Inhibition Of Mitogen-activated Protein Kinase Activation Research Articles

Anti-inflammatory effects of the extract of indigo naturalis in human neutrophils

Ethnopharmacological relevance Indigo naturalis is used by traditional Chinese medicine to treat various inflammatory diseases. Aim of the study Topical indigo naturalis ointment showed efficacy in treating psoriasis in our previous clinical studies. In this study, we investigated the anti-inflammatory effects of the extract of indigo naturalis (QD) and its main components indirubin, indigo, and tryptanthrin in human neutrophils. Materials and methods Superoxide anion (O 2 −) generation and elastase release were measured by spectrophotometry. Some important signals including mitogen-activated protein kinase (MAPK), cAMP, and calcium were studied by Western blot analysis, an enzyme immunoassay, and spectrofluorometry. Results QD significantly inhibited O 2 − generation and elastase release in formyl- l-methionyl- l-leucyl- l-phenylalanine (FMLP)-activated human neutrophils in a concentration-dependent fashion, while neither indirubin, indigo, nor tryptanthrin produced a comparable result. QD attenuated the FMLP-induced phosphorylation of extracellular regulated kinase, p38 MAPK, and c- Jun N-terminal kinase. Furthermore, QD inhibited calcium mobilization caused by FMLP. However, QD did not affect cellular cAMP levels. On the other hand, neither indirubin, indigo, nor tryptanthrin produced similar changes in human neutrophils. Conclusions Taken collectively, these findings indicate that QD, but not indirubin, indigo, or tryptanthrin, inhibited O 2 − generation and elastase release in FMLP-induced human neutrophils, which was at least partially mediated by the inhibition of MAPK activation and regulation of calcium mobilization.

Recent Developments in Anti-Cancer Agents Targeting the Ras/Raf/ MEK/ERK Pathway

The Ras/Raf/MEK/ERK mitogen-activated protein kinase (MAPK) pathway mediates cellular responses to different growth signals and is frequently deregulated in cancer. There are three Raf kinases-A-Raf, B-Raf, and C-Raf; however, only B-Raf is frequently mutated in various cancers. The most common B-Raf mutation involves a substitution of a glutamic acid residue to a valine moiety at codon 600. Subsequently, the MAPK pathway is constitutively activated, even in the absence of any growth signals. Although early attempts to target Ras have not yielded any viable drug candidates, many novel compounds inhibiting the activities of Raf and MEK have been developed and investigated in clinical trials in recent years. The first MEK inhibitor (CI-1040) lacked efficacy in clinical trials, but its low toxicity has encouraged the search for novel compounds with enhanced target potency to inhibit MAPK activation at low nanomolar concentrations. In this review, we will discuss new patents or patent applications related to inhibitors of the Ras/Raf/MEK/ERK pathway.

DUSP1 Is Controlled by p53 during the Cellular Response to Oxidative Stress

p53 controls the cellular response to genotoxic stress through multiple mechanisms. We report here that p53 regulates DUSP1, a dual-specific threonine and tyrosine phosphatase with stringent substrate specificity for mitogen-activated protein kinase (MAPK). DUSP1 is a potent inhibitor of MAPK activity through dephosphorylation of MAPK. In a colon cancer cell line containing inducible ectopic p53, DUSP1 protein level is significantly increased upon activation of p53, leading to cell death in response to nutritional stress. In mouse embryo fibroblast cells, DUSP1 protein abundance is greatly increased after oxidative stress in a p53-dependent manner and also when apoptosis is triggered. We show that p53 induces the activity of a human DUSP1 regulatory region. Furthermore, p53 can physically interact with the DUSP1 regulatory region in vivo, and p53 binds to a 10-bp perfect palindromic site in this DUSP1 regulatory region. We show that overexpression of DUSP1 or inhibition of MAPK activity significantly increases cellular susceptibility to oxidative damage. These findings indicate that p53 is a transcriptional regulator of DUSP1 in stress responses. Our results reveal a mechanism whereby p53 selectively regulates target genes and suggest a way in which subgroups of those target genes might be controlled independently.

Reversal of the Estrogen Receptor–Negative Phenotype in Breast Cancer and Restoration of Antiestrogen Response

In breast cancer, the presence of estrogen receptor alpha (ER) denotes a better prognosis and response to antiestrogen therapy. Lack of ERalpha correlates with overexpression of epidermal growth factor receptor or c-erbB-2. We have shown that hyperactivation of mitogen-activated protein kinase (MAPK) directly represses ERalpha expression in a reversible manner. In this study, we determine if inhibition of MAPK in established ERalpha(-) breast cancer cell lines and tumors results in reexpression of ERalpha, and further, if reexpression of ERalpha in these ERalpha(-) tumors and cell lines could restore antiestrogen responses. Established ERalpha(-) breast cancer cell lines, ERalpha(-) breast tumors, and tumor cell cultures obtained from ERalpha(-) tumors were used in this study. Inhibition of hyperactive MAPK was accomplished via the MAPK/ERK kinase 1/2 inhibitor U0126 or via upstream inhibition with Iressa or Herceptin. Western blotting or reverse transcription-PCR for ERalpha was used to assess the reexpression of ERalpha in cells treated with U0126. Growth assays with WST-1 were done to assess restoration of antiestrogen sensitivity in these cells. Inhibition of MAPK activity in ERalpha(-) breast cancer cell lines results in reexpression of ERalpha; upstream inhibition via targeting epidermal growth factor receptor or c-erbB-2 is equally effective. Importantly, this reexpressed ERalpha can now mediate an antiestrogen response in a subset of these ERalpha(-) breast cancer cell lines. Treatment of ERalpha(-) tumor specimens with MAPK inhibitors results in restoration of ERalpha mRNA, and similarly in epithelial cultures from ERalpha(-) tumors, MAPK inhibition restores both ERalpha protein and antiestrogen response. These data show both the possibility of restoring ERalpha expression and antiestrogen responses in ERalpha(-) breast cancer and suggest that there exist ERalpha(-) breast cancer patients who would benefit from a combined MAPK inhibition/hormonal therapy.

AMP N1‐Oxide, a Unique Compound of Royal Jelly, Induces Neurite Outgrowth from PC12 Vells via Signaling by Protein Kinase A Independent of that by Mitogen‐Activated Protein Kinase

Earlier we identified adenosine monophosphate (AMP) N1-oxide as a unique compound of royal jelly (RJ) that induces neurite outgrowth (neuritegenesis) from cultured rat pheochromocytoma PC12 cells via the adenosine A2A receptor. Now, we found that AMP N1-oxide stimulated the phosphorylation of not only mitogen-activated protein kinase (MAPK) but also that of cAMP/calcium-response element-binding protein (CREB) in a dose-dependent manner. Inhibition of MAPK activation by a MEK inhibitor, PD98059, did not influence the AMP N1-oxide-induced neuritegenesis, whereas that of protein kinase A (PKA) by a selective inhibitor, KT5720, significantly reduced neurite outgrowth. AMP N1-oxide also had the activity of suppressing the growth of PC12 cells, which correlated well with the neurite outgrowth-promoting activity. KT5720 restored the growth of AMP N1-oxide-treated PC12 cells. It is well known that nerve growth factor suppresses proliferation of PC12 cells before causing stimulation of neuronal differentiation. Thus, AMP N1-oxide elicited neuronal differentiation of PC12 cells, as evidenced by generation of neurites, and inhibited cell growth through adenosine A2A receptor-mediated PKA signaling, which may be responsible for characteristic actions of RJ.

Material-based regulation of the myofibroblast phenotype

Fibroblast growth factor receptor (FGFR) activation by basic fibroblast growth factor (FGF-2) serves to naturally repress the myofibroblast activation of valvular interstitial cells (VICs). Co-receptors for FGF-2, the heparan sulfate proteoglycans (HSPGs), are key participants in the formation of active FGF-2 signaling complexes. Bioactive environments regulating the myofibroblast phenotype were created by utilizing heparin glycosaminoglycan as a competitive inhibitor of HSPGs. First, soluble heparin was delivered to compete with cell-surface HSPG for the binding of FGF-2. Exogenous soluble heparin prevented serum-dependent activation of the classic mitogen-activated protein kinase (MAPK) and induced myofibroblast alpha smooth muscle actin ( αSMA) expression and collagen production. Next, heparin-functionalized hydrogel cell substrates were polymerized from vinyl-modified precursors and rendered adhesive through incorporation of RGDS peptide. Culture of VICs on heparin-modified gels induced αSMA expression and inhibited MAPK activity compared to control gel substrates lacking heparin. Additionally, heparin-functionalized gels continued to induce αSMA expression in serum-free culture conditions, suggesting that bioactivity was independent of exogenous soluble mediators. Biomaterial scaffolds targeting cell surface growth factor receptors are a promising new direction for regulating cell functions in tissue-engineering applications.

Calpain Cleaves a Memory Inhibitor

Calpain, a calcium-dependent protease, has been implicated in neuronal responses to various stimuli, and Shimizu et al . provide evidence that calpain contributes to certain types of learning and memory. Shimizu et al . (see commentary by Bolshakov) found that suprachiasmatic nucleus circadian oscillatory protein (SCOP) was abundant in the hippocampus of mice and rats. Overexpression of SCOP in cultured hippocampal neurons decreased the expression of an adenosine 3′,5′-monophosphate (cAMP) response element (CRE)-regulated reporter gene stimulated by brain-derived neurotrophic factor (BDNF), whereas knockdown of the endogenous protein using RNA interference enhanced reporter gene expression in the presence or absence of BDNF. CRE-mediated gene expression is downstream of activation of mitogen-activated protein kinases (MAPKs) and contributes to synaptic changes involved in learning and memory. SCOP was previously reported to inhibit K-Ras by sequestering the nucleotide-free form, thereby inhibiting MAPK activation. The authors found that SCOP abundance was decreased in cultured hippocampal neurons stimulated with BDNF, the glutamate receptor agonist NMDA, or KCl. Assays for SCOP cleavage in cell-free extracts in the presence of various protease pharmacological inhibitors indicated that calpains, not caspase 3 or the proteasome, cleaved SCOP and that SCOP cleavage required calcium. In cultured hippocampal neurons, calpain inhibition blocked SCOP degradation and decreased the phosphorylation (and activation) of the MAPK ERK-2 stimulated by BDNF. In mice, memory for new objects was associated with a decrease in the abundance of SCOP (in whole hippocampal extracts). Mice overexpressing SCOP using an inducible promoter showed decreased memory for new objects 24 hours after object training but no deficiency in short-term memory. Consistent with a role for calpain in memory, administration of a calpain inhibitor into the hippocampus blocked new object memory after 24 hours but had no effect on short-term memory measured after 8 minutes. K. Shimizu, T. Phan, I. M. Mansuy, D. R. Storm, Proteolytic degradation of SCOP in the hippocampus contributes to activation of MAP kinase and memory. Cell 128 , 1219-1229 (2007). [Online Journal] V. Y. Bolshakov, SCOPing out proteases in long-term memory. Cell 128 , 1029-1030 (2007). [Online Journal]

MAPK‐dependent actin cytoskeletal reorganization underlies BK channel activation by insulin

Numerous brain regions are enriched with insulin and insulin receptors, and several lines of evidence indicate that insulin is an important modulator of neuronal function. Indeed, recent studies have demonstrated that insulin inhibits hippocampal epileptiform-like activity, in part by activating large-conductance Ca2+-activated potassium (BK) channels. Moreover, the mitogen-activated protein kinase (MAPK) signalling cascade has been found to couple insulin to BK channel activation. However, the cellular events downstream of MAPK that underlie this action of insulin are unknown. Here we demonstrate that in hippocampal neurons, BK channel activation by insulin is blocked by actin filament stabilization, suggesting that this process is dependent on the actin cytoskeleton. Stabilizing actin filaments also markedly attenuated the ability of insulin to inhibit the aberrant hippocampal synaptic activity evoked following Mg2+ removal. Insulin also promoted rapid reorganization of fluorescently labelled polymerized actin filaments; an action that was prevented by inhibitors of MAPK activation. Moreover, in parallel studies, insulin increased the level of phospho-MAPK immunostaining in hippocampal neurons. These data are consistent with BK channel activation by insulin involving MAPK-dependent alterations in actin dynamics. This process may have important implications for the role of insulin in regulating hippocampal excitability.

Metaphase Arrest by Cyclin E-Cdk2 Requires the Spindle-Checkpoint Kinase Mps1

Cytostatic factor (CSF) arrests vertebrate eggs in metaphase of meiosis II through several pathways that inhibit activation of the anaphase-promoting complex/cyclosome (APC/C). In Xenopus, the Mos-MEK1-MAPK-p90(Rsk) cascade utilizes spindle-assembly-checkpoint components to effect metaphase arrest. Another pathway involves cyclin E-Cdk2, and sustained cyclin E-Cdk2 activity in egg extracts causes metaphase arrest in the absence of Mos; this latter finding suggests that an independent pathway contributes to CSF arrest. Here, we demonstrate that metaphase arrest with cyclin E-Cdk2, but not with Mos, requires the spindle-checkpoint kinase monopolar spindles 1 (Mps1), a cyclin E-Cdk2 target that is also implicated in centrosome duplication. xMps1 is synthesized and activated during oocyte maturation and inactivated upon CSF release. In egg extracts, CSF release by calcium was inhibited by constitutively active cyclin E-Cdk2 and delayed by wild-type xMps1. Ablation of cyclin E by antisense oligonucleotides blocked accumulation of xMps1, suggesting that cyclin E-Cdk2 controls Mps1 levels. During meiosis II, activated cyclin E-Cdk2 significantly inhibited the APC/C even in the absence of the Mos-MAPK pathway, but this inhibition was not sufficient to suppress S phase between meiosis I and II. These results uniquely place xMps1 downstream of cyclin E-Cdk2 in mediating a pathway of APC/C inhibition and metaphase arrest.

Therapeutic Implications of a Human Neutralizing Antibody to the Macrophage-Stimulating Protein Receptor Tyrosine Kinase (RON), a c-MET Family Member

RON is a member of the c-MET receptor tyrosine kinase family. Like c-MET, RON is expressed by a variety of epithelial-derived tumors and cancer cell lines and it is thought to play a functional role in tumorigenesis. To date, antagonists of RON activity have not been tested in vivo to validate RON as a potential cancer target. In this report, we used an antibody phage display library to generate IMC-41A10, a human immunoglobulin G1 (IgG1) antibody that binds with high affinity (ED50 = 0.15 nmol/L) to RON and effectively blocks interaction with its ligand, macrophage-stimulating protein (MSP; IC50 = 2 nmol/L). We found IMC-41A10 to be a potent inhibitor of receptor and downstream signaling, cell migration, and tumorigenesis. It antagonized MSP-induced phosphorylation of RON, mitogen-activated protein kinase (MAPK), and AKT in several cancer cell lines. In HT-29 colon, NCI-H292 lung, and BXPC-3 pancreatic cancer xenograft tumor models, IMC-41A10 inhibited tumor growth by 50% to 60% as a single agent, and in BXPC-3 xenografts, it led to tumor regressions when combined with Erbitux. Western blot analyses of HT-29 and NCI-H292 xenograft tumors treated with IMC-41A10 revealed a decrease in MAPK phosphorylation compared with control IgG-treated tumors, suggesting that inhibition of MAPK activity may be required for the antitumor activity of IMC-41A10. To our knowledge, this is the first demonstration that a RON antagonist and specifically an inhibitory antibody of RON negatively affects tumorigenesis. Another major contribution of this report is an extensive analysis of RON expression in approximately 100 cancer cell lines and approximately 300 patient tumor samples representing 10 major cancer types. Taken together, our results highlight the potential therapeutic usefulness of RON activity inhibition in human cancers.

Regulation of Integrin Activity by MIA

MIA (melanoma inhibitory activity) has been identified as a small protein secreted from malignant melanoma cells, which interacts with extracellular matrix proteins including fibronectin. Here, we show that MIA negatively regulates the activity of the mitogen-activated protein kinase pathway in malignant melanoma. Using far Western blotting and co-immunoprecipitation we searched for MIA-binding cell surface proteins. We found that MIA interacts with integrin alpha4beta1 and alpha5beta1, leading to down-regulation of integrin activity and reduction of mitogen-activated protein kinase signaling. These findings also suggest that MIA may play a role in tumor progression and the spread of malignant melanomas via mediating detachment of cells from extracellular matrix molecules by modulating integrin activity. Inhibiting MIA functions in vivo may therefore provide a novel therapeutic strategy for metastatic melanoma disease.

Distinct β-Arrestin- and G Protein-dependent Pathways for Parathyroid Hormone Receptor-stimulated ERK1/2 Activation

Parathyroid hormone (PTH) regulates calcium homeostasis via the type I PTH/PTH-related peptide (PTH/PTHrP) receptor (PTH1R). The purpose of the present study was to identify the contributions of distinct signaling mechanisms to PTH-stimulated activation of the mitogen-activated protein kinases (MAPK) ERK1/2. In Human embryonic kidney 293 (HEK293) cells transiently transfected with hPTH1R, PTH stimulated a robust increase in ERK activity. The time course of ERK1/2 activation was biphasic with an early peak at 10 min and a later sustained ERK1/2 activation persisting for greater than 60 min. Pretreatment of HEK293 cells with the PKA inhibitor H89 or the PKC inhibitor GF109203X, individually or in combination reduced the early component of PTH-stimulated ERK activity. However, these inhibitors of second messenger dependent kinases had little effect on the later phase of PTH-stimulated ERK1/2 phosphorylation. This later phase of ERK1/2 activation at 30-60 min was blocked by depletion of cellular beta-arrestin 2 and beta-arrestin 1 by small interfering RNA. Furthermore, stimulation of hPTH1R with PTH analogues, [Trp1]PTHrp-(1-36) and [d-Trp12,Tyr34]PTH-(7-34), selectively activated G(s)/PKA-mediated ERK1/2 activation or G protein-independent/beta-arrestin-dependent ERK1/2 activation, respectively. It is concluded that PTH stimulates ERK1/2 through several distinct signal transduction pathways: an early G protein-dependent pathway meditated by PKA and PKC and a late pathway independent of G proteins mediated through beta-arrestins. These findings imply the existence of distinct active conformations of the hPTH1R responsible for the two pathways, which can be stimulated by unique ligands. Such ligands may have distinct and valuable therapeutic properties.

Peanut lectin stimulates proliferation of colon cancer cells by interaction with glycosylated CD44v6 isoforms and consequential activation of c-Met and MAPK: functional implications for disease-associated glycosylation changes

Peanut agglutinin lectin (PNA) binds the Thomsen-Friedenreich (TF) oncofetal carbohydrate antigen (galactose beta1-3N-acetylgalactosamine alpha) that shows increased expression in colon cancer, adenomas, and inflammatory bowel disease. PNA is mitogenic, both in vitro and in vivo, for colon epithelial cells. In these cells, PNA binds predominantly to cell-surface TF antigen expressed by high molecular weight isoforms of the transmembrane glycoprotein CD44 that are generated in inflamed and neoplastic colonic epithelia by altered RNA splicing. Our aim was to identify the signaling mechanism underlying the proliferative response to PNA. This was investigated in HT29, T84, and Caco2 colon cancer cells. Parallel lectin and immunoblotting of PNA affinity-purified HT29 cell membrane extracts showed PNA binding to high molecular weight CD44v6 isoforms. Within 5 min, PNA (25 microg/mL) caused a 6-fold increase in phosphorylation of hepatocyte growth factor receptor c-Met, known to co-associate with CD44v6. This was followed by the downstream activation of p44/p42 mitogen-activated protein kinase (MAPK) over 15-20 min. The presence of 100 microg/mL asialofetuin, a TF antigen-expressing glycoprotein, blocked both PNA-induced c-Met and MAPK activation. A similar PNA-induced c-Met and MAPK phosphorylation was also seen in T84 cells that express CD44v6 but not in Caco2 cells that lack CD44v6. PNA-induced cell proliferation was completely blocked by 1 microM PD98059, an inhibitor of MAPK activation (p < 0.0001). The expression of TF antigen by CD44 isoforms in colonic epithelial cells allows lectin-induced mitogenesis that is mediated by phosphorylation of c-Met and MAPK. It provides a mechanism by which dietary, microbial, or endogenous galactose-binding lectins could affect epithelial proliferation in the cancerous and precancerous colon.

Involvement of mitogen-activated protein kinase in 2-hydroxyestradiol-17β-induced oocyte maturation in the catfish Heteropneustes fossilis and a note on possible interaction with protein phosphatases

Mitogen-activated protein kinase (MAPK) was demonstrated in the postvitellogenic follicles (theca-granulosa and oocyte) of catfish by Western blotting using a polyclonal anti-rabbit serum, which recognized both ERK1 and ERK2. Two distinct protein bands resolved in the 46–48 kDa range of 12% SDS–PAGE were immunoblotted. Incubation of the follicles with 5 μM 2-OHE 2 elicited GVBD significantly in a duration-dependent manner with a concomitant increase in the expression of MAPK (ERK1 and ERK2). Densitometric analysis of the immunoblots showed significant variations in the intensity of staining. The ERK1 expression increased significantly from 6 h onwards but the changes were less pronounced. On the other hand, ERK2 registered a sharp significant increase after 3 h, which paralleled the GVBD response. The MEK inhibitor PD098059 alone did not induce GVBD. Co-incubation of the follicles with 2-OHE 2 and PD098059 significantly inhibited the steroid-induced GVBD at all concentrations. Immunoblot analysis showed that PD098059 inhibited MAPK activity significantly compared to the 2-OHE 2 group. The addition of okadaic acid (OA) in the incubation medium containing both 2-OHE 2 and PD098059 reversed the inhibitory effect of the latter and GVBD was elevated significantly over that of the 2-OHE 2 group but significantly lower than that of the 2-OHE 2 + OA group. The results suggest an involvement of MAPK in meiotic maturation but the site(s) of action: oocyte, follicular envelope or both needs further investigation.

O.189 Interferon regulatory factor-1 promoter polymorphism and the outcome of hepatitis C virus infection

IFNλ is important for epidermal defense against viruses. It is produced by, and acts on, keratinocytes, whereas fibroblasts were previously considered to be unresponsive to this type III IFN. Herein we report findings revealing cell type-specific differences in IFNλ signaling and function in skin resident cells. In dermal fibroblasts, IFNλ induced the expression of myxovirus protein A (MxA), a potent antiviral factor, but not other IFN signature genes as it does in primary keratinocytes. In contrast to its effect on keratinocytes, IFNλ did not phosphorylate signal transducer and activator of transcription 1 in fibroblasts, but instead activated mitogen activated protein kinases (MAPK). Accordingly, inhibition of MAPK activation (p38 and p42/44) blocked the expression of MxA protein in fibroblasts but not in keratinocytes. Functionally, IFNλ inhibited proliferation in keratinocytes but not in fibroblasts. Moreover, IFNλ upregulated the expression of Tumor growth factor beta 1 (TGFβ1)-induced collagens in fibroblasts. Taken together, our findings identify primary human dermal fibroblasts as responder cells to IFNλ. Our study shows cutaneous cell type-specific IFN signaling and suggests that IFNλ, although important for epidermal antiviral competence, may also have a regulatory role in the dermal compartment balancing type I IFN-induced inhibition of tissue repair processes.

O.190 Inhibition of hepatitis C virus replication by tumor suppressor p53: Novel prospects of p53 in antiviral defense

IFNλ is important for epidermal defense against viruses. It is produced by, and acts on, keratinocytes, whereas fibroblasts were previously considered to be unresponsive to this type III IFN. Herein we report findings revealing cell type-specific differences in IFNλ signaling and function in skin resident cells. In dermal fibroblasts, IFNλ induced the expression of myxovirus protein A (MxA), a potent antiviral factor, but not other IFN signature genes as it does in primary keratinocytes. In contrast to its effect on keratinocytes, IFNλ did not phosphorylate signal transducer and activator of transcription 1 in fibroblasts, but instead activated mitogen activated protein kinases (MAPK). Accordingly, inhibition of MAPK activation (p38 and p42/44) blocked the expression of MxA protein in fibroblasts but not in keratinocytes. Functionally, IFNλ inhibited proliferation in keratinocytes but not in fibroblasts. Moreover, IFNλ upregulated the expression of Tumor growth factor beta 1 (TGFβ1)-induced collagens in fibroblasts. Taken together, our findings identify primary human dermal fibroblasts as responder cells to IFNλ. Our study shows cutaneous cell type-specific IFN signaling and suggests that IFNλ, although important for epidermal antiviral competence, may also have a regulatory role in the dermal compartment balancing type I IFN-induced inhibition of tissue repair processes.

The Cannabinoid CB1 Receptor Antagonist Rimonabant (SR141716) Inhibits Cell Proliferation and Increases Markers of Adipocyte Maturation in Cultured Mouse 3T3 F442A Preadipocytes

Adipocyte cell proliferation is an important process in body fat mass development in obesity. Adiponectin or Acrp30 is an adipocytokine exclusively expressed and secreted by adipose tissue that regulates lipid and glucose metabolism and plays a key role in body weight regulation and homeostasis. Adiponectin mRNA expression in adipose tissue and plasma level of adiponectin are decreased in obesity and type 2 diabetes. In obese rodents, the selective CB(1) receptor antagonist rimonabant reduces food intake and body weight and improves lipid and glucose parameters. We have reported previously that rimonabant stimulated adiponectin mRNA expression in adipose tissue of obese fa/fa rats, by a direct effect on adipocytes. We report here that rimonabant (10-400 nM) inhibits cell proliferation of cultured mouse 3T3 F442A preadipocytes in a concentration-dependent manner. In parallel to this inhibitory effect on preadipocyte cell proliferation, rimonabant (25-100 nM) stimulates mRNA expression and protein levels of two late markers of adipocyte differentiation (adiponectin and glyceraldehyde-3-phosphate dehydrogenase) with a maximal effect at 100 nM, without inducing the accumulation of lipid droplets. Furthermore, treatment of mouse 3T3 F442A preadipocytes with rimonabant (100 nM) inhibits basal and serum-induced p42/44 mitogen-activated protein (MAP) kinase activity. These results suggest that inhibition of MAP kinase activity by rimonabant may be one of mechanisms involved in the inhibition of 3T3 F442A preadipocyte cell proliferation and stimulation of adiponectin and GAPDH expression. The inhibition of preadipocyte cell proliferation and the induction of adipocyte late "maturation" may participate in rimonabant-induced antiobesity effects, particularly the reduction of body fat mass.

Phosphodiesterase 3A binds to 14-3-3 proteins in response to PMA-induced phosphorylation of Ser428

PDE3A (phosphodiesterase 3A) was identified as a phosphoprotein that co-immunoprecipitates with endogenous 14-3-3 proteins from HeLa cell extracts, and binds directly to 14-3-3 proteins in a phosphorylation-dependent manner. Among cellular stimuli tested, PMA promoted maximal binding of PDE3A to 14-3-3 proteins. While p42/p44 MAPK (mitogen-activated protein kinase), SAPK2 (stress-activated protein kinase 2)/p38 and PKC (protein kinase C) were all activated by PMA in HeLa cells, the PMA-induced binding of PDE3A to 14-3-3 proteins was inhibited by the non-specific PKC inhibitors Ro 318220 and H-7, but not by PD 184352, which inhibits MAPK activation, nor by SB 203580 and BIRB0796, which inhibit SAPK2 activation. Binding of PDE3A to 14-3-3 proteins was also blocked by the DNA replication inhibitors aphidicolin and mimosine, but the PDE3A-14-3-3 interaction was not cell-cycle-regulated. PDE3A isolated from cells was able to bind to 14-3-3 proteins after in vitro phosphorylation with PKC isoforms. Using MS/MS of IMAC (immobilized metal ion affinity chromatography)-enriched tryptic phosphopeptides and phosphospecific antibodies, at least five sites on PDE3A were found to be phosphorylated in vivo, of which Ser428 was selectively phosphorylated in response to PMA and dephosphorylated in cells treated with aphidicolin and mimosine. Phosphorylation of Ser428 therefore correlated with 14-3-3 binding to PDE3A. Ser312 of PDE3A was phosphorylated in an H-89-sensitive response to forskolin, indicative of phosphorylation by PKA (cAMP-dependent protein kinase), but phosphorylation at this site did not stimulate 14-3-3 binding. Thus 14-3-3 proteins can discriminate between sites in a region of multisite phosphorylation on PDE3A. An additional observation was that the cytoskeletal cross-linker protein plectin-1 coimmunoprecipitated with PDE3A independently of 14-3-3 binding.

FoxO-dependent and -independent mechanisms mediate SirT1 effects on IGFBP-1 gene expression

Sirtuin 1 (SirT1), an NAD-dependent deacetylase that is important for promoting longevity during caloric restriction, can deacetylate and enhance the function of forkhead box transcription factors, O subfamily (FoxO). We examined the effect of SirT1 on the regulation of insulin-like growth factor-binding protein 1 (IGFBP-1), a known target of FoxO proteins that is increased in fasting. Co-transfection with a SirT1 expression vector dose-dependently stimulated IGFBP-1 promoter activity and a heterologous reporter gene construct containing three FoxO-binding sites linked to a minimal promoter. This effect is mimicked by 20 μM resveratrol, a potent SirT1 activator, and immunoprecipitation and Western blotting confirm that SirT1 and FoxO1 interact in cells. Interestingly, mutation of FoxO-binding sites in the IGFBP-1 promoter reduces, but does not completely disrupt, the stimulatory effect of SirT1 on promoter activity. We found that overexpression of SirT1 is accompanied by enhanced mitogen-activated protein kinase (MAPK) activation. Treatment of SirT1-cotransfected cells with PD98059, which inhibits MAPK activation, decreased IGFBP-1 promoter activity by ∼50%, in a FoxO-binding site-independent manner, and disrupts the residual effect of SirT1. These results indicate that SirT1 stimulates IGFBP-1 promoter activity through FoxO-dependent and -independent mechanisms, and provides the first evidence that activation of MAPK contributes to effects of SirT1 on gene expression.

Inhibition of MAP kinases by crude extract and pure compound isolated from Commiphora mukul leads to down regulation of TNF-α, IL-1β and IL-2

The anti-inflammatory effect of the medicinal plant, Commiphora mukul gum was studied in peripheral blood mononuclear cells (PBMC). Bioassay-guided fractionation using conventional solvent extraction procedures, subsequent column fractionation, followed by monitoring specific activity in PBMC led to the isolation of a lead compound. Both crude ethyl acetate extract and the lead compound, thus isolated, showed inhibitory effect on proliferative response of PBMC in mitogenic lymphocyte proliferation and MLR assays. Further studies on inflammatory mediators such as IFN-γ, IL-12, TNF-α, IL-1β and NO showed down regulation, whereas no inhibition was observed in the case of anti-inflammatory cytokine IL-10. Immunoblot analysis revealed the inhibitory effect of crude ethyl acetate extract on phosphorylation of all the three mitogen activated protein kinases (MAPK) such as ERK, JNK and p38 MAPK. In contrast treatment with pure compound showed no inhibitory effect on ERK. c-fos and c-jun mRNA levels were also reduced in PMA stimulated cells on treatment with crude extract and pure compound. This reduction in c-fos and c-jun levels, when taken together with inhibition of MAPK activation, provides a possible mechanism by which both crude ethyl acetate extract and purified compound isolated from C. mukul exert its action.