Enhanced proliferation of cultured human vascular smooth muscle cells linked to increased function of RNA-binding protein HuR

Abstract

In dividing cells, the RNA-binding protein HuR associates with and stabilizes labile mRNAs encoding proliferative proteins, events that are linked to the increased cytoplasmic presence of HuR. Here, assessment of HuR levels in various vascular pathologies (intimal hyperplasia, atherosclerosis and neointimal proliferation, sclerosis of arterialized saphenous venous graft, and fibromuscular dysplasia) revealed a distinct increase in HuR expression and cytoplasmic abundance within the intima and neointima layers. On the basis of these observations, we postulated a role for HuR in promoting the proliferation of vascular smooth muscle cells. To test this hypothesis directly, we investigated the expression, subcellular localization, and proliferative influence of HuR in human vascular smooth muscle cells (hVSMCs). Treatment of hVSMCs with platelet-derived growth factor increased HuR levels in the cytoplasm, thereby influencing the expression of metabolic, proliferative, and structural genes. Importantly, knockdown of HuR expression by using RNA interference caused a reduction of hVSMC proliferation, both basally and following platelet-derived growth factor treatment. We propose that HuR contributes to regulating hVSMC growth and homeostasis in pathologies associated with vascular smooth muscle proliferation.

FIG. 1.

Immunohistochemical detection of HuR in healthy and diseased vascular tissues. A, HuR expression (brown signal) was examined in the indicated vascular specimens (left) at either ×20 or ×40. Corresponding hematoxylin-eosin (HE) staining is shown (right). B, positive controls for HuR signals are shown.

FIG. 2.

Left, subcellular distribution of HuR in hVSMCs as detected by immunofluorescence at the times shown after stimulation with 10 ng/ml PDGF. Control heterogeneous nuclear ribonucleoprotein C1/C2 (hnRNP C1/C2), also detected by immunofluorescence, remained in the nucleus throughout the treatment. Right, quantitation of HuR immunofluorescent signals; data represent the means ± S.E. from three independent experiments. Significant differences (p < 0.05) were found at 3 and 6 h.

FIG. 3.

Silencing of HuR expression in hVSMCs by RNA interference. Following transfection of hVSMCs with 10 μM HuR siRNA, HuR levels were monitored by Western blot analysis. β-Actin signals served to monitor the evenness in sample loading and transfer.

FIG. 4.

Validation of HuR target mRNAs showing differential regulation by PDGF. A, reverse transcription + real-time PCR analysis of the levels of mRNAs encoding SAT, CDK2, SLC7A7, OSBLP2, and p21 (positive control). Initial normalization was performed by comparing the levels of these mRNAs with those of housekeeping gene SDHA for each treatment group (Ctrl. siRNA, HuR siRNA, each following either no treatment or PDGF stimulation). The percentage difference in mRNA abundance in PDGF-treated relative to untreated cultures was then calculated for each transfection group. The data reflect the means ± S.E. from three independent experiments. Ctrl., control. B, biotin pull-down assays using hVSMC whole-cell ly-sates (details under “Experimental Procedures”). C, Western blot of HuR in the cytoplasm of WI-38 cells that were either untreated (Unt.) or treated with PDGF for 8 h. D, biotin pull-down assay using cytoplasmic lysates from WI-38 cells that were either left untreated or were stimulated with PDGF for 8 h.

FIG. 5.

Impact of HuR knockdown on PDGF-mediated proliferation of hVSMCs. A, changes in cell numbers after incubation of cell populations in each transfection group with PDGF for 3 days. B, incorporation of [³H]thymidine for 16 h in populations expressing either wild-type (Ctrl. siRNA) or reduced (HuR siRNA) HuR levels. The data represent the means ± S.E. from three independent experiments. Unt., untreated.