TY - JOUR
T1 - Hypoxia and hypoxia mimetics inhibit TNF-dependent VCAM1 induction in the 5A32 endothelial cell line via a hypoxia inducible factor dependent mechanism
AU - Cartee, Todd V.
AU - White, Kellie J.
AU - Newton-West, Marvin
AU - Swerlick, Robert A.
N1 - Funding Information:
The demonstration that three distinct mechanisms, hypoxia, iron chelation, and α-KG antagonism, all repress VCAM expression strongly implicates an Fe(II)/α-KG dioxygenase, such as the HIF-PHDs, in VCAM regulation. Hypoxia mimetics, such as DMOG and iron chelators, simulate hypoxia through the inhibition of the PHDs involved in the modification and degradation of HIF. A role for HIF is supported by gene silencing experiments which resulted in partial restoration of VCAM-1 induction. Of note, silencing of both transcriptionally active isoforms of HIF was necessary to observe this rescue. Presumably, each isoform can fully compensate for the absence of the other in HDMEC at least vis-à-vis VCAM-1 inhibition.
PY - 2012/2
Y1 - 2012/2
N2 - Background: We previously reported that iron chelators inhibit TNFα-mediated induction of VCAM-1 in human dermal microvascular endothelial cells. We hypothesized that iron chelators mediate inhibition of VCAM-1 via inhibition of iron-dependent enzymes such as those involved with oxygen sensing and that similar inhibition may be observed with agents which simulate hypoxia. Objective: We proposed to examine whether non-metal binding hypoxia mimetics inhibit TNFα-mediated VCAM-1 induction and define the mechanisms by which they mediate their effects on VCAM-1 expression. Methods: These studies were undertaken in vitro using immortalized dermal endothelial cells, Western blot analysis, ELISA, immunofluorescence microscopy, quantitative real-time PCR, and chromatin immunoprecipitation. Results: Hypoxia and the non-iron binding hypoxia mimetic dimethyl oxallyl glycine (DMOG) inhibited TNFα-mediated induction of VCAM-1. DMOG inhibition of VCAM-1 was dose-dependent, targeted VCAM-1 gene transcription independent of NF-κB nuclear translocation, and blocked TNFα-mediated chromatin modifications of relevant elements of the VCAM-1 promoter. Combined gene silencing of both HIF-1α and HIF-2α using siRNA led to a partial rescue of VCAM expression in hypoxia mimetic-treated cells. Conclusion: Iron chelators, non-metal binding hypoxia mimetics, and hypoxia all inhibit TNFα-mediated VCAM-1 expression. Inhibition is mediated independent of nuclear translocation of NF-κB, appears to target TNFα-mediated chromatin modifications, and is at least partially dependent upon HIF expression. The absence of complete VCAM-1 expression rescue with HIF silencing implies an important regulatory role for an Fe(II)/α-ketoglutarate dioxygenase distinct from the prolyl and asparagyl hydroxylases that control HIF function. Identification of this dioxygenase may provide a valuable target for modulating inflammation in human tissues.
AB - Background: We previously reported that iron chelators inhibit TNFα-mediated induction of VCAM-1 in human dermal microvascular endothelial cells. We hypothesized that iron chelators mediate inhibition of VCAM-1 via inhibition of iron-dependent enzymes such as those involved with oxygen sensing and that similar inhibition may be observed with agents which simulate hypoxia. Objective: We proposed to examine whether non-metal binding hypoxia mimetics inhibit TNFα-mediated VCAM-1 induction and define the mechanisms by which they mediate their effects on VCAM-1 expression. Methods: These studies were undertaken in vitro using immortalized dermal endothelial cells, Western blot analysis, ELISA, immunofluorescence microscopy, quantitative real-time PCR, and chromatin immunoprecipitation. Results: Hypoxia and the non-iron binding hypoxia mimetic dimethyl oxallyl glycine (DMOG) inhibited TNFα-mediated induction of VCAM-1. DMOG inhibition of VCAM-1 was dose-dependent, targeted VCAM-1 gene transcription independent of NF-κB nuclear translocation, and blocked TNFα-mediated chromatin modifications of relevant elements of the VCAM-1 promoter. Combined gene silencing of both HIF-1α and HIF-2α using siRNA led to a partial rescue of VCAM expression in hypoxia mimetic-treated cells. Conclusion: Iron chelators, non-metal binding hypoxia mimetics, and hypoxia all inhibit TNFα-mediated VCAM-1 expression. Inhibition is mediated independent of nuclear translocation of NF-κB, appears to target TNFα-mediated chromatin modifications, and is at least partially dependent upon HIF expression. The absence of complete VCAM-1 expression rescue with HIF silencing implies an important regulatory role for an Fe(II)/α-ketoglutarate dioxygenase distinct from the prolyl and asparagyl hydroxylases that control HIF function. Identification of this dioxygenase may provide a valuable target for modulating inflammation in human tissues.
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U2 - 10.1016/j.jdermsci.2011.10.003
DO - 10.1016/j.jdermsci.2011.10.003
M3 - Article
C2 - 22093255
AN - SCOPUS:84856441550
SN - 0923-1811
VL - 65
SP - 86
EP - 94
JO - Journal of Dermatological Science
JF - Journal of Dermatological Science
IS - 2
ER -