Transcription factors in normal and neoplastic pituitary tissues

Transcription Factors in Normal and NeoplasticPituitary TissuesRICARDO V. LLOYD1* AND ROBERT Y. OSAMURA2

1Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, Minnesota 559052Tokai University School of Medicine, Isehara City, Japan

KEY WORDS transcription factors; DNA; proteins; pituitary tissues

ABSTRACT Transcription factors are proteins that bind to regulatory elements in DNA andhave critical roles in gene regulation during development, in cellular growth and differentiation. Thefour major groups of transcription factors have been classified according to the motif in theDNA-binding domains and include: (1) the helix-turn-helix group, which includes the Pit-1/GHF-1(Pit-1) transcription factor; (2) the zing finger group, which includes estrogen and other steroidhormone receptors; (3) the leucine zipper group, which includes c-fos protooncogene, and (4) thehelix-loop-helix group, which includes the c-myc oncogene. Members of all four groups have beendescribed in normal and neoplastic anterior pituitary gland tissues. Pit-1 has been shown toregulate prolactin (PRL), growth hormone (GH), and thyroid-stimulating hormone (TSH) cellsduring development and differentiation. Genetic defects in this transcription factor have led tospecific diseases in rodents and humans such as dwarfism and cretinism. Estrogen receptor (ER)protein plays a critical role in the regulation of gene expression in some anterior pituitary cells.There is a differential distribution of ER in anterior pituitary cells and tumors; PRL, gonadotroph,and null cell tumors are the principal adenomas expressing ER. The protooncogene c-fos is regulatedby estrogen in various tissues, linking the regulation of one transcription factor by anothertranscription factor with a different motif. The c-myc oncogene has been detected in the pituitarygland and in some pituitary tumors, although the exact role of this oncogene in pituitary tumordevelopment is uncertain. Because of the critical role that transcription factors play in pituitary celldevelopment and differentiation, we can anticipate many more studies to elucidate theirmany functions in normal and neoplastic pituitary tissues. Microsc. Res. Tech. 39:168–181,1997. r 1997 Wiley-Liss, Inc.

INTRODUCTIONTranscription factors are proteins that bind to regula-

tory elements in the promoter and enhancer regions ofDNA and play pivotal roles in regulating gene expres-sion (Latchman, 1991; Pabo and Sauer, 1992; Pa-pavassiliou, 1995; Rosenthal, 1994; Vellanoweth et al.,1994). These factors interact with DNA and stimulate(or inhibit) gene transcription and mRNA production.The DNA-binding domain of transcription factors usu-ally forms an alpha helix adjacent to or within posi-tively charged amino acids (Pabo and Sauer, 1992;Papavassiliou, 1995; Vellanoweth et al., 1994). Theactivity of transcription factors is regulated by variousmodifications including phosphorylation. Transcriptionfactors have critical regulatory roles in developmentand differentiation.

The four general motifs in the DNA-binding domainsof transcription factors include (1) helix-turn-helix, (2)zinc finger, (3) leucine zipper, and (4) helix-loop-helix(Table 1 and Fig. 1).

HELIX-TURN-HELIXThe transcription factors of the homeobox or homeodo-

main group have the helix-turn-helix motif. Homeoboxgenes encode highly conserved sequences of 60 aminoacid residues forming a domain in a set of proteinsbinding to specific sites on the DNA target gene. Four

helices usually constitute the polypeptide chain in thehomeodomain and one of these chains recognizes aspecific DNA sequence. Homeobox genes were firstdescribed in Drosophila but are critical for embryonicdevelopment of multicellular organisms including mam-mals (Graham et al., 1989). These transcription factorscontain a conserved homeodomain, the POU homeodo-main, linked to another conserved domain, the POU-specific domain (Fig. 2) (Andersen and Rosenfeld, 1994).These two domains are linked together by a poorlyconserved linker sequence and this gene family or POU(Pit-1, Oct-1, Oct-2, unc 86) plays critical roles in theregulation of gene development of the central nervoussystem, pituitary, and immune system (Andersen andRosenfeld, 1994; Theill and Karin, 1993). The Pit-1/GHF-1 (referred to as Pit-1), transcription factor wasinitially described and characterized in two laborato-ries (Andersen and Rosenfeld, 1994; He et al., 1989;Theill and Karin, 1993).

The Pit-1 gene has 291 amino acid residues and ishighly conserved with a 96% sequence protein identity

*Correspondence to: R.V. Lloyd, M.D., Dept. of Laboratory Medicine andPathology, Mayo Clinic, 200 First Street, SW, Rochester, MN 55905. Fax: (507)284-1599.

Received 10 February 1995; accepted in revised form 12 May 1995

MICROSCOPY RESEARCH AND TECHNIQUE 39:168–181 (1997)

r 1997 WILEY-LISS, INC.

and a 90% identity at the DNA level in rodents andprimates.

The homeodomain of Pit-1 has a 73% match to aconsensus homeodomain sequence and is essential forbinding to DNA. The POU-specific domain has a groupof amino acids (about 27 of 81) that are constant amongPOU proteins, indicating a conserved structure andfunction of this domain. Extensive studies have shownthat Pit-1 expression is largely restricted to the ante-rior pituitary gland (reviewed by Andersen and Rosen-feld, 1994; Theill and Karin, 1993).

Pit-1 has a DNA-binding region that is composed of a60–amino acid homeodomain linked to a 75–amino acidPOU-specific domain. The POU homeodomain is simi-

lar to the helix-turn-helix of other homeodomains. TheDNA-binding sites for Pit-1 contains a core sequence,TATNCAT, or highly related octamer site. The GH andPRL regulatory regions also contain an AT-rich stretchadjacent to this core sequence. Pit-1 binds cooperativelyto the response elements as a dimer that requires thePOU domain that is important in gene activation(Andersen and Rosenfeld, 1994).

Activation of the Pit-1 gene occurs by various mecha-nisms. Pit-1 is phosphorylated in vivo at Ser 115 andThr 220 in response to cAMP or phorbol ester, implicat-ing protein kinase A (PKA) and protein kinase C (PKC)

Fig. 1. Protein structures of the major transcription factors includ-ing the transcriptional activating domains, which are linked to aDNA-binding domain. A dimerization domain is present with thehelix-turn-helix (HTH), leucine zipper (LZ), and helix-loop-helix (HLH)

motifs. Zn, zinc; A, aminoacids cystine or histidine; L, leucine. The plussign in the LZ and HLH indicate a positive charge from basic aminoacids.

Fig. 2. Pit-1/GHF-1 protein representing the helix-turn-helix tran-scription factor motif. The POU domain composed of the POU-specificand POU homeodomain is responsible for DNA binding.

TABLE 1. Classification of transcription factors1

Class ExamplesExamples in

pituitary

Helix-turn-helix Homeobox or homeodomain Pit-1/GHF-1Zinc finger Steroid hormone receptors Estrogen receptor

Thyroid hormone receptorRetinoic acid receptor

Leucine zipper c-Jun and c-fos protoonco-genes

CREB

c-Jun and c-fosCREBTEF

Helix-loop-helix Immunoglobulin geneenhancers

c-myc

Muscle transcription fac-tors

c-Myc genes1CREB, cyclic AMP response element binding protein; TEF, thyrotroph embry-onic factor.

169TRANSCRIPTION FACTORS IN PITUITARY TISSUES

activation pathways, although this phosphorylationdecreases the ability of Pit-1 to bind proximal PRL andGH DNA target sites. One possibility is that Pit-1 mayrecruit other factors in a PKA- or PKC-dependentmanner to the Pit-1-binding sites (Kapiloft et al., 1991).Precedent for this interaction is observed with coopera-tion of Pit-1 with Oct-1 or ER to activate the rPRLpromoter and interaction with Zn 15 and thyroid hor-mone receptor to activate the rGH promoter and inter-action with an activator protein-1- (AP-1) like factor tomediate basal and PKC-regulated human TSHb pro-moter activity (Kim et al., 1993).

In experiments with transgenic mice, a 14.8-Kbportion of 58-flanking Pit-1 DNA sequence is sufficientto target expression to the anterior pituitary (Andersenand Rosenfeld, 1994). However, an enhancer with fivePit-1-binding sites, a vitamin D3 response element, anda retinoic acid response element, which is composed of390 base pairs, are located about 10 Kb 58 upstream ofthe transcription start site. A 15-Kb Pit-1 58 flankingsequence linked to be SV40 large T oncogene producedPit-1-expressing tumors in mice, but the tumors did notproduce PRL, GH, or TSH, suggesting that it was aprecursor cell type (Lew et al., 1993).

The major Pit-1 proteins are 31 and 33 kDa in size.They are generated by the alternative use of transla-tional initiation sites. Several variants of Pit-1 of lowabundance have also been reported (Andersen andRosenfeld, 1994; Bodner et al., 1988; Day and Day,1994; Gutierrez-Hartman, 1994; Haugen et al., 1994;

Okimira et al., 1994). Pit-1 protein is phosphorylated attwo sites in response to treatment with cAMP andphorbol esters. Phosphorylation can alter the ability ofPit-1 to bind to certain DNA-binding sites. Some recentstudies suggest that an alternatively spliced form ofPit-1 is a potential mediator of repression of PRL geneexpression (Andersen and Rosenfeld, 1994; Day andDay, 1994).

Evidence implicating Pit-1 in pituitary cell prolifera-tion was reported by Castrillo et al. (1991). Theseinvestigators used antisense constructs to show thatPit-1 has a stimulatory effect on cell proliferation in theanterior pituitary.

Various studies have shown that the GH, PRL, andTSHb genes are the direct targets of Pit-1. Evidence forthese conclusions is supported by the absence of thesethree genes in Pit-1-defective mice (Camper et al., 1990;Li et al., 1990; Sen et al., 1990; Voss and Rosenfeld,1992). The Snell and Jackson dwarf mutants, whichhave virtually no PRL, GH in TSH cells, and markedlyhypoplastic anterior pituitary glands, have Pit-1 genemutations. The Jackson mutation is a rearrangementwhile the Snell dwarf has a missense mutation in thePit-1 POU homeodomain. The growth hormone-releas-ing hormone (GHRH) receptor gene is another targetgene for Pit-1. Cloning of the GHRH receptor (Mayo,1992) has revealed a mutation in the Little mouse inwhich the somatotrophs are decreased by 90%. Thesefindings support the concept that GHRH receptor was atarget for Pit-1-induced GH cell proliferation.

Developmental studies in rodents have outlined aspecific sequence of pituitary cell development: alphasubunit of glycoprotein hormone-expressing cells fol-lowed by proopiomelanocortin- (POMC) expressing cell,then TSHb and follicle-stimulating hormone/luteiniz-ing hormone (FSH/LH) cells followed by GH and PRLcells (Voss and Rosenfeld, 1992). Pit-1 primarily affectsdevelopment of PRL, GH, and TSHb cells in the caudo-medial, but not in the rostral tip of the developingpituitary gland (Andersen and Rosenfeld, 1994). How-ever, other transcription factors are needed for pitu-itary cell development. For example, the PRL distalenhancer requires estrogen receptor in addition to Pit-1while the GH promoter requires several transcriptionfactors in addition to Pit-1, which may include thethyroid hormone receptor and Zn 15, a zinc fingerprotein that binds between the Pit-1 elements in theGH gene (Andersen and Rosenfeld, 1994). The TSHbgene may require synergy with an activator protein-1-(AP-1) like factor and Pit-1 (Andersen and Rosenfeld,1994). Another transcription factor thyrotroph embry-onic factor (TEF), which is a basic leucine repeattranscription factor, is needed for TSHb expression inthe rostral tip of the developing pituitary gland (Droletet al., 1991).

Localization of Pit-1 ProteinVarious immunohistochemical studies have been done

to localize Pit-1 protein in rodent and human pituitar-ies. Simmons et al. (1990) observed Pit-1 protein expres-sion in GH, PRL, and TSH cells in the rat pituitary, butnot in the ACTH or gonadotroph cells. Interestingly,Pit-1 mRNA was localized in all five cell types by in situhybridization. Osamura and his colleagues (Osamuraet al., 1993; Sanno et al., 1994a,b; Umemura et al.,

Fig. 3. Immunohistochemical localization of various hormones inthe pituitary adenomas of a 10-month-old male growth hormonereleasing hormone (GHRH) transgenic mouse. The adenoma (A) showspositive cells for hGHRH, GH, PRL, and TSH beta subunit. Theadenoma is negative for alpha subunit and ACTH. H, hyperplasia.(3300.)

170 R.V. LLOYD AND R.Y. OSAMURA

1995) studied Pit-1 expression in GHRH transgenicmice using a synthetic tetradecapeptide (KVR-RIKLGYTQTNV) to produce antibodies. The sequencecorresponded to amino acids 147–162. Pit-1 antibodiesproduced in rabbits stained hyperplastic pituitary cellsand adenomas with more intense staining in the adeno-mas compared to hyperplastic pituitaries (Osamura etal., 1993) (Figs. 3 and 4).

Several investigators have localized Pit-1 protein inhuman pituitary tumors (Table 2, Figs. 5 and 6). GH,mixed GH, PRL, and PRL adenomas are consistentlypositive usually with strong nuclear staining whileadrenocorticotropic hormone (ACTH) and gonadotrophtumors are usually negative for Pit-1 immunoreactiv-ity. The findings in human pituitary tumors are thussimilar to these in the rodent pituitary (Asa et al., 1993;

Delhase et al., 1993; Friend et al., 1993; Hoggard et al.,1993; Inada et al., 1993; Sanno et al., 1993).

Analysis of Pit-1 mRNA expression in rodent pitu-itary has been reported by various investigators (Lloydet al., 1993b; Simmons et al., 1990). A double localiza-tion study by Lloyd et al. (1993b) using in situ hybridiza-tion and immunostaining showed 72% of GH cells, 45%PRL cells, 38% of TSH cells, and 23% of LH cellsexpressing Pit-1 mRNA confirming the earlier observa-tion of Simmons et al. (1990) that Pit-1 mRNA was alsoexpressed in cell types other than GH, PRL, and TSH inthe rodent pituitary.

Many investigators have examined Pit-1 mRNA ex-pression in human pituitaries using a variety of meth-ods including in situ hybridization, Northern hybridiza-tion, and the reverse-transcription polymerase chain

Fig. 4. Immunohistochemical localization Pit-1 protein in the portion of pituitary tissue with theadenoma (A) and with the hyperplasia (H) in the pituitary gland of the transgenic mouse in Figure 1(3200). Note more intense immunolocalization of Pit-1 in the adenoma cells, which suggests a role infunctional differentiation (inset 3600).

TABLE 2. PIT-1/GHF-1 protein expression in human pituitary adenomas1

Reference Technique

Adenoma type

GH GH/PRL PRL TSH NF Null GTH ACTH

Sanno et al. (1994a,b) IHC 11 ND 11 111 1 ND 0 0Asa et al. (1993) IHC 111 11 111 ND ND 1a 1a 0Friend et al. (1993) IHC 111 111 111 ND ND 1 0 0Hoggard et al. (1993) Western blotting 111 ND 111 ND 111 ND ND NDDelhase et al. (1993) IHC 111 111 111 111 ND 0 0 01a, focal staining reported; NF, nonfunctional adenomas; 0, negative; 1, few cells positive or weak signal; 11, moderate number of cell positive; 111, many cellspositive; ND, not done.


reaction (RT-PCR) (Asa et al., 1993; Delhase et al.,1993; Friend et al., 1993; Hoggard et al., 1993; Lloyd etal., 1993a; Pellegrini, 1994) (Figs. 7–9). These studieshave localized Pit-1 to GH, PRL, mixed GH/PRL, andTSH tumors. Some null cell adenomas also expressPit-1 mRNA while gonadotroph cell, ACTH tumorshave very low levels or absent Pit-1 mRNA transcripts(Table 3). The principal Pit-1 mRNA transcript is a2.4-Kb species with smaller amounts of larger (3.4, 4.5Kb) and smaller (1.2 and 1.6 Kb) transcripts. In studiesin which both normal pituitaries and adenomas wereexamined, the tumors consistently expressed morePit-1 mRNA compared to the normal pituitary, suggest-ing increased expression of Pit-1 with neoplastic devel-opment.

As previously discussed, dwarfism in mice has beenassociated with specific genetic changes in the Pit-1gene. The Snell mouse has a single-base substitutionwith a T for G in codon 261, which changed it fromtryptophan to cysteine in the POU homeodomain. TheJackson mutation is caused by a rearrangement. Thesemutations result in low to absent levels of GH and PRLcells as well as decreased or absent TSH cells. TheAmes dwarf mutation produces a phenotype very simi-lar to the Jackson and Snell mutations in the Pit-1 gene(Camper et al., 1990; Li et al., 1990; Voss and Rosenfeld,1993).

Pit-1 mutations in humans have been reported byvarious investigators after the human Pit-1 gene wascloned (Lew and Elsholtz, 1991; Ohta et al., 1992a;

Radovick et al., 1992; Tatsumi et al., 1992a,b). Muta-tions in codons 271, 158, 172, and 24 have all beenobserved and are associated with dwarfism and/orcretinism. In the study of Pfaffle et al. (1992), a genedosage effect and disruption of the predicted alpha-helical structure in the POU-specific domain resultingin a compromised transactivation of a subset of Pit-1target genes was observed. Deficiencies of GH, PRL,and TSH have been observed in humans with Pit-1mutations, while the LH and ACTH responses to stimu-lation are usually within normal ranges (Ohta et al.,1992b; Parks et al., 1993; Radovick et al., 1992; Tat-sumi et al., 1992a). Identification of specific mutationsin Pit-1 has broad clinical implications, since it canidentify the causes of hormonal deficiency that areobserved clinically and can also be used to identifycarriers and for presymptomatic diagnosis of yet uniden-tified members in the same family.

ZINC FINGER GROUPThe zinc finger group represents a second motif of

transcription factors, which consists of either a pair ofhistidine and a pair of cysteines or two pairs of cys-teines that together bind a zinc ion. Zinc finger transcrip-tion factors function by binding as dimers to theupstream response elements. These include steroidhormone receptors, thyroid hormone receptors, retinoicacid, and vitamin D receptors (Papavassiliou, 1995).

Fig. 5. Immunohistochemical localization of Pit-1 protein in normal human pituitary gland. Thehuman Pit-1 protein is localized in the nuclei of GH, PRL, and TSH cells (3300).


Estrogen ReceptorsEstrogen receptor (ER) is a ligand-activated transcrip-

tion factor that binds a cis-acting DNA sequence, theestrogen response element (ERE) after binding estro-gen. This interaction usually enhances transcription ofestrogen responsive genes (Carson-Jurica et al., 1990;Green, 1990; Pakdel and Katzenellenbogen, 1992; Wei,1993). Estrogen binding to the ER usually activatesmitogenesis in various cell types. Two domains of theER have been conserved in several species, the DNA-binding and hormone-binding regions. The functionalparts of the receptor include ligand binding, dimeriza-tion, DNA binding, and transcription activation. Theestrogen-bound ER activates specific gene transcrip-tion by binding as a dimer of receptor-hormone com-plexes to a DNA enhancer sequence or ERE. Estrogeninduces the expression of some early response genessuch as c-fos, and the c-fos gene has an ERE in its58-flanking region (Weisz and Rosales, 1990).

ER protein has been studied in the pituitary ofexperimental animals (Keefer et al., 1976; Thieulantand Duval, 1985) and various workers have shown thatestradiol regulates the transcription of the prolactingene (Maurer, 1982; Shull and Gorski, 1985). In thepituitary gland of animals, ER is localized primarily toPRL, GH, and gonadotroph cells (Fig. 10). In humanpituitary and pituitary adenomas, a few studies haveexamined ER (Fig. 11). Pinchon et al. (1980) foundestrogen binding in 14 of 23 adenomas with the higher

levels in PRL tumors, but GH and chromophobic adeno-mas also expressed ER. Nakao et al. (1989) found ERprotein in 10 of 56 adenomas with the highest levels inPRL adenomas, gonadotroph adenomas, and combinedGH/PRL adenomas. ER was not detected in pure GHtumors or in nonfunctioning tumors. Recent studieshave used immunohistochemistry with highly specificanti-ER antibodies and/or molecular techniques to local-ize ER in pituitary tumors (Friend et al., 1994; Stefane-anu et al., 1994). Stefaneanu et al. (1994) localized ERby in situ hybridization in 109 surgically resectedadenomas and 9 non-tumorous pituitaries. ER mRNAwas present in all adenoma types, but the strongestsignal was present in PRL tumors. Prior bromocriptinetreatment in pituitary adenomas markedly reduced thehybridization signal, suggesting that the ER gene has arole in inhibiting PRL synthesis and tumor growth.Friend et al. (1994) used a combination of techniquesincluding immunostaining with a recently developedmonoclonal antibody for ER (ER1D5), which is moresensitive than the Abbott H222 rat monoclonal ERantibody, Northern analysis, ribonuclease protectionassays, and (3H) estrogen binding to analyze ER in 41human pituitary adenomas. ER was detected in 2 of 2PRL adenomas and in 2 of 5 mixed GH/PRL adenomasand in 10 of 17 gonadotroph adenomas while all 4 pureGH adenomas were negative for ER mRNA. ER immu-nochemistry in 14 cases showed a 100% correlationwith ribonuclease protection assay results.

Fig. 6. Immunohistochemical localization of Pit-1 protein in a GH-secreting human pituitaryadenoma. Note intense immunolocalization in many adenoma cells (3300).


The effects of estrogens on cultured pituitary cellsmay be related to the expression of ER. In a cell culturestudy of six human pituitary adenomas, Caronti et al.(1993) observed a stimulatory effect of estradiol on cellproliferation, which appeared to correlate with specificreceptor expression. Tamoxifen had an inhibitory effecton cell proliferation and this effect appeared to beindependent of ER content. The role of estrogen and ERin pituitary tumor development is uncertain. In a studyof 67 men treated with diethylstilbestrol at the MayoClinic, Scheithauer et al. (1989) found no statisticallysignificant differences in PRL cell hyperplasias or adeno-mas in treated patients compared to controls. However,they did observe a correlation between the duration ofestrogen treatment and the total number of pituitaryadenomas including those composed of prolactin cells.

Only anecdotal reports of estrogen treatment leading toprolactinoma have been reported (Gooren et al., 1988).

The observation of patients with ER-positive breastcarcinomas that behave like ER-negative tumors hasled investigators to analyze the molecular mechanismsin order to explain the apparent paradox in theseneoplasms (Fuqua et al., 1992; McGuire et al., 1991).This has led to the description of ER variants in breastcarcinoma including those that act as dominant-positive receptors that are transcriptionally active inthe absence of estrogen and dominant-negative recep-tors that are transcriptionally inactive but prevent theaction of normal estrogen receptors. These investiga-tors suggested that elevated levels of expression ofvariant ER, which interferes with normal ER function,could make a tumor unresponsive to estrogen andanti-estrogens, or to become functionally receptor nega-tive. Estrogen receptor heterogeneity has been de-scribed in the rat pituitary (Geffrey-Roisne et al., 1992)with a 65-kDa form predominating in gonadotroph-enriched cell population and a 50 kDa-form predominat-ing in PRL and GH immunoreactive cells. However, it isnot known if these variant forms are present in thehuman pituitary and the significance of variant formsof ER in pituitary diseases is uncertain.

LEUCINE ZIPPER GROUPTranscription factors with a leucine-zipper motif

consist of an extended alpha helix in which leucineoccupies every seventh position (Papavassiliou, 1995).The interaction of these periodic leucine residues alongone face of the alpha helix with the corresponding helixof the other chain results in an intermolecular coil thatstabilizes the two proteins with the leucine-zippermotif. These proteins are made up of homodimer orheterodimers (Papavassiliou et al., 1992; Papavassil-iou, 1995). The protooncogenes products Jun and fosand the AP-1 transcriptor factor have specific effects inthe pituitary gland (Fig. 12). c-Fos is an intermediateearly gene whose product has been implicated in theregulation of transcription of a number of hormonegenes, including several pituitary hormones. Recentstudies have shown that c-fos must dimerize to amember of the c-jun family (jun A, jun B, or jun D) tobind DNA and modulate transcription (Vogt and Bos,1990).

Billestrup et al. (1987) studied the expression of c-fosin primary pituitary cell cultures and noted that c-fosmRNAwas increased within 20–60 minutes after stimu-lation with the hypothalamic peptide growth hormone-releasing hormone. Both PKA (forskolin) and PKC(phorbol 12, 13 dibutyrate) induced c-fos gene andincreased fos protein, which was localized in the cellnuclei. Somatostatin partially inhibited the GRF-induced c-fos expression. Similar findings by Boutillieret al. (1991) on the induction of c-fos mRNA accumula-tion in response to corticotrophin-releasing factor (CRF)in the AtT 20 cell line have been reported. Theseinvestigators observed that c-fos transcription wasmediated by a PKA activation mechanism. As discussedearlier, estrogen also has a role in c-fos mRNA regula-tion (Insel, 1990; Loose-Mitchell et al., 1988) in theuterus and in the brain. Similar stimulation by thyrotro-pin-releasing hormone on c-fos mRNA has been ob-served in the GH3 transplantable pituitary cell line by

Fig. 7. Northern hybridization analysis of Pit-1 (A), GH (B), and bactin mRNA (C) in a cultured GH adenoma. Lane 1 is untreatedcontrol; lane 2 is GRF-treated cells; lane 3 is phorbol 12-myristrate 13acetate-treated cells; and lane 4 is dexamethasone-treated cells.Twenty micrograms of total RNA was used in each lane, and b-actinwas used to normalize the blot. Blots were washed between eachhybridization experiment, and autoradiograms were analyzed bydensitometry. (Reproduced from Lloyd et al. 1993a with permission ofthe publisher.)


Weisman et al. (1987). Pessegue et al. (1994) recentlyreported that thyrotropin-releasing hormone increasedc-fos mRNA levels in the GH3B6 pituitary cell line. Inaddition, jun B mRNA was also stimulated by thyrotro-pin-releasing hormone and the heterodimer of JunB/c-Fos was thought to participate in PRL secretion andgene transcription. Estrogen treatment also increasedc-fos mRNA in the rat pituitary gland (Chernavsky etal., 1993a; Szijan et al., 1992).

c-Fos in Human AdenomasProtooncogene expression in human pituitary tumors

has been examined in a few recent studies. In a study of30 tumors by Chernavsky et al. (1993b), these investiga-tors found no differences in c-fos mRNA expression inthe various tumor types including 12 GH, 7 PRL, and 11nonfunctional adenomas, although all tumors ex-pressed c-fos mRNA. In an immunohistochemical studyof pituitary adenomas from 33 patients, Raghavan etal. (1994) found fos immunoreactivity within the nucleusin 27/33 (82%). In this same study localization of Jun,another transcription factor with the leucine-zippermotif, was present in 21/33 cases (64%). In a relatedstudy of 30 pituitary adenomas by Woloschak andcolleagues (1994) using the sensitive RNase protectionassay, these investigators found c-fos mRNAoverexpres-sion in only 1 of 30 tumors, which was a silent ACTHadenoma (in which the patient did not have Cushing’s

disease). A related study by Boggild et al. (1994) usingloss of heterozygosity and PCR in 88 adenomas foundno amplification of c-fos mRNA, indicating that amplifi-cation of this protooncogene is indeed uncommon.

cAMP Response Binding ElementCyclic adenosine 3858-monophosphate (cAMP) regu-

lates a large number of genes and its action is mediatedby members of the activator protein-2 (AP-2) or cAMPresponse element-binding protein (Imagawa et al., 1987;Meyer and Habener, 1993) both of which are membersof the leucine-zipper class of DNA-binding proteins.Various investigators have shown that cyclic adenosine3858 monophosphate-responsive element binding pro-tein(s) (CREB) is important in the regulation of thePRL and GH genes (Keech et al., 1992; McCormick etal., 1990; Shepard et al., 1994; Struthers et al., 1991).CREB binds to the Pit-1 promoter (McCormick et al.,1990) and regulates the Pit-1 gene. After cAMP-dependent protein kinase A (PKA) is activated, there issubsequent phosphorylation and activation of CREB bythe catalytic subunits of PKA. In the pituitary glandcAMP has a mitogenic effect in GH cells (Billestrup etal., 1988). Struthers et al. (1991) developed transgenicmice expressing a transcriptionally inactive mutant ofCREB, which could not be phosphorylated in the ante-

Fig. 8. In situ hybridization localizing Pit-1 mRNA in a PRL secreting adenoma with an oligonucleo-tide probe labeled with 35S. The positive cells are covered with silver grains (3400).


rior pituitary (CREBMI), so it competed with wild typeCREB activity and blocked the response to cAMP. Thisresulted in a dwarf phenotype with an atrophic anteriorpituitary, which was deficient in GH cells, but ex-pressed all of the other anterior pituitary cell typesnormally. The appearance of normal PRL cells in theseexperiments was surprising, since most PRL cells arethought to be derived from GH cells (Frawley andBoockfor, 1991). It has been suggested by Struthers et

al. (1991) that hyperplasia of GH cells may be related tooverstimulation of cAMP, and that the previously de-scribed mutations in the Gs protein (Vallar et al., 1987)may be due in part to a constitutive activation of CREB.

HELIX-LOOP-HELIX GROUPThe myc gene belongs to the helix-loop-helix group of

transcription factors. Both the helix-loop-helix and the

Fig. 9. In situ hybridization localizing Pit-1 mRNA in a GH adenoma with an oligonucleotide probelabel with 35S. A strong hybridization signal is present in the positive cells (3400).

TABLE 3. PIT-1/GHF-1 mRNA expression in human pituitary adenomas1

ReferenceTechnique

used

mRNAsize(Kb)

Adenoma typea

GH PRL TSH Null NF GTH ACTH

Lloyd et al. (1993a) ISH — 111 111 111 1 ND 1 1Northern 2.4

(1.2)Asa et al. (1993) ISH 2.4 111 111 111 1 ND 0 0

Northern (3.4)Friend et al. (1993) Northern 2.4 111 111 111 1 ND 0 0

RNAse (1.6)Protection (3.4)

(4.5)Hoggard et al. (1993) RT-PCR — 111 111 ND ND 11 ND ND

Get mobility shiftDelhase et al. (1993) ISH — 111 111 111 0 ND 0 0Pellegrini et al. (1994) RT-PCR 2.4 111 111 1 ND 0 0 ND

Northern (4.5)1ISH, in situ hybridization; RT-PCR, reverse transcription-polymerase chain reaction; NF, nonfunctional adenoma; a, mixed GH-PRL producing tumors areconsistently positive; ND, not done; 0, negative; 1, few cells positive; 11, moderate number of cells positive; 111, many cells positive.


leucine-zipper groups have a region of positively chargedamino acids that makes contact with DNA and anadjoining region that mediates dimer formation and theactivity is regulated by heterodimer formation (Pa-pavassiliou, 1995).

The myc proteins are associated with the nuclearmatrix (Eisenman et al., 1985; Slamon et al., 1986) (Fig.13). Previous studies have shown that the fixative used

can determine the cellular localization of this protein(Loke et al., 1988). In frozen tissues, c-myc was local-ized in the nucleus of mouse tissues, but in formalin-fixed, paraffin-embedded tissue sections there was lossof nuclear staining and appearance of immunoreactiv-ity in the cytoplasm.

The c-myc oncogene has been implicated in thepathogenesis of various human tumors (Erisman et al.,

Fig. 10. Estrogen receptor localization in cultured GH3 pituitary cells that secrete PRL and GH.Hybridization was performed with a digoxigenin-labeled oligonucleotide probe. The antisense probe onthe left shows a positive hybridization signal in the cytoplasm while the sense control probe on the right isnegative (3300).

Fig. 11. In situ hybridization localizing estrogen receptor mRNA in a PRL-secreting adenoma. There isa strong positive hybridization signal using a 35S-labeled oligonucleotide probe for estrogen receptor(3300).


1985; Little et al., 1983). Recent studies on c-mycexpression in the rat pituitary showed that estrogenincreased c-myc levels implicating c-myc in estrogen-induced PRL cell proliferation (Szijan et al., 1992;Chernavsky et al., 1993a,b).

Myc in Human Pituitary TumorsIn a study of 30 adenomas by Chernavsky et al.

(1993a,b), these investigators found higher levels of

c-myc mRNA in GH tumors compared to PRL andnonfunctioning tumors. However, in a study by Ragha-van et al. (1994), using a pan myc antibody to stain 33pituitary adenomas, myc immunoreactivity was foundonly in the solitary ACTH tumor examined (1/33 cases).The staining was described as strong and diffuse cyto-plasmic and perinuclear but not nuclear. In the analy-sis of Boggild et al. (1994), amplification or rearrange-ment of c-myc was not observed in an analysis of 88

Fig. 12. Immunohistochemical localization of fos protein in the nucleus of cultured GH3 pituitary cellsusing diaminobenzidine chromogen (3350).

Fig. 13. Immunohistochemical localization of myc protein in the nucleus of cultured GH3 pituitarycells using diaminobenzidine chromogen (3350).


tumors. However, using a sensitive RNase protectionassay Woloschak et al. (1994) reported overexpressionof c-myc in 9 of 30 tumors including nonfunctional,ACTH, PRL, and GH adenomas. The amount of c-mycamplification was 4 to 9 times greater than that de-tected in normal postmortem pituitary samples. How-ever, there was no relationship between tumor invasive-ness and c-myc overexpression. Because of thesecontradictory findings the significance of c-myc overex-pression in human pituitary adenomas is uncertain.

Other Transcription Factors in the PituitaryVarious investigators have reported other putative

transcription factors in the pituitary that have not beencompletely characterized. Riegel et al. (1990) demon-strated that a factor (PO-B) detected in mammalian celllines binds specifically between the TATA box and thetranscription initiation site of the POMC gene andproposed that this factor may facilitate POMC geneexpression by interacting with components of the tran-scription initiation complex. Roberson et al. (1995)recently described a pituitary-specific enhancer withinthe 58-flanking region of the mouse glycoprotein alpha-subunit gene, which was present in gonadotrope andthyrotrope lineages. They isolated a LIM homeodomaintranscription factor protein, which was expressed in thecentral nervous system and the anterior pituitarygonadotrope and thyrotrope cells. Because this factorwas expressed in alpha subunit producing cells only,they suggested that it may play a role in cell lineagedetermination in the pituitary.

The thyrotroph embryonic factor (TEF) described byDrolet et al. (1991) is a member of the leucine zippertranscription group. It appears to be important forTSHb gene expression during embryogenesis but subse-quently becomes widely expressed (Gutierrez-Hart-man, 1994).

This overview of the major groups of transcriptionfactors affecting the pituitary gland illustrates theimportance of these genes in regulating pituitary devel-opment, growth, and function. Genetic alterations insome of these transcription factors can lead to endo-crine disorders such as dwarfism and cretinism fromPit-1 deficiency. Multiple genetic defects and otherfactors probably contribute to pituitary tumorigenesis(Prager and Melmed, 1993), so the role of specifictranscription factors in pituitary tumorigenesis re-mains to be elucidated.

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Transcription factors in normal and neoplastic pituitary tissues