Prolactin Receptor Messenger Ribonucleic Acid in Normal and Neoplastic Human Pituitary Tissues

0021-972X/97/$03.00/0 Journal of Clinical Endocrinology and Metabolism Copyright © 1997 by The Endocrine Society Vol. 82, No. 3 Printed in U.S.A. Prolactin Receptor Messenger Ribonucleic Acid in Normal and Neoplastic Human Pituitary Tissues* LONG JIN, XIANG QIAN, ELZBIETA KULIG, BERNARD W. SCHEITHAUER, ROCIO CALLE-RODRIGUE, CHARLES ABBOUD, DUDLEY H. DAVIS, KALMAN KOVACS, AND RICARDO V. LLOYD ABSTRACT We examined the specific cell types in normal human pituitaries that expressed PRL receptor (PRL-R) messenger ribonucleic acid (mRNA) by combined in situ hybridization and immunohistochemistry. The distribution of PRL-R mRNA in 28 pituitary adenomas was examined by in situ hybridization and reverse transcription-PCR in 12 cases of adenomas. In another set of experiments, 34 PRL adenomas from men, women, and bromocriptine-treated patients were analyzed for PRL-R by in situ hybridization. In the normal pituitary, PRL- and LH-producing cells had significantly more mean grain counts per cell and higher percentages of cells positive for PRL-R than GH and TSH cells. PRL-R mRNA was present in all groups of adenomas by in situ hybridization and reverse transcription-PCR. PRL adenomas had a significantly higher density of labeling compared to other adenoma types. Although there was no difference in the levels of PRL-R mRNA in PRL adenomas from men and premenopausal and postmenopausal women, patients treated with bromocriptine before pituitary surgery had significantly lower levels of PRL-R compared to all other groups. These results indicate that in the normal pituitary, PRL and LH cells have the highest level of PRL-R mRNA, whereas PRL adenomas have significantly higher levels of PRL-R mRNA than other types of adenomas, and bromocriptine treatment decreases the levels of PRL-R mRNA in PRL adenomas. (J Clin Endocrinol Metab 82: 963–968, 1997) P Subjects and Methods RL EXERTS a wide variety of biological functions in many tissues, including effects on lactation, reproduction, growth, metabolism, osmoregulation, immunomodulation, and behavior (1–5). PRL action is mediated via hormone binding to the PRL receptor (PRL-R) on the cell surface. The PRL-R is a member of the cytokine/GH/PRL receptor superfamily based on conserved sequences in their extracellular domain (5–7). PRL-R is widely distributed in many tissues and is present as a long and a short form in some species, such as rats and mice (5– 8). A long form of the human PRL-R consisting of 598 amino acids in its mature form has been characterized (9). PRL-R has been examined in some human tissues, including breast (10), placenta, decidua (11, 12), digestive tissues (13), and lymphoid cells (14). PRL-R has also been examined in human pituitary adenomas by radioreceptor assay (15). However, the distribution of PRL-R messenger ribonucleic acid (mRNA) has not been previously reported in normal or neoplastic human pituitaries. In this study we examine the distribution of PRL-R mRNA in normal and neoplastic human pituitary tissues. Differences in PRL-R mRNA distribution in pituitary tissues from men and women and from patients treated with bromocriptine were also analyzed. Study groups Formalin-fixed, paraffin-embedded tissue sections of normal pituitaries and pituitary adenomas retrieved from the files of the Mayo Clinic were used for these studies. Three nonneoplastic pituitaries obtained within 8 h of death were studied by combined PRL-R in situ hybridization and immunostaining for pituitary hormones to localize the specific cell types with PRL-R gene expression. Twenty-eight pituitary adenomas were used for the PRL-R in situ hybridization study in the first set of experiments; these included PRL (n 5 6), GH (n 5 6), ACTH (n 5 3), FSH/LH (n 5 6), null cell adenomas (n 5 6), and a TSH adenoma. Pituitary adenomas were characterized by immunostaining in all cases and by ultrastructural studies in some cases. In another set of experiments, only PRL adenomas from 34 patients were used. The PRL adenoma cases included men (n 5 10), reproductive age women ranging in age from 24 – 40 yr (n 5 9), postmenopausal women ranging in age from 43– 68 yr (n 5 10), and a final group of patients who had been treated with bromocriptine before transsphenoidal surgery (n 5 5). Frozen tissues from portions of 12 pituitary adenomas and 2 normal pituitary tissues were used for RNA extraction and reverse transcription-PCR (RT-PCR) studies. In situ hybridization (ISH) The oligonucleotide probes for human PRL-R were synthesized with an automated DNA synthesizer at the Mayo Foundation from the published sequences (9). PRL-R mRNA expression was analyzed by ISH with 35S-labeled probes to human PRL-R gene (Table 1). The probes to human PRL-R was used for all tissues in this study, and a sense probe was used as a control. The specificity of the probes was verified by a GenBank search. The oligonucleotide probes were labeled at the 39-end with 35S as previously described (16, 17). Sections were hybridized with 3 3 106 cpm/slide at 42 C for 18 h, followed by washings with 0.5–2 3 SSC (standard saline citrate) and autoradiography for 2–3 weeks. ISH analysis for all cases from one set of experiments was performed to- Received September 10, 1996. Revision received November 18, 1996. Accepted November 22, 1996. Address all correspondence and requests for reprints to: R. V. Lloyd, M.D., Department of Laboratory Medicine and Pathology, Mayo Clinic and Mayo Foundation, 200 First Street SW, Rochester, Minnesota 55905. * This work was supported in part by NIH Grant CA-42951. 963 Department of Laboratory Medicine and Pathology (L.J., X.Q., E.K., B.W.S., R.C.-R., R.V.L.), Division of Endocrinology/Metabolism and Internal Medicine (C.A.), and the Department of Neurologic Surgery (D.H.D.), Mayo Clinic and Mayo Foundation, Rochester, Minnesota 55905; and the Department of Pathology, St. Michaels Hospital (K.K.), Toronto, Canada 964 JCE & M • 1997 Vol 82 • No 3 JIN ET AL. TABLE 1. Oligonucleotide probes and primers for PRL-R and for glyceraldehyde phosphate dehydrogenase (GAPDH) Oligonucleotides hPRL-R (sense) hPRL-R (antisense) hPRL-R (internal) GAPDH (sense) GAPDH (antisense) Location in gene (nucleotides) Sequence 59-GTG 59-CCA 59-CCT 59-ATG 59-GTT GCA CAT GTG GTG GTC TCT GGA GTA AAG ATG GCA GGT AGT GTC GAT TABLE 2. PRL-R mRNA expression in normal pituitary cells MGC PRL GH ACTH LH TSH 22.7 6 2.0 14.3 6 1.8a 19.7 6 3.7 26.3 6 2.6 11.0 6 0.6a Combined ISH and immunohistochemistry were performed in normal pituitary as described in Materials and Methods, and the MGC for each cell type with PRL-R mRNA was expressed as the mean 6 SEM for three pituitaries. a P , 0.05 compared to PRL cells. P , 0.05 compared to LH cells. gether. Negative control for ISH consisted of pretreating tissues with 200 mg/mL ribonuclease A (Sigma) before hybridization and using a sense control probe for PRL-R. Formalin-fixed, paraffin-embedded sections of liver tissues were used (...truncated)