Project Title

Comparison Of Two Rat Somatotroph Cell Lines To Examine Tissue-Specific Transcription Of Growth Hormone

Authors' Affiliations

Concentration in Pharmacy Research, and Department of Pharmaceutical Sciences, Bill Gatton College of Pharmacy, ETSU, Johnson City, TN 37614.

Location

Ballroom

Start Date

4-5-2018 8:00 AM

End Date

4-5-2018 12:00 PM

Poster Number

99

Name of Project's Faculty Sponsor

David L. Hurley

Faculty Sponsor's Department

Dept. Pharmaceutical Sciences, Bill Gatton College of Pharmacy

Type

Poster: Competitive

Classification of First Author

Pharmacy Student

Project's Category

Biomedical and Health Sciences

Abstract Text

Growth Hormone (GH), also known as somatotrophin, is of cardinal importance in the regulation of somatic growth. Decreased GH expression results in short stature, whereas increased levels of GH lead to disorders such as gigantism or acromegaly. Mutations in the transcription factor Pit-1 have been shown to decrease GH, as well as prolactin (PRL; Parks et al., JCEM, 1999). However, understanding the regulation by selective GH transcription regulators, such as Zn16, a protein encoding 16 zinc fingers that binds to the GH promotor DNA (Wojtkiewicz et al., Endocrine, 2002), will require comparison of currently available rodent cell lines that express GH. Two current rat somatotroph cell lines are the somatotroph MtT/S and lactosomatotroph GH3 cell lines, both of which have been implemented in studies on the regulation of GH expression (Schaaf et al., Endocr Relat Cancer, 2009). MtT/S cells almost exclusively express GH, whereas GH3 cells are less differentiated and co-express PRL as well as GH. GH3 cells have been available longer, and thus are more frequently used for in vitro GH experiments, but there may be some increased utility in utilizing MtT/S cells. MtT/S cells were procured from Riken Cell Bank in Japan, and GH3 cells were acquired from the ATCC. These lines were cultured, then secretion of GH and PRL was examined after Insulin-like Growth Factor (IGF-1) treatment, which is inhibitory to GH and PRL secretion. Further, both cell lines were treated with stimulatory factors GH releasing hormone (GHRH); retinoic acid; cortisone; GH Releasing Peptide-6; and both cortisone and GHRH. GH secretion was on average higher in MtT/S cells than in GH3 cells. On the other hand, PRL secretion was extremely lower in MtT/S cells than in GH3 cells. This result confirms that the MtT/S cells are further differentiated as somatotrophs than GH3 cells. Although hormone release in response to treatment did not appear different, the overall difference in GH vs. PRL secretion may be useful for evaluating the role of Zn16 in selective control of the GH promoter. Therefore, mRNA levels of GH, PRL, Pit-1 and Zn16 in these cell lines are currently being measured using quantitative real-time PCR with ribosomal protein L19 (RPL19) expression as a standard. With the investigation of the characteristics of these differentiated pituitary cell types, we hope to advance the knowledge of GH transcription and regulation, particularly the study of Zn16 effects during pituitary development. Recently it was observed that Zn16 may have a role in a tumor suppressor gene that undergoes mutation leading to a form of colorectal cancer and childhood leukemia. This augments the utility of determining the proper cell lines to examine Zn16, and its role in tumor suppression and cell division as well as gene expression during hypophyseal development.

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Apr 5th, 8:00 AM Apr 5th, 12:00 PM

Comparison Of Two Rat Somatotroph Cell Lines To Examine Tissue-Specific Transcription Of Growth Hormone

Ballroom

Growth Hormone (GH), also known as somatotrophin, is of cardinal importance in the regulation of somatic growth. Decreased GH expression results in short stature, whereas increased levels of GH lead to disorders such as gigantism or acromegaly. Mutations in the transcription factor Pit-1 have been shown to decrease GH, as well as prolactin (PRL; Parks et al., JCEM, 1999). However, understanding the regulation by selective GH transcription regulators, such as Zn16, a protein encoding 16 zinc fingers that binds to the GH promotor DNA (Wojtkiewicz et al., Endocrine, 2002), will require comparison of currently available rodent cell lines that express GH. Two current rat somatotroph cell lines are the somatotroph MtT/S and lactosomatotroph GH3 cell lines, both of which have been implemented in studies on the regulation of GH expression (Schaaf et al., Endocr Relat Cancer, 2009). MtT/S cells almost exclusively express GH, whereas GH3 cells are less differentiated and co-express PRL as well as GH. GH3 cells have been available longer, and thus are more frequently used for in vitro GH experiments, but there may be some increased utility in utilizing MtT/S cells. MtT/S cells were procured from Riken Cell Bank in Japan, and GH3 cells were acquired from the ATCC. These lines were cultured, then secretion of GH and PRL was examined after Insulin-like Growth Factor (IGF-1) treatment, which is inhibitory to GH and PRL secretion. Further, both cell lines were treated with stimulatory factors GH releasing hormone (GHRH); retinoic acid; cortisone; GH Releasing Peptide-6; and both cortisone and GHRH. GH secretion was on average higher in MtT/S cells than in GH3 cells. On the other hand, PRL secretion was extremely lower in MtT/S cells than in GH3 cells. This result confirms that the MtT/S cells are further differentiated as somatotrophs than GH3 cells. Although hormone release in response to treatment did not appear different, the overall difference in GH vs. PRL secretion may be useful for evaluating the role of Zn16 in selective control of the GH promoter. Therefore, mRNA levels of GH, PRL, Pit-1 and Zn16 in these cell lines are currently being measured using quantitative real-time PCR with ribosomal protein L19 (RPL19) expression as a standard. With the investigation of the characteristics of these differentiated pituitary cell types, we hope to advance the knowledge of GH transcription and regulation, particularly the study of Zn16 effects during pituitary development. Recently it was observed that Zn16 may have a role in a tumor suppressor gene that undergoes mutation leading to a form of colorectal cancer and childhood leukemia. This augments the utility of determining the proper cell lines to examine Zn16, and its role in tumor suppression and cell division as well as gene expression during hypophyseal development.