P19 Embryonal Cells Research Articles

Effects of lamellar microstructure of retinoic acid loaded-matrixes on physicochemical properties, migration, and neural differentiation of P19 embryonic carcinoma cells

Abstract In this study, retinoic acid loaded-PLGA-gelatin matrixes were prepared with both freeze-casting and freeze-drying techniques. Herein, the effect of unidirectional microstructure with tunable pores on release profile, cellular adhesion, migration, and differentiation was compared. Morphological observation determined that highly interconnected porous structure can be formed, but lamellar pore channels were observed in freeze-casting prepared constructs. The absorption ratio was increased, and the biodegradation rate was decreased as a function of the orientation of microstructure. The in-vitro release study illustrated non-Fickian release mechanism in both methods, so that erosion has predominated over diffusion. Accordingly, PLGA-gelatin scaffolds prepared with freeze-drying technique showed no adequate erosion due to the rigid structure, while freeze-casting one presented more favorable erosion. Microscopic observations of adhered P19 embryonic cells on the scaffolds showed that the freeze-casting matrixes with unidirectional pores provide a more compatible microenvironment for cell attachments and spreading. Besides, it facilitated cell migration and penetration inside the structure and may act as guidance for neuron growth. Improvement in the expression of neural genes in unidirectionally oriented pores proved the decisive role of contact guidance for nerve healing. It seems that the freeze-cast PLGA-gelatin-retinoic acid scaffolds have initial features for nerve tissue regeneration studies.

The two splice variant forms of Cdc42 exert distinct and essential functions in neurogenesis

The small GTPase cell division cycle 42 (CDC42) plays essential roles in neurogenesis and brain development. Previously, using murine embryonic P19 cells as a model system, we showed that CDC42 stimulates mTOR complex 1 (mTORC1) activity and thereby up-regulates transcription factors required for the formation of neural progenitor cells. However, paradoxically, although endogenous CDC42 is required for both the initial transition of undifferentiated P19 cells to neural progenitors and their ultimate terminal differentiation into neurons, ectopic CDC42 overexpression promotes only the first stage of neurogenesis (i.e. the formation of neuroprogenitors) and not the second phase (differentiation into neurons). Here, using both P19 cells and mouse embryonic stem cells, we resolve this paradox, demonstrating that two splice variants of CDC42, differing only in nine amino acid residues in their very C-terminal regions, play distinct roles in neurogenesis. We found that a CDC42 splice variant that has a ubiquitous tissue distribution, termed here as CDC42u, specifically drives the formation of neuroprogenitor cells, whereas a brain-specific CDC42 variant, CDC42b, is essential for promoting the transition of neuroprogenitor cells to neurons. We further show that the specific roles of CDC42u and CDC42b in neurogenesis are due to their opposing effects on mTORC1 activity. Specifically, CDC42u stimulated mTORC1 activity and thereby induced neuroprogenitor formation, whereas CDC42b worked together with activated CDC42-associated kinase (ACK) in down-regulating mTOR expression and promoting neuronal differentiation. These findings highlight the remarkable functional specificities of two highly similar CDC42 splice variants in regulating distinct stages of neurogenesis.

LncRNA-uc.40 silence promotes P19 embryonic cells differentiation to cardiomyocyte via the PBX1 gene.

Uc.40 is a long noncoding RNA that is highly conserved among different species, although its function is unknown. It is highly expressed in abnormal human embryonic heart. We previously reported that overexpression of uc.40 promoted apoptosis and inhibited proliferation of P19 cells, and downregulated PBX1, which was identified as a potential target gene of uc.40. The current study evaluated the effects of uc40-siRNA-44 (siRNA against uc.40) on the differentiation, proliferation, apoptosis, and mitochondrial function in P19 cells, and investigated the relationship between uc.40 and PBX1 in cardiomyocytes. The uc.40 silencing expression was confirmed by quantitative real-time polymerase chain reaction (RT-PCR). Observation of morphological changes in transfected P19 cells during different stages of differentiation revealed that uc40-siRNA-44 increased the number of cardiomyocyes. There was no significant difference in the morphology or time of differentiation between the uc40-siRNA-44 group and the control group. uc40-siRNA-44 significantly promoted proliferation of P19 cells and inhibited serum starvation-induced apoptosis. There was no significant difference in mitochondrial DNA copy number or cellular ATP level between the two groups, and ROS levels were significantly decreased in uc40-siRNA-44-transfected cells. The levels of PBX1 and myocardial markers of differentiation were examined in transfected P19 cells; uc40-siRNA-44 downregulated myocardial markers and upregulated PBX1 expression. These results suggest that uc.40 may play an important role during the differentiation of P19 cells by regulation of PBX1 to promote proliferation and inhibit apoptosis. These studies provide a foundation for further study of uc.40/PBX1 in cardiac development.

Bilobalide induces neuronal differentiation of P19 embryonic carcinoma cells via activating Wnt/β-catenin pathway.

Bilobalide, a natural product extracted from Ginkgo biloba leaf, is known to exhibit a number of pharmacological activities. So far, whether it could affect embryonic stem cell differentiation is still unknown. The main aim of this study was to investigate the effect of bilobalide on P19 embryonic carcinoma cells differentiation and the underlying mechanisms. Our results showed that bilobalide induced P19 cells differentiation into neurons in a concentration- and time-dependent manner. We also found that bilobalide promoted neuronal differentiation through activation of Wnt/β-catenin signaling pathway. Exposure to bilobalide increased inactive GSK-3β phosphorylation, further induced the nuclear accumulation of β-catenin, and also up-regulated the expression of Wnt ligands Wnt1 and Wnt7a. Neuronal differentiation induced by bilobalide was totally abolished by XAV939, an inhibitor of Wnt/β-catenin pathway. These results revealed a novel role of bilobalide in neuronal differentiation from P19 embryonic cells acting through Wnt/β-catenin signaling pathway, which would provide a better insight into the beneficial effects of bilobalide in brain diseases.

P2X receptors in maintenance and differentiation of neural progenitor cells.

P2X receptors in maintenance and differentiation of neural progenitor cells.

Directed differentiation of embryonic P19 cells and neural stem cells into neural lineage on conducting PEDOT–PEG and ITO glass substrates

Differentiation of pluripotent and lineage restricted stem cells such as neural stem cells (NSCs) was studied on conducting substrates of various nature without perturbation of the genome with exogenous genetic material or chemical stimuli. Primary mouse adult neural stem cells (NSCs) and P19 pluripotent embryonal (P19 EC) carcinoma cells were used. Expression levels of neuronal markers β-III-tubulin and neurofilament were evaluated by immunochemistry and flow cytometry. It was shown that the ability of the substrate to induce differentiation directly correlated with its conductivity. Conducting substrates (conducting oxides or doped π-conjugated organic polymers) with different morphology, structure, and conductivity mechanisms all promoted differentiation of NSC and P19 cells into neuronal lineage to a similar degree without use of additional factors such as poly-l-ornithine coating or retinoic acid, as verified by their morphology and upregulation of the neuronal markers but not astrocyte marker GFAP. However, substrates with low conductance below ca. 10−4Scm−2 did not show this ability. Morphology of differentiating cells was visualized by atomic force microscopy. NSCs cells increased β-III-tubulin expression by 95% and P19 cells by over 30%. Our results suggest that the substrate conductivity is a key factor governing the cell fate. Differentiation of P19 cells into neuronal lineage on conducting substrates was attributed to downregualtion of Akt signaling pathway and increase in expression of dual oxidase 1 (DUOX 1).

CCN2/CTGF silencing blocks cell aggregation in embryonal carcinoma P19 cell

Connective tissue growth factor (CCN2/CTGF) is a matricellular-secreted protein involved in extracellular matrix remodeling. The P19 cell line is an embryonic carcinoma line widely used as a cellular model for differentiation and migration studies. In the present study, we employed an exogenous source of CCN2 and small interference RNA to address the role of CCN2 in the P19 cell aggregation phenomenon. Our data showed that increasing CCN2 protein concentrations from 0.1 to 20 nM decreased the number of cell clusters and dramatically increased cluster size without changing proliferation or cell survival, suggesting that CCN2 induced aggregation. In addition, CCN2 specific silencing inhibited typical P19 cell aggregation, which could be partially rescued by 20 nM CCN2. The present study demonstrates that CCN2 is a key molecule for cell aggregation of embryonic P19 cells.

Oxytocin-Gly-Lys-Arg: A Novel Cardiomyogenic Peptide

BackgroundOxytocin (OT), synthesized in the heart, has the ability to heal injured hearts and to promote cardiomyogenesis from stem cells. Recently, we reported that the OT-GKR molecule, a processing intermediate of OT, potently increased the spontaneous formation of cardiomyocytes (CM) in embryonic stem D3 cells and augmented glucose uptake in newborn rat CM above the level stimulated by OT. In the present experiments, we investigated whether OT-GKR exists in fetal and newborn rodent hearts, interacts with the OT receptors (OTR) and primes the generation of contracting cells expressing CM markers in P19 cells, a model for the study of early heart differentiation.Methodology/Principal FindingsHigh performance liquid chromatography of newborn rat heart extracts indicated that OT-GKR was a dominant form of OT. Immunocytochemistry of mouse embryos (embryonic day 15) showed cardiac OT-GKR accumulation and OTR expression. Computerized molecular modeling revealed OT-GKR docking to active OTR sites and to V1a receptor of vasopressin. In embryonic P19 cells, OT-GKR induced contracting cell colonies and ventricular CM markers more potently than OT, an effect being suppressed by OT antagonists and OTR-specific small interfering (si) RNA. The V1a receptor antagonist and specific si-RNA also significantly reduced OT-GKR-stimulated P19 contracting cells. In comparison to OT, OT-GKR induced in P19 cells less α-actinin, myogenin and MyoD mRNA, skeletal muscle markers.Conclusions/SignificanceThese results raise the possibility that C-terminally extended OT molecules stimulate CM differentiation and contribute to heart growth during fetal life.

Neuronal Differentiation of P19 Embryonal Cells in Polyhydroxybutyrate Scaffold as a Model for Nerve Tissue Engineering

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Pharmacological properties of purinergic receptors and their effects on proliferation and induction of neuronal differentiation of P19 embryonal carcinoma cells

We have used P19 embryonal carcinoma cells as in vitro model for early neurogenesis to study ionotropic P2X and metabotropic P2Y receptor-induced Ca2+ transients and their participation in induction of proliferation and differentiation. In embryonic P19 cells, P2Y1, P2Y2 and P2X4 receptors or P2X-heteromultimers with similar P2X4 pharmacology were responsible for ATP and ATP analogue-induced Ca2+ transients. In neuronal-differentiated cells, P2Y2, P2Y6, P2X2 and possibly P2X2/P2X6 heteromeric receptors were the major mediators of the elevations in intracellular free calcium concentration [Ca2+]i. We have collected evidence for the involvement of metabotropic purinergic receptors in proliferation induction of undifferentiated and neural progenitor cells by using a BrdU-incorporation assay. ATP-, UTP-, ADP-, 2-MeS-ATP- and ADP-βS-induced proliferation in P19 cells was mediated by P2Y1 and P2Y2 receptors as judged from pharmacological profiles of receptor responses. ATP-provoked acceleration of neuronal differentiation, determined by analysis of nestin and neuron-specific enolase gene and protein expression, also resulted from P2Y1 and P2Y2 receptor activation. Proliferation- and differentiation-induction involved the activation of inositol-trisphosphate sensitive intracellular Ca2+ stores.

HP1α guides neuronal fate by timing E2F-targeted genes silencing during terminal differentiation

A critical step of neuronal terminal differentiation is the permanent withdrawal from the cell cycle that requires the silencing of genes that drive mitosis. Here, we describe that the alpha isoform of the heterochromatin protein 1 (HP1) protein family exerts such silencing on several E2F-targeted genes. Among the different isoforms, HP1alpha levels progressively increase throughout differentiation and take over HP1gamma binding on E2F sites in mature neurons. When overexpressed, only HP1alpha is able to ensure a timed repression of E2F genes. Specific inhibition of HP1alpha expression drives neuronal progenitors either towards death or cell cycle progression, yet preventing the expression of the neuronal marker microtubule-associated protein 2. Furthermore, we provide evidence that this mechanism occurs in cerebellar granule neurons in vivo, during the postnatal development of the cerebellum. Finally, our results suggest that E2F-targeted genes are packaged into higher-order chromatin structures in mature neurons relative to neuroblasts, likely reflecting a transition from a 'repressed' versus 'silenced' status of these genes. Together, these data present new epigenetic regulations orchestrated by HP1 isoforms, critical for permanent cell cycle exit during neuronal differentiation.

Reelin signals survival through Src‐family kinases that inactivate BAD activity

Reelin plays an important role in the migration of embryonic neurons, but its continuing presence suggests additional functions in the brain. We now report a novel function where reelin protects P19 embryonal cells from apoptosis during retinoic acid-induced neuronal differentiation. This increased survival is associated with reelin activation of the phosphatidyl-inositol-3-kinase (PI3 K)/Akt pathway. When PI3 K was inhibited with LY294002, reelin failed to protect against this retinoic acid-induced apoptosis. The protective effect of reelin includes activating the Src-family kinases/PI3 K/Akt pathway which then led to selective phosphorylation of Bcl-2/Bcl-XL associated death promoter (BAD) at serine-136, while the phosphorylation-incompetent mutation of BAD (S136A) suppressed this protection. These and additional studies define a novel pathway where reelin binds apoE receptors, significantly activates the PI3 K/Akt pathway causing phosphorylation of BAD which helps to protect cells from apoptosing, thus serving an important role in promoting the survival of maturing neurons in the brain.

P19 embryonal carcinoma cells as in vitro model for studying purinergic receptor expression and modulation of N-methyl- d-aspartate–glutamate and acetylcholine receptors during neuronal differentiation

The in vitro differentiation of P19 murine embryonal carcinoma cells to neurons resembles developmental stages which are encountered during neuronal development. Three days following induction to neuronal differentiation by retinoic acid, most cells of the P19 population lost expression of the stage specific embryonic antigen (SSEA-1) and expressed the neural progenitor cell specific antigen nestin. Beginning from day 4 of differentiation nestin expression was down-regulated, and expression of neuron-specific enolase as marker of differentiated neurons increased. The molecular mechanisms underlying neuronal differentiation are poorly understood. We have characterized the participation of purinergic ionotropic (P2X) and metabotropic (P2Y) receptors at mRNA transcription and protein levels as well as ATP-induced Ca 2+ transients during neuronal differentiation of P19 cells. Gene and protein expression of P2X 2, P2X 6, P2Y 2, and P2Y 6 receptors increased during the course of differentiation, whereas P2X 3, P2X 4, P2Y 1 and P2Y 4 receptor expression was high in embryonic P19 cells and then decreased following induction of P19 cells to differentiation. P2X 1 receptor protein expression was only detected on days 2 and 4 of differentiation. Although P2X 5 and P2X 7 mRNA transcription was present, no protein expression for this receptor subunit could be detected throughout the differentiation process. In undifferentiated cells, mainly ionotropic P2X receptors contributed to the ATP-induced Ca 2+-response. In neuronal-differentiated P19 cells, the ATP-induced Ca 2+-response was increased and the metabotropic component predominated. Purinergic receptor function is implicated to participate in neuronal maturation, as cholinergic and glutamate– N-methyl- d-aspartate (NMDA) induced calcium responses were affected when cells were differentiated in the presence of purinergic receptor antagonists pyridoxalphosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS), suramin or reactive blue-2. Our data suggest that inhibition of P2Y 1 and possibly P2X 2 receptors led to a loss of NMDA receptor activity whereas blockade of possibly P2X 2 and P2Y 2 purinergic receptors during neuronal differentiation of P19 mouse led to inhibition of cholinergic receptor responses.

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.

SAGE identification of differentiation responsive genes in P19 embryonic cells induced to form cardiomyocytes in vitro

Transcriptome profiling facilitates the identification of developmentally regulated genes. To quantify the functionally active genome of P19 embryonic carcinoma (EC) cells induced to form cardiomyocytes, we employed serial analysis of gene expression (SAGE) to sequence and compare a total of 171,735 SAGE tags from three libraries (undifferentiated P19 EC cells, differentiation days 3+0.5 and 3+3.0). After in vitro differentiation, only 3.1% of the gene products demonstrated significant ( P<0.05) changes in expression. The most highly significant changes ( P<0.01) involved altered expression of 410 genes encoding predominantly transcription factors, differentiation factors and growth regulators. Quantitative polymerase chain reaction analysis and in situ hybridization revealed five growth regulators (Dlk1, Igfbp5, Hmga2, Podxl and Ptn) and two unknown ESTs with expression profiles similar to known cardiac transcription factors, implicating these growth regulators in cardiac differentiation. These SAGE libraries thus serve as a reference resource for understanding the role of differentiation-dependent genes in embryonic stem cell models induced to form cardiomyocytes in vitro.

YY1 activates Msx2 gene independent of bone morphogenetic protein signaling

Msx2 is a homeobox gene expressed in multiple embryonic tissues which functions as a key mediator of numerous developmental processes. YY1 is a bi-functional zinc finger protein that serves as a repressor or activator to a variety of promoters. The role of YY1 during embryogenesis remains unknown. In this study, we report that Msx2 is regulated by YY1 through protein-DNA interactions. During embryogenesis, the expression pattern of YY1 was observed to overlap in part with that of Msx2. Most notably, during first branchial arch and limb development, both YY1 and Msx2 were highly expressed, and their patterns were complementary. To test the hypothesis that YY1 regulates Msx2 gene expression, P19 embryonal cells were used in a number of expression and binding assays. We discovered that, in these cells, YY1 activated endogenous Msx2 gene expression as well as Msx2 promoter-luciferase fusion gene activity. These biological activities were dependent on both the DNA binding and activation domains of YY1. In addition, YY1 bound specifically to three YY1 binding sites on the proximal promoter of Msx2 that accounted for this transactivation. Mutations introduced to these sites reduced the level of YY1 transactivation. As bone morphogenetic protein type 4 (BMP4) regulates Msx2 expression in embryonic tissues and in P19 cells, we further tested whether YY1 is the mediator of this BMP4 activity. BMP4 did not induce the expression of YY1 in early mouse mandibular explants, nor in P19 cells, suggesting that YY1 is not a required mediator of the BMP4 pathway in these tissues at this developmental stage. Taken together, these findings suggest that YY1 functions as an activator for the Msx2 gene, and that this regulation, which is independent of the BMP4 pathway, may be required during early mouse craniofacial and limb morphogenesis.

Ajuba, a cytosolic LIM protein, shuttles into the nucleus and affects embryonal cell proliferation and fate decisions.

Cellular adhesive events affect cell proliferation and differentiation decisions. How cell surface events mediating adhesion transduce signals to the nucleus is not well understood. After cell-cell or cell-substratum contact, cytosolic proteins are recruited to clustered adhesion receptor complexes. One such family of cytosolic proteins found at sites of cell adhesion is the Zyxin family of LIM proteins. Here we demonstrate that the family member Ajuba was recruited to the cell surface of embryonal cells, upon aggregate formation, at sites of cell-cell contact. Ajuba contained a functional nuclear export signal and shuttled into the nucleus. Importantly, accumulation of the LIM domains of Ajuba in the nucleus of P19 embryonal cells resulted in growth inhibition and spontaneous endodermal differentiation. The differentiating effect of Ajuba mapped to the third LIM domain, whereas regulation of proliferation mapped to the first and second LIM domains. Ajuba-induced endodermal differentiation of these cells correlated with the capacity to activate c-Jun kinase and required c-Jun kinase activation. These results suggest that the cytosolic LIM protein Ajuba may provide a new mechanism to transduce signals from sites of cell adhesion to the nucleus, regulating cell growth and differentiation decisions during early development.

Segmental expression of Hoxb2 in r4 requires two separate sites that integrate cooperative interactions between Prep1, Pbx and Hox proteins.

Direct auto- and cross-regulatory interactions between Hox genes serve to establish and maintain segmentally restricted patterns in the developing hindbrain. Rhombomere r4-specific expression of both Hoxb1 and Hoxb2 depends upon bipartite cis Hox response elements for the group 1 paralogous proteins, Hoxal and Hoxbl. The DNA-binding ability and selectivity of these proteins depend upon the formation of specific heterodimeric complexes with members of the PBC homeodomain protein family (Pbx genes). The r4 enhancers from Hoxb1 and Hoxb2 have the same activity, but differ with respect to the number and organisation of bipartite Pbx/Hox (PH) sites required, suggesting the intervention of other components/sequences. We report here that another family of homeodomain proteins (TALE, Three-Amino acids-Loop-Extension: Prep1, Meis, HTH), capable of dimerizing with Pbx/EXD, is involved in the mechanisms of r4-restricted expression. We show that: (1) the r4-specific Hoxb1 and Hoxb2 enhancers are complex elements containing separate PH and Prep/Meis (PM) sites; (2) the PM site of the Hoxb2, but not Hoxb1, enhancer is essential in vivo for r4 expression and also influences other sites of expression; (3) both PM and PH sites are required for in vitro binding of Prepl-Pbx and formation and binding of a ternary Hoxbl-Pbxla (or 1b)-Prepl complex. (4) A similar ternary association forms in nuclear extracts from embryonal P19 cells, but only upon retinoic acid induction. This requires synthesis of Hoxbl and also contains Pbx with either Prepl or Meisl. Together these findings highlight the fact that PM sites are found in close proximity to bipartite PH motifs in several Hox responsive elements shown to be important in vivo and that such sites play an essential role in potentiating regulatory activity in combination with the PH motifs.

Differential Expression of Calcitonin and Parathyroid Hormone/Parathyroid Hormone-Related Protein Receptors in P19 Embryonic Carcinoma Cells Treated with Retinoic Acid*

Mouse embryonic carcinoma P19 cell aggregates treated with retinoic acid (RA) sequentially differentiate into neurons and astrocytes, whereas attached cells develop a mesodermal phenotype. The expression of calcitonin (CT) and PTH/PTH-related protein (PTHrP) receptors was investigated in embryonic cells, and during neural and mesodermal differentiation. In embryonic P19 cells, specific binding of [125I]salmon (s) CT(1-32) ([125I]sCT(1-32)) was 56 fmol/mg protein, and of [125I]chicken (ch) [Tyr36]PTHrP(1-36) amide ([125I]chPTHrP(1-36)) < 0.5 fmol/mg protein. Correspondingly, cAMP was maximally stimulated 47-fold by sCT(1-32) (EC50 0.05 nM) and 3-fold by chPTHrP(1-36) (EC50 1.3 nM). Receptor autoradiography revealed specific binding of [125I]sCT(1-32) to the undifferentiated P19 cells, but not to RA induced neurons and astrocytes. At the same time, [125I]sCT(1-32) binding and cAMP accumulation by sCT were gradually decreased. But, specific binding of [125I]chPTHrP(1-36) was raised at least 6-fold compared with embryonic cells to 3 fmol/mg protein, in parallel with a 10-fold higher maximal cAMP accumulation. A similar, but delayed suppression of CT and stimulation of PTH/PTHrP receptor expression was observed during mesodermal cell differentiation. The results indicate that CT receptors are associated with undifferentiated P19 cells, whereas PTH/PTHrP receptors are expressed in RA induced neural and mesodermal cells.

Differential Expression of Calcitonin and Parathyroid Hormone/Parathyroid Hormone-Related Protein Receptors in P19 Embryonic Carcinoma Cells Treated with Retinoic Acid

Mouse embryonic carcinoma P19 cell aggregates treated with retinoic acid (RA) sequentially differentiate into neurons and astrocytes, whereas attached cells develop a mesodermal phenotype. The expression of calcitonin (CT) and PTH/PTH-related protein (PTHrP) receptors was investigated in embryonic cells, and during neural and mesodermal differentiation. In embryonic P19 cells, specific binding of[ 125I]salmon (s) CT(1–32) ([125I]sCT(1–32)) was 56 fmol/mg protein, and of[ 125I]chicken (ch) [Tyr36]PTHrP(1–36) amide ([125I]-chPTHrP(1–36)) < 0.5 fmol/mg protein. Correspondingly, cAMP was maximally stimulated 47-fold by sCT(1–32) (EC50 0.05 nm) and 3-fold by chPTHrP(1–36) (EC50 1.3 nm). Receptor autoradiography revealed specific binding of [125I]sCT(1–32) to the undifferentiated P19 cells, but not to RA induced neurons and astrocytes. At the same time, [125I]sCT(1–32) binding and cAMP accumulation by sCT were gradually decreased. But, specific binding of [125I]chPTHrP(1–36) was raised at least 6-fold compared with embryonic cells to 3 fmol/mg protein, in parallel with a 10-fold higher maximal cAMP accumulation. A similar, but delayed suppression of CT and stimulation of PTH/PTHrP receptor expression was observed during mesodermal cell differentiation. The results indicate that CT receptors are associated with undifferentiated P19 cells, whereas PTH/PTHrP receptors are expressed in RA induced neural and mesodermal cells.