Upregulation of Suppressor of Cytokine Signaling 3 in Microglia by Cinnamic Acid

Abstract

Background: Neuroinflammation plays an important role in the pathogenesis of various neurodegenerative diseases including Alzheimer's disease (AD). Suppressor of cytokine signaling 3 (SOCS3) is an anti-inflammatory molecule that suppresses cytokine signaling and inflammatory gene expression in different cells including microglia.

Objective: The pathways through which SOCS3 could be upregulated are poorly described. Cinnamic acid is a metabolite of cinnamon, a natural compound that is being widely used all over the world as a spice or flavoring agent. Here, we examined if cinnamic acid could upregulate SOCS3 in microglia.

Method: Microglia and astroglia isolated from mouse brain as well as BV-2 microglial cells were treated with cinnamic acid followed by monitoring the level of SOCS3 and different proinflammatory molecules by RT-PCR and real-time PCR. To nail down the mechanism, we also performed ChIP analysis to monitore the recruitment of cAMP response element binding (CREB) to the socs3 gene promoter and carried out siRNA knockdown of CREB.

Results: Cinnamic acid upregulated the expression of SOCS3 mRNA and protein in mouse BV-2 microglial cells in dose- and time-dependent manner. Accordingly, cinnamic acid also increased the level of SOCS3 and suppressed the expression of inducible nitric oxide synthase and proinflammatory cytokines (TNFα, IL-1β and IL-6) in LPSstimulated BV-2 microglial cells. Similar to BV-2 microglial cells, cinnamic acid also increased the expression of SOCS3 in primary mouse microglia and astrocytes. We have seen that cAMP response element is present in the promoter of socs3 gene, that cinnamic acid induces the activation of CREB, that siRNA knockdown of CREB abrogates cinnamic acid-mediated upregulation of SOCS3, and that cinnamic acid treatment leads to the recruitment of CREB to the socs3 gene.

Conclusions: These studies suggest that cinnamic acid upregulates the expression of SOCS3 in glial cells via CREB pathway, which may be of importance in neuroinflammatory and neurodegenerative disorders.

Keywords: CREB; Cinnamic acid; SOCS3; glial cells; microglial cells; neuroinflammation..

Figure 1. Upregulation of SOCS3 by cinnamic acid in mouse BV-2 microglial cells

Cells were treated with 300 μM cinnamic acid (CA) for different time points (1, 2, 4, 6, and 18 h) in serum-free DMEM/F12 followed by monitoring the mRNA expression of SOCS3 by semi-quantitative RT-PCR (A) and real-time PCR (B). The protein level of SOCS3 was monitored by Western blot (C). Actin was run as a loading control. Bands were scanned and values (SOCS3/actin) presented as relative to control (D). Results are mean ± SD of at least three independent experiments. NS, not significant. Cells were treated with different doses of CA for 2 h followed by monitoring the mRNA expression of SOCS3 by semi-quantitative RT-PCR (E) and real-time PCR (F). The protein level of SOCS3 after 6 h of CA treatment was monitored by Western blot (G). Bands were scanned and values (SOCS3/actin) presented as relative to control (H). Cells were treated with 300 μM CA for 6 h under the same culture conditions and were double-labeled for SOCS3 and Iba1 (I). DAPI was used to stain nuclei. Results represent three independent experiments.

Figure 2. Cinnamic acid attenuates LPS-induced expression of proinflammatory molecules while upregulating SOCS3 in mouse BV-2 microglial cells

Cells pretreated with different concentrations of cinnamic acid (CA) for 2 h were stimulated with LPS (1 μg/ml) under serum-free conditions for 4 h followed by monitoring the mRNA expression of SOCS3, iNOS, TNFα, IL-1β, and IL-6 by semi-quantitative RT-PCR (A) and real-time PCR (B, SOCS3; C, iNOS; D, TNFα; E, IL-1β; F, IL-6). Results are mean ± SD of at least three independent experiments. ^ap < 0.001 vs control; ^bp < 0.001 vs LPS.

Figure 3. Upregulation of SOCS3 by cinnamic acid (CA) in primary mouse microglia

Primary microglia were treated with different concentrations of CA for 2 h under serum-free condition followed by monitoring the mRNA expression of SOCS3 by semi-quantitative RT-PCR (A) and real-time PCR (B). After 6 h of treatment with CA, the protein level of SOCS3 was monitored by Western blot (C). Actin was run as a loading control. Bands were scanned and values (SOCS3/actin) presented as relative to control (D). Results are mean ± SD of at least three independent experiments. Primary microglia were treated with 300 μM CA for 6 h under the same culture conditions followed by double-labeling for SOCS3 and Iba1 (E). Results represent three independent experiments. Primary microglia were treated with different concentrations of CA for 24 h followed by monitoring cell survival by MTT (F) and LDH (G). Results are mean ± SD of at least three independent experiments. NS, not significant.

Figure 4. Cinnamic acid (CA) upregulates SOCS3 in primary mouse astrocytes

Primary astrocytes were treated with different concentrations of CA for 2 h under serum-free condition followed by monitoring the mRNA expression of SOCS3 by semi-quantitative RT-PCR (A) and real-time PCR (B). After 6 h of treatment with CA, the protein level of SOCS3 was monitored by Western blot (C). Actin was run as a loading control. Bands were scanned and values (SOCS3/actin) presented as relative to control (D). Results are mean ± SD of at least three independent experiments. Primary astrocytes were treated with 300 μM CA for 6 h under the same culture conditions followed by double-labeling for SOCS3 and GFAP (E). DAPI was used to stain nuclei. Results represent three independent experiments.

Figure 5. Activation of CREB by cinnamic acid (CA) in mouse BV-2 microglial cells

(A) Position of CRE in the Socs3 gene promoter. Cells were treated with 300 μM CA for different minutes under serum-free conditions followed by monitoring the level of phospho-CREB (pCREB) by Western blot (B). Graph represents densitometric analysis of pCREB protein levels normalized to total CREB (loading control) (C). Results are mean ± SD of three independent experiments. ^ap < 0.05 vs control; ^bp < 0.01 vs control. Cells treated with 300 μM CA for 30 mins under serum-free condition were double-labeled for pCREB & Iba1 (D) and total CREB & Iba1 (E). DAPI was used to stain nuclei. Cells were treated with 300uM of CA under serum-free condition for 15, 30, 60, and 90 min followed by monitoring the DNA-binding activity of CREB by EMSA (F). Results represent three independent experiments. Upper, middle and bottom arrows indicate CREB DNA-binding, non-specific (NS) DNA-binding and unbound probe, respectively.

Figure 6. Effect of siRNA knockdown of CREB on cinnamic acid-mediated up regulation of SOCS3 in mouse BV-2 microglial cells

Cells were transfected with either control or CREB siRNA. Forty-eight h after transfection, level of CREB was monitored by Western blot (A). Graph represents densitometric analysis of CREB protein levels normalized to β-Actin (loading control) (B). Cells were transfected with either control or CREB siRNA. Forty-eight h after transfection, cells were treated with 200 and 300 μM CA for 6 h under serum-free condition followed by monitoring the level of SOCS3 by Western Blot (C). Graph represents densitometric analysis of SOCS3 protein levels normalized to β-Actin (loading control) (D). Results are mean ± SD of three independent experiments. NS, not significant.

Figure 7. Cinnamic acid (CA) treatment induces the recruitment of CREB to the socs3 gene promoter in mouse BV-2 microglial cells

(A) DNA sequence of the Socs3 promoter region containing the CRE with positions of the primers used for the ChIP analysis. Cells were stimulated with 300 μM CA for 1h under serum-free condition. The recruitment of CREB to the Socs3 promoter was monitored by ChIP PCR analysis (B) and ChIP real-time PCR (C). Normal IgG was used as control. Results are mean ± SD of three independent experiments.