Effects of prostaglandin F(2α) on adipocyte biology relevant to graves' orbitopathy

Abstract

Background: In Graves' orbitopathy (GO), increased proliferation, excess adipogenesis, and hyaluronan overproduction produce GO exophthalmos. Enophthalmos occurs in some glaucoma patients treated with Bimatoprost (prostaglandin F2α, PGF2α) eye drops. We hypothesized that enophthalmos is secondary to reductions in orbital tissue proliferation, adipogenesis, and/or increased lipolysis. We aimed to determine which of these is affected by PGF2α by using the 3T3-L1 murine preadipocyte cell line and primary human orbital fibroblasts (OFs) from GO patients (n=5) and non-GO (n=5).

Methods: 3T3-L1 cells and orbital OFs were cultured alone or with PGF2α (all experiments used 10(-8) to 10(-6) M) and counted on days 1/2/3 or 5, respectively; cell cycle analysis (flow cytometry) was applied. Adipogenesis (in the presence/absence of PGF2α) was evaluated (day 7 or 15 for 3T3-L1 and primary cells, respectively) morphologically by Oil Red O staining and quantitative polymerase chain reaction measurement of adipogenesis markers (glycerol-3-phosphate dehydrogenase and lipoprotein lipase, respectively). For lipolysis, in vitro-differentiated 3T3-L1 or mature orbital adipocytes were incubated with norepinephrine and PGF2α and free glycerol was assayed. Appropriate statistical tests were applied.

Results: The population doubling time of 3T3-L1 was 27.3±1.4 hours-significantly increased by dimethyl sulfoxide 0.02% to 44.6±4.8 hours (p=0.007) and further significantly increased (p=0.049 compared with dimethyl sulfoxide) by 10(-8) M PGF2α to 93.6±19.0 hours, indicating reduced proliferation, which was caused by prolongation of G2/M. GO OFs proliferated significantly more rapidly than non-GO (population doubling time 5.36±0.34 or 6.63±0.35 days, respectively, p=0.035), but the proliferation of both was significantly reduced (dose dependent from 10(-8) M) by PGF2α, again with prolongation of G2/M. Adipogenesis in 3T3-L1 cells was minimally affected by PGF2α when assessed morphologically, but the drug significantly reduced transcripts of the glycerol-3-phosphate dehydrogenase differentiation marker. GO OFs displayed significantly higher adipogenic potential than non-GO, but in both populations, adipogenesis, evaluated by all 3 methods, was significantly reduced (dose dependent from 10(-8) M) by PGF2α. There was no effect of PGF2α on basal or norepinephrine-induced lipolysis, in 3T3-L1 or human OFs, either GO or non-GO.

Conclusions: The results demonstrate that PGF2α significantly reduces proliferation and adipogenesis and that human OFs are more sensitive to its effects than 3T3-L1. Consequently, PGF2α could be effective in the treatment of GO.

FIG. 1.

Direct cell counting to assess the effects of PGF_2α on proliferation of 3T3-L1 cells cultured alone (black bars, DMSO control), with addition of 10⁻⁸ M PGF_2α on day 0 (stippled bars) or with daily addition of PGF_2α (gray bars). Results are expressed as mean±SEM of two individual experiments all performed in triplicate. *p<0.05; **p<0.01; ***p<0.001 compared with the control on respective days. DMSO, dimethyl sulfoxide; PGF_2α, prostaglandin F_2α.

FIG. 2.

Cell cycle analysis of 3T3-L1, to assess PGF_2α effects, presented as scatter plots (A, C) and histograms (B, D) in control (A, B) and 10⁻⁶ M PGF_2α-treated (C, D) cells. The x-axis on the scatter plot represents cell size where the arbitrary box was drawn to gate single cells, and the y-axis represents fluorescence intensity of DNA dye (propidium iodide). The histograms show the G1, S, and G2/M phases (figures above=percentage of cells) in (B) control (+DMSO) medium or (D) treated with PGF_2α. The histogram reports DNA content (x-axis) and cell number (y-axis). The figure is a representative experiment of two performed, both in duplicate.

FIG. 3.

In vitro–induced adipogenesis of 3T3-L1 cells assessed by Q-PCR measurement of GPDH transcripts expressed as TCN per 1000 copies of the acidic ribophosphoprotein (ARP) housekeeper gene after 7 days of exposure to control (DM+DMSO) and treatment (DM+PGF_2a). Data shown (mean±SEM) are from a representative experiment of two performed in duplicate. Error bar represents±SEM; **p<0.01. DM, differentiation medium; GPDH, glycerol-3-phosphate dehydrogenase; Q-PCR, quantitative polymerase chain reaction; TCN, transcript copy number.

FIG. 4.

Direct cell counting to assess the effects of PGF_2α on proliferation of human OFs cultured for 5 days alone (DMSO control) or with 10⁻⁸ to 10⁻⁶ M PGF_2α. Stippled bars are Graves' OFs (n=3) and gray bars non-GO (n=3). Results are presented as mean±SEM; *p<0.05 compared with the control on each respective day. OFs, orbital fibroblasts.

FIG. 5.

Cell cycle analysis was performed in OFs, to assess PGF_2α effects, presented as scatter plots (A, C) and histograms (B, D) in the control (A, B), and 10⁻⁶ M PGF_2α-treated (C, D) cells. The x-axis on the scatter plot represents cell size where the arbitrary box was drawn to gate single cells, and the y-axis represents fluorescence intensity of DNA dye (propidium iodide). The histograms show the G1, S, and G2/M (figures above=percent of cells) phases in (B) control (+DMSO) medium or (D) treated with PGF_2α. The histogram reports DNA content (x-axis) and cell number (y-axis). The figure presents data from Graves' OFs and is a representative experiment of two performed (using OFs from different donors), both in duplicate.

FIG. 6.

In vitro–induced adipogenesis (15 days in DM) in orbital preadipocytes in the control (DMSO), and PGF_2α-treated cells was assessed by (A) counting foci of differentiation, (B) Q-PCR measurement of LPL transcripts, and (C) quantification of Oil Red O staining. (A) Colony counts (n=3) are expressed as the mean±SEM from 4 representative quadrants of the well. (B) Q-PCR results (n=5) expressed as mean±SEM of TCN per 1000 copies of housekeeper gene (adenosine phosphoribosyl transferase, APRT). (C) Oil Red O staining (n=2) expressed as the mean±SEM of the OD₄₉₀ absorbance. Stippled bars are Graves' OFs and gray bars non-GO. In all cases, the bar above the control represents statistical comparison between TCN in GO and non-GO orbital cells; other comparisons are between treated and control. *p<0.05; **p<0.01; ***p<0.001. LPL, lipoprotein lipase.

FIG. 7.

The effects of PGF_2α on adipogenesis are reversible; confluent Graves' OFs (n=2) were treated with the differentiation medium alone or supplemented with PGF_2α 10⁻⁶ M for varying periods during differentiation as indicated in the graph. LPL transcripts were measured on day 15 and expressed as mean±SEM of transcript copy number (TCN) per 1000 copies of housekeeper gene (APRT). In all cases, comparisons are between treated and control. **p<0.01; ***p<0.001.