Transcriptional control of human antigen R by bone morphogenetic protein

Abstract

Human antigen R (HuR) is an RNA-binding protein with protective activities against cellular stress. This study considers the mechanisms by which HuR transcriptional regulation occurs in renal proximal tubule cells. Under basal conditions, HuR mRNA is expressed in two forms: one that contains a approximately 20-base 5'-untranslated region (UTR) sequence and one that contains a approximately 150-base, G+C-rich 5'-UTR that is inhibitory to translation. Recovery from cellular stresses such as thapsigargin and ATP depletion induced increased expression of the shorter, more translatable transcript and decreased expression of the longer form. Analysis of HuR upstream regions revealed sequences necessary for regulation of the shorter mRNA. Within the long, G+C-rich 5'-UTR exist multiple copies of the alternate Smad 1/5/8-binding motif GCCGnCGC. Recovery from ATP depletion induced increases in Smad 1/5/8 levels; further, gel shift and chromatin immunoprecipitation analyses demonstrated the ability of these Smads to bind to the relevant motif in the HuR 5'-UTR. Transfection of exogenous Smad 1 increased HuR mRNA expression. Finally, HuR mRNA expression driven by the Smad-binding sites was responsive to BMP-7, a protein with known protective effects against ischemic injury in kidney. These data suggest that transcriptional induction of a readily translatable HuR mRNA may be driven by a mechanism known to protect the kidney from injury and provides a novel pathway through which administration of BMP-7 may attenuate renal damage.

FIGURE 1.

Alternate transcriptional starts of HuR mRNA. A, effects of thapsigargin treatment on HuR mRNA expression were assessed by RPA. Lane 1, molecular size markers; lane 2, probe + yeast tRNA, undigested; lane 3, probe + yeast tRNA, digested; lane 4, mRNA from untreated cells; lane 5, mRNA from cells treated with thapsigargin for 1 h; lane 6, mRNA from cells treated with thapsigargin for 1 h and allowed to recover for 6 h. B, effects of ATP depletion/recovery on HuR mRNA as assessed by RPA. Lane 1, mRNA from untreated cells; lane 2, mRNA from cells ATP depleted for 2 h and allowed to recover for 6 h. C, graphical representation of increases in the smaller HuR transcript relative to the longer mRNA, as demonstrated by RPA analysis.

FIGURE 2.

Characterization of the HuR promoter and 5′-UTR. A, transcriptional activation was examined using promoter constructs containing various deletions of upstream sequences as well as deletion of the 5′-UTR. B, stable cell lines were cultured in normal growth medium or ATP depletion medium with or without recovery for 4 h. Total RNA was isolated and subjected to competitive RT-PCR for quantification of luciferase mRNA. The asterisks indicate statistical significance as determined by Student's t test (p < 0.05). C, murine and porcine versions of the HuR 5′-UTR show multiple copies of the Smad 1/5/8-binding site, GCCGnCGC. The solid boxes indicate perfect matches; the dashed boxes indicate motifs with one nucleotide variation.

FIGURE 3.

Distribution and expression of Smads 1/5/8 during ATP depletion and recovery. A, LLC-PK₁ cells were cultured in normal growth medium or ATP depletion medium for 2 h or allowed to recover for an additional 2 or 4 h. Immunocytochemical localization of HuR, total R-Smads, and pSmad 1/5 was determined. B, total R-Smad, Smad 1/5/8, and pSmad 1/5 were assayed by Western analysis. β-Actin was included as a loading control. Bands from multiple Western blots as shown were quantified and expressed in graphical form. C, gel mobility shift assays and ChIP were performed on nuclear extracts from untreated (unt) or ATP-depleted/recovered (rec) LLC-PK₁ cells. In gel mobility shift assays (top), one major band (designated by the arrow) was found to specifically bind Smad 1/5/8 consensus motifs in the porcine HuR 5′-UTR. The intensity of this band increased following ATP depletion/recovery. In ChIP assays (bottom panel), antibody against Smad 1/5/8 precipitated DNA corresponding to the HuR 5′-UTR. D, introduction of exogenous Smad 1 into LLC-PK₁ cells increased HuR mRNA expression, as determined by competitive RT-PCR. An empty vector control produced no effect. The asterisks indicate p < 0.005, as determined by Student's t test.

FIGURE 4.

BMP-7 induced Smad 1/5/8 activity during recovery from ATP depletion. A, immunolocalization of BMP-7 in LLC-PK₁ cells grown in normal medium or subjected to ATP depletion/recovery. B, ELISA of conditioned medium from normal LLC-PK₁ cells or cells subjected to 2 h ATP depletion alone or followed by recovery in normal growth media. The asterisk shows statistically a significant difference from 0 h control (p < 0.05). C, left panel, addition of exogenous BMP-7 to LLC-PK₁ cells induced a shift in HuR mRNA expression, as assessed by RPA. Lane 1, probe + yeast tRNA, undigested; lane 2, probe + yeast tRNA, digested; lane 3, mRNA from untreated cells; lane 4, mRNA from cells treated with BMP-7 in 0.2% fetal bovine serum for 6 h; lane 5, mRNA from cells ATP depleted for 2 h and then treated with BMP-7 in 0.2% serum; lane 6, mRNA from cells cultured for 6 h in 0.2% serum alone. Right panel, the relative increase in the smaller HuR transcript relative to the longer mRNA under ATP depletion/recovery or BMP-7 was quantified and graphed. D, treatment of LLC-PK₁ cells with exogenous BMP-7 induced luciferase expression in cells stably expressing constructs containing Smad 1/5/8-binding sites. The asterisks show statistically significant differences (p < 0.05). E, ATP depletion and recovery induced expression of ALK2 mRNA (left panel) and protein (right panel). A β-actin immunoblot is included as a loading control.