活化素抑制大鼠垂体GH_3细胞中人生长激素基因启动子的活性

本研究旨在探讨活化素(activin)对大鼠垂体GH3细胞中人生长激素(hGH)基因启动子活性的影响及其可能的调节机制。采用荧光素酶报告基因方法。首先建立含hGH基因启动子(-484～+30bp)和荧光素酶融合基因的稳定转染GH3细胞株,然后加入活化素或同时加入活化素与相关信号转导途径的激动剂,通过检测细胞培养液和细胞裂解液中GH的含量,以及GH3细胞内荧光素酶的变化,反映活化素对GH分泌、合成和hGH基因启动子活性的影响。将含不同长度hGH基因启动子序列的荧光素酶表达质粒分别转染GH3细胞,观察它们对活化素的反应,寻找活化素影响hGH基因启动子活性的关键DNA序列。结果表明,活化素(5,50nmol/L)能抑制大鼠垂体GH3细胞中GH的分泌和合成,活化素(5,50nmol/L)还能够抑制GH3细胞中hGH基因启动子的活性,使之仅达对照组的77%和69%;在胞内信号转导激动剂中,丝裂原活化蛋白激酶激酶(MAPKK/MEK)特异性激动剂C6ceramide(1μmol/L)完全取消了活化素对hGH基因启动子活性的抑制作用;活化素发挥抑制作用所需要的hGH基因启动子关键序列位于-132～-66bp之间。上述研究表明,活化素能抑制大鼠垂体GH3中hGH基因启动子的活性,它可能是通过抑制细胞内依赖MAPK的信号转导途径来完成的,同时hGH启动子上-132～-66bp的序列在其中发挥重要的作用。     

Analysis of rat prolactin promoter sequences that mediate pituitary-specific and 3',5'-cyclic adenosine monophosphate-regulated gene expression in vivo

To determine the rat PRL (rPRL) promoter sequences that mediate pituitary-specific and cAMP-induced gene expression in vivo, various lengths of the rPRL promoter were ligated to the luciferase reporter gene and introduced into pituitary and non-pituitary cell lines. A 30-fold increase in rPRL promoter activity was observed in GH4 rat pituitary tumor cells compared to nonpituitary Rat2 fibroblast and HeLa cervical carcinoma cells. About 45% of this cell-specific promoter activity was competed by a plasmid containing the -67 to -45 rPRL promoter region, which is the most proximal binding site for a lactotroph-specific factor. Compared to a -425 rPRL construct, transfection with rPRL 5'-end points of -212, -178, and -127 contained 23%, 45%, and 1%, respectively, of luciferase activity. Forskolin stimulation resulted in a 10-fold induction of all the rPRL promoter fragments tested. Of note, a -127 deletion which was devoid of any basal promoter activity was also induced 10-fold by forskolin. The forskolin effect was abolished when GH4 rat pituitary cells were cotransfected with a plasmid encoding a protein kinase A inhibitor, indicating protein kinase A is involved in the activation mechanism. These data document that both positive and negative effectors influence basal rPRL promoter activity. Furthermore, the minimum sequences required for pituitary-specific rPRL promoter activity are altered by intracellular cAMP levels. Taken together, the data indicate that hormone-activated and cell-specific factors may interact to establish a particular setpoint for rPRL gene expression.     

IFN-gamma increases the hGH gene promoter activity in rat GH3 cells

AIM: To study the effect(s) of interferon gamma (IFN-gamma) on the activity of human growth hormone (hGH) gene promoter in rat pituitary GH3 cells and the molecular mechanism underlying the effect(s). METHODS: Cell transfection and luciferase reporter gene were used. RESULTS: IFN-gamma (10(2) and 10(3) U/ml) increased the activity of hGH in GH3 cells. The addition of the mitogen-activated protein kinase inhibitor PD98059 (40 micromol/l) to the cells blocked the stimulatory effect of IFN-gamma. Neither overexpression of Pit-1 nor inhibiting Pit-1 expression affected IFN-gamma induction of hGH promoter activity. To identify the DNA sequence that mediated the effect of IFN-gamma, four deletion constructs of hGH gene promoter were created. The stimulatory effect of IFN-gamma was abolished following deletion of the -250 to -132 fragment. CONCLUSIONS: IFN-gamma increases the activity of hGH gene promoter in rat pituitary GH3 cells. This stimulatory effect of IFN-gamma appears to require the intracellular mitogen-activated protein kinase-dependent signaling pathway. The effect of IFN-gamma requires the promoter sequence that spans the -250 to -132 fragment of the gene, but is unrelated to Pit-1 protein.     

Cell-type-specific function of the C-type natriuretic peptide gene promoter in rat anterior pituitary-derived cultured cell lines.

The promoter function of the human C-type natriuretic peptide (CNP) gene in various cultured cells was examined by transient transfection assays. The CNP promoter functioned very effectively in GH3 cells, which originated from the growth hormone-producing tumor of the rat anterior pituitary and somatomammotroph phenotype, but functioned much less effectively in GH1 cells, another type of rat pituitary-derived cell with a somatotroph phenotype, and rat primary cardiocytes. The CNP promoter did not function at all in other cells, including AtT20 cells of murine pituitary corticotroph origin. Functional analyses of the deleted promoters with various 5' deletion breakpoints revealed the existence of at least two negative and one positive regulatory regions. Within the positive regulatory region (positions -54 to -19), which conferred 90% of the promoter activity in GH3 cells, two equipotent GC-rich cis elements (positions -49 to -45 and -40 to -35) were identified. Both sites shared half of the promoter activity and binding properties to the nuclear protein in GH3 cells. Rat anterior pituitary tissue contained the binding protein of the identified cis element, which was identical or similar to that of GH3 cells. With Southwestern (DNA-protein) analysis, a 70-kDa specific binding protein distinct from known factors such as SP-1, AP-2, and Pit-1 was identified in the nuclear extract of GH3 cells.     

Avian somatotrophs: differentiation, morphology, distribution, and regulation

Growth hormone (GH) is primarily synthesized, stored, and released by pituitary somatotrophs. These cells comprise a highly labile population that continuously undergoes proliferation, differentiation, and morphogenesis in response to changing physiological stimuli. They are also functionally and morphologically heterogeneous with distinct spatial and temporal distribution within the pituitary gland. The characteristics of these cells are discussed in this brief review.     

Changes in thyrotroph and somatotroph cell populations induced by stimulation and inhibition of their secretory activity

Summary The populations of cells which produce immunoreactive growth hormone (GH) and thyroid stimulating hormone (TSH) in the rat pituitary gland do not occur in fixed percentages but vary greatly under different physiological and experimental conditions. These variations can be directly correlated to the levels of stimulation and/or inhibition of the specific secretory activity. In both types of cell, sustained stimulation with trophic hormones or blockage of the feedback mechanisms induces remarkable growth in the specific cell population. Conversely, the interruption or inhibition of the stimulus thwarted the hormonal secretion and caused a massive degeneration of redundant cells. The stimulation of both GH and TSH cells is accompanied by an enhanced secretory activity as judged by their higher concentrations in serum and hypertrophy of the cytoplasmic organelles involved in synthesis and intracellular processing of the hormones. By contrast, interruption of the stimulus is followed by a variable degree of disruption of the cytoplasmic organization, including a sizable degeneration of cells. In stimulated rats, the concentrations of both GH and TSH decreased significantly in pituitary tissue due to mobilization of the hormonal stores contained in secretory granules. On the other hand, the withdrawal of stimuli blocked the hormonal release; this is reflected by the accumulation of both hormones and secretory granules in pituitary tissue. The strict correlation between the size of the GH and TSH populations with stimulation and inhibition of hormonal secretory activity reported in this investigation further supports the critical role played by the cell renewal process in endocrine secretion.     

Dynamics of stimulus-expression coupling as revealed by monitoring of prolactin promoter-driven reporter activity in individual, living mammotropes.

Single-cell paradigms have greatly expanded our knowledge about stimulus-secretion coupling, but the understanding of stimulus-gene expression coupling has lagged behind for lack of a dynamic model sufficiently sensitive to provide single-cell resolution. In the present study, we made continuous indirect measurements within individual, living cells of expression dynamics both before and after treatment with a gene-activating secretagogue. This was accomplished by transfecting (via microinjection) individual, primary mammotropes with a PRL promoter-driven luciferase reporter plasmid, and then quantifying the rate of photonic emissions (reflective of endogenous gene activity). We found that individual cells exhibit spontaneous, random, short-term fluctuations of basal reporter activity and are extremely heterogeneous in terms of responses to a stimulatory agent (TRH). In addition, we found that responses are affected by several factors including the secretory status of the pituitary donor, the manner in which the stimulus is presented, and by the initial level of reporter activity. Moreover, the responsiveness of an individual cell can fluctuate dramatically over time. These results invite speculation that a given cell can "sense" its gene activation state and regulate its response accordingly to satisfy requirements for the corresponding secretory product.     

Prolactin-releasing peptide activation of the prolactin promoter is differentially mediated by extracellular signal-regulated protein kinase and c-Jun N-terminal protein kinase

Regulation of the mitogen-activated protein kinase (MAPK) family by prolactin-releasing peptide (PrRP) in both GH3 rat pituitary tumor cells and primary cultures of rat anterior pituitary cells was investigated. PrRP rapidly and transiently activated extracellular signal-regulated protein kinase (ERK) in both types of cells. Both pertussis toxin, which inactivates G(i)/G(o) proteins, and exogenous expression of a peptide derived from the carboxyl terminus of the beta-adrenergic receptor kinase I, which specifically blocks signaling mediated by the betagamma subunits of G proteins, completely blocked the PrRP-induced ERK activation, suggesting the involvement of G(i)/G(o) proteins in the PrRP-induced ERK activation. Down-regulation of cellular protein kinase C did not significantly inhibit the PrRP-induced ERK activation, suggesting that a protein kinase C-independent pathway is mainly involved. PrRP-induced ERK activation was not dependent on either extracellular Ca(2+) or intracellular Ca(2+). However, the ERK cascade was not the only route by which PrRP communicated with the nucleus. JNK was also shown to be significantly activated in response to PrRP. JNK activation in response to PrRP was slower than ERK activation. Moreover, to determine whether a MAPK family cascade regulates rat prolactin (rPRL) promoter activity, we transfected the intact rPRL promoter ligated to the firefly luciferase reporter gene into GH3 cells. PrRP activated the rPRL promoter activity in a time-dependent manner. Co-transfection with a catalytically inactive form of a MAPK construct or a dominant negative JNK, partially but significantly inhibited the induction of the rPRL promoter by PrRP. Furthermore, co-transfection with a dominant negative Ets completely abolished the response of the rPRL promoter to PrRP. These results suggest that PrRP differentially activates ERK and JNK, and both cascades are necessary to elicit rPRL promoter activity in an Ets-dependent mechanism.     

Patterned response to odor in single neurones of goldfish olfactory bulb: influence of odor quality and other stimulus parameters

Responses of 75 single units in the goldfish olfactory bulb were analyzed in detail for their relationship to the time-course of the change in odor concentration during each odor stimulus. Odor stimuli were controlled for rise time, duration, and peak concentration by an apparatus developed for the purpose. This apparatus enabled aqueous odor stimuli to be interposed into a constant water stream without changes in flow rate. The time-course of the concentration change within the olfactory sac was inferred from conductivity measurements at the incurrent and excurrent nostrils. Temporal patterns of firing rate elicited by stimuli with relatively slow rising and falling phases could be quite complex combinations of excitation and suppression. Different temporal patterns were produced by different substances at a single concentration in most units. Statistical measures of the temporal pattern of response for a small number of cells at a given concentration were more characteristic of the stimulus substance than any of three measures of magnitude of response. The temporal patterns change when the peak concentration, duration, and rise time of the stimuli are varied. The nature of these changes suggests that the different patterns are due primarily to the combined influence of two factors: (a) a stimulus whose concentration varies over time and (b) a relationship between concentration and impulse frequency which varies from unit to unit. Some units produce patterns suggestive of influence by neural events of long time constant. The importance of temporal patterns in odor quality and odor intensity coding is discussed.