Molecular characterization of NF-HEV, a nuclear factor preferentially expressed in human high endothelial venules.
Lymphocyte homing to secondary lymphoid tissue and lesions of chronic inflammation is directed by multi-step interactions between the circulating cells and the specialized endothelium of high endothelial venules (HEVs). In this study, we used the PCR-based method of suppression subtractive hybridization (SSH) to identify novel HEV genes by comparing freshly purified HEV endothelial cells (HEVECs) with nasal polyp-derived microvascular endothelial cells (PMECs). By this approach, we cloned the first nuclear factor preferentially expressed in HEVECs, designated nuclear factor from HEVs (NF-HEV). Virtual Northern and Western blot analyses showed strong expression of NF-HEV in HEVECs, compared to human umbilical vein endothelial cells (HUVECs) and PMECs. In situ hybridization and immunohistochemistry revealed that NF-HEV mRNA and protein are expressed at high levels and rather selectively by HEVECs in human tonsils, Peyers's patches, and lymph nodes. The NF-HEV protein was found to contain a bipartite nuclear localization signal, and was targeted to the nucleus when ectopically expressed in HUVECs and HeLa cells. Furthermore, endogenous NF-HEV was found in situ to be confined to the nucleus of tonsillar HEVECs. Finally, threading and molecular modeling studies suggested that the amino-terminal part of NF-HEV (aa 1-60) corresponds to a novel homeodomain-like Helix-Turn-Helix (HTH) DNA-binding domain. Similarly to the atypical homeodomain transcription factor Prox-1, which plays a critical role in the induction of the lymphatic endothelium phenotype, NF-HEV may be one of the key nuclear factors that controls the specialized HEV phenotype.
Baekkevold
Espen S
ES
Laboratory for Immunohistochemistry and Immunopathology, University of Oslo, Rikshospitalet, Oslo, Norway.
Roussigné
Myriam
M
Yamanaka
Takeshi
T
Johansen
Finn-Eirik
FE
Jahnsen
Frode L
FL
Amalric
François
F
Brandtzaeg
Per
P
Erard
Monique
M
Haraldsen
Guttorm
G
Girard
Jean-Philippe
JP
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D004729:Endothelium, Lymphatic / Q000166:cytology / Q000502:physiology*; D004730:Endothelium, Vascular / Q000378:metabolism; D019077:Helix-Turn-Helix Motifs; D006801:Humans; D017403:In Situ Hybridization; D000067596:Interleukin-33; D007378:Interleukins; D008198:Lymph Nodes / Q000166:cytology / Q000378:metabolism; D008214:Lymphocytes; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D009693:Nucleic Acid Hybridization / Q000379:methods; D014066:Palatine Tonsil / Q000166:cytology / Q000378:metabolism; D010581:Peyer's Patches / Q000166:cytology / Q000378:metabolism; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D014699:Venules / Q000166:cytology / Q000378:metabolism*
C506536:IL33 protein, human; D000067596:Interleukin-33; D007378:Interleukins; D009687:Nuclear Proteins