P38 Mitogen-activated Protein Research Articles

Phosphatidylinositol-3-Kinase–Akt Kinase and p42/p44 Mitogen-Activated Protein Kinases Mediate Neurotrophic and Excitoprotective Actions of a Secreted Form of Amyloid Precursor Protein

The α-secretase-derived form of the amyloid precursor protein (sAPPα), which is released from neurons in an activity-dependent manner, has been shown to promote long-term survival of hippocampal and cortical neurons in culture and can protect those neurons against excitotoxic and ischemic injury in culture and in vivo. The signal transduction pathway(s) activated by sAPPα has not been established. We now report that sAPPα activates the phosphatidylinositol-3-kinase (PI3K)–Akt kinase signaling pathway in cultured hippocampal neurons. sAPPα also stimulates phosphorylation of p42 (ERK1) and p44 (ERK2) mitogen-activated protein (MAP) kinases by a PI3K-independent pathway. Treatment of neurons with sAPPα protects them against death induced by trophic factor deprivation and exposure to glutamate, and these survival-promoting effects of sAPPα are abolished or attenuated when either PI3K or p42/p44 MAP kinases are selectively blocked. Exposure of neurons to sAPPα resulted in a decrease in the level of IκBβ and an increase in NF-κB DNA binding activity, both of which were blocked by wortmannin, suggesting that the transcription factor NF-κB may be a downstream target of the PI3K–Akt pathway that may play a role in the cell survival-promoting action of sAPPα. These findings suggest that the PI3K–Akt pathway and p42/p44 MAP kinases mediate responses of neurons to sAPPα in physiological and pathological settings, with implications for synaptic plasticity and the pathogenesis of Alzheimer's disease.

Inhibition of the p38 MAP kinase pathway destabilizes smooth muscle length during physiological loading

We tested the hypothesis that mechanical plasticity of airway smooth muscle may be mediated in part by the p38 mitogen-activated protein (MAP) kinase pathway. Bovine tracheal smooth muscle (TSM) strips were mounted in a muscle bath and set to their optimal length, where the active force was maximal (F(o)). Each strip was then contracted isotonically (at 0.32 F(o)) with ACh (maintained at 10(-4) M) and allowed to shorten for 180 min, by which time shortening was completed and the static equilibrium length was established. To simulate the action of breathing, we then superimposed on this steady distending force a sinusoidal force fluctuation with zero mean, at a frequency of 0.2 Hz, and measured incremental changes in muscle length. We found that TSM strips incubated in 10 microM SB-203580-HCl, an inhibitor of the p38 MAP kinase pathway, demonstrated a greater degree of fluctuation-driven lengthening than did control strips, and upon removal of the force fluctuations they remained at a greater length. We also found that the force fluctuations themselves activated the p38 MAP kinase pathway. These findings are consistent with the hypothesis that inhibition of the p38 MAP kinase pathway destabilizes muscle length during physiological loading.

Target Depletion of Distinct Tumor Necrosis Factor Receptor Subtypes Reveals Hippocampal Neuron Death and Survival through Different Signal Transduction Pathways

Tumor necrosis factor receptor-I (TNFRI) and TNFRII are two TNFR subtypes in the immune system, but their roles in the brain remain unclear. Here we present a novel interaction between TNFR subtypes and TNF-alpha in the brain. Our studies on target-depleted TNFR in mice show that TNF-alpha has little effect on hippocampal neurons in which TNFRI, containing an "intracellular death domain," is absent (TNFRI -/-), whereas neurons from TNFRII knock-out mice are vulnerable to TNF-alpha even at low doses. Moreover, little nuclear factor-kappaB (NF-kappaB) translocation is induced by TNF-alpha in neurons of TNFRI -/-, whereas NF-kappaB subunit p65 is still translocated from the cytoplasm into the nucleus in neurons from wild-type and TNFRII -/- mice. Furthermore, p38 mitogen-activated protein (MAP) kinase activity is upregulated in neurons from both wild-type and TNFRI -/-, but no alteration of p38 MAP kinase was found in neurons from TNFRII. Results from overexpression of TNF receptors further support the above findings. NT2 neuronal-like cells transiently transfected with TNFRI are very sensitive to TNF-alpha, whereas TNF-alpha is not toxic and even seems to be trophic to the cells with TNFRII overexpression. Last, our radioligand-binding experiments demonstrate that TNF-alpha binds TNFRI with high affinity (K(d) of 0.6 nm), whereas TNFRII shows lower binding affinity (K(d) of 1.14 nm) to TNF-alpha in NT2 transfected cells. Together, these studies reveal novel neuronal responses of TNF-alpha in mediating consequences of TNF receptor activation differently. Subsequent neuronal death or survival may ultimately depend on a particular subtype of TNF receptor that is predominately expressed in neurons of the brain during neural development or with neurological diseases.

Target depletion of distinct tumor necrosis factor receptor subtypes reveals hippocampal neuron death and survival through different signal transduction pathways.

Tumor necrosis factor receptor-I (TNFRI) and TNFRII are two TNFR subtypes in the immune system, but their roles in the brain remain unclear. Here we present a novel interaction between TNFR subtypes and TNF-alpha in the brain. Our studies on target-depleted TNFR in mice show that TNF-alpha has little effect on hippocampal neurons in which TNFRI, containing an "intracellular death domain," is absent (TNFRI -/-), whereas neurons from TNFRII knock-out mice are vulnerable to TNF-alpha even at low doses. Moreover, little nuclear factor-kappaB (NF-kappaB) translocation is induced by TNF-alpha in neurons of TNFRI -/-, whereas NF-kappaB subunit p65 is still translocated from the cytoplasm into the nucleus in neurons from wild-type and TNFRII -/- mice. Furthermore, p38 mitogen-activated protein (MAP) kinase activity is upregulated in neurons from both wild-type and TNFRI -/-, but no alteration of p38 MAP kinase was found in neurons from TNFRII. Results from overexpression of TNF receptors further support the above findings. NT2 neuronal-like cells transiently transfected with TNFRI are very sensitive to TNF-alpha, whereas TNF-alpha is not toxic and even seems to be trophic to the cells with TNFRII overexpression. Last, our radioligand-binding experiments demonstrate that TNF-alpha binds TNFRI with high affinity (K(d) of 0.6 nm), whereas TNFRII shows lower binding affinity (K(d) of 1.14 nm) to TNF-alpha in NT2 transfected cells. Together, these studies reveal novel neuronal responses of TNF-alpha in mediating consequences of TNF receptor activation differently. Subsequent neuronal death or survival may ultimately depend on a particular subtype of TNF receptor that is predominately expressed in neurons of the brain during neural development or with neurological diseases.

P38 MAP kinase mediates platelet‐derived growth factor‐stimulated migration of hepatic myofibroblasts

Although the migration of hepatic myofibroblasts (HMFs) contributes to the development of fibrosis, the signals regulating migration of these cells are poorly understood. In this study, we tested the hypothesis that HMF migration is stimulated by platelet-derived growth factor-BB (PDGF-BB) through p38 mitogen-activated protein (MAP) kinase and extracellular signal-regulated kinase (ERK) signaling pathways. This hypothesis was addressed by directly visualizing the migration of cultured human HMFs into a wound. PDGF-BB stimulated membrane ruffling, migration, and proliferation. PDGF-BB also induced activation of p38 MAP kinase, its downstream effector, heat shock protein (HSP) 27, ERK 1 and ERK 2, and p125 focal adhesion kinase (FAK). Selective antagonism of p38 MAP kinase blocked PDGF-BB-stimulated HSP 27 phosphorylation, membrane ruffling, and migration, but did not alter PDGF-BB-induced proliferation. Selective antagonism of ERK kinase inhibited PDGF-BB-induced ERK phosphorylation and proliferation, but did not affect PDGF-BB-stimulated migration. Concentrations of PDGF-BB that stimulated migration and proliferation did not influence myosin-dependent contractility. Neither selective inhibition of p38 MAP kinase nor ERKs altered PDGF-BB-induced activation of FAK. In conclusion, these results provide novel evidence indicating that (1) HMF migration is stimulated by PDGF-BB through the regulation of membrane ruffling by a p38 MAP kinase signaling pathway, (2) whereas p38 MAP kinase mediates PDGF-BB-stimulated migration, but not proliferation, ERKs mediate PDGF-induced proliferation, but not migration, and (3) increases in myosin-dependent contractility are not required for PDGF-BB-stimulated migration.

Vascular smooth muscle cell proliferation requires both p38 and BMK1 MAP kinases

Vascular smooth muscle cell (VSMC) proliferation is a key event in the progression of atherosclerosis. Induction of both c-fos (through the transcription factor Elk-1) and c-jun, both immediate early genes, is important for the stimulation of VSMC proliferation and migration. It was earlier found that p38 mitogen-activated protein (MAP) kinase upregulates c-jun gene transcription through phosphorylation of two myocyte enhancer factor 2 (MEF2) family transcription factors, MEF2A and MEF2C, while big MAP kinase 1 (BMK1) may upregulate c-jun gene transcription through MEF2A, MEF2C, and also MEF2D. Here, we report that inhibition of BMK1 by a dominant negative form of MEK5 or pharmacologic inhibition of p38 by SB 203580 additively suppress serum-induced VSMC proliferation. This additive effect of p38 and BMK1 inhibition implies that these two kinases coordinately regulate MEF2 transcription factors. The exclusive activation of MEF2D by BMK1 appears required for this cooperative upregulation of c-jun in VSMC, and coactivation of p38 and BMK1 also has additive effects on the activation of a reporter gene linked to the c-jun promoter in our experimental system. Thus, coordinate activity of both the p38 and BMK1 pathways appears necessary for optimal transcription of c-jun and, pari pasu, VSMC proliferation. These results may have implications for the future design of pharmacologic agents for inhibition of VSMC growth.

ERK and p38 MAP kinase are required for rat renal development

We previously demonstrated that extracellular signal-regulated protein kinase (ERK) and p38 mitogen-activated protein (MAP) kinase (p38) are strongly expressed in the embryonic kidney. In the present study, we investigated the role of ERK and p38 during kidney development. Rat metanephroi were cultured from 15-day-old embryos, and exposed to inhibitors of MEK, an activator of ERK, PD98059 (300 micromol/L), U0126 (10 micromol/L), or a p38 inhibitor SB203580 (30 micromol/L) 24 to 120 hours after the start of culture. Growth of metanephroi was measured by surface area and thymidine incorporation. Ureteric buds and glomeruli were identified by labeling with Dolichos biflorus lectin and peanut agglutinin, respectively. PCNA staining and TUNEL assay were performed on kidney sections. The level of apoptosis was evaluated by examining DNA ladder formation. Growth of metanephroi was significantly inhibited by SB203580 but not by PD98059 or U0126. Ureteric bud branching was not affected by SB203580 or MEK inhibitors. Glomerular number was markedly reduced by SB203580 and to a lesser extent by U0126 (14 +/- 2 and 48 +/- 10% of controls, respectively). On histological examination, the number of tubuloglomerular structures was reduced in MEK inhibitor-treated metanephroi compared to controls. Very few mesenchymal condensates were observed in kidneys incubated with SB203580. PCNA-positive cells were reduced in SB203580-treated metanephroi compared to control and PD98059-treated kidneys. Apoptosis was increased in SB203580-treated kidneys and to a lesser extent in PD98059-treated cultures. Both ERK and p38 are required for renal development. ERK appears to play a role in nephrogenesis and p38 for kidney growth and nephrogenesis.

ERK and p38 MAP kinase are involved in arachidonic acid release induced by H(2)O(2) and PDGF in mesangial cells.

Increased prostaglandin production is implicated in the pathogenesis of glomerular disease. With this consideration, we examined the combined effects of reactive oxygen species and platelet-derived growth factor (PDGF), which might initiate glomerular dysfunction, on arachidonic acid release and cytosolic phospholipase A(2) (cPLA(2)) activation in rat mesangial cells. H(2)O(2)-induced release of arachidonic acid was enhanced by PDGF, which by itself had little effect on the release, and the enhancement was completely inhibited by a cPLA(2) inhibitor. The phosphorylation of cPLA(2), extracellular signal-regulated kinase (ERK), and p38 mitogen-activated protein (MAP) kinase was upregulated by H(2)O(2) or PDGF alone and except for ERK was enhanced further by the two in combination. The release of arachidonic acid induced by PDGF together with H(2)O(2) was inhibited partially by an inhibitor of ERK or p38 MAP kinase and completely when the two inhibitors were combined; the inhibitory pattern was similar to that for the phosphorylation of cPLA(2). These results suggest that the ERK and p38 MAP kinase pathways are involved in the increase in cPLA(2) activation and arachidonic acid release induced by PDGF together with H(2)O(2).

Insulin signaling in the transcriptional and posttranscriptional regulation of CYP2E1 expression

Diabetes has been reported to increase the expression of cytochrome P450 (CYP) 2E1 messenger RNA (mRNA) and protein several-fold, and enhanced expression has been associated with elevated ketone bodies. Primary cultured rat hepatocytes were used to explore ketone body and insulin regulation of CYP2E1 expression. Hydroxybutyrate and acetoacetate (AC), alone or in combination, either failed to affect or decreased CYP2E1 mRNA levels by up to 90% relative to untreated hepatocytes. Insulin produced a concentration-dependent decrease in CYP2E1 mRNA levels, and insulin receptor immunoprecipitation showed a correspondence between receptor phosphorylation and the decrease in CYP2E1 mRNA levels at physiologic levels of insulin. Phosphatase inhibitors decreased CYP2E1 mRNA levels by greater than 95%. The phosphatidylinositol 3-kinase (PI3-kinase) inhibitors wortmannin or LY294002 and rapamycin, an inhibitor of p70 S6 kinase phosphorylation, ameliorated the insulin-mediated decrease in CYP2E1 mRNA levels. Geldanamycin, which inhibits Src kinase, also abrogated the insulin-mediated decrease in CYP2E1 mRNA levels. In contrast, the protein kinase C (PKC) inhibitor bisindolylmaleimide, the mitogen-activated protein kinase kinase (MEK) inhibitor PD98059, and the p38 mitogen-activated protein (MAP) kinase inhibitor SB202190 did not affect the insulin-mediated decrease in CYP2E1. CYP2E1 mRNA half-life decreased from ∼48 hours in the absence of insulin to ∼15 hours at 10 nmol/L insulin, and this decrease was prevented by wortmannin. The half-life of CYP2B mRNA was increased by insulin, whereas that of CYP3A was unaffected. Analysis of CYP2E1 gene transcription using heterogeneous nuclear RNA (hnRNA) showed that insulin suppressed CYP2E1 transcription. In conclusion, these data show involvement of transcriptional and posttranscriptional mechanisms in the insulin-mediated regulation of CYP2E1 and implicate PI3-kinase, p70 S6 kinase, and Src kinase in mediating these effects. (HEPATOLOGY .)

Activation of p42 mitogen-activated protein kinase (MAPK), but not c-Jun NH(2)-terminal kinase, induces phosphorylation and stabilization of MAPK phosphatase XCL100 in Xenopus oocytes.

Dual-specificity protein phosphatases are implicated in the direct down-regulation of mitogen-activated protein kinase (MAPK) activity in vivo. Accumulating evidence suggests that these phosphatases are components of negative feedback loops that restore MAPK activity to low levels after diverse physiological responses. Limited information exists, however, regarding their posttranscriptional regulation. We cloned two Xenopus homologs of the mammalian dual-specificity MAPK phosphatases MKP-1/CL100 and found that overexpression of XCL100 in G2-arrested oocytes delayed or prevented progesterone-induced meiotic maturation. Epitope-tagged XCL100 was phosphorylated on serine during G2 phase, and on serine and threonine in a p42 MAPK-dependent manner during M phase. Threonine phosphorylation mapped to a single residue, threonine 168. Phosphorylation of XCL100 had no measurable effect on its ability to dephosphorylate p42 MAPK. Similarly, mutation of threonine 168 to either valine or glutamate did not significantly alter the binding affinity of a catalytically inactive XCL100 protein for active p42 MAPK in vivo. XCL100 was a labile protein in G2-arrested and progesterone-stimulated oocytes; surprisingly, its degradation rate was increased more than twofold after exposure to hyperosmolar sorbitol. In sorbitol-treated oocytes expressing a conditionally active DeltaRaf-DD:ER chimera, activation of the p42 MAPK cascade led to phosphorylation of XCL100 and a pronounced decrease in the rate of its degradation. Our results provide mechanistic insight into the regulation of a dual-specificity MAPK phosphatase during meiotic maturation and the adaptation to cellular stress.

The involvement of reactive oxygen species (ROS) and p38 mitogen-activated protein (MAP) kinase in TRAIL/Apo2L-induced apoptosis

To determine the apoptotic signaling pathway which tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/Apo2L) induced, we investigated the contribution of reactive oxygen species (ROS), p38 mitogen-activated protein (MAP) kinase and caspases in human adenocarcinoma HeLa cells. Here we show that upon TRAIL/Apo2L exposure there was pronounced ROS accumulation and activation of p38 MAP kinase, and that activation of caspases and apoptosis followed. Pretreatment with antioxidants such as glutathione or estrogen attenuated TRAIL/Apo2L-induced apoptosis through a reduction of ROS generation and diminished p38 MAP kinase and caspase activation. The p38 MAP kinase inhibitor SB203580 prevented apoptosis through a blockage of caspase activation, although ROS generation was not attenuated. Furthermore, the pan-caspase inhibitor Z-Val-Ala- DL-Asp-fluoromethyl ketone fully prevented apoptosis, while neither ROS accumulation nor p38 MAP kinase activation were affected. Therefore, our results suggest that TRAIL/Apo2L-induced apoptosis is mediated by ROS-activated p38 MAP kinase followed by caspase activation in HeLa cells.

Hepatitis C Virus Core Protein Induces Cell Proliferation and Activates ERK, JNK, and p38 MAP Kinases Together with the MAP Kinase Phosphatase MKP-1 in a HepG2 Tet-Off Cell Line

Hepatitis C virus (HCV) core protein is a multifunctional protein interacting with cellular and viral proteins and promoters. A tetracycline-regulated system was used to generate a HepG2 Tet-Off cell line allowing regulated expression of a full-length (191 aa) and an Nc-truncated core protein (160 aa). In this system HCV core protein expression activates extracellular signal-regulated kinase (ERK), c-jun N-terminal kinase (JNK), and p38 mitogen-activated protein (MAP) kinase, induces MAP kinase phosphatase MKP-1 expression, and increases cell proliferation. This was accompanied by an activation of c-Jun and ATF-2, but not Elk-1 and c-Fos. Furthermore, AP-1 activation was independent of c-Fos. Full-length and Nc-truncated HCV core proteins exerted similar effects.

Cytokine-driven proliferation and differentiation of human naive, central memory, and effector memory CD4(+) T cells.

Memory T lymphocytes proliferate in vivo in the absence of antigen maintaining a pool of central memory T cells (T(CM)) and effector memory T cells (T(EM)) with distinct effector function and homing capacity. We compared human CD4(+) naive T, T(CM), and T(EM) cells for their capacity to proliferate in response to cytokines, that have been implicated in T cell homeostasis. Interleukin (IL)-7 and IL-15 expanded with very high efficiency T(EM), while T(CM) were less responsive and naive T cells failed to respond. Dendritic cells (DCs) and DC-derived cytokines allowed naive T cells to proliferate selectively in response to IL-4, and potently boosted the response of T(CM) to IL-7 and IL-15 by increasing the expression of the IL-2/IL-15Rbeta and the common gamma chain (gamma(c)). The extracellular signal regulated kinase and the p38 mitogen-activated protein (MAP) kinases were selectively required for TCR and cytokine-driven proliferation, respectively. Importantly, in cytokine-driven cultures, some of the proliferating T(CM) differentiated to T(EM)-like cells acquiring effector function and switching chemokine receptor expression from CCR7 to CCR5. The sustained antigen-independent generation of T(EM) from a pool of T(CM) cells provides a plausible mechanism for the maintenance of a polyclonal and functionally diverse repertoire of human CD4(+) memory T cells.

Secretory phospholipase A(2) elicits proinflammatory changes and upregulates the surface expression of fas ligand in monocytic cells: potential relevance for atherogenesis.

Type IIA secretory phospholipase A(2) (sPLA(2)) is an acute-phase reactant that plays a role in atherogenesis and is expressed in atherosclerotic arterial walls displaying inflammatory features. This generates a relevant question addressing the biological effects of this enzyme on monocytic cells, in view of the role of these cells in the inflammatory process associated with atherosclerosis. sPLA(2) produced a mild activation of the p42 mitogen-activated protein module of the mitogen-activated protein kinase (MAPK) cascade and a prominent activation of c-Jun N-terminal kinase in THP-1 monocytes. This activation showed both an early and a late peak, different from that elicited by tumor necrosis factor-alpha (TNF-alpha), which only showed the first peak. This was accompanied by activation of arachidonate metabolism, as judged from both the activation of the cytosolic phospholipase A(2) (cPLA(2)) and the induction of cyclooxygenase-2 (COX-2) expression. sPLA(2) also elicited the production of monocyte chemoattractant protein-1 (MCP-1) and showed a synergistic effect with TNF-alpha on both COX-2 induction and MCP-1 production. sPLA(2) upregulated the expression of Fas ligand at the cell surface, but it did not influence Fas expression nor cell survival of monocytes. In summary, these data indicate that some of the atherogenic effects of sPLA(2) can be exerted by engagement of an sPLA(2)-binding structure on monocytic cells, most probably the M-type receptor for sPLA(2), which produces the activation of the MAPK cascade, induces a proinflammatory phenotype, and upregulates the cell surface expression of Fas ligand.

Inhibition by adenylyl cyclase-cAMP system of ET-1-induced HSP27 in osteoblasts.

We have previously reported that endothelin-1 (ET-1) stimulates heat shock protein (HSP) 27 induction in osteoblast-like MC3T3-E1 cells and that p38 mitogen-activated protein (MAP) kinase acts at a point downstream from protein kinase C (PKC) in HSP27 induction. In the present study, we investigated the effect of the adenylyl cyclase-cAMP system on ET-1-stimulated induction of HSP27 in MC3T3-E1 cells. Dibutyryl-cAMP (DBcAMP) dose dependently inhibited the HSP27 accumulation stimulated by ET-1. Forskolin and cholera toxin significantly suppressed the ET-1-stimulated accumulation of HSP27. However, dideoxyforskolin, a forskolin derivative that does not activate cAMP, failed to suppress the ET-1-induced HSP27 accumulation. Forskolin reduced the p38 MAP kinase phosphorylation induced by ET-1 or 12-O-tetradecanoylphorbol-13-acetate (TPA). PGE(1), an extracellular agonist that activates cAMP production, reduced the ET-1-induced HSP27 accumulation. In addition, the phosphorylation of p38 MAP kinase induced by ET-1 or TPA was suppressed by PGE(1). Forskolin, DBcAMP, and PGE(1) suppressed the ET-1-stimulated increase in the mRNA level for HSP27. These results indicate that the adenylyl cyclase-cAMP system has an inhibitory role in ET-1-stimulated HSP27 induction in osteoblasts and that the effect is exerted at the point between PKC and p38 MAP kinase in osteoblasts.

Mechanisms underlying reduced responsiveness of neonatal neutrophils to distinct chemoattractants

Potential mechanisms underlying impaired chemotactic responsiveness of neonatal neutrophils were investigated. Two distinct chemoattractants were compared: bacterially derived N-formyl-methionyl-leucyl-phenylalanine (fMLP) and a unique chemotactic monoclonal antibody, designated DL1.2, which binds to a neutrophil antigen with an apparent molecular mass of 120 kDa. Chemotaxis of neutrophils toward fMLP, as well as DL1.2, was reduced in neonates when compared with adult cells. This did not appear to be a result of decreased fMLP receptor or DL1.2 antigen expression by neonatal neutrophils. fMLP, but not DL1.2, induced a rapid increase in intracellular calcium in adult and neonatal cells, which reached a maximum within 30 s. The calcium response of cells from neonates to fMLP was reduced when compared with adult cells, and an unresponsive subpopulation of neonatal neutrophils was identified. NF-kappaB nuclear binding activity induced by fMLP and DL1.2, as well as expression of the p65 NF-kappaB subunit and IkappaB-alpha, was also significantly reduced in neonatal cells, when compared with adult cells. In contrast, although fMLP, but not DL1.2, activated p42/44 and p38 mitogen-activated protein (MAP) kinases in neutrophils, no differences were observed between adults and neonates. Chemotaxis of adult and neonatal neutrophils toward fMLP and DL1.2 was also blocked to a similar extent by inhibitors of phosphatidylinositol 3-kinase, as well as an inhibitor of NF-kappaB. These findings indicate that reduced chemotactic responsiveness in neonatal neutrophils is a result of, at least in part, aberrations in chemoattractant-induced signaling. However, the biochemical pathways mediating this defect appear to be related to the specific chemoattractant.

Tumor Necrosis Factor α Is a Negative Regulator of Resistin Gene Expression and Secretion in 3T3-L1 Adipocytes

Resistin has recently been implicated as an adipocytokine leading to insulin resistance and, therefore, potentially linking obesity and diabetes. To further characterize the regulation of this fat-secreted protein by insulin sensitivity-modulating hormones, 3T3-L1 adipocytes were treated with tumor necrosis factor (TNF) α, angiotensin (AT) 2, as well as growth hormone (GH), and resistin gene expression and protein secretion were determined by quantitative real-time reverse transcription-polymerase chain reaction and Western blotting. Interestingly, both, resistin mRNA expression and protein secretion, were inhibited by 70–90% after TNFα-treatment whereas AT2 and GH did not have any effect. The inhibitory effect of TNFα was time- and dose-dependent with significant inhibition occurring as early as 4 h after effector addition and at concentrations as low as 1 ng/ml TNFα. Pharmacological inhibition of protein kinase A (PKA), p44/42, and p38 mitogen-activated protein (MAP) kinase did not reverse the inhibitory effect of TNFα suggesting that neither of these signaling molecules is involved in suppression of resistin gene expression by TNFα. Furthermore, suppression of resistin mRNA levels could be completely reversed to control levels by withdrawal of TNFα for 24 h. Taken together, these results suggest that TNFα is a pivotal negative regulator of resistin gene expression. This may have important implications for the pathogenesis of insulin resistance and its link to obesity.

Getting First Dibs on Calcium

Calcium ions (Ca 2+ ) that enter a cell through L-type voltage-activated channels (LTCs) are linked to expression of certain genes involved in key neuronal functions, whereas Ca 2+ entering the cell by other means can exert different effects. Dolmetsch et al . (Perspective by Ikeda) use a clever strategy to explore this perplexing aspect of specificity in cell signaling. They first engineered an LTC that is resistant to a channel inhibitor and then expressed and analyzed it in cultured primary neurons that were treated with the inhibitor to eliminate endogenous channel function. A calmodulin-binding region of the LTC is essential for Ca 2+ -induced activation of the transcription factors CREB and MEF-2. Thus, the Ca 2+ entering though LTCs is apparently sensed by calmodulin molecules already positioned at the mouth of the channel. This activation of calmodulin is required for sustained activation of the p42 and p44 mitogen-activated protein kinases, which in turn convey the signal on into the nucleus. R. E. Dolmetsch, U. Pajvani, K. Fife, J. M. Spotts, M. E. Greenberg, Signaling to the nucleus by an L-type calcium channel-calmodulin complex through the MAP kinase pathway. Science 294 , 333-339 (2001). [Abstract] [Full Text] S. R. Ikeda, Calcium channels--link locally, act globally. Science 294 , 318-319 (2001). [Summary] [Full Text]

PDEγ, A Link Between Receptors and MAPK?

The inhibitory γ subunit of the rod cyclic guanosine monophosphate phosphodiesterase (PDE) is expressed in several nonphotoreceptive cells, including lung, kidney, heart, testis, and smooth muscle and the human embryonic kidney cell line HEK293. Wan et al. found that expression of antisense for PDEγ inhibited epidermal growth factor (EGF)-stimulated or thrombin-stimulated phosphorylation of the p44 and p42 mitogen-activated protein kinases (p42 MAPK and p44 MAPK ). Furthermore, overexpression of either the rod or cone forms of PDEγ enhanced p42/p44 MAPK phosphorylation stimulated by these two ligands. EGF receptors are tyrosine kinase growth factor receptors, and the thrombin receptor is a G protein-coupled receptor (GPCR). Internalization of both receptor tyrosine kinases and GPCRs may be important for efficient MAPK activation. Internalization of the receptors may involve G protein-coupled receptor kinase 2 (GRK2). PDEγ is a substrate for GRK2, and phosphorylation of PDEγ is enhanced by the addition of Gβγ in vitro. Expression of a form of PDEγ not phosphorylated by GRK2 inhibited EGF-stimulated phosphorylation of p42/p44 MAPK , suggesting a link between GRK2 and coupling of EGF to MAPK. In thrombin-treated cells, PDEγ coprecipitates with dynamin II, a guanosine triphosphatase (GTPase) involved in clathrin-mediated endocytosis, and may stimulate dyanmin II's GTPase activity, further supporting a link between receptor internalization, PDEγ, and MAPK signaling. K. F. Wan, B. S. Sambi, M. Frame, R. Tate, N. J. Pyne, The inhibitory γ subunit of the type 6 retinal cyclic guanosine monophosphate phosphodiesterase is a novel intermediate regulating p42/p44 mitogen-activated protein kinase signaling in human embryonic kidney 293 cells. J. Biol. Chem. 276 , 37802-27808 (2001). [Abstract] [Full Text]

PDE , A Link Between Receptors and MAPK?

The inhibitory γ subunit of the rod cyclic guanosine monophosphate phosphodiesterase (PDE) is expressed in several nonphotoreceptive cells, including lung, kidney, heart, testis, and smooth muscle and the human embryonic kidney cell line HEK293. Wan et al. found that expression of antisense for PDEγ inhibited epidermal growth factor (EGF)-stimulated or thrombin-stimulated phosphorylation of the p44 and p42 mitogen-activated protein kinases (p42MAPK and p44MAPK). Furthermore, overexpression of either the rod or cone forms of PDEγ enhanced p42/p44MAPK phosphorylation stimulated by these two ligands. EGF receptors are tyrosine kinase growth factor receptors, and the thrombin receptor is a G protein-coupled receptor (GPCR). Internalization of both receptor tyrosine kinases and GPCRs may be important for efficient MAPK activation. Internalization of the receptors may involve G protein-coupled receptor kinase 2 (GRK2). PDEγ is a substrate for GRK2, and phosphorylation of PDEγ is enhanced by the addition of Gβγ in vitro. Expression of a form of PDEγ not phosphorylated by GRK2 inhibited EGF-stimulated phosphorylation of p42/p44MAPK, suggesting a link between GRK2 and coupling of EGF to MAPK. In thrombin-treated cells, PDEγ coprecipitates with dynamin II, a guanosine triphosphatase (GTPase) involved in clathrin-mediated endocytosis, and may stimulate dyanmin II's GTPase activity, further supporting a link between receptor internalization, PDEγ, and MAPK signaling. K. F. Wan, B. S. Sambi, M. Frame, R. Tate, N. J. Pyne, The inhibitory γ subunit of the type 6 retinal cyclic guanosine monophosphate phosphodiesterase is a novel intermediate regulating p42/p44 mitogen-activated protein kinase signaling in human embryonic kidney 293 cells. J. Biol. Chem. 276, 37802-27808 (2001).[Abstract][Full Text]