Estrogenic effects of two derivatives of icariin on human breast cancer MCF-7 cells

The aims of the present study were to determine the estrogenic activities of icariin (ICA) and its derivatives and their structure–estrogenic activity relationship. Therefore, icaritin (ICT) and desmethylicaritin (DICT) were derived from ICA. The estrogenic activities of ICA, ICT and DICT were examined by cell proliferation and progestogen receptor mRNA expression of estrogen-receptor-positive MCF-7 cells. Current studies exhibited that ICT and DICT both markedly enhanced the proliferation of MCF-7 cells; as compared to estradiol (100%), their relative proliferative effects (RPE) were 90% and 94%, respectively. Cell proliferation induced by ICT and DICT was completely antagonized by ICI182,780. ICT and DICT increased progestogen receptor (PR) at mRNA levels at 48 h after treatment, although the effects were not as prominent as 17β-estradiol (E₂). These phenomena were not observed with ICA. Results demonstrate that ICT and DICT (nonconjugated forms) possess estrogen-like activity; however, ICA appears to have no estrogenicity in the MCF-7 cell line model in vitro.     

Distinct regulation of mitogen-activated protein kinase activities is coupled with enhanced cardiac differentiation of human embryonic stem cells

Improving cardiac differentiation of human pluripotent stem cells is mandatory to provide functional heart muscle cells for novel therapies. Here, we have investigated the enhancing effect of the small molecule SB203580, a p38 MAPK inhibitor, on cardiomyogenesis in hESC by monitoring the phosphorylation patterns of the major MAPK pathway components p38, JNK and ERK by western immunoblotting. A remarkable drop in phosphorylation levels of all three MAPK pathways was induced after overnight embryoid body (EB) formation. Upon further differentiation, phosphorylation dynamics in EBs were specifically altered by distinct inhibitor concentrations. At 5μM of SB203580, cardiomyogenesis was most efficient and associated with the expected p38 pathway inhibition. In parallel, JNK activation was observed suggesting a regulatory interlink between these pathways in hESC ultimately supporting cardiac differentiation. In contrast, moderately elevated SB203580 concentrations (15-30μM) resulted in complete disruption of cardiomyogenesis which was associated with prominent inhibition of ERK and further elevated JNK activity. We propose that a tightly-balanced pattern in MAPK phosphorylation is important for early mesoderm and subsequent cardiomyocyte formation. Our data provide novel insights into molecular consequences of small molecule supplementation in hESC differentiation, emphasizing the role of MAPK-signaling.     

Signaling pathway of MAPK/ERK in cell proliferation,differentiation, migration,senescence and apoptosis

Abstract

The generic mitogen-activated protein kinases (MAPK) signaling pathway is shared by four distinct cascades, including the extracellular signal-related kinases (ERK1/2), Jun amino-terminal kinases (JNK1/2/3), p38-MAPK and ERK5. Mitogen-activated protein kinases/extracellular signal-regulated kinase (MAPK/ERK) pathway is reported to be associated with the cell proliferation, differentiation, migration, senescence and apoptosis. The literatures were searched extensively and this review was performed to review the role of MAPK/ERK signaling pathway in cell proliferation, differentiation, migration, senescence and apoptosis.     

Redox-dependent MAP kinase signaling by Ang II in vascular smooth muscle cells: role of receptor tyrosine kinase transactivation

We investigated the role of receptor tyrosine kinases in Ang II-stimulated generation of reactive oxygen species (ROS) and assessed whether MAP kinase signaling by Ang II is mediated via redox-sensitive pathways. Production of ROS and activation of NADPH oxidase were determined by DCFDA (dichlorodihydrofluorescein diacetate; 2 micromol/L) fluorescence and lucigenin (5 micromol/L) chemiluminescence, respectively, in rat vascular smooth muscle cells (VSMC). Phosphorylation of ERK1/2, p38MAP kinase and ERK5 was determined by immunoblotting. The role of insulin-like growth factor-1 receptor (IGF-1R) and epidermal growth factor receptor (EGFR) was assessed with the antagonists AG1024 and AG1478, respectively. ROS bioavailability was manipulated with Tiron (10(-5) mol/L), an intracellular scavenger, and diphenylene iodinium (DPI; 10(-6) mol/L), an NADPH oxidase inhibitor. Ang II stimulated NADPH oxidase activity and dose-dependently increased ROS production (p < 0.05). These actions were reduced by AG1024 and AG1478. Ang II-induced ERK1/2 phosphorylation (276% of control) was decreased by AG1478 and AG1024. Neither DPI nor tiron influenced Ang II-stimulated ERK1/2 activity. Ang II increased phosphorylation of p38 MAP kinase (204% of control) and ERK5 (278% of control). These effects were reduced by AG1024 and AG1478 and almost abolished by DPI and tiron. Thus Ang II stimulates production of NADPH-inducible ROS partially through transactivation of IGF-1R and EGFR. Inhibition of receptor tyrosine kinases and reduced ROS bioavaliability attenuated Ang II-induced phosphorylation of p38 MAP kinase and ERK5, but not of ERK1/2. These findings suggest that Ang II activates p38MAP kinase and ERK5 via redox-dependent cascades that are regulated by IGF-1R and EGFR transactivation. ERK1/2 regulation by Ang II is via redox-insensitive pathways.     

Albumin prevents reactive oxygen species-induced mitochondrial damage, autophagy, and apoptosis during serum starvation

Aberrant levels of reactive oxygen species (ROS) rapidly generated from NADPH oxidase (NOX) activation can be cytotoxic due to activating pro-apoptotic signals. However, ROS also induce pro-survival autophagy through the engulfment of damaged mitochondria. This study is aimed at investigating the cytoprotective role of albumin against NOX/ROS-induced autophagy and apoptosis under serum starvation. Serum starvation induced apoptosis following a myeloid cell leukemia sequence 1 (Mcl-1)/Bax imbalance, loss of the mitochondrial transmembrane potential, and caspase activation accompanied by pro-survival autophagy following canonical inhibition of mammalian target of rapamycin complex 1 (mTORC1). Aberrant ROS generation, initially occurring through NOX, facilitated mitochondrial damage, autophagy, and apoptosis. Autophagy additionally regulated the accumulation of ROS-generating mitochondria. NOX/ROS permitted p38 mitogen-activated protein kinase (p38 MAPK)-regulated mitochondrial apoptosis, accompanied by non-canonical induction of autophagy. In addition, activation of glycogen synthase kinase (GSK)-3β by NOX/ROS-inactivated Akt facilitated a decrease in Mcl-1, followed by mitochondrial apoptosis as well as autophagy. Restoring albumin conferred an anti-oxidative effect against serum starvation-deregulated NOX, p38 MAPK, and Akt/GSK-3β/Mcl-1/caspase-3 signaling. Albumin also prevented autophagy by sustaining mTORC1. These results indicate an anti-oxidative role for albumin via preventing NOX/ROS-mediated mitochondrial signaling to stimulate apoptosis as well as autophagy. Autophagy, initially induced by canonical inhibition of mTORC1 and enhanced by non-canonical mitochondrial damage, acts physically as a pro-survival mechanism.     

p38 and ERK1/2 coordinate cellular migration and proliferation in epithelial wound healing: evidence of cross-talk activation between MAP kinase cascades

One important action of growth factors is their participation in tissue repair; however, the signaling pathways involved are poorly understood. In a model of corneal wound healing, we found that two paracrine growth factors, hepatocyte growth factor (HGF) and keratinocyte growth factor (KGF), induced rapid and marked activation and prompt nuclear accumulation of phospho-p38 (p-p38) and -ERK1/2 (p-ERK1/2), but not of JNK (p-JNK1/2), in corneal epithelial cells. Interruption of p38 and ERK1/2 signaling pathways by pretreatment with inhibitors SB203580 and PD98059 and subsequent stimulation with HGF or KGF abolished the activation and nuclear localization. Inhibition of either one of these mitogen-activated protein kinases, p38 or ERK1/2, induced a robust cross-activation of the other. In immunofluorescence studies of wounded cornea, p-p38, unlike p-ERK1/2, was immediately detectable in epithelium after injury. Inhibition of p38 by SB203580 blocked migration of epithelial cells almost completely. In contrast, PD98059 seemed to slightly increase the migration, through concomitant activation of p38. Unlike ERK1/2, p38 did not significantly contribute to proliferation of epithelial cells. Inhibition of either the ERK1/2 or p38 pathway resulted in delayed corneal epithelial wound healing. Interruption of both signaling cascades additively inhibited the wound-healing process. These findings demonstrate that both p38 and ERK1/2 coordinate the dynamics of wound healing: while growth factor-stimulated p38 induces epithelial migration, ERK1/2 activation induces proliferation. The cross-talk between these two signal cascades and the selective action of p38 in migration appear to be important to corneal wound healing, and possibly wound healing in general, and may offer novel drug targets for tissue repair.     

Role of the actin cytoskeleton in angiotensin II signaling in human vascular smooth muscle cells

Angiotensin II (Ang II) regulates vascular smooth muscle cell (VSMC) function by activating signaling cascades that promote vasoconstriction, growth, and inflammation. Subcellular mechanisms coordinating these processes are unclear. In the present study, we questioned the role of the actin cytoskeleton in Ang II mediated signaling through mitogen-activated protein (MAP) kinases and reactive oxygen species (ROS) in VSMCs. Human VSMCs were studied. Cells were exposed to Ang II (10-7 mol/L) in the absence and presence of cytochalasin B (10-6 mol/L, 60 min), which disrupts the actin cytoskeleton. Phosphorylation of p38MAP kinase, JNK, and ERK1/2 was assessed by immuno blotting. ROS generation was measured using the fluoroprobe chloromethyl-2',7'-dichlorodihydrofluorescein diacetate (4 micromol/L). Interaction between the cytoskeleton and NADPH oxidase was determined by evaluating the presence of p47phox in the Triton X-100 insoluble membrane fraction. Ang II significantly increased phosphorylation of p38MAP kinase, JNK, and ERK1/2 (two- to threefold above control, p < 0.05). Cytochalasin B pretreatment attenuated p38MAP kinase and JNK effects (p < 0.05) without altering ERK1/2 phosphorylation. ROS formation, which was increased in Ang II stimulated cells, was significantly reduced by cytochalasin B (p < 0.01). p47phox, critically involved in NADPH oxidase activation, colocalized with the actin cytoskeleton in Ang II stimulated cells. Our data demonstrate that Ang II mediated ROS formation and activation of p38MAP kinase and JNK, but not ERK1/2, involves the actin cytoskeleton in VSMCs. In addition, Ang II promotes interaction between actin and p47phox. These data indicate that the cytoskeleton is involved in differential MAP kinase signaling and ROS generation by Ang II in VSMCs. Together, these studies suggest that the cytoskeleton may be a central point of crosstalk in growth- and redox-signaling pathways by Ang II, which may be important in the regulation of VSMC function.     

NADPH oxidase and eNOS control cardiomyogenesis in mouse embryonic stem cells on ascorbic acid treatment

Ascorbic acid (AA) increases cardiomyogenesis of embryonic stem (ES) cells. Herein we show that treatment of mouse ES cells with AA enhanced cardiac differentiation accompanied by an upregulation of the NADPH oxidase isoforms NOX2 and NOX4, phosphorylation of endothelial nitric oxide synthase (eNOS), and cyclic GMP (cGMP) formation, indicating that reactive oxygen species (ROS) as well as nitric oxide (NO) may be involved in cardiomyogenesis. In whole mount embryoid bodies as well as isolated Flk-1-positive (Flk-1⁺) cardiovascular progenitor cells ROS elevation by AA was observed in early stages of differentiation (Days 4-7), and absent at Day 10. In contrast NO generation following incubation with AA was absent at Day 4 and increased at Days 7 and 10. AA-mediated cardiomyogenesis was blunted by the NADPH oxidase inhibitors diphenylen iodonium (DPI) and apocynin, the free radical scavengers N-(2-mercaptopropionyl)-glycine (NMPG) and ebselen, and the NOS inhibitor L-NAME. Downregulation of NOX4 by short hairpin RNA (shRNA) resulted in significant inhibition of cardiomyogenesis and abolished the stimulation of MHC-ß and MLC2v gene expression observed on AA treatment. Our data demonstrate that AA stimulates cardiomyocyte differentiation from ES cells by signaling pathways that involve ROS generated at early stages and NO at late stages of cardiomyogenesis.     

Rit contributes to nerve growth factor-induced neuronal differentiation via activation of B-Raf-extracellular signal-regulated kinase and p38 mitogen-activated protein kinase cascades

Rit is one of the original members of a novel Ras GTPase subfamily that uses distinct effector pathways to transform NIH 3T3 cells and induce pheochromocytoma cell (PC6) differentiation. In this study, we find that stimulation of PC6 cells by growth factors, including nerve growth factor (NGF), results in rapid and prolonged Rit activation. Ectopic expression of active Rit promotes PC6 neurite outgrowth that is morphologically distinct from that promoted by oncogenic Ras (evidenced by increased neurite branching) and stimulates activation of both the extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein (MAP) kinase signaling pathways. Furthermore, Rit-induced differentiation is dependent upon both MAP kinase cascades, since MEK inhibition blocked Rit-induced neurite outgrowth, while p38 blockade inhibited neurite elongation and branching but not neurite initiation. Surprisingly, while Rit was unable to stimulate ERK activity in NIH 3T3 cells, it potently activated ERK in PC6 cells. This cell type specificity is explained by the finding that Rit was unable to activate C-Raf, while it bound and stimulated the neuronal Raf isoform, B-Raf. Importantly, selective down-regulation of Rit gene expression in PC6 cells significantly altered NGF-dependent MAP kinase cascade responses, inhibiting both p38 and ERK kinase activation. Moreover, the ability of NGF to promote neuronal differentiation was attenuated by Rit knockdown. Thus, Rit is implicated in a novel pathway of neuronal development and regeneration by coupling specific trophic factor signals to sustained activation of the B-Raf/ERK and p38 MAP kinase cascades.     

Salvicine inactivates beta 1 integrin and inhibits adhesion of MDA-MB-435 cells to fibronectin via reactive oxygen species signaling

Integrin-mediated adhesion to the extracellular matrix plays a fundamental role in tumor metastasis. Salvicine, a novel diterpenoid quinone compound identified as a nonintercalative topoisomerase II poison, possesses a broad range of antitumor and antimetastatic activity. Here, the mechanism underlying the antimetastatic capacity of salvicine was investigated by exploring the effect of salvicine on integrin-mediated cell adhesion. Salvicine inhibited the adhesion of human breast cancer MDA-MB-435 cells to fibronectin and collagen without affecting nonspecific adhesion to poly-l-lysine. The fibronectin-dependent formation of focal adhesions and actin stress fibers was also inhibited by salvicine, leading to a rounded cell morphology. Furthermore, salvicine down-regulated beta(1) integrin ligand affinity, clustering and signaling via dephosphorylation of focal adhesion kinase and paxillin. Conversely, salvicine induced extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) phosphorylation. The effect of salvicine on beta(1) integrin function and cell adhesion was reversed by U0126 and SB203580, inhibitors of MAPK/ERK kinase 1/2 and p38 MAPK, respectively. Salvicine also induced the production of reactive oxygen species (ROS) that was reversed by ROS scavenger N-acetyl-l-cysteine. N-acetyl-l-cysteine additionally reversed the salvicine-induced activation of ERK and p38 MAPK, thereby maintaining functional beta(1) integrin activity and restoring cell adhesion and spreading. Together, this study reveals that salvicine activates ERK and p38 MAPK by triggering the generation of ROS, which in turn inhibits beta(1) integrin ligand affinity. These findings contribute to a better understanding of the antimetastatic activity of salvicine and shed new light on the complex roles of ROS and downstream signaling molecules, particularly p38 MAPK, in the regulation of integrin function and cell adhesion.     

Signaling pathways activated by PACAP in MCF-7 breast cancer cells

PACAP has opposing roles ranging from activation to inhibition of tumor growth and PACAP agonists/antagonists could be used in tumor therapy. In this study, the effect of PACAP stimulation on signaling pathways was investigated in MCF-7 human adenocarcinoma breast cancer cells. Results showed that MCF-7 cells express VPAC1 and VPAC2, but not PAC1, receptors. In addition, PACAP increased the phosphorylation levels of STAT1, Src and Raf within seconds, confirming their involvement in early stages of PACAP signaling whereas maximal phosphorylation of AKT, ERK and p38 was reached 10 to 20 min later. Moreover, selective inhibition of Src or PI3K resulted in a significant decrease in the phosphorylation of ERK and AKT, but not p38, demonstrating that PACAP signaling follows Src/Raf/ERK and PI3K/AKT pathways. On the other hand, selective inhibition of PLC or PKA resulted in a significant decrease in the phosphorylation of p38, but not AKT or ERK, indicating that PACAP signaling also follows the PLC and PKA/cAMP pathways. Furthermore, PACAP induced ROS through H₂O₂ production whereas pretreatment with NAC inhibitor decreased AKT and ERK phosphorylation, but not p38. Selective NOX2 inhibition affected Src/Raf/Erk and PI3K/Akt pathways, without affecting the p38/PLC/PKA pathway whereas other inhibitors (ML171, VAS2870) had no effect on PACAP induced ROS generation. On the other hand, PACAP induced calcium release, which was decreased by pretreatment with PLC inhibitor. Finally, PACAP stimulation promoted apoptosis by increasing Bax and decreasing Bcl2 expression. In conclusion, we demonstrated that PACAP signaling in MCF-7 cells follows the Src/Raf/ERK and PI3K/AKT pathways and is VPAC1 dependent in a ROS dependent manner, whereas it follows PLC and PKA/cAMP pathways and is VPAC2 dependent through p38 MAP kinase activation involving calcium.     

NKCC activity restores muscle water during hyperosmotic challenge independent of insulin,ERK, and p38 MAPK

In isosmotic conditions, insulin stimulation of PI 3-K/Akt and p38 MAPK pathways in skeletal muscle inhibits Na(+)-K(+)-2Cl(-) cotransporter (NKCC) activity induced by the ERK1,2 MAPK pathway. Whether these signaling cascades contribute to NKCC regulation during osmotic challenge is unknown. Increasing osmolarity by 20 mosM with either glucose or mannitol induced NKCC-mediated (86)Rb uptake and water transport into rat soleus and plantaris skeletal muscle in vitro. This NKCC activity restored intracellular water. In contrast to mannitol, hyperosmolar glucose increased ERK1,2 and p38 MAPK phosphorylation. Glucose, but not mannitol, impaired insulin-stimulated phosphorylation of Akt and p38 MAPK in the plantaris and soleus muscles, respectively. Hyperosmolarity-induced NKCC activation was insensitive to insulin action and pharmacological inhibition of ERK1,2 and p38 MAPK pathways. Paradoxically, cAMP-producing agents, which stimulate NKCC activity in isosmotic conditions, suppressed hyperosmolar glucose- and mannitol-induced NKCC activity and prevented restoration of muscle cell volume in hyperosmotic media. These results indicate that NKCC activity helps restore muscle cell volume during hyperglycemia. Moreover, hyperosmolarity activates NKCC regulatory pathways that are insensitive to insulin inhibition.     

The relationship between p53/p21/Rb and MAPK signaling pathways in human endometrium-derived stem cells under oxidative stress

Human endometrium-derived mesenchymal stem cells (hMESC) under the sublethal oxidative stress induced by H₂O₂ activate both the p53/p21/Rb and p38/MAPKAPK-2 pathways that are responsible for the induction of hMESC premature senescence (Borodkina et al., 2014). However, the interrelations between the p53/p21/Rb and MAPK signaling pathways, including ERK1/2, p38, and JNK, remain yet unexplored. Here, we used the specific inhibitors—pifithrin-α (PFT), U0126, SB203580, and SP600125 to “switch off” one of the proteins in these cascades and to evaluate the functional status alterations of the rest of the proteins. Each MAPK suppression significantly increased the p53 phosphorylation level, as well as p21 protein expression followed by Rb hypophosphorylation. On the other hand, PFT-induced p53 inhibition enhanced mostly the ERK1/2 activation rather than p38 and JNK. These results suggest the existence of a reciprocal negative regulation between p53- and MAPK-dependent signaling pathways. By analyzing the possible interactions among the members of the MAPK family, we showed that p38 and JNK can function as ERK antagonists: JNK is able to activate ERK, while p38 may block ERK activation. Together, these results demonstrate the existence of complex links between different signaling cascades in stressed hMESC, implicating ERK, p38, and JNK in regulation of premature senescence via the p53/p21/Rb pathway.     

Apoptosis induced by capsaicin in prostate PC-3 cells involves ceramide accumulation,neutral sphingomyelinase,and JNK activation

Numerous studies have recently focused on the anticarcinogenic, antimutagenic, or chemopreventive activities of the main pungent component of red pepper, capsaicin (N-vanillyl-8-methyl-1-nonenamide). We have previously shown that, in the androgen-independent prostate cancer PC-3 cells, capsaicin inhibits cell growth and induces apoptosis through reactive oxygen species (ROS) generation [Apoptosis 11 (2006) 89–99]. In the present study, we investigated the signaling pathways involved in the antiproliferative effect of capsaicin. Here, we report that capsaicin apoptotic effect was mediated by ceramide generation which occurred by sphingomyelin hydrolysis. Using siRNA, we demonstrated that N-SMase expression is required for the effect of capsaicin on prostate cell viability. We then investigated the role of MAP kinase cascades, extracellular signal-regulated protein kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK, in the antiproliferative effect of capsaicin, and we confirmed that capsaicin could activate ERK and JNK but not p38 MAPK. Pharmacological inhibition of JNK kinase, as well as inhibition of ROS by the reducing agent N-acetylcysteine, prevented ceramide accumulation and capsaicin-induced cell death. However, inhibition of ceramide accumulation by the SMase inhibitor D609 did not modify JNK activation. These data reveal JNK as an upstream regulator of ceramide production. Capsaicin-promoted activation of ERK was prevented with all the inhibitors tested. We conclude that capsaicin induces apoptosis in PC-3 cells via ROS generation, JNK activation, ceramide accumulation, and second, ERK activation.     

p38 Mitogen-Activated Protein Kinase Functionally Contributes to Chondrogenesis Induced by Growth/Differentiation Factor-5 in ATDC5 Cells

Recent studies of intracellular signal transduction mechanisms for the transforming growth factor-beta (TGF-beta) superfamily have focused on Smad proteins, but have paid little attention to mitogen-activated protein (MAP) kinase cascades. Here we demonstrate that growth/differentiation factor-5 (GDF-5), but neither bone morphogenetic protein-2 (BMP-2) nor TGF-beta1, fully promotes the early phase of the chondrogenic response by inducing cellular condensation followed by cartilage nodule formation in a mouse chondrogenic cell line, ATDC5. We investigated which, if any, of the three major types of MAP kinase plays a functional role in the promotion of chondrogenesis induced by GDF-5. GDF-5 induced phosphorylation of p38 MAP kinase and extracellular signal-regulated kinase (ERK) but not that of c-Jun N-terminal kinase (JNK). The phosphorylation of p38 MAP kinase was also induced by BMP-2 and TGF-beta1. An inhibitor of p38 and p38 beta MAP kinase, SB202190, showed complete inhibition of cartilage nodule formation but failed to affect alkaline phosphatase (ALP) activity induced by GDF-5. Expression of the type II collagen gene, a hallmark of chondrogenesis in vertebrates, was also induced by GDF-5 treatment and strongly suppressed by SB202190. On the other hand, although an inhibitor of MAP/ERK kinase, PD98059, inhibited the rapid phosphorylation of ERK by GDF-5, it inhibited neither ALP activity nor cartilage nodule formation induced by GDF-5. These results strongly suggest that the p38 MAP kinase cascade is involved in GDF-5 signaling pathways and that a role of the p38 MAP kinase pathway is necessary over a longer period to promote chondrogenesis in ATDC5 cells.