Soluble LDL-receptor is induced by TNF-α and inhibits hepatocytic clearance of LDL-cholesterol

Abstract

Defective LDL-C clearance and hence its elevation in the circulation is an established risk factor for cardiovascular diseases (CVDs) such as myocardial infarction (MI). A soluble LDL-receptor (sLDL-R) has been detected in human plasma which correlates strongly with circulating LDL-C and classical conditions that promote chronic inflammation. However, the mechanistic interplay between sLDL-R, inflammation, and CVDs remains to be investigated. Here, we report that stimulation of HepG2 cells with TNF-α induces the release of sLDL-R into culture supernatants. In addition, TNF-α induces gene expression of peptidases ADAM-17 and MMP-14 in HepG2 cells, and inhibiting these peptidases using TMI 1 significantly reduces the TNF-α induced sLDL-R release. We found that a soluble form of recombinant LDL-R (100 nM) can strongly bind to LDL-C and form a stable complex (KD = E-12). Moreover, incubation of HepG2 cells with this recombinant LDL-R resulted in reduced LDL-C uptake in a dose-dependent manner. In a nested case-control study, we found that baseline sLDL-R in plasma is positively correlated with plasma total cholesterol level. Furthermore, a twofold increase in plasma sLDL-R was associated with a 55% increase in the risk of future MI [AOR = 1.55 (95% CI = 1.10-2.18)]. Nevertheless, mediation analyses revealed that a significant proportion of the association is mediated by elevation in plasma cholesterol level (indirect effect β = 0.21 (95% CI = 0.07-0.38). Collectively, our study shows that sLDL-R is induced by a pro-inflammatory cytokine TNF-α via membrane shedding. Furthermore, an increase in sLDL-R could inhibit hepatic clearance of LDL-C increasing its half-life in the circulation and contributing to the pathogenesis of MI. KEY MESSAGES: TNF-α causes shedding of hepatocytic LDL-R through induction of ADAM-17 and MMP-14. sLDL-R binds strongly to LDL-C and inhibits its uptake by hepatocytic cells. Plasma sLDL-R is positively correlated with TNF-α and cholesterol. Plasma sLDL-R is an independent predictor of myocardial infarction (MI). Plasma cholesterol mediates the association between sLDL-R and MI.

Keywords: ADAM-17; Chronic inflammation; Hypercholesterolemia; MMP-14; Mediation analyses; Myocardial infarction.

Fig. 1

Flowchart showing the inclusion of study participants. The time from screening to incident MI in MONICA cohort ranged from 0.01 to 20 years with median of 6.9 years. The cases included in the current study were chosen depending on shorter duration between screening and incident MI (≤ 7 years), and one of the two matched controls of each case was randomly selected

Fig. 2

TNF-α induces release of soluble LDL-R (sLDL-R) in HepG2 cells. A Bar graphs showing a dose-dependent release of sLDL-R from HepG2 cells treated with TNF-α (24 h and 48 h) into culture supernatants. B A representative histogram and a bar graph showing the surface expression of LDL-R on HepG2 cells following TNF-α treatment (50 ng/ml for 48 h). C A representative histogram and a bar graph depicting the LDL-C uptake by HepG2 cells following TNF-α treatment (50 ng/ml for 48 h). D Representative microscopic images and quantification of LDL-C uptake by HepG2 cells treated or not with TNF-α (50 ng/ml) for 48 h. **p < 0.01, ***p < 0.001. AU arbitrary unit, FMO fluorescence minus one, MFI median fluorescence intensity, ns non-significant

Fig. 3

TNF-α induces the gene expression of LDL-R and surface protein peptidases in HepG2 cells. Bar graphs depicting gene expression of LDL-R variants in HepG2 cells treated with TNF-α (50 ng/ml) using primers targeting exons 1–2 (A), 15–16 (B), and 17–18 (C). Bar graphs showing gene expression of peptidases ADAM-17 (D), MMP14 (E), and BMP1 (F) in HepG2 cells treated with TNF-α (50 ng/ml). G Bar graph showing the level of sLDL-R released from HepG2 cells stimulated with TNF-α (50 ng/ml) in the absence or presence of peptidase inhibitors UK383367 (1 µM) and TMI 1 (1 µM). *p < 0.05, **p < 0.01, ***p < 0.001. AU arbitrary unit, ns non-significant

Fig. 4

Recombinant human LDL-R strongly binds to LDL-C and inhibits its uptake by HepG2 cells. A Representative Sensogram and a plot of KD values from 3 independent analyses. B A representative histogram and a bar graph depicting the LDL-C uptake by HepG2 cells in the presence of rhLDL-R. C Representative microscopic images and quantification of LDL-C uptake by HepG2 cells in the presence of rhLDL-R. *p < 0.05, **p < 0.01. AU arbitrary unit, FMO fluorescence minus one, ns non-significant

Fig. 5

Plasma sLDL-R level and its relationship with clinical characteristics of study participants at baseline. A A scatter plot showing the distribution/comparison of plasma sLDL-R in categories of clinical characteristics of study participants at baseline. Scatter plots showing the correlation between plasma sLDL-R concentration and total cholesterol (B) TNF-α (C). NPX normalized protein expression

Fig. 6

Plasma sLDL-R level is associated with increased risk of future MI, and its association is mediated by increase in plasma cholesterol level. A Scatter plot showing the distribution of plasma sLDL-R concentration in cases and controls (n = 584). B Schematic illustration of the direct and indirect effects of plasma sLDL-R on risk of future MI (n = 543)