GRP78 upregulation by atheroprone shear stress via p38-, alpha2beta1-dependent mechanism in endothelial cells

Abstract

Objective: The initiation of atherosclerosis is in part dependent on the hemodynamic shear stress environment promoting a proinflammatory phenotype of the endothelium. Previous studies demonstrated increased expression of ER stress protein and unfolded protein response (UPR) regulator, GRP78, within all vascular cells in atherosclerotic lesions and its regulation in the endothelium by several atherosclerotic stressors; however, regulation of GRP78 by shear stress directly has not been established.

Method and results: Using an in vitro model to simulate human arterial shear stress waveforms, atheroprone or atheroprotective flow was applied to human endothelial cells. GRP78 was found to be significantly upregulated (3-fold) in a sustained manner under atheroprone, but not atheroprotective flow up to 24 hours. This response was dependent on both sustained activation of p38, as well integrin alpha2beta1. Increased GRP78 correlated with the activation of the ER stress sensing element (ERSE1) promoter by atheroprone flow as a marker of the UPR. Shear stress regulated GRP78 through increased protein stability when compared to other flow regulated proteins, such as connexin-43 and vascular cell adhesion molecule (VCAM)-1. Increased endothelial expression of GRP78 was also observed in atheroprone versus atheroprotective regions of C57BL6 mice.

Conclusions: This study supports a role of the hemodynamic environment in preferentially inducing GRP78 and the UPR in atheroprone regions, before lesion development, and suggests a potential atheroprotective (ie, prosurvival), compensatory effect in response to ER stress within atherosclerotic lesions.

Figure 1. GRP78 is differentially expressed in atheroprone areas and within atherosclerotic lesions of mice aorta

Histological sections of the aorta were stained for GRP78 (red) and matrix (green/autoflorescence) in C57BL/6 (A-F) or ApoE-/- (G-H) mice. Representative images of the aortic arch of C57BL/6 mice show the inner arch (atheroprone) and the outer arch (atheroprotective) (A-D). White arrows (100× images) indicate individual ECs. Staining was compared to protected regions of the thoracic aorta (E-F). GRP78 expressed in a 20-week lesion (G) and 8 week cross-section (H) along the descending aorta (intersecting at an intercostal branch) of an ApoE-/- mouse.

Figure 2. Atheroprone shear stress differentially regulates GRP78 and activates the UPR pathway

(A) Human hemodynamic shear stress profiles from an atheroprone and atheroprotective regions were applied to EC monolayers in a cyclical manner (waveform from one cardiac cycle shown). (B) Representative blots of GRP78 protein expression at flow onset (t0) and 24-hours static time-matched (Tm), atheroprone, or atheroprotective flow normalized to total actin. (C) Changes in protein expression of GRP78 as a function of exposure to atheroprone or protective flow from 4 to 24-hours or Tm control were analyzed using densitometry (# p=.058, * p<0.05, ** p≤0.002 when compared to protective flow or T0, n=3-6). (D) Changes in the p90-ATF6 following exposure to atheroprone flow from 10 minutes to 24-hours relative to time zero (* p<0.05, ** p<0.005; n=3-8). (E) Increase in ERSE1 promoter-luciferase activity derived from either the GRP78 or CHOP genes compared to initial (t0) static controls following 6-hours of atheroprone or atheroprotective flow (normalized to mutant ERSE1 under the same conditions, * p<0.05; n=3-6). (F) Relative changes in GRP78 protein expression was measured after exposure to cytokines IL-1β (5ng/ml) or TNF-α (10ng/ml) from 1 to 24-hours (n=3).

Figure 3. Shear stress patterns differentially regulate protein stability

(A) GRP78 mRNA levels were measured by real time RT-PCR following exposure to atheroprone or atheroprotective flow from 1 to 16-hours (n=3-6). (B) ECs were preconditioned with 4-hours of atheroprone or atheroprotective flow followed by exposure to DMSO control or cycloheximide (CHX; 10ug/ml) for 2 additional hours under flow. Representative western blots for GRP78, Cx43, and VCAM1 after the combined 6-hours of flow and normalized to actin from three independent experiments; mean±se. (*p<0.05, **p<0.001)

Figure 4. Regulation of GRP78 under atheroprone flow is dependent on p38

(A) Early and (B) late transient activation of p38 phosphorylation following exposure to atheroprone or atheroprotective flow. Data reported as fold change relative to initial p38 activity and normalized to total p38. (C) Pretreatment with specific p38 inhibitor, SB202190, blocked the induction of GRP78 following 24-hours of atheroprone flow. (D) GRP78 protein expression in ECs following exposure to ER stressors DTT, thapsigarin (Tg), or tunicamycin (Tm) for 6 or 24-hours in the presence or absence of SB202190. (n=5-6, ** p<0.001, *p<0.02).

Figure 5. Upregulation of GRP78 and p38 by atheroprone flow is dependent on α2β1

R2-8C8, an α2β1 blocking antibody, blocked atheroprone flow induced increase in GRP78 after 24-hours when compared to the non-blocking control, 12F1 as seen in representative western blots (A) or quantified using densitometry (B). (C) Reduction in p38 phosphorylation following 24-hours of atheroprone flow in the presence of blocking antibody R28C8, but not the control 12F1 (* p<0.02; NS, not significant n=6-9). (D) After treatment with R2-8C8 or 12F1, GRP78 expression was measured when challenged with ER stressors DTT, thapsigarin (Tg), or tunicamycin (Tm) for 6 or 24-hours.