INTRODUCTION

The human pituitary adenomas have been classified according to their function, such as production of growth hormone (GH), prolactin (PRL), thyroid-stimulating hormone (TSH), adrenocorticotropic hormone (ACTH), follicle-stimulating hormone (FSH), and α-glycoprotein subunit (α-SU). GH, PRL, and TSH have been expressed as a group among the functioning adenomas and the role of Pit-1 has been emphasized (1, 2, 3, 4, 5, 6). Synergistic action of Pit-1 with various receptors, such as those on the cell membrane and nuclear receptor super families, has also been reported (7, 8, 9, 10, 11). Although the mechanisms of functional expression toward GH, PRL, and TSH have been investigated in detail with pituitary adenomas, their mechanisms toward the expression of other anterior pituitary hormones have not been studied extensively. Recently, several transcription factors relative to pituitary development have been introduced. Especially, transcription factors transiently expressed during pituitary development include the prophet of Pit-1 (Prop-1) and Rpx/Hesx-1 (12, 13, 14). It is known that Prop-1 acts as an early enhancer of Pit-1. Prop-1 is expressed before Pit-1 from embryonic day 10 to 10.5 (e10 to 10.5) and expression of Prop-1 peaks on e12. Pit-1 is first expressed from e13.5, and expression of Prop-1 decreases as the expression of Pit-1 increases, whereas no Prop-1 expression is seen in adults (12). Rpx/Hesx-1, a homeo box gene, is also expressed transiently from e9 to 14.5 during differentiation of the pituitary gland, although its function is not yet fully clarified (14). Rpx/Hesx-1 has been reported to form a heterodimer with Prop-1 and to inhibit Pit-1 activation by Prop-1 (12), which was inferred from the finding that Rpx/Hesx-1 was expressed throughout embryonic days in Prop-1-deficient mice (12, 15). Because it has also been reported that pituitary hypoplasia occurs in transgenic mice with Rpx/Hesx-1 expression throughout embryonic days, disappearance of Rpx/Hesx-1 seems to be necessary for proper differentiation of the various types of pituitary cells (16). Therefore, the activation of Pit-1 by Prop-1 may be triggered through the disappearance of Rpx/Hesx-1. On the other hand, a novel transcription factor pituitary homeo box 1 (Ptx1) has been initially introduced as a factor toward the expression of proopiomelanocortin (POMC) (17). But, in the studies on rat pituitary glands and cultured cells, Ptx1 has been expressed not only in the POMC secreting cells but also in the other hormone secreting cells of the anterior lobe as well as in the cell lines of various different hormone productions (16). This study is aimed to elucidate the expression of Ptx1 in the human pituitary adenomas of various types in order to clarify the role of Ptx1 in the functional differentiation in the neoplastic conditions.

MATERIALS AND METHODS

Patients

Five human non-neoplastic pituitaries were obtained from autopsy within 4 hours postmortem of the patients without endocrinologic abnormalities, and seventy-three of pituitary adenomas (male 30, female 43; age range, 16 to 80 years) were obtained by the transsphenoidal surgery. The clinical and endocrinologic features were as follows: 14 patients with GH-secreting adenomas and symptoms of acromegaly, 12 with PRL-secreting adenomas whose serum PRL levels ranged from 120 to 2560 ng/mL, 20 with ACTH-secreting adenomas and typical Cushing’s syndrome, seven with TSH-secreting adenomas and hyperthyroidism, and 20 with nonfunctioning adenomas that clinically presented no evidence of anterior pituitary hormone excess and did not show high serum concentrations of any of the anterior pituitary hormone except mild hyperprolactinemia (less than 100 ng/mL). Among those 20 cases of nonfunctioning adenomas, 12 cases involved visual disturbance, four cases involved headache, and four cases were incidentally shown to have pituitary tumors by computed tomography or magnetic resonance imaging. Most of seven cases with TSH-secreting adenomas were previously reported using immunohistochemistry and in situ hybridization by Sanno et al. (18, 19). These adenomas were classified based on their clinical manifestations, biological functions, and ultrastructures (20, 21).

Tissue Preparation and Antibodies

The tissues were routinely fixed in 10% formalin or 4% paraformaldehyde for 8 to 24 hours and embedded in paraffin. Serial sections were prepared for hematoxylin-eosin staining, avidin-biotin complex peroxidase (ABC) method, or the indirect method. Polyclonal antibody for Ptx1 was raised against synthetic peptide containing the following murine Ptx1 amino acid residues: Ptx1 31 to 50 (FHLARAADPREPLENSASES) (22). This antibody had homology only to 20% with murine Ptx2 sequence, but to 90% with human Ptx1 sequence (23).

The anti-anterior-pituitary-hormone antibodies and their dilutions used in this study were: anti-human (h) GH (1:800), anti-hPRL (1:600), anti-hACTH (1:800) monoclonal antibodies (DAKO, Carpinteria, CA), and anti-h-FSHβ (1:200), anti-h-LHβ (1:200), anti-h-TSHβ (1:200) monoclonal antibodies (Immunotech S.A., France), and anti-α-subunit of glycoprotein (α-SU; 1:100) monoclonal antibodies (Chemicon International, Inc., Temecula, CA).

Light Microscopic Immunohistochemistry

Immunohistochemical study was performed by the ABC method (24). The deparaffinized and rehydrated specimens were incubated with 0.3% hydrogen peroxidase in methanol for 30 min to block endogenous peroxidase activity. Then the specimens were incubated with primary antibody for 12 h, rinsed in PBS, and subsequently incubated with biotinylated anti-rabbit immunoglobulin (Vector Laboratories, UK). Then the sections were rinsed and incubated with avidin-biotin complex (Vector Laboratories). The reaction was visualized by incubation with 3,3′-diaminobenzidine tetrahydrochloride for 3 min, which resulted in a brown color. In the normal human pituitary glands and pituitary adenomas, the double immunostaining method was applied to determine the structural relationship between Ptx1 immunoreactivity and anterior pituitary hormones as reported previously (18, 25). In brief, after visualizing the immunoreactivity of the Ptx1 by diaminobenzidine, the antibodies were removed by rinsing the sections in a glycine-HCl buffer, pH 2.2, for 2 h. Then the tissue sections were incubated with anterior pituitary hormone antibodies for 1 h, followed by incubation with alkaline-phosphatase-conjugated second antibodies (Amersham International, UK) and visualized with fast blue.

Western Blotting for Ptx1 in the Pituitary Adenomas

To confirm the specificity of Ptx1 antibodies, Western blotting for Ptx1 was performed in the representative GH-secreting adenoma and PRL-secreting adenoma, which were immunohistochemically positive for Ptx1 protein. These tissues were homogenized in ice-cold 50 mm Tris-HCl, pH 7.5, containing 2 mm ethylene glycol tetraacetic acid (EGTA), 1 mm dithiothreitol (DTT), and 0.001% leupeptin. Each tissue homogenate was centrifuged at 100,000 × g at 4° C for 1 h and the supernatants were used for electrophoresis. The protein concentration of each sample was measured and the amount of each sample applied to polyacrylamide gel was adjusted for equal loading. After electrophoresis, each sample was transferred to nitrocellulose membrane. For Western blotting of Ptx1 protein, anti-Ptx1 polyclonal antibodies (1:250 diluted) and subsequently HRP-labeled anti-rabbit immunoglobulin (donkey, 1:400, diluted in Tween 20-PBS; Amersham International) were applied to each lane as the primary and secondary antibodies, respectively. Anti-Ptx1 polyclonal antibodies used for Western blotting are the same as the ones used for light immunohistochemistry. Immunoreactivity for Ptx1 protein was visualized by incubation with 3,3′-diaminobenzidine tetrahydrochloride for 3 min, which resulted in a brown color.

RT-PCR for Ptx1

Total RNA extraction was performed by the single-step method (TRIzol reagent kit, Life Technologies, Gaithersburg, MD) from a representative non-neoplastic pituitary and 18 pituitary adenomas and total RNA was treated by Deoxyribonuclease I (Life Technologies). First-strand complementary DNA (cDNA) was prepared from total RNA with Deoxyribonuclease I treatment using a T-Primed First-Strand kit (Amershan Pharmacia Biotech Inc., Uppsala, Sweden). The RT reaction was performed at 37° C for 60 min in a final volume of 50 μL with 5 μg total RNA. For PCR, the following oligonucleotide primers were used to identify human Ptx1: up-stream 5′-TGG CTA CGT GCC GCA GTT CA-3′ and down-stream 5′-GCT GTT GTA CTG GCA CGC GT-3′, which generate a RT-PCR product of 470 bp. These primers were synthesized on the basis of the reported sequence of human Ptx1 (23). The integrity of RNA from each specimen was verified by RT-PCR for β-actin using the up-stream primer 5′-GAT ATC GCC GCG CTC GTC GTC-3′ and the down-stream primer 5′-GGC TGG GGT GTT GAA GGT CTC-3′, which generated a RT-PCR product of 381 bp. These primers were previously reported by Fields et al. (26). The PCR was carried out in 100 μL final reaction volumes containing 1 μL RT reaction product as template DNA, corresponding to cDNA synthesized from 500 ng total RNA, 1 × PCR buffer II, 1.0 mmol/L MgCl2, 0.2 mmol/L of each deoxynucleotide, 0.4 μmol/L each up-stream and down-stream primer for human Ptx1, and 2.5 U AmpliTaq Gold (Perkin-Elmer, Norwalk, CT). Programmable temperature cycling (Perkin-Elmer, GeneAmp PCR System 9700) was performed with the following cycle profile: 95° C for 10 min as an initial denaturing step, followed by 95° C for 1 min as an amplification, and 64° C for 1 min as an annealing and extension (40 cycles) for human Ptx1; and 95° C for 10 min as an initial denaturing step, followed by 95° C for 1 min as an amplification, and 60° C for 1 min as an annealing and extension (40 cycles) for β-actin, respectively. Samples of the reaction products (10 μL each) were electrophoresed through 2% agarose gels with ethidium bromide and photographed under ultraviolet light. In RT-PCR experiments, total RNA from the human non-neoplastic pituitary gland without reverse transcriptase was included as a negative control for human Ptx1.

RESULTS

Immunohistochemical Findings and Western Blotting

In the five human non-neoplastic pituitary glands, Ptx1 protein was expressed in 60 to 70% of the nuclei in the anterior pituitary cells (Fig. 1A). By double staining for the anterior pituitary hormones, Ptx1 immunoreactivity was observed in all types of anterior pituitary cells (Fig. 1, B–H). The endocrine cells immunoreactive for Ptx1 protein in the anterior lobe of human pituitary gland were summarized in Table 1.

FIGURE 1
figure 1

Immunohistochemistry for Ptx1 protein in human non-neoplastic pituitary. A, expression of Ptx1 protein is observed in the nuclei of anterior pituitary cells in a brown color. The immunopositive cells were observed about 60 to 70% of the nuclei in the anterior pituitary cells (original magnification, 500 ×). B-H, double immunohistochemical staining in human non-neoplastic pituitary indicating that Ptx1 immunopositivity (brown) does frequently co-localized in GH (B), ACTH (D), and α-SU (H) immunopositive cells (blue), and does occasionally co-localized in PRL (C), FSHβ (E), LHβ (F), and TSHβ (G) immunopositive cells (blue) (original magnification, 600 ×).

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TABLE 1 Results of Immunohistochemical Co-Localization with the Anterior Pituitary Hormones and Ptx1 Protein in Human Non-Neoplastic Pituitaries
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Seventy-three pituitary adenomas have been classified histologically on the basis of cytoplasmic staining affinities into categories of eosinophilic, basophilic, and chromophobe, and have been immunohistochemically confirmed by their hormonal expression (Table 2). Ten of 20 clinically nonfunctioning adenomas were expressed one or more of FSHβ, LHβ or α-SU, and were subclassified as gonadotropin-subunit-positive adenomas. Immunohistochemical detection of Ptx1 protein was observed in 63 (86.3%) of 73 cases of human pituitary adenomas and was expressed in the nuclei of each adenoma cells. In the pituitary adenomas that were immunohistochemically positive for Ptx1 protein, the Ptx1 immunopositivity was observed from scattered cells to about 70% adenoma cells, and the number of immunopositive cells varied from case to case (Table 2). The results of the immunohistochemical examination of the different types of adenoma are summarized in Table 3. Ptx1 protein was detected in 10 (71.4%) of 14 cases with GH-secreting adenomas, all of 12 cases (100%) with PRL-secreting adenomas, 18 (90.0%) of 20 cases with ACTH-secreting adenomas, six (85.7%) of seven cases with TSH-secreting adenomas, 17 (85.0%) of 20 cases with nonfunctioning adenomas. In ten cases of gonadotropin-subunit-positive adenomas that were categorized in the clinically nonfunctioning adenomas, nine cases (90%) were positive for Ptx1 protein by immunohistochemistry. In Ptx1 positive ACTH-secreting adenomas, which were only positive for ACTH by immunohistochemistry in most of cases, Ptx1 immunopositivity was often localized in the ACTH-secreting cells (Fig. 2). In PRL-secreting adenomas, which are characterized by a “Golgi pattern” of PRL localized and only positive for PRL by immunohistochemistry in this series, Ptx1 immunopositivity were occasionally expressed in PRL-secreting cells (Fig. 3 ). On the other hand, in GH-secreting adenomas, which were all positive for GH and PRL, and were occasionally positive for α-SU by the immunohistochemistry in this series, Ptx1 immunopositivity were coexpressed not only for GH positive adenoma cells but also for PRL and α-SU positive adenoma cells (Fig. 4, A–C ). In gonadotropin-subunit-positive adenomas, which were immunohistochemically positive for one or more of FSHβ, LHβ or α-SU, Ptx1 protein was coexpressed for gonadotropin-subunit-positive adenoma cells (Fig. 5, A–B). Thus, Ptx1 protein was expressed in the pituitary adenoma cells that produced various pituitary hormones and in the GH-secreting adenomas and gonadotropin-subunit-positive adenomas that were immunohistochemically and frequently multihormonal.

TABLE 2 Results of Immunohistochemical Analysis for Anterior Pituitary Hormones and Ptx1 in 73 Pituitary Adenomas
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TABLE 3 Immunohistochemical Detection of Ptx1 Protein in Human Non-Neoplastic Pituitaries and Pituitary Adenomas
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FIGURE 2
figure 2

Immunohistochemical detection of Ptx1 protein in ACTH-secreting adenomas. Immunoreactivity for Ptx1 protein is observed in the nuclei of adenoma cells and often co-localized with ACTH positive cells (original magnification, 600 ×).

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FIGURE 3
figure 3

Immunohistochemical detection of Ptx1 protein in PRL-secreting adenomas. Immunoreactivity for Ptx1 protein is observed in the nuclei of adenoma cells and occasionally co-localized with PRL positive cells (arrows) (original magnification, 600 ×).

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FIGURE 4
figure 4

Immunohistochemical detection of Ptx1 protein in GH-secreting adenomas. Immunoreactivity for Ptx1 protein is observed in the nuclei of adenoma cells and often co-localized with GH positive cells (A), and occasionally co-localized with PRL (arrow) (B), and α-SU (arrow) (C) (original magnification, 600 ×).

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FIGURE 5
figure 5

Immunohistochemical detection of Ptx1 protein in gonadotropin-subunit-positive adenomas. In this case, Ptx1 immunopositivity was occasionally co-localized with α-SU (arrow) (A), and FSHβ positive cells (B) (original magnification, 600 ×).

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To confirm the specificity of Ptx1 antibodies, Western blotting for Ptx1 was performed in the representative GH-secreting adenoma and PRL-secreting adenoma, indicating the case of No. 1 and 19, respectively, in Table 2, which were strongly positive for Ptx1 protein by immunohistochemistry. The Ptx1 antibodies revealed a band of 34 kDa in an immunoblot of both GH-secreting adenoma and PRL-secreting adenoma (Fig. 6).

FIGURE 6
figure 6

Western blotting for Ptx1 protein in human pituitary adenomas. The Ptx1 antibodies detected a band of 34 kDa in immunoblot of both GH-secreting adenoma and PRL-secreting adenoma. These adenomas were indicated by the case of No. 1 and 19 respectively in the Table 2. (Lane 1, GH-secreting adenoma; Lane 2, PRL-secreting adenoma).

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RT-PCR Analysis

In five human non-neoplastic pituitaries and 73 pituitary adenomas, a representative human non-neoplastic pituitary and 18 pituitary adenomas, including the six GH-secreting adenomas, two PRL-secreting adenomas, two ACTH-secreting adenomas, two TSH-secreting adenomas, two null cell adenomas, and four gonadotropin-subunit-positive adenomas, were available for RT-PCR in this study. Table 4 indicates the clinical and endocrinologic feature in 18 pituitary adenomas examined for RT-PCR, and two GH-secreting adenomas were immunohistochemically negative for Ptx1 protein in this series. The results of RT-PCR are shown to Figure 7. Analysis of human Ptx1 messenger RNA (mRNA) indicated that the expected 470 bp PCR product was detected by ethidium bromide staining in one human non-neoplastic pituitary and in all of 18 pituitary adenomas. Interestingly, two GH-secreting adenomas indicating lane G4 and G6, which were negative for Ptx1 protein by immunohistochemistry, were expressed Ptx1 mRNA. The control RT-PCR experiment, using total RNA extracted from human non-neoplastic pituitary without reverse transcriptase, was negative for human Ptx1 mRNA.

TABLE 4 Summary of Representative 18 Pituitary Adenomas for RT-PCR Analysis
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FIGURE 7
figure 7

RT-PCR of human non-neoplastic pituitary and pituitary adenomas for Ptx1 mRNA. One non-neoplastic pituiary and all eighteen pituitary adenomas, containing six GH-secreting adenomas (G1-G6), two PRL-secreting adenomas (P1, P2), two ACTH-secreting adenomas (A1, A2), two TSH-secreting adenomas (T1, T2), two null cell adenomas (Nu1, Nu2), and four gonadotropin-subunit-positive adenomas (Gn1-Gn4), have expression of Ptx1 mRNA. Two of six GH-secreting adenomas (G4, G6) which were immunohistochemically negative for Ptx1 protein, also have expression of Ptx1 mRNA, but one of its two cases (G6) were faint signal for Ptx1 mRNA. RT(-) indicates a negative control without reverse transcription in human non-neoplastic pituitary.

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DISCUSSION

The pituitary gland develops from Rathke’s pouch and its primordium appears on e8.5 in mice. It has been suggested that the hormone-producing cells of the pituitary gland initially appear as α-SU positive cells on e11 and subsequently differentiate into anterior pituitary hormone secreting cells (27). Particularly, the mechanism by which anterior pituitary hormone secreting cells undergo function-ally specific differentiation has been the major issue in pituitary research. As a key to answer this question, considerable attention has been paid to transcription factors that are specific to the pituitary gland. In 1988, Bodner et al. (28) and Ingraham et al. (29) separately reported the presence of a pituitary specific transcription factor (Pit-1/GHF-1). Pit-1 was found in the nuclear extract of rat pituitary cells and was shown to enhance the transcription of GH and PRL (30). In addition, Pit-1 was found to activate transcription of the gene for TSHβ (31, 32, 33) and to play a role in the functional differentiation of these cells as well as in the maintenance of their viability and proliferation (34, 35, 36). There have been several studies on the expression of Pit-1 in human pituitary adenomas (1, 2, 3, 4, 5, 6).

It has been recently shown that another transcription factor is also involved in the functional differentiation of pituitary cells. Pituitary homeo box 1 (Ptx1) is a transcription factor first reported by Lamonerie et al. (17) that has been suggested to play a role in the transcription of POMC because it specifically binds to the nucleus of AtT20 cells, a murine corticotroph cell line. Ptx1 is closely related to the mammalian Otx gene, which is expressed in the rostral brain during the process of development (37, 38), and Otx2 is a fundamental factor related to forebrain and midbrain development in mice (39). Ptx1 mRNA is strongly expressed in adult corticotroph cells, which secrete POMC, whereas its expression in Rathke’s pouch has also been detected using the in situ hybridization technique (17). Thus, there remains a possibility that Ptx1 is expressed in pituitary cells other than corticotrophs, so that its activities need further investigation, including the relationship with other transcription factors related to pituitary cells.

Recent investigations using cultured cells have shown that Ptx1 is not only involved with POMC transcription in corticotrophs but also with the transcription of other anterior pituitary hormones. Tremblay et al. (16) investigated Ptx1 expression in AtT20 cells, a corticotroph cell line used for Ptx1 cloning, as well as in αT3–1 cells (gonadotroph precursor), αTSH cells (thyrotroph precursor), GHFT1.5 cells (somatolactotroph precursor), GH3 cells (a somatolactotroph cell line), GH4C1 cells, and TtT-97 cells (thyrotroph tumor). They showed by Northern blotting that the Ptx1 mRNA level was higher in αT3–1, αTSH, and GHFT1.5 cells than in AtT20 cells, indicating the possibility that Ptx1 is actively involved in pituitary cells other than corticotrophs (16). More recently, it has been found that Ptx1 is expressed in all pituitary cells, but is differentially expressed in different lineages at both the mRNA and protein levels. Especially, the highest levels of Ptx1 expression were observed in α-SU positive cells (40). However, previous studies were performed in mammalians (such as rats) or on cultured cells, and no studies have been performed extensively on the human tissues. Only Ptx1 mRNA expression was demonstrated by Pellegrini-Bouiller et al. (41) using RT-PCR methods in human pituitary adenomas. In the present study, we first report Ptx1 protein expression by immunohistochemistry in normal human pituitary tissues and pituitary adenomas as well as its mRNA expression by RT-PCR. We detected Ptx1 protein in the nuclei of all types of anterior pituitary hormone secreting cells, indicating that Ptx1 is a nuclear binding protein. This protein was also observed extensively in all types of pituitary adenoma, although some GH-secreting adenomas, ACTH-secreting adenomas, TSH-secreting adenomas, and nonfunctioning adenomas were negative for Ptx1 protein. This may indicate that another transcription factor, such as Pit-1, is also involved synergistically in functional expression of the adenoma cells, although technical problems on antigen preservation in the specimens may also have to be considered. In our investigation of Ptx1 mRNA by RT-PCR, this mRNA was detected in all patients analyzed, including two GH-secreting adenomas negative for Ptx1 protein. Based on the above results, it seems that Ptx1 has no hormone-related specificity and is involved in the transcription of all types of anterior pituitary cells. This finding appears to be consistent with the previous report by Pellegrini-Bouiller et al. (41).

Regarding the role of Ptx1 in the functional differentiation of pituitary cells, an in vitro study has suggested that at least one Ptx1 binding site exists in the promoter region for anterior pituitary hormones such as POMC and that Ptx1 enhances the activity of such promoters (16). The synergistic actions between Ptx1 and transcription factors for other types of pituitary cells have also been reported, particularly Ptx1 synergy with NeuroD1/β2, which is the transcription factor containing a helix-loop-helix heterodimer to activate POMC transcription (42, 43, 44, 45). On the other hand, Ptx1 is thought to be in synergy with SF-1/Ad4BP, which is a transcription factor regulating the P-450 gene, to enhance the promoter activity of LHβ (16, 46, 47, 48, 49). Lastly, synergistic action of Pit-1 with Ptx1 has also been reported in the case of PRL promoter activity (16, 50). In the present study, Ptx1 expression was observed in 100% of PRL-secreting adenomas, suggesting that Ptx1 may be of synergy with Pit-1 in the functional differentiation of these adenomas.

The evidence that has been accumulated regarding the involvement of Ptx1 in the transcription of α-SU and P-Lim/Lhx-3 seems to be involved in α-SU transcription as a cofactor of Ptx1 (51). P-Lim/Lhx-3 mRNA initially appears in Rathke’s pouch on e8.5 to 9, and it is known that four types of anterior pituitary cells including α-SU are completely depleted in P-Lim/Lhx-3 deficient knockout mice (52). An experiment using a Ptx1 knockout cell line showed that expression of the Lhx-3 gene was decreased remarkably compared with that of the α-SU gene (16), and this indicates that Ptx1 is essential for Lhx-3 gene expression. Although P-Lim/Lhx-3 was not analyzed in the present study, it appears necessary to investigate the expression of P-Lim/Lhx-3 and its association with Ptx1 in pituitary adenomas, especially in the α-SU positive adenomas.

As a summary, Ptx1 is expressed in all types of pituitary adenomas and is speculated to play a role in the transcription factor in synergy with the other factors to define the specific hormone production. Therefore, Ptx1 is one of the recently reported and inevitable transcription factors in pituitary tumors. Along with the advances of molecular biology in recent years, many factors involved in pituitary cell function have been identified. These factors appear to interact with each other in a complex manner and it would be of great interest if such interactions could be clarified further.