Massive elimination of multinucleated osteoclasts by eupatilin is due to dual inhibition of transcription and cytoskeletal rearrangement

Abstract

Osteoporosis is an aging-associated disease requiring better therapeutic modality. Eupatilin is a major flavonoid from Artemisia plants such as Artemisia princeps and Artemisia argyi which has been reported to possess various beneficial biological effects including anti-inflammation, anti-tumor, anti-cancer, anti-allergy, and anti-oxidation activity. Complete blockade of RANK-dependent osteoclastogenesis was accomplished upon stimulation prior to the receptor activator of nuclear factor κB (RANK)-ligand (RANKL) treatment or post-stimulation of bone marrow macrophages (BMCs) in the presence of RANKL with eupatilin. This blockade was accompanied by inhibition of rapid phosphorylation of Akt, GSK3β, ERK and IκB as well as downregulation of c-Fos and NFATc1 at protein, suggesting that transcriptional suppression is a key mechanism for anti-osteoclastogenesis. Transient reporter assays or gain of function assays confirmed that eupatilin was a potent transcriptional inhibitor in osteoclasts (OC). Surprisingly, when mature osteoclasts were cultured on bone scaffolds in the presence of eupatilin, bone resorption activity was also completely blocked by dismantling the actin rings, suggesting that another major acting site of eupatilin is cytoskeletal rearrangement. The eupatilin-treated mature osteoclasts revealed a shrunken cytoplasm and accumulation of multi-nuclei, eventually becoming fibroblast-like cells. No apoptosis occurred. Inhibition of phosphorylation of cofilin by eupatilin suggests that actin may play an important role in the morphological change of multinucleated cells (MNCs). Human OC similarly responded to eupatilin. However, eupatilin has no effects on osteoblast differentiation and shows cytotoxicity on osteoblast in the concentration of 50 μM. When eupatilin was administered to LPS-induced osteoporotic mice after manifestation of osteoporosis, it prevented bone loss. Ovariectomized (OVX) mice remarkably exhibited bone protection effects. Taken together, eupatilin is an effective versatile therapeutic intervention for osteoporosis via; 1) transcriptional suppression of c-Fos and NFATc1 of differentiating OC and 2) inhibition of actin rearrangement of pathogenic MNCs.

Reversion of in vivo fibrogenesis by novel chromone scaffolds

“…Taken together, these data suggest that eupatilin inhibits proliferation and drives morphological change in DHLFs by negatively regulating the transdifferentiation into myofibroblasts. In previous work, we demonstrated that eupatilin does not promote cell death [18]. Unexpectedly, our findings indicated that the HLEC/TGF-β axis does not contribute to IPF.…”

“…Formation of latent TGF-β complex loaded with processed active TGF-β, along with LTBP1 or LTBP4, integrin αvβ3 (or other integrin β family member), and LAP1, in the ECM is prerequisite for activation of TGFR [35]. Integrin αvβ3 is anchored to actin filaments at focal adhesions [36], and we showed previously that eupatilin has potent actin depolymerizing activity [18]. To explore the anti-fibrotic potential of eupatilin, we conducted a series of ICC experiments involving inhibition of actin depolymerization, which results in dismantling of the latent TGF-β complex.…”

“…Chromone, 1,4-benzopyrone-4-one, is a central chemical scaffold in flavones [16], and chromone derivatives (CDs) are a diverse family of compounds in which branching chemical residues are coupled to this core chemical framework [17]. We recently reported that eupatilin, a CD from an Artemisia species, dramatically inhibited osteoclastogenesis via actin depolymerization in cells differentiated from bone marrow–derived macrophages in the presence of macrophage-colony stimulating factor (M-CSF) and receptor activator of nuclear factor kappa-Β ligand (RANKL) [18], and a vast majority of the downregulated genes associated with the epithelial mesenchymal transition (EMT), which is a hallmark of fibrosis. Specifically, 24 of the 50 top genes differentially regulated by eupatilin during osteoclastogenesis are associated with EMT, prompting us to hypothesize that eupatilin could prevent fibrosis (Fig.…”

Chromone scaffold–mediated reprogramming of the epithelial–mesenchymal transition prevents fibrosis

“…Taken together, these data suggest that eupatilin inhibits proliferation and drives morphological change of DHLFs, thereby negatively regulating EMT. In previous work, we demonstrated that eupatilin does not promote cell death (10).…”

A noble TGF beta biogenesis inhibitor exhibits both potent anti-fibrotic and anti-inflammatory capabilities

“…10 Due to their anti-oxidant and anti-inflammatory activities, flavones have long been used to treat inflammatory diseases such as arthritis and asthma 11 Chromone, 1,4-benzopyrone-4-one, is a central chemical scaffold for flavones, 12 with chromone scaffoldcontaining derivatives (CSD) being a diverse family, with branching chemical residues coupled to this core chemical scaffold. 13 We recently reported that eupatilin, a CSD from an Artemisia species, dramatically inhibits lipopolysaccharide (LPS)-induced osteoporosis, partially via actin depolymerization 14 and the downregulation of multiple genes associated with the epithelial-mesenchymal transition (EMT). The present study shows that a noble CSD, ONG41008, can block the induction of Type 2 EMT, also called fibrogenesis, arrest cell growth and alter cell morphology, thereby substantially ameliorating lung fibrosis.…”