FOXP3 can modulate TAL1 transcriptional activity through interaction with LMO2.

T-cell acute lymphoblastic leukemia (T-ALL) frequently involves aberrant expression of TAL1 (T-cell acute lymphocytic leukemia 1) and LMO2, oncogenic members of the TAL1 transcriptional complex. Transcriptional activity of the TAL1-complex is thought to have a pivotal role in the transformation of thymocytes and is associated with a differentiation block and self-renewal. The transcription factor Forkhead Box P3 (FOXP3) was recently described to be expressed in a variety of malignancies including T-ALL. Here we show that increased FOXP3 levels negatively correlate with expression of genes regulated by the oncogenic TAL1-complex in human T-ALL patient samples as well as a T-ALL cell line ectopically expressing FOXP3. In these cells, FOXP3 expression results in altered regulation of cell cycle progression and reduced cell viability. Finally, we demonstrate that FOXP3 binds LMO2 in vitro, resulting in decreased interaction between LMO2 and TAL1, providing a molecular mechanism for FOXP3-mediated transcriptional modulation in T-ALL. Collectively, our findings provide initial evidence for a novel role of FOXP3 as a tumor suppressor in T-ALL through modulation of TAL1 transcriptional activity.

Cytokine-specific transcriptional regulation through an IL-5Ralpha interacting protein.

Cytokine receptors consist of multiple subunits, which are often shared between different receptors, resulting in the functional redundancy sometimes observed between cytokines. The interleukin 5 (IL-5) receptor consists of an IL-5-specific alpha-subunit (IL-5Ralpha) and a signal-transducing beta-subunit (betac) shared with the IL-3 and granulocyte-macrophage colony-stimulating factor (GM-CSF) receptors. In this study, we sought to find a role for the cytoplasmic domain of IL-5Ralpha. We show that syntenin, a protein containing PSD-95/Discs large/zO-1 (PDZ) domains, associates with the cytoplasmic tail of the IL-5Ralpha. Syntenin was found to directly associate with the transcription factor Sox4. Association of syntenin with IL-5Ralpha was required for IL-5-mediated activation of Sox4. These studies identify a mechanism of transcriptional activation by cytokine-specific receptor subunits.

Forkhead transcription factor FKHR-L1 modulates cytokine-dependent transcriptional regulation of p27(KIP1).

Interleukin-3 (IL-3), IL-5, and granulocyte-macrophage colony-stimulating factor regulate the survival, proliferation, and differentiation of hematopoietic lineages. Phosphatidylinositol 3-kinase (PI3K) has been implicated in the regulation of these processes. Here we investigate the molecular mechanism by which PI3K regulates cytokine-mediated proliferation and survival in the murine pre-B-cell line Ba/F3. IL-3 was found to repress the expression of the cyclin-dependent kinase inhibitor p27(KIP1) through activation of PI3K, and this occurs at the level of transcription. This transcriptional regulation occurs through modulation of the forkhead transcription factor FKHR-L1, and IL-3 inhibited FKHR-L1 activity in a PI3K-dependent manner. We have generated Ba/F3 cell lines expressing a tamoxifen-inducible active FKHR-L1 mutant [FKHR-L1(A3):ER*]. Tamoxifen-mediated activation of FKHR-L1(A3):ER* resulted in a striking increase in p27(KIP1) promoter activity and mRNA and protein levels as well as induction of the apoptotic program. The level of p27(KIP1) appears to be critical in the regulation of cell survival since mere ectopic expression of p27(KIP1) was sufficient to induce Ba/F3 apoptosis. Moreover, cell survival was increased in cytokine-starved bone marrow-derived stem cells from p27(KIP1) null-mutant mice compared to that in cells from wild-type mice. Taken together, these observations indicate that inhibition of p27(KIP1) transcription through PI3K-induced FKHR-L1 phosphorylation provides a novel mechanism of regulating cytokine-mediated survival and proliferation.

Identification of cytokine-regulated genes in human leukocytes in vivo.

BACKGROUND: Human polymorphic nuclear granulocytes (PMNs) such as neutrophils and eosinophils play a critical role in mediating inflammatory responses to microbial and parasitic infections. Exposure of these leukocytes to cytokines leads to an amplification of granulocyte effector functions by a mechanism termed "priming." Although many studies have investigated the effects of granulocyte priming, little is known concerning the molecular mechanisms that lead to this phenomenon. OBJECTIVE: The purpose of this study was to identify potential markers for granulocyte priming and thus also to gain further insight into the pathogenesis of inflammatory responses. METHODS: We used a modified differential display technique, random arbitrary primed-PCR to identify genes regulated during the priming of human polymorphic nuclear granulocytes by GM-CSF in vitro. Genes identified were validated by Northern blot analysis of in vitro and in vivo primed leukocytes. RESULTS: Several genes were identified and their expression characterized in vitro. One of these genes, 5-lipoxygenase-activating protein, was also found to be up-regulated in leukocytes isolated after allergen challenge of allergic asthmatic patients. CONCLUSION: The use of differential display technology is a rapid and effective means of identifying genes whose expression is regulated by priming in vitro and in vivo. Further analysis will lead to a better understanding of the priming phenotype and may provide further insight into the pathologic mechanisms of inflammatory processes.

Stimulation of receptor protein-tyrosine phosphatase alpha activity and phosphorylation by phorbol ester.

Receptor Protein-Tyrosine Phosphatase alpha (RPTP alpha) is a transmembrane protein with two cytoplasmic catalytic protein-tyrosine phosphatase (PTP) domains and a relatively short (123 amino acids) extracellular domain. Here we report that treatment of transfected cells that express RPTP alpha with the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate, a direct activator of protein kinase C, induced a rapid, transient increase in RPTP alpha activity due to a 2- to 3-fold increase in substrate affinity. A transient increase in RPTP alpha serine phosphorylation was concomitant with the enhanced activity. Tryptic phosphopeptide mapping of RPTP alpha demonstrated that phosphorylation of three tryptic peptides was enhanced in response to phorbol ester. In vitro dephosphorylation of RPTP alpha from phorbol ester-treated cells reduced RPTP alpha activity to prestimulation levels, indicating that enhanced serine phosphorylation directly accounted for the increase in activity. Our results demonstrate that serine phosphorylation may play a key role in the regulation of the activity of transmembrane PTPs.

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.

Receptor protein tyrosine phosphatase alpha activates pp60c-src and is involved in neuronal differentiation.

Here we report that protein tyrosine phosphatases (PTPases), like their enzymatic counterpart the protein tyrosine kinases, can play an important role in cell differentiation. Expression of the transmembrane PTPase receptor protein tyrosine phosphatase alpha (RPTP alpha) is transiently enhanced during neuronal differentiation of embryonal carcinoma (EC) and neuroblastoma cells. Retinoic acid induces wild type P19 cells to differentiate into endoderm- and mesoderm-like cells. By contrast, retinoic acid treatment leads to neuronal differentiation of P19 cells, ectopically expressing functional RPTP alpha, as illustrated by their ability to generate action potentials. Endogenous pp60c-src kinase activity is enhanced in the RPTP alpha-transfected cells, which may be due to direct dephosphorylation of the regulatory Tyr residue at position 527 in pp60c-src by RPTP alpha. Our results demonstrate that RPTP alpha is involved in neuronal differentiation and imply a role for pp60c-src in the differentiation process.

Differential expression of a novel murine non-receptor protein tyrosine phosphatase during differentiation of P19 embryonal carcinoma cells.

Protein phosphorylation on tyrosine residues is one of the major mechanisms of cell signal transduction and is regulated by protein tyrosine kinases and protein tyrosine phosphatases. Here we report the molecular cloning of an additional member of the protein tyrosine phosphatase-family from differentiated murine P19 embryonal carcinoma cells. This non-receptor protein tyrosine phosphatase, P19-PTP, does not contain regulatory sequences, homologous to the ones found in other non-receptor PTPases. P19-PTP is differentially expressed during in vitro differentiation of P19 EC cells, in that P19-PTP mRNA could only be detected in embryoid bodies, derived from P19 cells.

Characterization of the mouse junD promoter--high basal level activity due to an octamer motif.

The product of the junD gene belongs to the Jun/Fos family of nuclear DNA binding transcription factors. This family regulates the expression of TPA responsive genes by binding to the TPA responsive element (TRE). Unlike its counterparts c-jun and junB, junD expression is hardly inducible by growth factors and phorbol esters. In fact, junD is constitutively expressed at high levels in a wide variety of cells. To unravel the molecular mechanisms underlying constitutive junD expression, we have cloned and characterized the mouse junD promoter. We show that the high constitutive expression is caused by multiple cis-acting elements in its promoter, including an SP1 binding site, an octamer motif, a CAAT box, a Zif268 binding site and a TRE-like sequence. The octamer motif is the major determinant of junD promoter activity, while somewhat smaller contributions are made by the TRE and Zif268 binding site. The SP1 and CAAT box are shown to be of minor importance. The junD TRE is in its behavior indistinguishable from previously identified TREs. However, the junD promoter is not TPA inducible due to the presence of the octamer motif.

Transcriptional control of c-jun by retinoic acid.

The proto-oncogene c-jun, a major component of transcription factor AP-1, is expressed at very low levels in undifferentiated embryonal carcinoma (EC) end embryonic stem (ES) cells. Retinoic acid (RA) induced differentiation causes a strong increase in the levels of c-jun mRNA. In this paper we report the cloning and characterization of the mouse c-jun promoter. Our results show that RA treatment causes a strong enhancement in c-jun promoter activity, an effect probably mediated by the RA-receptor beta (RAR beta). Sequences located between -329 and -293 are responsible for the observed RA effect, and bind at least five different protein complexes, of which three are decreased upon RA treatment. These protein binding sites do not resemble RA-responsive elements (RARE's) found in the promoters of retinoic acid receptor beta (RAR beta) and laminin B1. Furthermore, we could not detect a direct interaction of RAR alpha and RAR beta to these sequences, indicating that RA-induced c-jun expression is an indirect effect of RAR action.

Evidence for the involvement of tyrosine-69 in the control of stereospecificity of porcine pancreatic phospholipase A2.

We have studied the role of Tyr-69 of porcine pancreatic phospholipase A2 in catalysis and substrate binding, using site-directed mutagenesis. A mutant was constructed containing Phe at position 69. Kinetic characterization revealed that the Phe-69 mutant has retained enzymatic activity on monomeric and micellar substrates, and that the mutation has only minor effects on kcat and Km. This shows that Tyr-69 plays no role in the true catalytic events during substrate hydrolysis. In contrast, the mutation has a profound influence on the stereospecificity of the enzyme. Whereas the wild-type phospholipase A2 is only able to catalyse the degradation of sn-3 phospholipids, the Phe-69 mutant hydrolyses both the sn-3 isomers and, at a low (1-2%) rate, the sn-1 isomers. Despite the fact that the stereospecificity of the mutant phospholipase has been altered, Phe-69 phospholipase still requires Ca2+ ions as a cofactor and also retains its specificity for the sn-2 ester bond. Our data suggest that in porcine pancreatic phospholipase A2 the hydroxyl group of Tyr-69 serves to fix and orient the phosphate group of phospholipid monomers by hydrogen bonding. Because no such interaction can occur between the Phe-69 side-chain and the phosphate moiety of the substrate monomer, the mutant enzyme loses part of its stereospecificity but not its positional specificity.