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Different Mechanisms of Calcitonin, Calcitonin Gene-Related Peptide

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Different Mechanisms of Calcitonin, Calcitonin Gene-Related Peptide Henry Ford Hospital Medical Journal Volume 35 Number 2 Second International Workshop on Article 20 MEN-2 6-1987 Different Mechanisms of Calcitonin, Calcitonin Gene-Related Peptide, and Somatostatin Regulation by Glucocorticoids in a Cell Culture of Human Medullary Thyroid Carcinoma Gilbert J. Cote Robert F. Gagel Follow this and additional works at: https://scholarlycommons.henryford.com/hfhmedjournal Part of the Life Sciences Commons, Medical Specialties Commons, and the Public Health Commons Recommended Citation Cote, Gilbert J. and Gagel, Robert F. (1987) "Different Mechanisms of Calcitonin, Calcitonin Gene-Related Peptide, and Somatostatin Regulation by Glucocorticoids in a Cell Culture of Human Medullary Thyroid Carcinoma," Henry Ford Hospital Medical Journal : Vol. 35 : No. 2 , 149-152. Available at: https://scholarlycommons.henryford.com/hfhmedjournal/vol35/iss2/20 This Article is brought to you for free and open access by Henry Ford Health System Scholarly Commons. It has been accepted for inclusion in Henry Ford Hospital Medical Journal by an authorized editor of Henry Ford Health System Scholarly Commons. Different Mechanisms of Calcitonin, Calcitonin Gene-Related Peptide, and Somatostatin Regulation by Glucocorticoids in a Cell Culture of Human Medullary Thyroid Carcinoma Gilbert J. Cote, and Robert F. Gagel* We have employed the TT cell line, a model for the human medullary thyroid carcinoma cell, lo study the regulation of peptide hormone production by glucocorticoids. Complementary DNA probes were used to measure the calcitonin (CT), CT gene-related peptide (CGRP), and somatostatin (SRIF) mRNA levels. Dose-response experiments in serum-free medium showed that dexamethasone (six-day treatment) lowered somatostatin (to 1% of basal) and CGRP mRNA (to 50% of basal) and stimulated CT mRNA (threefold to thirteenfold) with a half-maximal effective concentration of 10 " M. Time course studies for cells continuously exposed to 70" M dexamethasone showed a rapid (within hours) lowering of SRIF mRNA. whereas the effects on CT and CGRP mRNA required six to eight days. These results demonstrate the presence of two mechanisms, transcriptional (somatostatin) and posttranscriptional (the RNA splice decision to make CT or CGRP mRNA). that can be hormonally regulated. (Henry Ford Hosp MedJ 1987;35:149-52) he TT cell line is a continuous cell line originally derived viously described (6). All steroids were added to the medium as Tfrom the needle biopsy of a human medullary thyroid car­ 25 mM stocks prepared in ethanol. Control cells received an cinoma (1). We have employed this cell line to study the produc­ equal volume of ethanol. tion of the peptide hormones calcitonin (CT), CT gene-related peptide (CGRP), and somatostatin (SRIF), Two precursor pep­ Preparation of RNA tides for CT and CGRP are produced from the CT gene by alter­ The TT cells were plated at an initial density of ~3 x 10*" native RNA processing, whereas the SRIF gene codes for the cells/100 mm dish. The cells were allowed to attach for 24 hours production of a single precursor peptide (2,3). Because the TT before treatment. Total cellular RNA was isolated from various cell line produces all three of these peptides in significant quan­ cell types by the guanidine isothiocyanate procedure (8), with tities, it provides a useful model system for studying the regula­ the previously described modifications (5). Total RNA was tion ofthe two genes. We have previously demonstrated an stored dissolved in sterile distilled H^O in the presence of effect of the synthetic glucocorticoid dexamethasone on vanadium-riboside complex (9) at -70°C. production of SRIF (4), as well as CT and CGRP (5). In this pa­ per we present and compare the effects of dexamethasone treat­ Hybridizations ment on CT, CGRP and SRIF mRNA in the TT cell line. The RNA dot blot hybridizations were performed by immobiliza­ results demonstrate the existence of two distinct mechanisms of tion of purified total RNA onto nitrocellulose paper as described action for dexamethasone: a rapid transcriptional effect seen on by Thomas (10). The SRIF-, CT-, and CGRP-specific probes SRIF, and a more slowly developing posttranscriptional effect were prepared as previously described (4,5). Prehybridization, on the processing of primary RNA transcript derived from the hybridization, and washing procedures were identical to those of CT gene. Thomas (10). Autoradiographs were quantitated by computer- assisted densitometric scanning with a video camera as de­ Experimental Procedures scribed by Mariash et al (11). Cell culture methodology The TT cells were maintained in RPMI 1640 medium supple­ Results mented with 10% fetal calf semm (Gibco Laboratories, Grand Fig 1 shows the effect of a pharmacologic dose of dexa­ Island, NY), using previously described techniques (6). In methasone (10-* M) on CT, CGRP, and SRIF mRNA levels. experiments using semm-free medium, cells were subcultured 48 hours before treatment in serum-containing medium and then switched to semm-free medium 24 hours before the onset Submitted for publication: March 3, 1987, of treatment. Serum-free defined medium was prepared as Accepted for publication: April 29, 1987, previously described for a rat C-cell line (7), Cell counts were •Address correspondence to Dr Gagel, Laboratory for Molecular and Cellular Endo­ crinology IIIE, Veterans Administration Medical Center. 2002 Holcombe Blvd, Houston, determined with the aid of a Coulter Counter (model ZF), as pre­ TX 772II, Henry Ford Ho,sp Med J—Vol 35, Nos 2&3, 1987 Peptide Hormone Regulation in TT Cells—Cote & Gagel 149 300 ' 250 -1 coc 200 t- z o o 1 50 ' ••" CALCITONIN mRNA ••- CALCITONIN mRNA 1- z UJ CGRP mRNA CGRP mRNA o cc 1 00 1 UJ ••- SOMATOSTATIN mRNA 50 10 10"° 4 5 6 DEXAMETHASONE (M) TIME (DAYS) Fig 1—Time course of dexamethasone treatment on CT. CGRP. and SRIF mRNA production in the TT cells. The TT cells were plated at an initial density of ~3 X 70* cells/100 mm dish and Fig 2—Dose-response curve for the effect of dexamethasone on allowed to attach in growth medium (24 hours) before changing CT. CGRP, and SRIF mRNA production. The TT cells were to medium containing I x 70 * M dexamethasone. Medium plated at an initial density of ~3 X ICP cells/100 mm dish in was changed at 48-hour intervals. RNA measurements were serum-containing medium for 24 hours, then switched to serum- made at indicated time points after the initiation of treatment by free medium for 24 hours before treatment. Cells were then removal of medium and addition of guanidine isothiocyanate. grown in the presence of the indicated concentration of dexa­ The values represent the average hybridization value of two methasone in serum-free medium for six days with medium dishes as determined by dot hybridization and quantitated by a changed at 48-hour intervals. The mRNA levels were deter­ computer-assisted densitometer. mined as described in Fig 1. Dexamethasone treatment affected the levels of each mRNA dif­ mechanisms in the human medullary thyroid carcinoma TT cell ferently. The levels of SRIF mRNA showed a rapid decrease line. Three possible points of regulation are transcription, RNA (levels reach 50% of control by 12 hours) and were barely detect­ processing, and mRNA degradation. Fig 3 schematically dem­ able by 48 hours. While CGRP mRNA levels also declined as a onstrates the production of CT, CGRP and SRIF mRNA from result of dexamethasone treatment, a six-day treatment was re­ their respective genes. The SRIF gene is transcribed, the tran­ quired to achieve a 50% reduction. Unlike SRIF and CGRP, CT script undergoes RNA processing in the nucleus to produce ma­ mRNA showed an increase over two to eight days following ture mRNA, and then the mRNA is typically degraded in the treatment with dexamethasone. Although this increase could be cytoplasm after several rounds of translation. Unlike the SRIF explained by an enhancement of transcription, the rate ofthe re­ gene, the CT gene's primary transcript is altematively processed sponse and the simultaneous decrease in CGRP mRNA levels to produce one of two possible mRNAs, one coding for CT and suggest a more complex explanation. the other for CGRP Therefore, RNA processing becomes a key To determine whether glucocorticoids might play a physio­ regulatory step in the production of CT and CGRP mRNA, logically significant role in the regulation of these genes, we ex­ amined the dose-dependent effects of dexamethasone on CT, Effect of dexamethasone on SRIF mRNA levels CGRP and SRIF mRNA levels (Fig 2). Again, both SRIF and We have shown that dexamethasone rapidly lowered SRIF CGRP mRNA levels were depressed, while CT levels were mRNA levels. Because the ~ 12-hour half-life of the SRIF enhanced. The TT cells are typically grown in the presence of mRNA (data not shown) approximates the rate of decline in 10% fetal bovine serum, which we have found to contain ap­ SRIF mRNA following dexamethasone treatment, we favor a proximately 10 M endogenous steroid. For this reason, the model whereby dexamethasone acts to inhibit directiy the tran­ dose-response curves were generated in serum-free medium. scription ofthe SRIF gene. The possibility that dexamethasone The half-maximal doses of dexamethasone (10' M for CT, 10'* affects SRIF mRNA stability, though unlikely, has not been M for CGRP, and 10" M for SRIF mRNA) are shifted excluded. two orders of magnitude to the left, compared to our previous findings in the presence of serum (4,5), and now approach phys­ Effect of dexamethasone on CT and CGRP mRNA levels iologically significant levels (12). Dexamethasone acts to enhance CT mRNA levels and de­ crease CGRP mRNA levels. Regulation of this event must occur Discussion at either the RNA processing step or at the level of mRNA deg­ Differences between regulation ofthe CT and SRIF genes radation.
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