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.