Loss of expression of the ubiquitous transcription factor cAMP response element-binding protein (CREB) and compensatory overexpression of the activator CREM tau in the human adrenocortical cancer cell line H295R

The pituitary hormone ACTH, acting through the cAMP pathway, plays a key ro le in proliferation and differentiation of the adrenal cortex. CAMP respons e element (CRE)-binding protein (CREB) is an ubiquitous transcription facto r that binds to the CRE present in the promoter of numerous genes and media tes transcription stimulation by cAMP. Characterization of CRE-binding prot eins was performed in the H295R cell line, which is considered a model for human adrenocortical tumor studies. Western blot and RT-PCR studies demonstrated that CREB is not expressed in the human adrenocortical cancer cell line H295R, whereas it is expressed in normal adrenal. During transient transfection experiments, cAMP stimulatio n of two reporter genes containing canonical CRE was maintained. Cotransfec tion of the dominant negative inhibitor A-CREB, which prevents transcriptio n factors containing a CREB-like leucine zipper domain to bind DNA, complet ely inhibited cAMP-induced stimulation of CRE activity. Western blot and RT -PCR studies showed that activating transcription factor-1 (ATF-1), CRE mod ulator-alpha/gamma (CREM alpha/gamma), and CREM tau 2 alpha are expressed i n H295R cells. High amounts of CREM proteins were present in H295R, demonst rating an overexpression of this transcription factor in the absence of CRE B. Furthermore, expression of the activator isoform CREM tau was very high in H295R compared to normal adrenal cortex. Transfection assays demonstrate d that CREM tau 2 alpha is a potent stimulator of CRE activity in H295R. Fi nally, gel retardation assays showed that CREM and ATF-1 are the nuclear pr oteins that specifically bind the CRE in H295R cells, whereas CREM binding to CRE is not observed in a CREB-expressing cell line. H295R cells are the first established nontransgenic cell line that does not express the ubiquitous transcription factor CREB. H295R demonstrates that CREM tau up-regulation can compensate for CREB deficiency to maintain CRE r egulation by cAMP and is a model of compensation mechanisms between the mem bers of the CREB/CREM/ATF-1 family of transcription factors. This loss of C REB expression and the overexpression of CREM could be linked to cellular t ransformation, as the normal adrenal cortex express high levels of CREB and no or low levels of CREM tau.