StarD5 Plays a Critical Role in the Hepatocyte ER Stress Survival Response

Abstract

The unfolded protein response (UPR) is a highly orchestrated survival response initiated in cells under endoplasmic reticulum (ER) stress. Steroidogenic acute regulatory-related lipid transfer domain 5 (StarD5) is an ER stress-responsive, cholesterol-binding protein under the regulation of IRE1. Based upon in vitro findings, StarD5 delivers a protective response by translocating ER cholesterol to the plasma membrane (PM) and accompanying protective changes in PM fluidity. The study aimed to determine if StarD5's ability to provide in vitro hepatocyte protective responses is translatable to in vivo conditions. ER stress in mouse livers was induced by intraperitoneal injection of tunicamycin (Tm). Adenovirus was used to restore the expression of StarD5 in the livers of StarD5^-/- mice. Immunoblotting, histological analysis, and lipid measurements were performed. Induction of ER stress led to increased expression of StarD5 and steatosis in the livers of wild-type (WT) mice, while in StarD5^-/- mice, steatosis and apoptosis were more acute compared to WT mice, as evidenced by increased lipid accumulation and cleavage of PARP, respectively. Selectively restoring StarD5 expression to ER-stressed StarD5^-/- mice blunted the effects of tunicamycin. StarD5 appears to play a critical role in the ER stress survival response through its ability to regulate intracellular cholesterol homeostasis.

Keywords: StarD5; apoptosis; endoplasmic reticulum stress; liver steatosis; unfolded protein response.

Figure 1

Tunicamycin-induced ER stress in vivo increases StarD5 expression in the liver. (A) Representative immunoblots of StarD5 and β-actin (as loading control) were performed on liver homogenates from WT and StarD5^−/− mice injected with vehicle or tunicamycin for 1 or 5 days. Relative expression levels were determined by densitometry of StarD5 relative to GAPDH. n = 6. * p < 0.05. ** p < 0.01. (B) Sections from WT and StarD5^−/− mouse livers were immunostained for the detection of StarD5 and stained with hematoxilin. Images were visualized using 20× objective and 60× objective. Representative images from three mice are shown. Note the increased staining following tunicamycin injection after 5 days.

Figure 2

Tunicamycin-induced ER stress in vivo leads to weight loss and increases lipid accumulation in the livers of StarD5^−/− mice compared to wild type. (A) Percentage of body weight loss in WT and StarD5^−/− mice injected with tunicamycin for 1 or 5 days; n = 3. (B) Total cholesterol and triglycerides were determined in livers of WT and StarD5^−/− mice injected with vehicle or tunicamycin for 1 or 5 days. n = 3. * p < 0.05. (C) Sections from WT and StarD5^−/− mouse livers were stained with H&E and visualized using a 40× objective. Representative images from three mice are shown. Note the extensive cytoplasmic vacuolization in the livers from StarD5^−/− animals. n = 3.

Figure 3

Tunicamycin-induced ER stress in vivo increases apoptosis in livers of StarD5^−/− mice compared to wild type livers. Representative immunoblots of StarD5, BiP, PARP, and GAPDH (loading control) in livers of WT and StarD5^−/− mice injected with vehicle or tunicamycin (Tm) for 1 or 5 days.

Figure 4

StarD5 overexpression in StarD5^−/− mice reduces apoptosis following tunicamycin-induced ER stress in vivo. StarD5^−/− mice were injected with an adenovirus encoding either StarD5 or β-gal (control) to overexpress StarD5, and after a week injected with different amounts of tunicamycin (Tm) to induce ER stress. Immunoblots of StarD5 and BiP (A) and PARP (B) with GAPDH (as loading control) in livers of WT and StarD5^−/− mice were obtained.