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 10−8 M. Time course studies for cells continuously exposed to 10−6 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

Cote, Gilbert J.

Gagel, Robert F.

English

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Henry Ford Hospital Medical Journal Volume 35 Number 2 Second International Workshop on MEN-2 Article 20 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) The TT cell line is a continuous cell line originally derived from the needle biopsy of a human medullary thyroid car-cinoma (1). We have employed this cell line to study the produc-tion of the peptide hormones calcitonin (CT), CT gene-related peptide (CGRP), and somatostatin (SRIF), Two precursor pep-tides for CT and CGRP are produced from the CT gene by alter-native RNA processing, whereas the SRIF gene codes for the production of a single precursor peptide (2,3). Because the TT cell line produces all three of these peptides in significant quan-tities, it provides a useful model system for studying the regula-tion ofthe two genes. We have previously demonstrated an effect of the synthetic glucocorticoid dexamethasone on production of SRIF (4), as well as CT and CGRP (5). In this pa-per we present and compare the effects of dexamethasone treat-ment on CT, CGRP and SRIF mRNA in the TT cell line. The results demonstrate the existence of two distinct mechanisms of action for dexamethasone: a rapid transcriptional effect seen on SRIF, and a more slowly developing posttranscriptional effect on the processing of primary RNA transcript derived from the CT gene. Experimental Procedures Cell culture methodology The TT cells were maintained in RPMI 1640 medium supple-mented with 10% fetal calf semm (Gibco Laboratories, Grand Island, NY), using previously described techniques (6). In 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 of treatment. Serum-free defined medium was prepared as previously described for a rat C-cell line (7), Cell counts were determined with the aid of a Coulter Counter (model ZF), as pre-viously described (6). All steroids were added to the medium as 25 mM stocks prepared in ethanol. Control cells received an equal volume of ethanol. Preparation of RNA The TT cells were plated at an initial density of ~3 x 10*" cells/100 mm dish. The cells were allowed to attach for 24 hours before treatment. Total cellular RNA was isolated from various cell types by the guanidine isothiocyanate procedure (8), with the previously described modifications (5). Total RNA was stored dissolved in sterile distilled H^O in the presence of vanadium-riboside complex (9) at -70°C. Hybridizations RNA dot blot hybridizations were performed by immobiliza-tion of purified total RNA onto nitrocellulose paper as described by Thomas (10). The SRIF-, CT-, and CGRP-specific probes were prepared as previously described (4,5). Prehybridization, hybridization, and washing procedures were identical to those of Thomas (10). Autoradiographs were quantitated by computer-assisted densitometric scanning with a video camera as de-scribed by Mariash et al (11). Results Fig 1 shows the effect of a pharmacologic dose of dexa-methasone (10-* M) on CT, CGRP, and SRIF mRNA levels. Submitted for publication: March 3, 1987, Accepted for publication: April 29, 1987, •Address correspondence to Dr Gagel, Laboratory for Molecular and Cellular Endo-crinology IIIE, Veterans Administration Medical Center. 2002 Holcombe Blvd, Houston, TX 772II, Henry Ford Ho,sp Med J—Vol 35, Nos 2 & 3 , 1987 Peptide Hormone Regulation in TT Cells—Cote & Gagel 149 3 0 0 ' 2 5 0 - 1 o cc t -2 0 0 z o o 1 50 ' 1 -z UJ o cc UJ 1 00 1 50 ••" CALCITONIN mRNA CGRP mRNA • • - SOMATOSTATIN mRNA 10 10"° DEXAMETHASONE (M) 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 allowed to attach in growth medium (24 hours) before changing to medium containing I x 70 * M dexamethasone. Medium was changed at 48-hour intervals. RNA measurements were made at indicated time points after the initiation of treatment by removal of medium and addition of guanidine isothiocyanate. The values represent the average hybridization value of two dishes as determined by dot hybridization and quantitated by a computer-assisted densitometer. • • - CALCITONIN mRNA CGRP mRNA 4 5 6 TIME (DAYS) Fig 2—Dose-response curve for the effect of dexamethasone on CT. CGRP, and SRIF mRNA production. The TT cells were plated at an initial density of ~3 X ICP cells/100 mm dish in serum-containing medium for 24 hours, then switched to serum-free medium for 24 hours before treatment. Cells were then grown in the presence of the indicated concentration of dexa-methasone in serum-free medium for six days with medium changed at 48-hour intervals. The mRNA levels were deter-mined as described in Fig 1. Dexamethasone treatment affected the levels of each mRNA dif-ferently. The levels of SRIF mRNA showed a rapid decrease (levels reach 50% of control by 12 hours) and were barely detect-able by 48 hours. While CGRP mRNA levels also declined as a result of dexamethasone treatment, a six-day treatment was re-quired to achieve a 50% reduction. Unlike SRIF and CGRP, CT mRNA showed an increase over two to eight days following treatment with dexamethasone. Although this increase could be explained by an enhancement of transcription, the rate ofthe re-sponse and the simultaneous decrease in CGRP mRNA levels suggest a more complex explanation. To determine whether glucocorticoids might play a physio-logically significant role in the regulation of these genes, we ex-amined the dose-dependent effects of dexamethasone on CT, CGRP and SRIF mRNA levels (Fig 2). Again, both SRIF and CGRP mRNA levels were depressed, while CT levels were enhanced. The TT cells are typically grown in the presence of 10% fetal bovine serum, which we have found to contain ap-proximately 10 M endogenous steroid. For this reason, the dose-response curves were generated in serum-free medium. The half-maximal doses of dexamethasone (10' M for CT, 10'* M for CGRP, and 10" M for SRIF mRNA) are shifted two orders of magnitude to the left, compared to our previous findings in the presence of serum (4,5), and now approach phys-iologically significant levels (12). Discussion Differences between regulation ofthe CT and SRIF genes The results presented in this and previous papers (4,5) sug-gest that dexamethasone acts to regulate mRNA levels by several mechanisms in the human medullary thyroid carcinoma TT cell line. Three possible points of regulation are transcription, RNA processing, and mRNA degradation. Fig 3 schematically dem-onstrates the production of CT, CGRP and SRIF mRNA from their respective genes. The SRIF gene is transcribed, the tran-script undergoes RNA processing in the nucleus to produce ma-ture mRNA, and then the mRNA is typically degraded in the cytoplasm after several rounds of translation. Unlike the SRIF gene, the CT gene's primary transcript is altematively processed to produce one of two possible mRNAs, one coding for CT and the other for CGRP Therefore, RNA processing becomes a key regulatory step in the production of CT and CGRP mRNA, Effect of dexamethasone on SRIF mRNA levels We have shown that dexamethasone rapidly lowered SRIF mRNA levels. Because the ~ 12-hour half-life of the SRIF mRNA (data not shown) approximates the rate of decline in SRIF mRNA following dexamethasone treatment, we favor a model whereby dexamethasone acts to inhibit directiy the tran-scription ofthe SRIF gene. The possibility that dexamethasone affects SRIF mRNA stability, though unlikely, has not been excluded. Effect of dexamethasone on CT and CGRP mRNA levels Dexamethasone acts to enhance CT mRNA levels and de-crease CGRP mRNA levels. Regulation of this event must occur at either the RNA processing step or at the level of mRNA deg-radation. Transcriptional regulation alone would result in par-allel changes in CT and CGRP mRNA levels, such as seen after phorbol ester or cAMP analog treatment (12), and not the diver-150 Henry Ford Hosp Med J—Vol 35, Nos 2 & 3, 1987 Peptide Hormone Regulation in TT Cells—Cote SL Gagel A. SOMATOSTATIN TRANSCRIPT POLYADENYLATION SPLICING OF ^ INTPON SOMATOSTATIN mRNA 5 I 1 , 1 - PO'V * 3' FACTOR BINDING INTRON ^ FACTOR BINDING M T O POLY A SITE CGRP CGRP NON CODING O 3-Fig 4—Proposed model for CGRP mRNA production. B CALCITONIN PRIMARY BNA 5 TRANSCRIPT CT CGRP CGRP NON CODING -cm OT^-—H mi^-— POLYAQENYLATION ^ t A 9iia POLYADENVLATION CGRP CGOP NONCODING — H CZ>DOIf A SPLICING OF ^ INTFtONS CALCITONIN mRNA C ALIERNATE SPLICE TQ REMOVE CT EXON 3 CGRP CGRP NON CODING 1-DOIv A SPLICING OF V INTFIONS CGRP mRNA I I I ~ M | f-f>oi> Fig 3—Schematic outline comparing RNA processing of the pri-mary transcript from the SRIF gene and the CT gene: A) pro-cessing ofthe SRIF gene primary transcript to SRIF mRNA, and B) processing of the CT gene primary transcript into CT and CGRP mRNA. gence seen here. We have previously demonstrated that dexa-methasone has no effect on the half-life of either CT or CGRP mRNA (5). Therefore, we proposed that dexamethasone must be acting to affect the RNA processing of the primary transcript. How might dexamethasone alter the RNA processing of the primary transcript of the CT gene? The first possibility to con-sider is a direct effect of dexamethasone on RNA processing. The dexamethasone-glucocorticoid receptor complex, or some other dexamethasone-binding protein with regulatory abilities, might affect RNA processing directly. Glucocorticoid receptors have been demonstrated to bind RNA (13), and RNA processing is brought about by protein-RNA complexes (14). However, it is unlikely that dexamethasone is acting through one of these di-rect mechanisms, because the time course for the effect is too slow, and a similar effect can be produced in the absence of any steroids by long-term culturing of the cells without subculturing (as described by A. deBustros et al at these proceedings). There-fore, we propose that dexamethasone is acting in an indirect fashion to alter RNA processing. Model for the regulation of alternative RNA processing by dexamethasone To explain the regulation of the altemative processing of the CT gene primary transcript, we favor a model where the produc-tion of CT mRNA is the unregulated pathway or "null" choice, and production of CGRP mRNA requires the presence of spe-cific factor(s). This model is based on the available information regarding the processing of the CT gene transcript (15) and on current theories of RNA processing (14). From this model we predict that in the thyroid C-cell, cleavage and polyadenylation at the site immediately following the CT exon are the early steps which necessitate an exon 3 to CT exon splice (Fig 3). If our CT "null" choice is correct, then the production of CGRP mRNA would require that: 1) polyadenylation immediately after the CT exon be blocked; and 2) because the blockage of cleavage fol-lowing the CT exon would be insufficient to ensure CGRP mRNA production, a mechanism must exist to prevent an exon 3 to CT exon splice. This is most easily accomplished either by preventing branch point formation within the third intron or by blocking the accessibility of the 3' splice junction (studies have shown that spliceosome binding to the 3' splice site is re-quired for 5' cleavage) (14). This blockage would then allow preferential usage of the next available 3' splice junction (at the CGRP exon). This model would predict the existence of protein or RNA factor(s) in CGRP-producing cell types functioning to block polyadenylation immediately after the CT exon and removal of the intron immediately before the CT exon. Whether this is ac-complished by a single factor, several factors, or a complex re-mains to be elucidated. One such way in which the interaction could occur is diagramed in Fig 4, where separate binding sites cause the RNA to loop out while forcing CGRP mRNA pro-duction. If the production of this complex was inhibited by dexamethasone, cell cycle-dependent or cell type-dependent mechanisms, then the production of CT would be favored. Acknowledgments This work was supported by USPHS Grant AM 31307 and grants from the Veterans Administration. References 1. Leong SS, Horoszewicz JS, Shimaoka K, etal. A new cell line for study of human medullary thyroid carcinoma. In: Advances in thyroid neoplasia. Rome: Field Educational Italia, 1981:95-108. 2. Rosenfeld MG, Amara SG, Evans RM. Alternative RNA processing: Determining neuronal phenotype. Science 1984;225:1315-20. 3. Shen LP, Rutter WJ. Sequence of the human somatostatin I gene. Science 1984;224:168-71. 4. Cote GJ, Palmer WN, Leonhart K, Leong SS, Gagel RF. The regulation of somatostatin production in human medullary thyroid carcinoma cells by dexamethasone. J Biol Chem 1986;261:12930-5. 5. Cote GJ, Gagel RF. Dexamethasone differentially affects the levels of calcitonin and calcitonin gene-related peptide mRNAs expressed in a human medullary thyroid carcinoma cell line. J Biol Chem I986;261:15524-8. 6. Gagel RF, Zeytinoglu FN, Voelkel EF, Tashjian AH Jr Establishment of a calcitonin-producing rat medullary thyroid carcinoma cell line: I I . Secretory studies of the tumor and cells in culture. Endocrinology 1980;107:516-23. 7. Muszynski M, Birnbaum RS, Roos BA. Glucocorticoids stimulate the Henry Ford Hosp Med J—Vol 35, Nos 2 & 3, 1987 Peptide Hormone Regulation in TT Cells—Cote & Gagel 151 production of preprocalcitonin-derived secretory peptides by a rat medullary thyroid carcinoma cell line, J Biol Chem 1983;258:11678-83. 8, Chirgwin JM, Przybyala AE, MacDonald RJ, Rutter WJ, Isolation of biologically active ribonucleic acid from source enriched in ribonuclease. Biochemistry 1979;18:5294-9, 9, Berger SL, Birkenmeier CS, Inhibition of intractable nucleases with ribonucleoside-vanadyl complexes: Isolation of mes.senger ribonucleic acid from resting lymphocytes. Biochemistry 1979;18:5143-9, 10, Thomas PS, Hybridization of denatured RNA and small DNA fragments transferred to niuocellulose, Proc Natl Acad Sci USA 1980;77:5201-5, 11, Mariash CN, Seelig S, Oppenheimer JH, A rapid, inexpensive, quan-titative technique for the analysis of two-dimensional electrophoretograms. Anal Biochem 1982;121:388-94, 12, deBusU-os A, Baylin SB, Levine MA, Nelkin BD, Cyclic AMP and phor-bol esters separately induce growth inhibition, calcitonin secretion, and calcitonin gene transcription in cultured human medullary thyroid carcinoma, J Biol Chem 1986;261:8036-41, 13, Ali M, Vedeckis WV, The glucocorticoid receptor protein binds to ttans-fer RNA, Science 1987:235:467-70, 14, Padgett RA, Grabowski PJ, Konarska MM, Seiler S, Sharp PA, Splicing of messenger RNA precursors, Annu Rev Biochem 1986;55:1119-50, 15, Leff SE, Rosenfeld MG, Evans RM, Complex transcriptional units: Diversity in gene expression by altemative RNA processing, Annu Rev Bio-chem 1986;55:1091-117, 152 Henry Ford Hosp Med J—Vol 35, Nos 2 & 3, 1987 Peptide Hormone Regulation in TT Cells—Cote & Gagel 

Different Mechanisms of Calcitonin, Calcitonin Gene-Related Peptide, and Somatostatin Regulation by Glucocorticoids in a Cell Culture of Human Medullary Thyroid Carcinoma

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Different Mechanisms of Calcitonin, Calcitonin Gene-Related Peptide, and Somatostatin Regulation by Glucocorticoids in a Cell Culture of Human Medullary Thyroid Carcinoma

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