Recently, a pituitary-specific enhancer was identified within the 5' flanking region of The glycoprotein hormones are a family of heterodimeric proteins which consist of a common a subunit noncovalently associated with a hormone-specific 13 subunit (37). The glycoprotein hormones include the pituitary hormones, luteinizing hormone, follicle-stimulating hormone, and thyroid-stimulating hormone. In addition, some species also synthesize a chorionic gonadotropin within the placenta. Within a species, the glycoprotein hormones all share a common a subunit, while the unique ,B subunits specify the biological activity of the heterodimer. In the pituitary gland, luteinizing hormone and follicle-stimulating hormone are synthesized within cells which are designated gonadotropes, while thyroid-stimulating hormone is synthesized in thyrotropes. Thus, the glycoprotein hormone a-subunit gene is expressed within two different cell types in the pituitary and in some species within the placenta. The mechanisms mediating the tissue-specific expression of the a-subunit gene have been the focus of a large number of studies. While a number of DNA elements which are important for at-subunit expression in the placenta have been identified (3,4,11,12,25,28,34,46), much less is known concerning requirements for expression in the pituitary. It has been demonstrated that reporter genes containing various * Corresponding author. Mailing address:

Inhibin gene expression in the ovary is stimulated by FSH, which uses cAMP as an intracellular second messenger. To examine further the transcriptional regulation of the alpha inhibin gene by FSH and cAMP, we have isolated and characterized a genomic clone that contains the entire rat alpha inhibin gene. Sequence analysis of the alpha inhibin promoter region revealed several potential cAMP response elements (CREs) and transcription factor AP2-binding sites that might mediate cAMP regulation. To determine the functional importance of these sequences, fusion genes including the alpha inhibin 5' flanking region linked to a luciferase reporter gene were transiently transfected into primary granulosa cells isolated from immature rats. These fusion genes were both expressed and regulated by the adenylyl cyclase activator forskolin in transfected granulosa cells. Analysis of a series of 5' deletion mutants indicated that a construct containing as little as 170 basepairs up-stream of the alpha inhibin start site, which includes a single imperfect CRE and no AP2 sites, was regulated by forskolin. DNAse footprinting was used to demonstrate that bacterially expressed CRE-binding protein (CREB) binds to this CRE located 122 basepairs up-stream of the alpha inhibin gene transcriptional start site. To investigate further the role of this CRE in alpha inhibin gene expression, site-specific mutagenesis of the CRE was performed. The alpha inhibin promoter containing a mutated CRE was not regulated by forskolin in granulosa cells and did not bind the CREB protein. Interestingly, mutation of the CRE also substantially reduced basal expression of the alpha inhibin promoter. Lastly, a gel mobility shift assay was used to examine CRE-binding proteins from granulosa cell extracts. Granulosa cells contain a protein that specifically interacts with CRE-containing oligonucleotides or with the alpha inhibin promoter and that is recognized by antibodies against the CREB protein. Our results suggest that CREB or related transcription factors play an important role in both basal and cAMP-regulated expression of the alpha inhibin gene in ovarian granulosa cells.

Transient transfection studies have been used to determine the DNA sequences of the glycoprotein hormone alpha-subunit gene that are required for tissue-specific expression. In the initial phase of these studies, a variant mouse alpha gene was identified which contains a fully palindromic cAMP response element (CRE). The corresponding region of a previously cloned and sequenced mouse alpha gene contains a single point mutation that disrupts the symmetrical nature of this element. DNase footprint studies demonstrate that the fully palindromic CRE binds the CRE-binding protein with much higher affinity than the imperfect palindrome. Transfection experiments using both mouse alpha gene variants demonstrate differences in basal and cAMP-induced expression. Studies of the cAMP response of the human alpha gene indicated that this gene contains sequences other than the known CRE that are sufficient to permit a transcriptional response to cAMP in both placental and pituitary cells. Expression of human and mouse alpha-subunit genes has been examined in cells of the gonadotrope, thyrotrope, and trophoblast lineages to identify DNA sequences that mediate selective transcription of the alpha gene in these cells. The results demonstrate that sequences between about -500 and -200 are important for expression in the pituitary, but not the placenta. Clustered point mutations were used to further characterize sequences required for expression in the pituitary. Two regions, one at positions -445 to -438 and one at positions -337 to -330, were required for expression in cells of the gonadotrope lineage. One of these regions, at -337 to -330, is also important for expression in thyrotropes. When linked to a minimal promoter, multiple copies of the -344 to -300 region had transcriptional enhancer activity in gonadotropes and thyrotropes, but not in several other cell types. These results are consistent with a model involving different combinations of regulatory elements that determine cell-specific alpha expression in gonadotropes and thyrotropes.

ABSTRACT. Experiments performed in vitro have demonstrated that leukocyte neutral proteases produce an important mediator of inflammation, C5a, by proteolysis of the C5 component of the complement system. Cystic fibrosis (CF) lung fluids were characterized by high levels of neutrophils (39% of total cells versus 2% in normals) and contained significantly elevated amounts of elastolytic activity (mean 17.7 ng/pg total protein) compared to the lung fluids obtained from normal volunteers (0.2 ng elastolytic activitylpg protein, p = 0.001). The objective of these studies was to determine if complement activation and complement-derived chemotactic activity are present in CF lung fluids. C3c peptide representing activation of C3 could not be identified in the bronchial-alveolar lung lavage fluids of normal subjects but was readily identified by means of crossed immunoelectrophoresis "in CF lung fluids (n = 9, mean 49% of C3); the mean level of C3 was decreased in CF lung specimens. Chemotactic activity was significantly elevated in lung fluids of the CF patients when compared to normal lung fluids. Using gel-filtration chromatography and a sensitive radioimmunoassay the chemotaxin present in CF specimens was identified as the anaphylatoxin C5a. C5a levels in the bronchial-alveolar lavage fluids of CF patients was inversely related to volume in liters expired in 1 s of a forced expiratory maneuver expressed as a percent of vital capacity determined from a forced expiratory maneuver ( r = -0.72). Because there was a direct relationship between the total elastolytic activity present in CF airways and the concentration of C5a ( r = 0.97, p = 0.03), it was postulated that airway proteases with elastolytic activity also cleave C5, nonimmunologically producing C5a. Detailed inhibition assays revealed that much of the total elastolytic activity had the inhibition profile of a serine proteinase. The levels of the serine proteinases were closely correlated with the numbers of neutrophilic leukocytes present per ml of lavage fluid ( r = 0.7, p = 0.05).However, inhibitors of leukocyte serine proteases did not prevent the generation of additional chemotactic activity