Characterization of glycoprotein hormone free alpha-subunit from human pituitary and placenta extracts

Standard alpha-subunit dissociated from glycoprotein hormones differs from individual (free) alpha-subunit found in sera or in cell culture media; secreted free alpha-subunit is larger, more acidic and lacks the ability of recombining in vitro with standard hCG-beta. It is unclear whether the large free alpha-subunit is only a secretory product or whether it is also present in tissue. Herein were studied the molecular size, the isoelectric pH, and the recombining activity of free alpha-subunit obtained from pituitary and placenta extracts. Sephadex exclusion chromatography showed the presence of both a large and a small form, and a changing large/small free alpha-subunit ratio in the various extracts. Most of the large form obtained from placenta extracts electrofocused into two peaks of pI 4.8 and 5.1. The large form showed no recombining activity with standard hCG-beta while the small free alpha-subunit recombined as well as did standard hCG-alpha. The observation of three common characteristics (a larger size, a pI 4.8, and a lack of recombining activity) suggests a similarity between the large secreted form and a fraction of the free alpha-subunit in tissue.     

Accelerated evolution of the pituitary adenylate cyclase-activating polypeptide precursor gene during human origin

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide abundantly expressed in the central nervous system and involved in regulating neurogenesis and neuronal signal transduction. The amino acid sequence of PACAP is extremely conserved across vertebrate species, indicating a strong functional constraint during the course of evolution. However, through comparative sequence analysis, we demonstrated that the PACAP precursor gene underwent an accelerated evolution in the human lineage since the divergence from chimpanzees, and the amino acid substitution rate in humans is at least seven times faster than that in other mammal species resulting from strong Darwinian positive selection. Eleven human-specific amino acid changes were identified in the PACAP precursors, which are conserved from murine to African apes. Protein structural analysis suggested that a putative novel neuropeptide might have originated during human evolution and functioned in the human brain. Our data suggested that the PACAP precursor gene underwent adaptive changes during human origin and may have contributed to the formation of human cognition.     

Expression and regulation of the pituitary- and placenta-specific human glycoprotein hormone alpha-subunit gene is restricted to the pituitary in transgenic mice.

Expression of the glycoprotein hormone alpha subunit occurs in both the pituitary and placenta in humans. However, this study found that expression of this subunit is restricted to the pituitary in mice. An interspecies analysis of human alpha-subunit gene regulation was undertaken, using the transgenic-mouse approach. In mice transgenic for a genomic clone containing the complete human alpha-subunit gene and several kilobases of 5'- and 3'-flanking sequences, cell-type-specific expression and hormonal regulation of the human alpha-subunit transgene occurred in the mouse pituitary, whereas no expression of the transgene was detectable in the mouse placenta. These findings provide strong evidence that a common trans-acting factor(s) regulates glycoprotein hormone alpha-subunit gene expression in the human and mouse pituitaries; however, this factor(s) or a unique factor(s), though functional in the human placenta, is either nonfunctional or absent in the mouse placenta.     

Neural-restrictive silencers in the regulatory mechanism of pituitary adenylate cyclase-activating polypeptide gene expression

Pituitary adenylate cyclase-activating polypeptide (PACAP) is known as a pleiotropic neuropeptide and is present abundantly in central nervous system. During a detailed analysis of the 5'-flanking region of the mouse PACAP gene, we found and characterized two negative regulatory elements, which are homologous to the neural-restrictive silencer element, and are termed neural-restrictive silencer-like elements 1 and 2 (NRSLE1 and NRSLE2). Their sequence and position were significantly conserved among mouse, human, and rat PACAP genes. In the electrophoretic mobility shift assay (EMSA) with nuclear extracts of Swiss-3T3 cells and individual oligonucleotide probes for NRSLE1 and NRSLE2, a specific complex was observed to have the same migration as compared with the NRSE probe of rat type II sodium channel gene (NaII). Furthermore, these complexes were efficiently competed by the unlabeled NaII probe. In the luciferase reporter assay, the reporter gene constructs containing NRSLEs, driven by heterologous SV40 promoter, exhibited repression of luciferase activity almost equal to basal level in Swiss-3T3 cells. In contrast, the repression was not observed in differentiated PC12 cells with NGF. These results suggested that the neural-restrictive silencer system might be involved in the regulatory mechanism of neuron-specific PACAP gene expression.     

Early multipotential pituitary focal hyperplasia in the alpha-subunit of glycoprotein hormone-driven pituitary tumor-transforming gene transgenic mice

Pituitary tumor-transforming gene (PTTG), a securin protein isolated from pituitary tumor cell lines, is highly expressed in invasive tumors and exhibits characteristics of a transforming gene. To determine the role of PTTG in pituitary tumorigenesis, transgenic human PTTG1 was targeted to the mouse pituitary using the alpha-subunit of glycoprotein hormone. Males showed plurihormonal focal pituitary transgene expression with LH-, TSH-, and, unexpectedly, also GH-cell focal hyperplasia and adenoma, associated with increased serum LH, GH, testosterone, and/or IGF-I levels. MRI revealed both pituitary and prostate enlargement at 9-12 months. Urinary obstruction caused by prostatic hyperplasia and seminal vesicle hyperplasia, with renal tract inflammation, resulted in death by 10 months in some animals. Pituitary PTTG expression results in plurihormonal hyperplasia and hormone-secreting microadenomas with profound peripheral growth-stimulatory effects on the prostate and urinary tract. These results provide evidence for early pituitary plasticity, whereby PTTG overexpression results in a phenotype switch in early pituitary stem cells and promotes differentiated polyhormonal cell focal expansion.     

Phorbol esters enhance basal and stimulated adenylate cyclase activity in a pituitary cell line

We reported in anterior pituitary cells that hormone stimulation of cyclic AMP levels is amplified by agents that activate protein kinase C (e.g., phorbol esters). We utilized the 235-1 pituitary cell line to explore the mechanism of this response. PGE1- and forskolin-stimulated cyclic AMP accumulation and adenylate cyclase activity are enhanced by exposing viable cells to phorbol esters. Adenylate cyclase activity in the presence of PGE1 demonstrated a biphasic stimulatory, then inhibitory response to increasing GTP concentrations; phorbol esters attenuated this inhibition. These data support the hypothesis that protein kinase C can covalently change the functional state of the adenylate cyclase holoenzyme, amplifying its response to certain hormones.     

An upstream regulator of the glycoprotein hormone alpha-subunit gene mediates pituitary cell type activation and repression by different mechanisms.

Targeting of alpha-subunit gene expression within the pituitary is influenced by an upstream regulatory region that directs high level expression to thyrotropes and gonadotropes of transgenic mice. The same region also enhanced the activity of the proximal promoter in transfections of pituitary-derived alpha-TSH and alpha-T3 cells. We have localized the activating sequences to a 125-bp region that contains consensus sites for factors that also play a role in proximal promoter activity. Proteins present in alpha-TSH and alpha-T3 cells as well as those from GH3 somatotrope-derived cells interact with this region. The upstream area inhibited proximal alpha-promoter activity by 80% when transfected into GH3 cells. Repression in GH3 cells was mediated through a different mechanism than enhancement, as supported by the following evidence. Reversing the orientation of the area resulted in a loss of proximal promoter activation in alpha-TSH and alpha-T3 cells but did not relieve repression in GH3 cells. Mutation of proximal sites shown to be important for activation had no effect on repression. Finally, bidirectional deletional analysis revealed that multiple elements are involved in activation and repression and, together with the DNA binding studies, suggests that these processes may be mediated through closely juxtaposed or even overlapping elements, thus perhaps defining a new class of bifunctional gene regulatory sequence.     

Tissue-specific enhancer of the human glycoprotein hormone alpha-subunit gene: dependence on cyclic AMP-inducible elements.

We identified and characterized elements which confer tissue specificity and cyclic AMP (cAMP) responsiveness to the human glycoprotein alpha-subunit gene. An enhancer containing an 18-base-pair repeat conferred cAMP responsiveness in a non-tissue-specific fashion. DNase I protection assays revealed DNA-binding factors that bound to this element in both placental and nonplacental cells. It also enhanced the alpha-subunit promoter in a tissue-specific manner but had a negligible effect on a heterologous promoter. A unique element found upstream of this enhancer had no independent activity but, in combination with the cAMP-responsive enhancer, distinctly increased the tissue-specific activity of both the alpha-subunit promoter and a heterologous promoter. A factor that bound to this upstream element was found in placental but not nonplacental cells. We conclude that this novel element acts, perhaps through a specific trans-acting factor, in concert with a cAMP-responsive enhancer to confer tissue specificity to the alpha-subunit gene.     

Expression and distribution of pituitary adenylate cyclase-activating peptide in human prostate and prostate cancer tissues

The therapeutic potential of the intestinotrophic mediator glucagon-like peptide-2 (1-33) [GLP-2 (1-33)] has increased interest in the pharmacokinetics of the peptide. This study was undertaken to investigate whether the primary degradation product GLP-2 (3-33) interacts with the GLP-2 receptor. Functional (cAMP) and binding in vitro studies were carried out in cells expressing the transfected human GLP-2 receptor. Furthermore, a biologic response of GLP-2 (3-33) was tested in vivo. Mice were allocated to groups treated for 10 days (twice daily) with: (1) 5 microg GLP-2 (1-33), (2) 25 microg GLP-2 (3-33), (3) 5 microg GLP-2 (1-33)+100 microg GLP-2 (3-33), or (4) 5 microg GLP-2 (1-33)+500 microg GLP-2 (3-33). The intestine was investigated for growth changes. GLP-2 (3-33) bound to the GLP-2 receptor with a binding affinity of 7.5% of that of GLP-2 (1-33). cAMP accumulation was stimulated with an efficacy of 15% and a potency more than two orders of magnitude lower than that of GLP-2 (1-33). Increasing doses of GLP-2 (3-33) (10(-7)-10(-5) M) caused a shift to the right in the dose-response curve of GLP-2 (1-33). Treatment of mice with either GLP-2 (1-33) or (3-33) induced significant growth responses in both the small and large intestines, but the response induced by GLP-2 (3-33) was much smaller. Co-administration of 500 microg of GLP-2 (3-33) and 5 microg GLP-2 (1-33) resulted in a growth response that was smaller than that of 5 microg GLP-2 (1-33) alone. Consistent with the observed in vivo activities, our functional studies and binding data indicate that GLP-2 (3-33) acts as a partial agonist with potential competitive antagonistic properties on the GLP-2 receptor.