HYPOXIC REGULATION OF LACTATE-DEHYDROGENASE-A - INTERACTION BETWEEN HYPOXIA-INDUCIBLE FACTOR-1 AND CAMP RESPONSE ELEMENTS

The oxygen-regulated control system responsible for the induction of e rythropoietin (Epo) by hypoxia is present in most (if not all) cells a nd operates on other genes, including those involved in energy metabol ism. To understand the organization of cis-acting sequences that are r esponsible for oxygen-regulated gene expression, we have studied the 5 ' flanking region of the mouse gene encoding the hypoxically inducible enzyme lactate dehydrogenase A (LDH). Deletional and mutational analy sis of the function of mouse LDH-reporter fusion gene constructs in tr ansient transfection assays defined three domains, between -41 and -84 base pairs up stream of the transcription initiation site, which were crucial for oxygen-regulated expression, The most important of these, although not capable of driving hypoxic induction in isolation, had t he consensus of a hypoxia-inducible factor 1 (HIF-1) site, and cross-c ompeted for the binding of HIF-1 with functionally active Epo and phos phoglycerate kinase-1 sequences. The second domain was positioned clos e to the HIF-1 site, in an analogous position to one of the critical r egions in the Epo 3' hypoxic enhancer, The third domain had the motif of a cAMP response element (CRE), Activation of cAMP by forskolin had no effect on the level of LDH mRNA in normoxia, but produced a magnifi ed response to hypoxia that was dependent upon the integrity of the CR E, indicating an interaction between inducible factors binding the HIF -1 and CRE sites.