RXRalpha regulates the pregnancy-specific glycoprotein 5 gene transcription through a functional retinoic acid responsive element.

Human pregnancy-specific glycoproteins (PSG) are major placental polypeptides encoded by eleven highly conserved genes expressed by the syncytiotrophoblast. The minimal promoter region of all PSG genes contains a putative Retinoic Acid Responsive Element (RARE) though the ability of retinoids to regulate PSG gene expression has not been established. Retinoid signaling pathway plays a key role for overall placenta biology and is essential for trophoblast differentiation. In this work, we investigated the participation of the RARE motif in the regulation of PSG5 gene transcription by retinoic acid and its receptors. The minimal promoter region of PSG5 gene was activated by RXRalpha but not by RARalpha, in a ligand-dependent manner. The RARE sequence of PSG5 gene promoter was recognized by endogenous RXRalpha present in placental nuclear extracts as well as by RXRalpha either over expressed in cultured non-placental cells or in vitro translated. Mutations at specific nucleotides within the RARE motif abrogated both RXRalpha DNA binding and transcriptional activation of PSG5 promoter mediated by RXRalpha. Moreover, endogenous PSG expression was significantly induced in trophoblast-derived Jeg-3 cells upon 9-cis retinoic acid treatment. Interestingly, the induction level was higher following methotrexate-induced differentiation of Jeg-3 cells to syncytiotrophoblast-like structures. Altogether, these data provide the first evidences demonstrating that transcriptional activity of PSG5 gene is responsive to an external signal involving the retinoids-RXRalpha axis through a conserved RARE motif shared by all PSG gene family members.

A new death domain associated with gestational trophoblastic diseases induces apoptosis in distinct cell types.

Gestational trophoblastic diseases, like the complete hydatidiform mole (CHM), are a group of human interrelated neoplasms whose etiology and progression is poorly understood at the molecular level. We have previously reported the cloning and expression of a new tumor necrosis factor receptor (TNF-R) related transcript, named CHMS-1 that encodes a potential death domain. Here we show that ectopic expression of the putative CHMS-1 death domain specifically induced apoptosis in a dose-dependent manner, in trophoblastic (JEG-3) and non-trophoblastic (COS-7) cells. We also investigated the expression of apoptosis-related molecules such as Bcl-2 and p53 and demonstrated that Bcl-2 is repressed in CHM while p53 is overexpressed in CHM compared with persistent gestational trophoblastic tumors. Altogether, these data indicate that the CHMS-1 death domain is able to trigger apoptosis, thus suggesting that this new entity might be an important inducer of molar regression mechanisms in women.

The nuclear orphan receptor TR4 promotes proliferation of myeloid progenitor cells.

Nuclear receptors represent key regulators in cell proliferation, differentiation, and development. Here we demonstrate that the nuclear orphan receptor TR4 is highly expressed in hematopoietic cells and tissues and have analyzed the impact of TR4 in this cell compartment. We show that TR4, when ectopically expressed in bone marrow cells via retrovirus vector, promotes proliferation of myeloid progenitor cells. Cells represent promyelocytes as judged by morphological features, expression of cell surface molecules, and specific markers like Mim-1 and CAAT/enhancer binding protein beta. We also demonstrate that the growth promoting activity of TR4 is not exclusively dependent on its association with DNA, because expression of a mutated TR4 version devoid of its DNA binding domain exhibits a similar proliferative potential as wild-type TR4. In conclusion, these data position the orphan receptor TR4 as an important regulator of myeloid progenitor cell proliferation and development.

Molecular cloning, expression and regulation of the avian tubby-like protein 1 (tulp1) gene.

The tubby-like protein 1 (tulp1) gene is a member of the tubby multigene family which includes tub, tulp1, tulp2 and tulp3. Human and mouse tulp1 genes were cloned and mutations in tulp1 have been implicated in retinitis pigmentosa in man. Here we report on the cDNA cloning of the chicken tulp1 homologue and its protein product deduced from the nucleotide sequence. The chicken Tulp1 protein comprises 358 amino acids with a calculated molecular mass of 40 kDa. The overall structure of Tub and Tulp proteins, exemplified by the highly conserved C-terminal domain of 255 amino acids and the signature motif KLACE, is also preserved in chicken Tulp1. Phylogenetic analysis demonstrates that chicken tulp1 cDNA and protein are closely related to human and mouse tulp1. In addition, chicken tulp1 mRNA is abundantly expressed in retina similar to tulp1 expression in human and mouse. Two tulp1-specific transcripts of 2 and 3 kb in size were identified that showed differential regulation during embryonic and postnatal development. Finally, tulp1 mRNA was found to be expressed in chicken erythroid cells and upregulated by ligand-activated thyroid hormone receptor (TR alpha/c-erbA).

Thyroid hormone regulates the obesity gene tub.

Thyroid hormone T3/T4 is a major regulator of energy metabolism in vertebrates, and defects in thyroid status are frequently associated with changes in body weight. It is demonstrated here that thyroid hormone regulates in vivo and in vitro the tub gene, which when mutated in tubby mice causes obesity, insulin resistance and sensory deficits. Hypothyroidism in rats altered tub mRNA and protein in discrete brain areas. These changes could be attributed to thyroid hormone deficiency since T3/T4 treatment restored normal tub expression. T3 also upregulated tub mRNA within 4-6 h in neuronal cells in culture, suggesting that T3 is a positive regulator of tub gene expression. Thus, these results establish a novel pathway of T3 action and provide an important molecular link between thyroid status and the tubby-associated syndrome.

Transcription of genes encoding pregnancy-specific glycoproteins is regulated by negative promoter-selective elements.

The human pregnancy-specific glycoprotein (PSG) genes comprise a family of 11 highly conserved members whose expression is maximal in placental cells and marginal in other cell types. We have investigated here the molecular basis of PSG regulation by analysing a large regulatory region of the PSG-5 gene in cells that do and do not express these genes. The promoter region (-254 to -43), which does not contain a TATA-box, large GC-rich sequences or a classical initiator, was active in all cell types analysed. Additional upstream sequences up to position -3204 repressed promoter activity. Two independent repressor regions were identified and found to operate effectively in HeLa, COS-7 and HTR8/SVneo placental cells. More significantly, these negatively acting modules failed to repress a heterologous TATA-containing thymidine kinase promoter. Detailed transcriptional and DNA-protein analyses of the proximal repressor region (-605 to -254) revealed the presence of both negative and positive cis-acting elements. Disruption of the repressive functions resulted in an enhanced transcription of the reporter constructs. In conclusion, these results demonstrate that PSG-5 gene transcription is highly repressed by promoter-selective negative regulatory regions and the relief of repression allows enhanced PSG-5 gene transcription irrespective of the cell type. Furthermore, our findings suggest that PSG genes are expressed mainly through a derepression mechanism.

The Krüppel-like core promoter binding protein gene is primarily expressed in placenta during mouse development.

The human core promoter binding protein (hCPBP) has been identified as a DNA-binding protein involved in the regulation of TATA box-less genes like those encoding the pregnancy-specific glycoproteins. Structurally, hCPBP contains three zinc fingers in the C-terminal domain, which is highly conserved in a number of proteins that constitute the Krüppel-like family of transcription factors. In the present work, we report the molecular cloning of the mouse CPBP (mCPBP) and its expression pattern during development as well as in adult tissues. The mouse cDNA encodes a protein of 283 amino acids that share 94.4% of identity with the hCPBP. The highest level of mCPBP transcript was detected in placenta, and its expression was lower in total embryos and in adult tissues. We also show by in situ hybridization that during embryonic development the mCPBP gene is mainly expressed in extra-embryonic structures throughout gestation; essentially no specific expression was detected in embryonic tissues. Our data demonstrate that CPBP transcript is enriched in the trophoblastic tissue and strongly suggest that its encoded polypeptide regulates target genes involved in placental development and pregnancy maintenance.

The fibroblast growth factor receptor FGFR-4 acts as a ligand dependent modulator of erythroid cell proliferation.

Receptor and non-receptor tyrosine kinases constitute a large family of proteins that play a pivotal role in hematopoiesis. Here we conducted a comprehensive survey of tyrosine kinase gene expression in primary erythroid progenitor cells from bone marrow by employing a PCR-based strategy that targets the conserved kinase encoding region. We demonstrate that erythroid progenitor cells express several receptor and non-receptor tyrosine kinases, like c-kit, Jak1, Ryk, FAK, Syk, Arg, Csk and members of the insulin receptor family. Specific changes in the expression profile of tyrosine kinases were observed following differentiation induction. We also report on the identification of a new ligand dependent modulator of erythropoiesis, fibroblast growth factor receptor-4 (FGFR-4). FGFR-4 is effectively expressed in erythroid progenitors and downregulated when cells differentiate. Furthermore, the FGFR-4 ligand, basic fibroblast growth factor (bFGF), enhanced erythroid cell proliferation induced by SCF or insulin, and thus modulated both erythroid proliferation and differentiation in vitro.

Polarised expression pattern of focal contact proteins in highly motile antigen presenting dendritic cells.

Dendritic cells are professional antigen presenting cells that capture antigens and migrate to lymphoid tissues to elicit specific T cell responses. Here we used an in vitro differentiation system for generating highly motile dendritic cells from chicken bone marrow progenitors by employing the conditional v-Rel estrogen receptor (ER) fusion protein v-RelER. Molecular mechanisms of dendritic cell motility were investigated. Differentiation of v-relER progenitors into dendritic cells is associated with a reduction in cell-cell and cell-extracellular matrix interactions as cells acquire motility. We demonstrate that v-relER progenitors and dendritic cells express several adhesion receptors and components of adhesion complexes. Differentiation of v-relER cells was accompanied by downregulation of focal adhesion kinase (FAK), a key molecule of adhesion complexes, but ectopic FAK expression did not affect cell adhesion and motility. Interestingly, v-relER dendritic cells exhibit a polarised expression pattern of actin and vimentin, with actin being highly concentrated at the leading edge of the cells where lamellipodia are formed. FAK, paxillin and tyrosine phosphorylated proteins are found at both poles of the cell and colocalise with actin at the leading edge, while surface beta1 integrin is confined to the uropod at the rear. CD34(+ )stem cell-derived human dendritic cells also exhibited an elongated bipolar morphology, mode of migration and a polarised pattern of actin-vimentin expression similar to v-relER dendritic cells.

Molecular cloning, expression and evolutionary analysis of the avian tyrosine kinase JAK1.

The Janus protein tyrosine kinases (JAK) constitute a protein family that plays a pivotal role in signalling of a large number of cytokine receptors. The cDNA of the chicken homologue of JAK1 was cloned and its nucleotide sequence determined. Chicken JAK1 protein comprises 1150 amino acids as deduced from its cDNA sequence with a calculated molecular mass of 133kDa. The overall structure of JAK proteins exemplified by the JAK homology domains JH1-JH7 is also preserved in chicken JAK1. Additionally, phylogenetic analysis demonstrates that chicken JAK1 is more closely related to mammalian JAK1 than to those of fish, exhibiting 80%, 79% and 63% identity in amino acid sequence to human, mouse and zebrafish JAK1, respectively. JAK1 proteins were found to be most conserved in the kinase (JH1) and pseudokinase (JH2) domains. This data is supported by Southern hybridization studies of ZOO blots. Chicken JAK1 shows a ubiquitous expression pattern and is transcribed as a 5.5kb mRNA in various tissues and cell types. JAK1 expression was particularly high in lymphoid cells.

Differential expression of a tumor necrosis factor receptor-related transcript in gestational trophoblastic diseases in women.

Gestational trophoblastic diseases comprise a group of interrelated neoplasms, including complete hydatidiform mole (CHM), persistent gestational trophoblastic tumor (GTT), and choriocarcinoma. To better define the molecular features of these diseases, a CHM cDNA library was constructed and a novel cDNA sequence, named CHMS-1, was isolated by differential screening. The CHMS-1 sequence showed a 62% homology with the tumor necrosis factor receptor (TNF-R2) cDNA, and its amino acid deduced sequence shared a high level of homology with the "death domain" region found in various proteins, including two members of the TNF receptor superfamily, the TNF-R1 and Fas. We also determined the CHMS-1, TNF-R1, and TNF-R2 expression patterns among different CHM tissues and cell lines of trophoblastic (JEG-3) and nontrophoblastic (HeLa and COS-7) origin. Our results indicated that the CHMS-1 transcript is highly expressed in CHM in comparison with both normal early and term placenta and that it exhibits an expression profile identical to that of TNF-R1. Furthermore, the CHMS-1 transcript was undetectable in CHM-derived GTT and in the human choriocarcinoma-derived JEG-3 cells, suggesting that its expression is down-regulated in the malignant transformation of trophoblast. The presence of a potential "death domain" in CHMS-1, together with its high expression level in CHM, strongly suggests that the CHMS-1 gene encodes a protein that might be involved in tumor regression processes occurring at later stages of molar development.

A novel human zinc finger protein that interacts with the core promoter element of a TATA box-less gene.

We describe a novel human cDNA isolated by target site screening of a placental expression library, using as a probe, an essential element of a TATA box-less promoter corresponding to a pregnancy-specific glycoprotein gene. The cDNA encoded a predicted protein of 290 amino acids, designated core promoter-binding protein (CPBP), which has three zinc fingers (type Cys2-His2) at the end of its C-terminal domain, a serine/threonine-rich central region and an acidic domain lying within the N-terminal region. Additional sequence analysis and data base searches revealed that only the zinc finger domains are conserved (60-80% identity) in other transcription factors. In cotransfection assays, CPBP increased the transcription from a minimal promoter containing its natural DNA-binding site. Moreover, a chimeric protein between CPBP and Gal4 DNA binding domain also increased the activity of an heterologous reporter gene containing Gal4 DNA binding sites. The tissue distribution analysis of CPBP mRNA revealed that it is differentially expressed with an apparent enrichment in placental cells. The DNA binding and transcriptional activity of CPBP, in conjunction with its expression pattern, strongly suggests that this protein may participate in the regulation and/or maintenance of the basal expression of PSG and possibly other TATA box-less genes.

Analyses of cis-acting and trans-acting elements that are crucial to sustain pregnancy-specific glycoprotein gene expression in different cell types.

Pregnancy-specific beta 1 glycoprotein genes (PSG) are mainly expressed during human placental development, though their expression has been reported in other normal and pathological tissues, e.g. hydatidiform mole (HM), of distinct origins. However, the molecular components implicated in the regulation of PSG are not well understood. To identify some of the regulatory elements involved in the transcriptional control of PSG expression, the DNA-protein interactions and the basal activities of the TATA-box-less PSG5 promoter were determined in different tissues and cell types. In DNAse-I protection assays, DNA-binding proteins from human term placenta (HTP) protected a region of 27 bp located from nucleotides --150 to --124, overlapping the farthest 5' upstream cap site and resembling an initiator-like element. In electrophoretic mobility shift assays (EMSA), three complexes were detected using nuclear extracts from HTP and an oligonucleotide containing the 27-bp motif. In situ ultraviolet crosslinking analysis of the specific complexes revealed that two proteins of 78.0 kDa and 53.0 kDa are involved in such interactions, in accordance with the bands of 80.0 kDa and 57.5 kDa observed by Southwestern blotting. Competitive EMSA using mutant oligonucleotides with the substitution of 5'ACCCAT3' by 5'GATATC3' within the 27-bp motif revealed that this sequence is fundamental for the formation of the specific DNA-protein complexes. We show in transient transfection experiments performed in HeLa, COS-7 and JEG-3 cells, that such mutation completely abolished the transcriptional activity of the PSG5 promoter, independently of the cell type. Moreover, this mutation disrupted the formation of the specific DNA-protein complexes which were essentially the same as those displayed by HTP. We also determined the binding activities of nucleoproteins derived from placental tissues in earlier developmental and pathological stages, i.e. first trimester placenta (1-TRIM) and HM, respectively, showing that the DNA-binding patterns were different from each other and distinct from those elicited by HTP. Our results indicate that the cis-acting and trans-acting elements analyzed are indispensable to support PSG5 promoter activity in cell lines which do or do not produce PSG. In addition, these elements appear to play a role in the mechanisms involved in PSG basal expression during placental development and differentiation.