CO-TRANSACTIVATION OF THE 3' ERYTHROPOIETIN HYPOXIA INDUCIBLE ENHANCER BY THE HIF-1 PROTEIN

Erythropoietin (Epo) is a glycoprotein hormone that is the primary reg ulator of red blood cell production, Epo production increases in respo nse to tissue hypoxia. This increase occurs primarily at the transcrip tional level. Hypoxia inducible factor (KIF-I) is a DNA binding protei n that binds to a hypoxia inducible enhancer in the 3' flanking sequen ce of the Epo gene. HIF-1 is a heterodimer that consists of an a and b eta subunit, I-T[F-l DNA binding activity is induced in response to. h ypoxia. In order to determine if one or both HIF-1 subunits is capable of ligand binding, subsequently leading to Epo production we performe d co-transactivation experiments, Transfections were performed in Hep 3B, an Epo producing human hepatoma cell line and Cos-7, a non-Epo pro ducing monkey kidney cell Line, Cells were co-transfected with the 38 bp Epo enhancer fragment bearing the HIF-1 binding motif, subcloned in the luciferase reporter plasmid and either the HIF-1 alpha cDNA, HIF- 1 beta cDNA, HIF-1 alpha and HIF-1 beta cDNAs or pREP-4 respectively. Cells were incubated in an hypoxic (1%O-2) or normoxic (21%O-2) enviro nment and assayed for luciferase activity. Epo levels were measured in the culture media from the transfected plates by an ELISA assay. Unde r hypoxic conditions Hep 3B cells transfected with the HIF-1 alpha cDN A alone showed a 2.2 fold increase in luciferase activity, HIF-1 beta showed a 3.4 fold increase and cells transfected with HlF-1 alpha and beta showed a 6.9 fold increase in activity over cells transfected wit h pREP-4. The baseline luciferase activity in transfected 3B cells inc ubated in normoxia was very low. However, a similar fold increase in l uciferase activity in cells transfected with both HIF-1 alpha and beta was noted. Under normoxic or hypoxic conditions in Cos-7 cells, a 1.5 fold increase was obtained with the HIF-1 alpha and beta constructs t ransfected independently and a 3.5 fold increase was noted in cells tr ansfected with both constructs, Epo levels increased several fold in a ll Hep 3B cells that were incubated in hypoxic conditions. However, th ere was no additional increase in Epo levels in transfected Hep 3B cel ls, We therefore conclude that although the HIF-1 alpha and beta subun its can independently co-transactivate the Epo enhancer, binding of bo th subunits and a hypoxic environment is necessary for maximal transac tivation. Overexpression of the HIF-1 protein alone in normoxic or hyp oxic conditions is insufficient for an increase in Epo secretion. Acti vation/inactivation and interaction of other tissue specific factors i s necessary for an increase in Epo gene expression in response to hypo xia.