Murine Embryonic Palate Mesenchymal Research Articles

Associations between the proliferation of palatal mesenchymal cells, Tgfβ2 promoter methylation, Meg3 expression, and Smad signaling in atRA-induced cleft palate

All-trans retinoic acid (atRA) is a teratogen that can induce cleft palate formation. During palatal development, murine embryonic palate mesenchymal (MEPM) cell proliferation is required for the appropriate development of the palatal frame, with Meg3 serving as a key regulator of the proliferative activity of these cells and the associated epithelial-mesenchymal transition process. DNA methylation and signaling via the TGFβ/Smad pathway are key in regulating embryonic development. Here, the impact of atRA on MEPM cell proliferation and associations between Tgfβ2 promoter methylation, Meg3, and signaling via the Smad pathway were explored using C57BL/6 N mice treated with atRA (100 mg/kg) to induce fetal cleft palate formation. Immunohistochemistry and BrdU assays were used to detect MEPM proliferation and DNA methylation assays were performed to detect Tgfβ2 promoter expression. These analyses revealed that atRA suppressed MEPM cell proliferation, promoted the upregulation of Meg3, and reduced the levels of Smad2 and Tgfβ2 expression phosphorylation, whereas Tgfβ2 promoter methylation was unaffected. RNA immunoprecipitation experiments indicated that the TgfβI receptor is directly targeted by Meg3, suggesting that the ability of atRA to induce cleft palate may be mediated through the Tgfβ/Smad signaling pathway.

The mechanisms governing mouse embryonic palate mesenchymal cells' proliferation associated with atRA-induced cleft palate in mice: insights from integrated transcriptomic and metabolomic analyses.

While exposure to high levels of all-trans retinoic acid (atRA) during pregnancy is known to suppress murine embryonic palate mesenchymal (MEPM) cells proliferation and to result in cleft palate (CP) development, the underlying mechanisms are poorly understood. Accordingly, this study was designed with the goal of clarifying the etiological basis for atRA-induced CP. A murine model of CP was established via the oral administration of atRA to pregnant mice on gestational day (GD) 10.5, after which transcriptomic and metabolomic analyses were performed with the goal of clarifying the critical genes and metabolites associated with CP development through an integrated multi-omics approach. MEPM cells proliferation was altered by atRA exposure as expected, contributing to CP incidence. In total, 110 genes were differentially expressed in the atRA treatment groups, suggesting that atRA may influence key biological processes including stimulus, adhesion, and signaling-related activities. In addition, 133 differentially abundant metabolites were identified including molecules associated with ABC transporters, protein digestion and absorption, mTOR signaling pathway, and the TCA cycle, suggesting a link between these mechanisms and CP. Overall, combined analyses of these transcriptomic and metabolomic results suggested that the MAPK, calcium, PI3K-Akt, Wnt, and mTOR signaling pathways are particularly important pathways enriched in the palatal cleft under conditions of atRA exposure. Together, these integrated transcriptomic and metabolomic approaches provided new evidence with respect to the mechanisms underlying altered MEPM cells proliferation and signal transduction associated with atRA-induced CP, revealing a possible link between oxidative stress and these pathological changes.

Intracellular dynamics of Smad‐mediated TGFβ 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.

Nuclear convergence of the TGFβ and cAMP signal transduction pathways in murine embryonic palate mesenchymal cells

Transforming growth factors β (TGFβ) and cyclic AMP (cAMP) both participate in growth and differentiation of the developing mammalian secondary palate and elicit similar biological responses. Cross-talk between these two signal transduction pathways in cells derived from the embryonic palate has been demonstrated previously. In the present study, we have examined nuclear convergence of these signalling pathways at the level of transcriptional complex formation. Biotinylated oligonucleotides encoding a consensus Smad binding element (SBE), or a cyclic AMP response element (CRE), were mixed with cell extracts from murine embryonic palate mesenchymal (MEPM) cells that were treated with either TGFβ or forskolin. Protein–oligonucleotide complexes were precipitated with streptavidin–agarose, and analysed by Western blotting to identify proteins in the complex bound to each consensus oligonucleotide. TGFβ treatment of MEPM cells increased the levels of phosphorylated Smad2, phosphorylated cAMP response element binding protein (CREB), and the coactivator, CREB binding protein (CBP), that were part of a complex bound to the SBE. Treatment of cells with forskolin, a stimulator of adenylate cyclase, increased the amount of phosphorylated CREB and CBP, but not the amount of phosphorylated Smad2 bound in a complex to the SBE. Additionally, the presence of the co-repressors, c-Ski and SnoN, was demonstrated as part of a complex bound to the SBE (but not the CRE). Amounts of c-Ski and SnoN found in the SBE-containing complex increased in response to either TGFβ or forskolin. These results demonstrate that phosphorylated CREB forms a complex with the co-activator CBP, phosphorylated Smad2 and the co-repressors c-Ski and SnoN on a consensus SBE. This suggests cooperative regulation of genes with SBE-containing promoters by the cAMP and TGFβ signalling pathways in the developing palate.

Phosphatase regulation of gene expression during development of the palate

In mammalian cells, including those of the embryonic palate, the level of phosphorylation of cellular proteins at any given time reflects the activities of protein kinases and protein phosphatases. Both protein phosphatase-1 (PP-1) and PP-2A inhibit cAMP-mediated increases in transcription by dephosphorylating CREB at ser-133. Western blot analysis indicated that protein phosphatase 1 (PP-1) was expressed constitutively in palatal tissue during its development. Expression of PP-2A was regulated developmentally with maximal expression on gestational day (gd) 14. Densitometric scanning revealed a 30% increase in expression from gd 13 to gd 14. Virtually all phosphatase activity in the tissue extracts could be inhibited by 5 μM okadaic acid, demonstrating that PP-1 and PP-2A account for all detectable ser/thr protein phosphatase activity present in the developing palate. Moreover, no significant differences in PP-1 and PP-2A activities were observed during the period of palate development. Treatment of primary cultures of murine embryonic palate mesenchymal (MEPM) cells with forskolin (20 μM) to elevate intracellular cAMP levels, resulted in a time-dependent increase in CREB ser-133 phosphorylation and a corresponding time dependent decrease in PP-1 and PP-2A levels. Moreover, treatment of MEPM cells with okadaic acid resulted in a dramatic increase in basal CREB ser-133 phosphorylation. This suggests that PP-1 activity may contribute to transcriptional regulation of CREB and that PP-1 and PP-2A are regulated differentially by cAMP. Treatment of MEPM cells with TGFβ1 (1 ng/ml) under conditions of TGFβ-induced CREB phosphorylation resulted in no effect on the expression of either PP-1 or PP-2A proteins and no significant alterations in total basal protein phosphatase activity. These results demonstrate that transcriptional regulation of CREB in embryonic palatal issue is dependent on the coordinate activity of specific kinases and phosphatases.

Convergence of c AMP, TGF-β and retinoic acid signaling pathways in cells of the embryonic palate

We have previously described bi-directional cross-talk between the retinoic acid (RA) and transforming growth factor beta (TGF-β) signal transduction pathways in primary cultures of murine embryonic palate mesenchymal (MEPM) cells. In this paper we identify interactions between the TGF-β1, cyclic adenosine 3′, 5′-monophosphate (cAMP) and RA signaling systems. TGF-β1 and forskolin, an activator of the cAMP pathway, inhibited RA-induced expression of RAR-β mRNA in MEPM cells, though only TGF-β1 inhibited RA-induced RAR-β protein expression. Forskolin, but not TGF-β1, abrogated RA-induced expression of a reporter construct containing 900 base pair (bp) of the RAR-β gene promoter, transfected into MEPM cells, suggesting that this portion of the promoter contains the forskolin-responsive, but not the TGF-β-responsive, element. Thus, a putative TGF-β Inhibitory Element (TIE) adjacent to the retinoic acid response element (RARE) in the RAR-β promoter is either non-functional, or requires promoter/enhancer elements not present in the promoter construct used in these experiments. These studies further clarify the complex interactions among signal transduction pathways in the regulation of retinoic acid receptor gene expression.

Differential expression and biological activity of retinoic acid-induced TGFbeta isoforms in embryonic palate mesenchymal cells.

The effect of retinoic acid (RA) on TGF-beta mRNA expression and protein production in murine embryonic palate mesenchymal (MEPM) cells was examined by Northern blotting and TGF-beta bioassay in association with TGF-beta isoform-specific neutralizing antibodies. Heat or acid activation was used to distinguish between latent and active TGF-beta protein released into the culture medium. RA had little or no effect on TGF-beta1 mRNA expression and protein production. In contrast, RA increased TGF-beta2 and beta3 protein released into the culture medium, the protein being mostly in an inactive or latent form. The amount of active TGF-beta released was increased relative to the total increase in TGF-beta released, suggesting that RA treatment stimulated activation of latent TGF-beta. RA also increased TGF-beta2 mRNA expression; we have previously shown that RA upregulates TGF-beta3 mRNA in these cells. RA and TGF-beta 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-beta2 or beta3-specific neutralizing antibodies partially relieved the inhibitory effect on 3H-thymidine incorporation, suggesting that this proliferative response was due to RA-induced TGF-beta. Simultaneous treatment with RA and TGF-beta also stimulated gycosaminoglycan (GAG) synthesis to an extent greater than that seen with TGF-beta treatment alone, this despite the ability of RA to inhibit GAG synthesis. These data demonstrate a role for RA and RA-induced TGF-beta in the regulation of palate cell proliferation and GAG synthesis and suggest a role for TGF-beta in retinoid-induced cleft palate.

TGF-β Signaling in Murine Embryonic Palate Cells Involves Phosphorylation of the CREB Transcription Factor

A number of studies over the last several years have demonstrated a crucial role for TGF-β in epithelial and mesenchymal differentiation during development of the embryonic palate. Molecular mechanism(s) of signal transduction responsible for eliciting these responses remain unresolved. Since cAMP signaling also modulates the same tissue differentiation in the developing palate and palate-derived cells, we hypothesized that TGF-β activity may be mediated through cAMP-inducible pathways. We thus examined the effects of TGF-β on activation of the cAMP regulatory element binding protein CREB, a nuclear transcription factor which mediates transcription of genes containing CRE recognition sequences in their promoters. We examined the ability of TGF-β-treated murine embryonic palate mesenchymal (MEPM) cells to phosphorylate CREB on the amino acid residue serine 133, phosphorylation of which is indispensable for transcriptional activation. TGF-β treatment led to increasedphosphorylation of CREB ser-133 in a time- and dose-dependent manner. Inhibition of serine-threonine phosphatases by okadaic acid enhanced but did not prolong this response. TGF-β failed to induce the activity of protein kinase A (PKA), a known CREB kinase. Inhibition of either PKA or calcium/calmodulin kinase II (CaMK II) did not abrogate phosphorylation of CREB by TGF-β. TGF-β treatment also did not induce phosphorylation of mitogen-activated protein kinases, erk-1 and erk-2, on tyrosine 185, suggesting that these kinases do not mediate CREB phosphorylation by TGF-β. Additionally, TGF-β had no effect on CREB binding to known CREB DNA consensus recognition sequences, CRE and TRE. Together, these data suggest an alternative or novel CREB kinase in MEPM cells through which TGF-β acts to induce CREB ser-133 phosphorylation and subsequent activation of CRE-containing genes.

Antisense oligonucleotides to CRABP I and II alter the expression of TGF-beta 3, RAR-beta, and tenascin in primary cultures of embryonic palate cells.

The cellular retinoic acid-binding proteins (CRABPs) are thought to modulate the responsiveness of cells to retinoic acid (RA). We have previously shown that primary cultures of murine embryonic palate mesenchymal (MEPM) cells express both CRABP-I and CRABP-II genes and that this expression is regulated by RA and transforming growth factor beta (TGF-beta). These cells also express high levels of TGF-beta 3, which is also regulated by RA and TGF-beta. We have used an antisense strategy to investigate the role of the CRABPs in retinoid-induced gene expression. Subconfluent cultures of MEPM cells were treated for several days with phosphorothioate modified 18-mer oligonucleotides antisense to CRABP-I or CRABP-II and then with all-trans-retinoic acid at a concentration of 3.3 microM or 0.33 microM for 5 or 22 h. Total RNA was then extracted and the expression of TGF-beta 3, retinoic acid receptor beta (RAR-beta), and tenascin was assessed by northern blot analysis. Antisense oligonucleotides to CRABP-I partially inhibited the RA-induced TGF-beta 3, RAR-beta, and tenascin mRNA expression. The corresponding mis-sense oligonucleotides were without effect. Antisense oligonucleotides to CRABP-II also partially inhibited RA-induced expression of these genes. As with the CRABP-I antisense, mis-sense oligonucleotides to CRABP-II had no effect. These data suggest that both CRABPs modulate the responsiveness of MEPM cells to retinoic acid. Inhibition of endogenous CRABP expression renders MEPM cells less responsive to RA with respect to induction of TGF-beta 3, RAR-beta, and tenascin gene expression.(ABSTRACT TRUNCATED AT 250 WORDS)

Patterns of cyclic AMP‐dependent protein kinase gene expression during ontogeny of the murine palate

Normal growth and differentiation of embryonic palatal tissue depends on regulated levels of intracellular cAMP. Cyclic AMP-dependent protein kinases (PKA) act to mediate the biological activities of cAMP. PKA isozyme protein profiles demonstrate a clear pattern of temporal alterations in embryonic palatal tissue during its development. In order to ascertain the molecular basis for changing PKA isozyme profiles during palatal ontogeny, the spatial and temporal expression of mRNAs for regulatory (RI alpha, RII alpha, and RII beta) and catalytic (C alpha) subunits of PKA was examined. RNA extracted from murine embryonic palatal tissue (days 12-14 of gestation) was examined by Northern blot analysis. Significant levels of constitutively expressed RI alpha and C alpha mRNA were seen on all days of gestation examined. RI alpha transcripts were substantially less abundant in palate mesenchymal cells in vitro than in palatal tissue in vivo. Levels of RII alpha and RII beta mRNA were highest on gestational day (GD) 12, a period characterized by pronounced palatal tissue growth. In addition, patterns of tissue distribution of RII beta, not previously described, were examined in the developing embryonic palate. A dramatic developmental shift in tissue distribution of RII beta was seen. The isozyme was evenly distributed between palatal epithelial and mesenchymal cells on GD 12 but by GD 14, RII beta was predominantly localized to palatal epithelial cells. Direct activation of adenylate cyclase with forskolin in murine embryonic palate mesenchymal (MEPM) cells resulted in an increase in RII alpha mRNA levels but had no effect on steady state levels of RII beta or C alpha mRNA. In addition, elevation of intracellular levels of cAMP resulted in a shift in the transcriptional profile of RI alpha mRNAs. Results of this study document specific patterns of expression for the genes encoding the various cAMP-dependent protein kinase regulatory and C alpha subunits in murine embryonic palatal tissue. In addition, we have demonstrated adaptational changes of this kinase in MEPM cells in response to conditions of increased intracellular levels of cAMP.

Interactions between the transforming growth factor beta (TGFβ) and retinoic acid signal transduction pathways in murine embryonic palatal cells

Regulation of expression of transforming growth factor-beta 3 (TGF- β3) and the cellular retinoic acid-binding proteins-I and II (CRABP-I, -II) by retinoic acid (RA) and TGF- β was examined in primary cultures of murine embryonic palate mesenchymal (MEPM) cells. Northern blot hybridization revealed that RA and TGF- β1, β2 and β3 stimulated the expression of TGF- β3 mRNA within 24 hours of treatment. RA down-regulated the expression of CRABP-I mRNA and up-regulated the expression of CRABP-II mRNA in a time- and dose-dependent fashion. TGF- β1, β2 and β3 also down-regulated the expression of CRABP-I mRNA, while epidermal growth factor (EGF) and transforming growth factor alpha (TGF- α) were without effect. TGF- β1 also stimulated a dose-dependent increase in the expression of CRABP-II mRNA. Again EGF and TGF- α were without effect. Basic fibroblast growth factor (bFGF) elicited a slight inhibitory effect on CRABP-II and a slight stimulatory effect on CRABP-I mRNA expression. Thus, cells derived from the mammalian developing palate express CRABP-I and CRABP-II mRNAs, both of which may be regulated by RA and TGF- β. These data constitute the first demonstration of an effect of TGF- β on the expression of CRABP-I and CRABP-II and provide further evidence for cross-talk between RA and TGF- β signal transduction pathways.

Regulation of TGFβ3 gene expression in embryonic palatal tissue

The TGF beta family of genes has been shown to play an important role in regulating various aspects of development, although the mechanisms by which TGF beta exerts its effects have not yet been clarified. Growth and differentiation of both murine embryonic palate mesenchymal (MEPM) cells and palatal epithelium can be regulated by the TGF beta s. We therefore examined the expression of mRNAs encoding TGF beta 1, TGF beta 2, and TGF beta 3 in developing embryonic palatal tissue as well as factors that modulate their levels of expression. Northern blot analysis of RNA isolated from murine embryonic palatal tissue on gestational days (GD) 12, 13, and 14 demonstrated the presence of one mRNA transcript for TGF beta 1 (2.5 kb), two transcripts for TGF beta 2 (4.4 kb, 6.0 kb), and one transcript for TGF beta 3 (3.5 kb). Although steady-state levels of TGF beta 1 mRNA showed no changes during development of the palate, TGF beta 2 mRNA levels were maximal on both GD13 and GD14 and TGF beta 3 mRNA levels transiently increased on GD 13. In addition, levels of TGF beta 3 mRNA seemed much higher than either TGF beta 1 or TGF beta 2. both TGF beta 1 and TGF beta 2 were able to increase, in a dose-related manner, the expression of TGF beta 3 mRNA in murine embryonic palate mesenchymal cells in vitro. In contrast, epidermal growth factor (EGF) down-regulated the expression of TGF beta 3 mRNA even in the presence of TGF beta 1 or TGF beta 2.(ABSTRACT TRUNCATED AT 250 WORDS)

TGFβ1 regulation of collagen metabolism by embryonic palate mesenchymal cells

Proper metabolism of the extracellular matrix (ECM) in mammalian embryonic palatal tissue is required for normal development of the palate. In particular, perturbation of collagen metabolism in the embryonic orofacial region results in the production of cleft palate. Although several types of collagen have been localized in the embryonic palate, factors responsible for regulating their synthesis have not been identified. Transforming growth factor beta (TGF beta), shown to be capable of modulating ECM metabolism in other tissues, has been localized in the developing palate. Thus, we examined the ability of TGF beta 1 to modulate collagen synthesis and degradation in murine embryonic palate mesenchymal (MEPM) cells in vitro. Immunohistochemical analysis confirmed that type III collagen was predominant in the mesenchyme of the embryonic palate, whereas type I collagen was ubiquitous throughout palatal epithelium and mesenchyme. Total collagen production by TGF beta-treated confluent MEPM cells in serum-free conditioned medium was determined by measuring incorporation of L-[2-3-4-5-3H]proline into hydroxyproline. Treatment for 24 hr with TGF beta 1 stimulated incorporation into both cell layer and medium fractions. Quantification of collagen types by ELISA indicated that TGF beta 1 stimulated the accumulation of type III collagen as early as 3 hr after treatment. Northern blot analysis of MEPM cells treated with TGF beta 1 revealed that steady-state levels of mRNA encoding for procollagen alpha 1 (I) and alpha 1 (III) were increased and that these effects were ablated by cycloheximide but not actinomycin. The effects of TGF beta treatment on MEPM cell collagen levels also reflected alterations in collagen degradation. TGF beta-treated MEPM cells exhibited a significant diminution of total protease activity. Moreover, analysis by substrate gel electrophoresis indicated specific decreases in vertebrate collagenase and stromelysin. These data represent the first report of changing proteolytic profiles during palatogenesis. Thus, TGF beta regulates the amount of collagen present in embryonic palatal tissue at the level of synthesis and degradation.

Transforming growth factor-β receptor profiles of human and murine embryonic palate mesenchymal cells

Cell signalling in the developing mammalian palate appears to involve various growth factors and hormones. An important developmental role for the transforming growth factor-β (TGF-β) class of growth factors is suggested by the immunolocalization of TGF-β1 in the palate during its ontogeny. This study examined the effects of TGF-β stimulation of, as well as TGF-β receptor profiles in, murine embryonic palate mesenchymal (MEPM) and human embryonic palate mesenchymal (HEPM) cells. Results showed that TGF-β1 (1 ng/ml) stimulated proliferation of HEPM cells and inhibited proliferation of MEPM cells in a dose-dependent manner. The time course of 125I-TGF-β1 binding to specific receptors was determined by incubating cells in the presence of 170 p M 125I-TGF-β1 for up to 4 h. In both cell types, at 37 °C, the binding of 125I-TGF-β decreased linearly over 4 h, while at 4 °C, binding increased with time of incubation. Incubation of both cell types at 4 °C for 4 h, with increasing concentrations of 125I-TGF-β1, resulted in binding which demonstrated saturation kinetics. Scatchard analyses revealed one class of receptors for HEPM (K 32.3 p M) and MEPM (K 26.3 p M). However, SDS-PAGE analyses of 125I-TGF-β chemically crosslinked to specific receptor sites revealed that both cell types contained the types I (65,000 M r) and III (230,000 M r) TGF-β receptors while MEPM also contained the type II (86,000 M r) receptor. Binding studies further demonstrated the ability of platelet-derived growth factor to transmodulate TGF-β binding. These results indicate that the HEPM cell line and primary cultures of MEPM cells, although obtained from palates at similar developmental stages, are dramatically different in their responsiveness to TGF-β and have disparate TGF-β receptor profiles.

Effects of ethanol on cAMP production in murine embryonic palate mesenchymal cells

Ethanol affected the ability of murine embryonic palate mesenchymal (MEPM) cells to produce cAMP in response to hormone treatment. Acute exposure to ethanol resulted in an increase in hormone-stimulated cAMP levels, while chronic ethanol treatment led to decreased sensitivity to hormone. Forskolin-stimulated cAMP levels were decreased by both acute and chronic ethanol treatment, while the cells' response to cholera toxin was unchanged by ethanol treatment. The lack of sensitivity of the cholera toxin response to ethanol suggests that, in contrast to what has been observed in other systems, ethanol does not affect the production or activity of Gαs in MEPM cells. These results suggest a possible explanation for the molecular basis for the craniofacial abnormalities observed in the fetal alcohol syndrome.

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.

Characterization of a polyamine transport system in murine embryonic palate mesenchymal cells.

Polyamines (putrescine, spermidine, and spermine) are normal cellular constituents able to modulate cellular proliferation and differentiation in a number of developing systems. Ornithine decarboxylase (ODC), the rate-limiting enzyme in the polyamine biosynthetic pathway, has been shown to be causally related to an increase in glycosaminoglycan synthesis in murine embryonic palatal mesenchyme cells (MEPM). In order to understand other mechanisms that exist to regulate polyamine levels in cells derived from the developing craniofacial area, the present study investigated the capacity of MEPM cells to accumulate exogenous putrescine and tests the hypothesis that polyamine transport can serve as an adaptational response of MEPM cells to a change in their ability to synthesize polyamines. Transport was initiated in confluent cultures of MEPM cells by the addition of 0.1 microCi/ml of 14C-putrescine. The rate of transport, monitored for 20-120 minutes, was found to be a time-dependent saturable process. The rate of initial transport, determined by incubating MEPM cells for 15 minutes in the presence of different concentrations (1.0-20.0 microM) of 14C-putrescine, was also found to be saturable, suggesting a carrier-mediated event. Lineweaver-Burk analysis of these data revealed an apparent Km of 5.78 microM and a Vmax of 2.63 nmol/mg protein/15 minutes. Transport measured either at 4 degrees C or in the presence of 2-4 DNP was dramatically inhibited. Thus, putrescine transport is an active process, dependent upon metabolic energy. Conditions in which 1) NaCl was iso-osmotically replaced with choline chloride or 2) the Na+-electrochemical gradient was dissipated with Na+, K+-specific ionophores resulted in a decreased rate of transport indicating that putrescine transport in these cells is Na+ dependent. Noncompetitive inhibition assays utilizing sulfhydryl reagents that blocked sulfhydryl groups inhibited putrescine transport, suggesting that sulfhydryl groups are important for putrescine uptake. Competitive inhibition assays demonstrated that while spermidine and spermine inhibited putrescine uptake, ornithine did not inhibit transport. Spermidine, spermine, and putrescine thus appear to share a common transport system that is separate from that for ornithine. Putrescine transport is subject to adaptive regulation in both exponentially growing and confluent cultures of MEPM cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Epidermal growth factor potentiates the induction of ornithine decarboxylase activity by prostaglandins in embryonic palate mesenchymal cells: Effects on cell proliferation and glycosaminoglycan synthesis

Epidermal growth factor (EGF) and prostaglandins (PGs) have been implicated in the regulation of a number of developmental processes in the mammalian embryonic palate. Normal palatal ontogenesis is dependent on the presence and quite possibly on the interaction of various hormones and growth factors. The interaction between EGF and PGs in regulation of murine embryonic palate mesenchymal (MEPM) cell growth and differentiation was therefore investigated by monitoring the activity of ornithine decarboxylase (ODC), the principle and rate limiting enzyme of polyamine biosynthesis. ODC activity is tightly coupled to the proliferative and differentiative state of eukaryotic cells and therefore serves as a reliable indicator of such cellular functions. Treatment of confluent cultures of MEPM cells with EGF (1–50 ng/ml) resulted in a dose-related increase in ODC activity, while similar treatment with either PGE 2 or PGF 2α (at concentrations up to 1 μ M) did not elicit a dose-dependent increase in enzyme activity. Concurrent treatment of MEPM cells with EGF (20 ng/ml) and either PGE 2 or PGF 2α (0.1–10000 n M) resulted in a marked prostaglandin dose-dependent induction of ODC activity, suggesting a strong cooperative interaction between these factors. ODC activity was maximal by 4 to 8 hr and could be completely inhibited by preincubation of the cells with actinomycin D or cycloheximide, indicating that de novo synthesis of RNA and protein is necessary for enzyme induction. Stimulation of ODC activity by EGF and PGE 2 in these cells was not positively correlated with the level of cellular DNA synthesis but did result in a ninefold increase in the synthesis of extracellular glycosaminoglycans (GAGs), a key macromolecular family implicated in palatal morphogenesis. Stimulation of GAG synthesis was significantly inhibited by the administration of 5 m M DFMO (an irreversible inhibitor of ODC), indicating that the marked increase in GAG production was dependent, in part, on the induction of ODC activity by EGF and PGE 2. Qualitative analysis of the palatal GAGs indicated that synthesis of several major classes of GAGs was stimulated. Collectively these data demonstrate a cooperative interaction between EGF and PGs in the induction of ODC activity. Such activity may serve to regulate the synthesis of GAGs, which are instrumental in mammalian palatal ontogenesis.

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 E 1-induced elevation of intracellular levels of cAMP, and (2) the responsiveness of cells to calcium ionophore (A23187) and melittin-induced synthesis of prostaglandins E 2 and F 2α. 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.