The novel MKL target gene myoferlin modulates expansion and senescence of hepatocellular carcinoma.

Megakaryoblastic Leukemia 1 and 2 (MKL1/2) are transcriptional coactivators of Serum Response Factor (SRF) with an essential role for hepatocellular carcinoma (HCC) growth and oncogene-induced senescence. In this report, we identified myoferlin as a novel MKL/SRF target gene by gene expression profiling and verification in vivo in HCC xenografts. Myoferlin was overexpressed in human and murine HCCs triggered by conditional expression of constitutively active SRF-VP16 protein in hepatocytes. Furthermore, myoferlin was required for HCC cell invasion, proliferation and anchorage-independent cell growth. We provide evidence that myoferlin is a crucial gene target of MKL1/2 mediating its effect on oncogene-induced senescence by modulating the activation state of the EGFR and downstream MAPK and p16-/Rb pathways. Depletion of myoferlin in tumour cells from SRF-VP16-derived murine HCCs induced a senescence phenotype. These findings identify MKL1/2 and myoferlin as novel therapeutic targets to treat human HCC by a senescence-inducing strategy.

Downstream of FGF during mesoderm formation in Xenopus: the roles of Elk-1 and Egr-1.

Signalling by members of the FGF family is required for induction and maintenance of the mesoderm during amphibian development. One of the downstream effectors of FGF is the SRF-interacting Ets family member Elk-1, which, after phosphorylation by MAP kinase, activates the expression of immediate-early genes. Here, we show that Xenopus Elk-1 is phosphorylated in response to FGF signalling in a dynamic pattern throughout the embryo. Loss of XElk-1 function causes reduced expression of Xbra at neurula stages, followed by a failure to form notochord and muscle and then the partial loss of trunk structures. One of the genes regulated by XElk-1 is XEgr-1, which encodes a zinc finger transcription factor: we show that phosphorylated XElk-1 forms a complex with XSRF that binds to the XEgr-1 promoter. Superficially, Xenopus tropicalis embryos with reduced levels of XEgr-1 resemble those lacking XElk-1, but to our surprise, levels of Xbra are elevated at late gastrula stages in such embryos, and over-expression of XEgr-1 causes the down-regulation of Xbra both in whole embryos and in animal pole regions treated with activin or FGF. In contrast, the myogenic regulatory factor XMyoD is activated by XEgr-1 in a direct manner. We discuss these counterintuitive results in terms of the genetic regulatory network to which XEgr-1 contributes.

[PI3-kinase inhibitors LY294002 and wortmannin have different effects on proliferation of murine embryonic stem cells].

Murine embryonic stem (mES) cells can proliferate independently of the presence of growth factors in the medium. It is yet unknown what intrinsic activity triggers cell cycle events in mES cells. Here we investigated the contribution of the PI3-kinase cascade to autonomous proliferation of mES cell using PI3-kinase inhibitors wortmannin and LY294002. Wortmannin displays a weaker inhibitory effect on phosphorylation of PI3-kinase pathway target PKB as compared with LY294002, and does not downregulate mES cells proliferation, while LY294002 causes a strong decrease in the share of cells in S-phase and accumulation of cells in G1 phase. Both inhibitors cause significant decrease in cyclin D1 amount. The treatment with LY294002, rather than with wortmannin results in a decrease of cyclin E amount and cyclin E-assossiated kinase activity. In mES cells, inactivation of PI3-kinase-dependent pathway and G1 arrest are not accompanied by induction of p27kip 1 transcription and accumulation of this inhibitor of cyclin-cdk complexes at the protein level, implying that these events accomplished by some p27kip 1-independent mechanism. Both LY294002 and wortmannin cause apoptotic death of mES cells and downregulate the growth of population. Thus, inactivation of PI3-kinase in mES cells may lead to apoptosis rather than to cell cycle arrest.

Tissue-specific expression of the Ets gene Xsap-1 during Xenopus laevis development.

We report the cloning of Xenopus laevis Xsap-1 cDNA, encoding a member of the ternary complex factor subfamily of ETS transcription factors. The expression pattern of Xsap-1 was examined during Xenopus embryogenesis using whole-mount in situ hybridization. Spatial expression of Xsap-1 mRNA is first detected at the animal pole at the mid-blastula stage. During neurulation Xsap-1 is expressed in cells participating in neural tube formation, in the sensorial layer of the epidermal ectoderm, and in an anterior region of the ventral mesoderm. Later, Xsap-1 expression is observed in the eye, ear vesicle, branchial arches, heart, pronephros, in the somites, and the developing nervous system, such as fore-, mid-, and hindbrain as well as in the cranial ganglion X.

Transforming growth factor beta regulates parathyroid hormone-related protein expression in MDA-MB-231 breast cancer cells through a novel Smad/Ets synergism.

The majority of breast cancers metastasizing to bone secrete parathyroid hormone-related protein (PTHrP). PTHrP induces local osteolysis that leads to activation of bone matrix-borne transforming growth factor beta (TGF beta). In turn, TGF beta stimulates PTHrP expression and, thereby, accelerates bone destruction. We studied the mechanism by which TGF beta activates PTHrP in invasive MDA-MB-231 breast cancer cells. We demonstrate that TGF beta 1 up-regulates specifically the level of PTHrP P3 promoter-derived RNA in an actinomycin D-sensitive fashion. Transient transfection studies revealed that TGF beta 1 and its effector Smad3 are able to activate the P3 promoter. This effect depended upon an AGAC box and a previously described Ets binding site. Addition of Ets1 greatly enhanced the Smad3/TGF beta-mediated activation. Ets2 had also some effect, whereas other Ets proteins, Elf-1, Ese-1, and Erf-1, failed to cooperate with Smad3. In comparison, Ets1 did not increase Smad3/TGF beta-induced stimulation of the TGF beta-responsive plasminogen activator inhibitor 1 (PAI-1) promoter. Smad3 and Smad4 were able to specifically interact with the PTHrP P3-AGAC box and to bind to the P3 promoter together with Ets1. Inhibition of endogenous Ets1 expression by calphostin C abrogated TGF beta-induced up-regulation of the P3 transcript, whereas it did not affect the TGF beta effect on PAI expression. In TGF beta receptor II- and Ets1-deficient, noninvasive MCF-7 breast cancer cells, TGF beta 1 neither influenced endogenous PTHrP expression nor stimulated the PTHrP P3 promoter. These data suggest that TGF beta activates PTHrP expression by specifically up-regulating transcription from the PTHrP P3 promoter through a novel Smad3/Ets1 synergism.

Cleavage of AML1/MTG8 by asymmetric hammerhead ribozymes.

The chromosomal translocation t(8;21) is one of the most frequent aberrations associated with acute myeloid leukaemia. It joins the 5' section of the AML1 gene with the almost complete open reading frame of MTG8 (ETO). The resulting fusion RNA represents a leukaemia-specific target for antisense/ribozyme inhibition. We tested several asymmetric hammerhead ribozymes targeted against the fusion site for their ability to cleave the AML1/MTG8 RNA at low magnesium concentrations. One ribozyme cleaves AML1/MTG8 RNA with high catalytic efficiency without binding or cleaving the wild-type AML1 transcript. The presence of cellular RNA does not affect the cleavage. Injection of AML1/MTG8 RNA and ribozyme RNA into Xenopus eggs or oocytes causes a specific reduction of AML1/MTG8 protein expression. Asymmetric anti-AML1/MTG8 ribozymes may be valuable modulators of AML1/MTG8 expression in leukaemic cells.

Serum response factor is required for immediate-early gene activation yet is dispensable for proliferation of embryonic stem cells.

Addition of serum to mitogen-starved cells activates the cellular immediate-early gene (IEG) response. Serum response factor (SRF) contributes to such mitogen-stimulated transcriptional induction of many IEGs during the G0-G1 cell cycle transition. SRF is also believed to be essential for cell cycle progression, as impairment of SRF activity by specific antisera or antisense RNA has previously been shown to block mammalian cell proliferation. In contrast, Srf(-/-) mouse embryos grow and develop up to E6.0. Using the embryonic stem (ES) cell system, we demonstrate here that wild-type ES cells do not undergo complete cell cycle arrest upon serum withdrawal but that they can mount an efficient IEG response. This IEG response, however, is severely impaired in Srf(-/-) ES cells, providing the first genetic proof that IEG activation is dependent upon SRF. Also, Srf(-/-) ES cells display altered cellular morphology, reduced cortical actin expression, and an impaired plating efficiency on gelatin. Yet, despite these defects, the proliferation rates of Srf(-/-) ES cells are not substantially altered, demonstrating that SRF function is not required for ES cell cycle progression.

Srf(-/-) ES cells display non-cell-autonomous impairment in mesodermal differentiation.

The serum response factor (SRF) transcription factor is essential for murine embryogenesis. SRF+(-/-) embryos stop developing at the onset of gastrulation, lacking detectable mesoderm. This developmental defect may reflect cell-autonomous impairment of SRF(-/-) embryonic cells in mesoderm formation. Alternatively, it may be caused by a non-cell-autonomous defect superimposed upon inappropriate provision of mesoderm-inducing signals to primitive ectodermal cells. We demonstrate that the ability of SRF(-/-) embryonic stem (ES) cells to differentiate in vitro into mesodermal cells is indeed impaired. However, this impairment can be modulated by external, cell-independent factors. Retinoic acid, but not dimethylsulfoxide, permitted activation of the mesodermal marker gene T(Bra), which was also activated when SRF was expressed in SRF(-/-) ES cells. Embryoid bodies from SRF(-/-) ES cell aggregates also activated mesodermal marker genes, but displayed unusual morphologies and impairment in cavitation. Finally, in nude mice, Srf(-/-) ES cells readily differentiated into mesodermal cells of SRF(-/-) genotype, including cartilage, bone or muscle cells. We demonstrate that SRF contributes to mesodermal gene expression of ES cells and that SRF(-/-) ES cells display a non-cell-autonomous defect in differentiation towards mesoderm.

Evaluation of the effectiveness of DNA-binding drugs to inhibit transcription using the c-fos serum response element as a target.

Previous work has demonstrated that sequence-selective DNA-binding drugs can inhibit transcription factors from binding to their target sites on gene promoters. In this study, the potency and effectiveness of DNA-binding drugs to inhibit transcription were assessed using the c-fos promoter's serum response element (SRE) as a target. The drugs chosen for analysis included the minor groove binding agents chromomycin A(3) and Hoechst 33342, which bind to G/C-rich and A/T-rich regions, respectively, and the intercalating agent nogalamycin, which binds G/C-rich sequences in the major groove. The transcription factors targeted, Elk-1 and serum response factor (SRF), form a ternary complex (TC) on the SRE that is necessary and sufficient for induction of c-fos by serum. The drugs' abilities to prevent TC formation on the SRE in vitro were nogalamycin > Hoechst 33342 > chromomycin. Their potencies in inhibiting cell-free transcription and endogenous c-fos expression in NIH3T3 cells, however, were chromomycin > nogalamycin > Hoechst 33342. The latter order of potency was also obtained for the drugs' cytotoxicity and inhibition of general transcription as measured by [(3)H]uridine incorporation. These systematic analyses provide insight into how drug and transcription factor binding characteristics are related to drugs' effectiveness in inhibiting gene expression.

Improved sensitivity proteomics by postharvest alkylation and radioactive labelling of proteins.

We describe approaches to improve the detection of proteins by postharvest alkylation and subsequent radioactive labeling with either [3H]iodoacetamide or 125I. Database protein sequence analysis suggested that cysteine is not suitable for detection of the entire proteome, but that cysteine alkylating reagents can increase the number of proteins able to be detected by iodination chemistry. Proteins were alkylated with beta-(4-hydroxyphenyl)ethyl iodoacetamide, or with 1,5-l-AEDANS (the Hudson Weber reagent). Subsequent iodination using the Iodo-Gen system was found to be most efficient. The enhanced sensitivity obtainable by using these approaches is expected to be sufficient for visualization of the lowest copy number proteins from human cells, such as from clinical samples. However, we argue that significantly improved methods of protein separation will be necessary to resolve the large number of proteins expected to be detectable with this sensitivity.

Artefactual gene induction during preparation of Xenopus laevis animal cap explants.

The animal cap assay in Xenopus laevis was used to study the induction and regulation of the mesoderm-specific gene Xegr-1, a homolog of the mammalian egr-1 genes. Egr-1 is an immediate-early gene whose growth factor-stimulated transcriptional induction displays a transient activity profile and occurs independent of protein synthesis. The Xegr-1 promoter contains multiple serum response elements (SREs). In this paper we show that Xegr-1 is induced unspecifically during the process of animal cap preparation. Transcripts of Xegr-1 appear already 30 min after cutting of animal caps. Xfos, another SRE-regulated immediate-early gene, is induced with the same kinetics as Xegr-1. In contrast, the Xbra gene is not induced under the experimental conditions used. Xfos and Xegr-1 transcripts are not rapidly down-regulated after mechanical stimulation, but can be detected for up to 4 h later. Wounding-dependent Xegr-1 induction is reduced by injection of either mRNA coding for the dominant inhibitory forms of both the FGF receptor and the transcription factor Elk-1. Xegr-1 expression can be reinduced by mesoderm-inducing factors. These results led us to develop a new protocol for animal cap preparation, which circumvents the observed undesired artefactual gene activation events.

Ca2+-induced p38/SAPK signalling inhibited by the immunosuppressant cyclosporin A in human peripheral blood mononuclear cells.

To understand the effects of the immunosuppressant cyclosporin A (CsA) on Ca2+-mediated intracellular signalling pathways in human peripheral blood mononuclear cells (PBMCs), we investigated its effects on the activity profiles of mitogen-activated protein kinase (MAPK) cascades. PBMCs, or subpopulations thereof, were simultaneously stimulated with a phorbol ester and the calcium ionophore ionomycin, in the presence or absence of therapeutic concentrations of CsA. In these primary human cells, CsA significantly inhibited PMA/ionomycin-mediated and ionomycin-mediated activation of the MAPK kinase MKK6, as well as its downstream kinases SAPK2a (p38alpha) and MAPKAP-K2. PMA/ionomycin treatment also mediated activation of SAPK1 (JNKs) which was inhibited by CsA. Treatment with ionomycin alone also resulted in CsA-sensitive activation of SAPK1. With regard to transcription factors targeted by the Ca2+-induced MAPK signalling network, we found CsA to inhibit the ionomycin-mediated phosphorylation of ATF2 at Thr71. We identified the heterodimeric transcription factor ATF2/CREB as constitutively binding to the essential cAMP response element (CRE) site within the Ca2+-regulated DNA polymerase beta promoter and contributing to the activation of this promoter. Our data implicate ATF2 phosphorylation status as a nuclear sensor within PBMCs that monitors converging intracellular Ca2+-signalling pathways.

Cyclosporin A inhibits Ca2+-mediated upregulation of the DNA repair enzyme DNA polymerase beta in human peripheral blood mononuclear cells.

Alterations in gene expression may represent an underlying cause of undesired side-effects mediated by the immunosuppressant cyclosporin A (CsA). We employed the method of differential display PCR to identify new genes whose expression is modulated by CsA. Human peripheral blood mononuclear cells (PBMCs), or subpopulations thereof, were simultaneously stimulated with the phorbol ester 4beta-phorbol 12-myristate 13-acetate (PMA) and the calcium ionophore ionomycin, in the presence or absence of therapeutic concentrations of CsA. We identify the gene encoding the DNA repair enzyme DNA polymerase beta (Pol beta) as a novel CsA-sensitive transcription unit. Our data show that transcription of pol beta mRNA is induced by Ca2+ and that CsA significantly inhibits PMA/ionomycin- and ionomycin-mediated upregulation of both pol beta mRNA and Pol beta protein. The CsA-mediated inhibition of pol beta upregulation is maintained for at least 21 h after gene activation and is exerted via the phosphatase calcineurin. FK506, another immunosuppressant that targets calcineurin, also inhibits pol beta upregulation, while rapamycin competes with FK506 action. This work identifies Ca2+ as an inducer of pol beta gene activity in primary blood cells. The demonstrated CsA sensitivity of this process suggests a novel molecular mechanism that may contribute to the increased tumor incidence in patients receiving CsA treatment.

Id genes are direct targets of bone morphogenetic protein induction in embryonic stem cells.

Bone morphogenetic proteins (BMPs) are morphogenetic signaling molecules essential for embryonic patterning. To obtain molecular insight into the influence of BMPs on morphogenesis, we searched for new genes directly activated by BMP signaling. In vitro cultured mouse embryonic stem (ES) cells were used, cultivated in chemically defined growth medium (CDM). CDM-cultured ES cells responded very selectively to stimulation by various mesoderm inducers (BMP2/4, activin A, and basic fibroblast growth factor). BMP2/4 rapidly induced transcript levels of the homeobox genes Msx-1 and Msx-2 and the proto-oncogene JunB, whereas c-jun transcripts displayed delayed albeit prolonged increase. Using differential display cDNA cloning, six direct BMP target genes were identified. These include Id3, which showed strong mRNA induction, and the moderately induced Cyr61, DEK, and eIF4AII genes, as well as a gene encoding a GC-binding protein. Besides Id3, also the Id1 and Id2 genes were activated by BMP4 in both ES cells and a range of different cell lines. Id genes encode negative regulators of basic helix-loop-helix transcription factors. In vivo we observed local ectopic expression of Id3 and Msx-2 mRNAs in Ft/+ embryos at overlapping regions of ectopic Bmp4 misexpression. We therefore propose that the Msx and Id genes are direct target genes of embryonic BMP4 signaling in vivo.

MAPKAP kinase 2 phosphorylates serum response factor in vitro and in vivo.

Several growth factor- and calcium-regulated kinases such as pp90(rsk) or CaM kinase IV can phosphorylate the transcription factor serum response factor (SRF) at serine 103 (Ser-103). However, it is unknown whether stress-regulated kinases can also phosphorylate SRF. We show that treatment of cells with anisomycin, arsenite, sodium fluoride, or tetrafluoroaluminate induces phosphorylation of SRF at Ser-103 in both HeLa and NIH3T3 cells. This phosphorylation is dependent on the kinase p38/SAPK2 and correlates with the activation of MAPKAP kinase 2 (MK2). MK2 phosphorylates SRF in vitro at Ser-103 with similar efficiency as the small heat shock protein Hsp25 and significantly better than CREB. Comparison of wild type murine fibroblasts with those derived from MK2-deficient mice (Mk(-/-)) reveals MK2 as the major SRF kinase induced by arsenite. These results demonstrate that SRF is targeted by several signal transduction pathways within cells and establishes SRF as a nuclear target for MAPKAP kinase 2.

Regulation of the human interleukin-5 promoter by Ets transcription factors. Ets1 and Ets2, but not Elf-1, cooperate with GATA3 and HTLV-I Tax1.

Interleukin-5 (IL-5), expressed primarily by type-2 T helper (Th2) cells, plays an important role in the development of allergic diseases, such as allergic asthma. Studying the regulation of IL-5 gene expression by Ets transcription factors, we found that Ets1 and Ets2, but not Elf-1, were able to activate the human IL-5 promoter in Jurkat T-cells. This required the presence of either phorbol 12-myristate acetate (PMA) plus ionomycin or PMA plus the viral protein HTLV-I Tax1. By mutation studies, it could be shown that Ets1 and Ets2 exerted their effects on the IL-5 promoter through a GGAA motif within the Cle0 element. In myeloid Kasumi cells, Ets1 and Ets2 failed to stimulate IL-5 promoter activity, unless the T-cell specific transcription factor GATA3 was added. These results show, for the first time, that Ets1 and Ets2 are able to cooperate with GATA3. Both ionomycin and Tax1 increased the combined effect of GATA3 with Ets1 and Ets2 in the presence of PMA. The data further demonstrate that, in addition to Ets1, Ets2 is also able to functionally cooperate with Tax1. The synergism of GATA3 with either Ets1 or Ets2 may play an important role in calcium- or Tax1-dependent regulation of IL-5 expression in Th2 cells or in HTLV-I transformed adult T-cell leukemia cells, respectively.

Characterization of the human elk-1 promoter. Potential role of a downstream intronic sequence for elk-1 gene expression in monocytes.

To characterize the human elk-1 promoter, we mapped the transcriptional start site and isolated elk-1-specific genomic phage clones that contained extensive upstream and downstream sequences. A TATA-like motif was identified immediately upstream of the transcriptional start site. Functional analyses of DNA fragments containing the TATA element and the identification of a DNase I-hypersensitive chromatin site (HS 1) in close proximity to the TATA box suggest that the identified TATA motif is important for elk-1 transcription in vivo. Sequences upstream and downstream from the TATA box were found to contribute to elk-1 promoter activity. A second hypersensitive site (HS 2) was identified within the first intron in pre-monocytic cells, which express Elk-1 only when differentiating to monocytes. In a variety of other cell types, which display a constitutive Elk-1 expression, HS 2 did not exist, suggesting that inducibility of elk-1 expression is associated with the presence of HS 2. Egr-1 and the serum response factor were found to interact specifically with the intronic sequence at +265 and +448, respectively. Because Egr-1 mRNA and protein levels were observed to increase significantly before induction of elk-1 expression, we propose that Egr-1 is important for the regulation of elk-1 transcription in differentiating monocytes.

Serum response factor is essential for mesoderm formation during mouse embryogenesis.

The transcription factor serum response factor (SRF), a phylogenetically conserved nuclear protein, mediates the rapid transcriptional response to extracellular stimuli, e.g. growth and differentiation signals. DNA- protein complexes containing SRF or its homologues function as nuclear targets of the Ras/MAPK signalling network, thereby directing gene activities associated with processes as diverse as pheromone signalling, cell-cycle progression (transitions G0-G1 and G2-M), neuronal synaptic transmission and muscle cell differentiation. So far, the activity of mammalian SRF has been studied exclusively in cultured cells. To study SRF function in a multicellular organism we generated an Srf null allele in mice. SRF-deficient embryos (Srf -/-) have a severe gastrulation defect and do not develop to term. They consist of misfolded ectodermal and endodermal cell layers, do not form a primitive streak or any detectable mesodermal cells and fail to express the developmental marker genes Bra (T), Bmp-2/4 and Shh. Activation of the SRF-regulated immediate early genes Egr-1 and c-fos, as well as the alpha-Actin gene, is severely impaired. Our study identifies SRF as a new and essential regulator of mammalian mesoderm formation. We therefore suggest that in mammals Ras/MAPK signalling contributes to mesoderm induction, as is the case in amphibia.

The Spemann organizer-expressed zinc finger gene Xegr-1 responds to the MAP kinase/Ets-SRF signal transduction pathway.

The transcriptional activity of a set of genes, which are all expressed in overlapping spatial and temporal patterns within the Spemann organizer of Xenopus embryos, can be modulated by peptide growth factors. We identify Xegr-1, a zinc finger protein-encoding gene, as a novel member of this group of genes. The spatial expression characteristics of Xegr-1 during gastrulation are most similar to those of Xbra. Making use of animal cap explants, analysis of the regulatory events that govern induction of Xegr-1 gene activity reveals that, in sharp contrast to transcriptional regulation of Xbra, activation of Ets-serum response factor (SRF) transcription factor complexes is required and sufficient for Xegr-1 gene expression. This finding provides the first indication for Ets-SRF complexes bound to serum response elements to be activated during gastrulation. MAP kinase signalling cascades can induce and sustain expression of both Xegr-1 and Xbra. Ectopic Xbra can induce Xegr-1 transcription by an indirect mechanism that appears to operate via primary activation of fibroblast growth factor secretion. These findings define a cascade of events that links Xbra activity to the signal-regulated control of Xegr-1 transcription in the context of early mesoderm induction in Xenopus laevis.