Shear stress activation of nuclear receptor PXR in endothelial detoxification

Abstract

Endothelial cells (ECs) are constantly exposed to xenobiotics and endobiotics or their metabolites, which perturb EC function, as well as to shear stress, which plays a crucial role in vascular homeostasis. Pregnane X receptor (PXR) is a nuclear receptor and a key regulator of the detoxification of xeno- and endobiotics. Here we show that laminar shear stress (LSS), the atheroprotective flow, activates PXR in ECs, whereas oscillatory shear stress, the atheroprone flow, suppresses PXR. LSS activation of PXR in cultured ECs led to the increased expression of a PXR target gene, multidrug resistance 1 (MDR1). An in vivo study using rats showed that the expression of MDR1 was significantly higher in the endothelium from the descending thoracic aorta, where flow is mostly laminar, than from the inner curvature of aortic arch, where flow is disturbed. Functionally, LSS-activated PXR protects ECs from apoptosis triggered by doxorubicin via the induction of MDR1 and other detoxification genes. PXR also suppressed the expression of proinflammatory adhesion molecules and monocyte adhesion in response to TNF-α and lipopolysaccharide. Overexpression of a constitutively active PXR in rat carotid arteries potently attenuated proinflammatory responses. In addition, cDNA microarray revealed a large number of the PXR-activated endothelial genes whose products are responsible for major steps of detoxification, including phase I and II metabolizing enzymes and transporters. These detoxification genes in ECs are induced by LSS in ECs in a PXR-dependent manner. In conclusion, our results indicate that PXR represents a flow-activated detoxification system to protect ECs against damage by xeno- and endobiotics.

Keywords: endothelial homeostasis; gene regulation; hemodynamics; nuclear hormone receptor.

Fig. 1.

Shear stress regulates PXR activity in ECs. (A) BAECs were cotransfected with the PXR overexpression plasmid together with pCYP3A4XREM-362/+53, which is the plasmid for PXRE-driven luciferase reporter. Transfected cells were exposed to LSS or OSS or kept static for 18 h. Rifampicin (Rif, 20 μM) was used as a positive control. (B) MDR1 expression was analyzed by qRT-PCR in HUVECs exposed to LSS or OSS for 0, 1, 3, 6, and 24 h. (C and D) HUVECs were pretreated with SFN or DMSO for 24 h (C) or transfected with PXR siRNA or control siRNA for 48 h (D) and then exposed to laminar flow for another 6 h (C) or 24 h (D), mRNA levels of MDR1 were analyzed by qRT-PCR. (E) BAECs were transfected with the GAL4 reporter plasmid together with Gal4-PXR-LBD or Gal4-PXR-LBDmutant. Cells were then exposed to LSS or kept under static condition for 24 h. Rifampicin was used as a positive control. The results are expressed as fold change in luciferase activities compared with static control. Luciferase activity was assayed, normalized, and expressed as fold induction compared with static control. (F) MDR1 mRNA levels from intima at TA and AA were analyzed by qRT-PCR. Data are shown as mean ± SEM of three independent experiments. *P < 0.05 vs. control. ^#P < 0.05 vs. static (B) or control and with LSS treatment (C and D).

Fig. 2.

PXR activation attenuates EC apoptosis. HUVECs were pretreated by rifampicin (20 μM) or vehicle (DMSO) for 24 h before exposed to doxorubicin (A) (5 μM, 24 h) or staurosporine (B) (500 nM, 6 h). Apoptosis was assessed by using flow cytometry for annexin V-FITC. (C) Intracellular level of doxorubicin was measured by using flow cytometry for fluorescence intensity in HUVECs pretreated with rifampicin (with or without SFN) in the presence or absence of PSC833 before exposure to doxorubicin. (D) Caspase 3 cleavage was detected with Western blotting. (E) HUVECs were treated with LSS or kept static for 12 h before exposure to doxorubicin for 12 h or staurosporine for 6 h. (F) HUVECs were transfected with PXR or control siRNA for 48 h and exposed to LSS or kept static for another 12 h. Apoptosis was detected after exposure to doxorubicin or staurosporine. Data shown are as mean ± SEM of three independent experiments. *P < 0.05 vs. control. ^#P < 0.05 vs. DMSO (A and B) or Rif (C).

Fig. 3.

PXR activation ameliorates proinflammatory response in ECs. (A) HUVECs were pretreated by rifampicin (20 μM) or DMSO for 24 h before exposure to TNF-α (10 ng/mL, 4 h). Then ECs were incubated with fluorescence-labeled THP-1 cells (5 × 10⁵ cells per mL) for 30 min. THP-1 adhesions were counted under a fluorescence microscope. (B) Cells were infected with Ad-VP-PXR or mock infection before TNF-α stimulation. (C) VCAM-1 and E-selectin mRNA level was analyzed by using qRT-PCR in VP-PXR-infected or mock-infected ECs. (D) Protein levels of VCAM-1 and E-selectin were detected using Western blotting. (E and F) Surface expressions of VCAM-1 and E-selectin were examined using flow cytometry, and relative levels were quantified and expressed as bar graphs (Lower). (G) HUVECs were transfected with PXR or control siRNA and exposed 48 h later to LSS or static condition for 24 h. After stimulation with TNF-α for 2 h, RNA was extracted and analyzed by qRT-PCR. (H) Cells were stimulated with TNF-α for 4 h and incubated with THP-1 cells. THP-1 adhesions were counted; representative images are shown. Data are shown as mean ± SEM of three independent experiments. *P < 0.05 vs. control. ^#P < 0.05, Rif vs. DMSO (A) or VP-PXR vs. mock (B).

Fig. 4.

PXR inhibits NF-κB and vascular inflammation. (A) BAECs were cotransfected with a reporter plasmid NF-κB×5-luc together with a VP-PXR expression plasmid or vector control (pcDNA3.1). Data are shown as mean ± SEM of three independent experiments. *P < 0.05 vs. control. (B) Protein levels of p65 and IκBα in the nucleus and cytosol were analyzed using Western blotting in VP-PXR or mock-infected HUVECs after exposed to TNF-α. Histone and β-actin were used as internal controls for nuclear and cytosolic protein, respectively. (C) Rat carotid arteries were infected with Ad-VP-PXR or Ad-LacZ. Immunofluorescence staining shows overexpression of VP-PXR in endothelium 24 h after the infection. Expression of VCAM-1 and E-selectin after LPS challenge for 24 h was detected with en face staining. Nuclei were counterstained with DAPI. Microphotographs are representative of 3 rats in each group.

Fig. 5.

Gene profile of flow-induced PXR targets in ECs. (A) Heat map of the detoxification-related genes in VP-PXR-infected or mock-infected ECs. (B) PXR up-regulated genes were validated with the use of qRT-PCR in HUVECs. (C–E) Relative mRNA levels of selected PXR targets in ECs exposed to LSS for 6 h with the pretreatment of SFN or DMSO. (F) Relative mRNA levels of the representative PXR targets in mouse AA and TA. Data are shown as mean ± SEM of three independent experiments. *P < 0.05 vs. control. ^#P < 0.05 vs. DMSO and with LSS treatment.