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. 2015 Jul;99(Pt B):219-29.
doi: 10.1016/j.steroids.2015.02.022. Epub 2015 Mar 7.

Rapid proteasomal elimination of 3-hydroxy-3-methylglutaryl-CoA reductase by interferon-γ in primary macrophages requires endogenous 25-hydroxycholesterol synthesis

Hongjin Lu  1 , Simon Talbot  1 , Kevin A Robertson  2 , Steven Watterson  3 , Thorsten Forster  2 , Douglas Roy  1 , Peter Ghazal  4
Affiliations

Rapid proteasomal elimination of 3-hydroxy-3-methylglutaryl-CoA reductase by interferon-γ in primary macrophages requires endogenous 25-hydroxycholesterol synthesis

Hongjin Lu et al. Steroids. 2015 Jul.

Abstract

Interferons (IFNs) play a central role in immunity and emerging evidence suggests that IFN-signalling coordinately regulates sterol biosynthesis in macrophages, via Sterol Regulatory Element-Binding Protein (SREBP) dependent and independent pathways. However, the precise mechanisms and kinetic steps by which IFN controls sterol biosynthesis are as yet not fully understood. Here, we elucidate the molecular circuitry governing how IFN controls the first regulated step in the mevalonate-sterol pathway, 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), through the synthesis of 25-Hydroxycholesterol (25-HC) from cholesterol by the IFN-inducible Cholesterol-25-Hydroxylase (CH25H). We show for the first 30-min of IFN stimulation of macrophages the rate of de novo synthesis of the Ch25h transcript is markedly increased but by 120-min becomes transcriptionally curtailed, coincident with induction of the Activating Transcription Factor 3 (ATF3) repressor. We demonstrate ATF3 induction by Toll-like receptors is strictly dependent on IFN-signalling. While the SREBP-pathway dependent rates of de novo transcription of Hmgcr are relatively unchanged in the first 90-min of IFN treatment, we find HMGCR enzyme levels undergo a rapid proteasomal-mediated degradation, defining a previously unappreciated SREBP-independent mechanism for IFN-action. These events precede a sustained marked reduction in Hmgcr RNA levels involving SREBP-dependent mechanisms. We demonstrate that HMGCR proteasomal-degradation by IFN strictly requires the synthesis of endogenous 25-HC and functionally couples HMGCR to CH25H to coordinately suppress sterol biosynthesis. In conclusion, we quantitatively delineate proteomic and transcriptional levels of IFN-mediated control of HMGCR, the primary enzymatic step of the mevalonate-sterol biosynthesis pathway, providing a foundational framework for mathematically modelling the therapeutic outcome of immune-metabolic pathways.

Keywords: 25-Hydroxycholesterol; CH25H; Cholesterol biosynthesis; Immunity; Infection; Macrophages.

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Figures

Fig. 6
Fig. 6
Schematic representation of the dual role of 25-HC in the sterol biosynthesis pathway and the IFN-γ-regulated innate immune response using SBGN system (inset shows key).
Fig. 1
Fig. 1
Synthesis rates of de novo transcription of Atf3 and Ch25h following IFN-γ treatment and RNA abundance of Irf1, Ch25h and Atf3 upon Poly (I:C) treatment. (a) Measurement of de novo transcribed RNA of Atf3 and Ch25h upon IFN-γ treatment in wild-type BMDMs (relative to control treated) in Medium A over an 8-h period. Each point represents transcript synthesis in control treated vs IFN-γ during a 30-min period. Normalised log fold change values were calculated by subtracting the control treated from the IFN-γ treated signal values. Atf3: p = ns; Ch25h: p=1.91E−12. (b) Irf1 abundance in wild-type and Ifnb−/− BMDMs (relative to control treated t = 0) in Medium A following 10 μg/mL Poly (I:C) treatment. (c) Ch25h abundance in wild-type and Ifnb−/− BMDMs (relative to control treated t = 0) in Medium A following 10 μg/mL Poly (I:C) treatment. (d) Atf3 abundance in wild-type and Ifnb−/− BMDMs (relative to control treated t = 0) in Medium A following 10 μg/mL Poly (I:C) treatment.
Fig. 2
Fig. 2
HMGCR protein levels following mevastatin and 25-HC treatments. (a) Western blot analysis of the HMGCR protein level in mevastatin-treated wild-type BMDMs. Cells were treated overnight with various concentrations of mevastatin in Medium B. (b) Intensity values of HMGCR to β-actin were calculated, based on which the EC50 value of mevastatin was then generated. Data are mean ± SEM (n = 3). (c) Western blot analysis of the endogenous HMGCR protein level in 25-HC-treated wild-type BMDMs. Cells were treated with various concentrations of 25-HC for 24 h in Medium C. (d) Intensity values of HMGCR to β-actin were calculated, based on which the IC50 value of 25-HC was then generated. Data are mean ± SEM (n = 4). (e) Western blot analysis of the endogenous HMGCR protein level in 25-HC-treated wild-type BMDMs. Cells were pre-treated with Medium C overnight and then treated with various concentrations of 25-HC in Medium C for another 24 h. (f) Intensity values of HMGCR to β-actin were calculated, based on which the IC50 value of 25-HC was then generated. Data are mean ± SEM (n = 4).
Fig. 3
Fig. 3
Time-course showing the Hmgcr mRNA and HMGCR protein levels following IFN-γ treatment. (a) Temporal alterations in abundance of Hmgcr transcript in IFN-γ treated BMDMs (relative to control treated) in Medium A over an 8-h period. Each point represents overall abundance of transcript at the indicated time. Relative log fold change values were calculated by subtracting the control treated from the IFN-γ-treated signal values. p = 2.72E−05. (b) Temporal alterations in de novo synthesis rate of Hmgcr transcript in IFN-γ-treated BMDMs (relative to control treated) in Medium A over an 8-h period. Each point represents transcript synthesis rate in control treated vs IFN-γ during a 30-min period. Normalised log fold change values were calculated by subtracting the control treated from the IFN-γ-treated signal values. p = 2.93E−11. (c) Half-life values of Hmgcr at indicated period time. Based on the RNA abundance and newly transcribed RNA levels of Hmgcr, the half-lives of Hmgcr for each period were calculated as previously described . (d) Wild-type BMDMs were treated with 2.5 μM of 25-HC in Medium C at multiple time points and Hmgcr mRNA level was determined by qRT-PCR. Data are mean ± SEM (n = 3). (e) Western blot analysis of the HMGCR protein level with 2.5 μM of 25-HC treatment in Medium C at multiple time points. (f) Intensity values of HMGCR to β-actin. Data are mean ± SEM (n = 3). (g) Wild-type BMDMs were treated with 5 ng/mL of IFN-γ in Medium C at multiple time points and Hmgcr mRNA level was determined by qRT-PCR. Data are mean ± SEM (n = 3). (h) Western blot analysis of HMGCR protein level with 5 ng/mL of IFN-γ treatment in Medium C at multiple time points. (i) Intensity values of HMGCR to β-actin. Data are mean ± SEM (n = 3). ∗p < 0.05, ∗∗p < 0.01, determined with an unpaired Student’s t test.
Fig. 4
Fig. 4
IFN-γ induces proteasomal degradation of HMGCR. (a) Wild-type BMDMs were pre-treated with MG132 in Medium C for 1 h and then treated with 25-HC or IFN-γ in the same culture medium for another 6 h. Western blot was performed to determine HMGCR protein levels. (b) Intensity values of HMGCR to tubulin. As MG132 has an effect on β-actin protein levels, tubulin was used as the internal control. Data are mean ± SEM (n = 4). ∗p < 0.05, determined with an unpaired Student’s t test. (c) Intensity values of HMGCR to tubulin. Data are mean ± SEM (n = 4). ∗∗p < 0.01, determined with an unpaired Student’s t test.
Fig. 5
Fig. 5
Comparison of Hmgcr mRNA and HMGCR protein levels following 25-HC or IFN-γ treatment in wild-type and Ch25h−/− BMDMs. (a) Western blot analysis of HMGCR with 2.5 μM of 25-HC or 5 ng/mL of IFN-γ treatment in Medium C at 4-h and 9-h time points in wild-type BMDMs. (b) Intensity values of HMGCR to β-actin. Bars present the mean ± SEM (n = 3). (c) Western blot analysis of HMGCR with 2.5 μM of 25-HC or 5 ng/mL of IFN-γ treatment in Medium C at 4-h and 9-h time points in Ch25h−/− BMDMs. (d) Intensity values of HMGCR to β-actin. Bars present the mean ± SEM (n = 3). (e) HMGCR transcript abundance in wild-type and Ch25h−/− BMDMs with 2.5 μM of 25-HC or 5 ng/mL of IFN-γ treatment for 24 h in Medium A. The 25-HC-treated groups were normalised by the Vehicle and the IFN-γ-treated groups were normalised by the control treated samples, respectively. Bars present the mean ± SEM (n = 9). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, determined with an unpaired Student’s t test.
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