F9 Cells Research Articles

Simultaneous evaluation of radiation-induced apoptosis and micronuclei in five cell lines

Purpose: This study was conducted to clarify the relationship among the frequencies of micronuclei (MN) and apoptosis, and clonogenic cell survival after irradiation. Materials and methods: The frequencies of MN and apoptosis were compared in the surviving fraction in three human tumour cell lines and two rodent cell lines at various irradiation doses. Results: The SHIN-3, DU-145 and CHO-K1 cells showed dosedependent increases of MN per binucleate cell and an excellent correlation between the MN frequency and surviving fraction after irradiation. The F9 and COLO 320DM cells did not show this correlation. The number of apoptotic cells increased according to the increase in radiation dose in the F9 and COLO 320DM cells, but not in the SHIN-3, DU-145 or CHO-K1 cells. Conclusions: The detection of the MN frequency alone is insufficient to measure cellular intrinsic radiosensitivity. The simultaneous use of the MN assay and the detection of apoptotic cells would be more reliable as a method for predicting cell survival after radiation.

Identification of a Retinoic Acid Response Domain Involved in the Activation of the β 1-Adrenergic Receptor Gene by Retinoic Acid in F9 Teratocarcinoma Cells

The density of β 1-adrenergic receptors (β 1-AR) is up-regulated upon differentiation of embryonic F9 teratocarcinoma cells by retinoic acid (RA) to the primitive endodermal phenotype. To identify the domains involved in RA-mediated activation of β 1-AR gene transcription, three kb of 5′-flanking sequence of the β 1-AR gene were ligated to a luciferase reporter gene and transiently transfected into F9 cells that were pre-exposed to 100 nM RA for 2 days. By generating deletions in the β 1-AR promoter, a region between −125 and −100 was found to mediate a 3-fold induction in cells exposed to RA for an additional 2 days. Through site-directed mutagenesis of this region, it was determined that the RA responsive element (RARE) was organized as a direct repeat separated by 5 nucleotides in which the 5′-most AGGTCG half-site was between nucleotides −106 and −101 and the 3′-most AGGTCA half-site was between nucleotides −117 and −112. The RA receptor α (RARα) isoform bound to the oligomer representing the sequences between −125 and −100 as a heterodimer complex with the retinoid X receptor α (RXRα). In a separate study, it was determined that the nucleotides between −125 and −100 are involved in thyroid hormone-mediated activation of the β 1-AR gene in ventricular myocytes. Therefore, transcriptional activation of the β 1-AR gene by thyroid hormone or RA involves a single binding site in the promoter.

Vinculin and cell-cell adhesion.

Vinculin, a 117-kDa protein, is a constituent of adhesion plaques and adherence junctions in non-muscle cells. We investigated the role of vinculin on the physical strength of cell-cell adhesion by conducting disaggregation assays on aggregates of parental wild-type F9 mouse embryonal carcinoma cells (clone BIM), two vinculin-depleted F9 cell lines, gamma 227 and gamma 229, and a reconstituted gamma 229 cell line (R3) that re-express vinculin. Immunoblotting demonstrated that the four cell lines used in the study had similar expressions of the cell-cell adhesion molecule E-cadherin and associated membrane proteins alpha- and beta-catenin. Double immunofluorescence analysis showed that, in contrast to the vinculin-null cell lines. BIM and R3 cells expressed abundant vinculin at the cell margins in adhesion plaques and in cell-cell margins that also contained actin. Laminar flow assays showed that both the vinculin-positive and vinculin-negative cell aggregates that were formed in culture in the course of 24 to 48 hours largely remained intact despite the imposition of shear flow at high shear rates. Since laminar flow imposed on cell aggregates act to separate cells from each other, our data indicate that F9 cells that were adherent to a substrate formed strong cell-cell adhesion bonds independent of vinculin expression. On the other hand, aggregates of vinculin-depleted gamma 229 and gamma 227 cells that were formed in suspension during a two-hour static incubation at 37 degrees C were desegregated more easily with the imposition of shear flow than the BIM and R3 cell aggregates formed under identical conditions. Loss of vinculin was associated with a reduction in cell-cell adhesion strength only among those cells lacking contact to a substrate. Overall, the results indicate that vinculin is not needed for forming strong cell-cell adhesion bonds between neighboring carcinoma cells which are adherent to the basal lamina.

Retinoic Acid Differentially Regulates Retinoic Acid Receptor-Mediated Pathways in the HEP3B Cell Line

Retinoic acid (RA) up-regulates retinoic acid receptor β (RARβ) gene expression in a variety of cell lines. Whether up-regulation of the RARβ gene reflects increased activity in a RARβ-mediated biological process is unclear since RARβ tends to heterodimerize with retinoid x receptor (RXR). In F9 teratocarcinoma cell line, RA-induced differentiation is accompanied by increased expression of the RARβ, RXRα, and α-fetoprotein (AFP) genes. Previously, we have shown that the RA-mediated regulation of the AFP gene is through RXRα homodimers. In contrast to F9 cells, Hep3B is unique in that the AFP gene is down-regulated by RA in a manner reminiscent of down-regulation of AFP in postfetal liver. In this paper, we have examined the RA-mediated regulation of the RAR, RXR, peroxisome proliferator-activated receptor (PPAR), and AFP genes in Hep3B cells. RA induced the expression of RARα, β, and γ mRNA in Hep3B cells. However, the expression of RXRα mRNA was down-regulated, and the levels of RXRβ and RXRγ mRNA remained unchanged after RA treatment. In addition, the expression of the PPARα, β, and γ genes was also unchanged. Gel retardation assays demonstrated that RA decreased the overall binding of nuclear receptors to the RA and PPAR response elements. By super-shift assays using specific anti-RAR and -RXR antibodies, RA treatment decreased the amount of RXRα while increasing the amount RARβ bound to retinoic acid response element-DR1 (direct repeat with spacer of one nucleotide), indicating the levels of RAR/RXR heterodimer, RXR/RXR homodimer, or RAR/RAR homodimers were altered upon RA treatment of Hep3B cells. In addition, the RA-mediated reduction of RXRα in part results in down-regulation of the AFP gene. Our data indicates that RA exerts its effects by differentially regulating its own receptor gene expression.

An alternative splice variant of the tissue specific transcription factor HNF4alpha predominates in undifferentiated murine cell types.

The transcription factor hepatocyte nuclear factor 4alpha (HNF4alpha) is a tissue specific transcription factor mainly expressed in the liver, kidney, intestine and the endocrine pancreas, but is also an essential regulator for early embryonic events. Based on its protein structure HNF4alpha is classified as an orphan member of the nuclear receptor superfamily. Comparing HNF4alpha transcription factors in the differentiated and dedifferentiated murine hepatocyte cell line MHSV-12 we identified in dedifferentiated cells the novel splice variant HNF4alpha7. This variant is characterized by an alternative first exon and has a lower transactivation potential in transient transfection assays using HNF4 dependent reporter genes. HNF4alpha7 mRNA and the corresponding protein are expressed in the undifferentiated pluripotent embryonal carcinoma cell line F9, whereas HNF4alpha1 only appears after differentiation of F9 cells to visceral endoderm. HNF4alpha7 mRNA is also found in totipotent embryonic stem cells. However, the function of HNF4alpha7 seems not to be restricted to embryonic cells as the HNF4alpha7 mRNA is also present in adult tissues, most notably the stomach. All these features suggest that the presence of distinct splice variants of HNF4alpha modulates the activity of HNF4alphain a cell type specific way.

Increased probability of expression from modified retroviral vectors in embryonal stem cells and embryonal carcinoma cells.

Gene expression from the Moloney murine leukemia retrovirus (Mo-MuLV) is highly restricted in embryonic carcinoma (EC) and embryonic stem (ES) cells. We compared levels of expression in PA317 fibroblasts, F9 (EC) cells, and CCE (ES) cells by Mo-MuLV-based vectors and vectors based on our previously reported MND backbone, which has alterations to address three viral elements implicated as repressors of expression by Mo-MuLV: the enhancer, the primer binding site, and the negative-control region. Expression was evaluated with three reporter genes, the chloramphenicol acetyltransferase (CAT) gene, whose expression was measured by enzymatic assay and by Northern blotting; a truncated nerve growth factor receptor (tNGFR), whose expression was measured by fluorescence-activated cell sorting (FACS) as a cell surface protein; and the enhanced green fluorescent protein (EGFP), whose expression was measured intracellularly by flow cytometry. We found significantly higher levels of CAT activity (5- to 300-fold) and greater quantities of vector-specific transcripts in ES and EC cells transduced with the modified MND-CAT-SN vector than in those transduced with L-CAT-SN. Northern blot analysis indicated that long terminal repeat transcripts from MND-CAT-SN are >80 times more abundant than the L-CAT-SN transcripts. FACS analysis of tNGFR expression from a pair of vectors, L-tNGFR-SN and MND-tNGFR-SN, indicated that only 1.04% of the CCE cells containing the L-tNGFR-SN vector expressed the cell surface reporter, while the MND-tNGFR-SN vector drove expression in 99.54% of the CCE cells. Of the F9 cells containing the L-tNGFR-SN vector, 13.32% expressed tNGFR, while 99.89% of the F9 cells transduced with MND-tNGFR-SN showed expression. Essentially identical results were produced with an analogous pair of vectors encoding EGFP. In unselected pools of F9 cells 48 h posttransduction, the L-EGFP-SN vector drove expression in only 5% of the population while the MND-EGFP-SN vector drove expression in 88% of the cells. After more than 3 weeks in culture without selection, the proportion of cells showing expression from L-EGFP-SN decreased slightly to 3% while expression from the MND-EGFP-SN vector persisted in 80% of the cells. Interestingly, in the few ES and EC cells which did show expression from the L-tNGFR-SN or L-EGFP-SN vectors, the magnitude of reporter expression was similar to that from the MND-tNGFR-SN or MND-EGFP-SN vector in nearly all cells, suggesting that the MND vectors are far less susceptible to position-dependent variegation of expression than are the Mo-MuLV-based vectors. Therefore, the modified retroviral vector, MND, achieves higher net levels of expression due to a greater frequency of expression, which may be useful for the expression of exogenous genes in EC and ES cells.

Increased expression of c-jun, but not retinoic acid receptor β, is associated with F9 teratocarcinoma stem cell differentiation induced by polyamine depletion

alpha-Difluoromethylornithine (DFMO), an enzyme-activated irreversible inhibitor of ornithine decarboxylase, and all-trans-retinoic acid (RA) are known to induce F9 teratocarcinoma stem cell differentiation. Both compounds induce the formation of the same cell type, i.e., parietal endoderm-like cells expressing tissue plasminogen activator and collagen type IV alpha-1. The present study shows that DFMO and RA induce terminal differentiation of F9 cells through different pathways. Thus, retinoic acid receptor (RAR) alpha mRNA is weakly expressed during DFMO treatment, but strongly induced during an early phase of RA treatment. RAR beta mRNA is not detectable in DFMO-treated cells, but very strongly induced by RA and maintained at a high level throughout the differentiative process. RAR gamma mRNA is relatively strongly expressed in untreated control cells and remains at approximately the same level during DFMO-induced differentiation. In RA-treated cells, however, RAR gamma mRNA is rapidly down-regulated and becomes nondetectable during the final course of differentiation. These experiments show that the differentiation of F9 cells into parietal endoderm-like cells does not necessarily involve changes in any of the RAR mRNA subtypes. Even though the steady-state levels of the RAR alpha and RAR gamma transcripts may be sufficient to support the differentiative process, our data clearly show that induction of RAR beta mRNA transcription is neither a prerequisite for F9 cell differentiation, nor an absolute consequence of the elevated c-jun mRNA expression that is consistently observed during the course of parietal endoderm differentiation.

Increased expression of c‐jun, but not retinoic acid receptor β, is associated with F9 teratocarcinoma stem cell differentiation induced by polyamine depletion

α-Difluoromethylornithine (DFMO), an enzyme-activated irreversible inhibitor of ornithine decarboxylase, and all-trans-retinoic acid (RA) are known to induce F9 teratocarcinoma stem cell differentiation. Both compounds induce the formation of the same cell type, i.e., parietal endoderm-like cells expressing tissue plasminogen activator and collagen type IV α-1. The present study shows that DFMO and RA induce terminal differentiation of F9 cells through different pathways. Thus, retinoic acid receptor (RAR) α mRNA is weakly expressed during DFMO treatment, but strongly induced during an early phase of RA treatment. RAR β mRNA is not detectable in DFMO-treated cells, but very strongly induced by RA and maintained at a high level throughout the differentiative process. RAR γ mRNA is relatively strongly expressed in untreated control cells and remains at approximately the same level during DFMO-induced differentiation. In RA-treated cells, however, RAR γ mRNA is rapidly down-regulated and becomes nondetectable during the final course of differentiation. These experiments show that the differentiation of F9 cells into parietal endoderm-like cells does not necessarily involve changes in any of the RAR mRNA subtypes. Even though the steady-state levels of the RAR α and RAR γ transcripts may be sufficient to support the differentiative process, our data clearly show that induction of RAR β mRNA transcription is neither a prerequisite for F9 cell differentiation, nor an absolute consequence of the elevated c-jun mRNA expression that is consistently observed during the course of parietal endoderm differentiation. J. Cell. Biochem. 67:378–385, 1997. © 1997 Wiley-Liss, Inc.

Differential effects of ascorbate depletion and α,α′-dipyridyl treatment on the stability, but not on the secretion, of type IV collagen in differentiated F9 cells

Ascorbic acid stimulates secretion of type I collagen because of its role in 4-hydroxyproline synthesis, but there is some controversy as to whether secretion of type IV collagen is similarly affected. This question was examined in differentiated F9 cells, which produce only type IV collagen, by labeling proteins with [14C]proline and measuring collagen synthesis and secretion. Hydroxylation of proline residues in collagen was inhibited to a greater extent in cells treated with the iron chelator alpha,alpha'-dipyridyl (97.7%) than in cells incubated without ascorbate (63.1%), but both conditions completely inhibited the rate of collagen secretion after 2-4 h, respectively. Neither treatment affected laminin secretion. Collagen synthesis was not stimulated by ascorbate even after treatment for 2 days. On SDS polyacrylamide gels, collagen produced by alpha,alpha'-dipyridyl-treated cells consisted mainly of a single band that migrated faster than either fully (+ ascorbate) or partially (- ascorbate) hydroxylated alpha 1(IV) or alpha 2(IV) chains. It did not contain interchain disulfide bonds or asn-linked glycosyl groups, and was completely digested by pepsin at 15 degrees C. These results suggested that it was a degraded product lacking the 7 S domain and that it could not form a triple helical structure. In contrast, the partially hydroxylated molecule contained interchain disulfide bonds and it was cleaved by pepsin to collagenous fragments similar in size to those obtained from the fully hydroxylated molecule, but at a faster rate. Kinetic experiments and monensin treatment suggested that completely unhydroxylated type IV collagen was degraded intracellularly in the endoplasmic reticulum or cis Golgi. These studies indicate that partial hydroxylation of type IV collagen confers sufficient helical structure to allow interchain disulfide bond formation and resistance to pepsin and intracellular degradation, but not sufficient for optimal secretion.

Differential effects of ascorbate depletion and α,α′‐dipyridyl treatment on the stability, but not on the secretion, of type IV collagen in differentiated F9 cells

Ascorbic acid stimulates secretion of type I collagen because of its role in 4-hydroxyproline synthesis, but there is some controversy as to whether secretion of type IV collagen is similarly affected. This question was examined in differentiated F9 cells, which produce only type IV collagen, by labeling proteins with [14C]proline and measuring collagen synthesis and secretion. Hydroxylation of proline residues in collagen was inhibited to a greater extent in cells treated with the iron chelator α,α′-dipyridyl (97.7%) than in cells incubated without ascorbate (63.1%), but both conditions completely inhibited the rate of collagen secretion after 2–4 h, respectively. Neither treatment affected laminin secretion. Collagen synthesis was not stimulated by ascorbate even after treatment for 2 days. On SDS polyacrylamide gels, collagen produced by α,α′-dipyridyl-treated cells consisted mainly of a single band that migrated faster than either fully (+ ascorbate) or partially (− ascorbate) hydroxylated α1(IV) or α2(IV) chains. It did not contain interchain disulfide bonds or asn-linked glycosyl groups, and was completely digested by pepsin at 15°C. These results suggested that it was a degraded product lacking the 7 S domain and that it could not form a triple helical structure. In contrast, the partially hydroxylated molecule contained interchain disulfide bonds and it was cleaved by pepsin to collagenous fragments similar in size to those obtained from the fully hydroxylated molecule, but at a faster rate. Kinetic experiments and monensin treatment suggested that completely unhydroxylated type IV collagen was degraded intracellularly in the endoplasmic reticulum or cis Golgi. These studies indicate that partial hydroxylation of type IV collagen confers sufficient helical structure to allow interchain disulfide bond formation and resistance to pepsin and intracellular degradation, but not sufficient for optimal secretion. J Cell. Biochem. 67:338–352, 1997. Published 1997 Wiley-Liss, Inc.

Specific and redundant functions of retinoid X Receptor/Retinoic acid receptor heterodimers in differentiation, proliferation, and apoptosis of F9 embryonal carcinoma cells.

We have generated F9 murine embryonal carcinoma cells in which either the retinoid X receptor (RXR)alpha and retinoic acid receptor (RAR)alpha genes or the RXRalpha and RARgamma genes are knocked out, and compared their phenotypes with those of wild-type (WT), RXRalpha-/-, RARalpha-/-, and RARgamma-/- cells. RXRalpha-/-/ RARalpha-/- cells were resistant to retinoic acid treatment for the induction of primitive and parietal endodermal differentiation, as well as for antiproliferative and apoptotic responses, whereas they could differentiate into visceral endodermlike cells, as previously observed for RXRalpha-/- cells. In contrast, RXRalpha-/-/RARgamma-/- cells were defective for all three types of differentiation, as well as antiproliferative and apoptotic responses, indicating that RXRalpha and RARgamma represent an essential receptor pair for these responses. Taken together with results obtained by treatment of WT and mutant F9 cells with RAR isotype- and panRXR-selective retinoids, our observations support the conclusion that RXR/ RAR heterodimers are the functional units mediating the retinoid signal in vivo. Our results also indicate that the various heterodimers can exert both specific and redundant functions in differentiation, proliferation, and apoptosis. We also show that the functional redundancy exhibited between RXR isotypes and between RAR isotypes in cellular processes can be artifactually generated by gene knockouts. The present approach for multiple gene targeting should allow inactivation of any set of genes in a given cell.

Phosphorylation of activation functions AF-1 and AF-2 of RAR alpha and RAR gamma is indispensable for differentiation of F9 cells upon retinoic acid and cAMP treatment.

The role of RAR alpha 1 and RAR gamma 2 AF-1 and AF-2 activation functions and of their phosphorylation was investigated during RA-induced primitive and parietal differentiation of F9 cells. We found that: (i) primitive endodermal differentiation requires RAR gamma 2, whereas parietal endodermal differentiation requires both RAR gamma 2 and RAR alpha 1, and in all cases AF-1 and AF-2 must synergize; (ii) primitive endodermal differentiation requires the proline-directed kinase site of RAR gamma 2-AF-1, whereas parietal endodermal differentiation additionally requires that of RAR alpha 1-AF-1; (iii) the cAMP-induced parietal endodermal differentiation also requires the protein kinase A site of RAR alpha-AF-2, but not that of RAR gamma; and (iv) the AF-1-AF-2 synergism and AF-1 phosphorylation site requirements for RA-responsive gene induction are promoter context-dependent. Thus, AF-1 and AF-2 of distinct RARs exert specific cellular and molecular functions in a cell-autonomous system mimicking physiological situations, and their phosphorylation by kinases belonging to two main signalling pathways is required to enable RARs to transduce the RA signal during F9 cell differentiation.

A Regulatory Element within a Coding Exon Modulates Keratin 18 Gene Expression in Transgenic Mice

Multiple tissue-specific, DNase-hypersensitive sites are correlated with known or potential regulatory regions of the human keratin 18 (K18) gene. One of these sites is found within exon 6, close to a potential AP-1 binding site. Footprint analysis confirmed that this site is capable of binding c-Jun and c-Fos in vitro. However, exon 6 can stimulate expression of a reporter gene driven by the K18 proximal promoter independent of AP-1 in F9 cells and additionally modulates AP-1 responsiveness when in combination with an intron enhancer. Analysis in transgenic mice and by transient transfections of mutant forms of the K18 gene showed that exon 6 contributes to the expression of the K18 gene. However, substitution of part of exon 6 with the corresponding part of the keratin 19 gene which lacks an AP-1 site decreased but did not destroy the regulatory activity of the exon. Furthermore, this mutation did not alter either the tissue specificity or the position-independent and copy number-dependent behavior of the K18 gene. In contrast, a frameshift mutation within exon 6 dramatically decreased the expression of the gene. K18 RNA expression from the frameshift mutation was less than 10% of the wild type K18 transgene. This decline in expression was the result of a combination of decreased stability of mutant K18 RNA and the creation of a negative regulatory element that can interact with the first intron regulatory elements and actively suppress K18 expression. These results demonstrate that a protein-coding portion of the K18 gene also has a regulatory function.

The C-terminal ligand-binding domain of Clostridium difficile toxin A (TcdA) abrogates TcdA-specific binding to cells and prevents mouse lethality

We have investigated the ability of a recombinant protein (REP231), derived from Clostridium difficile toxin A C-terminal domain, to protect against toxin A (TcdA) intoxication in vitro and in vivo. REP231 was cloned, expressed and purified by thyroglobulin affinity chromatography, and demonstrated identical binding properties to TcdA. Immunofluorescence experiments and in vitro cytotoxicity assays using mouse teratocarcinoma cells F9 showed that specific binding of TcdA to F9 cells through its C-terminal domain is essential for producing cytotoxic effects. TcdA binding and cytotoxicity was inhibited by REP231 and a monoclonal antibody directed against the C-terminal domain. Toxin B did not bind to F9 cells and was consequently inactive in cytotoxicity assays. Inhibition studies with lectins and a Le x-specific antibody supported earlier findings that a terminal galactose is part of the bound saccharide but excluded Le x as a receptor for TcdA. Mice immunised with REP231 were protected against a threefold lethal dose of TcdA. Thus, REP231 appeared to be a suitable candidate to develop an alternative therapeutic agent, which is able to neutralise carbohydrate-mediated TcdA binding and might act as a vaccine.

DNA binding and transactivation properties of Fos variants with homodimerization capacity.

The mammalian Fos and Fos-related proteins are unable to form homodimers and to bind DNA in the absence of a second protein, like c-Jun for example. In order to study the implications of hydrophobic point mutations in the c-Fox leucine zipper on DNA binding of the entire c-Fos protein, we have constructed and purified a set of Fos mutant proteins harboring one or several isoleucine or leucine residues in the five Fos zipper a positions. We show that a single point mutation in the hydrophobic interface of the c-Fos leucine zipper enables the c-Fos mutant protein to bind specifically to an oligonucleotide duplex harboring the TRE consensus sequence TGA(C/G)TCA. This point mutation (Thr196-->Ile) is situated in the a position of the second heptade (a2) of the Fos zipper. The introduction of additional isoleucine residues in the other a positions progressively increases the DNA binding affinity of these homodimerizing Fos zipper variants. Heterodimerization of these c-Fos variants with c-Jun reveals a complex behavior, in that the DNA binding affinity of these heterodimers does not simply increase with the number of isoleucine side chains in position a. For example, a c-Fos variant harboring a wild-type Thr in position a1 aad Ile in the four other a positions (c-Fos4I) interacts more tightly with c-Jun than a variant harboring Ile in all five a positions (c-Fos5I). The same holds true for the corresponding leucine variants, suggesting that the wild-type a1 residue of the Fox zipper (Thr162) is thermodynamically relevant for Fos-Jun heterodimer formations and DNA binding. The c-Fos4I variant forms heterodimers with c-Jun slightly better than the wild-type zipper protein, suggesting that the driving force for Fos-Jun heterodimerization is not the simple fact that the Fos protein is unable to form homodimers. These c-Fos variants were further tested for their transactivation properties in F9 and NIH3T3 cells. At low expression levels the most efficiently homodimerizing variant (c-Fos5I) activates transcription in F9 cells about 6-fold. However part of this activation may be due to the formation of heterodimers with a member of the Jun family (like JunD for example), since a wild type c-Fos expression vector confers a 3-fold activation under these conditions. In the case of the homodimerizing c-Fos variants however, this activation is abrogated at higher expression levels due to a strong inhibition of basal transcription activity.

Identification of an intronic enhancer that nullifies upstream repression of SPARC gene expression.

The SPARC gene 5'flanking sequence has been shown to contain enhancer elements, but also negative control elements immediately upstream of the enhancer elements. Although these 5'enhancer elements are active in F9 and PYS-2 cells, their activities are nullified by the 5'repressor activity. In the present study we have identified within intron 1 between nucleotides (nt) +5000 and +5150 of the SPARC gene an enhancer element that bound to two transcription factors of 48 and 52 kDa and between nt +5000 and +5523 a DNase I hypersensitive site. Furthermore, a region containing the 3'intron 1 enhancer element, together with the 5'enhancer elements, neutralized the 5'repressor activity and stimulated efficient transcription. The resulting SPARC promoter activity is about equal in F9, differentiated F9 and PYS-2 cells. We consistently found that the rate of SPARC transcription is nearly the same in F9 and PYS-2 cells. Association of the 3'enhancer element in intron 1 with the DNase I hypersensitive site suggests that both play a role in regulating SPARC expression in vivo .

Triple synergism of human T-lymphotropic virus type 1-encoded tax, GATA-binding protein, and AP-1 is required for constitutive expression of the interleukin-5 gene in adult T-cell leukemia cells.

Accumulated evidence demonstrates that adult T-cell leukemia (ATL) is frequently associated with eosinophilia, and human T-lymphotropic virus type 1 (HTLV-1)-infected cells frequently express interleukin-5 (IL-5). However, the molecular mechanism of constitutive IL-5 expression in HTLV-1-infected cells remains unclear. To clarify the mechanism of aberrant IL-5 expression in HTLV-1-infected cells, we investigated the response of the human IL-5 promoter to the HTLV-1-encoded protein Tax. Cotransfection experiments using Jurkat cells revealed that Tax is incapable of activating the IL-5 promoter by itself but that it synergistically transactivates the promoter with GATA-binding protein (GATA-4) and 12-O-tetradecanoylphorbol-13-acetate (TPA) stimulation. By introducing a series of mutations within the IL-5 promoter, we found that conserved lymphokine element 0 (CLE0) is responsible for mediating the signal induced by Tax-TPA. A deletion construct of the promoter indicated that the -75 GATA element and CLE0 are sufficient to mediate synergistic activation of the IL-5 promoter. Electrophoretic mobility shift assays using Jurkat cell nuclear extracts demonstrated that TPA induces a transcription factor to bind CLE0, and an experiment using JPX-9 cell nuclear extracts showed that Tax enhances this binding activity. An antibody supershift experiment revealed that this band consists of c-Jun and JunD. However, among the Jun family members, only c-Jun is able to cooperate with Tax and GATA-4 to activate the IL-5 promoter. We have determined the minimum factors required for IL-5 gene activation by reconstituting the IL-5 promoter activity in F9 cells. This is the first report to demonstrate the functional involvement of Tax protein in IL-5 gene regulation and to suggest the functional triple synergism among Tax, GATA-4, and AP-1, which disrupts regulated control of the gene and leads to constitutive expression of the IL-5 gene.

Alternative signaling mechanism of leukemia inhibitory factor responsiveness in a differentiating embryonal carcinoma cell.

Leukemia inhibitory factor (LIF) is a cytokine that plays an important role during mouse embryogenesis. We showed that adenovirus E1A represses the interleukin-6 signal transduction pathway that uses the same JAK tyrosine kinase and STAT (signal transducer and activator of transcription) transcription factor as LIF. Here, we report that the LIF-JAK-STAT signal transduction pathway is blocked in cellular E1A-expressing undifferentiated F9 cells, and that the block is overcome by retinoic acid-induced differentiation. LIF failed to stimulate the expression of the acute phase response element (APRE)-driven luciferase gene in undifferentiated F9 cells, whereas the luciferase activity was remarkably increased by LIF treatment in differentiated F9 (dF9) cells. We analyzed the mechanism of the APRE regulation and found that the LIF-induced APRE-binding activity was regulated in a differentiation-dependent manner. The protein levels and the tyrosine phosphorylation of JAK1, JAK2, and STAT3 in F9 cells were not different from those in dF9 cells. The exogenous expression of activated c-Ha-ras partially recovered the LIF responsiveness of the APRE-luciferase gene in F9 cells, but the dominant negative ras N-17 did not repress the LIF-induced activation of APRE-luciferase in dF9 cells. These results suggested that an unknown coactivation process that is partially compensated by Ras is required for STAT3-APRE binding in F9 cells.

A Role forIndian hedgehogin Extraembryonic Endoderm Differentiation in F9 Cells and the Early Mouse Embryo

Hedgehoggenes in Drosophila and vertebrates control patterning of a number of different structures during embryogenesis. They code for secreted signaling proteins that are cleaved into an active aminopeptide and a carboxypeptide. The aminopeptide can mediate local and long range events and can act as a morphogen, inducing differentiation of distinct cell types in a concentration-dependent manner. We demonstrate here that the expression ofIndian hedgehogmRNA and protein is upregulated dramatically as F9 cells differentiate in response to retinoic acid, into either parietal endoderm or embryoid bodies, containing an outer visceral endoderm layer. The ES cell line D3 forms embryoid bodies in suspension culture without addition of retinoic acid and also upregulatesIndian hedgehogexpression. RT-PCR analysis of blastocyst outgrowth cultures demonstrates that whereas little or noIndian hedgehogmessage is present in blastocysts, significant levels appear upon subsequent days of culture, coincident with the emergence of parietal endoderm cells.In situhybridization analysis forIndian hedgehogmRNA expression demonstrates the presence of elevated levels of message in the outer visceral endoderm cells relative to the core cells in mature embryoid bodies and in the visceral endoderm of Day 6.5 embryos. Whole-mountin situhybridization analysis of Day 7.5 and 8.5 embryos indicates thatIndian hedgehogexpression is highest in the visceral yolk sac at this stage. F9 cell lines expressing a full lengthIndian hedgehogcDNA express a number of characteristics of differentiated cells, in the absence of retinoic acid. Taken together, these data suggest thatIndian hedgehogis involved in mediating differentiation of extraembryonic endoderm during early mouse embryogenesis.

Evidence for Serum Response Factor-Mediated Regulatory Networks GoverningSM22αTranscription in Smooth, Skeletal, and Cardiac Muscle Cells

SM22α is an adult smooth muscle-specific protein that is expressed in the smooth, cardiac, and skeletal muscle lineages during early embryogenesis before becoming restricted specifically to all vascular and visceral smooth muscle cells (SMC) in late fetal development and adulthood. We have used theSM22αgene as a marker to define the regulatory mechanisms that control muscle-specific gene expression in SMCs. Previously, we reported that the 445-base-pair promoter ofSM22αwas sufficient to direct transcription of a lacZ reporter gene in early cardiac and skeletal muscle cell lineages and in a subset of arterial SMCs, but not in venous nor visceral SMCs in transgenic mice. Here we describe two evolutionarily conserved CArG (CC(A/T)6GG) boxes in theSM22αpromoter, both of which are essential for full promoter activity in cultured SMCs. In contrast, only the promoter-proximal CArG box is essential for specific expression in developing smooth, skeletal, and cardiac muscle lineages in transgenic mice. Both CArG boxes bind serum response factor (SRF), but SRF binding is not sufficient forSM22αpromoter activity, since overexpression of SRF in the embryonal teratocarcinoma cell line F9, which normally expresses low levels of SRF, fails to activate the promoter. However, a chimeric protein in which SRF was fused to the transcription activation domain of the viral coactivator VP16 is able to activate theSM22αpromoter in F9 cells. These results demonstrate the SM22α promoter-proximal CArG box is a target for the regulatory programs that confer smooth, skeletal, and cardiac muscle specificity to theSM22αpromoter and they suggest that SRF activatesSM22αtranscription in conjunction with additional regulatory factors that are cell type-restricted.