Activin synergistically increased c-jun mRNA in P19 embryonal carcinoma cells in the presence of retinoic acid.

Activin and retinoids, which are involved in the induction and regulation of the early differentiation process in vertebrate embryogenesis, synergistically increased the amount of c-jun mRNA in P19 embryonal carcinoma cells, but activin alone had no effect. Among the retinoids, all-trans-retinoic acid most effectively increased c-jun mRNA. Activin (lng/ml) was sufficient to induce the synergistic increase of c-jun mRNA in P19 EC cells with all-trans-retinoic acid. The synergistic increase of the amount of c-jun mRNA by their cooperative action may be important in vertebrate development.     

Suppression of PTEN expression during aggregation with retinoic acid in P19 mouse embryonal carcinoma cells

Apoptosis is thought to be involved in the maintenance of cellular homeostasis, as well as various pathological processes. However, little information is available about the regulation of apoptosis during the aggregation stage of P19 embryonal carcinoma (EC) cells. Here we report that aggregation-induced apoptosis is markedly attenuated by treatment with retinoic acid (RA). PTEN (phosphatase and tensin homolog deleted on chromosome 10) expression was down-regulated during the aggregation phase of P19 EC cells in the presence, but not in the absence, of RA. Suppression of PTEN expression during the aggregation was accompanied by increased phosphorylation of serine/threonine kinase Akt and glycogen synthase kinase-3beta (GSK-3beta). Our results suggest that RA attenuates the induction of apoptosis during the aggregation phase of P19 EC cells, probably by suppressing PTEN expression.     

Cell density and cell cycle effects on retinoic acid-induced embryonal carcinoma cell differentiation.

P19 embryonal carcinoma cells can be induced to differentiate with a pulse of only 4 hr in retinoic acid (RA). The efficiency of RA-induced differentiation is independent of the position of P19 cells in the cell cycle but is critically dependent on the ratio between the number of cells and the number of moles of RA in the culture medium. P19 cultures at lower cell density are more efficiently induced to differentiate than cultures containing cells at higher cell densities. This effect is not mediated by cell-to-cell contact but may be related to the rapid metabolism of RA by the cells. Individual clones of differentiating P19 cells can develop into at least three different cell types suggesting that each cell in the population of embryonal carcinoma cells retains pluripotency.     

Neuronal differentiation of P19 embryonal carcinoma cells modulates kinin B2 receptor gene expression and function

Kinins are vasoactive oligopeptides generated upon proteolytic cleavage of low and high molecular weight kininogens by kallikreins. These peptides have a well established signaling role in inflammation and homeostasis. Nevertheless, emerging evidence suggests that bradykinin and other kinins are stored in the central nervous system and may act as neuromediators in the control of nociceptive response. Here we show that the kinin-B2 receptor (B2BKR) is differentially expressed during in vitro neuronal differentiation of P19 cells. Following induction by retinoic acid, cells form embryonic bodies and then undergo neuronal differentiation, which is complete after 8 and 9 days. Immunochemical staining revealed that B2BKR protein expression was below detection limits in nondifferentiated P19 cells but increased during the course of neuronal differentiation and peaked on days 8 and 9. Measurement of [Ca(2+)](i) in the absence and presence of bradykinin showed that most undifferentiated cells are unresponsive to bradykinin application, but following differentiation, P19 cells express high molecular weight neurofilaments, secrete bradykinin into the culture medium, and respond to bradykinin application with a transient increase in [Ca(2+)](i). However, inhibition of B2BKR activity with HOE-140 during early differentiation led to a decrease in the size of embryonic bodies formed. Pretreatment of differentiating P19 cells with HOE-140 on day 5 resulted in a reduction of the calcium response induced by the cholinergic agonist carbamoylcholine and decreased expression levels of M1-M3 muscarinic acetylcholine receptors, indicating crucial functions of the B2BKR during neuronal differentiation.     

Smooth muscle actin expression during P19 embryonal carcinoma differentiation in cell culture

P19 embryonal carcinoma (EC) cells can be induced in vitro to differentiate into cells resembling those normally formed in the embryo. Among these cell types is one whose morphology is fibroblast-like. Using indirect immunofluorescence and Western blot analysis with antibodies directed against various isoforms of actin, many of these fibroblast-like cells were found to express smooth muscle actin isoforms. Northern blot analysis of RNA indicated the presence of a smooth muscle-specific isoform of myosin heavy-chain mRNA in immortal lines of these fibroblast-like cells. These results suggest that these fibroblast-like cells resemble fetal myofibroblastic or myoepithelial cells, which have a wide distribution during embryonic development.     

Regulation of expression of sulfoglucuronyl carbohydrate (HNK-1), Amphoterin and RAGE in retinoic acid-differentiated P19 embryonal carcinoma cells

HNK-1 antibody reactive sulfoglucuronyl carbohydrate (SGC) and SSEA-1 antibody reactive Lewis X (Lex) epitope are expressed on several glycolipids, glycoproteins, and proteoglycans of the nervous system and have been implicated in cell-cell recognition, neurite outgrowth, and/or neuronal migration during development. Interaction of SGC with its binding protein Amphoterin and interaction of Amphoterin with a cell-signaling molecule, receptor for advance glycation end product (RAGE) have been suggested to regulate neurite outgrowth and neuronal migration. The regulation of expression of SGC, Lex, Amphoterin, and RAGE was studied in embryonal carcinoma P19 cells after treatment with retinoic acid (RA). The untreated proliferating P19 cells strongly expressed the Lex epitope, which was mostly due to Lex-glycoproteins. P19 cells, when differentiated into neuron-like cells by RA, did not express the Lex epitope, but expressed increasing levels of SGC, with time in culture. Quantitative biochemical analyses showed that in the P19 cells after RA treatment, the amount of SGC-glycoproteins increased at a significantly higher level than sulfoglucuronyl glycolipid-1 (SGGL-1). The increase in the levels of SGGL-1 was due to 16-fold upregulation in the activity of lactosylceramide: N-acetylglucosaminyl-transferase (Lc3 synthase), which synthesizes the key intermediate lactotriosylceramide (Lc3Cer), for lacto- and neolacto-glycolipids. The large increase in the activity of Lc3 synthase appeared to regulate the levels of other neolacto glycolipids, such as Lc3Cer, nLc4Cer, nLc6Cer, disialosyl-nLc4Cer (LD1), and Lex-glycolipids. Strong upregulation of glucuronyl-transferase and modest twofold enhancement in the activity of the glucuronyl-sulfotransferase, which catalyze the final steps in the SGC synthesis, also would account for the large increase in the synthesis SGC-glycoproteins. RA also upregulated the synthesis of Amphoterin and RAGE in P19 cells. SGC, RAGE, and Amphoterin were co-localized in the RA-differentiated neurons. The initiation of neurite outgrowth along with co-ordinated upregulation of Amphoterin, RAGE, SGC-glycoproteins, and SGGLs in RA-treated P19 cells support the hypothesis that these molecules are involved in the neuronal process formation.     

Visceral-endoderm-like cell lines induce differentiation of murine P19 embryonal carcinoma cells

When P19 embryonal carcinoma (EC) cells were cocultured with cells from one of several established visceral-endoderm-like cell lines, the EC cells were rapidly induced to aggregate and differentiate, into cell types including mesoderm-derived cardiac and skeletal muscle. Neither parietal-endoderm- nor mesoderm-like cell lines induced aggregation or differentiation of EC cells in coculture, although a cell line with both parietal and visceral endoderm characteristics induced aggregation but not differentiation. Also, without the feeder cells aggregates of P19 failed to differentiate, provided that serum in the culture medium had been previously passed over dextran-coated charcoal to remove lipophilic substances, which may include endogenous retinoids. All experiments were carried out using serum treated in this way. Taken together, the results demonstrated that aggregation was necessary, but not sufficient, to make P19 EC cells differentiate. Direct contact between the two cell types was not necessary, since even when separated by an agar layer in cocultures, aggregates of P19 still differentiated. Medium conditioned by cells of the END-2 line, a visceral-endoderm-like derivative of P19, was particularly potent in inducing endodermal and mesodermal differentiation of single P19 aggregates, confirming the involvement of a diffusible factor secreted specifically by visceral-endoderm-like cells in this process.     

The cell cycle,cell death,and cell morphology during retinoic acid-induced differentiation of embryonal carcinoma cells

Time-lapse films were made of PC13 embryonal carcinoma cells, synchronized by mitotic shake off, in the absence and presence of retinoic acid. Using a method based on the transition probability model, cell cycle parameters were determined during the first five generations following synchronization. In undifferentiated cells, cell cycle parameters remained identical for the first four generations, the generation time being 11–12 hr. In differentiating cells, with retinoic acid added at the beginning of the first cycle, the first two generations were the same as controls. The duration of the third generation, however, was increased to 15.7 hr while the fourth and fifth generation were approximately 20 hr, the same as in exponentially growing, fully differentiated cells. The increase in generation time of dividing cells was principally due to an increase in the length of S phase. Cell death induced by retinoic acid also occurred principally in the third and subsequent generations. Cell population growth was then significantly less than that expected from the generation time derived from cycle analysis of dividing cells. Cells lysed frequently as sister pairs suggesting susceptibility to retinoic acid toxicity determined in a generation prior to death. Morphological differentiation, as estimated by the area of substrate occupied by cells, was shown to begin in the second cell cycle after retinoic acid addition. These results demonstrate that as in the early mammalian embryo, differentiation of embryonal carcinoma cells to an endoderm-like cell is also accompanied by a decrease in growth rate but that this is preceded by acquisition of the morphology characteristic of the differentiated progeny.     

MEKK4 mediates differentiation in response to retinoic acid via activation of c-Jun N-terminal kinase in rat embryonal carcinoma P19 cells

Differentiation of P19 embryonal carcinoma cells in response to the morphogen retinoic acid is regulated by Galpha(12/13) and is associated with activation of c-Jun N-terminal kinase. The role of MEKK1 and MEKK4 upstream of the c-Jun N-terminal kinase was investigated in P19 cells. P19 clones stably expressing constitutively active and dominant negative mutants of MEKK1 and MEKK4 were created and characterized. Expression of the constitutively active form of either MEKK1 or MEKK4 mimicked the action of retinoic acid, inducing these embryonal carcinoma cells to primitive endoderm. Expression of the dominant negative form of MEKK1 had no influence on the ability of retinoic acid to induce either JNK activation or primitive endoderm formation in P19 stem cells. Expression of the dominant negative form of MEKK4, in contrast, effectively blocks both morphogen-induced activation of JNK and cellular differentiation. These data identify MEKK4 as upstream of c-Jun N-terminal kinase in the pathway mediating differentiation of P19 stem cells to primitive endoderm.     

Brachyury regulatory region active in embryonal carcinoma P19 cells

Brachyury (T) is involved in mesoderm induction during early mouse development. We analyzed the region regulating expression of T in embryonal carcinoma P19 cells, which differentiate into mesoderm derivatives in vitro. Transfection of plasmids encoding reporter genes under the control of the 5'-flanking region showed positive regulatory elements between -298 and -129 bp are responsible for driving T expression in mesodermal cells.     

Directed differentiation of mouse P19 embryonal carcinoma cells to neural cells in a serum- and retinoic acid-free culture medium

P19 embryonal carcinoma cells (EC-cells) provide a simple and robust culture system for studying neural development. Most protocols developed so far for directing neural differentiation of P19 cells depend on the use of culture medium supplemented with retinoic acid (RA) and serum, which has an undefined composition. Hence, such protocols are not suitable for many molecular studies. In this study, we achieved neural differentiation of P19 cells in a serum- and RA-free culture medium by employing the knockout serum replacement (KSR) supplement. In the KSR-containing medium, P19 cells underwent predominant differentiation into neural lineage and by day 12 of culture, neural cells were present in 100% of P19-derived embryoid bodies (EBs). This was consistently accompanied by the increased expression of various neural lineage-associated markers during the course of differentiation. P19-derived neural cells comprised of NES⁺ neural progenitors (~?46%), TUBB3⁺ immature neurons (~?6%), MAP2⁺ mature neurons (~?2%), and GFAP⁺ astrocytes (~?50%). A heterogeneous neuronal population consisting of glutamatergic, GABAergic, serotonergic, and dopaminergic neurons was generated. Taken together, our study shows that the KSR medium is suitable for the differentiation of P19 cells to neural lineage without requiring additional (serum and RA) supplements. This stem cell differentiation system could be utilized for gaining mechanistic insights into neural differentiation and for identifying potential neuroactive compounds.