Abstract
Pregnenolone is a steroid hormone precursor that is synthesized in various steroidogenic tissues, in the brain, and in lymphocytes. In addition to serving as the precursor for other steroid hormones, pregnenolone exerts its own effect as an anti-inflammatory molecule to maintain immune homeostasis in various inflammatory conditions. Pregnenolone and its metabolic derivatives have been shown to have beneficial effects in the brain, including enhancing memory and learning, reversing depressive disorders, and modulating cognitive functions. A decreased level of pregnenolone has been observed in neuroinflammatory diseases, which emphasizes its role in neuroprotection and neuroregeneration. Although the anti-inflammatory property of pregnenolone was recognized several decades ago, its mechanism of action remains unknown. Here we report that pregnenolone promotes ubiquitination and degradation of the TLR2/4 adaptor protein TIRAP and TLR2 in macrophages and microglial cells. Pregnenolone and its metabolites suppressed the secretion of tumor necrosis factor α and interleukin-6 mediated through TLR2 and TLR4 signaling. Pregnenolone has been reported to induce activation of cytoplasmic linker protein 170, and this protein has recently been shown to promote targeted degradation of TIRAP. We observed enhanced degradation of TIRAP and TLR4 suppression by cytoplasmic linker protein 170 in the presence of pregnenolone. Our experimental data reveal novel nongenomic targets of pregnenolone and provide important leads to understand its role in restoring immune homeostasis in various inflammatory conditions.
Highlights
Pregnenolone is a steroid hormone precursor that is synthesized in various steroidogenic tissues, in the brain, and in lymphocytes
We investigated the effect of pregnenolone and its metabolic derivatives, such as pregnenolone sulfate, allopregnanolone, and progesterone, on secretion of TNF␣ by mouse and human macrophages and mouse microglial cells
RAW264.7 cells, bone marrow– derived macrophages (BMDMs), THP1 cells, or N9 microglial cells were treated with pregnenolone or its metabolites, followed by induction with LPS or Pam3CSK4 and quantification of TNF␣ by ELISA and quantitative PCR
Summary
Pregnenolone suppresses LPS and Pam3CSK4-induced TNF␣ and IL-6 in macrophages and microglial cells. We did not observe suppression of LPS-induced IFN- in RAW264.7 cells, which indicates that pregnenolone or it metabolites do not affect the MyD88-independent pathway of TLR4 signaling (Fig. S2A). Treatment with pregnenolone sulfate or allopregnenolone induced slight degradation, whereas progesterone or cholesterol did not affect the level of FLAG-TIRAP (Fig. 2, A and B). To further examine the degradation of TLR2, HEK293T cells overexpressing FLAG-TLR2 were treated with pregnenolone or its metabolic derivatives, followed by detection of FLAG-tagged proteins by immunoblotting. RAW264.7 macrophages overexpressing FLAG-TIRAP and MYC-CLIP170 were treated with pregnenolone or DMSO followed by immunoblotting to detect the level of FLAG-TIRAP. The degradation of FLAG-TIRAP appeared to be enhanced further in the presence of pregnenolone in MYC-CLIP170- and FLAG-TIRAP– overexpressing cells (Fig. 4A). Cells overexpressing CLIP170, which was treated with pregnenolone, exhibited more FLAG-TIRAP ubiquitination compared with DMSO-treated samples (Fig. 4B). The experimental data suggest that pregnenolone enhances the TLR4 suppression property of CLIP170 by promoting its activation
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