AS160 controls eukaryotic cell cycle and proliferation by regulating the CDK inhibitor p21

Abstract

AS160 (TBC1D4) has been implicated in multiple biological processes. However, the role and the mechanism of action of AS160 in the regulation of cell proliferation remain unclear. In this study, we demonstrated that AS160 knockdown led to blunted cell proliferation in multiple cell types, including fibroblasts and cancer cells. The results of cell cycle analysis showed that these cells were arrested in the G1 phase. Intriguingly, this inhibition of cell proliferation and the cell cycle arrest caused by AS160 depletion were glucose independent. Moreover, AS160 silencing led to a marked upregulation of the expression of the cyclin-dependent kinase inhibitor p21. Furthermore, whereas AS160 overexpression resulted in p21 downregulation and rescued the arrested cell cycle in AS160-depeleted cells, p21 silencing rescued the inhibited cell cycle and proliferation in the cells. Thus, our results demonstrated that AS160 regulates glucose-independent eukaryotic cell proliferation through p21-dependent control of the cell cycle, and thereby revealed a molecular mechanism of AS160 modulation of cell cycle and proliferation that is of general physiological significance.

Keywords: AS160/TBC1D4; G1/S; cell cycle; cell proliferation; p21.

Figure 1.

AS160 silencing suppresses cell proliferation and induces cell cycle arrest in the G1 phase in multiple cell types. (A) Levels of the indicated protein in 3T3-L1 fibroblasts infected with a scrambled (SCR) or AS160-specific shRNA (KD) were determined by performing protein gel blotting with duplicate sample loading; β-actin served as a loading control. (B) Proliferation of cells from (A) was determined by counting cells and normalizing the numbers relative to the initial cell numbers. Data represent mean ± s.e.m. (n = 3 represents 3 replicated experiments, same below); here and below, *p < 0.05 and **p < 0.01 compared to SCR, 2-tailed t test. (C) Western blots of MCF7 cells transfected with 2 AS160 siRNAs (KD1 and KD2) or scrambled siRNA (SCR), at 48 and 72 h post-transfection. (D) Proliferation levels of MCF7 cells from (C) were determined using the MTS assay and normalized relative to the respective initial OD values. Data represent mean ± s.e.m. (n = 3). (E) Western blots of Huh7 cells transfected with 2 AS160 siRNAs (KD1 and KD2) or scrambled siRNA (SCR), at 48 and 72 h post-transfection. (F) Proliferation levels of Huh7 cells from (E) were determined using the MTS assay and normalized relative to the respective initial OD values. Data represent mean ± s.e.m. (n = 3). (G) Cell cycle analysis of SCR and K_D 3T3-L1 fibroblasts. Results represent percentages of cells in G1, S, and G2/M phases for the representative experiment (left) and mean ± s.e.m. (right, n = 3); here and below, *p < 0.05 compared to SCR, t test. (H) Cell cycle analysis of MCF7 cells transfected with 2 AS160 siRNAs (KD1 and KD2) or scrambled siRNA (SCR).

Figure 2.

Altering glucose or lactate does not rescue AS160-depletion-induced blunted cell proliferation or cell cycle arrest in G1 in 3T3-L1 fibroblasts. (A) Representative images of oil-red-stained 3T3-L1 adipocytes infected with scrambled (SCR) or AS160-specific shRNA (KD). Quantified Results represent normalized mean±s .e.m. of OD values of oil-red staining (right, n = 3 represents 3 replicated experiments, same below); here and below, NS, not significant. (B) Representative GFP and Cy3 images of 3T3-L1 SCR and KD adipocytes electroporated with the HA-GLUT4-GFP construct and immunostained with Cy3-conjugated HA antibodies in the basal state. Quantified data represent normalized Cy3/GFP fluorescence ratio (right, n = 3). (C) Glucose uptake into 3T3-L1 adipocytes from (B), determined by measuring glucose in the supernatant and the cell numbers. Data represent normalized mean ± s.e.m. (n = 3); *p < 0.05, 2-tailed t test, same below. (D) Glucose uptake into 3T3-L1 SCR and KD fibroblasts in a 4-d period of cell proliferation. Data represent normalized mean ± s.e.m. (n = 3); here and below, *p < 0.05 compare to SCR, t test. (E) Glucose uptake into 3T3-L1 SCR and KD fibroblasts exposed to the indicated glucose concentration in the culture medium. The amounts of glucose taken up into the fibroblasts were equal when 10 mM glucose was included in the medium used for SCR cells and 5 mM glucose was added in the medium for KD cells. The result represent mean ± s.e.m. (n = 4). (F) Cell cycle analysis of 3T3-L1 fibroblasts from (E). Data shown are mean ± s.e.m. (n = 4); *p < 0.05, t test. G, Lactate levels in the supernatants of 3T3-L1 SCR and KD fibroblasts in a 4-d period of cell proliferation (n = 3). H, Lactate levels in the supernatants of 3T3-L1 SCR and KD fibroblasts in a 7-d period of cell proliferation. The lactate levels were equal in the supernatants of KD and SCR cells when the SCR cell-culture medium was supplemented an extra 0.6 mM lactate (n = 3); **p < 0.01, t test. I, Proliferation of 3T3-L1 fibroblast from (H) was quantified through cell counting and normalized relative to the initial cell numbers. Results represent normalized mean ± s.e.m. (n = 3).

Figure 3.

AS160 knockdown upregulates the CDK inhibitor p21 in multiple cell types. (A) Screening of mRNA levels of AS160, p16, p19, p21, and p27 in 3T3-L1 fibroblasts infected with scrambled (SCR) or AS160-specific (KD) shRNAs, at indicated times post-plating. Data shown are normalized mean ± s.e.m. (n = 3 represents 3 replicated experiments, same blow). (B) Levels of the indicated proteins in 3T3-L1 SCR and KD fibroblasts, determined with triplicated sample loading. Tubulin severed as the loading control. (C) Western blots of MCF7, HeLa, Huh7, and HEK-293 cells transfected with 2 AS160 siRNAs (KD1 and KD2) or a scrambled siRNA (SCR), at 48 and 72 h post-transfection. (D) Protein levels of phosphorylated-Rb and AS160 in 3T3-L1 SCR and KD fibroblasts.

Figure 4.

p21 silencing or AS160 overexpression in AS160-silenced cells rescues blunted cell cycle and cell proliferation caused by AS160 knockdown. (A) Levels of indicated proteins in MCF7 cells transfected with 2 AS160 siRNAs (AS160-KD1 and AS160-KD2) and/or p21 siRNAs (p21-KD1 and p21-KD2), scrambled siRNA (SCR), or a wild-type AS160 construct (WT AS160), as indicated. (B) Quantified levels of p21 and AS160 proteins from (A); the data were averaged and normalized relative to the scramble cells, respectively (n = 3 represents 3 replicated experiments, same below); here and below, NS, not significant; 2-tailed t test, *p < 0.05, **p < 0.01 compared to AS160 level of SCR; #p < 0.05 compared to p21 level of SCR; and p < 0.05. (C) Cell cycle analysis of MCF7 cells from (A and B). Results represent mean ± s.e.m. (n = 3). (D) Quantification of the percentage of cells in G1 phase from (C); n = 3, here and below, *p < 0.05 and **p < 0.01. (E) Proliferation of SCR and KD (AS160-KD1) MCF7 cells from (A–D) was measured using the MTS assay and normalized relative to the initial OD values. Data shown are mean ± s.e.m. (n = 3). (F) Proliferation of SCR and KD (AS160-KD2) MCF7 cells from (A–D) was measured using the MTS assay and normalized relative to the initial OD values. Data shown are mean ± s.e.m. (n = 3).