HLA-B35 upregulates endothelin-1 and downregulates endothelial nitric oxide synthase via endoplasmic reticulum stress response in endothelial cells

Abstract

The presence of the HLA-B35 allele has emerged as an important risk factor for the development of isolated pulmonary hypertension in patients with scleroderma, however the mechanisms underlying this association have not been fully elucidated. The goal of our study was to determine the molecular mechanisms that mediate the biological effects of HLA-B35 in endothelial cells (ECs). Our data demonstrate that HLA-B35 expression at physiological levels via adenoviral vector resulted in significantly increased endothelin-1 (ET-1) and a significantly decreased endothelial NO synthase (eNOS), mRNA, and protein levels. Furthermore, HLA-B35 greatly upregulated expression of chaperones, including heat shock proteins (HSPs) HSP70 (HSPA1A and HSPA1B) and HSP40 (DNAJB1 and DNAJB9), suggesting that HLA-B35 induces the endoplasmic reticulum (ER) stress and unfolded protein response in ECs. Examination of selected mediators of the unfolded protein response, including H chain binding protein (BiP; GRP78), C/Ebp homologous protein (CHOP; GADD153), endoplasmic reticulum oxidase, and protein disulfide isomerase has revealed a consistent increase of BiP expression levels. Accordingly, thapsigargin, a known ER stress inducer, stimulated ET-1 mRNA and protein levels in ECs. This study suggests that HLA-B35 could contribute to EC dysfunction via ER stress-mediated induction of ET-1 in patients with pulmonary hypertension.

FIGURE 1

Upregulation of PPET1 and dowregulation of eNOS mRNA after HLA-B35 (and HLA-B8) over-expression in ECV304 (A), HUVEC (B), and HDMEC (C) cells. In the top panel, we show the dose of Ad that expresses HLA-B35 at the physiological levels corresponding to those found in HLA-B35+ individual (gray column) in ECV304 (A), HUVECs (B), and HDMECs (C). Confluent dishes of ECs were transduced with 10 and 15 MOI (ECV304) or 5 and 10 MOI (HUVECs and HDMECs) of Ad encoding HLA-B35 and HLA-B8 for 48 h. Total RNA was extracted and mRNA levels of PPET1 (middle row) and eNOS (bottom row) were quantified by quantitative RT-PCR. Expression of the housekeeping gene β-actin served as an internal positive control in each assay performed. After measurement of the relative fluorescence intensity for each sample, the amount of each mRNA transcript was expressed as a threshold cycle value. *p = 0.05; **p = 0.001 Ad-B35/GFP (black column) versus Ad-B8/GFP (white column).

FIGURE 2

Expression of ET-1 and eNOS protein levels in ECV304 (A), HUVEC (B), and HDMEC (C) cells transduced with HLA-B35 or HLA-B8 Ads. ECs were transduced with 10 and 15 MOI (ECV304) or 5 and 10 MOI (HUVECs and HDMECs) of Ad-B35/GFP or Ad-B8/GFP for 48 h; 20 μg of total cellular proteins were separated via 15% SDS-PAGE for ET-1 (7.5% for eNOS) and transferred to a nitrocellulose membrane. The blots were probed overnight with primary Abs at 4°C. As a control for equal protein loading, membranes were stripped and reprobed for β-actin using a mAb to β-actin. Representative blots of at least three experiments are shown.

FIGURE 3

Expression of HSPs in ECV304 (A), HUVEC (B), and HDMEC (C) cells transduced with HLA-B35 (HLA-B8) Ads. Total RNA was isolated from ECs transduced with 10 and 15 MOI (ECV304) or 5 and 10 MOI (HUVECs and HDMECs) of Ad-B35/GFP or Ad-B8/GFP for 48 h. Quantitative RT-PCR was performed with SYBR Green and β-actin as an internal control. *p = 0.05; **p = 0.001 Ad-B35/GFP (black column) versus Ad-B8/GFP (white column).

FIGURE 4

Expression of HSP70 and HSP40 protein levels in ECV304 (A), HUVEC (B), and HDMEC (C) cells transduced with HLA-B35 (HLA-B8) Ads. The 30 μg total cellular proteins were separated via 10% SDS-PAGE and transferred to a nitrocellulose membrane. The blots were probed overnight with 1:1000 dilutions of primary Abs in 3% milk/Tween-Tris buffered saline at 4°C. As a control for equal protein loading, membranes were stripped and reprobed for β-actin using a mAb to β-actin. Representative blots of at least three experiments are shown.

FIGURE 5

Expression of UPR genes in ECV304 (A), HUVEC (B), and HDMEC (C) cells transduced with HLA-B35 (HLA-B8) Ads. Total RNA was isolated from ECs transduced with 10–15 (ECV304) or 5–10 (HUVECs and HDMECs) MOI of Ad-B35/GFP (Ad-B8/GFP) after 48 h. Quantitative RT-PCR was performed with SYBR Green and β-actin as an internal control. *p = 0.05 Ad-B35/GFP (black column) versus Ad-B8/GFP (white column).

FIGURE 6

Expression of BiP and CHOP protein levels in ECV304 (A), HUVEC (B), and HDMEC (C) cells transduced with HLA-B35 (HLA-B8) Ads. The 30 μg total cellular proteins were separated via 10% SDS-PAGE and transferred to a nitrocellulose membrane. The blots were probed overnight with 1:1000 dilutions of primary Abs in 3% milk/Tween-Tris buffered saline at 4°C. As a control for equal protein loading, membranes were stripped and reprobed for β-actin using a mAb to β-actin. Representative blots of at least three experiments are shown.

FIGURE 7

Upregulation of ET-1 and HSPA1A in ECV304 (A) and HDMEC (B) cells after TG treatment, total RNA was isolated from ECV304 and HDMEC cells and treated with TG for 24 h (1–5– 10 pM). Quantitative RT-PCR was performed with SYBR Green and β-actin as an internal control. The 30 μg total cellular proteins were separated via SDS-PAGE and transferred to a nitrocellulose membrane. The blots were probed overnight with primary Abs at 4°C. As a control for equal protein loading, membranes were stripped and reprobed for β-actin using a mAb to β-actin. *p = 0.05 cells after TG treatment versus untreated cells.