Pituitary-specific Gene Research Articles

Suppression of Pituitary Hormone Genes in Subjects Who Died From COVID-19 Independently of Virus Detection in the Gland

ContextInvolvement of the pituitary gland in SARS-CoV-2 infection has been clinically suggested by pituitary hormone deficiency in severe COVID-19 cases, by altered serum ACTH levels in hospitalized patients, and by cases of pituitary apoplexy. However, the direct viral infection of the gland has not been investigated.ObjectivesTo evaluate whether the SARS-CoV-2 genome and antigens could be present in pituitary glands of lethal cases of COVID-19, and to assess possible changes in the expression of immune-related and pituitary-specific genes.MethodsSARS-CoV-2 genome and antigens were searched in the pituitary gland of 23 patients who died from COVID-19 and, as controls, in 12 subjects who died from trauma or sudden cardiac death. Real-time RT-PCR, in situ hybridization, immunohistochemistry and transmission electron microscopy were utilized. Levels of mRNA transcripts of immune-related and pituitary-specific genes were measured by the nCounter assay.ResultsThe SARS-CoV-2 genome and antigens were detected in 14/23 (61%) pituitary glands of the COVID-19 group, not in controls. In SARS-CoV-2 positive pituitaries, the viral genome was consistently detected by PCR in the adeno- and the neurohypophysis. Immunohistochemistry, in situ hybridization and transmission electron microscopy confirmed the presence of SARS-CoV-2 in the pituitary. Activation of type I interferon signaling and enhanced levels of neutrophil and cytotoxic cell scores were found in virus-positive glands. mRNA transcripts of pituitary hormones and pituitary developmental/regulatory genes were suppressed in all COVID-19 cases irrespective of virus-positivity.ConclusionOur study supports the tropism of SARS-CoV-2 for human pituitary and encourage to explore pituitary dysfunction post-COVID-19.

Genomewide analysis of circular RNA in pituitaries of normal and heat-stressed sows

BackgroundAs a newly characterized type of noncoding RNA, circular RNA (circRNA) has been shown to have functions in diverse biological processes of animals. It has been reported that several noncoding RNAs may regulate animals’ response to heat stress which can be easily induced by hyperthermia in summer. However, the expression and functions of circRNAs in the pituitary of sows and whether they participate in heat stress adaption are still unclear.ResultsIn this study, we found that high temperature over the thermoneutral zone of sows during the summer increased the serum heat shock protein 70 (HSP70) level, decreased the superoxide dismutase (SOD) vitality and prolactin (PRL) concentration, and induced heat stress in sows. Then, we explored circRNA in the pituitary of heat-stressed and normal sows using RNA sequencing and bioinformatics analysis. In total, 12,035 circRNAs were detected, with 59 circRNAs differentially expressed, including 42 up-regulated and 17 down-regulated circRNAs in pituitaries of the heat-stressed sows. Six randomly selected circRNAs were identified through reverse transcription PCR followed by DNA sequencing and other 7 randomly selected differentially expressed circRNAs were verified by quantitative real-time PCR analysis. The predicted target genes regulated by circRNAs through sponging microRNAs (miRNAs) were enriched in metabolic pathway. Furthermore, the predicted circRNA–miRNA–mRNA interactions showed that some circRNAs might sponge miRNAs to regulate pituitary-specific genes and heat shock protein family members, indicating circRNA’s roles in pituitary hormone secretion and heat stress response.ConclusionsOur results provided a meaningful reference to understand the functions of circRNA in the porcine pituitary and the mechanisms by which circRNA may participate in animals’ response to heat stress.

Is it time to consider the expression of specific-pituitary hormone genes when typifying pituitary tumours?

The aim of the present study is to check whether we can replicate, in an independent series, previous results showing that the molecular study of pituitary-specific gene expression complements the inmunohistochemical identification of pituitary neuroendocrine tumours. We selected 112 patients (51 (46.4%) women; mean age 51.4±16 years; 102 macroadenomas (91.9%), 9 microadenomas (8.1%)) with complete clinical, radiological, immunohistochemical and molecular data from our data set of pituitary neuroendocrine tumours. Patients were different from those previously studied. We measured the expression of the pituitary-specific hormone genes and type 1 corticotrophin-releasing hormone and arginine vasopressin 1b receptors, by quantitative real-time polymerase chain reaction using TaqMan probes. Afterwards, we identified the different pituitary neuroendocrine tumour subtypes following the 2017 World Health Organization classification of pituitary tumours, calculating the concordance between their molecular and immuhistochemical identification. The concordance between molecular and immunohistochemical identification of functioning pituitary neuroendocrine tumours with the clinical diagnosis was globally similar to the previous series, where the SYBR Green technique was used instead of TaqMan probes. Our results also corroborated the poor correlation between molecular and immunohistochemical detection of the silent pituitary neuroendocrine tumour variants. This discrepancy was more remarkable in lactotroph, null-cell and plurihormonal pituitary neuroendocrine tumours. In conclusion, this study validates the results previously published by our group, highlighting a complementary role for the molecular study of the pituitary-specific hormone genes in the typification of pituitary neuroendocrine tumours subtypes.

Development and characterization of five rainbow trout pituitary single-cell clone lines capable of producing pituitary hormones

Five single-cell clone lines (mRTP1B, mRTP1E, mRTP1F, mRTP1K, and mRTP2A) have been developed from adult rainbow trout pituitary glands. These cell lines have been maintained in a CO(2)-independent medium supplemented with 10% fetal bovine serum (FBS) for more than 150 passages. At about 150 passages, the doubling time of each single-cell clone in a CO(2)-independent medium supplemented with 10% FBS at 20 degrees C was 3.6+/-0.7, 2.8+/-0.7, 3.2+/-0.8, 5.5+/-0.6, and 6.6+/-0.6 days respectively. Each single-cell clone contains 60+/-2 chromosomes, which is within the range of the 2N chromosome numbers reported for rainbow trout. Reverse transcription-PCR analysis revealed that in addition to expressing gh, prolactin (prl), and estradiol (E(2)) receptor alpha (e2ralpha or esr1) genes, each single-cell clone line also expressed other pituitary-specific genes such as tsh, gonadotropin 1 (gth-1 or fshb), gonadotropin 2 (gth-2 or lhb), somatolactin (sl or smtl), proopiomelanocortin-B (pomcb), and corticosteroid receptor (cr or nr3c1). Immunocytochemical analysis showed that all the five single-cell clones produced both Gh and Prl. Furthermore, the expression of gh and prl genes in the single-cell clone lines is responsive to induction by E(2), dexamethasone, and o,p'-dichlorodiphenyltrichloroethane. All together, these results confirm that each of the single-cell clones was derived from rainbow trout pituitary glands. These single-cell clone lines not only can be used to study factors that regulate the expression of pituitary hormone genes, but can also be developed as a rapid screening system for identifying environmental endocrine disruptors.

EXPRESSED SEQUENCE TAG ANALYSIS OF A 4 YEAR-OLD CHINESE STURGEON PITUITARY

Expressed sequence tag ( EST) analysis is an efficient tool for gene discovery and for profiling gene expression. In order to isolate specific functional genes involved in reproduction and endocrine regulation and to reveal their evolutionary mechanisms in Chinese sturgeon (Acipenser sinensis Gray), a chondrostean fish with a history of 140 million years, we constructed its pituitary cDNA library front a 4 year-old male. A total of 944 random clones were sequenced and compared with sequences in GenBank database. Among all the 944 EST clones, 802 (84.96%) clones were identified as 461 known genes, and additional 142 (15.04%) as unknown genes. Functional categorization indicated that the most abundantly expressed functional gene was the proopiomelanocortin ( POMC), which accounted for almost 10.17% of the overall expression, indicating its important function in the pituitary. Interestingly, the expression patterns of 7 unknown genes were analyzed in various tissues, such as heart, liver, spleen, kidney, muscle, testes, ovary and pituitary. Three different categories of expression patterns were observed from them. Several unknown ESTs, such as EG009334, EG009337, EG009338 and EG009340, were detected to be pituitary-specific, or pituitary and ovary-specific genes. Further studies on their functions will be very useful for better understanding the mechanisms of sturgeon reproduction biology and endocrinology.

Essential role of the homeodomain for pituitary homeobox 1 activation of mouse gonadotropin-releasing hormone receptor gene expression through interactions with c-Jun and DNA.

The gonadotropin-releasing hormone receptor (GnRHR) is expressed primarily in the gonadotropes of the anterior pituitary. Pituitary homeobox 1 (Pitx-1) has been shown to activate pituitary-specific gene expression by direct DNA binding and/or protein-protein interaction with other transcription factors. We hypothesized that Pitx-1 might also dictate tissue-specific expression of the mouse GnRHR (mGnRHR) gene in a similar manner. Pitx-1 activated the mGnRHR gene promoter, and transactivation was localized to sequences between -308 and -264. Pitx-1 bound to this region only with low affinity. This region includes an activating protein 1 (AP-1) site, which was previously shown to be important for mGnRHR gene expression. Further characterization indicated that an intact AP-1 site was required for full Pitx-1 responsiveness. Furthermore, Pitx-1 and AP-1 were synergistic in the activation of the mGnRHR gene promoter. A Pitx-1 homeodomain (HD) point mutation, which eliminated DNA binding ability, caused only a partial reduction of transactivation, whereas deletion of the HD completely prevented transactivation. Pitx-1 interacted directly with c-Jun, and the HD was sufficient for this interaction. While the point mutation in the Pitx-1 HD did not affect interaction with c-Jun, deletion of the HD eliminated the interaction. Taken together, our studies indicate that Pitx-1 can direct transactivation of the mGnRHR gene, in part by DNA binding and in part by an action of Pitx-1 as a cofactor for AP-1, augmenting AP-1 activity through a novel protein-protein interaction between c-Jun and the HD of Pitx-1.

Fgf3 signaling from the ventral diencephalon is required for early specification and subsequent survival of the zebrafish adenohypophysis.

The pituitary gland consists of two major parts: the neurohypophysis, which is of neural origin; and the adenohypophysis, which is of non-neural ectodermal origin. Development of the adenohypophysis is governed by signaling proteins from the infundibulum, a ventral structure of the diencephalon that gives rise to the neurohypophysis. In mouse, the fibroblast growth factors Fgf8, Fgf10 and Fgf18 are thought to affect multiple processes of pituitary development: morphogenesis and patterning of the adenohypophyseal anlage; and survival, proliferation and differential specification of adenohypophyseal progenitor cells. Here, we investigate the role of Fgf3 during pituitary development in the zebrafish, analyzing lia/fgf3 null mutants. We show that Fgf3 signaling from the ventral diencephalon is required in a non-cell autonomous fashion to induce the expression of lim3, pit1 and other pituitary-specific genes in the underlying adenohypophyseal progenitor cells. Despite the absence of such early specification steps, fgf3 mutants continue to form a distinct pituitary anlage of normal size and shape, until adenohypophyseal cells die by apoptosis. We further show that Sonic Hedgehog (Shh) cannot rescue pituitary development, although it is able to induce adenohypophyseal cells in ectopic placodal regions of fgf3 mutants, indicating that Fgf3 does not act via Shh, and that Shh can act independently of Fgf3. In sum, our data suggest that Fgf3 signaling primarily promotes the transcriptional activation of genes regulating early specification steps of adenohypophyseal progenitor cells. This early specification seems to be essential for the subsequent survival of pituitary cells, but not for pituitary morphogenesis or pituitary cell proliferation.

Transcriptional regulation of the Luteinizing Hormone Beta-Subunit (LHβ) gene by Ptx1

Objective: Pituitary homeobox 1 (Ptx1) is critical for normal pituitary development and has more recently been shown to be important for expression of pituitary-specific genes in the adult, including the LHβ gene. Separate studies have demonstrated that the orphan nuclear receptors, steroidogenic factor-1 (SF-1) and chicken ovalbumin upstream promoter-transcription factor (COUP-TF), also mediate LHβ gene expression. The specific objective of this study was to further characterize transcriptional regulation of the LHβ gene by Ptx1, alone and in conjunction with SF-1 and the COUP-TFs. Design: LHβ promoter activity was measured in the presence or absence of the transcription factors of interest using a transient transfection-reporter system. Electrophoretic mobility shift assay (EMSA) was used to evaluate Ptx1 binding to LHβ gene sequences. Materials and Methods: A) Monkey kidney fibroblast cells (CV-1) were transiently transfected with rat LHβ promoter sequences subcloned into the luciferase reporter vector, pXP2. Cells were cotransfected with CMV-driven expression vectors for Ptx1, SF-1 and/or COUP-TF. Results are expressed as fold-change relative to expression in cells receiving equivalent amounts of empty expression vector. Statistical significance was determined by ANOVA followed by Student’s t test with significance set at p < 0.05. B) EMSA was used to determine the ability of a GST-Ptx1 fusion protein or endogenous Ptx1 (from the mouse gonadotrope cell line αT3–1) to bind to various rat LHβ promoter sequences. Results: A) Consistent with previous reports by our group and others, addition of Ptx1 significantly increased expression of region −797/+5 of the rat LHβ promoter (6-fold). Introduction of SF-1 also stimulated LHβ promoter activity (37-fold), with marked synergy in the presence of both factors (over 200-fold) (p < 0.01). Mutation of a previously identified Ptx1 cis-element at position -101 decreased (2.6-fold), but did not eliminate, the Ptx1 effect in a −207/+5 construct (p < 0.005 versus control). The Ptx1-SF-1 synergy was lost in this mutated construct. 5’deletion constructs further localized the residual Ptx1 response to nucleotides −82/+5, a region with three potential Ptx-1 cis-elements based on sequence homology to the consensus site. Overexpression of either COUP-TFI or COUP-TFII blunted the Ptx1 response by 75% and 50%, the SF-1 response by 50% and 30%, and the SF-1/Ptx1 response by 85% and 70%, respectively (p < 0.0001 in the presence versus the absence of the COUP-TFs). B) By EMSA, both endogenous Ptx1 and GST-Ptx1 bind to an oligonucleotide spanning the -101 LHβ-Ptx1 cis-element as confirmed by supershift of the protein-DNA complex with a Ptx1-specific antibody. The Ptx1 fusion protein also bound to the 3’Ptx1 region, although with lower affinity than to the −101 element. Oligonucleotides containing mutations in the putative 3’Ptx1 sites demonstrated loss of binding with mutation at position −65, but not at the other two sites. Conclusions: Ptx1 increases transcription of the rat LHβ gene via two cis-elements. SF-1 synergizes with Ptx1 at the 5’element, but apparently not at the 3’ element. Overexpression of the COUP-TFs blunts both the SF-1 response and the Ptx1 response, although the mechanism by which this occurs remains to be elucidated. These data further our understanding of both stimulatory and inhibitory control of gonadotropin gene expression. Supported by: R01 HD38089 (LMH, CDH, ZH) and HD19938 (K-HJ, UBK).

Imaging the Localized Protein Interactions Between Pit-1 and the CCAAT/Enhancer Binding Protein α in the Living Pituitary Cell Nucleus

The homeodomain protein Pit-1 cooperates with the basic-leucine zipper protein CCAAT/enhancer binding protein alpha (C/EBPalpha) to control pituitary-specific prolactin gene transcription. We previously observed that C/EBPalpha was concentrated in regions of centromeric heterochromatin in pituitary GHFT1-5 cells and that coexpressed Pit-1 redistributed C/EBPalpha to the subnuclear sites occupied by Pit-1. Here, we used fluorescence resonance energy transfer microscopy to show that when C/EBPalpha was recruited by Pit-1, the average distance separating the fluorophores labeling the proteins was less than 7 nm. A mutation in the Pit-1 homeodomain, or truncation of the C/EBPalpha transactivation domain disrupted the redistribution of C/EBPalpha by Pit-1. Fluorescence resonance energy transfer analysis revealed that the mutant Pit-1 still associated with C/EBPalpha, and the truncated C/EBPalpha still associated with Pit-1, but these interactions were preferentially localized in regions of centromeric heterochromatin. In contrast, a truncation in C/EBPalpha that prevented DNA binding also blocked its association with Pit-1, suggesting that the binding of C/EBPalpha to DNA is a critical first step in specifying its association with Pit-1. These findings indicated that the protein domains that specify the interaction of Pit-1 and C/EBPalpha are separable from the protein domains that direct the positioning of the associated proteins within the nucleus. The intimate association of Pit-1 and C/EBPalpha at certain sites within the living cell nucleus could foster their combinatorial activities in the regulation of pituitary-specific gene expression.

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

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.

Pituitary homeobox 1 activates the rat FSHbeta (rFSHbeta) gene through both direct and indirect interactions with the rFSHbeta gene promoter.

Molecular mechanisms underlying gonadotrope-specific and hormonal regulation of FSHbeta gene expression remain largely unknown. We have studied the role of pituitary homeobox 1 (Ptx1), a transcription factor important for regulation of many pituitary-specific genes, in the regulation of rat FSHbeta (rFSHbeta) gene transcription. We demonstrate that Ptx1 activates the rFSHbeta gene promoter both basally and in synergy with GnRH. The effect of Ptx1 was localized to -140/-50, a region also important for basal activity of the promoter. Two putative Ptx1 binding sites (P1 and P2) homologous to consensus Ptx1 binding elements were identified in this region. We demonstrate specific binding of Ptx1 to the P2 but not to the P1 site. Furthermore, functional studies indicate that the P2 but not the P1 site mediates activation of the promoter by Ptx1. Residual activation of the promoter by Ptx1 was observed independent of the P2 site. However, no additional Ptx1 binding sites were identified in this region, indicating that the residual activation observed is likely independent of direct Ptx1 binding to the promoter. These results identify a functional Ptx1 binding site in the rFSHbeta gene promoter and suggest the presence of an additional activating pathway that is independent of direct binding of Ptx1 to the promoter.

Multiple Mechanisms for Pitx-1 Transactivation of a Luteinizing Hormone β Subunit Gene

The pituitary homeobox factor-1 (Pitx-1) transactivates a number of pituitary-specific genes through direct interaction with other specific transcription factors. We demonstrate here that Pitx-1 plays a crucial role in the regulation of the Chinook salmon luteinizing hormone beta gene promoter through a number of novel mechanisms. On the proximal promoter its action involves a synergistic effect with steroidogenic factor-1 (SF-1) alone or in combination with the estrogen receptor; promoter activity being induced by 9- or 35-fold over controls, respectively. Further upstream, a series of four Pitx-1 response elements (located between 1366 and 1506 bp from the transcriptional start site) is also involved in regulating the promoter activity. The two distal sequences have the greatest effect on the basal activity and are also essential for the gonadotropin-releasing hormone (GnRH) response. Mammalian two-hybrid assays revealed that Pitx-1 can homodimerize. Moreover, circular permutation assays indicate that binding of Pitx-1 to more than one response element induces conformational changes of the target DNA. This constitutes an additional mechanism through which Pitx-1 can mediate transactivation of this gene, allowing the demonstrated interaction of proximal response elements and distal enhancers, thus facilitating the maximal GnRH response that was seen in the longer promoter constructs. Our research also indicates that Pitx-1 is phosphorylated on three residues when bound to the DNA.

Expression profile of active genes in the human pituitary gland.

To characterize transcripts abundantly expressed in the human pituitary gland in general as well as to isolate novel transcripts expressed specifically in this gland, we generated an expression profile of the active genes transcribed in it. A total of 1015 randomly collected 3prime prime or minute expressed sequence tags (ESTs) (gene signatures, GSs) were grouped into 527GS species. The results showed the relative expression levels of genes in the pituitary gland. The genes comprising more than 1% of total mRNA were prolactin, growth hormone and chromogranin B genes. When known genes were categorized, the genes for pituitary hormones were the most actively transcribed, followed by the genes for ribosomal proteins, nuclear proteins and secretory granule proteins. Through comparison of this gene expression profile with the BodyMap database containing profiles generated from 63 other human tissues, we obtained 11 genes which appeared to be specifically expressed in the pituitary gland. In addition to the eight known genes, we identified three novel pituitary-specific transcripts which encode putative proteins: pituitary gland specific factor 1a (PGSF1a), PGSF1b and PGSF2. This expression profile method is a novel approach to the isolation of pituitary-specific genes that may have important functions.

Pituitary Adenylate Cyclase-activating Polypeptide and Cyclic Adenosine 3′,5′-Monophosphate Stimulate the Promoter Activity of the Rat Gonadotropin-releasing Hormone Receptor Gene via a Bipartite Response Element in Gonadotrope-derived Cells

Specific type I receptors for pituitary adenylate cyclase-activating polypeptide (PACAP) are present in gonadotrope cells of the anterior pituitary gland. By transient transfection of mouse gonadotrope-derived alphaT3-1 cells, which are direct targets for PACAP and express gonadotropin-releasing hormone receptor (GnRH-R), a marker of the gonadotrope lineage, we provide the first evidence that PACAP stimulates rat GnRH-R gene promoter activity. The EC(50) of this stimulation is compatible with a mediation via activation of the cyclic AMP-dependent signaling pathway and, consistently, co-transfection of an expression vector expressing the protein kinase A inhibitor causes reduction in PACAP as well as cholera toxin-stimulated promoter activity. Deletion and mutational analyses indicate that PACAP activation necessitates a bipartite response element that consists of a first region (-272/-237) termed PACAP response element (PARE) I that includes a steroidogenic factor-1 (SF-1)-binding site and a second region (-136/-101) referred to as PARE II that contains an imperfect cyclic AMP response element. Gel shift experiments indicate the specific binding of the SF-1 and a potential SF-1-interacting factor to PARE I while a protein immunologically related to the cyclic AMP response element-binding protein interacts with PARE II. These findings suggest that PACAP might regulate the GnRH-R gene at the transcriptional level, providing novel insights into the regulation of pituitary-specific genes by hypothalamic hypophysiotropic signals.

Dual regulation of somatostatin receptor subtype 1 gene expression by pit-1 in anterior pituitary GH3 cells.

Somatostatin represents a major release inhibiting factor for hypophyseal hormones and mediates its action via five receptor subtypes, sst1-sst5, that are all present in the anterior pituitary. The pituitary specific transcription factor Pit-1 is essential for the pituitary development and pituitary-specific gene expression. Here the transcriptional regulation of the sst1 gene, which contains putative Pit-1-binding sites, was studied in anterior pituitary GH3 cells. We found that a fragment of 2 kb suffices to drive the expression of a reporter gene specifically in this cell line. Positive and negative cis-regulatory elements contributed to the promoter activity. Among these elements two functional binding sites for Pit-1 were identified. While the proximal site mediated transcriptional activation, the distal site attenuated transcription of reporter gene constructs. Mutations of the proximal Pit-1 site prevented expression of the reporter gene. Targeting Pit-1 mRNA by antisense oligonucleotides caused inhibition of transcription of reporter gene constructs containing the proximal Pit-1-binding site. Moreover, the expression of the endogenous sst1 gene in GH3 anterior pituitary cells was blocked. This resulted in reduced sst1 levels at the plasma membrane. Reduced sst1 levels were associated with a diminished antisecretory response to the sst1-specific agonist CH-275 and somatostatin. These results demonstrate the importance of Pit-1 for the expression of the sst1 gene, which hence is placed under common genetic control with the genes for hypophysiotropic hormones and the gene for the receptor of GH-releasing hormone.

Human growth disorders: molecular genetics of the growth hormone-insulin-like growth factor I axis.

The critical role of the growth hormone-insulin-like growth factor I axis in controlling somatic growth in humans and other vertebrate species has been known for many years. Through molecular cloning and other related techniques many of the components of this axis have been characterized, with the most recent additions being key transcription factors required for pituitary development and for pituitary-specific gene expression. Several of these genes have been shown to be mutated in familial and sporadic human growth deficiency syndromes, thereby validating the central roles of the encoded proteins in the endocrine pathways regulating somatic growth. The purpose of this review is to highlight these recent advances from the perspective of the molecular genetics of human growth disorders.

The PTX family of homeodomain transcription factors during pituitary developments

A subfamily of bicoid-related homeodomain factors was recently discovered through its involvement in transcription of pituitary-specific genes. We isolated the first member of this family, Ptx1 (pituitary homeobox 1), through its DNA binding properties whereas a second related gene, Ptx2 (RIEG), was identified by positional cloning as the causative gene for Rieger's syndrome. The mechanisms of Ptx action on its target genes as well as its putative roles during development are reviewed with particular emphasis on its role in pituitary function.

Differential regulation of pituitary-specific gene expression by insulin-like growth factor 1 in rat pituitary GH4C1 and GH3 cells.

We have compared the influence of insulin-like growth factor 1 (IGF-1) on pituitary gene expression in the rat cell lines GH4C1 and GH3. Incubation with IGF-1 increased PRL messenger RNA (mRNA) levels in GH4C1 cells by 4- to 5-fold but decreased the levels of PRL transcripts in GH3 cells. In addition, the levels of GH-mRNA that were not affected by IGF-1 in GH4C1 cells were significantly inhibited by the growth factor in GH3 cells. IGF-1 also decreased PRL and GH-mRNA response to T3, retinoic acid, and Fk in GH3 cells. Stability of PRL or GH transcripts was not altered by IGF-1 in GH3 cells, suggesting that the inhibitory effect is exerted at a transcriptional level. The pituitary-specific transcription factor GHF-1/Pit-1 activates both the GH and PRL promoters. As analyzed by Western blot, IGF-1 did not alter GHF-1/Pit-1 protein levels in GH4C1 cells but reduced the levels of the transcription factor in GH3 cells. This decrease is secondary to a reduction of GHF-1/Pit-1 transcripts in IGF-1-treated GH3 cells. Thus, a different effect of IGF-1 on the expression of GHF-1/Pit-1 in GH3 and GH4C1 cells is likely involved in the different regulation of GH and PRL gene in both cell types. IGF-1 increases the activity of the PRL promoter in transient transfection assays in GH4C1 cells by a Ras-dependent mechanism. Expression of oncogenic Ras(Val12) mimics the effect of IGF-1, and the dominant negative Ras(Asn17) blocks IGF-1-mediated stimulation of the PRL promoter in GH4C1 cells. Although IGF-1 did not stimulate the PRL promoter in GH3 cells, Ras(Val12) strongly activated the promoter in these cells. Hence, the machinery to activate Ras-dependent signaling is intact in GH3 cells. Moreover, IGF-1 stimulates the mitogen-activated protein kinase in GH3 cells, showing that the components linking the IGF-1 receptor to Ras are also active. These results suggest that, in addition to the Ras/mitogen-activated protein kinase pathway, IGF-1 could activate a different pathway and that the combination of both is required to elicit PRL gene expression by the growth factor. This second pathway may be defective in GH3 cells that respond to Ras but not to IGF-1.

Differential Regulation of Pituitary-Specific Gene Expression by Insulin-Like Growth Factor 1 in Rat Pituitary GH4C1 and GH3 Cells

Differential Regulation of Pituitary-Specific Gene Expression by Insulin-Like Growth Factor 1 in Rat Pituitary GH4C1 and GH3 Cells

PIT-1 Protein Localization at Different Optical Sections in a Single Living Cell Using FRET Microscopy and Green Fluorescent Proteins

Abstract The pituitary specific transcription factor Pit-1 is required for transcriptional activity of the prolactin (PRL) gene. The Pit-1 protein is a member of the POU homeodomain transcription factors that is expressed in several different anterior pituitary cell types, where it functions as an important determinant of pituitary-specific gene expression. The Pit-1 protein generally interacts with DNA elements in the PRL gene promoter as a dimer, and has been demonstrated to associate with other transcription factors. The objective of our research is to define the critical molecular events involved in transcriptional regulation of the PRL gene in living cells. Methods that allow monitoring of the intimate interactions between protein partners in living cells provide an unparalleled perspective on these biological processes. Using the jellyfish green fluorescent protein (GFP) as a tag, we applied the fluorescence resonance energy transfer (FRET) technique to visualize where and when the Pit-1 protein interacts in the living cell. FRET is a quantum mechanical effect that occurs between donor (D) and acceptor (A) fluorophores provided: (i) the emission energy of D is coincident with the energy required to excite A, and (ii) the distance that separating the two fluorophores is 10-100 Å. Mutant forms of GFP that fluoresce either green or blue (BFP) have excitation and emission spectra that are suitable for FRET imaging.