ALBERT

Notes: 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 μM 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 μM) 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 β-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.

Notes: 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 μCi/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 μM) 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 μM and a Vmax of 2.63 nmol/mg protein/15 minutes. Transport measured either at 4 deg;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. DFMO, an irreversible inhibitor of ODC, which depletes endogenous pools of putrescine, stimulated putrescine uptake with an apparent Km and Vmax of 12.58 μM and 3.51 nmol/mg protein/ 15 minutes, respectively, while preloading MEPM cells with putrescine inhibited uptake. The ability of exogenous putrescine to reverse the inhibition of MEPM cellular proliferation by DFMO suggests that MEPM cells can utilize putrescine transport to regulate cellular proliferation. These data indicate that MEPM cells have the capacity to regulate intracellular polyamine levels in an alternative and/or adjunctive manner to de novo synthesis, via transportation of extracellular polyamines. Furthermore, the rate of putrescine transport apears to be adaptively regulated by the intracellular content of polyamines, which may contribute to the homeostatic regulation of cellular polyamine levels and cellular proliferation.

Notes: 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 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 μM (Km) and 23.6 nmol/mg protein/15 minutes (Vmax) in nonstimulated cells to 4.48 μM (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 μg/ml) and actinomycin D (50 μg/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 α-aminoisobutryic 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.

Notes: 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 (Rlα, Rllα, and Rllβ) and catalytic (Cα) 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 Rlα and Cα mRNA were seen on all days of gestation examined. Rlα transcripts were substantially less abundant in palate mesenchymal cells in vitro than in palatal tissue in vivo. Levels of Rllα and Rllβ mRNA were highest on gestational day (GD) 12, a period characterized by pronounced palatal tissue growth. In addition, patterns of tissue distribution of Rllβ, not previously described, were examined in the developing embryonic palate. A dramatic developmental shift in tissue distribution of Rllβ was seen. The isozyme was evenly distributed between palatal epithelial and mesenchymal cells on GD 12 but by GD 14, Rllβ 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 Rllα mRNA levels but had no effect on steady state levels of Rllβ or Cα mRNA. In addition, elevation of intracellular levels of cAMP resulted in a shift in the transcriptional profile of Rlα mRNAs. Results of this study document specific patterns of expression for the genes encoding the various cAMP-dependent protein kinase regulatory and Cα 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. © 1995 Wiley-Liss, Inc.

Notes: Cyclic AMP, via activation of cAMP-dependent protein kinase (PKA) and subsequent protein phosphorylation, regulates a number of cellular and tissue responses that are critical to normal development of the mammalian palate. The present study examines the expression, distribution, and phosphorylation in the developing murine palate of a substrate for PKA known as the cAMP-response element binding protein (CREB). This 43 × 103 Mr protein functions as a regulator of cAMP-inducible gene expression. CREB is expressed constituitively throughout the palatal morphogenetic period and is ubiquitously distributed throughout palatal tissue. Immunofluorescent staining of palatal cells and tissues with an anti-CREB antibody revealed CREB to be localized to cell nuclei. Western blot analysis of extracts of staged palatal shelves with an antibody specific for phospho-ser 133-CREB demonstrated a steady increase in CREB phosphorylation at this residue during palate development. These observations show a temporal correlation with expression levels of cAMP-regulated genes in palate cells. The data indicate that CREB activity in the developing palate is most likely to be regulated at the level of protein phosphorylation as opposed to changes in levels of CREB protein expression. © 1995 Wiley-Liss, Inc.