Stress-activated Protein kinase/c-Jun N-terminal Kinase Research Articles

Resveratrol improves sperm parameter and testicular apoptosis in cisplatin-treated rats: Effects on ERK1/2, JNK, and Akt pathways

ABSTRACTThis study aimed to investigate the protective role of resveratrol (RES) against cisplatin (Cis)-induced testicular damage and reproductive dysfunction in rats and to examine the underlying mechanisms of protection including its effect on endoplasmic reticulum (ER) stress, P53, extracellular signal-regulated kinase (ERK)-1/2, stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK), and Protein kinase B (Akt) signaling. Eight-week-old Rats were divided into four groups (n = 12 each) of 1) control group: received normal saline (i.p.) as vehicle for 45 days, 2) RES-treated group: received RES (20 mg/kg, i.p) for 45 days, 3) Cis-treated group: received Cis (3 mg/kg) for 3 days and then continued on normal saline, and 4) Cis + RES-treated group: received Cis for the first 3 days and then continued on RES for the next 45 days. Serum sex hormones levels, sperm parameters, and levels of testicular antioxidant potential and inflammatory mediators were assessed in all rats. In addition, activation of ER stress, P53, ERK1/2, JNK, and Akt and markers of apoptosis were evaluated in rats’ testis. Cis lowered sperm count and motility and increased sperm morphological abnormalities. Testis of Cis-treated rats had low expression of antioxidant enzymes including SOD, CAT, and GPx and decreased the level of GSH. Concomitantly, Cis upregulated levels of cleaved caspase-3, P53, calpain-1/cleaved caspase-12, p-ERK1/2, and p-SAPK/p-JNK. However, RES administration post-Cis administration restored all sperm parameters and prevented testicular apoptosis mediated by inhibition of all above-mentioned apoptotic pathways. Moreover, RES enhanced testosterone, FSH, and LH levels and upregulated p-Akt/p-Bad levels in both control and Cis-treated rats. In conclusion, RES protects against Cis-induced testicular damage and reproductive dysfunction via improving testosterone levels, increasing sperm count, reducing testicular apoptosis via an antioxidant potential, inhibition of ER stress, P53, ERK1/2, JNK, and activation of Akt.Abbreviations: RES: resveratrol, Cis: cisplatin; ER: endoplasmic reticulum; ERK1/2: extracellular signal-regulated kinase1/2; SAPK/JNK: stress-activated protein kinase/c-Jun N-terminal kinase; Akt: protein kinase B; HPG axis: hypothalamic-pituitary-gonadal axis; PUFAs: polyunsaturated fatty acids; FSH: Follicular stimulating hormone; LH: Luteinizing hormone; PBS: phosphate buffered saline; GSH: reduced glutathione; GSSG: glutathione disulfide; TNF-α: tumor necrosis factor-α; IL-6: interleukin-6; GRx: glutathione reductase; SOD: superoxide dismutase; CAT: catalase; 4HNE: 4-hydroxynonenal.

The Role of p38 and CK2 Protein Kinases in the Response of RAW 264.7 Macrophages to Lipopolysaccharide

The role of protein kinases p38 and CK2 (casein kinase II) in the response of RAW 264.7 macrophages to the lipopolysaccharide (LPS) from gram-negative bacteria was studied. Using specific p38 and CK2 inhibitors (p38 MAP kinase Inhibitor XI and casein kinase II Inhibitor III, respectively), we investigated the effects of these protein kinases on (i) LPS-induced activation of signaling pathways involving nuclear factor κB (NF-κB), stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK), p38, and interferon regulatory factor 3 (IRF3); (ii) expression of Toll-like receptor 4 (TLR4) and inducible heat-shock proteins HSP72 and HSP90; and (iii) production of interleukins IL-1α, IL-1β, IL-6, tumor necrosis factor α, and IL-10. Activation of the proapoptotic signaling in the macrophages was evaluated from the ratio between the active and inactive caspase-3 forms and p53 phosphorylation. Six hours after LPS addition (2.5 μg/ml) to RAW 264.7 cells, activation of the TLR4 signaling pathways was observed that was accompanied by a significant increase in phosphorylation of IκB kinase α/β, NF-κB (at both Ser536 and Ser276), p38, JNK, and IRF3. Other effects of macrophage incubation with LPS were an increase in the contents of TLR4, inducible heat-shock proteins (HSPs), and pro- and anti-inflammatory cytokines, as well as slight activation of the pro-apoptotic signaling in the cells. Using inhibitor analysis, we found that during the early response of macrophages to the LPS, both CK2 and p38 modulate activation of MAP kinase and NF-κB signaling pathways and p65 phosphorylation at Ser276/Ser536 and cause accumulation of HSP72, HSP90 and the LPS-recognizing receptor TLR4. Suppression of the p38 MAP kinase and CK2 activities by specific inhibitors (Inhibitor XI and Inhibitor III, respectively) resulted in the impairment of the macrophage effector function manifested as a decrease in the production of the early-response proinflammatory cytokines and disbalance between the pro- and anti-apoptotic signaling pathways leading presumably to apoptosis development. Taken together, our data indicate the inefficiency of therapeutic application of p38 and CK2 inhibitors during the early stages of inflammatory response.

Inhibitors of heat shock protein 90 augment endothelin‑1‑induced heat shock protein 27 through the SAPK/JNK signaling pathway in osteoblasts.

It has been previously reported that endothelin‑1 (ET‑1) stimulates the induction of heat shock protein (HSP) 27 through the activation of p38 mitogen‑activated protein (MAP) kinase and stress‑activated protein kinase/c‑Jun N‑terminal kinase (SAPK/JNK) in osteoblast‑like MC3T3‑E1 cells. The present study investigated whether HSP90, a high‑molecular‑weight HSP, was implicated in the ET‑1‑stimulated HSP27 induction in MC3T3‑E1 cells. The effects of HSP90 inhibitors on the induction of HSP27 were examined. The HSP90 inhibitors geldanamycin and 17‑demethoxygeldanamycin (17‑DMAG) significantly amplified HSP27 induction stimulated by ET‑1 in a dose‑dependent manner. In addition, onalespib (another HSP90 inhibitor) significantly strengthened the ET‑1‑induced HSP27 protein levels. The ET‑1‑stimulated phosphorylation of p38 MAP kinase was minimally affected by geldanamycin, 17‑DMAG or onalespib. Onalespib and 17‑DMAG significantly enhanced the ET‑1‑induced phosphorylation of SAPK/JNK. In addition, SP600125, a SAPK/JNK inhibitor, notably reduced the amplification by onalespib of ET‑1‑induced HSP27. These results suggest that HSP90 limits ET‑1‑stimulated HSP27 induction at a point upstream of SAPK/JNK in osteoblasts. These results suggest that HSP90 may be a novel clinical target for metabolic bone diseases, including osteoporosis.

Balanites aegyptiaca ameliorates insulin secretion and decreases pancreatic apoptosis in diabetic rats: Role of SAPK/JNK pathway

SAPK-JNK pathway performs a significant role in the pathogenesis of type 2 diabetes. Balanites aegyptiaca (BA) is used as an anti-diabetic agent in folk medicine however its hypoglycemic mechanism is not fully elucidated. The current study aimed to evaluate the effect of crude extract, butanol, and dichloromethane fractions from BA on the stress-activated protein kinase/c-Jun N-terminal kinase (SAPK-JNK) pathway in experimental diabetic rats. Six groups of male Wistar rats were included: normal control, diabetic, diabetic rats treated with crude, butanol or dichloromethane fraction from BA (50 mg/kg BW) and diabetic rats treated with gliclazide as a reference drug for one month. Our results suggested a protective role of treatment of diabetic rats with BA against oxidative stress-induced SAPK-JNK pathway. Moreover, BA treatment produced a reduction in plasma glucose, HbA1c, lactic acid, lipid profile, malondialdehyde levels and produced an increase in insulin, reduced glutathione levels, catalase and superoxide dismutase activities compared with untreated diabetic rats. Moreover, it decreased apoptosis signal-regulating kinase 1, c-Jun N-terminal kinase 1, protein 53 and increased insulin receptor substrate 1 in rat pancreas while it increased glucose transporter 4 in rat muscle. Analysis of BA extracts by LC-HRMS revealed the presence of different saponins with reported hypoglycemic effect. In conclusion, BA exerted hypoglycemic, hypolipidemic, insulinotropic and antioxidant effects. Additionally, it reduced apoptosis in pancreatic β-cells and increased glucose uptake in muscle. These results suggest that the hypoglycemic effect of BA is due to the inhibition of the SAPK-JNK pathway.

Anticancer effect of acid ceramidase inhibitor ceranib-2 in human breast cancer cell lines MCF-7, MDA MB-231 by the activation of SAPK/JNK, p38 MAPK apoptotic pathways, inhibition of the Akt pathway, downregulation of ERα.

Acid ceramidase is the key enzyme of the ceramide metabolic pathway, which plays a vital role in regulating ceramide - sphingosine-1-phosphate rheostat. Ceramide acts as a proapoptotic molecule, but its metabolite sphingosine-1-phosphate, in contrast, signals for cell proliferation, cell survival, and angiogenesis. Acid ceramidase is highly upregulated in breast tumors and treatment with an acid ceramidase inhibitor, ceranib-2, significantly induced apoptosis in human breast cancer cell lines. However, the mechanisms underlying the induction of apoptosis remain ambiguous to date. Hence, in the present study, we have explored ceranib-2-mediated apoptotic signaling pathways in human breast cancer cell lines. MCF-7 and MDA MB-231 cells were treated with IC50 doses of ceranib-2 and tamoxifen. Nuclear changes showed the apoptotic effect of ceranib-2 in both the cell lines. Loss in the mitochondrial membrane potential was observed only in ceranib-2-treated MCF-7 cells. Ceranib-2 activated intrinsic and extrinsic apoptotic pathways in MCF-7 cells, but only the extrinsic apoptotic pathway was activated in MDA MB-231 cells. Further, ceranib-2 induced apoptosis by activating SAPK/JNK (stress-activated protein kinase/c-Jun N-terminal kinase), p38 MAPK (mitogen-activated protein kinase) apoptotic pathways and by inhibiting the Akt (antiapoptotic) pathway in both the cell lines. Most importantly, ERα (estrogen receptor-α) expression was highly downregulated after ceranib-2 treatment and a docking study predicted the highest binding affinity of ceranib-2 than tamoxifen with ERα in MCF-7 cells. Hence, ceranib-2 may have potential as a chemotherapeutic drug of breast cancer.

Heat shock protein 27 (HSPB1) suppresses the PDGF-BB-induced migration of osteoblasts.

Heat shock protein 27 (HSP27/HSPB1), one of the small heat shock proteins, is constitutively expressed in various tissues. HSP27 and its phosphorylation state participate in the regulation of multiple physiological and pathophysiological cell functions. However, the exact roles of HSP27 in osteoblasts remain unclear. In the present study, we investigated the role of HSP27 in the platelet-derived growth factor-BB (PDGF-BB)-stimulated migration of osteoblast-like MC3T3-E1 cells. PDGF-BB by itself barely upregulated the expression of HSP27 protein, but stimulated the phosphorylation of HSP27 in these cells. The PDGF-BB-induced cell migration was significantly downregulated by HSP27 overexpression. The PDGF-BB-induced migrated cell numbers of the wild-type HSP27-overexpressing cells and the phospho-mimic HSP27-overexpressing (3D) cells were less than those of the unphosphorylatable HSP27-overexpressing (3A) cells. PD98059, an inhibitor of MEK1/2, SB203580, an inhibitor of p38 mitogen-activated protein kinase, and SP600125, an inhibitor of stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) reduced the PDGF-BB-induced migration of these cells, whereas Akt inhibitor or rapamycin, an inhibitor of upstream kinase of p70 S6 kinase (mTOR), barely affected the migration. However, the PDGF-BB-induced phosphorylation of p44/p42 MAPK, p38 MAPK and SAPK/JNK was not affected by HSP27 overexpression. There were no significant differences in the phosphorylation of p44/p42 MAPK, p38 MAP kinase or SAPK/JNK between the 3D cells and the 3A cells. These results strongly suggest that HSP27 functions as a negative regulator in the PDGF-BB-stimulated migration of osteoblasts, and the suppressive effect is amplified by the phosphorylation state of HSP27.

3,5,4\u2032-trihydroxy-6,7,3\u2032-trimethoxyflavone protects against beta amyloid-induced neurotoxicity through antioxidative activity and interference with cell signaling

BackgroundAlzheimer’s disease is a neurodegenerative disease, characterized by progressive decline in memory and cognitive functions, that results from loss of neurons in the brain. Amyloid beta (Aβ) protein and oxidative stress are major contributors to Alzheimer’s disease, therefore, protecting neuronal cells against Aβ-induced toxicity and oxidative stress might form an effective approach for treatment of this disease. 3,5,4′-trihydroxy-6,7,3′-trimethoxyflavone (TTF) is a flavonoid we have purified from the plant Achillea fragrantissima; and the present study examined, for the first time, the effects of this compound on Aβ-toxicity to neuronal cells.MethodsVarious chromatographic techniques were used to isolate TTF from the plant Achillea fragrantissima, and an N2a neuroblastoma cell line was used to study its activities. The cellular levels of total and phosphorylated stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) and of total and phosphorylated extracellular signal-regulated kinase (ERK 1/2) were determined by enzyme-linked immunosorbent assay (ELISA). Intracellular reactive oxygen species (ROS) levels were measured by using 2′,7′-dichlorofluorescein diacetate (DCF-DA). Cytotoxicity and cell viability were assessed by using lactate dehydrogenase (LDH) activity in cell-conditioned media, or by crystal violet cell staining, respectively.ResultsTTF prevented the Aβ-induced death of neurons and attenuated the intracellular accumulation of ROS following treatment of these cells with Aβ. TTF also inhibited the Aβ-induced phosphorylation of the signaling proteins SAPK/JNK and ERK 1/2, which belong to the mitogen-activated protein kinase (MAPK) family.ConclusionTTF should be studied further as a potential therapeutic means for the treatment of Alzheimer’s disease.

Amplification by (‑)‑epigallocatechin gallate of prostaglandin F2α‑stimulated synthesis of osteoprotegerin in osteoblasts.

(‑)‑Epigallocatechin gallate (EGCG) and chlorogenic acid (CGA), major flavonoids in green tea, and coffee, respectively, are recognized as possessing potential benefits in a multitude of human health conditions, including bone disorders. We have previously demonstrated that prostaglandin F2α (PGF2α), a potent bone remodeling mediator, stimulates the synthesis of osteoprotegerin (OPG) through the activation of p44/p42 mitogen‑activated protein kinase (MAPK), p38 MAPK and stress activated protein kinase/c‑Jun N‑terminal kinase (SAPK/JNK) in osteoblast‑like MC3T3‑E1 cells. In the present study, the effects of EGCG and CGA on PGF2α‑stimulated OPG synthesis in MC3T3‑E1 cells were investigated. EGCG significantly upregulated PGF2α‑stimulated OPG release, whereas CGA did not affect OPG release. The PGF2α‑induced expression level of OPG mRNA was enhanced by EGCG. Regarding the intracellular signaling underlying the effect of EGCG, EGCG failed to affect PGF2α‑stimulated phosphorylation of p44/p42 MAPK, p38 MAPK or SAPK/JNK. EGCG by itself markedly induced the phosphorylation of p44/p42 MAP kinase for up to 10min and the status decreased subsequently, whereas EGCG did not significantly affect the phosphorylation status of p38 MAPK or SAPK/JNK within 60min. These results indicated that EGCG, but not CGA amplifies the PGF2α‑stimulated OPG synthesis in osteoblasts.

Attenuation of prostaglandin E1‑induced osteoprotegerin synthesis in osteoblasts by normoxic HIF inducers.

Mimosine, which is a natural plant amino acid present in the Leucaena genus, is able to induce hypoxia‑inducible factors (HIFs). Previous evidence has indicated that HIF regulates angiogenesis‑osteogenesis coupling in bone metabolism, and it has previously been reported that mimosine inhibits prostaglandin (PG)F2α‑induced osteoprotegerin (OPG) synthesis without affecting interleukin‑6 (IL‑6) production in osteoblast‑like MC3T3‑E1 cells. In addition, PGE1 has been demonstrated to induce OPG synthesis via activation of p38 mitogen‑activated protein (MAP) kinase and stress‑activated protein kinase/c‑Jun N‑terminal kinase (SAPK/JNK) in these cells, and PGE1 stimulates IL‑6 production via the activation of protein kinase A. In the present study, the effects of mimosine on the PGE1‑stimulated synthesis of OPG and IL‑6 were investigated in osteoblast‑like MC3T3‑E1 cells. The concentrations of OPG and IL‑6 were measured using relevant ELISA kits. OPG mRNA was measured by semi‑quantitative reverse transcription polymerase chain reaction. The phosphorylation of p38 MAP kinase and SAPK/JNK was analyzed by western blotting. Mimosine significantly reduced PGE1‑induced release of OPG and OPG mRNA expression levels without affecting the release of IL‑6. In addition, deferoxamine, which is also a normoxic HIF inducer, significantly inhibited PGE1‑induced OPG release and OPG mRNA expression levels; however, it had little effect on IL‑6 release. Furthermore, mimosine and deferoxamine failed to affect PGE1‑stimulated phosphorylation of p38 MAP kinase or SAPK/JNK. These results strongly suggest that normoxic HIF inducers attenuate PGE1‑stimulated OPG synthesis without affecting IL‑6 production in osteoblasts.

BPGAP1 spatially integrates JNK/ERK signaling crosstalk in oncogenesis.

Simultaneous hyperactivation of stress-activated protein kinase/c-Jun N-terminal protein kinase (SAPK/JNK) and mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (MEK/ERK) signaling cascades has been reported in carcinogenesis. However, how they are integrated to promote oncogenesis remains unknown. By analyzing breast invasive carcinoma database (The Cancer Genome Altas), we found that the mRNA expression levels of both JNK1 and ERK2 are positively correlated with the mRNA level of EEA1, an endosome associated protein, indicating the potential JNK/ERK crosstalk at endosome. Unbiased screen of different endosome-associated Rab GTPases reveals that late endosome serves as a unique platform to integrate JNK/ERK signaling. Furthermore, we identify that BPGAP1 (a BCH domain-containing, Cdc42GAP-like Rho GTPase-activating protein) promotes MEK partner 1 (MP1)-induced ERK activation on late endosome through scaffolding MP1/MEK1 complex. This regulatory function requires phosphorylation of BPGAP1 by JNK at its C terminal tail (Ser424) to unlock its autoinhibitory conformation. Consequently, phosphorylated BPGAP1 facilitates endosomal ERK signaling transduction to the nucleus, driving cell proliferation and transformation via the ERK-Myc-CyclinA axis. BPGAP1 therefore provides a crucial spatiotemporal checkpoint where JNK and MP1/MEK1 work in concert to regulate endosomal and nuclear ERK signaling in cell proliferation control.

(-)-Epigallocatechin gallate synergistically potentiates prostaglandin E2-stimulated osteoprotegerin synthesis in osteoblasts

(-)-Epigallocatechin gallate (EGCG), the most abundant flavonoid in green tea, and chlorogenic acid, the main polyphenol found in coffee, attract significant attention owing to health benefits. We have previously demonstrated that prostaglandin E2 (PGE2) stimulates osteoprotegerin synthesis through the activation of p38 mitogen-activated protein (MAP) kinase and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the effects of EGCG or chlorogenic acid on the PGE2-stimulated osteoprotegerin synthesis in MC3T3-E1 cells. EGCG significantly amplified the PGE2-induced release. EGCG markedly enhanced the expression levels of osteoprotegerin mRNA induced by PGE2. On the contrary, chlorogenic acid had no effect on the PGE2-stimulated release of osteoprotegerin. EGCG significantly strengthened the PGE2-induced phosphorylation of p38 MAP kinase and SAPK/JNK, whereas chlorogenic acid failed to affect them. BIRB0796 and SP600125, a p38 MAP kinase inhibitor and a SAPK/JNK inhibitor, respectively, markedly reduced the amplification by EGCG of the PGE2-stimulated osteoprotegerin release. These results strongly suggest that EGCG synergistically enhances the PGE2-stimulated osteoprotegerin synthesis via potentiation of p38 MAP kinase and SAPK/JNK in osteoblasts. Our present findings could present a new significant aspect in the favorable effect of EGCG on the prevention of osteoporotic bone loss and fracture especially in elderly people since osteoprotegerin secreted from osteoblasts is well-recognized to act as a suppressor of osteoclastic bone resorption.

Inhibition of EZH2 via activation of SAPK/JNK and reduction of p65 and DNMT1 as a novel mechanism in inhibition of human lung cancer cells by polyphyllin I

BackgroundPolyphyllin I (PPI), a bioactive phytochemical extracted from the Rhizoma of Paris polyphylla, has been reported to exhibit anti-cancer activity. However, the detailed mechanism underlying this remains to be elucidated.MethodsCell viability and cell cycle distribution were measured using a 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) and flow cytometry assays, respectively. The expression of enhancer of zeste homolog 2 (EZH2) mRNA was measured by quantitative real time PCR (qRT-PCR). Western blot analysis was performed to examine the phosphorylation and protein expression of stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK), p65, DNA methyltransferase 1 (DNMT1) and EZH2. Exogenous expression of p65, DNMT1, and EZH2 were carried out by transient transfection assays. Promoter activity of EZH2 gene was determined using Secrete-Pair Dual Luminescence Assay Kit. A xenografted tumor model in nude mice and bioluminescent imaging system were used to further test the effect of PPI in vivo.ResultsWe showed that PPI significantly inhibited growth and induced cell cycle arrest of non-small cell lung cancer (NSCLC) cells in a dose-dependent manner. Mechanistically, we found that PPI increased the phosphorylation of SAPK/JNK, reduced protein expression of p65 and DNMT1. The inhibitor of SAPK/JNK (SP600125) blocked the PPI-inhibited p65 and DNMT1 protein expression. Interestingly, exogenously expressed p65 overcame PPI-inhibited protein expression of DNMT1. Moreover, PPI reduced EZH2 protein, mRNA, and promoter activity; overexpression of EZH2 resisted the PPI-inhibited cell growth, and intriguingly, negative feedback regulation of SAPK/JNK signaling. Finally, exogenous expression of DNMT1 antagonized the PPI-suppressed EZH2 protein expression. Consistent with this, PPI inhibited tumor growth, protein expression levels of p65, DNMT1 and EZH2, and increased phosphorylation of SAPK/JNK in vivo.ConclusionOur results show that PPI inhibits growth of NSCLC cells through SAPK/JNK-mediated inhibition of p65 and DNMT1 protein levels, subsequently; this results in the reduction of EZH2 gene expression. The interactions among p65, DNMT1 and EZH2, and feedback regulation of SAPK/JNK by EZH2 converge on the overall responses of PPI. This study reveals a novel mechanism for regulating EZH2 gene in response to PPI and suggests a new strategy for NSCLC associated therapy.

Possible involvement of AMP-activated protein kinase in PGE1-induced synthesis of osteoprotegerin in osteoblasts.

AMP-activated protein kinase (AMPK) is firmly established as a central regulator of cellular energy homeostasis. We have previously reported that prostaglandin E1 (PGE1) stimulates the synthesis of osteoprotegerin through p38 mitogen-activated protein (MAP) kinase and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) in osteoblast-like MC3T3-E1 cells. The present study investigated the involvement of AMPK in PGE1-induced osteoprotegerin synthesis in MC3T3-E1 cells. The levels of osteoprotegerin were measured using an enzyme-linked immunosorbent assay, while the phosphorylation of AMPK, acetyl-CoA carboxylase, p38 MAP kinase and SAPK/JNK were analyzed by western blotting. In addition, the mRNA expression levels of osteoprotegerin were determined by a reverse transcription-quantitative polymerase chain reaction. It was revealed that PGE1 significantly induced the phosphorylation of the α and β subunits of AMPK in a time-dependent manner (P<0.05). In addition, acetyl-CoA carboxylase, a direct substrate of AMPK, was significantly phosphorylated by PGE1 (P<0.05). Compound C, an AMPK inhibitor, was revealed to suppress the phosphorylation of acetyl-CoA carboxylase, which significantly reduced the release and mRNA expression levels of PGE1-stimulated osteoprotegerin (P<0.05). However, the PGE1-induced phosphorylation of p38 MAP kinase and SAPK/JNK were not affected by compound C. The results of the present study indicated that AMPK may positively regulate PGE1-stimulated osteoprotegerin synthesis in osteoblasts; thus providing novel insight into the regulatory mechanisms underlying bone metabolism.

Regulation by AMP-activated protein kinase of PGE2-induced osteoprotegerin synthesis in osteoblasts.

Adenosine monophosphate-activated protein kinase (AMPK) is currently recognized to act as a key sensing enzyme in the regulation of cellular energy homeostasis. It has been previously demonstrated that prostaglandin E2 (PGE2) stimulates the synthesis of osteoprotegerin (OPG) through the activation of p38 mitogen-activated protein (MAP) kinase, p44/p42 MAP kinase and stress-activated protein kinase/c‑Jun N‑terminal kinase (SAPK/JNK) in osteoblast‑like MC3T3‑E1 cells. In the present study, it was investigated whether AMPK is implicated in the PGE2‑induced OPG synthesis in MC3T3‑E1 cells. PGE2 was observed to induce the phosphorylation of AMPKα (Thr‑172) and AMPKβ (Ser‑108) in a time‑dependent manner. PGE2 additionally induced the phosphorylation of acetyl‑coenzyme A (CoA) carboxylase, a direct substrate of AMPK. Compound C, an inhibitor of AMPK, which attenuated the phosphorylation of acetyl‑CoA carboxylase, significantly suppressed the PGE2‑stimulated OPG release and the mRNA expression level. Compound C failed to affect the PGE2‑stimulated phosphorylation of p38 MAP kinase or p44/p42 MAP kinase. On the contrary, the phosphorylation of SAPK/JNK was markedly attenuated by compound C. The results of the current study suggest that AMPK acts as a positive regulator in PGE2-stimulated OPG synthesis via SAPK/JNK signaling in osteoblasts.

Mimosine suppresses the PGF2α-induced synthesis of osteoprotegerin but not interleukin-6 in osteoblasts.

Mimosine, a plant amino acid, is known to act as a normoxic inducer of hypoxia-inducible factor (HIF). Previous research has suggested that HIF plays important roles in angiogenesis-osteogenesis coupling and bone metabolism. We previously reported that prostaglandin F2α (PGF2α) induced osteoprotegerin synthesis through p38 mitogen-activated protein (MAP) kinase, p44/p42 MAP kinase and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) in osteoblast-like MC3T3-E1 cells. We have also demonstrated that PGF2α induced the synthesis of interleukin-6 (IL-6) via p38 MAP kinase and p44/p42 MAP kinase but not SAPK/JNK in these cells. In the present study, we investigated the effects of mimosine on the PGF2α-induced synthesis of osteoprotegerin or IL-6 in MC3T3-E1 cells. We found that deferoxamine, another inducer of HIF, as well as mimosine, upregulated the protein levels of HIF-1α. Both mimosine and deferoxamine significantly suppressed the PGF2α-induced release of osteoprotegerin, and the mRNA expression level, without markedly affecting PGF2α-induced IL-6 release. Both mimosine and deferoxamine, by themselves, induced the release of vascular endothelial growth factor. The phosphorylation of p38 MAP kinase, p44/p42 MAP kinase or SAPK/JNK induced by PGF2α was not markedly affected by either mimosine or deferoxamine. Thus, the results of the present study strongly suggest that mimosine, a normoxic inducer of HIF, inhibits the PGF2α‑induced osteoprotegerin synthesis without affecting the IL-6 synthesis in osteoblasts.

Inactivation of mitogen-activated protein kinase signaling pathway reduces caspase-14 expression in impaired keratinocytes.

Several investigations have revealed that caspase-14 is responsible for the epidermal differentiation and cornification, as well as the regulation of moisturizing effect. However, the precise regulation mechanism is still not clear. This study was aimed to investigate the expression of caspase-14 in filaggrin-deficient normal human epidermal keratinocytes (NHEKs) and to explore the possible mechanism that contributes to the regulation of caspase-14. The filaggrin-deficient NHEKs were induced by transfection with lentivirus (LV) vector encoding small hairpin RNAs (shRNA). The inhibitors SB203580, PD98059 and SP600125 were used for suppressing the expression of p38 mitogen-activated protein kinase (MAPK), p44/42 MAPK and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK). The expression of filaggrin, p38 MAPK, p44/42 MAPK and SAPK/JNK, caspase-14, keratin1and keratin2 were detected by western blot. In filaggrin-deficient NHEKs, the expression of p38, p44/42 MAPK and SAPK/JNK and caspase-14 were significantly decreased. The inhibition of p38 and SAPK/JNK reduced the expression of caspase-14, while the p44/42 MAPK showed no consistent effects. Moreover, the filaggrin knockdown decreased the expression of keratin2, but had no effects on the level of keratin1. The decreased expression of caspase-14 in filaggrin-deficient NHEKs may be induced by the inactivation of MAPK signaling pathway. These provide a novel perspective to understand the mechanism for the protective effects of filaggrin and caspase-14 on skin barrier function.

CAMP/PKA enhances interleukin-1β-induced interleukin-6 synthesis through STAT3 in glial cells

We previously reported that interleukin (IL)-1β induces IL-6 synthesis via activation of the IκB/NFκB pathway, p38 mitogen-activated protein (MAP) kinase, stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK), and signal transducer and activator of transcription (STAT)3, but not p44/p42 MAP kinase in rat glioma cell line, C6 cells and that cAMP enhances the IL-6 synthesis. However, the details behind enhancement of IL-1β-induced IL-6 synthesis by cAMP remain to be elucidated. In the present study, we investigated the exact mechanism of cAMP underlying the amplification of IL-1β-induced IL-6 synthesis in C6 cells. 8-Bromo cAMP significantly enhanced IL-1β-induced STAT3 phosphorylation without affecting phosphorylation of IκB, p38 MAP kinase or SAPK/JNK. In addition, we found that forskolin, a direct activator of adenylyl cyclase, significantly enhanced IL-1β-induced STAT3 phosphorylation. Janus family of tyrosine kinase (JAK) inhibitor I markedly suppressed the amplification by 8-bromo cAMP of IL-1β-induced IL-6 release. IL-1β induced JAK2 phosphorylation, and FLLL32, a specific JAK2 inhibitor, significantly reduced IL-1β-stimulated IL-6 release. 4-Cyano-3-methylisoquinoline, an inhibitor of protein kinase A (PKA), significantly attenuated the enhancing effect of 8-bromo cAMP on IL-1β-induced STAT3 phosphorylation. 8-Bromo cAMP markedly induced JAK2 phosphorylation. PKA siRNA transfection reduced enhancement of IL-1β-induced IL-6 release by 8-bromo cAMP. In conclusion, our results strongly suggest that the adenylyl cyclase/cAMP/PKA pathway upregulates IL-1β-induced IL-6 synthesis through enhancement of the JAK2/STAT3 pathway in C6 glioma cells.

Activation of SAPK/JNK mediated the inhibition and reciprocal interaction of DNA methyltransferase 1 and EZH2 by ursolic acid in human lung cancer cells.

BackgroundUrsolic acid (UA), a pentacyclic triterpenoid, is known to have anti-tumor activity in various cancers including human non small cell lung cancer (NSCLC). However, the molecular mechanisms underlying the action of UA remain largely unknown.MethodsCell viability was measured by MTT assays. Apoptosis was analyzed with Annexin V-FITC/PI Apoptosis Detection Kit by Flow cytometry. Western blot analysis was performed to measure the phosphorylation and protein expression of stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK), DNMT1 [DNA (cytosine-5)-methyltransferase 1], enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2) and SP1. Exogenous expression of SP1 and DNMT1 was carried out by transient transfection assays.ResultsWe showed that UA inhibited the growth and induced apoptosis of NSCLC cells in the dose- and time-dependent fashion. Furthermore, we found that UA induced phosphorylation of SAPK/JNK and suppressed the protein expression of DNMT1 and EZH2. The inhibitor of SAPK/JNK (SP600125) blocked the UA-reduced expression of DNMT1 and EZH2. In addition, UA suppressed the expression of SP1 protein. Conversely, overexpression of SP1 reversed the effect of UA on DNMT1 and EZH2 expression, and feedback attenuated UA-induced phosphorylation of SAPK/JNK. Moreover, exogenous expression of DNMT1 antagonized the effect of UA on SAPK/JNK signaling, EZH2 protein expression, and NSCLC cell growth.ConclusionOur results show that UA inhibits growth of NSCLC cells through SAPK/JNK-mediated inhibition of SP1; this in turn results in inhibition the expression of DNMT1 and EZH2. Overexpression of DNMT1 diminishes UA-reduced EZH2 protein expression. The negative feedback regulation of SAPK/JNK signaling by SP1 and DNMT1, and the reciprocal interaction of EZH2 and DNMT1 contribute to the overall effects of UA. This study leads to important new insights into the mechanisms by which UA controls growth of NSCLC cells.

PGD2 stimulates osteoprotegerin synthesis via AMP-activated protein kinase in osteoblasts: Regulation of ERK and SAPK/JNK

AMP-activated protein kinase (AMPK), a key enzyme sensing cellular energy metabolism, is currently known to regulate multiple metabolic pathways. Osteoprotegerin plays a pivotal role in the regulation of bone metabolism by inhibiting osteoclast activation. We have previously reported that prostaglandin D2 (PGD2) stimulates the synthesis of osteoprotegerin through the activation of p38 mitogen-activated protein (MAP) kinase, p44/p42 MAP kinase and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) in osteoblast-like MC3T3-E1 cells. On the basis of these findings, we herein investigated the implication of AMPK in PGD2-stimulated osteoprotegerin synthesis in these cells. PGD2 induced the phosphorylation of AMPKα (Thr-172) and AMPKβ (Ser-108), and the phosphorylation of acetyl-coenzyme A carboxylase, a direct AMPK substrate. Compound C, an AMPK inhibitor, which suppressed the phosphorylation of acetyl-coenzyme A carboxylase, significantly attenuated both the release and the mRNA levels of osteoprotegerin stimulated by PGD2. The PGD2-induced phosphorylation of p44/p42 MAP kinase and SAPK/JNK but not p38 MAP kinase were markedly inhibited by compound C. These results strongly suggest that AMPK regulates the PGD2-stimulated osteoprotegerin synthesis at a point upstream of p44/p42 MAP kinase and SAPK/JNK in osteoblasts.

Mitogen‐activated kinase pathway activation in epidermal lamellae in the acute stages of carbohydrate overload laminitis models and the effect of regional deep hypothermia on signalling pathways

In sepsis models, mitogen-activated protein kinases (MAPKs) are reported to incite inflammatory injury to tissues and are purported to be a therapeutic target. To assess MAPK signalling in lamellae in sepsis-related laminitis (SRL) at different time points after induction of laminitis via carbohydrate overload, and to determine the effect of regional deep hypothermia (RDH) on MAPK signalling. In vitro study using archived tissue samples. Lamellar concentrations of MAPKs were assessed in archived lamellar samples from 2 studies: 1) the starch gruel model of SRL with 3 groups (n=6/group) of horses (control, onset of fever [DEV] Obel Grade 1 lameness [OG1]); and 2) from limbs maintained at ambient (AMB) and hypothermic (ICE) temperatures (n=6/group) in animals given a bolus of oligofructose.Immunoblotting and immunolocalisation were used to assess lamellar concentrations and cellular localisation of total and activated (phosphorylated) forms of p38 MAPK, extracellular-regulated kinase (ERK) 1/2, and stress-activated protein kinase/c-jun N terminal kinase (SAPK/JNK) 1/2. Lamellar samples had statistically significant increased concentrations of activated ERK 1/2 at the onset of OG1 laminitis (vs. control) in the starch gruel model, but showed no significant change between ICE and AMB limbs in the RDH model. Phospho-SAPK/JNK 1/2 exhibited a similar significant increase in the OG1 samples, but was also increased in ICE (vs. AMB) limbs. No statistically significant changes in lamellar p38 MAPK concentrations were noted. Increased concentrations of activated ERK 1/2 and SAPK/JNK in the acute stages of SRL indicate a possible role of these signalling proteins in lamellar injury. Signalling related to ERK 1/2 and SAPK/JNK 1/2 pathways should be further investigated to determine if these play a detrimental role in laminitis and may be therapeutic targets to be manipulated independently of RDH.