Wiebe Kruijer

Identification and functional characterization of a Smad binding element (SBE) in the JunB promoter that acts as a transforming growth factor-beta, activin, and bone morphogenetic protein-inducible enhancer.

Smad proteins have been identified as mediators of intracellular signal transduction by members of the transforming growth factor-β (TGF-β) superfamily, which affect cell proliferation, differentiation, as well as pattern formation during early vertebrate development. Following receptor activation, Smads are assembled into heteromeric complexes consisting of a pathway-restricted Smad and the common Smad4 that are subsequently translocated into the nucleus where they are thought to play an important role in gene transcription. Here we report the identification of Smad Binding Elements (SBEs) composed of the sequence CAGACA in the promoter of theJunB gene, an immediate early gene that is potently induced by TGF-β, activin, and bone morphogenetic protein (BMP) 2. TwoJunB SBEs are arranged as an inverted repeat that is transactivated in response to Smad3 and Smad4 co-overexpression and shows inducible binding of a Smad3- and Smad4-containing complex in nuclear extracts from TGF-β-treated cells. Bacterial-expressed Smad proteins bind directly to the SBE. Multimerization of the SBE creates a powerful TGF-β-inducible enhancer that is also responsive to activin and BMPs. The identification of the sequence CAGACA as a direct binding site for Smad proteins will facilitate the identification of regulatory elements in genes that are activated by members of the TGF-β superfamily.

Cloning and expression during development of three murine members of the COUP family of nuclear orphan receptors.

We have isolated the murine homologs of the members of the COUP-family of steroid hormone receptors, COUP-TF1, ARP-1 and EAR2. The proteins encoded by the murine genes appeared to be highly conserved when compared to their human counterparts. The expression of COUP-TF1 and ARP-1 was induced during differentiation of P19 embryonal carcinoma (EC) cells into derivatives of all three germ layers. Retinoic acid (RA) treatment rapidly induced expression of both genes, while other methods of differentiation were less effective. Undifferentiated P19 cells were found to express EAR2 mRNA and the expression level was only slightly elevated by RA-treatment. In addition, we analyzed the expression in P19 cells of three members of the retinoid X receptor (RXR) family, which have been shown to heterodimerize with members of the COUP-family. During RA mediated differentiation of P19 cells, RXR alpha expression was induced while RXR beta expression was not modulated and RXR gamma expression was down regulated. Gel shift analysis revealed that in P19 cells the members of the COUP-family comprise the major portion of proteins binding to a RA-responsive direct repeat of the consensus steroid hormone receptor binding half site (AGGTCA) spaced by one nucleotide (DR + 1). The members of the COUP-family appeared to down regulate RA-induced activation of RA-response element-containing reporter constructs in a promoter context-dependent manner. The expression patterns of COUP-TF1, ARP-1 and EAR2 during development were investigated by in situ hybridization. In agreement with the results obtained in vitro, the three genes appeared to be expressed in tissues derived from all three germ layers. However, COUP-TF1 and ARP-1 were found to be expressed predominantly in the developing central nervous system in mutually exclusive domains. Furthermore, strong ARP-1 expression was detected in lung and kidney. Our data strongly suggest an important role for the members of the COUP-family in the hormonal control of gene expression regulating embryogenesis.

Characterization of a negative retinoic acid response element in the murine Oct4 promoter.

Expression of Oct4 in embryonic stem cells is controlled by a distal upstream stem cell-specific enhancer that is deactivated during retinoic acid (RA)-induced differentiation by an indirect mechanism not involving binding of RA receptors (H. Okazawa, K. Okamoto, F. Ishino, T. Ishino-Kaneko, S. Takeda, Y. Toyoda, M. Muramatsu, and H. Hamada, EMBO J. 10:2997-3005, 1991). Here we report that in RA-treated P19 embryonal carcinoma cells the Oct4 promoter is also subject to negative regulation by RA. The minimal Oct4 promoter sequence mediating repression consists of a promoter-proximal sequence containing a GC-rich SP1 consensus-like sequence and several hormone response element half-sites that can be arranged into direct repeats with different spacing. The GC box binds a nuclear factor that is invariably present in undifferentiated and RA-treated differentiated P19 cells. By contrast, the hormone response element-containing sequence binds factors that are induced following RA treatment. Mutational analysis and competition experiments show that the functional entity binding the RA-induced factor is a direct repeat sequence with a spacing of one nucleotide, previously shown to be a binding site for COUP transcription factors (COUP-TFs). Cotransfected orphan receptors COUP-TF1, ARP-1, and EAR-2 were able to repress the activity of Oct4 promoter-driven reporters in P19 EC cells, albeit with different efficiencies. Furthermore, the negative transcriptional effect of COUP-TFs is dominant over the activating effect of the Oct4 embryonic stem cell-specific enhancer. These results show that negative regulation of Oct4 expression during RA-induced differentiation of embryonic stem cells is controlled by two different mechanisms, including deactivation of the embryonic stem cell-specific enhancer and promoter silencing by orphan nuclear hormone receptors.

Nuclear responses to protein kinase C signal transduction are sensitive to gravity changes

A number of studies have suggested that gravity changes may influence mammalian cell growth and differentiation. To obtain insight in the molecular mechanisms underlying these effects, we have studied immediate early gene expression in response to activation of cytoplasmic signal transduction under microgravity conditions. In this paper we show that epidermal growth factor (EGF)- and 12-O-tetradecanoyl-phorbol-13-acetate (TPA)-induced expression of the c-fos and c-jun protooncogenes is decreased in microgravity, while no effect of gravity changes was observed on A23187- and forskolin-induced expression of these genes. These decrease in c-fos expression was not due to delayed kinetics under microgravity. These results demonstrate that gravity differentially modulates distinctive signal transduction pathways.

Effects of retinoic acid on the expression of retinoic acid receptors during zebrafish embryogenesis

The cDNAs encoding the zebrafish homologs of retinoic acid receptor alpha(zRAR alpha) and gamma (zRAR gamma) were isolated and their expression studied in normal and retinoic acid (RA) treated embryos. Expression boundaries in the central nervous system are clearly different from those observed in the mouse, which can only partly be explained by morphogenetic differences. Treatment of embryos with RA induces ectopic zRAR gamma expression in anterior brain structures and both zRAR alpha and zRAR gamma expression in the eyes. Furthermore, striking differences occur in the zRAR gamma expression pattern in pharyngeal arch mesenchyme. Since the development of all of these structures has been shown to be affected by exogenous RA, our data suggest a role for zRAR alpha and zRAR gamma in the establishment of the RA phenotype in zebrafish.

Expression of transforming growth factor β2 during the differentiation of murine embryonal carcinoma and embryonic stem cells

Abstract Transforming growth factor β 2 (TGF β 2 ) mRNA expression was studied by Northern blot analysis in a range of feeder-independent murine embryonal carcinoma (EC) cells and in feeder-dependent EC and embryonic stem (ES) cells. TGF β 2 transcripts were not detected in any undifferentiated cells including P19, F9, PC13, C1003, PSA-1, P10, and ES. Following induction of differentiation, however, TGF β 2 became expressed, independently of the cell type formed. Retinoic acid (RA) addition and/or deprivation of the differentiation inhibiting activity of feeder cells resulted in the appearance of TGF β 2 transcripts within 2 days. These kinetics correlated entirely with the first appearance of the protein; an anti-peptide antibody specifically recognizing TGF β 2 did not stain P19 EC cells by immunofluorescence but 2–3 days after RA addition, a significant proportion of the population was strongly labeled. In addition, primitive endoderm cells emerging from the inner cell mass of substrate attached blastocysts stained brightly with anti-TGF β 2 , while the undifferentiated inner cell mass cells did not. Although all trophectoderm cells at the mid-blastocyst stage were stained, few had detectable levels of TGF β 2 after plating on a substrate. Neither TGF β 1 nor TGF β 2 affect growth of EC cells, but a range of differentiated derivatives were all inhibited, with TGF β 2 being marginally more effective than TGF β 1 at the same concentration.

Aggregation and cell cycle dependent retinoic acid receptor mRNA expression in P19 embryonal carcinoma cells

Differentiation of P19 EC cells along different pathways into derivatives resembling cells of the three embryonic germ layers is accompanied by characteristic differences in modulation of expression of each of the three retinoic acid receptor genes, RAR alpha, -beta and -gamma. Differentiation induced by addition of RA to P19 EC cells cultured in monolayer is accompanied by a rapid increase in expression of both RAR alpha and -beta. Induction of RAR beta occurs in a characteristic biphasic manner, suggesting that multiple factors and/or different mechanisms are involved in controlling its expression. RAR beta mRNA is induced to a far higher level during early aggregation in the presence of RA than during early differentiation in monolayer, suggesting that the direction of differentiation depends on the number and/or ratio of alpha and beta type of RA receptors. Aggregation of P19 EC cells in the presence of RA, but not DMSO, is accompanied by repression of RAR gamma, suggesting that the expression of RAR beta and RAR gamma during neuroectodermal differentiation is mutually exclusive. The effects of RA on RAR expression are significantly greater in G1 than in S-phase of the cell cycle. These results extend previous observations that commitment to differentiation is cell cycle dependent and indicates that critical target gene regulation in response to RA has to take place in G1 for differentiation to occur.

Transcriptional regulation of retinoic acid receptor β in retinoic acid-sensitive and -resistant P19 embryocarcinoma cells

As in other embryocarcinoma (EC) cell lines retinoic acid (RA) rapidly induces expression of the nuclear retinoic acid receptor (RAR) beta in murine P19 EC cells, while RAR alpha is expressed constitutively. In the RA-resistant P19 EC-derived RAC65 cells, however, there is no such induction and an aberrant (smaller) RAR alpha transcript is expressed. RAR gamma 1 is expressed at low levels in both cell lines. To study the regulation of the RAR beta gene and the possible involvement of RAR alpha protein in transcriptional activation of the RAR beta gene we transfected these cells with a construct containing a 1.6 kb promoter fragment of the human RAR beta gene fused to the CAT gene. Upon transient assays in P19 EC cells CAT activity is enhanced rapidly by RA, to more than 100-fold in a concentration-dependent fashion. On the contrary no activity can be observed in the RA-resistant RAC65 cells; however, co-transfection of hRAR alpha, hRAR beta or hRAR gamma 1 restores the RA-dependent induction of CAT activity. These results clearly show that RAR alpha and RAR gamma 1 can transactivate the RAR beta gene; that RAR beta can stimulate its own expression and that resistance to RA in RAC65 cells is probably due to the altered RAR alpha transcript present in these cells.

Expression of growth factors during the differentiation of embryonic stem cells in monolayer

Embryonic stem (ES) cells, derived in culture directly from the inner cells mass (ICM) of blastocysts, more closely resemble their embryonic counterparts than the more commonly used embryonal carcinoma (EC) cells derived from teratocarcinomas. In view of the potential role of growth factors in early development, we have now followed changes in the expression of transforming growth factor beta (in particular TGF beta 1, beta 3, beta 4), platelet-derived growth factor (PDGF-A, PDGF-B) and insulin-like growth factor (IGF II) during the differentiation of ES cells in monolayer. When maintained in medium conditioned by Buffalo rat liver cells (BRL-CM) to inhibit differentiation, ES cells expressed 2.5 and 1.8 kb transcripts for TGF beta 1, as well as transcripts for TGF beta 4, PDGF-A, and low levels of PDGF-B, but not TGF beta 3 or IGF II. After formation of parietal endoderm-like cells by addition of retinoic acid (RA) to BRL-CM, the 1.8-kb transcript of TGF beta 1 and PDGF-A expression were reduced, IGF II mRNA and a single TGF beta 3 transcript of 3.8 kb were induced while PDGF-B and TGF beta 4 remained virtually unchanged. By contrast, in ES cells induced to differentiate by the absence of BRL-CM, unusual transcripts for TGF beta 3 of 3.0 and 6.0 kb became detectable and PDGF-B expression increased. The changes in growth factor expression in ES cells are compared with those in F9 and P19 EC cells induced to differentiate in monolayer by RA.

Octamer-dependent regulation of the kFGF gene in embryonal carcinoma and embryonic stem cells

Expression of kFGF, which belongs to the family of fibroblast growth factor genes, is restricted to undifferentiated embryonal carcinoma and embryonic stem cells. Stem cell specific expression of kFGF is controlled by a distally localized enhancer, conferring both positive and negative regulation to the kFGF and tk promoters. This enhancer contains a consensus octamer binding sequence that controls positive regulation in EC and ES cells. The octamer sequence binds Oct1 and Oct4 in nuclear extracts from undifferentiated EC cells, while only Oct1 is bound in nuclear extracts from RA differentiated cells. These results suggest that the kFGF gene is a target for positive regulation by Oct4 and implicate Oct4 as target for regulation by the retinoic acid receptors.