title: DDX5 is a multifunctional co-activator of steroid hormone receptors.
abstract: The vitamin D receptor (VDR), an evolutionarily conserved member of the nuclear receptor superfamily, links the metabolically activated vitamin D ligand, calcitriol, with its vitamin D-responsive target genes that are implicated in diverse physiological processes. By genome-wide protein-protein interaction screening of a keratinocyte cDNA library using VDR as bait, we found that the DEAD box RNA helicase p68, also referred to as DDX5, directly interacts with VDR. Domain analysis reveals that the ligand-binding domain of VDR is responsible for the binding, an interaction typical of NR co-activators. Interestingly, the VDR interacting domain of DDX5 lacks a LXXLL-motif and interaction analysis of helix 12 VDR mutants E420K, E420Q and L417S, known to decrease binding affinity of LxxLL motif-containing co-activators showed no change in their interactions. As further support that this novel interactor might be involved in vitamin D-stimulated transcriptional regulation, we demonstrate that VDR and DDX5 co-localize within the nuclei of HaCaT keratinocytes and sub-cellular protein fractions. In vivo validation studies demonstrate, that overexpression of DDX5 has the capability to enhance both, calcitriol-dependent transcription of known response genes and an extrachromosomal DR3-type reporter response. In agreement with this, shRNA based knock-down of DDX5 in keratinocytes compensates for this particular response. Finally, our findings reveal parallels between the VDR-DDX5 interaction and the well-characterized interaction between DDX5 and human estrogen receptor α and the androgen receptor, thus underscoring the physiological significance of the novel protein-protein interaction.
authors.affiliation: Division of Molecular Dermatology, Department of Dermatology, Paracelsus Medical University (PMU), Salzburg, Austria. ma.wagner@salk.at
authors.forename: Martin
authors.initials: M
authors.lastname: Wagner
authors.forename: Raphaela
authors.initials: R
authors.lastname: Rid
authors.forename: Christina J
authors.initials: CJ
authors.lastname: Maier
authors.forename: Richard H
authors.initials: RH
authors.lastname: Maier
authors.forename: Martin
authors.initials: M
authors.lastname: Laimer
authors.forename: Helmut
authors.initials: H
authors.lastname: Hintner
authors.forename: Johann W
authors.initials: JW
authors.lastname: Bauer
authors.forename: Kamil
authors.initials: K
authors.lastname: Onder
mesh_terms: D002117:Calcitriol / Q000494:pharmacology; D002460:Cell Line; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D002877:Chromosomes, Human / Q000378:metabolism; D053487:DEAD-box RNA Helicases / Q000235:genetics / Q000378:metabolism*; D055785:Gene Knockdown Techniques; D017930:Genes, Reporter; D015894:Genome, Human / Q000235:genetics; D006801:Humans; D011485:Protein Binding / Q000187:drug effects; D017434:Protein Structure, Tertiary; D021381:Protein Transport / Q000187:drug effects; D034741:RNA, Small Interfering / Q000378:metabolism; D018167:Receptors, Calcitriol / Q000737:chemistry / Q000378:metabolism; D011987:Receptors, Steroid / Q000378:metabolism*; D013347:Subcellular Fractions / Q000187:drug effects / Q000378:metabolism; D015534:Trans-Activators / Q000378:metabolism*; D014158:Transcription, Genetic / Q000187:drug effects; D020798:Two-Hybrid System Techniques
chemical_list: D034741:RNA, Small Interfering; D018167:Receptors, Calcitriol; D011987:Receptors, Steroid; D015534:Trans-Activators; C506749:Ddx5 protein, human; D053487:DEAD-box RNA Helicases; D002117:Calcitriol