Neuronal differentiation in response to epidermal growth factor of transfected murine P19 embryonal carcinoma cells expressing human epidermal growth factor receptors

The human epidermal growth factor receptor (hEGF-R) was introduced into murine P19 embryonal carcinoma (EC) cells, which do not express endogenous EGF-R. Undifferentiated stable P19 EC transfectants containing multiple copies of the hEGF-R complementary DNA were isolated. These cells express functional EGF-R, exhibiting characteristic biphasic EGF binding and intrinsic tyrosine protein kinase activity. Whereas normally EGF induces the expression of multiple nuclear protooncogenes, only junB expression is induced by EGF in the HER-transfected cells. This indicates that undifferentiated P19 EC cells contain at least part of a signal transduction machinery capable of coupling to the ectopically expressed hEGF-R. Interestingly, neuronal differentiation is induced in these cells in response to EGF under culture conditions resembling those during early preimplantation embryogenesis. These results indicate that neuronal differentiation of pluripotent P19 EC cells can be induced via activation of a tyrosine protein kinase signaling pathway.     

Clonal variants of differentiated P19 embryonal carcinoma cells exhibit epidermal growth factor receptor kinase activity

Differentiated clonal cell lines were isolated from pluripotent P19 embryonal carcinoma (EC) cells treated as aggregates with retinoic acid. Two were characterized in detail. The lines differ in morphology, proliferation rate, the production of plasminogen activator, and in their mitogenic response to insulin but both produce extracellular matrix proteins and can be serially passaged over extended periods, in contrast to differentiated derivatives of many other EC lines. Further, both lines have receptors for and respond mitogenically to epidermal growth factor (EGF). Endogenous phosphorylation of several proteins, including the EGF receptor (150 kDa) and a 38-kDa protein, is induced by EGF in membranes isolated from these cells. Preincubation of membranes with EGF renders them able to catalyze phosphorylation of tyrosine residues in exogenously added peptide substrates. High voltage electrophoresis confirmed the tyrosine specificity of the phosphorylation on the 150- and 38-kDa bands. By contrast, similar experiments in undifferentiated cells showed that intact P19 EC neither bind nor respond to EGF mitogenically and EGF induces no changes in phosphorylation in isolated membranes.     

Establishment of a differentiated mesodermal line from P19 EC cells expressing functional PDGF and EGF receptors

Aggregation of pluripotent P19 embryonal carcinoma (EC) cells in the presence of DMSO induces differentiation to various mesodermal cell types, including spontaneously contracting muscle. We have established clonal cell lines from these cultures and characterized one (MES-1) in particular for its response to growth factors. In contrast to the undifferentiated stem cells, but as a number of myoblast and muscle cell lines, MES-1 cells respond to both carbachol and bradykinin by the rapid release of Ca2+ from intracellular stores. In addition, MES-1 express receptors for and respond mitogenically to epidermal growth factor (EGF) and platelet-derived growth factor (PDGF). Isolated membranes from these cells retain the capacity to bind both ligands; addition of EGF to membranes induces endogenous phosphorylation of several proteins, including the EGF receptor itself and a 38 kD protein, while addition of PDGF specifically induces phosphorylation of the PDGF receptor. By contrast, other derivatives of P19, isolated from retinoic acid (RA)-treated aggregates and resembling neuroectodermal or endodermal cell types respond only to EGF; PDGF neither binds nor induces phosphorylation and a mitogenic response in these cells. During differentiation from EC cells therefore MES-1 cells developed a combination of growth factor receptor characteristics typical of somatic mesodermal cells and indicate that such receptors on EC-derived mesodermal cells are also functional.     

Isolation and characterization of activin receptor from mouse embryonal carcinoma cells. Identification of its serine/threonine/tyrosine protein kinase activity.

The activin receptor protein was isolated from the mouse embryonal carcinoma (EC) cell line P19 by three cycles of affinity chromatography on an activin A-immobilized column. The purified receptor had a specific and high affinity for activins A, AB, and B (Kd = 345 pM), but not for transforming growth factor beta. The purified activin receptor was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and ligand blotting analysis as a single protein of 70 kDa. The amino acid sequence of the first 18 NH2-terminal residues revealed that the receptor is a member of the activin receptor family. The purified receptor phosphorylated itself and exogenous substrate proteins on serine, threonine, and tyrosine residues, indicating that the activin receptor is a transmembrane serine/threonine/tyrosine protein kinase. These results suggest that signal transduction of activin employs a novel pathway via a new class of cellular receptor in EC P19 cells.     

Functional reconstitutional of the human epidermal growth factor receptor system in Xenopus oocytes

We have expressed the human EGF receptor (hEGF-R) in Xenopus oocytes by injecting mRNA synthesized in vitro using SP6 vectors containing receptor cDNAs. Each oocyte could express over 1 x 10(10) receptors of a single affinity class and these were able to bind and rapidly internalize EGF. Occupancy resulted in receptor tyrosine autophosphorylation, downregulation, and release of intracellular calcium. Occupied receptors also rapidly induced meiotic maturation in stage VI oocytes. Receptors lacking tyrosine kinase activity bound EGF normally, but did not downregulate or induce any biological responses. The rate of oocyte maturation was proportional to hEGF-R occupancy and was significantly faster than progesterone-induced maturation at nanomolar EGF concentrations. Mutant hEGF-R truncated at residue 973 displayed identical phenotypes in both mammalian cells and oocytes in that they were defective in their ability to release intracellular calcium, undergo ligand induced internalization and receptor downregulation. However, these receptors were fully capable of inducing oocyte maturation. The remarkable retention of specific biological activities of different hEGF-R in the context of oocytes suggests that this receptor system interacts with generally available cellular components that have been conserved during evolution. In addition, it suggests that cell surface tyrosine kinase activity may play an important role in regulating resumption of the cell cycle.     

Protein kinase C and phorbol ester receptor expression related to growth and differentiation of nullipotent and pluripotent embryonal carcinoma cells

We have characterized effects of phorbol, 12-myristate 13 acetate (PMA) on growth and differentiation in a nullipotent embryonal carcinoma (EC) cell line, F9, in a pluripotent EC line, P19, and in the differentiated derivatives of these cells, In P19EC and F9EC PMA addition resulted in inhibition of growth, while in the differentiated derivates PMA was mitogenic. PMA did not induce differentiation in EC cells but potentiated the retinoic acid (RA) induced differentiation in P19EC, although, not in F9EC. Rapid morphological changes by PMA were seen in P19EC and two differentiated derivatives which represent different stages of differentiation. In F9 no rapid morphological changes were induced by PMA. Using [3H]phorbol dibutyrate as a ligand we showed that during differentiation into endoderm-like cells the number of phorbol ester receptors increases, while in epithelial-like derivatives no increase is found. In differentiated cells with an increased number of phorbol ester receptors, the cytoplasmic Ca2+- and phospholipid-dependent protein kinase (the putative receptor for phorbol esters) activity was also increased. Only in those derivatives where the number of phorbol ester receptors is increased, is the binding of epidermal growth factor (EGF) inhibited by PMA. These results suggest a relationship between levels of expression of phorbol ester receptors, cytoplasmic protein kinase C and biological effects, namely rapid morphological changes, altered growth, potentiation of RA induced differentiation, and inhibition of EGF binding.     

The Na+/H+ exchanger is constitutively activated in P19 embryonal carcinoma cells, but not in a differentiated derivative. Responsiveness to growth factors and other stimuli

We have examined the functional properties and growth factor responsiveness of the plasma membrane Na+/H+ exchanger in pluripotent P19 embryonal carcinoma (EC) cells and in a differentiated mesodermal derivative (MES-1) by analyzing the recovery of cytoplasmic pH (pHi) from an acute acid load under bicarbonate-free conditions. In the absence of exogenous growth factors, the mean steady-state pHi of undifferentiated P19 cells (7.49 +/- 0.03) is 0.55 unit higher than the value of differentiated MES-1 cells (6.94 +/- 0.01). In both cell types, recovery of pHi from an NH+4-induced acid load follows an exponential time course and is entirely mediated by the amiloride-sensitive Na+/H+ exchanger in the plasma membrane. Kinetic analysis indicates that the higher steady-state pHi in P19 EC cells is due to an alkaline shift in the pHi sensitivity of the Na+/H+ exchange rate, as compared to that in MES-1 cells. The Na+/H+ exchanger of MES-1 cells is responsive to epidermal growth factor, platelet-derived growth factor, serum, phorbol esters, and diacylglycerol, as shown by a rapid amiloride-sensitive rise in pHi of 0.15-0.35 unit. This mitogen-induced alkalinization is attributable to an alteration in the pHi sensitivity of the exchanger. In contrast, the Na+/H+ exchanger of P19 EC cells fails to respond to any of these stimuli. Similarly, hypertonic medium rapidly activates the Na+/H+ exchanger in MES-1, but not in P19 EC cells. We conclude that the Na+/H+ exchanger in undifferentiated P19 EC stem cells is maintained in a fully activated state which is unaffected by extracellular stimuli, as if signal pathways normally involved in growth factor action are constitutively operative.     

Extrinsic factors derived from mouse embryonal carcinoma cell lines maintain pluripotency of mouse embryonic stem cells through a novel signal pathway

Embryonic carcinoma (EC) cells, which are malignant stem cells of teratocarcinoma, have numerous morphological and biochemical properties in common with pluripotent stem cells such as embryonic stem (ES) cells. However, three EC cell lines (F9, P19 and PCC3) show different developmental potential and self‐renewal capacity from those of ES cells. All three EC cell lines maintain self‐renewal capacity in serum containing medium without Leukemia Inhibitory factor (LIF) or feeder layer, and show limited differentiation capacity into restricted lineage and cell types. To reveal the underlying mechanism of these characteristics, we took the approach of characterizing extrinsic factors derived from EC cells on the self‐renewal capacity and pluripotency of mouse ES cells. Here we demonstrate that EC cell lines F9 and P19 produce factor(s) maintaining the undifferentiated state of mouse ES cells via an unidentified signal pathway, while P19 and PCC3 cells produce self‐renewal factors of ES cells other than LIF that were able to activate the STAT3 signal; however, inhibition of STAT3 activation with Janus kinase inhibitor shows only partial impairment on the maintenance of the undifferentiated state of ES cells. Thus, these factors present in EC cells‐derived conditioned medium may be responsible for the self‐renewal capacity of EC and ES cells independently of LIF signaling.     

Actions of epidermal growth factor receptor/mitogen-activated protein kinase and protein kinase C signaling in granulosa cells from Gallus gallus are dependent upon stage of differentiation

Studies in both mammalian and nonmammalian ovarian model systems have demonstrated that activation of the mitogen-activated protein kinase (MAPK) and protein kinase C (PKC) signaling pathways modulates steroid biosynthesis during follicle development, yet the collective evidence for facilitory versus inhibitory roles of these pathways is inconsistent. The present studies in the hen ovary describe the changing role of MAPK and PKC signaling in the regulation of steroidogenic acute regulatory protein (STAR) expression and progesterone production in undifferentiated granulosa cells collected from prehierarchal follicles prior to follicle selection versus differentiated granulosa from preovulatory follicles subsequent to selection. Treatment of undifferentiated granulosa cells with a selective epidermal growth factor receptor (EGFR) and ERBB4 receptor tyrosine kinase inhibitor (AG1478) both augments FSH receptor (Fshr) mRNA expression and initiates progesterone production. Conversely, selective inhibitors of both EGFR/ERBB4 and MAPK activity attenuate steroidogenesis in differentiated granulosa cells subsequent to follicle selection. In addition, inhibition of PKC signaling with GF109203X augments FSH-induced Fshr mRNA plus STAR protein expression and initiates progesterone synthesis in undifferentiated granulosa cells, but inhibits both gonadotropin-induced STAR expression and progesterone production in differentiated granulosa. Granulosa cells from the most recently selected (9- to 12-mm) follicle represent a stage of transition as inhibition of MAPK signaling promotes, while inhibition of PKC signaling blocks gonadotropin-induced progesterone production. Collectively, these data describe stage-of-development-related changes in cell signaling whereby the differentiation-inhibiting actions of MAPK and PKC signaling in prehierarchal follicle granulosa cells undergo a transition at the time of follicle selection to become obligatory for gonadotropin-stimulated progesterone production in differentiated granulosa from preovulatory follicles.     

Receptor protein tyrosine phosphatase alpha activates pp60c-src and is involved in neuronal differentiation.

Here we report that protein tyrosine phosphatases (PTPases), like their enzymatic counterpart the protein tyrosine kinases, can play an important role in cell differentiation. Expression of the transmembrane PTPase receptor protein tyrosine phosphatase alpha (RPTP alpha) is transiently enhanced during neuronal differentiation of embryonal carcinoma (EC) and neuroblastoma cells. Retinoic acid induces wild type P19 cells to differentiate into endoderm- and mesoderm-like cells. By contrast, retinoic acid treatment leads to neuronal differentiation of P19 cells, ectopically expressing functional RPTP alpha, as illustrated by their ability to generate action potentials. Endogenous pp60c-src kinase activity is enhanced in the RPTP alpha-transfected cells, which may be due to direct dephosphorylation of the regulatory Tyr residue at position 527 in pp60c-src by RPTP alpha. Our results demonstrate that RPTP alpha is involved in neuronal differentiation and imply a role for pp60c-src in the differentiation process.     

Tyrosine kinase-regulated and inositol phosphate-independent Ca2+ elevation and mobilization in T cells.

Perturbation of the T cell antigen-specific receptor leads to a series of signaling events that includes a rapid increase in phosphoinositide hydrolysis, intracellular Ca2+, and tyrosine phosphorylation. We have examined the function of tyrosine phosphorylation in isolation by introducing the v-src tyrosine kinase into a T cell hybridoma. T cell receptor-mediated increases in phosphoinositide hydrolysis and, in particular the generation of inositol 1,4,5-trisphosphate, were comparable between v-src+ and v-src- cells. Unexpectedly, the v-src+ cells exhibited spontaneously elevated intracellular Ca2+ and exaggerated Ca2+ increases when stimulated via the T cell receptor. The enhanced Ca2+ response was not due to tyrosine phosphorylation of the T cell receptor itself, since the phenotype was evident in T cell receptor zeta chain-/v-src+ cells as well. These results demonstrate that an active protein tyrosine kinase can markedly affect intracellular Ca2+ handling by a process independent of inositol 1,4,5-trisphosphate production and T cell receptor tyrosine phosphorylation and raise the possibility that tyrosine kinases may directly regulate T cell receptor-mediated changes in intracellular Ca2+.     

Cell-type-specific roles of IGF-1R and EGFR in mediating Zn2+-induced ERK1/2 and PKB phosphorylation

Zn²⁺ exerts insulin-mimetic and antidiabetic effects in rodent models of insulin resistance, and activates extracellular-signal-regulated kinases 1 and 2 (ERK1/2) and protein kinase B (PKB), key components of the insulin signaling pathway. Zn²⁺-induced signaling has been shown to be associated with an increase in the tyrosine phosphorylation of insulin receptor (IR), as well as of insulin-like growth factor 1 receptor (IGF-1R) and epidermal growth factor receptor (EGFR) in several cell types. However, the specific contribution of these receptor protein tyrosine kinases (R-PTKs) in mediating Zn²⁺-induced responses in a cell-specific fashion remains to be established. Therefore, using a series of pharmacological inhibitors and genetically engineered cells, we have investigated the roles of various R-PTKs in Zn²⁺-induced ERK1/2 and PKB phosphorylation. Pretreatment of Chinese hamster ovary (CHO) cells overexpressing a human IR (CHO-HIR cells) with AG1024, an inhibitor for IR protein tyrosine kinase (PTK) and IGF-1R-PTK, blocked Zn²⁺-induced ERK1/2 and PKB phosphorylation, but AG1478, an inhibitor for EGFR, was without effect in CHO cells. On the other hand, both of these inhibitors were able to attenuate Zn²⁺-induced phosphorylation of ERK1/2 and PKB in A10 vascular smooth muscle cells. In addition, in CHO cells overexpressing tyrosine kinase deficient IR, Zn²⁺ was still able to induce the phosphorylation of these two signaling molecules, whereas the insulin effect was significantly attenuated. Furthermore, both Zn²⁺ and insulin-like growth factor 1 failed to stimulate ERK1/2 and PKB phosphorylation in IGF-1R knockout cells. Also, Zn²⁺-induced responses in CHO-HIR cells were not associated with an increase in the tyrosine phosphorylation of the IR β-subunit and insulin receptor substrate 1 in CHO-HIR cells. Taken together, these data suggest that distinct R-PTKs mediate Zn²⁺-evoked ERK1/2 and PKB phosphorylation in a cell-specific manner.     

Sphingosine 1-phosphate activates Erk-1/-2 by transactivating epidermal growth factor receptor in rat-2 cells

We investigated a signaling pathway leading to activation of extracellular signal-regulated protein kinase (Erk) 1 and 2 in Rat-2 cells stimulated with sphingosine 1-phosphate (S1P). S1P treatment transiently activated Erk-1/-2 in a dose-dependent manner, and its activation was blocked by pertussis toxin, expression of RasN17, or inhibition of Raf or MEK-1/-2. S1P-induced activation of Erk-1/-2 was also suppressed by the inhibition of epidermal growth factor (EGF) receptor tyrosine kinase with the specific inhibitor AG1478, suggesting that activation of EGF receptor tyrosine kinase was involved in the signaling pathway. S1P-induced Erk-1/-2 activation was enhanced up to 2-fold by inhibiting protein kinase C (PKC) with GF109203X, and PKC inhibition in the absence of S1P treatment also activated Erk-1/-2. The stimulatory effects of Erk-1/-2 activation by PKC inhibition was blocked by treating cells with AG1478, suggesting the involvement of PKC in the regulation of EGF receptor tyrosine kinase activation that leads to Erk-1/-2 activation. Together, these results suggest that S1P activates the EGF receptor through a PKC-dependent pathway that links Ras signaling to the activation of Erk-1/-2 in Rat-2 cells.     

An embryonic DNA-binding protein specific for a region of the human IFN beta 1 promoter.

Embryonal carcinoma (EC) cells are unable to make interferon in response to inducing agents. This block disappears after differentiation. We have found that nuclear extracts from undifferentiated P19 EC cells contain a DNA-binding activity which specifically recognizes a region within the human interferon-beta 1 promoter. This activity is absent from differentiated cell types, both of EC and non-EC origin. The binding of the factor in undifferentiated EC cells leads to dramatic changes in the overall protein binding pattern of the interferon promoter as compared with differentiated cells, and may be responsible for repression of the endogenous interferon-beta gene prior to differentiation.