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

Christophe Mariller, Monique Benaïssa, Stephan Hardivillé, Mathilde Breton, Guillaume Pradelle, Joël Mazurier, Annick Pierce

Research output: Contribution to journalArticlepeer-review

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-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.

Original languageEnglish (US)
Pages (from-to)2038-2053
Number of pages16
JournalFEBS Journal
Volume274
Issue number8
DOIs
StatePublished - Apr 2007

Keywords

  • Cell cycle progression
  • Delta-lactoferrin
  • Proteasome
  • Skp1
  • Transcription factor

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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