BackgroundMagnolin is a natural compound abundantly found in Magnolia flos, which has been traditionally used in oriental medicine to treat headaches, nasal congestion and anti-inflammatory reactions. Our recent results have demonstrated that magnolin targets the active pockets of ERK1 and ERK2, which are important signaling molecules in cancer cell metastasis. The aim of this study is to evaluate the effects of magnolin on cell migration and to further explore the molecular mechanisms involved.MethodsMagnolin-mediated signaling inhibition was confirmed by Western blotting using RSK2+/+ and RSK2−/− MEFs, A549 and NCI-H1975 lung cancer cells, and by NF-κB and Cox-2 promoter luciferase reporter assays. Inhibition of cell migration by magnolin was examined by wound healing and/or Boyden Chamber assays using JB6 Cl41 and A549 human lung cancer cells. The molecular mechanisms involved in cell migration and epithelial-to-mesenchymal transition were determined by zymography, Western blotting, real-time PCR and immunocytofluorescence.ResultsMagnolin inhibited NF-κB transactivation activity by suppressing the ERKs/RSK2 signaling pathway. Moreover, magnolin abrogated the increase in EGF-induced COX-2 protein levels and wound healing. In human lung cancer cells such as A549 and NCI-H1975, which harbor constitutive active Ras and EGFR mutants, respectively, magnolin suppressed wound healing and cell invasion as seen by a Boyden chamber assay. In addition, it was observed that magnolin inhibited MMP-2 and −9 gene expression and activity. The knockdown or knockout of RSK2 in A549 lung cancer cells or MEFs revealed that magnolin targeting ERKs/RSK2 signaling suppressed epithelial-to-mesenchymal transition by modulating EMT marker proteins such as N-cadherin, E-cadherin, Snail, Vimentin and MMPs.ConclusionsThese results demonstrate that magnolin inhibits cell migration and invasion by targeting the ERKs/RSK2 signaling pathway.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1580-7) contains supplementary material, which is available to authorized users.

In this study, we provide critical evidence that STAT2 stability regulation plays an essential role in melanoma cell proliferation and colony growth. We found that the interaction of FBXW7 and STAT2 induced STAT2 destabilization via a ubiquitination-mediated proteasomal degradation pathway. Notably, GSK3β-mediated STAT2 phosphorylation facilitated STAT2–FBXW7 interactions via the DNA binding domain of STAT2 and domains 1, 2, 6, and 7 of FBXW7 WD40. Importantly, the inverse correlation between protein levels of STAT2 and FBXW7 were observed not only in human melanoma cells but also in a human skin cancer tissue array. The relationship between protein levels of STAT2 and FBXW7, cell proliferation, and colony growth were similarly observed in the melanoma cell lines SK-MEL-2, -5, and -28. Moreover, STAT2 knockdown in melanoma cells suppressed melanoma cell proliferation and colony formation. These data demonstrated that FBXW7-mediated STAT2 stability regulation plays an essential role in melanoma cell proliferation and cancer growth.

Rheumatoid arthritis (RA) is a systemic inflammatory disease that mainly affects the synovial joints. Although involvement of the fibroblast growth factor (FGF) signaling pathway has been suggested as an important modulator in RA development, no clear evidence has been provided. In this study, we found that synovial fluid basic FGF (bFGF) concentration was significantly higher in RA than in osteoarthritis (OA) patients. bFGF stimulates proliferation and migration of human fibroblast-like synoviocytes (FLSs) by activation of the bFGF-FGF receptor 3 (FGFR3)-ribosomal S6 kinase 2 (RSK2) signaling axis. Moreover, a molecular docking study revealed that kaempferol inhibited FGFR3 activity by binding to the active pocket of the FGFR3 kinase domain. Kaempferol forms hydrogen bonds with the FGFR3 backbone oxygen of Glu555 and Ala558 and the side chain of Lys508. Notably, the inhibition of bFGF-FGFR3–RSK2 signaling by kaempferol suppresses the proliferation and migration of RA FLSs and the release of activated T-cell-mediated inflammatory cytokines, such as IL-17, IL-21, and TNF-α. We further found that activated phospho-FGFR3 and -RSK2 were more highly observed in RA than in OA synovium. The hyperplastic lining and sublining lymphoid aggregate layers of RA synovium showed p-RSK2-expressing CD68+ macrophages with high frequency, while pRSK2-expressing CD4+ T-cells was observed at a lower frequency. Notably, kaempferol administration in collagen-induced arthritis mice relieved the frequency and severity of arthritis. Kaempferol reduced osteoclast differentiation in vitro and in vivo relative to the controls and was associated with the inhibition of osteoclast markers, such as tartrate-resistant acid phosphatase, integrin β3, and MMP9. Conclusively, our data suggest that bFGF-induced FGFR3–RSK2 signaling may play a critical role during the initiation and progression of RA in terms of FLS proliferation and enhanced osteoclastogenesis, and that kaempferol may be effective as a new treatment for RA.

Ribosomal S6 kinase 2 (RSK2), regulated by Ras/Raf/MEKs/ERKs, transmits upstream activation signals to downstream substrates including kinases and transcription and epigenetic factors. We observed that ELK members, including ELK1, 3, and 4, highly interacted with RSK2. We further observed that the RSK2-ELK3 interaction was mediated by N-terminal kinase and linker domains of RSK2, and the D and C domains of ELK3, resulting in the phosphorylation of ELK3. Importantly, RSK2-mediated ELK3 enhanced c-fos promoter activity. Notably, chemical inhibition of RSK2 signaling using kaempferol (a RSK2 inhibitor) or U0126 (a selective MEK inhibitor) suppressed EGF-induced c-fos promoter activity. Moreover, functional deletion of RSK2 by knockdown or knockout showed that RSK2 deficiency suppressed EGF-induced c-fos promoter activity, resulting in inhibition of AP-1 transactivation activity and Ras-mediated foci formation in NIH3T3 cells. Immunocytofluorescence assay demonstrated that RSK2 deficiency reduced ELK3 localization in the nucleus. In MDA-MB-231 breast cancer cells, knockdown of RSK2 or ELK3 suppressed cell proliferation with accumulation at the G1 cell cycle phase, resulting in inhibition of foci formation and anchorage-independent cancer colony growth in soft agar. Taken together, these results indicate that a novel RSK2/ELK3 signaling axis, by enhancing c-Fos-mediated AP-1 transactivation activity, has an essential role in cancer cell proliferation and colony growth.

Our previous studies demonstrated that RSK2 plays a key role in cell proliferation and transformation induced by tumor promoters such as epidermal growth factor (EGF) in mouse and human skin cells. However, no direct evidence has been found regarding the relationship of RSK2 and cell survival. In this study, we found that RSK2 interacted and phosphorylated GSK3β at Ser9. Notably, GSK3β phosphorylation at Ser9 was suppressed in RSK2(-/-) MEFs compared with RSK2(+/+) MEFs by stimulation of EGF and calcium ionophore A23187, a cellular calcium stressor. In proliferation, we found that RSK2 deficiency suppressed cell proliferation compared with RSK2(+/+) MEFs. In contrast, GSK3β(-/-) MEFs induced the cell proliferation compared with GSK3β(+/+) MEFs. Importantly, RSK2(-/-) MEFs were induced severe cellular morphology change by A23187 and enhanced G1/G0 and sub-G1 accumulation of the cell cycle phase compared with RSK2(+/+) MEFs. The sub-G1 induction in RSK2(-/-) MEFs by A23187 was correlated with increase of cytochrome c release, caspase-3 cleavage and apoptotic DNA fragmentation compared with RSK2(+/+) MEFs. Notably, return back of RSK2 into RSK2(-/-) MEFs restored A23187-induced morphological change, and decreased apoptosis, apoptotic DNA fragmentation and caspase-3 induction compared with RSK2(-/-)/mock MEFs. Taken together, our results demonstrated that RSK2 plays an important role in stress-tolerance and cell survival, resulting in cell proliferation and cancer development.