Mammalian unfolded protein response inhibits cyclin D1 translation and cell-cycle progression

Abstract

Alterations in normal protein biogenesis and the resulting accumulation of improperly folded proteins in the endoplasmic reticulum (ER) trigger a stress response that up-regulates the expression of ER chaperones, while coordinately repressing overall protein synthesis and causing cell-cycle arrest. Activation of this unfolded protein response (UPR) in mouse NIH 3T3 fibroblasts with the glycosylation inhibitor tunicamycin led to a decline in cyclin D- and E-dependent kinase activities and to G(1) phase arrest. Cyclin D1 protein synthesis was rapidly inhibited by tunicamycin treatment. However, the drug did not significantly affect the mitogen-dependent activities of the extracellular signal-activated protein kinases ERK1 and ERK2 or the level of cyclin D1 mRNA until much later in the response. Therefore, the UPR triggers a signaling pathway that blocks cyclin D1 translation despite continuous mitogenic stimulation. Enforced overexpression of cyclin D1 in tunicamycin-treated cells maintained cyclin D- and E-dependent kinase activities and kept cells in cycle in the face of a fully activated UPR. Translational regulation of cyclin D1 in response to ER stress is a mechanism for checkpoint control that prevents cell-cycle progression until homeostasis is restored.

Figure 1

Loss of cyclin D1 correlates with tunicamycin-induced G₁ arrest. (A) NIH 3T3 cells treated with 0.5 μg/ml tunicamycin in complete serum-containing medium (Left) or transferred to medium containing 0.1% FCS but no drug (Right) were assayed for DNA content by flow cytometry at the indicated times. Cells with a 2N DNA content (abscissa) are in G₀/G₁, whereas those with a 4N DNA content have completed S phase and are in G₂ or M phase. The S phase fraction is represented by cells whose DNA content is between 2N and 4N. (B) Cells treated as above were lysed, and after separation of equal amounts of lysate proteins on denaturing polyacrylamide gels and transfer to nitrocellulose membranes, the indicated proteins were detected by direct immunoblotting by using cognate antibodies. All proteins were visualized by enhanced chemiluminescence, and exposures for Left and Right are matched.

Figure 2

Enforced overexpression of cyclin D1 prevents tunicamycin-induced growth arrest. (A) After treatment of parental NIH 3T3 (Left), D1-3T3 (Middle), or D1(T286A)-3T3 (Right) cells with 0.5 μg/ml tunicamycin in complete medium, cells were harvested at the indicated times and assayed for DNA content by flow cytometry. (B) NIH 3T3 (Left) and D1(T286A)-3T3 (Right) cells were treated with the indicated doses of tunicamycin and thapsigargin for 20 h and then assayed for DNA content by flow cytometry.

Figure 3

Constitutive expression of cyclin D1(T286A) prevents loss of cyclin A and CDK2 catalytic activity. (A) Parental NIH 3T3 (Left) or D1(T286A)-3T3 (Right) cells treated with 0.5 μg/ml tunicamycin in complete serum-containing medium were assayed for expression of cyclin D1, BiP, CHOP, cyclin A, and CDK2 by immunoblotting. Immune complexes recovered with antibodies to cyclin D1 (B) or to CDK2 (C) were assayed for protein kinase activity by using retinoblastoma protein or histone H1 as substrates, respectively. (D) Lysates from untreated NIH 3T3 cells (0) or those treated with tunicamycin for 20 h (20) were precipitated with antibodies to CDK2. Denatured immune complexes were then separated on gels and blotted with antibodies to p27^Kip1 or p21^Cip1 (indicated at left) to score for complexes containing the CKIs. Although cyclin A levels fell in drug-treated cells (A), the recovery of equivalent amounts of Cip/Kip proteins after tunicamycin treatment suggests that the ratio of CKIs to cyclin bound CDK2 was increased, consistent with the observed inhibition of CDK2 kinase activity (C).

Figure 4

Tunicamycin does not inhibit mitogenic signaling but blocks cyclin D1 translation. (A) After treatment of NIH 3T3 cells with tunicamycin or with medium containing 0.1% FCS, the levels of phosphorylated ERKs (Upper) were compared with the total ERK pool (Lower) by immunoblotting with antibodies that detect phosphorylated or all ERK isoforms, respectively. (B) After tunicamycin treatment of NIH 3T3 cells for the same time intervals, mRNAs encoding cyclin D1, CHOP, BiP, or actin were detected by Northern blotting performed with the cognate radiolabeled cDNA probes. (C) NIH 3T3 cells were left untreated (diamonds) or treated with 0.5 μg/ml tunicamycin for 2 h (squares), 4 h (triangles), or 12 h (circles) and then pulse-labeled for the indicated periods of time with Tran-³⁵S-label (ICN). Cyclin D1 was immunoprecipitated from lysates, separated on a denaturing gel, and the rate of cyclin D1 synthesis was quantified by scanning the autoradiographs.