Sterol regulatory element-binding proteins (SREBPs) are major transcription factors activating the expression of genes involved in biosynthesis of cholesterol, fatty acid and triglyceride. In this study, we identified a small molecule, betulin, that specifically inhibited the maturation of SREBP by inducing interaction of SREBP cleavage activating protein (SCAP) and Insig. Inhibition of SREBP by betulin decreased the biosynthesis of cholesterol and fatty acid. In vivo, betulin ameliorated diet-induced obesity, decreased the lipid contents in serum and tissues, and increased insulin sensitivity. Furthermore, betulin reduced the size and improved the stability of atherosclerotic plaques. Our study demonstrates that inhibition SREBP pathway can be employed as a therapeutic strategy to treat metabolic diseases including type II diabetes and atherosclerosis. Betulin, which is abundant in birch bark, could be a leading compound for development of drugs for hyperlipidemia.

Hedgehog (Hh) has been known as the only cholesterol-modified morphogen playing pivotal roles in development and tumorigenesis. A major unsolved question is how Hh signaling regulates the activity of Smoothened (SMO). Here, we performed an unbiased biochemical screen and identified that SMO was covalently modified by cholesterol on the Asp95 (D95) residue through an ester bond. This modification was inhibited by Patched-1 (Ptch1) but enhanced by Hh. The SMO(D95N) mutation, which could not be cholesterol modified, was refractory to Hh-stimulated ciliary localization and failed to activate downstream signaling. Furthermore, homozygous Smo (the equivalent residue in mouse) knockin mice were embryonic lethal with severe cardiac defects, phenocopying the Smo mice. Together, the results of our study suggest that Hh signaling transduces to SMO through modulating its cholesterylation and provides a therapeutic opportunity to treat Hh-pathway-related cancers by targeting SMO cholesterylation.

Activation of NF-kB and MAPK/activator protein 1 (AP-1) signaling pathways by receptor activator NF-kB ligand (RANKL) is essential for osteoclast activity. Targeting NF-kB and MAPK/AP-1 signaling to modulate osteoclast activity has been a promising strategy for osteoclast-related diseases. In this study we examined the effects of maslinic acid (MA), a pentacyclic triterpene acid that is widely present in dietary plants, on RANKL-induced osteoclastogenesis, osteoclast function, and signaling pathways by in vitro and in vivo assay systems. In mouse bone marrow monocytes (BMMs) and RAW264.7 cells, MA inhibited RANKL-induced osteoclastogenesis in a dosedependent manner within nongrowth inhibitory concentration, and MA decreased osteoclastogenesis-related marker gene expression, including TRACP, MMP9, c-Src, CTR, and cathepsin K. Specifically, MA suppressed osteoclastogenesis and actin ring formation at early stage. In ovariectomized mice, administration of MA prevented ovariectomy-induced bone loss by inhibiting osteoclast activity. At molecular levels, MA abrogated the phosphorylation of MAPKs and AP-1 activity, inhibited the IkBa phosphorylation and degradation, blocked NF-kB/p65 phosphorylation, nuclear translocation, and DNA-binding activity by downregulating RANK expression and blocking RANK interaction with TRAF6. Together our data demonstrate that MA suppresses RANKL-induced osteoclastogenesis through NF-kB and MAPK/AP-1 signaling pathways and that MA is a promising agent in the treatment of osteoclast-related diseases such as osteoporosis. ß

Receptor activator of NF-kB ligand (RANKL) stimulation leads to the activation of mitogen-activated protein kinase (MAPK)/AP-1 and Ca 2þ -nuclear factor of activated T-cells cytoplasmic 1 (NFATc1) signaling pathways in osteoclastogenesis. Targeting these pathways has been an encouraging strategy for bone-related diseases, such as postmenopausal osteoporosis. In this study, we examined the effects of caffeic acid 3,4-dihydroxy-phenethyl ester (CADPE) on osteoclastogenesis. In mouse bone marrow monocytes (BMMs) and RAW264.7 cells, CADPE suppressed RANKL-induced osteoclast differentiation and actin-ring formation in a dose-dependent manner within non-growth inhibitory concentrations at the early stage, while CADPE had no effect on macrophage colony-stimulating factor (M-CSF)-induced proliferation and differentiation. At the molecular level, CADPE inhibited RANKL-induced phosphorylation of MAPKs, including extracellular signal-regulated kinases 1/2 (ERK1/2), p38, and c-Jun N-terminal kinase (JNK), without significantly affecting the NF-kB signaling pathway. CADPE abrogated RANKL-induced activator protein 1 (AP-1)/FBJ murine osteosarcoma viral oncogene homolog (c-Fos) nuclear translocation and activation. Overexpression of c-Fos prevented the inhibition by CADPE of osteoclast differentiation. Furthermore, CADPE suppressed RANKL-induced the tumor necrosis factor receptor associated factor 6 (TRAF6) interaction with c-src tyrosine kinase (c-Src), blocked RANKL-induced the phosphorylation of protein kinase B (AKT), and inhibited RANKL-induced Ca 2þ oscillation. As a result, CADPE decreased osteoclastogenesis-related marker gene expression, including NFATc1, TRAP, cathepsin K, and c-Src. To test the effects of CADPE on osteoclast activity in vivo, we showed that CADPE prevented ovariectomyinduced bone loss by inhibiting osteoclast activity. Together, our data demonstrate that CADPE suppresses osteoclastogenesis and bone loss through inhibiting RANKL-induced MAPKs and Ca 2þ -NFATc1 signaling pathways. CADPE is a novel agent in the treatment of osteoclast-related diseases, such as osteoporosis. ß

Statins are inhibitors of HMG-CoA reductase, the rate-limiting enzyme of cholesterol biosynthesis, and have been clinically used to treat cardiovascular disease. However, a paradoxical increase of reductase protein following statin treatment may attenuate the effect and increase the side effects. Here we present a previously unexplored strategy to alleviate statin-induced reductase accumulation by inducing its degradation. Inspired by the observations that cholesterol intermediates trigger reductase degradation, we identify a potent degrader, namely Cmpd 81, through structure–activity relationship analysis of sterol analogs. Cmpd 81 stimulates ubiquitination and degradation of reductase in an Insig-dependent manner, thus dramatically reducing protein accumulation induced by various statins. Cmpd 81 can act alone or synergistically with statin to lower cholesterol and reduce atherosclerotic plaques in mice. Collectively, our work suggests that inducing reductase degradation by Cmpd 81 or similar chemicals alone or in combination with statin therapy can be a promising strategy for treating cardiovascular disease.