J Korean Med Sci.  2008 Apr;23(2):262-269. 10.3346/jkms.2008.23.2.262.

Regulation of Inhibitors of Differentiation Family Proteins by Thyroid-Stimulating Hormone in FRTL-5 Thyroid Cells

• Jo YS
• Hwang ES
• Lee JH
• Lee Y
• Kim SY
• Choi YS
• Bai YS
• Hong JH
• Kim YJ
• Lee IS
• Rha SY
• Ro HK
• Shong M

• KMID: 1713459
• DOI: http://doi.org/10.3346/jkms.2008.23.2.262

Abstract

Members of the inhibitors of differentiation (Id) family of helix-loop-helix (HLH) proteins are known to play important roles in the proliferation and differentiation of many cell types. Thyroid-stimulating hormone (TSH) regulates proliferation and differentiation by activating TSH receptor (TSHR) in thyrocytes. In this study, we found that Id2, one of the Id family proteins, is a major target for regulation by TSH in FRTL-5 thyroid cells. TSH rapidly increases the Id2 mRNA level in FRTL-5 thyroid cells but the Id2 protein showed biphasic regulatory patterns, being transiently reduced and subsequently induced by TSH treatment. Transient reduction of Id2 protein was noted within 2 hr of TSH treatment and was mediated by proteasomal degradation. Moreover, reduced Id2 expression correlated with the activity of the phosphatidylinositol 3 kinase pathway, which is activated by TSH. Although TSH increases the activity of the Id2 promoter, TSH-induced activation of this promoter was independent of c-Myc. Id2 did not alter TTF-1- and Pax-8-mediated effects on the regulation of the Tg promoter. Thus, in summary, we found that TSH regulates Id2 expression, but that Id2 does not alter the expression of thyroid-specific genes, such as Tg, in FRTL-5 thyroid cells.

Keyword

Thyroid Gland; Thyrotropin; Id2 protein

MeSH Terms

1-Phosphatidylinositol 3-Kinase/metabolism
Animals
Cattle
Cell Differentiation
Cell Proliferation
*Gene Expression Regulation
Inhibitor of Differentiation Protein 2/metabolism
Insulin/metabolism
Paired Box Transcription Factors/metabolism
Promoter Regions, Genetic
Proto-Oncogene Proteins c-myc/metabolism
Rats
Thyroglobulin/metabolism
Thyroid Gland/*cytology
Thyrotropin/*metabolism

Figure

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  Fig. 1 Effects of TSH and insulin on Id family RNA levels in cultured FRTL-5 cells. A, B FRTL-5 thyroid cells were grown to near confluency in Coon's modified Ham's F-12 medium containing 5% (v/v) calf serum. The cells were starved for 6 days with 0H medium and 5% calf serum which did not contain hormones. The cells were treated with TSH 1 mU/mL (A) and insulin 10 µg/mL (B). Total RNA was isolated from 1 to 12 hr after the final treatment and subjected to Northern analysis (20 µg/lane) using Id family and β-actin cDNA probes as described in Materials and Methods. The amount of RNA in each lane was monitored using β-actin. The results are representative of a minimum of three independent experiments.

• 

  Fig. 2 Effects of TSH, forskolin, and insulin on Id family protein levels in cultured FRTL-5 cells. A, B, C FRTL-5 thyroid cells were grown to near confluency in Coon's modified Ham's F-12 medium containing 5% (v/v) calf serum. The cells were starved for 6 days with 0H medium and 5% calf serum which did not contain hormones and were then treated with TSH 1 mU/mL (A), forskolin 10 µM (B), and insulin 10 µg/mL (C). The samples were subjected to SDS-PAGE and transferred to a nitrocellulose membrane. The protein levels of Id2 were detected using a specific antibody. β-actin was used to monitor protein loading. The phosphorylation of S6 ribosomal protein (S6P) reflects the activity of S6 kinase, which is activated by TSH, forskolin, and insulin in thyroid cells. The results are representative of a minimum of three independent experiments.

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  Fig. 3 TSH-induced Id2 degradation by the proteosomal pathway. A, B FRTL-5 thyroid cells were grown to near confluency in Coon's modified Ham's F-12 medium containing 5% (v/v) calf serum. The cells were starved for 6 days with 0H medium and 5% calf serum which did not contain hormones. The cells were pretreated without or with MG132 (10 µM) for 2 hr, incubated for an additional indicated time with TSH (1 mU/mL) (A) or forskolin (10 µM) (B). Cell lysates were separated by SDS-PAGE and transferred to a nitrocellulose membrane. The protein levels of Id2 were detected using an Id2-specific antibody. β-actin was used to monitor protein loading. The results are representative of a minimum of three independent experiments.

• 

  Fig. 4 Involvement of PI3 kinase in TSH-induced Id2 proteosomal degradation. FRTL-5 thyroid cells were grown to near confluency in Coon's modified Ham's F-12 medium containing 5% (v/v) calf serum. The cells were starved for 6 days with 0H medium and 5% calf serum which did not contain hormones. (A, B) The cells were pretreated with PI3 kinase inhibitors LY294002 (500 nM) (A) or wortmannin (100 nM) (B) for 2 hr prior to being exposed to TSH (1 mU) or forskolin (10 µM). (C) Cells were pretreated with rapamycin (20 nM) for 2 hr and again were exposed to TSH (1 mU) or forskolin (10 µM) for 30 min. (D) Cells were pretreated with H89 (50 µM) for 2 hr and then incubated with TSH (1 mU) or forskolin (10 µM) for an additional 2 hr before being lysed. Cell lysates were separated by SDS-PAGE and transferred to a nitrocellulose membrane. The protein levels of Id2 were detected using an Id2-specific antibody. β-actin was used to monitor protein loading. LY, LY294002 (500 nM); W, Wortmannin (100 nM); R, Rapamycin (20 nM); H89 (50 µM). The results are representative of a minimum of three independent experiments.

• 

  Fig. 5 TSH-induced Id2 promoter activity does not require the Myc binding element. (A) Wild-type and deletion constructs of the Id2 promoter. (B) FRTL-5 thyroid cells were grown to near confluency in Coon's modified Ham's F-12 medium containing 5% (v/v) calf serum. FRTL-5 cells were starved for 6 days with 0H medium and 5% calf serum which did not contain hormones. The cells were transiently transfected with wild-type or deletion mutant reporter plasmids following which they were treated with TSH (1 mU/mL) for 12 hr and assayed for luciferase activity as described in Materials and Methods. The fold-induction was calculated by dividing the luciferase activity of the TSH-treated samples by the luciferase activity of the medium-treated sample. The results are represented as the mean ±SD from at least three experiments.

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  Fig. 6 Transactivation of Id family members by TSH in cultured FRTL-5 cells. FRTL-5 cells were starved for 6 days with 0H medium and 5% calf serum which did not contain hormones. pBind-1, pBind-Id2, pBind-Id3, or pBind-Id4 (400 ng) were transiently cotransfected with 400 ng of the reporter plasmid pG5-tk-luc into FRTL-5 cells. Cells were cultured in the presence or absence of TSH (1 mU/mL) for 12-24 hr and collected for luciferase assay. The fold-inductions are expressed relative to luciferase activity obtained after cotransfection of the GAL-empty vector, arbitrarily set at 1. The results are represented as the mean±SD from at least three experiments.

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  Fig. 7 Effect of overexpression of Id2 on thyroglobulin promoter activity. (A) After FRTL-5 cells were grown to near confluency in complet e 6H medium with 5% serum, the cells were starved for 6 days with 0H medium and serum. The -808 Tg-PGL3, -688 Tg-PGL3, and -207 Tg-PGL3 plasmids were co-transfected into FRTL-5 cells with the expression vector encoding pCDNA3-Id2. After transfection, the cells were cultured in 6H medium with 5% serum for 12 hr, and were subjected to luciferase assay. (B, C) Cos7 cells were transiently transfected with -808 Tg-PGL3, the expression vector encoding pRc/CMV-TTF-1 (B), or pRc/CMV-Pax8 (C) and increasing concentrations of Id2 as indicated. Cells were cultured in DMEM containing 10% FBS for 12-24 hr and assayed for luciferase activity as described in Materials and Methods. The fold-inductions are expressed relative to luciferase activity obtained after transfection of the -808 Tg-PGL3 vector, arbitrarily set at 1. The results are represented as the mean±SD from at least three experiments.

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