Leclerc Gilles M, Leclerc Guy J, Shorte Spencer L, Stephen Frawley L, Boockfor Fredric R
Laboratory of Molecular Dynamics, Department of Cell Biology and Anatomy, Medical University of South Carolina, Charleston 29425, USA.
Gen Comp Endocrinol. 2002 Oct 15;129(1):45-55. doi: 10.1016/s0016-6480(02)00509-9.
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 768bp encoding a protein of 256 residues with an estimated molecular weight of 29.5kDa. 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 20.8kDa. 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 (LbetaT2 cells), thyrotropes (TalphaT1 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.
众所周知，细胞内钙水平调控着多种细胞过程，如垂体中的基因表达和分泌。最近，在神经内分泌细胞中鉴定出一种新基因，它编码DREAM，一种钙结合蛋白，通过结合DNA上特定的下游调控元件（DRE）发挥转录抑制因子的作用。为了探究DREAM可能在大鼠垂体中表达并在内分泌活动中发挥作用的可能性，我们通过逆转录聚合酶链反应（RT-PCR）分析了其mRNA表达。使用源自小鼠DREAM cDNA的寡核苷酸引物，我们从大鼠垂体组织中扩增、克隆并鉴定了一个852bp的RT-PCR产物。鉴定出大鼠DREAM基因的两种剪接变体，它们相差四个核苷酸（四聚体ACAG）。ACAG(+)变体（ORF1）由768bp组成，编码一个256个残基的蛋白质，估计分子量为29.5kDa。对ORF1的氨基酸序列分析表明，它与人及小鼠的DREAM基因产物的同一性分别为92.6%和98.1%。第二个变体ACAG(-)（ORF2）长567bp，预计编码一个189个残基的肽，分子量约为20.8kDa。为了确定哪些垂体内分泌细胞表达DREAM，我们评估了几个不同的克隆群体，这些群体包含表达特定垂体激素的细胞。我们发现，在所检测的每种垂体细胞类型中均表达了两种DREAM剪接变体，这些细胞类型包括促乳素细胞（MMQ细胞）、生长激素细胞（GC细胞）、促乳生长激素细胞（GH(3)细胞）、促性腺激素细胞（LbetaT2细胞）、促甲状腺激素细胞（TalphaT1细胞）和促肾上腺皮质激素细胞（AtT-20细胞）。有趣的是，在所测试的细胞类型中，两种变体的水平有所不同，ACAG(+)变体在促乳素细胞、生长激素细胞、促乳生长激素细胞和促肾上腺皮质激素细胞中约占DREAM表达的三分之二，而在促甲状腺激素细胞和促性腺激素细胞中则不到二分之一。我们最初尝试鉴定受DREAM调控的垂体特异性基因，结果显示催乳素基因表达不受DREAM影响，这表明DREAM的作用可能涉及其他垂体激素或由其他细胞过程介导。综合来看，我们关于DREAM在垂体中以垂体内分泌细胞类型特异性方式表达的发现，增加了这种蛋白质可能在决定特定垂体细胞功能中发挥作用的可能性。
