Form Of Mitogen-activated Protein Kinase Research Articles

Wound healing effects of Asparagus lucidus Lindl extract through the phosphorylation of ERK1/2

BackgroundSkin is the outermost part of the human body and is essential in maintaining body homeostasis. In the event of skin injury, rapid wound repair is crucial to protect the body. In this study, we investigated the wound-healing properties of Asparagus lucidus Lindl extract by promoting keratinocyte proliferation.MethodsTo evaluate the effect of Asparagus lucidus Lindl extract on skin regeneration, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was used to measure keratinocyte proliferation, while an in vitro wound-healing assay was performed to evaluate wound closure through keratinocyte re-epithelialization. The intracellular mechanisms of the extract were studied using Western blot analysis to measure the phosphorylated forms of mitogen-activated protein kinases and protein kinase B. The mRNA expression of cell cycle-related genes was analyzed using quantitative real time-PCR analysis. A murine in vivo wound-healing assay was also conducted to observe the effect of the extract on wound closure.ResultsAsparagus lucidus Lindl extract induced 131.15% keratinocyte proliferation compared to the control in the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The in vitro wound-healing assay showed that the extract improved wound closure by 216.94% through keratinocyte re-epithelialization. Western blot analysis revealed that the phosphorylated form of extracellular signal-regulated kinase 1/2 was increased by extract treatment. Quantitative real time-PCR analysis showed a dose-dependent increase in the mRNA expression of c-fos, c-jun, and VEGF. The in vivo wound-healing assay showed a significant increase (22.13%) of wound closure compared to the control on day 5.ConclusionAsparagus lucidus Lindl extract promotes keratinocyte proliferation by activating the extracellular signal-regulated kinase 1/2 pathway and up-regulating the mRNA expression of c-fos, c-jun, and vascular endothelial growth factor.

Inhibiting the JNK/ERK signaling pathway with geraniol for attenuating the proliferation of human gastric adenocarcinoma AGS cells.

Geraniol, a natural compound found in the essential oils of various aromatic plants, has attracted attention for its probable anticancer effects. The molecular mechanisms of the cell proliferation suppression and apoptosis induction via geraniol in gastric cancer cells (AGS), however, remain unclear. Gastric cancer cells were treated with geraniol, and it was found that the IC50 values were 25 μM/ml, as determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Results showed that 20 and 25 μM geraniol-induced reactive oxygen species (ROS) production (2'-7'dichlorofluorescin diacetate staining) and decreased mitochondrial membrane potential (rhodamine 123 staining) in AGS cells. Then, it effectively inhibited cell growth and induced apoptosis, confirmed through acridine orange/ethidium bromide, 4',6-diamidino-2-phenylindole, and propidium iodidestaining and molecular marker analysis in AGS cells. Also, geraniol potently diminished caspase-9, Bax, Bcl-2, and caspase-3 expression in AGS cells. We also evaluated the essential mechanism of the cytotoxic effect of geraniol. Moreover, the present study depicted that geraniol-induced cell death through mitochondrial ROS production and inhibited the phosphorylation form of mitogen-activated protein kinase (p38, MAPK, JNK, and ERK1/2) signaling pathway. Taken together, these results concluded that geraniol has a novel therapeutic property against human stomach cancer.

Gonadoliberin – Synthesis, Secretion, Molecular Mechanisms and Targets of Action

Decapeptide gonadoliberin (GnRH) is the most important regulator of the hypothalamic-pituitary-gonadal (HPG) axis that controls the synthesis and secretion of the luteinizing and follicle-stimulating hormones by gonadotrophs in the adenohypophysis. GnRH is produced by the specialized hypothalamic neurons using the site-specific proteolysis of the precursor protein and is secreted into the portal pituitary system, where it binds to the specific receptors. These receptors belong to the family of G protein-coupled receptors, and they are located on the surface of gonadotrophs and mediate the regulatory effects of GnRH on the gonadotropins production. The result of GnRH binding to them is the activation of phospholipase C and the calcium-dependent pathways, the stimulation of different forms of mitogen-activated protein kinases, as well as the activation of the enzyme adenylyl cyclase and the triggering of cAMP-dependent signaling pathways in the gonadotrophs. The gonadotropins, kisspeptin, sex steroid hormones, insulin, melatonin and a number of transcription factors have an important role in the regulation of GnRH1 gene expression, which encodes the GnRH precursor, as well as the synthesis and secretion of GnRH. The functional activity of GnRH-producing neurons depends on their migration to the hypothalamic region at the early stages of ontogenesis, which is controlled by anosmin, ephrins, and lactosamine-rich surface glycoconjugate. Dysregulation of the migration of GnRH-producing neurons and the impaired production and secretion of GnRH, lead to hypogonadotropic hypogonadism and other dysfunctions of the reproductive system. This review is devoted to the current state of the problem of regulating the synthesis and secretion of GnRH, the mechanisms of migration of hypothalamic GnRH-producing neurons at the early stages of brain development, the functional activity of the GnRH-producing neurons in the adult hypothalamus and the molecular mechanisms of GnRH action on the pituitary gonadotrophs. New experimental data are analyzed, which significantly change the current understanding of the functioning of GnRH-producing neurons and the secretion of GnRH, which is very important for the development of effective approaches for correcting the functions of the HPG axis.

Divergent Effects of Metformin on an Inflammatory Model of Parkinson's Disease.

The oral antidiabetic drug metformin is known to exhibit anti-inflammatory properties through activation of AMP kinase, thus protecting various brain tissues as cortical neurons, for example. However, the effect of metformin on the substantia nigra (SN), the main structure affected in Parkinson’s disease (PD), has not yet been studied in depth. Inflammation is a key feature of PD and it may play a central role in the neurodegeneration that takes place in this disorder. The aim of this work was to determine the effect of metformin on the microglial activation of the SN of rats using the animal model of PD based on the injection of the pro-inflammogen lipopolysaccharide (LPS). In vivo and in vitro experiments were conducted to study the activation of microglia at both the cellular and molecular levels. Our results indicate that metformin overall inhibits microglia activation measured by OX-6 (MHCII marker), IKKβ (pro-inflammatory marker) and arginase (anti-inflammatory marker) immunoreactivity. In addition, qPCR experiments reveal that metformin treatment minimizes the expression levels of several pro- and anti-inflammatory cytokines. Mechanistically, the drug decreases the phosphorylated forms of mitogen-activated protein kinases (MAPKs) as well as ROS generation through the inhibition of the NADPH oxidase enzyme. However, metformin treatment fails to protect the dopaminergic neurons of SN in response to intranigral LPS. These findings suggest that metformin could have both beneficial and harmful pharmacological effects and raise the question about the potential use of metformin for the prevention and treatment of PD.

Intracellular inflammatory and antioxidant pathways in postmortem frontal cortex of subjects with major depression: effect of antidepressants

BackgroundStudies show that Toll-like receptors (TLRs), members of the innate immune system, might participate in the pathogenesis of the major depressive disorder (MDD). However, evidence of this participation in the brain of patients with MDD has been elusive.MethodsThis work explores whether the protein expression by immunodetection assays (Western blot) of elements of TLR-4 pathways controlling inflammation and the oxidative/nitrosative stress are altered in postmortem dorsolateral prefrontal cortex of subjects with MDD. The potential modulation induced by the antidepressant treatment on these parameters was also assessed. Thirty MDD subjects (15 antidepressant-free and 15 under antidepressant treatment) were matched for gender and age to 30 controls in a paired design.ResultsNo significant changes in TLR-4 expression were detected. An increased expression of the TLR-4 endogenous ligand Hsp70 (+ 33%), but not of Hsp60, and the activated forms of mitogen-activated protein kinases (MAPKs) p38 (+ 47%) and JNK (+ 56%) was observed in MDD. Concomitantly, MDD subjects present a 45% decreased expression of DUSP2 (a regulator of MAPKs) and reduced (− 21%) expression of the antioxidant nuclear factor Nrf2. Antidepressant treatment did not modify the changes detected in the group with MDD and actually increased (+ 25%) the expression of p11, a protein linked with the transport of neurotransmitters and depression.ConclusionData indicate an altered TLR-4 immune response in the brain of subjects with MDD. Additional research focused on the mechanisms contributing to the antidepressant-induced TLR-4 pathway modulation is warranted and could help to develop new treatment strategies for MDD.

Oocyte aging in a marine protostome worm: The roles of maturation-promoting factor and extracellular signal regulated kinase form of mitogen-activated protein kinase.

The roles of maturation-promoting factor (MPF) and an extracellular signal regulated kinase form of mitogen-activated protein kinase (ERK MAPK) are analyzed during oocyte aging in the marine protostome worm Cerebratulus. About a day after removal from the ovary, unfertilized metaphase-I-arrested oocytes of Cerebratulus begin to flatten and swell before eventually lysing, thereby exhibiting characteristics of a necroptotic mode of regulated cell death. Based on immunoblots probed with phospho-specific antibodies, MPF and ERK are initially active in freshly mature specimens. However, as oocytes age, both kinase activities decline, with ERK deactivation occurring well before MPF downregulation. Experiments using pharmacological modulators indicate that oocyte degradation is promoted by the maturation-initiated activation of ERK as well as by the deactivation of MPF that occurs in extensively aged specimens. The potential significance of these findings is discussed relative to previously published results for apoptotic eggs and oocytes of echinoderm and vertebrate deuterostomes.

Src Tyrosine Kinase Activation by 4-Hydroxynonenal Upregulates p38, ERK/AP-1 Signaling and COX-2 Expression in YPEN-1 Cells.

4-Hydroxynonenal (4-HNE), a major end product of lipid peroxidation, is highly reactive and involved in various cellular processes, such as inflammatory signaling. However, to date, the mechanistic roles of 4-HNE in inflammatory signaling related to protein tyrosine kinases have not been elucidated. In the present study, we investigated the interaction between 4-HNE and Src (a non-receptor tyrosine kinase) for its involvement in the molecular modulation of the inflammatory signaling pathway utilizing the YPEN-1 cell system. Immunoprecipitation experiments showed that 4-HNE phosphorylates (activates) Src at Tyr416 via adduct formation. In addition, LC-MS/MS and a docking simulation model revealed an addiction site at the Cys248 residue of Src, resulting in the stimulation of downstream p38, ERK/AP-1 and cyclooxygenase-2 (COX-2) signaling in YPEN-1 cells. The role of 4-HNE-activated Src in downstream inflammatory signaling was further investigated using dasatinib (a Src inhibitor) and by siRNA knockdown of Src. p38 and ERK were directly regulated by Src, as revealed by immunoblotting of the phosphorylated forms of mitogen-activated protein kinases (MAPKs), which are key elements in the signaling transduction pathway initiated by Src. The study also shows that Src modulates the HNE-enhanced activation of AP-1 and the expression of COX-2 (a target gene of AP-1). Together, the results of this study show that 4-HNE stimulates Src tyrosine kinase in activation of the inflammation process.

Loss of the m-AAA protease subunit AFG3L2 causes mitochondrial transport defects and tau hyperphosphorylation

The m-AAA protease subunit AFG₃L₂ is involved in degradation and processing of substrates in the inner mitochondrial membrane. Mutations in AFG₃L₂ are associated with spinocerebellar ataxia SCA28 in humans and impair axonal development and neuronal survival in mice. The loss of AFG₃L₂ causes fragmentation of the mitochondrial network. However, the pathogenic mechanism of neurodegeneration in the absence of AFG₃L₂ is still unclear. Here, we show that depletion of AFG₃L₂ leads to a specific defect of anterograde transport of mitochondria in murine cortical neurons. We observe similar transport deficiencies upon loss of AFG₃L₂ in OMA1-deficient neurons, indicating that they are not caused by OMA1-mediated degradation of the dynamin-like GTPase OPA1 and inhibition of mitochondrial fusion. Treatment of neurons with antioxidants, such as N-acetylcysteine or vitamin E, or decreasing tau levels in axons restored mitochondrial transport in AFG₃L₂-depleted neurons. Consistently, tau hyperphosphorylation and activation of ERK kinases are detected in mouse neurons postnatally deleted for Afg3l2. We propose that reactive oxygen species signaling leads to cytoskeletal modifications that impair mitochondrial transport in neurons lacking AFG₃L₂.

Zinc chloride exposure increases heme oxygenase-1 expression in MDPC-23 odontoblast-like cells

ObjectiveThe aim of this study is to clarify the effects of zinc chloride (ZnCl2) exposure on the induction of heme oxygenase-1 (HO-1) expression and its regulatory mechanisms in MDPC-23 mouse odontoblast-like cells. MethodsMDPC-23 cells were incubated with ZnCl2, and the levels of HO-1 protein, phosphorylated forms of mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated protein kinase (ERK), c-Jun NH2-terminal kinase (JNK), and p38, and phosphorylated forms of amino kinase terminal (Akt) and nuclear factor-κB (NF-κB) p65 were determined with western immunoblotting. The level of HO-1 mRNA was determined with RT-PCR analysis. After pretreatment with inhibitors of ERK, JNK, p38, phosphoinositide-3 kinase (PI3K), and NF-κB, HO-1 protein level was determined in MDPC-23 cells exposed to ZnCl2. ResultsFollowing exposure to 500μM ZnCl2, the levels of both HO-1 mRNA and protein were markedly increased. The phosphorylated forms of ERK, JNK, and p38 increased after ZnCl2 exposure. Furthermore, the expression of HO-1 was markedly suppressed by treatment with the p38 inhibitor, SB203580, and mildly suppressed by the MAPK/ERK kinase inhibitor, U0126. However, treatment with the JNK inhibitor, SP600125, did not suppress ZnCl2-induced HO-1 expression. In addition, the phosphorylated forms of Akt, a downstream kinase of PI3K, and NF-κB p65 increased after ZnCl2 exposure. Treatment with the PI3K inhibitor, LY294002, and the NF-κB inhibitor, Bay11-7082, suppressed ZnCl2-induced HO-1 expression. ConclusionThese results suggest that ZnCl2 exposure induces HO-1 expression via multiple intracellular signalling pathways, including p38, ERK, PI3K/Akt, and NF-κB, in this odontoblast-like cell line.

Altered phosphorylation of mitogen-activated protein kinases in dorsal root ganglia and sciatic nerve of rats with cisplatin-induced neuropathy

Background: Peripheral neuropathy is a major side effect of cisplatin. Cisplatin preferentially accumulates in the dorsal root ganglia (DRG) and causes neuronal apoptosis. In vitro studies have implicated mitogenactivated protein kinases (MAPKs) in cisplatin-induced apoptosis. However, this has not been confirmed in vivo. Objective: We studied the phosphorylation of MAPKs, ERK, JNK, and p38, in the DRG and sciatic nerve of rats treated with cisplatin, and correlated it with the neuropathic abnormalities. Methods: Cisplatin 2 mg/kg was intraperitoneally injected in rats twice a week for five consecutive weeks. Neuropathy was assessed by measuring hind-paw thermal and mechanical thresholds, sciatic motor nerve conduction velocity (MNCV) and morphometric evaluation of DRG and sciatic nerve at various time points after the start of cisplatin treatment. Western blot analysis was done to determine the ratio of phosphorylated to total forms of MAPKs in the DRG and sciatic nerve. Results: Cisplatin induced transient thermal hypoalgesia, late reduction in MNCV and histopathological abnormalities of DRG and sciatic nerve indicating the neuropathy. ERK was activated in the nerve and DRG in the eighth and twelfth weeks, respectively. Transient activation of JNK in the nerve and DRG was observed only in the first week. At the same time point to JNK, p38 was temporarily inhibited in the DRG. Late activation of ERK was correlated with the presence of pathological changes, suggesting the possible role of ERK in these abnormalities. No correlation between MAPKs and functional abnormalities was observed. Conclusion: MAPK ERK might play a role in cisplatin-induced structural alterations in the DRG and sciatic nerve and can be the therapeutic target. However, to prove this hypothesis, future studies using the ERK inhibitor must be done. Keywords: Cisplatin, dorsal root ganglia, MAPK, neuropathy, sciatic nerve

A novel method for phosphoprotein extraction from macroalgae

Abstract The extraction of proteins from macroalgae has always been difficult, primarily because of their high internal levels of polysaccharides and polyphenols. Several methods for protein extraction have been developed recently that allow the production of high quality extracts for 1-D SDS gel electrophoresis or 2-D SDS-polyacrylamide gel electrophoresis for proteomic analysis. However, none of the existing methods are applicable to the study of the phosphorylation state of kinases involved in stress responses of macroalgae, primarily because trichloroacetic acid and phenol are used for protein precipitation. In this work, we describe a novel method for protein extraction from macroalgae based on the counterintuitive use of commercial RNA purification kits; this method maintains the phosphorylation state of the kinases intact, as demonstrated by either polyclonal or monoclonal antibodies against the phosphorylated forms of mitogen-activated protein kinases using Western blot methodology. This method may be appropriate for wider use in the analysis of phosphoproteomes.

Changes in Expression of mRNA for Interleukin-8 and Effects of Interleukin-8 Receptor Inhibitor in the Spinal Dorsal Horn in a Rat Model of Lumbar Disc Herniation

Autologous nucleus pulposus obtained from coccygeal intervertebral discs was grafted on the proximal of L5 dorsal root ganglion. Pain behavior, mRNA expression of Interleukin-8 (IL-8), and immunohistochemical changes were assessed. The purpose of this study is to investigate temporal changes of IL-8 expression in the spinal cord and dorsal root ganglion and the pain-related behaviors with time course and to elucidate whether repertaxin (IL-8 receptor inhibitor) attenuates pain-related behaviors in a rat model of lumbar disc herniation. Inflammatory mediators like cytokines and chemokines have been implicated in radicular pain because of disc herniation. IL-8, known as CXCL8, is a chemokine, which has been reported to be associated with painful degenerative disc disorders and chronic inflammatory pain states. Lumbar disc herniated rat model was made by implantation of the autologous nucleus pulposus, harvested from the coccygeal vertebra of each tail, on the left L5 nerve root just proximal to the dorsal root ganglion. Rats were tested for mechanical allodynia and thermal hyperalgesia at 2 days before surgery, and on days 1, 5, 10, 20, 30, 40, 50, and 60 postoperatively. Experimental group was intrathecally injected with the IL-8 receptor inhibitor at L5 level on postoperative day 10. Mechanical allodynia of the plantar surface of both hindpaw was tested on 30 minutes, 1, 3 hours, 1, 3, 5, and 10 days after administration. For the staining of astrocytes and microglia, immunohistochemical study was done 20 days after surgery. Mechanical allodynia in ipsilateral hindpaw developed 1 day after surgery and lasted until 60 days and thermal withdrawal latency decreased significantly on the ipsilateral side 10 days after surgery and gradually increased through day 60. The IL-8 receptor inhibitor attenuated the mechanical allodynia caused by nucleus pulposus when it was administered on postoperative day 10 and reduced microglial activation and phosphorylated form of mitogen-activated protein kinase (pERK) expression in the spinal dorsal horn. IL-8 might be a potential therapeutic target in chronic radicular neuropathic pain because of disc herniation, CXCL8 inhibitor could be one of its promising therapeutic agents.

Effects of salubrinal on cadmium-induced apoptosis in HK-2 human renal proximal tubular cells

Cadmium exposure is known to cause endoplasmic reticulum (ER) stress. In our current study, we examined the effects of salubrinal, a selective inhibitor of eukaryotic translation initiation factor 2 subunit α (eIF2α) dephosphorylation, on apoptotic cell death and ER stress-signaling events in HK-2 human renal proximal tubular cells exposed to cadmium chloride (CdCl(2)). Using phase-contrast microscopy and a cell viability assay, we observed that salubrinal suppressed CdCl(2)-induced cellular damage and cell death. Treatment with salubrinal reduced the number of TUNEL-positive cells and the cleavages of caspase-3 and poly(ADP-ribose) polymerase, but not the cleavage of light chain 3B, indicating protection from CdCl(2)-induced apoptosis but not autophagy. Although eIF2α remained phosphorylated after CdCl(2) exposure to salubrinal-treated HK-2 cells, the expression of activating transcription factor 4 (ATF4) and the 78 kDa glucose-regulated protein (GRP78) was not increased. On the other hand, CdCl(2)-induced expression of C/EBP homologous protein (CHOP) was reduced by salubrinal treatment. Expression of ATF4, an upstream regulator of GRP78 and CHOP, appeared to be a prerequisite for full protection by salubrinal against cadmium cytotoxicity, because CdCl(2)-induced cellular damage was not fully suppressed in ATF4-deficient cells. Phosphorylated forms of mitogen-activated protein kinases (MAPKs), including c-Jun NH(2)-terminal kinase (JNK), p38, and extracellular signal-regulated protein kinase (ERK), increased after CdCl(2) exposure, whereas salubrinal suppressed the phosphorylation of JNK and p38 but not ERK. These results suggest that salubrinal protects CdCl(2)-exposed HK-2 cells from apoptosis by suppressing cell death signal transduction pathways.

The role of mitogen-activated protein kinases in crystalline silica-induced cyclooxygenase-2 expression in A549 human lung epithelial cells

We examined the role of mitogen-activated protein kinase (MAPK) signaling pathways in crystalline silica-induced expression of cyclooxygenase (COX)-2, an important mediator of airway inflammation, in A549 human lung epithelial cells. The levels of COX-2 mRNA increased after a 30-min exposure, and COX-2 protein increased after a 2-h exposure to crystalline silica. Both remained elevated at 8 h; however, no change was observed in the expression of the constitutive COX-1 isoform. The level of prostaglandin E2, a major product of COX enzymes, increased in response to crystalline silica exposure. Phosphorylated forms of MAPKs including extracellular signal-regulated protein kinase (ERK), c-Jun NH2-terminal kinase, and p38 were also increased after crystalline silica exposure. COX-2 expression was markedly suppressed by treatment with the p38 inhibitor, SB203580, and mildly suppressed by the MAPK/ERK kinase inhibitor, U0126. Treatment with the nuclear factor-κB (NF-κB) inhibitor, BAY11-7082, markedly suppressed silica-induced COX-2 expression. These results show that crystalline silica exposure induces COX-2 expression in A549 cells in a manner that is dependent on the MAPK and NF-κB pathways. Although a marked induction of MAPK phosphatase (MKP)-1 expression was observed in A549 cells exposed to crystalline silica, the silencing of MKP-1 expression using short interference RNA did not affect silica-induced COX-2 expression, suggesting that the down-regulation of COX-2 expression by MKP-1 is unlikely.

Cadmium induces phosphorylation and stabilization of c-Fos in HK-2 renal proximal tubular cells

We examined the effects of cadmium chloride (CdCl 2) exposure on the expression and phosphorylation status of members of the Fos family, components of the activator protein-1 transcription factor, in HK-2 human renal proximal tubular cells. Following the exposure to CdCl 2, the expression of c- fos, fosB, fra-1, and fra-2 increased markedly, with different magnitudes and time courses. The levels of Fos family proteins (c-Fos, FosB, Fra-1, and Fra-2) also increased in response to CdCl 2 exposure. Although the elevation of c- fos transcripts was transient, c-Fos protein levels increased progressively with lower electrophoretic mobility, suggesting stabilization of c-Fos through post-translational modifications. Consistently, we observed phosphorylation of c-Fos at Ser362 and Ser374 in HK-2 cells treated with CdCl 2. Phosphorylated forms of mitogen-activated protein kinases (MAPKs)—including extracellular signal-regulated protein kinase (ERK), c-Jun NH 2-terminal kinase, and p38—increased after CdCl 2 exposure, whereas treatment with the MAPK/ERK kinase inhibitor U0126 and the p38 inhibitor SB203580 suppressed the accumulation and phosphorylation of c-Fos. We mutated Ser362 to alanine (S362A), Ser374 to alanine (S374A), and both residues to alanines (S362A/S374A) to inhibit potential phosphorylation of c-Fos at these sites. S374A or double S362A/S374A mutations reduced c-Fos level markedly, but S362A mutation did not. On the other hand, S362A/S374A mutations induced a more pronounced reduction in c-Fos DNA-binding activity than S374A mutation. These results suggest that while Ser374 phosphorylation seems to play a role in c-Fos stabilization, phosphorylation at two C-terminal serine residues is required for the transcriptional activation of c-Fos in HK-2 cells treated with CdCl 2.

Fluoride-induced cyclooxygenase-2 expression and prostaglandin E2 production in A549 human pulmonary epithelial cells

To clarify the mechanisms of fluoride-induced airway diseases, we examined the expression of cyclooxygenase-2 (COX-2), an important mediator of airway inflammation, in A549 human pulmonary epithelial cells treated with sodium fluoride (NaF). Following exposure to 5mM NaF, COX-2 protein and COX-2 transcript increased markedly. However, no change was observed in COX-1 expression. NaF-induced accumulation of COX-2 transcript was abolished by actinomycin D, but not cycloheximide. The level of prostaglandin E(2), a major product of COX enzymes, increased in response to NaF exposure, and its production was abolished by the selective COX-2 inhibitor NS-398. Phosphorylated forms of mitogen-activated protein kinases (MAPKs)-including extracellular signal-regulated protein kinase (ERK), c-Jun NH(2)-terminal kinase, and p38-increased after NaF exposure, while treatment with the MAPK/ERK kinase inhibitor U0126 and the p38 inhibitor SB203580 markedly suppressed COX-2 expression. Furthermore, NaF-induced COX-2 expression was markedly suppressed by the Src family kinase (SFK) inhibitor PP2, but only partially suppressed by the epidermal growth factor receptor (EGFR) inhibitor PD153035. These results suggest that NaF induces COX-2 expression by transcriptional up-regulation via p38 and ERK pathways, at least in part, and that SFKs may be upstream tyrosine kinases responsible for NaF-induced COX-2 expression in A549 cells.

Signalling Pathway Involving GULP, MAPK and Rac1 for SR-BI-Induced Phagocytosis of Apoptotic Cells

Class B scavenger receptor type I (SR-BI) is a phosphatidylserine (PS)-recognizing receptor of testicular Sertoli cells responsible for the phagocytosis of spermatogenic cells undergoing apoptosis. Here, we determined signal mediators that compose a signalling pathway for SR-BI-induced phagocytosis. Results of a yeast two-hybrid analysis and a cell-free binding assay indicated that SR-BI binds to engulfment adapter protein (GULP) using the C-terminal intracellular domain. A co-immunoprecipitation analysis showed the existence of a complex of GULP and SR-BI in cells prior to the activation of SR-BI by PS. A reduction of GULP expression in phagocytes decreased the SR-BI-mediated phagocytosis of apoptotic cells. Administration to phagocytes of PS-containing liposomes increased the levels of the GTP-bound form of Rac1 and the phosphorylated forms of mitogen-activated protein kinases (MAPK) p38 and extracellular signal-related kinase 1 and 2. Finally, lowering the expression of GULP abrogated MAPK phosphorylation, and the presence of MAPK inhibitors reduced the level of GTP-bound Rac1 in PS-activated phagocytes. These results collectively suggested the following signalling pathway for the SR-BI-induced phagocytosis: (i) PS-recognizing SR-BI activates associated GULP; (ii) activated GULP induces MAPK phosphorylation; (iii) activated MAPK increases GTP-bound Rac1; and (iv) activated Rac1 induces a rearrangement of the actin cytoskeleton.

Regulation of floral organ abscission in Arabidopsis thaliana

Abscission is a developmental program that results in the active shedding of infected or nonfunctional organs from a plant body. Here, we establish a signaling pathway that controls abscission in Arabidopsis thaliana from ligand, to receptors, to downstream effectors. Loss of function mutations in Inflorescence Deficient in Abscission (IDA), which encodes a predicted secreted small protein, the receptor-like protein kinases HAESA (HAE) and HAESA-like 2 (HSL2), the Mitogen-Activated Protein Kinase Kinase 4 (MKK4) and MKK5, and a dominant-negative form of Mitogen-Activated Protein Kinase 6 (MPK6) in a mpk3 mutant background all have abscission-defective phenotypes. Conversely, expression of constitutively active MKKs rescues the abscission-defective phenotype of hae hsl2 and ida plants. Additionally, in hae hsl2 and ida plants, MAP kinase activity is reduced in the receptacle, the part of the stem that holds the floral organs. Plants overexpressing IDA in a hae hsl2 background have abscission defects, indicating HAE and HSL2 are epistatic to IDA. Taken together, these results suggest that the sequential action of IDA, HAE and HSL2, and a MAP kinase cascade regulates the programmed separation of cells in the abscission zone.

Phosphorylation of Mitogen‐Activated Protein Kinase 8 (MAPK8) Is Associated With Germ Cell Apoptosis and Redistribution of the Bcl2‐Modifying Factor (BMF)

Successful spermatogenesis requires that germ cells remain in physical contact with Sertoli cells until spermiation. Previous studies have shown that the Bcl2-modifying factor (BMF) is a proapoptotic protein found in many epithelial cells which, when phosphorylated by the active form of mitogen-activated protein kinase 8 (p-MAPK8), initiates apoptosis in response to loss of adhesion of the cells to their basal lamina. Based on this, we hypothesized that p-MAPK8 and BMF may play important roles in the apoptotic death of testicular germ cells in response to their detachment from Sertoli cells. Immunohistochemical analysis of the normal rat testis revealed p-MAPK8 expression in spermatocytes and elongated spermatids but not in round spermatids. This localization was opposite to that of BMF, which is expressed in round spermatids but not in spermatocytes or elongated spermatids. When freshly isolated germ cells were cultured in the absence of Sertoli cells, a condition in which there was widespread germ cell apoptosis, an increase in p-MAPK8 relative to overall MAPK8 protein, was seen by Western blot analysis. Additionally, immunocytochemical analysis showed an increase in immunoreactive p-MAPK8 in round spermatids and spermatocytes in association with BMF expression. From these correlative data, we propose that the activation of MAPK8 and redistribution of BMF may be integrally involved in the mechanism by which specific germ cells undergo programmed cell death in response to their detachment from Sertoli cells.

Endothelin‐1 induces proliferation of human lung fibroblasts and IL‐11 secretion through an ETA receptor‐dependent activation of map kinases

Endothelin-1 (ET-1) is implicated in the fibrotic responses characterizing interstitial lung diseases, as well as in the airway remodeling process occurring in asthma. Within such a context, the aim of our study was to investigate, in primary cultures of normal human lung fibroblasts (NHLFs), the ET-1 receptor subtypes, and the intracellular signal transduction pathways involved in the proliferative effects of this peptide. Therefore, cells were exposed to ET-1 in the presence or absence of an overnight pre-treatment with either ET(A) or ET(B) selective receptor antagonists. After cell lysis, immunoblotting was performed using monoclonal antibodies against the phosphorylated, active forms of mitogen-activated protein kinases (MAPK). ET-1 induced a significant increase in MAPK phosphorylation pattern, and also stimulated fibroblast proliferation and IL-6/IL-11 release into cell culture supernatants. All these effects were inhibited by the selective ET(A) antagonist BQ-123, but not by the specific ET(B) antagonist BQ-788. The stimulatory influence of ET-1 on IL-11, but not on IL-6 secretion, was prevented by MAPK inhibitors. Therefore, such results suggest that in human lung fibroblasts ET-1 exerts a profibrogenic action via an ET(A) receptor-dependent, MAPK-mediated induction of IL-11 release and cell proliferation.