Monique Benaïssa

Expression and prognostic value of lactoferrin mRNA isoforms in human breast cancer

We investigated the expression levels of human lactoferrin (Lf), a steroid hormone‐inducible gene product the expression of which is often altered during oncogenesis, and of Δ‐lactoferrin (ΔLf), its alternative isoform, which has been shown to be absent from tumor cell lines in commonly used human breast epithelial cell lines, using semiquantitative RT‐PCR. Both mRNAs were detected but with levels of expression lower than those found in normal breast epithelial cells. This downregulation was much more visible for ΔLf since its expression was either significantly diminished (BT‐20, MCF‐7 cell lines) or practically absent (MDA‐MB‐231, T‐47D, HBL 100 cell lines). In order to determine whether Lf gene products are useful prognosic tools, we further analyzed their expression levels in 99 primary breast cancer biopsies. ΔLf transcripts were found in all of the samples, whereas Lf transcripts were found in 88% of them. Lf and ΔLf expression levels were positively correlated (p = 0.003). Lf expression was related to tumor type with a higher recovery in lobular‐type tumors (p = 0.04). ΔLf expression was related to the histoprognostic grading (p = 0.02). In univariate analyses, ΔLf and Lf expressions were prognosis parameters, high concentrations being associated with a longer overall survival.

Lactoferrin is synthesized by mouse brain tissue and its expression is enhanced after MPTP treatment

The biological role and origin of human lactoferrin (Lf) within the brain in normal and disease processes are as yet uncharted. In this context the origin and expression of brain Lf in normal and MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine)-treated mice were investigated using immunohisto chemistry, PCR amplification and in situ hybridization. Lf immunostaining was observed both on sections of mouse lactating mammary gland, which was used as a positive control, and brains from young, adult and aged mice. Lf immunoreactivity was present in the pituitary gland, the hippocampus and the cortex of mouse brains and to a greater extent in older mice. After reverse transcription, Lf transcripts were also found in these brain sections. Lf distribution and expression in the MPTP-induced parkinsonian mouse model were next investigated. A marked depletion of dopamine and its metabolites: dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA) and 5-hydroxy indole acetic acid (5-HIAA) occurs in the high dose MPTP-treated mice. The level of Lf expression was found to be greatly increased in the same animals but Lf immunoreactivity detected in the same brain region was not found increased in the affected areas.

Tumor Necrosis Factor-α Increases Lactoferrin Transcytosis Through the Blood-Brain Barrier

Abstract : Lactoferrin (Lf) is an iron‐binding protein involved in host defense against infection and severe inflammation, which accumulates in the brain during neurodegenerative disorders. Prior to determining Lf function in pathological brain tissues, we investigated its transport through the blood‐brain barrier (BBB) in inflammatory conditions. For this purpose, we used a reconstituted BBB model consisting of the coculture of bovine brain capillary endothelial cells (BBCECs) and astrocytes in the presence of tumor necrosis factor‐α (TNF‐α). As TNF‐α can be either synthesized by brain glial cells or present in circulating blood, BBCECs were exposed to this cytokine at their luminal or abluminal side. We have been able to demonstrate that in the presence of TNF‐α, whatever the type of exposure, BBCECs were activated and Lf transport through the activated BBCECs was markedly increased. Lf was recovered intact at the abluminal side of the cells, suggesting that increased Lf accumulation may occur in immune‐mediated pathophysiology. This process was transient as 20 h later, cells were in a resting state and Lf transendothelial traffic was back to normal. The enhancement of Lf transcytosis seems not to involve the up‐regulation of the Lf receptor but rather an increase in the rate of transendothelial transport.

Human delta-lactoferrin is a transcription factor that enhances Skp1 (S-phase kinase-associated protein) gene expression.

Delta‐lactoferrin is a cytoplasmic lactoferrin isoform that can locate to the nucleus, provoking antiproliferative effects and cell cycle arrest in S phase. Using macroarrays, the expression of genes involved in the G1/S transition was examined. Among these, Skp1 showed 2–3‐fold increased expression at both the mRNA and protein levels. Skp1 (S‐phase kinase‐associated protein) belongs to the Skp1/Cullin‐1/F‐box ubiquitin ligase complex responsible for the ubiquitination of cellular regulators leading to their proteolysis. Skp1 overexpression was also found after delta‐lactoferrin transient transfection in other cell lines (HeLa, MDA‐MB‐231, HEK 293) at comparable levels. Analysis of the Skp1 promoter detected two sequences that were 90% identical to those previously known to interact with lactoferrin, the secretory isoform of delta‐lactoferrin (GGCACTGTAC‐S1Skp1, located at − 1067 bp, and TAGAAGTCAA‐S2Skp1, at − 646 bp). Both gel shift and chromatin immunoprecipitation assays demonstrated that delta‐lactoferrin interacts in vitro and in vivo specifically with these sequences. Reporter gene analysis confirmed that delta‐lactoferrin recognizes both sequences within the Skp1 promoter, with a higher activity on S1Skp1. Deletion of both sequences totally abolished delta‐lactoferrin transcriptional activity, identifying them as delta‐lactoferrin‐responsive elements. Delta‐lactoferrin enters the nucleus via a short bipartite RRSDTSLTWNSVKGKK(417–432) nuclear localization signal sequence, which was demonstrated to be functional using mutants. Our results show that delta‐lactoferrin binds to the Skp1 promoter at two different sites, and that these interactions lead to its transcriptional activation. By increasing Skp1 gene expression, delta‐lactoferrin may regulate cell cycle progression via control of the proteasomal degradation of S‐phase actors.

Two Distinct Regions of Cyclophilin B Are Involved in the Recognition of a Functional Receptor and of Glycosaminoglycans on T Lymphocytes

Cyclophilin B is a cyclosporin A-binding protein exhibiting peptidyl-prolyl cis/trans isomerase activity. We have previously shown that it interacts with two types of binding sites on T lymphocytes. The type I sites correspond to specific functional receptors and the type II sites to sulfated glycosaminoglycans. The interactions of cyclophilin B with type I and type II sites are reduced in the presence of cyclosporin A and of a synthetic peptide mimicking the N-terminal part of cyclophilin B, respectively, suggesting that the protein possesses two distinct binding regions. In this study, we intended to characterize the areas of cyclophilin B involved in the interactions with binding sites present on Jurkat cells. The use of cyclophilin B mutants modified in the N-terminal region demonstrated that the 3Lys-Lys-Lys5 and14Tyr-Phe-Asp16 clusters are probably solely required for the interactions with the type II sites. We further engineered mutants of the conserved central core of cyclophilin B, which bears the catalytic and the cyclosporin A binding sites as an approach to localize the binding regions for the type I sites. The enzymatic activity of cyclophilin B was dramatically reduced after substitution of the Arg62 and Phe67residues, whereas the cyclosporin A binding activity was destroyed by mutation of the Trp128 residue and strongly decreased after modification of the Phe67 residue. Only the substitution of the Trp128 residue reduced the binding of the resulting cyclophilin B mutant to type I binding sites. The catalytic site of cyclophilin B therefore did not seem to be essential for cellular binding and the cyclosporin A binding site appeared to be partially involved in the binding to type I sites.

Expression of delta-lactoferrin induces cell cycle arrest

Delta-lactoferrin (ΔLf) mRNA is the product of alternative splicing of the Lf gene. It has been found in normal tissues and was reported to be absent from their malignant counterparts. Our recent investigations have shown that ΔLf expression is a good prognostic indicator in human breast cancer. However, ΔLf has up till now only been identified as a transcript, and in order to characterize the ΔLf protein and determine its function we have used a ΔLf cDNA construct to produce the protein in vitro and in vivo.A 73 kDa protein was immunoprecipitated from in vitro translation products and this molecular weight is in accordance with the use of the first in frame AUG start codon located in exon 2. We also produced a cell line expressing ΔLf under doxycycline induction. Using this model we have been able to show that ΔLf is mainly distributed in the cytoplasm. Its expression induces cell cycle arrest and inhibits cell proliferation. Our results suggest that ΔLf may play an important role in the regulation of normal cell growth.

Structural Determination of Two N-Linked Glycans Isolated from Recombinant Human Lactoferrin Expressed in BHK Cells

A full‐length cDNA coding for human lactoferrin was isolated from a mammary gland library and the recombinant protein was expressed in BHK cells as described by Stowell K.M. et al. [1991, Biochem. J. 276, 349–355]. Two N‐linked glycans from purified recombinant lactoferrin were released by hydrazinolysis and analyzed by 400‐MHz 1H‐NMR spectroscopy. The identified structures corresponded to N‐acetyllactosaminic biantennary glycans and were α‐2,3‐disialylated forms (80%) or α‐2,3‐monosialylated (20%) forms. Moreover, 70% of total glycans were α‐1,6‐fucosylated at the GlcNAc residue linked to asparagine. In regard to its glycan moiety, the recombinant glycoprotein is close to native lactoferrins from milk or leucocytes but shows specific structural features which should be taken into account prior to in vivo and in vitro biological studies.

O-GlcNAcylation/Phosphorylation Cycling at Ser10 Controls Both Transcriptional Activity and Stability of Δ-Lactoferrin

Δ-Lactoferrin (ΔLf) is a transcription factor that up-regulates DcpS, Skp1, and Bax genes, provoking cell cycle arrest and apoptosis. It is post-translationally modified either by O-GlcNAc or phosphate, but the effects of the O-GlcNAc/phosphorylation interplay on ΔLf function are not yet understood. Here, using a series of glycosylation mutants, we showed that Ser10 is O-GlcNAcylated and that this modification is associated with increased ΔLf stability, achieved by blocking ubiquitin-dependent proteolysis, demonstrating that O-GlcNAcylation protects against polyubiquitination. We highlighted the 391KSQQSSDPDPNCVD404 sequence as a functional PEST motif responsible for ΔLf degradation and defined Lys379 as the main polyubiquitin acceptor site. We next investigated the control of ΔLf transcriptional activity by the O-GlcNAc/phosphorylation interplay. Reporter gene analyses using the Skp1 promoter fragment containing a ΔLf response element showed that O-GlcNAcylation at Ser10 negatively regulates ΔLf transcriptional activity, whereas phosphorylation activates it. Using a chromatin immunoprecipitation assay, we showed that O-GlcNAcylation inhibits DNA binding. Deglycosylation leads to DNA binding and transactivation of the Skp1 promoter at a basal level. Basal transactivation was markedly enhanced by 2–3-fold when phosphorylation was mimicked at Ser10 by aspartate. Moreover, using double chromatin immunoprecipitation assays, we showed that the ΔLf transcriptional complex binds to the ΔLf response element and is phosphorylated and/or ubiquitinated, suggesting that ΔLf transcriptional activity and degradation are concomitant events. Collectively, our results indicate that reciprocal occupancy of Ser10 by either O-phosphate or O-GlcNAc coordinately regulates ΔLf stability and transcriptional activity.

Proteomic approach to the identification of novel delta-lactoferrin target genes: Characterization of DcpS, an mRNA scavenger decapping enzyme.

The expression of the transcription factor DeltaLf is deregulated in cancer cells. Its overexpression provokes cell cycle arrest along with antiproliferative effects and we recently showed that the Skp1 gene promoter was a target of DeltaLf. Skp1 belongs to the Skp1/Cullin-1/F-box ubiquitin ligase complex responsible for the ubiquitination and the proteosomal degradation of numerous cellular regulators. The transcriptional activity of DeltaLf is highly controlled and negatively regulated by O-GlcNAc, a dynamic post-translational modification known to regulate the functions of many intracellular proteins. We, therefore, constructed a DeltaLf-M4 mutant corresponding to a constitutively active DeltaLf isoform in which all the glycosylation sites were mutated. In order to discover novel targets of DeltaLf transcriptional activity and to investigate the impact of the O-GlcNAc regulation on this activity in situ we compared the proteome profiles of DeltaLf- and DeltaLf-M4-expressing HEK293 cells versus null plasmid transfected cells. A total of 14 differentially expressed proteins were visualized by 2D electrophoresis and silver staining and eight proteins were identified by mass spectrometry analyses (MALDI-TOF; LC-MS/MS), all of which were upregulated. The identified proteins are involved in several processes such as mRNA maturation and stability, cell viability, proteasomal degradation, protein and mRNA quality control. Among these proteins, only DcpS and TCPB were also upregulated at the mRNA level. Analysis of their respective promoters led to the detection of a cis-regulating element in the DcpS promoter. The S1(DcpS) is 80% identical to the S1 sequence previously described by He and Furmanski [Sequence specificity and transcriptional activation in the binding of lactoferrin to DNA, Nature 373 (1995) 721-724]. Reporter gene analyses and ChIP assays demonstrated that DeltaLf interacts specifically with the DcpS promoter in vivo. These data established that DcpS, a key enzyme in mRNA decay, is a new target of DeltaLf transcriptional activity.

Expression of the lactotransferrin receptor during the differentiation process of the megakaryocyte Dami cell line

Summary— In order to determine whether the human lactotransferrin receptor recently described on platelets was also present onhematopoietic precursors, we investigated its presence and characteristics on the megakaryocytic Dami cell line. The reversible binding of human 5‐([2‐(carbo(hydrazino)methyl]thio)acetyl)aminofluorescein‐labeled lactotransferrin showed that such a receptor was only present on the subpopulation of the largest cells. The increase in numbers of large cells during culture was paralleled by a concurrent increase in lactotransferrin receptor positive cells. Scatchard analysis of the binding of [125I]‐labeled lactotransferrin showed that a single affinity class of binding site was present (Kd = 446 ± 40 nM) and that there were 52 ± 3 × 105 sites per cell. The mouse monoclonal antibody DP5B3G10, specific for the human lactotransferrin receptor, allowed its characterization as a 105 kDa protein on Western blots. The same monoclonal antibody was used to separate the small and large cell subpopulations of Dami cells by panning. Separate culture of the small cells showed that the receptor appeared prior to and independent from endomitosis. In contrast, GPIb was expressed only by large megakaryocytes. The use of conditioned medium from cultures of whole Darni cell populations indicated that a soluble factor is involved in differentiation, but not in the appearance of the lactotransferrin receptor.