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

Abstract

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 G(1)/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-S1(Skp1), located at - 1067 bp, and TAGAAGTCAA-S2(Skp1), 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 S1(Skp1). 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.

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

“…The p⌬Lf-N-EGFP vector was kindly provided by Dr. C. Teng (National Institutes of Health, Research Triangle Park, NC). Immunofluorescence and microscopy were performed as described (30). Fluorescent microscopy images were obtained at room temperature with a Zeiss Axioplan 2 imaging system (Carl-Zeiss S.A.S., Le Pecq, France) equipped with appropriate filter cubes using a ϫ40 objective lens.…”

“…qPCR Conditions-qPCR analyses were performed as described (30). The primer pairs used for the detection of ⌬Lf (forward, 5Ј-AAGCCAGTGGACAAGTTCA-3Ј; reverse, 5Ј-GCTTTGTTGGGATTTGTAGTT-3Ј; annealing temperature, 55°C), ribosomal protein, large, P0 (34), and hypoxanthine-guanine phosphoribosyltransferase (forward, 5Ј-GACCAGTCAACAGGGGACAT-3Ј; reverse, 5Ј-AAC-ACTTCGTGGGGTCCTTTTC-3Ј; annealing temperature, 55°C) were purchased from Eurogentec.…”

“…⌬Lf is transcribed from the alternative promoter P2 in the lactoferrin (Lf) gene (29), and the use of the first available AUG codon in frame produces an alternative N terminus. Thus, compared with Lf, its secretory counterpart, ⌬Lf is a 73-kDa cytoplasmic protein able to enter the nucleus (30). Potential DNA-binding domains have been suggested for Lf, implicating the strong concentration of positive charges at the C-terminal end of the first helix, which is truncated in ⌬Lf, and at the interlobe region (31,32).…”

“…It is a transcription factor interacting via a ⌬Lf response element (⌬LfRE) found in the Skp1 and DcpS promoters (30,34). ⌬Lf is also at the crossroads between cell survival and cell death because we recently linked ⌬Lf overexpression to up-regulation of the Bax promoter and apoptosis (35).…”

La lactoferrine : une protéine multifonctionnelle

“…La hLf possède la capacité de se fixer à des séquences spécifiques d'ADN dont l'une d'entre elles s'est révélée être un élément de réponse fonctionnel. Elle possède, en plus de la séquence NLS (séquence de localisation nucléaire) conservée parmi les Lf de différentes espèces et fonctionnelle dans la ΔLf, une séquence NLS qui lui est propre et qui correspond aux résidus 1-5 amino-terminaux [29,30]. Ces deux Lf ont été observées dans le noyau où elles exercent une activité de facteur de transcription activant pour la Lf le gène de l'IL-1b et pour la ΔLf les gènes Skp1, Rb, Bax, Fas et DcpS (decapping enzyme scavenger) [29,31,32].…”