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The Journal of Clinical Endocrinology & Metabolism Vol. 85, No. 1 345-354
Copyright © 2000 by The Endocrine Society

Original Studies

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 H295R1

Lionel Groussin, Jean Francis Massias, Xavier Bertagna and Jérôme Bertherat

Groupe d’Etude en Physiopathologie Endocrinienne, Centre National de la Recherche Scientifique, UPR1524, Institut Cochin de Génétique Moléculaire, Université René Descartes-Paris V, 75014 Paris, France

Address all correspondence and requests for reprints to: Jérôme Bertherat, M.D., Ph.D., Groupe d’Etude en Physiopathologie Endocrinienne, Centre National de la Recherche Scientifique, UPR1524, Institut Cochin de Génétique Moléculaire, Centre Hospitalier Universitaire Cochin, 24 rue du Fg. St. Jacques, 75014 Paris, France. E-mail: bertherat{at}icgm.cochin.inserm.fr

The pituitary hormone ACTH, acting through the cAMP pathway, plays a key role in proliferation and differentiation of the adrenal cortex. CAMP response element (CRE)-binding protein (CREB) is an ubiquitous transcription factor that binds to the CRE present in the promoter of numerous genes and mediates transcription stimulation by cAMP. Characterization of CRE-binding proteins 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 stimulation of two reporter genes containing canonical CRE was maintained. Cotransfection of the dominant negative inhibitor A-CREB, which prevents transcription factors containing a CREB-like leucine zipper domain to bind DNA, completely inhibited cAMP-induced stimulation of CRE activity. Western blot and RT-PCR studies showed that activating transcription factor-1 (ATF-1), CRE modulator-{alpha}/{gamma} (CREM{alpha}/{gamma}), and CREM{tau}2{alpha} are expressed in H295R cells. High amounts of CREM proteins were present in H295R, demonstrating an overexpression of this transcription factor in the absence of CREB. Furthermore, expression of the activator isoform CREM{tau} was very high in H295R compared to normal adrenal cortex. Transfection assays demonstrated that CREM{tau}2{alpha} is a potent stimulator of CRE activity in H295R. Finally, gel retardation assays showed that CREM and ATF-1 are the nuclear proteins 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 regulation by cAMP and is a model of compensation mechanisms between the members of the CREB/CREM/ATF-1 family of transcription factors. This loss of CREB expression and the overexpression of CREM could be linked to cellular transformation, as the normal adrenal cortex express high levels of CREB and no or low levels of CREM{tau}.




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