Madia Charlier

High homology between a trophoblastic protein (trophoblastin) isolated from ovine embryo and α‐interferons

Ovine trophoblastic protein B (oTPB), an embryonic protein, is a 20 kDa secretory protein which is synthesized by the ovine conceptus from days 12 to 22 of pregnancy. oTPB was purified by HPLC using ion‐exchange chromatography on a DEAE column and was subsequently chromatographed on a reversed‐phase column. Automated Edman degradation was then used to determine the N‐terminal amino acid sequence up to 45 residues. The sequence data reveal a significant homology between oTPB and bovine interferons α of class II: 64% of the amino acids are identical and 75% are homologous. A highly conserved region including residues 23–44 exhibits 82% homology. Identity between oTPB and either HuIFN‐α.9 or MuIFNα. 1 is 55%. These alignments between oTPB and IFNs occur at the N‐terminus of the mature proteins and proceed without deletion. These results suggest that oTPB is an embryonic interferon.

IFN-tau: a novel subtype I IFN1. Structural characteristics, non-ubiquitous expression, structure-function relationships, a pregnancy hormonal embryonic signal and cross-species therapeutic potentialities.

IFN-tau (IFN-tau) constitutes a new class of type I IFN which is not virus-inducible, unlike IFN-alpha and IFN-beta, but is constitutively produced by the trophectoderm of the ruminant conceptus during a very short period in early pregnancy. It plays a pivotal role in the mechanisms of maternal recognition of pregnancy in ruminants and it displays high antiviral and antiproliferative activities across species with a prominent lack of cytotoxicity at high concentrations in vitro in cell culture and possibly in vivo. It exhibits high antiretroviral activity against HIV and exhibits immunosuppressive activity in a multiple sclerosis model and reduces embryo and fetal mortality by stimulation of IL-10 production. In this review all the biochemical and para-hormonal properties of this novel IFN-tau are described in detail: structural characteristics of proteins and genes, trophoblast expression, regulation of its expression, structure of its gene promoter, its absence in human species and in non-ruminant animals, the evolution of the IFN-tau genes, its structure-function relationships with its three-dimensional structure, structural localization of biological activities, its lack of cytotoxicity and its receptor. Surprisingly, for an IFN, IFN-tau is also a pregnancy-embryonic signal with paracrine antiluteolytic activity. In order to maintain luteal progesterone secretion, IFN-tau inhibits PGF-2alpha pulsatile secretion and oxytocin uterine receptivity in early pregnancy. It is believed to suppress pulsatile release of endometrial PGF-2alpha by preventing oxytocin and estrogen receptor expression. Additionally, it directly regulates prostaglandin metabolism and possibly the PGE:PGF-2alpha ratio.

Messenger RNA Expression of Leptin and Leptin Receptors and their Prognostic Value in 322 Human Primary Breast Cancers

Purpose: Leptin and obesity are clearly related, and obesity is associated with an increased risk of breast cancer. We therefore measured the expression of leptin and its two main receptor isoforms, OBR-L and OBR-S, in 322 breast cancers. We analyzed their relations with the classical prognostic factors and with survival to establish their links with breast cancer. Experimental Design: The expression of leptin and its receptors was quantified by real-time reverse transcription-PCR, using TaqMan fluorogenic probes and an ABI PRISM 7700 sequence detector system (Applied Biosystems, Courtaboeuf, France). TATA box binding protein was used to normalize expression. The human breast cancer cell, SK-BR-3, expressing the three targets, was chosen as the calibrator sample (i.e., target expression = 1). Results: All the tumors expressed both receptors, and 318 of 322 expressed leptin. These three variables correlated positively with each other and with estradiol and progesterone receptors, whereas they correlated negatively with histoprognostic grading and tumor diameter. OBR-L/OBR-S expression was inversely correlated with progesterone receptors. Patients with elevated OBR-S expression had longer relapse-free survival (P = 0.008), whereas high OBR-L/OBR-S was associated with a shorter relapse-free survival (P = 0.05). In Cox multivariate analyses, OBR-S maintained its prognostic value (P = 0.02; relative risk, 0.51). Conclusions: This study shows that (a) almost all of the breast cancers coexpress leptin and its two main isoforms of receptors, suggesting that the human epithelial breast cancer cells respond to leptin acting via an autocrine pathway; (b) high expression levels of leptin and leptin receptors are biological markers of a more differentiated phenotype; and that (c) OBR-S is an independent prognostic factor.

Cloning and expression of cDNA encoding ovine trophoblastin: its identity with a class-II alpha interferon

The cDNAs encoding ovine trophoblastin (oTP) were isolated from an ovine embryo cDNA lambda gt 11 library by screening with a synthetic 29-mer oligodeoxynucleotide corresponding to amino acid (aa) residues 34 to 43 of oTP. The cDNA contained an open reading frame of 595 bp and the deduced amino acid sequence indicates a protein precursor of 195 aa. Nucleotide and amino acid sequence comparisons establish that oTP shares extensive homology with alpha-interferon (IFN-alpha) but is more closely related to the IFN-alpha sII subfamily. When the oTP cDNA was cloned into an eukaryotic expression vector and transfected in monkey COS cells, a high level of antiviral activity was detected. RNA blot analyses of total RNA reveal that the oTP-coding gene is expressed during a relatively short period (eleven to 21 days). The abundant expression of oTP mRNA corresponds closely to the time at which the embryo acts to extend luteal lifespan. RNAs homologous to oTP were also detected in goat and cow embryos at equivalent periods of their development, but not in the pig.

Transactivation of erbB2 by short and long isoforms of leptin receptors

We generated kinase‐positive and kinase‐negative erbB2 tagged with YFP and the long form of leptin receptor (LEPRb) tagged with CFP. Both were as active as their untagged analogs. Both short and long isoforms of leptin receptor phosphorylated and thereby activated erbB2 upon leptin binding and enhanced MAPK activity. Our results unveil a novel route by which leptin may provoke erbB2s phosphorylation and thus enhance its oncogenic potential independently of HER family ligands or its overexpression. Using FRET technology in living cells, we found no evidence of complex formation between erbB2 and prolactin or leptin receptors, indicating that the transactivation occurs through an indirect interaction.

Quantitative FRET imaging of leptin receptor oligomerization kinetics in single cells

Background Information. Leptin, an adipocyte‐secreted hormone, signals through activation of its membrane‐embedded receptor (LEPR). To study the leptin‐induced events occurring in short (LEPRa) and long (LEPRb) LEPRs in the cell membrane, by FRET (fluorescence resonance energy transfer) methodology, the respective receptors, tagged at their C‐terminal with CFP (cyan fluorescent protein) or YFP (yellow fluorescent protein), were prepared.

Addition of a dipeptide spacer significantly improves secretion of ovine trophoblast interferon in yeast.

Yeast has been analysed for its potential to secrete an ovine member of the type-I interferon (IFN) family, trophoblastin (oTP-1). The processing potential of the yeast KEX2 gene product (KEX2p) was evaluated using gene oTP-1 fused to the pre-pro sequence encoding the pre-pro peptide of the yeast alpha-factor precursor. High-level accumulation of nonprocessed (unmatured) recombinant oTP-1 (re-oTP-1) was observed in the medium. In order to short-circuit the limiting activity of KEX2p and to obtain a fully matured re-oTP-1, secretion was directed using a pre::oTP-1 fusion, relying only on signal peptidase-dependent processing. However, secretion of oTP-1 was impaired. High-level secretion was restored when the gene product contained a peptide spacer between oTP-1 and the signal peptidase cleavage site. The oTP-1 variant was shown to have an extended N terminus. An N-extended form was examined further and shown to have the correct size. Surprisingly, the variant retained its in vitro and in vivo biological activities. This system is likely to represent a general method for high-level secretion of type-I IFNs.

Increase in prolactin receptor (PRL-R) mRNA level in the mammary gland after hormonal induction of lactation in virgin ewes☆

In order to examine the hormonal regulation of the prolactin-receptor (PRL-R) gene expression during mammary gland development, ewes were treated to induce lactation via an estrogen-progesterone-hydrocortisone and ovine growth hormone treatment. In situ hybridization analysis was used and revealed that sex steroids increased PRL-R mRNA levels in the mammary gland. Using RNase protection assay we showed that the estradiol + progesterone treatment increased both the levels of the long and the short forms of PRL-R mRNA. Addition of hydrocortisone increased the level of alphaS1-casein transcripts and the level of the ratio of the long to the short form of the PRL-R mRNA. This ratio can be further enhanced by addition of ovine growth hormone to the latter treatment. This suggests a role of hydrocortisone and ovine growth hormone in the alternative splicing that leads to the preferential expression of the long form of the PRL-R mRNA. In conclusion, the present experiments suggest that estrogen, progesterone and hydrocortisone are the major regulators of the PRL-R gene expression during pregnancy and prepare the mammary gland for its differentiation.

DNA Methylation and Transcription in a Distal Region Upstream from the Bovine AlphaS1 Casein Gene after Once or Twice Daily Milking

Once daily milking (ODM) induces a reduction in milk production when compared to twice daily milking (TDM). Unilateral ODM of one udder half and TDM of the other half, enables the study of underlying mechanisms independently of inter-individual variability (same genetic background) and of environmental factors. Our results show that in first-calf heifers three CpG, located 10 kb upstream from the CSN1S1 gene were methylated to 33, 34 and 28%, respectively, after TDM but these levels were higher after ODM, 38, 38 and 33%, respectively. These methylation levels were much lower than those observed in the mammary gland during pregnancy (57, 59 and 50%, respectively) or in the liver (74, 78 and 61%, respectively). The methylation level of a fourth CpG (CpG4), located close by (29% during TDM) was not altered after ODM. CpG4 methylation reached 39.7% and 59.5%, during pregnancy or in the liver, respectively. CpG4 is located within a weak STAT5 binding element, arranged in tandem with a second high affinity STAT5 element. STAT5 binding is only marginally modulated by CpG4 methylation, but it may be altered by the methylation levels of the three other CpG nearby. Our results therefore shed light on mechanisms that help to explain how milk production is almost, but not fully, restored when TDM is resumed (15.1±0.2 kg/day instead of 16.2±0.2 kg/day, p<0.01). The STAT5 elements are 100 bp away from a region transcribed in the antisense orientation, in the mammary gland during lactation, but not during pregnancy or in other reproductive organs (ovary or testes). We now need to clarify whether the transcription of this novel RNA is a consequence of STAT5 interacting with the CSN1S1 distal region, or whether it plays a role in the chromatin structure of this region.

Cellular localization and evolution of prolactin receptor mRNA in ovine endometrium during pregnancy.

In this study, we have investigated the expression of the prolactin receptor gene in ovine endometrium during oestrus cycle and pregnancy. Using reverse transcription‐PCR analysis, we provided evidence that the prolactin receptor gene is specifically transcribed in this tissue. As shown by Northern blot analysis, the level of the prolactin receptor transcripts increased dramatically during late pregnancy. In situ hybridization experiments revealed that prolactin receptor mRNA was specifically expressed in the glandular compartment and confirmed the dramatic increase of its expression that occurs at the end of pregnancy. Taken together, these findings are consistent with a putative role of prolactin and/or related molecules in the regulation of the proliferation of the glandular compartment and/or in the control of the secretory activity of the endometrium.