J Korean Endocr Soc.  2007 Feb;22(1):35-44. 10.3803/jkes.2007.22.1.35.

Effects of Wnt-1 on the Growth and Apoptosis of FRTL-5 Cells

• Kim JM
• Kim TY
• Shong YK
• Rhee YS
• Park EJ
• Choi HC
• Kim WB

• KMID: 2178240
• DOI: http://doi.org/10.3803/jkes.2007.22.1.35

Abstract

BACKGROUND: Wnt proteins are major signaling molecules involved in embryonic induction, generation of cell polarity and the cell fate decision. A central player in the Wnt signaling pathways is beta-catenin. Several studies have suggested that the Wnt/beta-catenin signaling pathway may be involved in the physiologic/pathologic control of thyroid cell growth and function.
METHODS
We investigated the effect of thyroid-stimulating hormone (TSH) on the expression of Wnt proteins in FRTL-5 cells. To evaluate the effect of Wnt-1 on FRTL-5 cells growth, we isolated a stable cell line that overexpressed Wnt-1 (W1), and a vector-transfected cell clone (V3) was used as a control. We investigated the differences in the cellular growth rate, the cell cycle and cell apoptosis in the W1 and V3 cell lines.
RESULTS
TSH caused a significant increase in the Wnt-1 level and a pronounced decrease in both the active and total beta-catenin levels in the FRTL-5 cells. The growth rate, the percentage of cells in the S/G2/M phase and the c-myc level were significantly higher in the W1 cells compared with the V3 cells. There was no change in the beta-catenin level and the cyclin D1 level in the W1 cells compared with the V3 cells. The cellular apoptosis induced by actinomycin-D seemed to be significantly decreased because the level of bcl-2 was increased in the W1 cells compared with the V3 cells.
CONCLUSION
The FRTL-5 cells expressed Wnt-1 protein, and TSH increased the Wnt-1 expression, and it paradoxically decreased beta-catenin in the FRTL-5 cells. Overexpression of Wnt-1 in the FRTL-5 cells increased cell growth and it decreased apoptosis. Growth stimulation by Wnt-1 overexpression was not mediated by beta-catenin (the canonical Wnt pathway), but seemed to be mediated by activation of the Wnt/Ca2+ pathway, which involves an increased c-myc level. Suppression of apoptosis with Wnt-1 overexpression was due to the increased bcl-2 level.

Keyword

Beta-catenin; Thyroid; Wnt-1 protein

MeSH Terms

Apoptosis*
beta Catenin
Cell Cycle
Cell Line
Cell Polarity
Clone Cells
Cyclin D1
Embryonic Induction
Thyroid Gland
Thyrotropin
Wnt Proteins
Wnt Signaling Pathway
Wnt1 Protein
Cyclin D1
Thyrotropin
Wnt Proteins
Wnt1 Protein
beta Catenin

Figure

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  Fig. 1 Wnt-1 expression in FRTL-5 cells being increased by TSH. FRTL-5 cells were grown in the absence (5H media, '5H') or presence (6H media, '6H') of TSH for 5 days. A, Total cellular RNA was subjected to Northern blot analyses using Wnt-1 probe. B, 20 µg of cell lysates were subjected to SDS-PAGE and Western blot analysis using Wnt-1 antibody. MCF7, a breast cancer cell line known to over-produce Wnt-1 protein, was used as positive control.

• 

  Fig. 2 Total and active beta-catenin level in FRTL-5 cells being affected by TSH. A, Lysates of FRTL-5 cells transfected with Wnt-1, Wnt-3a or Wnt-5a expression vectors were subjected to SDS-PAGE and Western blot analysis using antibody against 'active beta-catenin'. B, Lysates of FRTL-5 cells grown in the absence (5H media, '5H') or presence (6H media, '6H') of TSH were subjected to SDS-PAGE and Western blot analysis using antibody against total beta-catenin. C, Lysates of FRTL-5 cells grown in the absence (5H media, '5H') or presence (6H media, '6H') of TSH were subjected to SDS-PAGE and Western blot analysis using antibody against active beta-catenin. Arrows indicate target protein (total or active beta-catenin).

• 

  Fig. 3 The growth rate of FRTL-5 cells stably transfected with Wnt-1. Growth rates of FRTL-5 cells stably transfected with Wnt-1 expression vector (W1) or control vector (V3) were compared at indicated time points using MTT assay. Background absorbance (cell culture medium only) was subtracted to obtain actual absorbance. Absorbance values were plotted relative to baseline absorbance (i.e., absorbance at day 0). All values are mean ± standard deviation.

• 

  Fig. 4 Cell cycle analysis in stable transfectant overexpressing Wnt-1. Cell cycle analysis of Wnt-1 overexpressing cells (W1) and vector-transfected cells (V3) was performed using flow cytometry. DNA content of cells stained with propidium iodide was measured and analyzed. Cell cycle distribution of V3 clone grown in 6H media (A), maintained in 5H media for 6 days (B), W1 clone grown in 6H media (C), and maintained in 5H media for 6 days (D) are shown. M1 indicates cells in G1 phase and M2 indicates cells in G2/S/M phase.

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  Fig. 5 Beta-catenin level in Wnt-1 transfected FRTL-5 cells. 20 µg of cell lysates of Wnt-1 transfected (W1) or vector-transfected (V3) FRTL-5 cells and FRTL-5 cells grown in the presence of TSH (6H media) were subjected to SDS-PAGE and Western blot analysis using antibodies against 'active beta-catenin' and 'total beta-catenin'.

• 

  Fig. 6 Effect of staurosporin, a PKC inhibitor, on the growth rate of FRTL-5 cells stably trasfected with Wnt-1 or control vector. Growth rates of FRTL-5 cells stably transfected with Wnt-1 expression vector (W1) or control vector (V3) were compared in the presence of staurosporin or control (DMSO) using MTT assay. Background absorbance (cell culture medium only) was subtracted to obtain actual absorbance. Absorbance values at 72 hours were plotted relative to baseline absorbance (i.e., absorbance at day 0). All values are mean ± standard deviation of 6 independent wells. * indicates P< 0.05 compared to V3 cells treated with the same concentration of staurosporin.

• 

  Fig. 7 Protein expressions of c-myc, cyclin D, and actin of Wnt-1 transfected (W1) and vector-transfected (V3) FRTL-5 cells. Expressions of c-myc, cyclin D1, and actin of W1 and V3 were measured by Western blot analysis.

• 

  Fig. 8 Effects of actinomycin-D on apoptosis of Wnt-1 transfected (W1) and vector-transfected (V3) FRTL-5 cells. Apoptosis was measured by Cell death detection ELISA plus kit (Roche Diagnostics). W1 and V3 were treated by 0, 200, and 400 µM of actinomycin-D for 48 hours, respectively.

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  Fig. 9 Protein expressions of bcl-2 and actin of Wnt-1 transfected (W1) and vector-transfected (V3) FRTL-5 cells. Expressions of bcl-2 and actin of W1 and V3 were measured by Western blot analysis.

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