. Simultaneous stimulation with TGF beta 1 and TGF beta 2 did not elicit a synergistic response. These studies demonstrate the ability of TGF beta, synthesized by embryonic palatal cells, to specifically stimulate GAG synthesis by MEPM cells. Other growth factors present in the developing craniofacial region may also modulate TGF beta-induced GAG synthesis, a biosynthetic process critical to normal development of the embryonic palate.     

Effect of retinoic acid on in vitro proliferation activity and glycosaminoglycan synthesis of mesenchymal cells from palatal shelves of mouse fetuses

To clarify the mechanism by which retinoid causes cleft palate, we investigated the effect of retinoic acid (RA) on proliferation activity and glycosaminoglycan (GAG) synthesis in mouse fetuses palatal mesenchymal (MFPM) cells. MFPM cells were incubated for 1-11 days with various concentrations of RA to examine its effect on growth rate. Also, confluent cultures were incubated with [3H]glucosamine or [35S]sulfate in the presence of various concentrations of RA to investigate the effect of RA on GAG synthesis. RA remarkably inhibited the growth of MFPM cells in a dose-dependent manner. RA also inhibited the synthesis of GAGs, with sulfated GAGs being more severely affected than hyaluronic acid. These data suggest that the inhibition of proliferation activity and GAG synthesis of palatal mesenchymal cells might be involved in the induction of cleft palate by retinoic acid.     

Effect of glucocorticoid on glycosaminoglycans synthesis in cultured mouse embryonic palatal mesenchymal cells

The effect of glucocorticoids (GC) on the synthesis of glycosaminoglycans (GAGs) in mesenchymal cells obtained from palatal shelves of mouse embryos was studied. Both hyaluronic acid (HA) synthesis and sulfated GAG synthesis were inhibited with triamcinolone acetonide (TA) in a concentration-dependent manner. However, the degree of inhibition of synthesis with TA was greater for HA than for the sulfated GAGs. To determine whether the inhibitory effects of TA on HA and sulfated GAGs synthesis were mediated through GC receptors, we performed experiments using progesterone, vitamin B6, and molybdate. The inhibitory effect of TA was antagonized by all three. The results obtained in the present study suggest that the inhibition of synthesis of HA and sulfated GAGs with GC is mediated through GC receptors and is involved in cleft palate induction by GC.     

Immunohistochemical localization of prostaglandins E and F2 alpha in the developing murine palate

Prostaglandins E2 and F2 alpha (PGE2 and PGF2 alpha) have been shown to cause changes in adenosine 3',5'-cyclic monophosphate (cAMP) levels in a wide variety of tissues. In particular, murine palatal mesenchyme responds to PGE2 stimulation with dose-dependent increases in intracellular cAMP levels. These same mesenchymal cells also synthesize PGE2 and PGF2 alpha. The purpose of this study is to localize PGE and PGF2 alpha in the developing murine palate by using immunohistochemical techniques. Fresh frozen cryostat sections of murine C57BL/6J embryo palates (days 12-14 of gestation) were incubated with anti-PGE or PGF2 alpha monoclonal antibodies. On day 12 of gestation, PGE and PGF2 alpha, identified as 3',3-diaminobenzidine (DAB) reaction products, were localized throughout palatal mesenchyme and epithelium; on day 13 of gestation, reaction product indicative of both PGE and PGF2 alpha was detectable primarily in mesenchyme subjacent to palatal epithelium. Extracellular spaces of the adjacent mesenchyme in the central region of the day 13 palate exhibited less reaction product. Palatal epithelium, particularly the medial edge epithelium, exhibited a diminished amount of reaction product for both prostaglandins on day 13 as compared to the underlying mesenchyme. After formation of a midline epithelial seam between homologous palatal processes on day 14 of gestation, medial edge, oral, and nasal epithelium exhibited light staining for PGE or PGF2 alpha. Palate mesenchymal cells subjacent to the midline seam exhibited a diminished amount of reaction product for both PGE and PGF2 alpha as compared to day 13 of gestation. Overall, the results show local and temporal changes in the distribution of prostaglandins in the developing murine palate.     

Synthesis of prostaglandins and cyclic AMP by cultured embryonic palate mesenchyme at various population densities

During embryonic development, facial and palate mesenchymal cells exhibit differential growth rates. Normal palatal growth is regulated in part by hormones and growth factors. Because hormonal responsiveness of some cells correlates with their cell density, we have investigated the relationship between embryonic palate mesenchymal cell population density and their ability to synthesize prostaglandins (PGs) and cyclic AMP. Primary cultures of palate mesenchymal cells exhibited typical lag, log, and stationary phases of growth with a doubling time of 32-34 hrs. The ability of cells to produce PGE2 in response to a calcium ionophore (A23187), an activator of phospholipase A2 (melittin), arachidonic acid, or serum was maximal during the period of early exponential growth. Prostaglandin F2 alpha synthesis in response to A23187 or arachidonic acid showed a similar transient increase also corresponding temporally to the period of early exponential growth. The ability to synthesize PGF2 alpha in response to melittin, however, failed to diminish after early exponential growth. The pattern of cAMP synthesis in response to isoproterenol and PGE1 was different from that seen for induced prostaglandin synthesis. A transient increase in sensitivity to isoproterenol and PGE1 was seen that corresponded temporally to the period of late exponential growth just prior to attainment of confluency. Decreased sensitivity to stimulation of either prostaglandin or cAMP production as the cells became confluent was shown to be a density-dependent phenomenon; confluent cultures that were subcultured to reestablish logarithmic growth exhibited density-dependent hormonal responses identical to those seen in primary cultures. The ability of palate mesenchymal cells to synthesize both prostaglandins and cAMP, thought to be critical for proper palatal development, might thus be related to local differential craniofacial growth rates.     

Selective modulation of MAP kinase in embryonic palate cells

Murine embryonic palate mesenchyme (MEPM) cells are responsive to a number of endogenous factors found in the local embryonic tissue environment. Recently, it was shown that activation of the cyclic AMP (cAMP) or the transforming growth factor β (TGFβ) signal transduction pathways modulates the proliferative response of MEPM cells to epidermal growth factor (EGF). Since the mitogen-activated protein kinase (MAPK) cascade is a signal transduction pathway that mediates cellular responsiveness to EGF, we examined the possibility that several signaling pathways which abrogate EGF-stimulated proliferation do so via the p42/p44 MAPK signaling pathway. We demonstrate that EGF stimulates MAPK phosphorylation and activity in MEPM cells maximally at 5 minutes. Tyrosine phosphorylation and activation of MAPK was unaffected by treatment of MEPM cells with TGFβ or cholera toxin. Similarly, TGFβ altered neither EGF-induced MAPK tyrosine phosphorylation nor activity. However, the calcium ionophore, A23187, significantly increased MAPK phosphorylation which was further increased in the presence of EGF, although calcium mobilization reduced EGF-induced proliferation. Despite the increase in phosphorylation, we could not demonstrate induction of MAPK activity by A23187. Like EGF, phorbol ester, under conditions which activate PKC isozymes in MEPM cells, increased MAPK phosphorylation and activity but was also growth inhibitory to MEPM cells. The MEK inhibitor, PD098059, only partially abrogated EGF-induced phosphorylation. Likewise, depletion of PKC isozymes partially abrogated EGF-induced MAPK phosphorylation. Inhibition of both MEK and PKC isozymes resulted in a marked decrease in MAPK activity, confirming that EGF uses multiple pathways to stimulate MAPK activity. These data indicate that the MAPK cascade does not mediate signal transduction of several agents that inhibit growth in MEPM cells, and that there is a dissociation of the proliferative response and MAP kinase activation. Furthermore, other signaling pathways known to play significant roles in differentiation of palatal tissue converge with the MAPK cascade and may use this pathway in the regulation of alternative cellular processes. J. Cell. Physiol. 176:266–280, 1998. © 1998 Wiley-Liss, Inc.     

Epidermal growth factor: modulator of murine embryonic palate mesenchymal cell proliferation, polyamine biosynthesis, and polyamine transport

Polyamines (putrescine, spermidine, and spermine) are normal cellular constituents able to modulate cellular proliferation and differentiation in a number of tissues and cell types. This investigation explores the response of murine embryonic palate mesenchymal (MEPM) cells to epidermal growth factor (EGF) in terms of biosynthesis of putrescine and its transport across the plasma membrane and tests the hypothesis that polyamine transport can serve as an alternative mechanism (other than biosynthesis) for elevating intracellular polyamines during stimulation of MEPM cellular proliferation. MEPM cells treated with EGF were stimulated to proliferate and showed a dose- and time-dependent stimulation of ornithine decarboxylase (ODC) which was maximal at 4-6 hours. EGF also stimulated the initial rate of putrescine transport in a dose- and time-dependent manner. This stimulation was found to be maximal 3 hours after treatment and specific for the putrescine transport system. The kinetic parameters of putrescine transport shifted from 2.52 microM (Km) and 23.6 nmol/mg protein/15 minutes (Vmax) in nonstimulated cells to 4.48 microM (Km) and 39.8 nmol/mg protein/15 minutes (Vmax) in EGF-treated cells. This kinetic shift did not require de novo protein or RNA synthesis, as cycloheximide (10 micrograms/ml) and actinomycin D (50 micrograms/ml) had little effect on the ability of EGF to stimulate the initial rate of putrescine uptake. The rate of transport, however, was found to be inversely related to cell density. The addition of exogenous putrescine concomitantly with EGF blocked the induction of ODC, while in the presence of difluoromethylornithine (DFMO) (irreversible inhibitor of ODC) the initial rate of putrescine transport remained elevated throughout the time course studied. This stimulation of putrescine uptake caused by polyamine deprivation was reversed by exogenous putrescine and Ca++ while alpha-aminoisobutyric acid (AIB) further stimulated the rate of uptake. EGF's ability to stimulate cellular DNA synthesis was inhibited by DFMO. If DFMO-treated cells were stimulated with EGF in the presence of exogenous putrescine, this stimulatory effect was preserved. These studies indicate that the rate of polyamine transportation is highly responsive to a signal which initiates biosynthesis of polyamines. Further, this transportation system provides a compensatory mechanism allowing the cell to increase intracellular levels of polyamines when environmental conditions inhibit biosynthesis or when polyamines are abundant.     

Intracellular dynamics of Smad-mediated TGFbeta signaling

The transforming growth factor-beta (TGFbeta) family represents a class of signaling molecules that plays a central role in morphogenesis, growth, and cell differentiation during normal embryonic development. Members of this growth factor family are particularly vital to development of the mammalian secondary palate where they regulate palate mesenchymal cell proliferation and extracellular matrix synthesis. Such regulation is particularly critical since perturbation of either cellular process results in a cleft of the palate. While the cellular and phenotypic effects of TGFbeta on embryonic craniofacial tissue have been extensively catalogued, the specific genes that function as downstream mediators of TGFbeta action in the embryo during palatal ontogenesis are poorly defined. Embryonic palatal tissue in vivo and murine embryonic palate mesenchymal (MEPM) cells in vitro secrete and respond to TGFbeta. In the current study, elements of the Smad component of the TGFbeta intracellular signaling system were identified and characterized in cells of the embryonic palate and functional activation of the Smad pathway by TGFbeta1, TGFbeta2, and TGFbeta3 was demonstrated. TGFbeta-initiated Smad signaling in cells of the embryonic palate was found to result in: (1) phosphorylation of Smad 2; (2) nuclear translocation of the Smads 2, 3, and 4 protein complex; (3) binding of Smads 3 and 4 to a consensus Smad binding element (SBE) oligonucleotide; (4) transactivation of transfected reporter constructs, containing TGFbeta-inducible Smad response elements; and (4) increased expression of gelatinases A and B (endogenous genes containing Smad response elements) whose expression is critical to matrix remodeling during palatal ontogenesis. Collectively, these data point to the presence of a functional Smad-mediated TGFbeta signaling system in cells of the developing murine palate.     

Catecholamine modulation of embryonic palate mesenchymal cell DNA synthesis

Development of the mammalian embryonic palate depends on the precise temporal and spatial regulation of growth. The factors and mechanisms underlying differential growth patterns in the palate remain elusive. Utilizing quiescent populations of murine embryonic palate mesenchymal (MEPM) cells in vitro, we have begun to investigate hormonal regulation of palatal cell proliferation. MEPM cells in culture were rendered quiescent by 48 hr serum deprivation and were subsequently released from growth arrest by readdition of medium containing 10% (v/v) serum. The progression of cells into S-phase of the cell cycle was monitored by autoradiographic analysis of tritiated thymidine incorporation. Palate mesenchymal cell entry into S-phase was preceded by a 6- to 8-hr prereplicative lag period, after which time DNA synthesis increased and cells reached a maximum labeling index by 22 hr. Addition of 10 microM isoproterenol to cell cultures at the time of release from growth arrest lengthened the prereplicative lag period and delayed cellular entry into S-phase by an additional 2 to 4 hr. The rate of cellular progression through S-phase remained unaltered. The inhibitory effect of isoproterenol on the initiation of MEPM cell DNA synthesis was abolished by pretreatment of cells with propranolol at a concentration (100 microM) that prevented isoproterenol-induced elevations of cAMP. Addition of PGE2 to cell cultures, at a concentration that markedly stimulates cAMP formation, mimicked the inhibitory effect of isoproterenol on cellular progression into S-phase. These findings demonstrate the ability of the beta-adrenergic catecholamine isoproterenol to modulate MEPM cell proliferation in vitro via a receptor-mediated mechanism and raise the possibility that the delayed initiation of DNA synthesis in these cells is a cAMP-dependent phenomenon.     

Effect of epidermal growth factor on synthesis of prostaglandins and cyclic AMP by embryonic palate mesenchymal cells

The influence of epidermal growth factor (EGF) on the ability of murine embryonic palate mesenchymal (MEPM) cells to be stimulated to synthesize cAMP and prostaglandins was investigated. Preincubation of MEPM cells with EGF enhanced, in a dose-dependent fashion, (1) the responsiveness of MEPM cells to prostaglandin E1-induced elevation of intracellular levels of cAMP, and (2) the responsiveness of cells to calcium ionophore (A23187) and melittin-induced synthesis of prostaglandins E2 and F2 alpha. Hormonal responsiveness of MEPM cells to EGF, prostaglandins and cAMP has been implicated as being involved in controlling various aspects of normal oro-facial development. We show here that EGF can potentiate hormonal responsiveness of these cells and thus allows consideration of EGF as a factor which may modulate hormonally regulated craniofacial growth and differentiation.     

Epidermal growth factor modulation of prostaglandins and nitrite biosynthesis in rat fetal membranes

The production of prostaglandins (PGs) and nitric oxide (NO) by amnion tissue may play a significant role in parturition. It is thought that epidermal growth factor (EGF) may be one of the fetal signals that governs the initiation of labor. The aim of the present study was to investigate the effect of EGF in vivo on the PGs and nitrite production of rat fetal membranes. We have evaluated the regulation of PGs and nitrite production in rat fetal membranes ex vivo. The intra-uterine administration of EGF 500 ng in day 21 of pregnancy induced increases in PGE(2) (P<0.001) and PGF(2alpha) (P<0.01) compared to the control fetal membranes from pregnant rats on day 22. Also, this dose of EGF diminished nitrate production significantly (P<0.01). We found that fetal membranes at term (days 18-22 of gestation) expressed EGF-R. The NO donor, nitroprussiate 300 and 600 microM, elicited an inhibitory effect on the PGE(2) and PGF(2alpha) stimulated synthesis. On the other hand, indomethacin 10(-6) and 10(-7)M, a non-selective cyclooxygenase inhibitor, reverted the inhibitory effect exerted by EGF. Hence, rat fetal membranes were found to express epidermal growth factor receptors and, under the effect of EGF, PGs and nitrites production pathways interact probably to prevent a toxic effect caused by an exacerbated synthesis of these mediators.     

Inductive effects of prostaglandins on alkaline phosphatase in osteoblastic cells, clone MC3T3-E1

The effects of prostaglandins (PGs) on the induction of alkaline phosphatase (ALP) were investigated in osteoblastic clone MC3T3-E1 cells cultured in serum-free medium. Prostaglandin E2 (PGE2) stimulated ALP activity in the cells in a dose-dependent fashion with a maximal effect which was about twice that in the control cells at concentrations of 100-500 ng/ml. Actinomycin D and cycloheximide inhibited the stimulative effect of PGE2 on ALP activity in the cells. PGE2-induced and native ALPs in the cells were of the same type as that in adult mouse calvaria, being heat-labile, L-homoarginine- and levamisole-sensitive, and L-phenylalanine-insensitive. Isobutyl methylxanthine (IBMX), a cAMP phosphodiesterase inhibitor, stimulated the inductive effect of PGE2 on ALP activity at 0.1 mM, at which concentration IBMX alone had little effect on the activity. PGE2 also increased the intracellular cAMP content in a dose-dependent fashion with a maximal effect at 100 ng/ml. PGE1, PGF1 alpha, and PGF2 alpha (primary PGs like PGE2) increased the activity. Our present results suggest that PGs stimulate the differentiation of osteoblasts and are involved in bone formation in vivo, as well as in bone resorption.     

Prostaglandin E2 enhances uterine stromal cell alkaline phosphatase activity in vitro

Prostaglandins have been implicated in the process of uterine decidualization in vitro, but sites of action are uncertain. Since one of the earliest changes in endometrial stroma following induction of decidualization is an increase in alkaline phosphatase activity, we have investigated the effects of PGs on stromal cell alkaline phosphatase activity in vitro. Immature rats were pretreated with hormones to sensitize their uteri for the decidual cell reaction. Endometrial stromal cells were isolated and cultured for up to 4 days with PGE2 (0-10 micrograms/ml) or PGF2 (0-10 micrograms/ml). Analysis of variance revealed a highly significant interaction between day of culture and concentration of PGE2 in medium (P less than 0.01). Stromal cell alkaline phosphatase activity decreased significantly with increasing culture duration (P less than 0.01). In the presence of PGE2, alkaline phosphatase activity was significantly higher (P less than 0.01) regardless of day of culture. In contrast, PGF2 alpha had only a small and inconsistent effect. These data indicate that PGs, and in particular PGE2, can act directly upon stromal cells.     

Effects of exogenous prostanoids on the proliferation of osteoblast-like cells in vitro

The effects of several prostaglandins on the proliferation of secondary cultures of osteoblast-like cells, as measured by the incorporation of [3H]-thymidine into DNA and total DNA content of the cultures, were studied. PGE2 in the concentration range of 10(-8) to 10(-5) M caused a direct, dose-related stimulation of proliferation, while PGF2 alpha and PGD2 were less effective. PGA2 and 6-keto-PGF1 alpha were inactive in the osteoblasts in concentrations of 10(-7) to 10(-6) M. A similar stimulation profile was observed for the induction of ornithine decarboxylase (ODC, L-ornithine decarboxy-lyase, EC 4.1.1.17): the order of potency of the different prostaglandins in the induction of the ODC activity was PGE2 greater than PGF2 alpha = PGD2; again, PGA2 and 6-keto-PGF1 alpha were without effect in concentrations up to 10(-6) M. These results show that the primary prostaglandins, in order of potency PGE2 greater than PGF2 alpha = PGD2, can have a direct, stimulatory effect on the proliferation of osteoblasts, which is closely related to the induction of ODC activity.     

Effect of prostaglandins on ornithine decarboxylase activity in the testis of immature rat

Intratesticular injection of prostaglandin E2 (PGE2) and F2 alpha (PGF2 alpha) caused stimulation of ornithine decarboxylase (ODC) activity in the testis of immature rats. PGE2 at a dose of 10 microgram per testis was maximally effective 2 hours after the injection. Dibutyryl cyclic AMP (cAMP) and 1 methyl, 3-isobutyl xanthine (MIX), a phosphodiesterase inhibitor, also stimulated ODC activity. Simultaneous injection of PGE2 and FSH or LH caused additional stimulation of ODC activity. Similarly injection of PGE2 in addition to cAMP or MIX also caused increased stimulation of ODC. Indomethacin (IM, 60 microgram/testis) inhibited LH, FSH or cAMP induced ODC activity. However, IM at the same dose inhibited the synthesis of total proteins. These results suggest that PGE2 and PGF2 alpha stimulate the activity of ODC. The action of prostaglandins may be independent of the action of gonadotropic hormones. cAMP appears to mediate the action of prostaglandins in the testis of rat.     

Differential expression and biological activity of retinoic acid–induced TGFβ isoforms in embryonic palate mesenchymal cells

The effect of retinoic acid (RA) on TGF-β mRNA expression and protein production in murine embryonic palate mesenchymal (MEPM) cells was examined by Northern blotting and TGF-β bioassay in association with TGF-β isoform-specific neutralizing antibodies. Heat or acid activation was used to distinguish between latent and active TGF-β protein released into the culture medium. RA had little or no effect on TGF-β1 mRNA expression and protein production. In contrast, RA increased TGF-β2 and β3 protein released into the culture medium, the protein being mostly in an inactive or latent form. The amount of active TGF-β released was increased relative to the total increase in TGF-β released, suggesting that RA treatment stimulated activation of latent TGF-β. RA also increased TGF-β2 mRNA expression; we have previously shown that RA upregulates TGF-β3 mRNA in these cells. RA and TGF-β individually inhibited ³H-thymidine incorporation into MEPM cell DNA, while, when administered simultaneously, they inhibited proliferative activity to a greater extent. Heat- or acid-activated conditioned medium (CM) from MEPM cells treated with RA was able to inhibit ³H-thymidine incorporation into MEPM cell DNA to an extent greater than seen with RA treatment alone. Coincubation of heat-activated CM from RA-treated MEPM cells with pan-specific or TGF-β2 or β3-specific neutralizing antibodies partially relieved the inhibitory effect on ³H-thymidine incorporation, suggesting that this proliferative response was due to RA-induced TGF-β. Simultaneous treatment with RA and TGF-β also stimulated gycosaminoglycan (GAG) synthesis to an extent greater than that seen with TGF-β treatment alone, this despite the ability of RA to inhibit GAG synthesis. These data demonstrate a role for RA and RA-induced TGF-β in the regulation of palate cell proliferation and GAG synthesis and suggest a role for TGF-β in retinoid-induced cleft palate. J. Cell. Physiol. 177:36–46, 1998. © 1998 Wiley-Liss, Inc.     

Phytoestrogens modulate prostaglandin production in bovine endometrium: cell type specificity and intracellular mechanisms

Prostaglandins (PGs) are known to modulate the proper cyclicity of bovine reproductive organs. The main luteolytic agent in ruminants is PGF2alpha, whereas PGE2 has luteotropic actions. Estradiol 17beta (E2) regulates uterus function by influencing PG synthesis. Phytoestrogens structurally resemble E2 and possess estrogenic activity; therefore, they may mimic the effects of E2 on PG synthesis and influence the reproductive system. Using a cell-culture system of bovine epithelial and stromal cells, we determined cell-specific effects of phytoestrogens (i.e., daidzein, genistein), their metabolites (i.e., equol and para-ethyl-phenol, respectively), and E2 on PGF2alpha and PGE2 synthesis and examined the intracellular mechanisms of their actions. Both PGs produced by stromal and epithelial cells were significantly stimulated by phytoestrogens and their metabolites. However, PGF2alpha synthesis by both kinds of cells was greater stimulated than PGE2 synthesis. Moreover, epithelial cells treated with phytoestrogens synthesized more PGF2alpha than stromal cells, increasing the PGF2alpha to PGE2 ratio. The epithelial and stromal cells were preincubated with an estrogen-receptor (ER) antagonist (i.e., ICI), a translation inhibitor (i.e., actinomycin D), a protein kinase A inhibitor (i.e., staurosporin), and a phospholipase C inhibitor (i.e., U73122) for 0.5 hrs and then stimulated with equol, para-ethyl-phenol, or E2. Although the action of E2 on PGF2alpha synthesis was blocked by all reagents, the stimulatory effect of phytoestrogens was blocked only by ICI and actinomycin D in both cell types. Moreover, in contrast to E2 action, phytoestrogens did not cause intracellular calcium mobilization in either epithelial or stromal cells. Phytoestrogens stimulate both PGF2alpha and PGE2 in both cell types of bovine endometrium via an ER-dependent genomic pathway. However, because phytoestrogens preferentially stimulated PGF2alpha synthesis in epithelial cells of bovine endometrium, they may disrupt uterus function by altering the PGF2alpha to PGE2 ratio.     

Prostaglandin F2 alpha stimulates phosphatidylinositol turnover and increases the cellular content of 1,2-diacylglycerol in confluent resting Swiss 3T3 cells

Prostaglandin F2 alpha (PGF2 alpha); which stimulates DNA synthesis in resting 3T3 cells, also stimulates the incorporation of [32P]PO4 into phosphatidylinositol. The effect is selective for PGF2 alpha when compared with PGE1, PGE2, and PGF2 beta. Epidermal growth factor (EGF) also stimulates DNA synthesis but does not affect phosphatidylinositol turnover. PGE1, which acts synergistically with PGF2 alpha to enhance DNA synthesis, does not affect the ability of PGF2 alpha, to enhance the incorporation of [32P]PO4 into phosphatidylinositol. PGF2 alpha, also causes a small increase in the cellular content of 1,2-diacylglycerol. This effect is not shared by EGF or PGE1. Stimulation of phosphatidylinositol metabolism resulting in an increase in the cellular content of 1,2-diacylglycerol may thus constitute an event in the pathway leading to the initiation of DNA synthesis in which PGF2 alpha differs in its action from EGF.     

Radioimmunoassay measurement of ACTH-facilitated PGE2 and PGF2alpha release from isolated cat adrenocortical cells.

Prostaglandin (PG) biosynthesis by trypsin-dispersed cat adrenocortical cells was studied by radioimmunoassay (RIA). Parallel assays of incubation media using PGF2alpha and PGF1alpha antisera established that PGF2alpha is the primary PGF released by feline cortical cells. Following the reduction of PGE to PGF with sodium borohydride (NaBH4) these same two antisera were also used to identify PGE2 as the primary PGE released. RIA using a PGE antiserum confirmed the presence of PGE in the incubation medium. Steroidogenic concentrations of ACTH (50-250muU) enhanced PGE and PGF release, and indomethacin suppressed the ACTH-facilitated release. These studies provide additional evidence for ACTH-induced PG synthesis by feline cortical cells, and support the hypothesis that PGs play some role in the steroidogenic action of ACTH.     

Quantification and localization of ornithine decarboxylase in the embryonic palate.

Ornithine decarboxylase (ODC; EC4.1.1.17), the key enzyme in polyamine biosynthesis, and intracellular polyamines increase rapidly and markedly in tissues and cells that are actively proliferating as well as differentiating and decrease as these processes cease. ODC activity has also been implicated as playing a role in the proliferation and differentiation of cells derived from the developing palate. Ornithine decarboxylase activity was thus quantified and ODC localized in the developing murine palate in vivo. Levels of ODC activity showed little variation during the ontogeny of the palate, averaging 126 pmol CO2/mg protein/hr. When difluoromethylornithine (DFMO), an irreversible inhibitor of ODC activity, was administered to pregnant mice throughout the period of palate development (days 11-14), palatal tissue ODC activity was reduced by 85%. No craniofacial malformations were observed, however. The lack of a teratogenic effect by DFMO treatment could be due to sufficient remaining ODC activity in craniofacial tissue and/or maintenance of intracellular polyamine levels by the activity of a polyamine transport system. The activity of this system was demonstrated by the ability of palatal tissue in vivo to take up radiolabeled putrescine. The presence of a polyamine transport system was previously suggested by the demonstration of such a system in palate mesenchymal cells in vitro. Dramatic temporal and spatial shifts in tissue patterns of immunolocalization for ODC in developing palatal tissue were also seen. Immunostaining for ODC was evenly distributed in oral, nasal, and medial edge palate epithelial cells on day 12 of gestation. The basal aspects of epithelial cells were, however, more intensely stained. Mesenchymal cells exhibited a peri-nuclear immunostaining pattern. On days 12 and 13 of gestation, the staining patterns for ODC in palate epithelial and mesenchymal cells were comparable. On day 14 of gestation, all regions of the palate epithelium, particularly the medial edge epithelia, were immunostained for ODC, whereas the intensity of staining in the mesenchymal cells was significantly reduced. This study represents essential initial observations toward understanding the role that ODC may play in normal craniofacial development.