Isolation and Characterization of a Prolactin-Requlating Factor (PRF) from a Mouse Pituitary Intermediate Lobe Cell Line
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University of Cincinnati / OhioLINK
2000
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ndltd-OhioLink-oai-etd.ohiolink.edu-ucin9641210622021-08-03T06:15:48Z Isolation and Characterization of a Prolactin-Requlating Factor (PRF) from a Mouse Pituitary Intermediate Lobe Cell Line Hnasko, Robert Michael Prolactin Pituitary Cell Liens Heparin-Binding Growth Factors Prolactin (PRL) is regulated by inhibitory and stimulatory factors from the hypothalamus and pituitary. Dopamine is the primary PRL-inhibitory factor, but acute elevations in PRL require stimulation by a PRL-releasing factor (PRF). PRF from the rat pituitary rapidly stimulated PRL release and localized to the intermediate lobe (IL). Partial purification of multiple PRF species from IL tumors differed in size and chromatographic properties. However, the primary structure of PRF has not been resolved. Our hypothesis was: a subpopulation of IL cells secretes a novel peptide that functions as a PRF. Transgenic POMC-Tag mice that develop IL tumors were used to investigate the following objectives: 1) determine if POMC-Tag mice with IL tumors develop hyperprolactinemia, 2) establish and characterize an IL cell line the produces PRF, 3) determine the biochemical properties of PRF, 4) purify PRF and determine its structure. Hyperprolactinemia was not observed in POMC-Tag mice from 20 to 120 days. Cells from two IL tumors were cloned into mIL cell lines that differed in cellular characteristics. As assessed by RT-PCR the mIL39, but not mIL5, cells expressed POMC and D2R gene products, characteristic of IL melanotrophs. The cell lineage of mIL5 cells remains undefined. Only the mIL5 cells, co-cultured with GH3/luc cells, stimulated PRL gene expression and release. PRF from mIL5 was secreted and bound heparin. Two heparin-binding proteins, FGF2 and HB-EGF, identified in mIL5 cells were potent PRL inducers. However, neither significantly contributed to the PRF activity from mIL5 as judged by Western blotting, heparin-affinity and immunoneutralization. Purification of PRF from mIL5 by heparin-affinity chromatography indicated multiple species that differed in chromatographic properties. Sequential chromatography was used to isolate PRF from cell extract and sequencing identified a peptide identical to an internal sequence of heparin-interacting protein (HIP/L29). The expression of HIP protein was ubiquitous and two HIP antibodies were ineffective in the attenuation of PRF activity from mIL5 cells. Isolated PRF from conditioned media was subjected to mass spectrometry that revealed a mass of 14,968 Da. N-terminal sequence analysis failed to resolve the primary structure of PRF. 2000 English text University of Cincinnati / OhioLINK http://rave.ohiolink.edu/etdc/view?acc_num=ucin964121062 http://rave.ohiolink.edu/etdc/view?acc_num=ucin964121062 unrestricted This thesis or dissertation is protected by copyright: all rights reserved. It may not be copied or redistributed beyond the terms of applicable copyright laws. |
| collection |
NDLTD |
| language |
English |
| sources |
NDLTD |
| topic |
Prolactin Pituitary Cell Liens Heparin-Binding Growth Factors |
| spellingShingle |
Prolactin Pituitary Cell Liens Heparin-Binding Growth Factors Hnasko, Robert Michael Isolation and Characterization of a Prolactin-Requlating Factor (PRF) from a Mouse Pituitary Intermediate Lobe Cell Line |
| author |
Hnasko, Robert Michael |
| author_facet |
Hnasko, Robert Michael |
| author_sort |
Hnasko, Robert Michael |
| title |
Isolation and Characterization of a Prolactin-Requlating Factor (PRF) from a Mouse Pituitary Intermediate Lobe Cell Line |
| title_short |
Isolation and Characterization of a Prolactin-Requlating Factor (PRF) from a Mouse Pituitary Intermediate Lobe Cell Line |
| title_full |
Isolation and Characterization of a Prolactin-Requlating Factor (PRF) from a Mouse Pituitary Intermediate Lobe Cell Line |
| title_fullStr |
Isolation and Characterization of a Prolactin-Requlating Factor (PRF) from a Mouse Pituitary Intermediate Lobe Cell Line |
| title_full_unstemmed |
Isolation and Characterization of a Prolactin-Requlating Factor (PRF) from a Mouse Pituitary Intermediate Lobe Cell Line |
| title_sort |
isolation and characterization of a prolactin-requlating factor (prf) from a mouse pituitary intermediate lobe cell line |
| publisher |
University of Cincinnati / OhioLINK |
| publishDate |
2000 |
| url |
http://rave.ohiolink.edu/etdc/view?acc_num=ucin964121062 |
| work_keys_str_mv |
AT hnaskorobertmichael isolationandcharacterizationofaprolactinrequlatingfactorprffromamousepituitaryintermediatelobecellline |
| _version_ |
1719433684648460288 |