ESSAY | TOWARD JES 100TH ANNIVERSARY
My Pituitary Research as Pathology and Endocrinology
2024 Volume 71 Issue 1 Pages 1-6
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2024 Volume 71 Issue 1 Pages 1-6
It was a great honor for me to receive the prestigious Distinguished Endocrinologist Award from the Japan Endocrine Society (JES) in 2009. For many years, I had the unique opportunity of working with a number of wonderful colleagues promoting pituitary research. Our research group expanded not only nationally but also internationally, attracting many young researchers who became interested in pituitary cells and pituitary tumors. During the time we were so excited about the latest topics in endocrinology and worked hard to contribute to the field to clarify many important scientific questions. I would like to share the pleasure of receiving the award with all of you, especially those of you who worked with me.
On the occasion of the 100th year of JES, I would like to take this opportunity to review my research life.
1) Immunohistochemistry
2) Immunoelectron Microscopy
3) In situ Hybridization (ISH)
4) Transcription Factors and the Development of Pituitary Research
5) Introduction of New Technology
6) Contribution to the WHO Classification of “Endocrine and Neuroendocrine Tumors”
After graduating from the Keio University School of Medicine in 1970, I moved to the United States to begin my pathology residency at the University of Colorado Medical Center where I studied immunohistochemistry under the tutelage of Professor Paul K. Nakane. Professor Nakane attracted worldwide attention when he published his enzyme-labeled antibody method for immunohistochemistry, at a time when the fluorescent antibody method had been the mainstream method. The new enzyme antibody method was applied to pituitary hormones, and I was instantly fascinated by this wonderful technique for detecting specific hormones at the cellular level. After returning to Japan in 1975, I immediately began to apply the enzyme antibody method to observe the localization of pituitary hormones and worked on its application to pituitary adenomas. At that time, I began research on the pituitary gland together with Dr. Akira Teramoto (Professor Emeritus, Nippon Medical School) with whom I clarified the pathology of pituitary tumors [1-3].
Dr. Paul K. Nakane (first from left in front row) and I (second from right in front row) in a group photograph at the Department of Pathology, University of Colorado, 1970.
Immunohistochemical staining of the human anencephalic pituitary (upper left) and adult rat pituitary gland (upper right). In both pituitary glands, ACTH was positively stained.
The enzyme-antibody method allowed electron microscopic observation, and we energetically worked on the electron microscopic localization of hormones. Here, we were interested in the secretory granules, a common feature of neuroendocrine cells, including pituitary cells, and devoted our energies to elucidating their role. Surprisingly, we found that PRL (prolactin) localized estrogen-dependently in the secretory granules accumulated by castration and in the lumen of the developing rough endoplasmic reticulum after degranulation by E2 (estradiol) administration, and that the localization of PRL changed dramatically upon stimulation. For the simultaneous release of the two hormones, GH and PRL, we successfully showed that they co-localized in the same secretory granule.
A non-radioactive method of proving mRNA on sections had been developed and is still being used extensively; Prof. Nakane developed his own method using T-T dimer, and several methods are now available as kits. Dr. Akira Matsuno [4-8] (currently a professor at International University of Health and Welfare), using pituitary hormones (e.g., GH, PRL) as an example, demonstrated that mRNA is stored on rough endoplasmic reticulum polyribosomes by a pre-embedding method and that hormones are stored in secretory granules, as revealed by post-embedding immunoelectron microscopy. The results revealed the dynamics and morphological changes of mRNAs and hormones in the secretory granules. The report is still widely cited as a groundbreaking finding.
Since the beginning of my research, I have been interested in why the certain pituitary cells produce certain hormones. Fortunately, this interest was rapidly clarified by the cloning of a transcription factor, Pituitary specific transcription factor-1 (Pit-1), cloned by Rosenfeld et al. and Karin in 1988. It was found to be involved in the functional differentiation of GH, PRL, and TSH, followed by the subsequent cloning of NeuroD1, Tpit, SF-1 which are the transcription factors involved in the differentiation of ACTH, FSH/LH, respectively. In our series of investigations in pituitary adenomas, pituitary adenoma cells contained transcription factors similar to those of normal cells, suggesting that the differentiated cells had become tumorigenic. In rare cases of translineage differentiation, such as ACTHoma producing GH and GHoma producing ACTH, we have shown that this is also an aberrant expression of transcription factors.
Members and collaborators in our research at the time when I received the Distinguished Endocrinologist Award from the JES in 2009.
Human GH cells often coexist with αSU, an original finding we discovered. It has been known that ACTH is the first hormone in human induced by TPIT, followed by SF-1 and PIT1 producing FSH/LH and GH/PRL/TSH respectively.
In order to recapitulate the translineage differentiation by introducing a new transcription factor, we introduced GFP-Pit-1 gene into ACTH-producing cell line AtT20 cells and induced the expression of GH. The cells were transfected with GFP-Pit-1 gene, which was found in the nucleus. The cells were shown to simultaneously produce ACTH and GH of different lineages.
Pituitary cells also have characteristic secretory granules, but the process of formation of these granules had long been unknown. In 2007, Dr. Chie Inomoto demonstrated the formation of granules by gene transfer of chromogranin A-GFP and revealed a part of the formation process of the downstream granules [20]. Dr. Osamu Shimomura and his colleagues were awarded the Nobel Prize for their work on GFP.
2. Genetically engineering in experimental animalsThe technology of genetically-engineered animals became available in the laboratory, and research on the etiology of pituitary tumors progressed rapidly. GHRH-transgenic mice formed a model of GH-producing adenomas, and Prop-1 transgenic mice formed a variety of Pit-1-associated tumors. ACTH-producing tumors of intermediate lobe origin were formed in p27KO RbKO mice. Although the tumorigenic mechanisms in the human pituitary adenomas are not yet completely clear, these models provide valuable information.
3. Transfection of Pit-1 into HP 75 cellsWe transfected HP 75 cells (derived from a human non-functioning pituitary adenoma that expressed alpha SU and LH beta) with Pit-1 by using an adenovirus FLAG-Pit-1 construct. Most of the transfected cells expressed GH mRNA, with fewer cells expressing PRL and TSH beta mRNA. The HP 75 cells expressed the genes for ER and GATA-2, thus allowing their expression of GH, PRL, and TSH beta mRNA in response to Pit-1. These results support the hypothesis that GH can be induced in cells that possess an active alpha SU gene and shed light on the basic molecular mechanism that drives the development of GH, PRL, and TSH beta expression in the alpha SU-based gonadotroph lineage [23].
4. Laser (capture) MicrodissectionThe technique of extracting DNA and RNA from specific cells in tissue sections and cell samples and analyzing their genetic changes is also indispensable for analysis at the individual cell level. This method has revealed that GH-producing pituitary adenomas exhibit differentiation into PRL by acquiring additional estrogen receptor (ER). In addition, paraffin sections have quantitatively proven various mRNAs from specific cells.
With the research background mentioned above, I have been involved in publishing the 4th edition of the WHO Classification of Tumors of Endocrine Organs (2017) and the 5th edition of the WHO Classification of Endocrine and Neuroendocrine Tumors (2022).
A tremendous amount of our findings are reflected in the WHO classification. First of all, the pituitary adenomas are now designated as pituitary neuroendocrine tumors (PitNET) which are regarded as potentially malignant tumors with invasion and rare metastases. The classification of PitNETs are now based on the cell lineages by transcription factors PIT1, TPIT, and SF1 as shown in the Fig. 3.
Differentiation of hormone producing cells is regulated by the corresponding transcription factors. This is a basis for 5th edition of the WHO Classification of pituitary neuroendocrine tumors (PitNETs) in 2022.
It is good to know that our research in “pathology and endocrinology” has contributed to the current WHO classification of the tumors.
As described above, pituitary research has progressed rapidly through the development of morphological and genetic research methods combined with the creation of transgenic cells and genetically engineered animals. I consider it a great honor to have been involved in this epoch-making transition in pituitary research together with many other researchers. I am especially grateful to Professor Akira Teramoto and Professor Akira Matsuno with whom I shared the dreams of pituitary research for many years. I would also like to express my gratitude to the Ministry of Education, Culture, Sports, Science and Technology (MEXT) for their long-term support of this research with a Grant-in-Aid for Scientific Research. I wish to conclude by expressing my sincere gratitude to all those who have shared their time and interest with me in the field of pituitary research.
Robert Yoshiyuki Osamura
Honorary Member
Professor Emeritus, Tokai University
Department of Diagnostic Pathology, Nippon Koukan Hospital
Visiting Professor, Keio University School of Medicine
Careers in JES
2014– Honorary Member
2011– Senior Councilor
2009–2011 Auditor
2007–2009 Director (Academic Affairs and Publication)
1999–2007 Director (Public Relations and Collaboration)
1997–1999 Director (General Affairs)
1989– Councilor
1977– Member
Activities in JES
2003 Chair, 21st JES Summer Seminar on Endocrinology & Metabolism
JES Awards
2009 Distinguished Endocrinologist Award
