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 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.
Mariller
Christophe
C
Unité de Glycobiologie Structurale et Fonctionnelle, Unité Mixte de Recherche 8576 CNRS-Université des Sciences et Technologies de Lille1, Villeneuve d'Ascq, France.
Benaïssa
Monique
M
Hardivillé
Stephan
S
Breton
Mathilde
M
Pradelle
Guillaume
G
Mazurier
Joël
J
Pierce
Annick
A
D000595:Amino Acid Sequence; D001483:Base Sequence; D002352:Carrier Proteins / Q000737:chemistry / Q000502:physiology*; D002478:Cells, Cultured; D005786:Gene Expression Regulation; D006801:Humans; D007781:Lactoferrin; D008969:Molecular Sequence Data; D019913:Nuclear Localization Signals; D011401:Promoter Regions, Genetic; D020218:Response Elements / Q000502:physiology; D044786:S-Phase Kinase-Associated Proteins / Q000235:genetics*; D014157:Transcription Factors / Q000502:physiology*
D002352:Carrier Proteins; C490098:LTF protein, human; D019913:Nuclear Localization Signals; D044786:S-Phase Kinase-Associated Proteins; D014157:Transcription Factors; D007781:Lactoferrin