Receptor activator of nuclear factor-kappaB is induced by a rottlerin-sensitive and p38 MAP kinase-dependent pathway during monocyte differentiation

Receptor activator of nuclear factor-kappaB (RANK) plays a central role in the regulation of osteoclast differentiation and activation, but the mechanisms underlying its expression remain to be elucidated. In the present study we showed that expression of RANK was strongly induced by phorbol-12-myristate-13-acetate (PMA) during monocyte differentiation of U937 cells, and was enhanced by concomitant treatment with vitamin D3. Induction was dramatically inhibited by protein kinase C (PKC) inhibitors such as rottlerin and G?6983, but not by G?6976. Interestingly, rottlerin, a selective inhibitor of PKCdelta, reduced PMA-induced RANK expression while having no effect on CD11b expression. However overexpression of wild type PKCdelta, or a kinase-inactive mutant, did not affect PMA-induction of RANK, suggesting that rottlerin inhibits PMA-induced expression of RANK via a PKCdelta-independent mechanism. Rottlerin also inhibited PMA-induced phosphorylation of p38 mitogen-activated protein kinase (p38MAPK), and the p38 MAPK inhibitor SB203580 inhibited induction of RANK. Rottlerin and SB203580 also substantially reduced RANK mRNA expression in mouse BMM cells stimulated with macrophage colony stimulating factor (M-CSF). Together, these results demonstrate that expression of RANK is dependent upon a rottlerin-sensitive and p38MAPK-dependent pathway during monocyte differentiation.     

Interruption of NFkappaB-STAT1 signaling mediates EGF-induced cell-cycle arrest

It is known that EGF induces the cell-cycle arrest in A431 cells that possess high numbers of EGF receptors and it was previously suggested that p21/WAF1 protein was a major effector molecule of the EGF-mediated cell-cycle arrest of A431 cells. Here, we further investigate this phenomenon using the decoy double-strand oligonucleotides for STAT-binding sequence (STAT decoy) and IkappaB, an inhibitor of the nuclear factor kappa B (NFkappaB). Addition of STAT decoy restored EGF-induced A431 cell-growth arrest. Interestingly, infection of adenovirus vectors to express IkappaB (AxIkappaBalphaDeltaN) as the inhibitor of NFkappaB also reversed the A431 cell-growth inhibition. The individual treatment of two inhibitors partially inhibited the WAF1 gene expression, whereas simultaneous treatment of two inhibitors exhibited more efficient inhibition. These observations suggest the activation of NFkappaB via IkappaB degradation and STAT1 via specific receptor kinase activity synergistically induce WAF1 gene expression in A431 cells. Thus, NFkappaB and STAT1 pathways mutually interact to play an important role in the EGF-induced intracellular reaction.     

Fatty acid-induced insulin resistance in L6 myotubes is prevented by inhibition of activation and nuclear localization of nuclear factor kappa B

Recent studies have implicated inhibitor of kappaB kinase (IKK) in mediating fatty acid (FA)-induced insulin resistance. How IKK causes these effects is unknown. The present study addressed the role of nuclear factor kappaB (NFkappaB), the distal target of IKK activity, in FA-induced insulin resistance in L6 myotubes, an in vitro skeletal muscle model. A 6-h exposure of myotubes to the saturated FA palmitate reduced insulin-stimulated glucose uptake by approximately 30%, phosphatidylinositol-3 kinase and protein kinase B phosphorylation by approximately 40%, and stimulated inhibitor of kappaBalpha degradation and the nuclear translocation of NFkappaB. On the other hand, the Omega-3 polyunsaturated FA linolenate neither induced insulin resistance nor promoted nuclear localization of NFkappaB. Supporting the hypothesis that IKK acts through NFkappaB to cause insulin resistance, the IKK inhibitors acetylsalicylate and parthenolide prevented FA-induced reductions in insulin-stimulated glucose uptake and NFkappaB nuclear translocation. Most importantly, NFkappaB SN50, a cell-permeable peptide that inhibits NFkappaB nuclear translocation downstream of IKK, was sufficient to prevent palmitate-induced reductions in insulin-stimulated glucose uptake. Acetylsalicylate, but not NFkappaB SN50, prevented FA effects on phosphatidylinositol-3 kinase activity and protein kinase B phosphorylation. We conclude that FAs induce insulin resistance and activates NFkappaB in L6 cells. Furthermore, inhibition of NFkappaB activation, indirectly by preventing IKK activation or directly by inhibiting NFkappaB nuclear translocation, prevents the detrimental effects of palmitate on the metabolic actions of insulin in L6 myotubes.     

Okadaic acid induces sustained activation of NFkappaB and degradation of the nuclear IkappaBalpha in human neutrophils

Human neutrophils differ from other cells by containing high amount of IkappaBalpha in the nucleus, and this increased nuclear IkappaBalpha accumulation is associated with the inhibition of NFkappaB activity and increased apoptosis. However, the mechanisms regulating NFkappaB activation and IkappaBalpha degradation in human neutrophils are little understood. The objective of this study was to provide a further insight into the mechanisms regulating NFkappaB activity and IkappaBalpha degradation in human neutrophils. We show that okadaic acid (OA), an inhibitor of protein phosphatases PP1 and PP2A, induces sustained activation of NFkappaB and degradation of the nuclear IkappaBalpha, and increases interleukin-8 expression in the neutrophils. Furthermore, inhibitors of protein kinase C-delta (PKCdelta) and IkappaB kinase (IKK) inhibit the OA-induced activation of NFkappaB. Collectively, our results indicate that in human neutrophils, the sustained activation of NFkappaB is regulated by a continuous phosphorylation and degradation of the nuclear IkappaBalpha.     

The P2X(7) receptor-mediated phospholipase D activation is regulated by both PKC-dependent and PKC-independent pathways in a rat brain-derived Type-2 astrocyte cell line, RBA-2.

The aim of this study was to characterize the regulatory mechanisms of the P2X(7) receptor (P2X(7)R)-mediated phospholipase D (PLD) activation in a rat brain-derived Type-2 astrocyte cell line, RBA-2. A time course study revealed that activation of P2X(7)R resulted in a choline and not phosphorylcholine formation, suggesting that activation of P2X(7)R is associated with the phosphatidylcholine-PLD (PC-PLD) in these cells. GF 109203X, a selective protein kinase C (PKC) inhibitor, partially inhibited the P2X(7)R-mediated PLD activation, while blocking the phorbol 12-myristate 13-acetate (PMA)-stimulated PLD activity. In addition, PMA synergistically activated the P2X(7)R-mediated PLD activity. Furthermore, genistein, a tyrosine kinase inhibitor, blocked the P2X(7)R-activated PLD, while KN62, a Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) inhibitor, was less effective, whereas the mitogen-activated protein kinase (MAPK) inhibitor PD98059 was ineffective. No additive inhibitory effects were found by simultaneous treatment of GF 109203X and KN62 on P2X(7)R-activated PLD. Taken together, these results demonstrate that both PKC-dependent and PKC-independent signaling pathways are involved in the regulation of P2X(7)R-mediated PLD activation. Additionally, CaMKII may participate in the PKC-dependent pathway, and tyrosine kinase may play a pivotal role on both PKC-dependent and PKC-independent pathways in the P2X(7)R-mediated PLD activation in RBA-2 cells.     

Regulation of cyclin D1/Cdk4 complexes by calcium/calmodulin-dependent protein kinase I

The selective inhibitor of the multifunctional calcium/calmodulin-dependent kinases (CaMK), KN-93, arrests a variety of cell types in G(1). However, the biochemical nature of this G(1) arrest point and the physiological target of KN-93 in G(1) remain controversial. Here we show that in WI-38 human diploid fibroblasts KN-93 reversibly arrested cells in late G(1) prior to detectable cyclin-dependent kinase 4 (cdk4) activation. At the KN-93 arrest point, we found that cyclin D1/cdk4 complexes had assembled with p21/p27, accumulated in the nucleus, and become phosphorylated on Thr-172, yet were relatively inactive. Additional examination of cdk4 complexes by gel filtration analysis demonstrated that, in late G(1), cyclin D1-containing complexes migrated toward lower molecular weight (M(r)) fractions and this altered migration was accompanied by the appearance of two peaks of cdk4 activity, at 150-200 and 70 kDa, respectively. KN-93 prevented both the activation of cdk4, and this shift in cyclin D1 migration and overexpression of cyclin D1/cdk4 overcame the KN-93 arrest. To determine which multifunctional CaMK acts in G(1), we expressed kinase-deficient forms of CaMKI and CaMKII. Overexpression of kinase-deficient CaMKI, but not CaMKII, prevented cdk4 activation, mimicking the KN-93 arrest point. Therefore, we hypothesize that KN-93 prevents a very late, uncharacterized step in cyclin D/cdk4 activation that involves CaMKI and follows complex assembly, nuclear entry, and phosphorylation.     

Reversible integration of the dominant negative retinoid receptor gene for ex vivo expansion of hematopoietic stem/progenitor cells

Proliferation of vascular smooth muscle cells (VSMC) contributes to the pathogenesis of atherosclerosis, and glycated serum albumin (GSA, Amadori adduct of albumin) might be a mitogen for VSMC proliferation, which may further be associated with diabetic vascular complications. In this study, we investigated the involvement of mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK), and protein kinase C (PKC), in GSA-stimulated mitogenesis, as well as the functional relationship between these factors. VSMC stimulation with GSA resulted in a marked activation of ERK. The MAPK kinase (MEK) inhibitor, PD98059, blocked GSA-stimulated MAPK activation and resulted in an inhibition of GSA-stimulated VSMC proliferation. GSA also increased PKC activity in VSMC in a dose-dependent manner. The inhibition of PKC by the PKC inhibitors, GF109203X and Rottlerin (PKCdelta specific inhibitor), as well as PKC downregulation by phorbol 12-myristate 13-acetate (PMA), inhibited GSA-induced cell proliferation and blocked ERK activation. This indicates that phorbol ester-sensitive PKC isoforms including PKCdelta are involved in MAPK activation. Thus, we show that the MAPK cascade is required for GSA-induced proliferation, and that phorbol ester-sensitive PKC isoforms contribute to cell activation and proliferation in GSA-stimulated VSMC.     

Regulation of CD93 cell surface expression by protein kinase C isoenzymes

Human CD93, also known as complement protein 1, q subcomponent, receptor (C1qRp), is selectively expressed by cells with a myeloid lineage, endothelial cells, platelets, and microglia and was originally reported to be involved in the complement protein 1, q subcomponent (C1q)-mediated enhancement of phagocytosis. The intracellular molecular events responsible for the regulation of its expression on the cell surface, however, have not been determined. In this study, the effect of protein kinases in the regulation of CD93 expression on the cell surface of a human monocyte-like cell line (U937), a human NK-like cell line (KHYG-1), and a human umbilical vein endothelial cell line (HUV-EC-C) was investigated using four types of protein kinase inhibitors, the classical protein kinase C (cPKC) inhibitor Go6976, the novel PKC (nPKC) inhibitor Rottlerin, the protein kinase A (PKA) inhibitor H-89 and the protein tyrosine kinase (PTK) inhibitor herbimycin A at their optimum concentrations for 24 hr. CD93 expression was analyzed using flow cytometry and glutaraldehyde-fixed cellular enzyme-linked immunoassay (EIA) techniques utilizing a CD93 monoclonal antibody (mAb), mNI-11, that was originally established in our laboratory as a CD93 detection probe. The nPKC inhibitor Rottlerin strongly down-regulated CD93 expression on the U937 cells in a dose-dependent manner, whereas the other inhibitors had little or no effect. CD93 expression was down-regulated by Go6976, but not by Rottlerin, in the KHYG-1 cells and by both Rottlerin and Go6976 in the HUV-EC-C cells. The PKC stimulator, phorbol myristate acetate (PMA), strongly up-regulated CD93 expression on the cell surface of all three cell-lines and induced interleukin-8 (IL-8) production by the U937 cells and interferon-gamma (IFN-gamma) production by the KHYG-1 cells. In addition, both Go6976 and Rottlerin inhibited the up-regulation of CD93 expression induced by PMA and IL-8 or IFN-gamma production in the respective cell-lines. Whereas recombinant tumor necrosis factor-alpha (rTNF-alpha) slightly up-regulated CD93 expression on the U937 cells, recombinant interleukin-1beta (rIL-1beta), recombinant interleukin-2 (rIL-2), recombinant interferon-gamma (rIFN-gamma) and lipopolysaccharide (LPS) had no effect. Taken together, these findings indicate that the regulation of CD93 expression on these cells involves the PKC isoenzymes.     

Role of protein kinase C in arginine vasopressin-stimulated ERK and p70S6 kinase phosphorylation

We previously showed in rat renal glomerular mesangial cells, that arginine vasopressin (AVP)-stimulated cell proliferation was mediated by epidermal growth factor receptor (EGF-R) transactivation, and activation (phosphorylation) of ERK1/2 and p70S6 kinase (Ghosh et al. [2001]: Am J Physiol Renal Physiol 280:F972-F979]. In this paper, we extend these observations and show that different protein kinase C (PKC) isoforms play different roles in mediating AVP-stimulated ERK1/2 and p70S6 kinase phosphorylation and cell proliferation. AVP treatment for 0-60 min stimulated the serine/threonine phosphorylation of PKC isoforms alpha, delta, epsilon, and zeta. The activation of PKC was dependent on EGF-R and phosphatidylinositol 3-kinase (PI3K) activation. In addition, inhibition of conventional and novel PKC isoforms by chronic (24 h) exposure to phorbol 12-myristate 13-acetate (PMA) inhibited AVP-induced activation of ERK and p70S6 kinase as well as EGF-R phosphorylation. Rottlerin, a specific inhibitor of PKCdelta, inhibited both ERK and p70S6 kinase phosphorylation and cell proliferation. In contrast, a PKCepsilon translocation inhibitor decreased ERK1/2 activation without affecting p70S6 kinase or cell proliferation, while a dominant negative PKCzeta (K281W) cDNA delayed p70S6 kinase activation without affecting ERK1/2. On the other hand, G?6976, an inhibitor of conventional PKC isoforms, did not affect p70S6 kinase, but stimulated ERK1/2 phosphorylation without affecting cell proliferation. Our results indicate that PKCdelta plays an important role in AVP-stimulated ERK and p70S6 kinase activation and cell proliferation.     

Conjugated linoleic acid promotes human adipocyte insulin resistance through NFkappaB-dependent cytokine production

We previously demonstrated that trans-10, cis-12 conjugated linoleic acid (CLA) reduced the triglyceride content of human adipocytes by activating mitogen-activated protein kinase kinase/extracellular signal-related kinase (MEK/ERK) signaling via interleukins (IL) 6 and 8. However, the upstream mechanism is unknown. Here we show that CLA increased (>or=6 h) the secretion of IL-6 and IL-8 in cultures containing both differentiated adipocytes and stromal vascular (SV) cells, non-differentiated SV cells, and adipose tissue explants. CLA isomer-specific induction of IL-6 and tumor necrosis factor-alpha was associated with the activation of nuclear factor kappaB (NFkappaB) as evidenced by 1) phosphorylation of IkappaBalpha, IkappaBalpha kinase, and NFkappaB p65, 2) IkappaBalpha degradation, and 3) nuclear translocation of NFkappaB. Pretreatment with selective NFkappaB inhibitors and the MEK/ERK inhibitor U0126 blocked CLA-mediated IL-6 gene expression. Trans-10, cis-12 CLA suppression of insulin-stimulated glucose uptake at 24 h was associated with decreased total and plasma membrane glucose transporter 4 proteins. Inhibition of NFkappaB activation or depletion of NFkappaB by RNA interference using small interfering NFkappaB p65 attenuated CLA suppression of glucose transporter 4 and peroxisome proliferator-activated receptor gamma proteins and glucose uptake. Collectively, these data demonstrate for the first time that trans-10, cis-12 CLA promotes NFkappaB activation and subsequent induction of IL-6, which are at least in part responsible for trans-10, cis-12 CLA-mediated suppression of peroxisome proliferator-activated receptor gamma target gene expression and insulin sensitivity in mature human adipocytes.     

胰岛素和佛波酯在蛋白质合成中经不同途径激活p70 S6激酶

为研究佛波酯 (PMA)和胰岛素在蛋白质合成中的信号传递 ,应用激酶活性测定和Western印迹等方法 ,分别检测mTOR(mammaliantargetofrapamycin)特异性抑制剂rapamycin或磷脂酰肌醇 3激酶 (PI3K)的特异性抑制剂LY2 94 0 0 2预处理、PMA或胰岛素处理的血清饥饿的中国仓鼠肺成纤维细胞 (CHL)中p70S6激酶 (p70S6K)和蛋白激酶B(PKB)的活性及表达 .结果显示 ,PMA或胰岛素刺激促进p70S6K的活化和表达 .而rapamycin预处理可阻断PMA和胰岛素对p70S6K的激活作用 ,表明PMA和胰岛素可能是通过mTOR 依赖性途径激活p70S6K .结果还显示 ,胰岛素刺激促进PKB的活化和表达 ,而PMA对PKB的活性和表达无影响 .LY2 94 0 0 2预处理可阻断胰岛素对p70S6K和PKB的激活作用 ,但不能抑制PMA刺激引起的p70S6K的活化 .表明胰岛素和PMA介导p70S6K活化的信号途径有所不同 ,胰岛素介导p70S6K的活化可能依赖于PI3K途径 ,而PMA介导p70S6K的活化不通过PI3K途径     

Calcium elevation at fertilization coordinates phosphorylation of XErp1/Emi2 by Plx1 and CaMK II to release metaphase arrest by cytostatic factor

BACKGROUND: Vertebrate oocytes are arrested at second meiotic metaphase by cytostatic factor (CSF) while awaiting fertilization. Accumulating evidence has suggested that inhibition of the anaphase-promoting complex/cyclosome (APC/C) is responsible for this arrest. Xenopus polo-like kinase 1 (Plx1) is required for activation of the APC/C at the metaphase-anaphase transition, and calcium elevation, upon fertilization/activation of eggs, acting through calmodulin-dependent kinase II (CaMKII) is sufficient to activate the APC/C and terminate CSF arrest. However, connections between the Plx1 pathway and the CaMKII pathway have not been identified. RESULTS: Overexpression of Plx1 causes CSF release in the absence of calcium, and depletion of Plx1 from egg extracts blocks induction of CSF release by calcium and CaMKII. Prior phosphorylation of the APC/C inhibitor XErp1/Emi2 by CaMK II renders it a good substrate for Plx1, and phosphorylation by both kinases together promotes its degradation in egg extracts. The pathway is enhanced by the ability of Plx1 to cause calcium-independent activation of CaMKII. The results identify the targets of CaMKII and Plx1 that promote egg activation and define the first known pathway of CSF release in which an APC/C inhibitor is targeted for degradation only when both CaMKII and Plx1 are active after calcium elevation at fertilization. CONCLUSIONS: Plx1 with an intact polo-box domain is necessary for release of CSF arrest and sufficient when overexpressed. It acts at the same level as CaMKII in the pathway of calcium-induced CSF release by cooperating with CaMKII to regulate APC/C regulator(s), such as XErp1/Emi2, rather than by directly activating the APC/C itself.