17beta-Estradiol utilizes the estrogen receptor to regulate CD16 expression in monocytes

Abstract

Estrogen can significantly influence CD16 expression and alter monocytic cytokine release upon CD16 receptor activation. However, the function of the estrogen receptor (ER) alpha and beta in this response is unclear. To test whether estrogen binds ERalpha and/or ERbeta to affect CD16 expression, monocytic cells were treated with and without physiological levels of 17beta-estradiol and various doses of the ERalpha and ERbeta antagonist fulvestrant followed by measurement of CD16 transcript levels. To determine how estrogen induced changes in TNF-alpha and IL-1beta release due to CD16 activation we quantitated the amount of cytokines after treatment with estrogen, fulvestrant and antibodies that specifically bind and activate the CD16 receptor. Interaction of ERalpha and the CD16 promoter was then determined by chromatin immunoprecipitation. Furthermore, specific promoter elements utilized by estrogen to control CD16 expression were mutated and expression from a luciferase reporter quantitated after transfection. Using the luciferase reporter construct containing a wild type CD16 promoter, the role of ERalpha and ERbeta in the estrogen response was tested by treating transfected monocytes with an ERalpha specific agonist or an ERbeta specific agonist and measuring expression. Our results show that CD16 transcript levels significantly decreased in monocytic cells due to estrogen and that the observed decrease in message was blocked by the antagonist fulvestrant. Estrogen reduced CD16 expression and decreased TNF-alpha and IL-1beta release upon CD16 activation but the administration of fulvestrant blocked this decrease. ERalpha was found to interact with a region 5' of the CD16 gene in the presence of estrogen, and site-directed mutational analysis of this region indicated the necessity for an estrogen response element in modulating estrogen effects on CD16 expression. Moreover, both an ERalpha and an ERbeta agonist reduced expression of the CD16 reporter construct suggesting both receptors can play a role in CD16 regulation. In conclusion, CD16 expression can be altered by the activity of ERalpha or ERbeta and our results also show that ERalpha can associate with a region within the CD16 promoter that is important in production of transcript.

Figure 1. Estrogen modulates CD16 transcript levels in THP-1 monocytes

In panel A, cells were treated with 1 × 10⁻⁸ M 17 β-estradiol (E₂+), and in panel B, cells were treated with 1 × 10⁻⁹ M 17 β-estradiol (E₂+). In a portion of the cultures treated with 17 β-estradiol, the hormone was withdrawn (w E₂). Samples were collected at 0, 12, 24 and 48 hours after hormonal treatment or withdrawal of hormone. Transcript levels for CD16 were analyzed using real-time PCR. Two-way analysis of variance (ANOVA) was completed for the data in panel A and panel B. ANOVA results indicate that the data for the two treatment groups (E₂+ and w E₂) in panel A and in panel B were significantly different p<0.05. Three experiments were performed for each treatment at each time point. Relative CD16 expression was calculated in reference to the amount of 18S RNA present within the cells. Values are the mean ± SEM.

Figure 2. ER antagonist fulvestrant effects CD16 levels in THP-1 monocytes treated with high physiological levels of estrogen

In panel A, monocytes were treated with 1 × 10⁻⁸ M 17 β-estradiol and three different concentrations of fulvestrant (i.e., ICI 182,780), starting at time zero. In panel B, monocytes were initially treated with 1 × 10⁻⁸ M 17 β-estradiol and then had 17 β-estradiol withdrawn concomitant with addition of fulvestrant. In panel C, monocytes were cultured in media without the addition of 17 β-estradiol, and fulvestrant was added. After the addition of fulvestrant cells were collected at 0, 12, 24 and 48 hours. CD16 transcript levels were determined by real-time PCR. Three experiments were performed for each treatment at each time. Relative CD16 was calculated in reference to the amount of 18S RNA present within the cells. Values are the mean ± SEM.

Figure 3. TNF-α expression in activated primary monocytes in the presence of estrogen and fulvestrant

Primary monocytes were cultured without (no E₂) or with (E₂+) 1 × 10⁻⁸ M 17 β-estradiol. A portion of these cultures was treated with 100 nm fulvestrant (i.e., ICI 182, 780). After 48 hours, antibody against CD16 (25 μg/ml) or an IgG1 isotype control antibody (25 μg/ml) was added. The supernate (A) and cell pellets (B) were collected 48 hours after addition of antibody and the amount of TNF-α quantitated in each by ELISA. Values represent the mean amount of TNF-α (pg) from 1 ml of cultured cells. * = P<0.05 comparing estrogen treated to non-estrogen treated cells. Four experiments were performed for each treatment group.

Figure 4. Chromatin immunoprecipitation (ChIP) analysis of the CD16 promoter in the monocytic cell line U937 and in primary monocytes

Chromatin was isolated from U937 cells (lanes 1 through 3) and from primary monocytes (lanes 4 through 15). Chromatin was isolated from cells treated for 24 hours with or without 1 × 10⁻⁸ M 17 β-estradiol, as indicated by “+” or “−“ below each lane. The first lane shows a 100 base pair ladder (M). In lane 1, chromatin was immunoprecipitated with a non-specific IgG control antibody, and in lane 2, chromatin was immunoprecipitated with an ERα specific antibody followed by PCR amplification of a promoter region of the CD16 gene. Lane 3 represents a negative control PCR reaction using no chromatin as a template. Additional PCR reactions used template from either total chromatin, as a positive control (lanes 4, 7, 8, 11, 14 and 15) or chromatin immunoprecipitated with an ERα specific antibody (lanes 2, 5, 6, 9, 10, 12 and 13). PCR products from the ChIP analysis were resolved on an agarose gel, stained with ethidium bromide and imaged. Images are representative of duplicate experiments. In panel A, the PCR product comprised bases −353 to −50 of the CD16 5′ flanking region. In panel B, the PCR product included bases −864 to −257, and in panel C, the PCR product included bases −1113 to −820 of the CD16 5′ flanking region. Panel D shows the upstream promoter regions of the CD16 gene from bases −386 through −74. Estrogen response elements (ERE) or estrogen response element half-sites (1/2ERE), as well as Sp1 consensus binding sequences, are shown as boxed regions. Arrow indicates the direction of transcription.

Figure 5. Expression of mutated CD16 promoter constructs

Six different DNA constructs were generated for transfection into the primary monocytes. One construct had an SV40 promoter driving luciferase expression (positive control), one construct had a 1,833 base region of the CD16 promoter driving luciferase expression, and the remaining four constructs had site-directed mutations of the 1,833 base CD16 promoter, as indicated in panel A. Two of the mutated regions have three simultaneous base changes, one has two simultaneous base changes, and one had a single base change. Arrowhead points to the wild type base that was mutated in the sense strand of the CD16 promoter sequence and the bold letter above the arrowhead is the mutated base that was incorporated. Relative positions of these mutations within the 1,833 base CD16 promoter are shown in panel B. Primary monocytes transfected with one of the six constructs shown in panel B were cultured with (E₂+) or without (no E₂) 1 × 10⁻⁸ M 17 β-estradiol. Luciferase expression from each construct in panel B is aligned horizontally with the bar graph in panel C. Letter “a” over each bar in panel C indicates a significant difference (p<0.05) between the luminescence of cells treated with or without 17 β-estradiol after transfection with a particular DNA construct. Letter “b” indicates a significant difference (p<0.05) between the luminescence of cells transfected with the mutant construct and cells transfected with the intact CD16 promoter fragment (−1833 to −1). In panel D the cells were transfected with a reporter construct containing the CD16 promoter driving luciferase expression. After transfection cells were treated with or without 1 × 10⁻⁸ M 17 β-estradiol or the ERα-specific agonist PPT or the ERβ-specific agonist DPN at a concentration of 1 × 10⁻⁹ M. Comparing treatment groups significant differences are indicated by * = P<0.05, *** = P<0.001. Relative light units (RLU) represent the luminescent signal from the firefly luciferase normalized to an internal control Renilla luciferase construct. Three experiments were performed for each treatment group transfected with the site directed mutagenesis constructs and four experiments were performed for each group treated with the estrogen receptor antagonists.