Migration of F9 parietal endoderm cells is regulated by the ERK pathway

Cell migration is regulated by the action of many signaling pathways that are activated in specific regions of migrating cells. Extracellular regulated kinase 1/2 (ERK) signaling can modulate the migration of cells by controlling the turnover of focal adhesions and the dynamics of actin polymerization. Focal adhesion turnover is necessary for cell migration, and the formation of strong actin stress fibers and mature focal adhesions puts the brakes on cell migration. We used F9 wild-type and vinculin null (vin-/-) parietal endoderm (PE) outgrowth to study the role of the ERK signaling pathway in cell migration. Upon plating of F9 embryoid bodies (EBs) onto laminin-coated dishes, PE cells migrate away from the EBs, providing an in vitro model for studying directed migration of this embryonic cell type. Our results suggest that the ERK pathway regulates PE cell migration by affecting the formation of focal adhesions and lamellipodia through the action of myosin light chain kinase (MLCK).     

Differential expression of fetomodulin and tissue plasminogen activator to characterize parietal endoderm differentiation of F9 embryonal carcinoma cells.

Fetomodulin is a surface marker protein of differentiated F9 embryonal carcinoma cells. Gene cloning has recently identified it as thrombomodulin which binds thrombin and proteolytically activates protein C. Activity assays and RNA blotting were adopted to analyze F9 cell differentiation with specific reference to another well-characterized marker, tissue plasminogen activator. Retinoic acid induced primitive endoderm differentiation of F9 cells and simultaneously activated tissue plasminogen activator synthesis. This differentiation, however, did not result in fetomodulin expression. When primitive endoderm cells were exposed to 1 mM dibutyryl cyclic AMP, the tissue plasminogen activator level rose further within 6 hr. In contrast, the cofactor activity of fetomodulin stayed below a detectable level for as long as 15 hr and then increased with time. Expression of the two marker proteins appeared to be regulated differently.     

Expression of c-fos in parietal endoderm, amnion and differentiating F9 teratocarcinoma cells

The expression of the cellular proto-oncogene, c-fos, in extra-embryonic tissues of the mouse was investigated using a v-fos DNA probe and an affinity-purified antiserum raised against a C-terminal synthetic peptide. At 13.5 days of development, parietal endoderm--a tissue not previously studied using these methods--was found to express c-fos RNA at a higher level than the amnion or placenta. The previously reported dramatic increase in c-fos RNA levels in extra-embryonic membranes during gestation was found to be confined to the amnion. The antipeptide serum specifically recovered proteins with Mr values of 46,000 and 39,000 from extracts of parietal endoderm and amnion cells labelled for 15 min with 35S-methionine. On sodium-dodecyl-sulphate/polyacrylamide gel electrophoresis these proteins co-migrated with proteins immunoprecipitated using serum from rats inoculated with FBJ-MuSV-transformed cells (tumour-bearing rat serum). Pulse-chasing and 32P-labelling experiments showed that the protein with an Mr of 46,000 was rapidly converted into higher-molecular-weight phosphorylated derivatives. F9 teratocarcinoma stem cells differentiated into parietal-endoderm-like cells in response to treatment with retinoic acid and dibutyryl cyclic AMP. However, this differentiation was not accompanied by any large transient increase in c-fos RNA expression.     

Neuron-like derivatives of cultured F9 embryonal carcinoma cells express characteristics of parietal endoderm cells

Murine F9 embryonal carcinoma cells exposed to retinoic acid and dibutyryl cyclic AMP gradually arborize and acquire a neuron-like morphology in monolayer culture. Whether F9 cells can be induced to differentiate into cells with features specific to neural cells is controversial. We analyzed the intermediate filament content and pericellular matrix proteins of F9 cells after exposing them to retinoic acid, dibutyryl cyclic AMP, and nerve growth factor. In long-term cultures, a great majority of the cells appeared neuron-like, but showed intra- and pericellular laminin and type IV collagen, and frequently cytokeratin filaments as well. Several monoclonal antibodies to neurofilaments did not react with these cells in immunofluorescence or immunoblotting, though they recognize either all or individual mouse neurofilament triplet proteins. Polyclonal antibodies to neurofilament proteins gave a diffuse, nonfibrillar, vinblastine-resistant fluorescence in the morphologically neuron-like cells, but in immunoblotting failed to reveal polypeptides compatible with neurofilament triplet proteins. In long-term cultures, most of the cells appeared to have partially or totally lost the intermediate filaments. This was confirmed with anti-IFA antibodies which normally react with all intermediate filament proteins. The F9-derived cells did not respond to nerve growth factor in any detectable way. We conclude that the morphologically neuron-like derivatives of F9 cells display characteristics of modified parietal endoderm-like cells and do not show unequivocal features of neural cells.     

CD9-alpha6beta1 interactions in migratory parietal endoderm cells

Tetraspanins modulate the function of a variety of membrane proteins, including integrin receptors. We show here that the tetraspanin CD9 preferentially coimmunoprecipitates with the alpha6beta1 integrin heterodimer in F9-derived parietal endoderm cells in comparison to F9 stem cells. We also show that CD9 function-blocking antibody inhibits parietal endoderm migration in an embryoid body outgrowth assay. In addition, both CD9 and alpha6beta1 colocalize with vinculin to apparent focal adhesion sites in parietal endoderm cells. The data presented here suggests a role for CD9 in localizing the integrin to the focal adhesion. In addition, the data suggest a role for CD9 in alpha6beta1 mediated migration of parietal endoderm.     

A role for p21 (WAF1) in the cAMP-dependent differentiation of F9 teratocarcinoma cells into parietal endoderm

Combined treatment of teratocarcinoma F9 cells with retinoic acid and dibutyryl-cAMP induces the differentiation into cells with a phenotype resembling parietal endoderm. We show that the levels of cyclin-dependent kinase inhibitor p21/WAF1/Cip1 (p21) protein and mRNA are dramatically elevated at the end of this differentiation, concomitantly with the appearance of p21 in the immunoprecipitated CDK2-cyclin E complex. The induction of differentiation markers could not be achieved by expression of ectopic p21 alone and still required treatment with differentiation agents. Clones of F9 cells transfected with sense or antisense p21 cDNA constructs revealed, upon differentiation, upregulated levels of mRNA for thrombomodulin, a parietal endoderm-specific marker, or increased fraction of cells in sub-G1 phase of the cell cycle, respectively. Consistent with this observation, whereas p21 was strictly nuclear in undifferentiated cells, a large proportion of differentiated cells had p21 localized also in the cytoplasm, a site associated with the antiapoptotic function of p21. Furthermore, p21 activated the thrombomodulin promoter in transient reporter assays and the p21 mutant defective in binding to cyclin E was equally efficient in activation. The promoter activity in differentiated cells was reduced by cotransfection of p21-specific siRNA or antisense cDNA. Coexpression of p21 increased the activity of the GAL-p300(1-1303) fusion protein on the GAL sites-containing TM promoter. This implies that p21 might act through a derepression of the p300 N-terminal-residing repression domain, thereby enhancing the p300 coactivator function. As differentiation of F9 cells into parietal endoderm-like cells requires the cAMP signaling, the results together suggest that the cyclin-dependent kinase inhibitor p21 may promote specifically this pathway in F9 cells.     

Early increase in histone H1(0) mRNA during differentiation of F9 cells to parietal endoderm.

We have isolated and characterized cDNA clones coding for the H1 histone subtype H1(0) in mouse teratocarcinoma cells. The mRNA is 2100 nt long and contains a coding sequence which is highly related to that of the human H1(0) gene. Using this cDNA as a probe, we have shown that, in comparison to undifferentiated F9 cells, differentiated F9 teratocarcinoma cells contain large amounts of H1(0) mRNA. This increase takes place very early during differentiation and does not correlate with changes in the rate of cell division. This indicates that the accumulation of H1(0) mRNA is not the result of reduced proliferation. Most likely on the contrary, the increase in the amount of H1(0) and the resulting effects on the formation of high order chromatin structures are parts of the differentiation program induced in F9 cells.     

Independent regulation of matrix metalloproteinases and plasminogen activators in human fibrosarcoma cells

Serine proteases and matrix metalloproteinases have been shown to often cooperate in multiple physiological and pathological processes associated with changes in the extracellular matrix (ECM). We have examined the interaction between the plasminogen activator (PA)-plasmin system and matrix metalloproteinases (MMPs) in HT1080 human fibrosarcoma cells treated with 12-O-tetradecanoyl-phorbol-13-acetate (TPA). While TPA treatment evoked a temporary increased expression of urokinase type PA (uPA), the production of both types of plasminogen activator inhibitors (PAI) was induced and sustained over 12 h by TPA treatment shifting the protease-protease inhibitors balance in favor of the inhibitors. TPA treatment of HT1080 cells induced the expression of interstitial collagenase (MMP-1) and increased the expression of gelatinase B (MMP-9), tissue inhibitor of metalloproteinases-1 (TIMP-1), and MT-MMP, a membrane-bound activator of progelatinase A (proMMP-2), while MMP-2 and TIMP-2 expression were decreased. Increased MT-MMP expression by TPA treatment was associated with increased activation of proMMP-2. These data show that the regulation of PA-plasmin and metalloproteinase and their specific inhibitors is uncoordinated. In addition, inhibition of the PA-plasmin system by PAI-2 or aprotinin did not prevent the activation of proMMP-2 by TPA, suggesting that plasmin is not involved in MT-MMP-mediated activation of proMMP-2. © 1996 Wiley-Liss, Inc.     

The role of extracellular matrix in the migration and differentiation of parietal endoderm from teratocarcinoma embryoid bodies

Embryoid bodies formed from teratocarcinoma stem cells differentiate an outer layer consisting of parietal and visceral endoderm or of visceral endoderm exclusively. We have previously shown that when these embryoid bodies are plated on collagen-coated substrates a parietal endoderm-like cell migrates onto the substrate, whereas all of the visceral endoderm remains associated with the stem cell mass, suggesting a role for substrate contact in parietal endoderm differentiation. We now identify fibronectin as the migration-promoting component in these cultures, and note that laminin and collagen type IV are 10-fold less effective at promoting both attachment and endoderm outgrowth. The RGDS tetrapeptide (arg-gly-asp-ser) from the cell attachment domain of fibronectin can specifically block attachment and outgrowth on both fibronectin- and laminin-coated substrates. In addition, the involvement of the 140-kD fibronectin receptor is demonstrated using an antibody directed against this molecule.     

Teratocarcinoma F9 cells induced to differentiate with sodium butyrate produce both tissue-type and urokinase-type plasminogen activators.

Sodium butyrate (NaB) can induce teratocarcinoma cell differentiation as retinoic acid (RA). However, the function of these two agents seems to be a little different [Kosaka et al., Exp Cell Res, 192:46-51, 1991]. F9 cells treated with NaB synthesize both tissue-type (tPA) and urokinase-type (uPA) plasminogen activator, though RA induces only tPA production. Urokinase-type PA is demonstrated to exist in association with membrane and to localize its activity to the close environment of the cell surface. This may cause the specific cell morphology and characteristics of differentiated F9 cells induced with NaB.     

Disorders in the release of plasminogen activators

Impairment of the release of plasminogen activator has been looked for in patients with a predisposition to vascular disease or venous thrombosis. In normal people the fibrinolytic activity of the blood rises sharply after strenuous physical exercise or after the administration of certain drugs, among which DDAVP. These measures fail to elicit a normal response in many of these patients. In most cases this turned out to be due to a high level of a circulating plasminogen activator inhibitor which suppresses the rise in fibrinolytic activity. Release of activator can only be demonstrated reliably by the assay of t-PA-antigen. An impaired release appears to be very rare and in the experience of the author it occurs with some regularity only in patients with terminal renal insufficiency.     

Evidence for the existence of an early common biochemical pathway in the differentiation of F9 cells into visceral or parietal endoderm: Modulation by cyclic AMP

The addition of dibutyryl cyclic AMP (dbcAMP) to aggregate cultures of F9 cells in medium containing retinoic acid (RA) directs the pathway of differentiation into parietal endoderm instead of visceral endoderm. We examined the levels of some of the markers that characterize the two pathways and studied the time of commitment of cells to either direction of differentiation by using immunoprecipitation and enzyme-linked immunosorbent assays (ELISA). For either pathway, the levels and patterns of laminin, type IV collagen, and fibronectin are the same on the first day of differentiation, characterized by slightly decreased levels of laminin and type IV collagen synthesis and an increased level of fibronectin synthesis. These levels reverse on the second day of culture when the pathways diverge markedly. The differentiation pathway, however, can be redirected into the alternate one; parietal endoderm cells become committed after 3 days, whereas visceral endoderm cells are able to change into parietal endoderm cells at any time. Thus, α-fetoprotein (AFP)-producing F9 embryoid bodies switched to dbcAMP-containing medium lose the capacity to synthesize AFP and start to express genes characteristic of parietal endoderm. Our results indicate that at least some visceral endoderm cells may redifferentiate into parietal endoderm cells. These phenomena thus mimic features of endoderm differentiation in the mouse embryo.     

Plasminogen activator expression in F9 teratocarcinoma embryoid bodies and their endoderm derivatives

Plasminogen activators are believed to play an important role in tissue remodeling and cell migration. During mouse embryogenesis, visceral endoderm secretes urokinase-type plasminogen activator (uPA) whereas parietal endoderm secretes tissue-type plasminogen activator (tPA). Visceral endoderm from F9 embryoid bodies can transdifferentiate into parietal endoderm under the appropriate culture conditions. We have examined at the protein and mRNA levels the type of plasminogen activator expressed in whole embryoid bodies, visceral endoderm and its parietal endoderm derivatives. Our experiments show that the visceral endoderm on F9 embryoid bodies synthesizes and secretes substantial amounts of both tPA and uPA. In contrast, the parietal endoderm derived directly from the visceral endoderm secretes dramatically increased levels of tPA and decreases production of uPA to low or below detectable levels. These data support the finding that visceral endoderm can transdifferentiate to parietal endoderm. In addition, this transition provides an excellent model for studying the molecular basis of the coincident down- and upregulation of the two plasminogen activators as well as their potential function during embryogenesis.     

Binding of mutant tissue-type plasminogen activators to human endothelial cells and their extracellular matrix

We previously demonstrated that tissue-type plasminogen activator (t-PA) specifically bound to its receptor (t-PAR) on human umbilical vein endothelial cells (HUVEC). In addition to analyses of t-PA binding to plasminogen activator inhibitor-1 (PAI-1) in the extracellular matrix (ECM) and to the t-PAR, we further evaluated the binding of three t-PA mutants, deltaFE1X t-PA lacking finger (F), epidermal growth factor-like (E) domains and one sugar chain at Asn177 thus comprising two kringles (K1 and K2) and protease (P) domains, deltaFE3X t-PA with three glycosylation sites deleted at Asn117, 184, and 448, and deltaFEK1 t-PA comprising K2 and P domains without glycosylation. Wild-type t-PA bound to ECM with high affinity, which was completely blocked by anti-PAI-1 IgG. Wild-type t-PA, deltaFE1X t-PA and deltaFEK1 t-PA bound to two classes of binding sites with high and low affinities on monolayer HUVEC. However, all t-PAs bound to a single class of binding site in the presence of anti-PAI-1 IgG. DeltaFEK1 t-PA bound t-PAR maximally among these t-PAs. These results suggested that the high affinity binding of t-PA mainly occurred with PAI-1 on ECM while the low affinity binding was with t-PAR. The deletion of F, E domains and sugar chains had no effect on binding with t-PAR. However, since only K1-missing t-PA (deltaFEK1) exhibited significantly increased binding sites among these t-PAs, it was suggested that the binding to t-PAR was mediated mainly by K2 domain and that the increase of binding was due to direct exposure of K2 domain.     

Interaction of heparin with plasminogen activators and plasminogen: effects on the activation of plasminogen

The amidolytic plasmin activity of a mixture of tissue plasminogen activator (tPA) and plasminogen is enhanced by heparin at therapeutic concentrations. Heparin also increases the activity in mixtures of urokinase-type plasminogen activator (uPA) and plasminogen but has no effect on streptokinase or plasmin. Direct analyses of plasminogen activation by polyacrylamide gel electrophoresis demonstrate that heparin increases the activation of plasminogen by both tPA and uPA. Binding studies show that heparin binds to various components of the fibrinolytic system, with tight binding demonstrable with tPA, uPA, and Lys-plasminogen. The stimulation of tPA activity by fibrin, however, is diminished by heparin. The ability of heparin to promote plasmin generation is destroyed by incubation of the heparin with heparinase, whereas incubation with chondroitinase ABC or AC has no effect. Also, stimulation of plasmin formation is not observed with dextran sulfate or chondroitin sulfate A, B, or C. Analyses of heparin fractions after separation on columns of antithrombin III-Sepharose suggest that both the high-affinity and the low-affinity fractions, which have dramatically different anticoagulant activity, have similar activity toward the fibrinolytic components.