Induction of Hypoxia Inducible Factor 1 Alpha (HIF1α) by Caffeic Acid Phenethyl Ester (CAPE) and Caffeic Acid Phenethyl Amide (CAPA) in Mouse Skin Fibroblasts

Abstract

Esters and amides of caffeic acid (CA) such as CAPE and CAPA exhibit significant cytoprotection of a variety of cell types from oxidative stress while CA does not. A role for HIF1α in cytoprotection has been described and to better understand the mechanism we investigated skin fibroblasts from the FVB.1 29S6‐Gt(ROSA)26Sortml (HIF1αluc)Kael/J (HIF1αLuc) inbred strain (Jackson Laboratory, Bar Harbor, ME). This mouse (RosaLuc) has been genetically engineered to express luciferase in conjunction with accumulation of HIF1α. RosaLuc mice skin fibroblasts (MSF) were obtained from skin specimens dissociated in 0.1% collagenase for 3 h and cultivated in 10% fetal bovine serum in alpha Minimal Essential Medium. Passages 1–5 were used for these studies and subcultivated into 96‐well multiplates. CAPA was synthesized in the lab and CAPE (Cayman Labs, Ann Arbor, MI) and deferoxamine (DEF), a well known inducer of HIF1α and an iron chelator, were dissolved in DMSO. MSF cells were treated for 6 h with these drugs and assayed for luciferase activity (Promega, Madison, WI). CA was inactive but CAPE and CAPA were significantly better than DEF in producing a luminescent signal from MSF. These results indicate that CAPE and CAPA may directly activate HIF1α by inhibiting prolyl hydroxylase thereby preventing the degradation of nascent HIF1α.