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. 2012 Feb;180(2):650-60.
doi: 10.1016/j.ajpath.2011.10.009. Epub 2011 Nov 24.

TGF-β promotes proliferation of thyroid epithelial cells in IFN-γ(-/-) mice by down-regulation of p21 and p27 via AKT pathway

Yujiang Fang  1 , Shiguang Yu, Helen Braley-Mullen
Affiliations

TGF-β promotes proliferation of thyroid epithelial cells in IFN-γ(-/-) mice by down-regulation of p21 and p27 via AKT pathway

Yujiang Fang et al. Am J Pathol. 2012 Feb.

Abstract

IFN-γ(-/-) NOD.H-2h4 mice develop an autoimmune disease characterized by hyperplasia and proliferation of thyroid epithelial cells (TEC H/P). Proliferating TECs produce TGF-β, and IFN-γ inhibits TEC H/P. In the present study, cultured TECs were used to directly determine the mechanisms by which these cytokines act on TECs to result in proliferation or inhibition of proliferation. With TECs from IFN-γ(-/-) NOD.H-2h4 mice or mice expressing the dominant negative TGF-β type II receptor on TECs, TGF-β was shown to promote TEC proliferation and IFN-γ was shown to inhibit TEC proliferation in vitro. TGF-β may promote TEC proliferation by down-regulating antiproliferative molecules p21 and p27, whereas IFN-γ may inhibit proliferation by up-regulating antiproliferative molecules p18 and p21 and down-regulating the pro-proliferative molecule cyclin D. Inhibition of AKT abolished the effect of TGF-β on p21 and p27, resulting in similar proliferation of TGF-β-treated and control TECs. Increased expression of proliferating cell nuclear antigen (PCNA), TGF-β, and p-AKT and decreased expression of p21 and p27 by proliferating TECs correlated with the proliferative state of TEC H/P. Taken together, the results suggest that TGF-β promotes TEC proliferation by down-regulating p21 and p27 via the AKT pathway in IFN-γ(-/-) NOD.H-2h4 mice, which may have significant implications for development of effective therapeutic strategies targeting the TGF-β and AKT pathways for treatment of hyperplasia and/or neoplasia.

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Figures

Figure 1
Figure 1
Effect of TGF-β and IFN-γ on TEC proliferation evaluated by IHC. Shown are IHC staining results for PCNA of cultured TECs from IFN-γ−/− (GKO) Tg− and Tg+ (dnTβRII) mice cultured with the indicated amounts of TGF-β (A) and results for PCNA of cultured TECs from GKO and IFN-γR−/− mice cultured with the indicated amounts of IFN-γ (B). Original magnification, ×400. C and D: PCNA+ cells (red) in five to six randomly selected high-power fields of three wells/group were counted using MetaMorph software. Summarized results are shown; 0.5 or 2 ng/mL TGF-β induced significant proliferation of TECs from Tg− mice but not from Tg+ mice (*P < 0.05), and 6 or 20 ng/mL IFN-γ inhibited proliferation of TECs from IFN-γ−/− mice (P < 0.05) but had no effect on TECs of IFN-γR−/− mice. The activity of IFN-γ is 5 U/ng.
Figure 2
Figure 2
Effect of TGF-β and IFN-γ on TEC proliferation determined using a proliferation kit. A: TGF-β promotes proliferation of cultured TECs from GKO Tg− mice but not from GKO Tg+ mice. B: IFN-γ inhibits proliferation of cultured TECs from GKO but not from IFN-γR−/− mice. Results are expressed as the mean optical density (OD) ± SEM of TECs from four to five wells/group and are representative of three independent experiments. *P < 0.05 versus medium (M) alone.
Figure 3
Figure 3
Effect of TGF-β on pro- and antiproliferative molecules in cultured TECs. mRNA and proteins were extracted as described under Materials and Methods. A–E: RT-PCR. F: Western blot. Results are expressed as the mean ratio of PCNA, pro- and antiproliferative molecule densitometric units/β-actin ± SEM (×100) and are representative of two to three independent experiments. *P < 0.05 versus medium (M) alone.
Figure 4
Figure 4
IFN-γ inhibits proliferation of cultured TECs by modulating pro- and antiproliferative molecules. RT-PCR results are expressed as the mean ratio of PCNA, pro- and antiproliferative molecule densitometric units/β-actin ± SEM (×100), and are representative of three independent experiments. *P < 0.05 versus medium (M) alone.
Figure 5
Figure 5
TGF-β-induced proliferation of TECs is associated with increased p-AKT, and AKT inhibitor inhibits TGF-β-induced proliferation of TECs. Primary cultures of TECs from dnTβRII Tg+ IFN-γ−/− NOD.H-2h4 mice and their Tg− littermates were established, and TGF-β (2 ng/mL) was added for 3 days. A: IHC results for p-AKT and PCNA. Primary cultures of TECs from dnTβRII Tg+ mice and their Tg− littermates were established, and TGF-β (2 ng/mL) or medium with or without AKT inhibitor (30 μmol/L) was added for 3 days. Original magnification: ×100 (p-AKT); ×400 (PCNA). B and C: p-AKT+ or PCNA+ cells (red) in five to six randomly selected high power fields of three wells/group were counted using MetaMorph software. Summarized results are shown. *P < 0.05 versus medium alone (B). *P < 0.05 versus TGF-β alone (C). D: A cell proliferation kit was used to evaluate the effect of AKT inhibitor on TGF-β-induced proliferation of TECs. Results are expressed as the mean OD ± SEM of TECs from four to five wells/group. *P < 0.05 versus TGF-β alone. Results are representative of two or three independent experiments.
Figure 6
Figure 6
TGF-β-induced proliferation of TECs is associated with increased p-AKT and AKT inhibitor reverses the ability of TGF-β to down-regulate antiproliferative molecules A: Western blot analysis. Results are expressed as the mean ratio of p-AKT densitometric units/β-actin ± SEM (×100). *P < 0.05 versus medium alone. B–D: Primary cultures of TECs from dnTβRII Tg+ mice and their Tg− littermates were established. TGF-β (2 ng/mL) with or without AKT inhibitor (30 μmol/L) was added for 3 days. mRNA expression of p21, p27, and PCNA in cultured TECs from dnTβRII Tg+ mice and their Tg− littermates was determined by RT-PCR. Results are expressed as the mean ratio of PCNA, pro- and antiproliferative molecule densitometric units/β-actin ± SEM (×100), and are representative of three independent experiments. Results are representative of two independent experiments. *P < 0.05 versus TGF-β alone.
Figure 7
Figure 7
Increased proliferation of TECs correlates with increased expression of TGF-β and p-AKT and decreased expression of P21 and P27 by TECs in vivo. Splenocytes from IFN-γ−/− donors with 5+ TEC H/P severity scores were cultured with mouse thyroglobulin and were transferred to SCID recipient mice. A: Results of H&E staining and IHC staining for PCNA, TGF-β, p-AKT, p21, and p27 in thyroids at 28 and 60 days after cell transfer. Original magnification, ×400. B–D: PCNA+ cells (red) or relative staining intensity in five to six randomly selected high-power fields of three slides/group were counted or analyzed using MetaMorph software. Summarized results are shown. *P < 0.05 versus day 28. E: Protein expression levels of p-AKT, p21, and p27 evaluated by Western blot. *P < 0.05 versus day 28. Results shown are representative of two or three independent experiments.
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