A translational rheostat for RFLAT-1 regulates RANTES expression in T lymphocytes

Abstract

Activation of T lymphocytes by specific antigen triggers a 3- to 7-day maturation process. Terminal differentiation begins late after T cell activation and involves expression of effector genes, including the chemokine RANTES and its major transcriptional regulator, RANTES factor of late-activated T lymphocytes-1 (RFLAT-1). In this article we demonstrate that RFLAT-1 expression is translationally regulated through its 5'-UTR and in a cell type-specific manner. Overexpression of the translation initiation factor eIF4E increases RFLAT-1 protein, while inhibition of Mnk1, which phosphorylates eIF4E, reduces RFLAT-1 production, indicating cap-dependent translational regulation. These events are regulated by ERK-1/2 and p38 MAP kinases and allow T cells to rapidly adjust RANTES expression in response to changes in the cellular environment, such as stress and/or growth factors. These findings provide a molecular mechanism for a rheostat effect of increasing or decreasing RANTES expression at sites of inflammation. Memory T cells, already poised to make RANTES, are finely regulated by translational control of the major transcription factor regulating RANTES expression. This is the first example of such a mechanism regulating a chemokine, but it seems likely that this will prove to be a general way for cells to rapidly respond to stress, cytokines, and other proinflammatory factors in their local environment.

Figure 1

RFLAT-1 5′-UTR inhibits translation in vitro and in vivo. (a) Absorbance profile (254 nm) of sucrose gradients from lysates of resting (D0) or PHA-activated (D1 and D5) PBLs. (b) Nine fractions were collected from each gradient, and equal volumes of each fraction were separated on an agarose gel and analyzed by Northern hybridization with an RFLAT-1 probe. 18S and 28S rRNA in each fraction were visualized by ethidium bromide staining. (c) RFLAT-1 expression constructs. (full-length [RF] or lacking the 5′-UTR [ΔU]) were subjected to in vitro transcription-translation in the presence of ³⁵S methionine. The products were detected by autoradiography. (d–g) pcDNA3.1 Luc (Luc) or pcDNA 3.1 5′-UTR Luc (5′-UTR Luc) were subjected to in vitro transcription-translation (d) or transiently transfected into Jurkat T cells (e and g) or HEK293 cells (f) and assayed for luciferase activity. The data are presented as relative luciferase activity where the activity of the 5′-UTR Luc construct is arbitrarily set to 1. *P < 0.05 (d, e, and f). Decreasing amounts of lysates from transfected Jurkat cells were subjected to an RPA (g) using luciferase (upper panel) and actin riboprobes (bottom panel). Lanes 2 and 5, 30 μl; 3 and 6, 15 μl; 4 and 7, 7.5 μl lysate.

Figure 2

Effect of uORFs on RFLAT-1 translation efficiency. (a) Sequence alignment of the 5′-UTR of human and mouse RFLAT-1. The positions of the uAUGs and the first methionine are depicted (shaded box). Underlined are the stop codons for each of the three uORFs. Clear boxes indicate identical nucleotides; dashed lines, missing nucleotides. (b) RFLAT-1 5′-UTR uORF’s, designated 1, 2, and 3, are schematically represented. (c and d) The 5′-UTR Luc (wild-type or mutant) was subjected to in vitro translation (c) or transiently transfected into Jurkat T cells (d) as in Figure 1.

Figure 3

The eIF4E overexpression increases RFLAT-1 protein levels. (a and b) PHA-activated PBLs were lysed at days 0, 1, 2, 3, and 5. Western blot analysis with anti–RFLAT-1 and anti–eIF4E Ab was performed. Relative amount of eIF4E protein was determined by densitometry followed by normalizing to the amount of α-actinin in the lane. Results are representative of three independent experiments (b). (c and d) S2-6 and S2-6-4E cells were transiently transfected with 1 μg of RFLAT-1 expression constructs in the presence of 1 μg pCMV β-gal. eIF4E was induced 48 hours after transfection, and the cells were harvested 24 hours later. Samples were subjected to Western blot analysis with anti–RFLAT-1 and anti-eIF4E Ab. C, no DNA transfection control. The amount of RFLAT-1 protein was determined by densitometry and normalized to Hsc70 in the lane and to the β-galactosidase activity of the sample (d). Results are representative of two independent experiments. *P < 0.05.

Figure 4

Rapamycin inhibits RFLAT-1 protein expression. (a and b) A CD8⁺ cell line was stimulated with rIL-2 and cultured with or without rapamycin for 0, 2, 4, and 6 hours. Lysates were subjected to Western blot analysis with anti–RFLAT-1 Ab, anti–phospho-4EBP-1 as control for the effect of rapamycin, and anti-Hsc70. The relative amount of RFLAT-1 protein in each sample was determined as above. Results are representative of two independent experiments. *P < 0.05. Rap, rapamycin.

Figure 5

RFLAT-1 expression is regulated by MAP kinases and Mnk1. (a) PHA-activated PBLs were treated with medium (C), 4 μM SB203580 (SB), 4 μM SB202474 (SB-C), 10 μM PD98058 (PD), or both SB203580 and PD98058 (SB+PD). Western blots with anti–RFLAT-1, anti–ERK-1/2, and phospho-specific anti–ERK-1/2 Ab were performed. (b and c) Jurkat cells were electroporated with 5 μg of the RFLAT-1 expression vector alone or in combination with 10 μg of constructs expressing Mnk1. Western blots were probed with anti–RFLAT-1, anti–FLAG M2, and anti-Hsc70, and the amount of RFLAT-1 in the samples was determined as above. C, no DNA control. Results are representative of two independent experiments. *P < 0.05.

Figure 6

IL-2 induces RANTES and RFLAT-1 expression. (a) IL-2–dependent T cells were stimulated with 1,000 U/ml rIL-2, and intracellular RANTES was measured by FACS. Dotted line, no stimulation; solid line, 4 hours after stimulation. (b and c) Aliquots of the cells were lysed and subjected to Western blot analysis using anti–RFLAT-1, anti-eIF4E, and anti-Hsc70 Ab. Results are representative of two independent experiments. *P < 0.05.