HuR inhibits apoptosis by amplifying Akt signaling through a positive feedback loop

Abstract

Human antigen R (HuR) is a post-transcriptional regulator of gene expression that plays a key role in stabilizing mRNAs during cellular stress, leading to enhanced survival. HuR expression is tightly regulated through multiple transcription and post-transcriptional controls. Although HuR is known to stabilize a subset of mRNAs involved in cell survival, its role in the survival pathway of PI3-kinase/Akt signaling is unclear. Here, we show that in renal proximal tubule cells, HuR performs a central role in cell survival by amplifying Akt signaling in a positive feedback loop. Key to this feedback loop is HuR-mediated stabilization of mRNA encoding Grb10, an adaptor protein whose expression is critical for Akt activation. Stimulation of Akt by interaction with Grb10 then activates NF-κB, which further enhances HuR mRNA and protein expression. This feedback loop is active in unstressed cells, but its effects are increased during stress. Therefore, this study demonstrates a central role for HuR in Akt signaling and reveals a mechanism by which modest changes in HuR levels below or above normal may be amplified, potentially resulting in cell death or cellular transformation.

Figure 1. HuR levels are regulated by PI3K/Akt signaling

(A) Apoptosis levels were assessed by measurements of caspase 3/7 activity in HK-2 cells treated either with vehicle or PI3K inhibitor LY-294002. *, P< 0.05; **, P<0.005. (B) Control or ATP-depleted HK-2 cells were immunolabeled with an antibody against total Akt. Arrows indicate plasma membrane distribution of Akt, which is indicative of its activation. Scale bars = 20 µm. (C) The effects of PI3K inhibition on HuR levels were measured by Western blot. β-actin was detected as a loading control.

Figure 2. HuR levels are regulated by NF-κB signaling

The effects of NF-κB inhibition on HuR expression were demonstrated by treatment of HK-2 cells with BAY11-7082. (A) HuR protein levels were assessed by Western analysis. A representative blot is shown that also detects β-actin as a loading control. (B) Competitive RT-PCR demonstrates that inhibition of NF-κB suppresses HuR mRNA levels. (C) HK-2 cells overexpressing wild-type (WT) or constitutively active (CA) Akt1 were treated with vehicle or BAY11-7082, as were untransfected controls. HuR levels were assessed by Western analysis, along with β-actin as a loading control. A Western of pAkt levels indicates Akt activation levels. Below, data from three independent experiments are represented graphically. *, P= 0.01.

Figure 3. HuR regulates Akt activation and Grb10 expression

(A) Western analysis demonstrates that ATP depletion increases HuR protein levels and Akt phosphorylation. siRNA-mediated knockdown of HuR strongly inhibits Akt activation without affecting total Akt levels. β-actin is shown as a loading control. (B) Competitive RT-PCR (top row) demonstrates that Grb10 mRNA levels increase during ATP depletion, but are diminished by HuR knockdown. Western analysis, below, shows similar results for Grb10 protein. β-actin is shown as a loading control. (C) HuR was immunoprecipitated from ATP-depleted or control cells, and associated RNA was purified. The presence of Grb10 mRNA was assessed by RT-PCR. Controls demonstrate specificity by showing that a lack of HuR antibody or reverse transcriptase (RT) in the procedure fails to produce Grb10 cDNA. (D) (top) A murine Grb10 cDNA used for overexpression is shown schematically, including the 174 bp 5’-UTR, 1626 bp coding region, and the 2954 bp 3’ UTR. For generation of a mutant cDNA, 3’ UTR sequences downstream of the XbaI site were deleted. Asterisks indicate potential regions of HuR binding, based on sequence analysis. (bottom) The role of the Grb10 3’ UTR in controlling its expression was demonstrated by both RT-PCR and Western blot. β-actin levels are included as a loading control for the Western blot. Lane 1: untransfected; lane 2: empty expression vector; lane 3: full-length murine Grb10; lane 4: murine Grb10 lacking 3’ UTR sequences; lane 5: full-length murine Grb10 + HuR; lane 6: murine Grb10 lacking 3’ UTR sequences + HuR.

Figure 4. siRNA-mediated knockdown of Grb10 increases apoptosis in ATP-depleted HK-2 cells

(A) Competitive RT-PCR demonstrates efficient knockdown of Grb10 mRNA with nanomolar levels of siRNA. (B) Western analysis demonstrates efficient knockdown of Grb10 protein during ATP depletion. β-actin levels are assessed as a loading control. (C) Caspase 3/7 assays show that knockdown of Grb10 increases apoptosis during ATP depletion. *, P < 0.05; **, P< 0.01.

Figure 5. Knockdown of Grb10 inhibits Akt activation and HuR expression

(A) Western analysis of HK-2 cells treated with control oligonucleotides or Grb10 siRNAs show that suppression of Grb10 results in strongly diminished Akt activation and HuR protein expression. (B) Competitive RT-PCR demonstrates that knockdown of Grb10 decreases HuR expression at the mRNA level. (C) Under normal growth conditions (top row), Grb10 and pAkt demonstrate almost complete overlap in the nucleus but show no discernable distribution at the plasma membrane (arrow). ATP depletion (middle row) results in movement of a subset of both proteins to the plasma membrane (arrow). SiRNA-mediated knockdown of Grb10 (bottom row) results in a marked loss of pAkt and no clear plasma membrane distribution of either protein. The periphery of a single cell is outlined and the plasma membrane of a second cell is indicated with an arrow. Scale bars = 20 µm.

Figure 6. Schematic of HuR’s role in promoting a positive feedback loop of Akt signaling

Based on data presented here and in the literature, we propose the following positive feedback loop for HuR in Akt signaling. (1) NF-κB promotes HuR expression, most likely through transcriptional control; (2) HuR binds to and stabilizes Grb10 mRNA, resulting in increased Grb10 expression; (3) Grb10 activates Akt by aiding in its transport to the plasma membrane where it is activated by PI3K; and (4) Akt activation stimulates NF-kB activity.