Inhibiting O-GlcNAcylation impacts p38 and Erk1/2 signaling and perturbs cardiomyocyte hypertrophy.

The dynamic cycling of O-linked GlcNAc (O-GlcNAc) on and off Ser/Thr residues of intracellular proteins, termed O-GlcNAcylation, is mediated by the conserved enzymes O-GlcNAc transferase (OGT) and O-GlcNAcase. O-GlcNAc cycling is important in homeostatic and stress responses, and its perturbation sensitizes the heart to ischemic and other injuries. Despite considerable progress, many molecular pathways impacted by O-GlcNAcylation in the heart remain unclear. The mitogen-activated protein kinase (MAPK) pathway is a central signaling cascade that coordinates developmental, physiological, and pathological responses in the heart. The developmental or adaptive arm of MAPK signaling is primarily mediated by Erk kinases, while the pathophysiologic arm is mediated by p38 and Jnk kinases. Here, we examine whether O-GlcNAcylation affects MAPK signaling in cardiac myocytes, focusing on Erk1/2 and p38 in basal and hypertrophic conditions induced by phenylephrine. Using metabolic labeling of glycans coupled with alkyne-azide "click" chemistry, we found that Erk1/2 and p38 are O-GlcNAcylated. Supporting the regulation of p38 by O-GlcNAcylation, the OGT inhibitor, OSMI-1, triggers the phosphorylation of p38, an event that involves the NOX2-Ask1-MKK3/6 signaling axis and also the noncanonical activator Tab1. Additionally, OGT inhibition blocks the phenylephrine-induced phosphorylation of Erk1/2. Consistent with perturbed MAPK signaling, OSMI-1-treated cardiomyocytes have a blunted hypertrophic response to phenylephrine, decreased expression of cTnT (key component of the contractile apparatus), and increased expression of maladaptive natriuretic factors Anp and Bnp. Collectively, these studies highlight new roles for O-GlcNAcylation in maintaining a balanced activity of Erk1/2 and p38 MAPKs during hypertrophic growth responses in cardiomyocytes.

CCL20 induced by visfatin in macrophages via the NF-κB and MKK3/6-p38 signaling pathways contributes to hepatic stellate cell activation.

Chemokines interact with hepatic resident cells during inflammation and fibrosis. CC chemokine ligand (CCL) 20 has been reported to be important in inflammation and fibrosis in the liver. We hypothesized that visfatin, an adipocytokine, could play a role in hepatic fibrosis via CCL20. We investigated the effect of visfatin on CCL20 in THP-1 human promonocytic cells and examined the molecular mechanisms involved. Following treatment of THP-1 cells with visfatin, CCL20 expression and secretion were assessed. We assessed the intracellular signaling molecules IKK/NF-κB, JAK2/STAT3, MAPKs, and MKK3/6 by western blotting. We treated THP-1 cells with visfatin and signaling inhibitors, and examined CCL20 mRNA and protein levels. To investigate the effect of visfatin-induced CCL20 expression in hepatic stellate cells (HSCs), LX-2 cells were co-cultured with the culture supernatant of THP-1 cells with or without anti-CCL20 neutralizing antibodies, and fibrosis markers were examined by RT-PCR and immunoblotting. In THP-1 cells, visfatin increased the CCL20 mRNA and protein levels. visfatin increased the activities of the NF-κB, p38, and MLK3/6 signaling pathways but not those of the JAK2/STAT3 and ERK pathways. Visfatin treatment together with an NF-κB, p38, or MLK3 inhibitor reduced the mRNA and protein levels of CCL20. The visfatin-induced CCL20 increased the expression of fibrosis markers and CCR6 in HSCs. Following neutralization of CCL20, the levels of fibrosis markers and CCR6 were decreased. Visfatin increases the expression of CCL20 via the NF-κB and MKK3/6-p38 signaling pathways in macrophages, and visfatin-induced CCL20 expression promotes the fibrosis markers in HSCs.

Therapeutic effect of Rhizoma Dioscoreae Nipponicae on gouty arthritis based on the SDF-1/CXCR 4 and p38 MAPK pathway: an in vivo and in vitro study.

Rhizoma Dioscoreae Nipponicae (RDN) is a widely used traditional Chinese herb, which is used to treat arthroncus, arthrodynia and arthritis. As is known to us, inflammatory mechanisms have played an important role in the occurrence, course and prognosis of gouty arthritis (GA). The aim of this study was to determine the characteristic expressed proteins of synovium in GA rat and synovial cell. The rat model of GA was induced by monosodium urate (MSU) crystal. Tissue samples were assayed by immunohistochemical method. The effects of RDN on Stromal cell-derived factor 1 (SDF-1), CXCR 4 and p38 mitogen-activated protein kinase (MAPK) were investigated in MSU crystal-induced rat. The levels of SDF-1 and mitogen-activated kinase kinase (MKK) 3/6 were measured by Western Blot in interleukin-1ß (IL-1ß) incubated fibroblast-like synoviocytes (FLS). A significant increase in the levels of SDF-1, CXCR 4 and p38 MAPK were observed in MSU crystal-induced rat. The increased SDF-1 and MKK 3/6 levels were observed in IL-1ß incubated FLS. With the treatment of RDN, the above changes were reverted back to near normal levels. RDN might have some therapeutic effects on GA through SDF-1/CXCR 4 and p38 MAPK pathway, and dioscin may be the active compound in RDN to exert therapeutic effect on GA.

Suppression of CGRP and TRPV1 Expression in Dorsal Root Ganglia of Knee Osteoarthritis Rats by Huojing Decoction via TrkA/MKK3/6/p38 Pathway.

Objective: Knee osteoarthritis (KOA) is a chronic condition characterized by persistent pain that can lead to severe disability. In this study, we primarily investigated the analgesic effect of Huojing decoction on MIA-induced knee arthritis. Methods: The network pharmacology method was employed to acquire target information of Huojing decoction and KOA. MIA was intratibially injected to induce KOA pain in rats. Huojing decoction was then administered once daily via intragastric administration for 14 days. Pain level was assessed by paw withdrawal threshold (PWT) and paw withdrawal latency (PWL). The levels of inflammatory cytokines were determined by ELISA and PCR. TRPV1 and CGRP were detected through immunohistochemistry. The protein expression of TrkA, MKK3/6 and p38 was assessed by Western blot. Results: Mechanical allodynia and thermal hyperalgesia were observed in KOA rats. The expression levels of inflammatory cytokines were significantly decreased after Huojing decoction infusion of KOA rats. TRPV1 and CGRP were reduced with treatment. Furthermore, the protein expressions of TrkA, MKK3/6 and p38 in the DRG of rats were significantly decreased. Conclusion: Our data suggested that Huojing decoction can alleviate inflammation in KOA pain rats. Additionally, it can inhibit the expression of TrKA, MKK3/6 and p38 signaling pathways, indicating its analgesic effect on KOA pain rats.

The aryl hydrocarbon receptor is a novel negative regulator of interleukin-17-mediated signaling and inflammation in vitro.

Interleukin (IL)-17 plays a critical role in the pathogenesis of inflammation and autoimmune diseases. The aryl hydrocarbon receptor (AHR) is a transcription factor responsible for the elimination of xenobiotic chemicals. However, it remains unknown whether AHR is involved in IL-17 signaling. Here, we demonstrate that knockdown of AHR significantly enhances, while overexpression or activation of AHR inhibits IL-17-induced inflammation in Hela cells. AHR specifically suppresses IL-17-induced p38 activation, and inhibition of p38 activity markedly reverses the effect of AHR knockdown on IL-17-induced inflammation. Mechanistically, AHR physically interacts with TAK1 and mitogen-activated protein kinase kinase 3/6 (MKK3/6) and disrupts TAK1-MKK3/6 interaction, leading to impaired IL-17 signaling. Thus, our study indicates that AHR negatively regulates IL-17-mediated signaling and inflammation at least partially through interfering with the interaction between TAK1 and MKK3/6.

Gossypetin is a novel MKK3 and MKK6 inhibitor that suppresses esophageal cancer growth in vitro and in vivo.

Gossypetin as a hexahydroxylated flavonoid found in many flowers and Hibiscus. It exerts various pharmacological activities, including antioxidant, antibacterial and anticancer activities. However, the anticancer capacity of gossypetin has not been fully elucidated. In this study, gossypetin was found to inhibit anchorage-dependent and -independent growth of esophageal cancer cells. To identify the molecular target(s) of gossypetin, various signaling protein kinases were screened and results indicate that gossypetin strongly attenuates the MKK3/6-p38 signaling pathway by directly inhibiting MKK3 and MKK6 protein kinase activity in vitro. Mechanistic investigations showed that arginine-61 in MKK6 is critical for binding with gossypetin. Additionally, the inhibition of cell growth by gossypetin is dependent on the expression of MKK3 and MKK6. Gossypetin caused G2 phase cell cycle arrest and induced intrinsic apoptosis by activating caspases 3 and 7 and increasing the expression of BAX and cytochrome c. Notably, gossypetin suppressed patient-derived esophageal xenograft tumor growth in an in vivo mouse model. Our findings suggest that gossypetin is an MKK3 and MKK6 inhibitor that could be useful for preventing or treating esophageal cancer.

Fermented Extraction of Citrus unshiu Peel Inhibits Viability and Migration of Human Pancreatic Cancers.

Pancreatic cancer is one of the most dangerous cancers with high mortality rates. Despite continuous efforts, there has been limited improvement in its prognosis. In this study, we prepared fermented extract of Citrus unshiu peel (fCUP) from the by-product after juice processing and then examined the anticancer effects of fCUP on human pancreatic cancer cells. Treatment with fCUP inhibited the growth of human pancreatic cancer cells through induction of caspase-3 cleavage both in vitro and in vivo. Treatment with fCUP also blocked the migration of human pancreatic cancer cells through activation of intracellular signaling pathways such as MKK3/6 and P38. In contrast, treatment with fCUP did not inhibit growth and migration of human umbilical vein endothelial cells. In addition, we found that fCUP mainly consisted of aboriginal compounds, narirutin and hesperidin, as well as newly generated compounds, naringenin and hesperetin. In silico analysis showed that naringenin and hesperetin were the unique modules related to anticancer effect. Furthermore, fCUP exhibited the anticancer effects in in vivo xenograft models. Collectively, these results suggest that fCUP might have the potential to be developed into an effective anticancer drug for pancreatic cancers without causing adverse side-effects.

Chloroform fraction of Solanum tuberosum L. cv Jayoung epidermis suppresses LPS-induced inflammatory responses in macrophages and DSS-induced colitis in mice.

In this study, the authors investigated the molecular mechanism underlying the antiinflammatory effects of the chloroform fraction of the peel of 'Jayoung' (CFPJ), a color-fleshed potato, on lipopolysaccharide (LPS)-induced RAW 264.7 macrophages and in mice with dextran sulfate sodium (DSS)-induced colitis. CFPJ inhibited the expressions of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) at the transcription level, and attenuated the transcriptional activity of nuclear factor-κB (NF-κB) by reducing the translocation of NF-κB depending on degradation of inhibitory κB-α (IκB-α). Furthermore, CFPJ attenuated the phosphorylations of mitogen-activated protein kinase kinases3/6 (MKK3/6) and of p38. In colitis model, CFPJ significantly reduced the severity of colitis and the productions and protein levels of pro-inflammatory mediators in colonic tissue. These results suggest that the anti-inflammatory effects of CFPJ are associated with the suppression of NF-κB and p38 activation in macrophages, and support its possible therapeutic role for the treatment of colitis.