Estradiol enhances cell-associated paraoxonase 1 (PON1) activity in vitro without altering PON1 expression

Abstract

PON1 is a high density lipoprotein-associated enzyme that plays an important role in organophosphate detoxification and prevention of atherosclerosis. In vivo animal and human studies have indicated that estradiol (E2) supplementation enhances serum PON1 activity. In this study, we sought to determine if E2 directly up-regulates cell-associated PON1 activity in vitro and to characterize the mechanism of regulation. In vitro E2 treatment of both the human hepatoma cell line Huh7 and normal rat hepatocytes resulted in a 2- to 3-fold increase in cell-associated PON1 catalytic activity. E2 potently induced PON1 activity with average EC(50) values of 15nM for normal hepatocytes and 68nM for Huh7. The enhancement of PON1 activity by E2 was blocked by the estrogen receptor (ER) antagonist ICI 182,780 indicating that E2 was acting through the ER. The up-regulation of PON1 activity by E2 did not involve enhancement of PON1 mRNA or protein levels and did not promote secretion of PON1. Thus, E2 can enhance cell-associated PON1 activity in vitro without altering PON1 gene expression or protein level. Our data suggest that E2 may regulate the specific activity and/or stability of cell surface PON1.

Fig. 1

The effect of E2 on cell-derived PON1 activity. Concentration response curves were generated for E2 by addition of the indicated concentrations of E2 to cells and cell-associated PON1 enzymatic activity measured (■). The resulting raw velocities (A), percent specific activities relative to controls (B,D) or background subtracted specific velocities (D) are shown. (A) Huh7 cells (8,000/well) were plated and treated with E2 in assay media containing 0.1% FCS. (B) Huh7 cells (4,000/well) were plated and treated with E2 in assay media containing 10% charcoal-stripped FCS. (C) Normal rat hepatocytes (5,000 - 8,000/well) were plated and treated with E2 in assay media containing 10% charcoal-stripped FCS. Cell viability (▲, for B and C) was assessed in parallel treated wells and normalized to controls. Activity in the presence of 2HQ or EDTA, as indicated, added immediately prior to activity assessment for cells treated with high concentrations of E2 (B and C) or 24 hr time (D) are shown (□), except for (A) which shows DMSO + EDTA background control. Data points were performed in triplicate determination and error bars represent standard deviations. Data are representative of at least three independent experiments, except for the time course which is representative of two experiments.

Fig. 2

Characterization of PON1 activity enhancement by estradiol in Huh7 cells. (A) Cells were treated with E2, E2 + ICI 182,780, ICI 182,780 alone or progesterone as indicated and total PON1 activity determined. Compound concentrations were 200 nM. Controls received DMSO without EDTA (untreated, UT) or with EDTA (as labeled) added just prior to DEPFMU addition. (B) After treatment with DMSO (untreated, UT) or 200 nM E2, supernatants from the wells were removed and tested for activity (secreted activity) and the remaining cells were also tested for PON1 activity (cell-associated activity). Data points were performed in triplicate determination and error bars represent standard deviations. Data are representative of three independent experiments.

Fig. 3

Gene expression analysis of E2-treated cells. Total RNA was isolated from Huh7 cells and normal hepatocytes treated with 200 nM E2 or DMSO. The RNA was converted to cDNA and subjected to real-time quantitative PCR using GAPDH as the normalizing endogenous control. Relative quantitation (RQ) values were determined relative to DMSO control (RQ = 1). Genes analyzed are indicated. Data represents average RQ values of three independent experiments.

Fig. 4

PON1 protein expression analysis. (A) Cells were plated in 6 well plates and treated with 200 nM E2 or DMSO in the absence of serum. Whole cell protein lysates were analyzed by western blot with anti-PON1 monoclonal antibody and the same blot stripped and re-probed with anti-β-actin monoclonal antibody. A representative western blot is shown (A) indicating PON1 bands and actin bands from sequential probing. (B) The net intensities of the bands were digitally analyzed and the ratio of PON1: β-actin bands were calculated and the fold difference (E2 treated/DMSO treated) determined for each experiment. The average fold difference of E2 treated cells relative to DMSO treated cells using data from all three independent experiments is graphically represented.

Fig. 4

PON1 protein expression analysis. (A) Cells were plated in 6 well plates and treated with 200 nM E2 or DMSO in the absence of serum. Whole cell protein lysates were analyzed by western blot with anti-PON1 monoclonal antibody and the same blot stripped and re-probed with anti-β-actin monoclonal antibody. A representative western blot is shown (A) indicating PON1 bands and actin bands from sequential probing. (B) The net intensities of the bands were digitally analyzed and the ratio of PON1: β-actin bands were calculated and the fold difference (E2 treated/DMSO treated) determined for each experiment. The average fold difference of E2 treated cells relative to DMSO treated cells using data from all three independent experiments is graphically represented.