Cloning and mRNA expression of the Ca 2+-binding DREAM protein in the pituitary

Abstract

It is well recognized that the level of intracellular calcium governs several cellular processes such as gene expression and secretion in the pituitary. Recently, a novel gene has been identified in neuroendocrine cells that encodes DREAM, a calcium-binding protein that acts as a transcriptional repressor by binding specific downstream regulatory elements (DRE) on DNA. To explore the possibility that DREAM may be expressed in the rat pituitary and may function in endocrine activity, we analyzed its mRNA expression by RT-PCR. Using oligonucleotide primers derived from the mouse DREAM cDNA, we amplified, cloned, and characterized a 852-bp RT-PCR product from rat pituitary tissue. Two splice variants of the rat DREAM gene differing by four nucleotides (tetramer ACAG) were identified. The ACAG + variant (ORF1) consisted of 768 bp encoding a protein of 256 residues with an estimated molecular weight of 29.5 kDa. Amino acid sequence analysis of ORF1 indicated 92.6% and 98.1% identity to the DREAM gene product from human and mouse, respectively. The second variant, ACAG − (ORF2), was 567-bp long and was predicted to encode a peptide of 189 residues with a molecular mass of about 20.8 kDa. To determine which endocrine pituitary cells were expressing DREAM, we evaluated several different clonal populations containing cells that expressed specific pituitary hormones. We found that both DREAM splice variants were expressed in each pituitary cell types examined, which included the mammotropes (MMQ cells), somatotropes (GC cells), mammosomatotropes (GH 3 cells), gonadotropes (LβT2 cells), thyrotropes (TαT1 cells), and corticotropes (AtT-20 cells). Interestingly, the levels of the two variants differed between the cell types tested with the ACAG + variant comprising about two-thirds of the DREAM expression for the mammotropes, somatotropes, mammosomatotropes, and corticotropes as compared to less than one-half for the thyrotropes and the gonadotropes. Our initial attempts to identify pituitary-specific genes regulated by DREAM revealed that prolactin gene expression was not influenced by DREAM suggesting that an action of DREAM may involve other pituitary hormones or be mediated by other cell processes. When taken together, our findings of DREAM expression in the pituitary in a manner specific to pituitary endocrine cell type raises the possibility that this protein may play a role in determining specific pituitary cell function.