SUMMARY
Mice deficient in the nuclear hormone receptor RORγt have defective development of thymocytes, lymphoid organs, Th17 cells and type 3 innate lymphoid cells. RORγt binds to oxysterols derived from cholesterol catabolism but it is not clear whether these are its natural ligands. Here, we show that sterol lipids are necessary and sufficient to drive RORγt-dependent transcription. We combined overexpression, RNA interference and genetic deletion of metabolic enzymes to study RORγ-dependent transcription. Our results are consistent with the RORγt ligand(s) being a cholesterol biosynthetic intermediate (CBI) downstream of lanosterol and upstream of zymosterol. Analysis of lipids bound to RORγ identified molecules with molecular weights consistent with CBIs. Furthermore, CBIs stabilized the RORγ ligand-binding domain and induced co-activator recruitment. Genetic deletion of metabolic enzymes upstream of the RORγt-ligand(s) affected the development of lymph nodes and Th17 cells. Our data suggest that CBIs play a role in lymphocyte development potentially through regulation of RORγt.
DEP domain containing mTOR-interacting protein (DEPTOR) inhibits the mechanistic target of rapamycin (mTOR) but its in vivo functions are unknown. Previous work indicates that Deptor is part of the Fob3a quantitative trait locus (QTL) linked to obesity/leanness in mice with Deptor expression being elevated in white adipose tissue (WAT) of obese animals. This relation is unexpected considering the positive role of mTOR in adipogenesis. Here, we dissected the Fob3a QTL and show that Deptor is the highest priority candidate promoting WAT expansion in this model. Consistently, transgenic mice overexpressing DEPTOR accumulate more WAT. Furtheremore, in humans, DEPTOR expression in WAT correlates with the degree of obesity. We show that DEPTOR is induced by glucocorticoids during adipogenesis and that its overexpression promotes, while its suppression blocks, adipogenesis. DEPTOR activates the pro-adipogenic Akt/PKB-PPAR-γ axis by dampening mTORC1-mediated feedback inhibition of insulin signaling. These results establish DEPTOR as a new regulator of adipogenesis.
Recognition of the lipopolysaccharide
(LPS), a major component
of the outer membrane of Gram-negative bacteria, by the Toll-like
receptor 4 (TLR4)-myeloid differentiation factor 2 (MD-2) complex
is essential for the control of bacterial infection. A pro-inflammatory
signaling cascade is initiated upon binding of membrane-associated
portion of LPS, a glycophospholipid Lipid A, by a coreceptor protein
MD-2, which results in a protective host innate immune response. However,
activation of TLR4 signaling by LPS may lead to the dysregulated immune
response resulting in a variety of inflammatory conditions including
sepsis syndrome. Understanding of structural requirements for Lipid
A endotoxicity would ensure the development of effective anti-inflammatory
medications. Herein, we report on design, synthesis, and biological
activities of a series of conformationally confined Lipid A mimetics
based on β,α-trehalose-type scaffold. Replacement of the
flexible three-bond β(1→6) linkage in diglucosamine backbone
of Lipid A by a two-bond β,α(1↔1) glycosidic linkage
afforded novel potent TLR4 antagonists. Synthetic tetraacylated bisphosphorylated
Lipid A mimetics based on a β–GlcN(1↔1)α–GlcN
scaffold selectively block the LPS binding site on both human and
murine MD-2 and completely abolish lipopolysaccharide-induced pro-inflammatory
signaling, thereby serving as antisepsis drug candidates. In contrast
to their natural counterpart lipid IVa, conformationally constrained
Lipid A mimetics do not activate mouse TLR4. The structural basis
for high antagonistic activity of novel Lipid A mimetics was confirmed
by molecular dynamics simulation. Our findings suggest that besides
the chemical structure, also the three-dimensional arrangement of
the diglucosamine backbone of MD-2-bound Lipid A determines endotoxic
effects on TLR4.
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