Stretch-induced retinal vascular endothelial growth factor expression is mediated by phosphatidylinositol 3-kinase and protein kinase C (PKC)-zeta but not by stretch-induced ERK1/2, Akt, Ras, or classical/novel PKC pathways.

Stretch-induced expression of vascular endothelial growth factor (VEGF) is thought to be important in mediating the exacerbation of diabetic retinopathy by systemic hypertension. However, the mechanisms underlying stretch-induced VEGF expression are not fully understood. We present novel findings demonstrating that stretch-induced VEGF expression in retinal capillary pericytes is mediated by phosphatidylinositol (PI) 3-kinase and protein kinase C (PKC)-zeta but is not mediated by ERK1/2, classical/novel isoforms of PKC, Akt, or Ras despite their activation by stretch. Cardiac profile cyclic stretch at 60 cpm increased VEGF mRNA expression in a time- and magnitude-dependent manner without altering mRNA stability. Stretch increased ERK1/2 phosphorylation, PI 3-kinase activity, Akt phosphorylation, and PKC-zeta activity. Signaling pathways were explored using inhibitors of PKC, MEK1/2, and PI 3-kinase; adenovirus-mediated overexpression of ERK, PKC-alpha, PKC-delta, PKC-zeta, and Akt; and dominant negative (DN) mutants of ERK, PKC-zeta, Ras, PI 3-kinase and Akt. Although stretch activated ERK1/2 through a Ras- and PKC classical/novel isoform-dependent pathway, these pathways were not responsible for stretch-induced VEGF expression. Overexpression of DN ERK and Ras had no effect on VEGF expression in these cells. In contrast, DN PI 3-kinase as well as pharmacologic inhibitors of PI 3-kinase blocked stretch-induced VEGF expression. Although stretch-induced PI 3-kinase activation increased both Akt phosphorylation and activity of PKC-zeta, VEGF expression was dependent on PKC-zeta but not Akt. In addition, PKC-zeta did not mediate stretch-induced ERK1/2 activation. These results suggest that stretch-induced expression of VEGF involves a novel mechanism dependent upon PI 3-kinase-mediated activation of PKC-zeta that is independent of stretch-induced activation of ERK1/2, classical/novel PKC isoforms, Ras, or Akt. This mechanism may play a role in the well documented association of concomitant hypertension with clinical exacerbation of neovascularization and vascular permeability.     

Cyclic stretch activates ERK1/2 via G proteins and EGFR in alveolar epithelial cells

Mechanical stimuli are transduced into intracellular signals in lung alveolar epithelial cells (AEC). We studied whether mitogen-activated protein kinase (MAPK) pathways are activated during cyclic stretch of AEC. Cyclic stretch induced a rapid (within 5 min) increase in extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation in AEC. The inhibition of Na(+), L-type Ca(2+) and stretch-activated ion channels with amiloride, nifedipine, and gadolinium did not prevent the stretch-induced ERK1/2 activation. The inhibition of Grb2-SOS interaction with an SH3 binding sequence peptide, Ras with a farnesyl transferase inhibitor, and Raf-1 with forskolin did not affect the stretch-induced ERK1/2 phosphorylation. Moreover, cyclic stretch did not increase Ras activity, suggesting that stretch-induced ERK1/2 activation is independent of the classical receptor tyrosine kinase-MAPK pathway. Pertussis toxin and two specific epidermal growth factor receptor (EGFR) inhibitors (AG-1478 and PD-153035) prevented the stretch-induced ERK1/2 activation. Accordingly, in primary AEC, cyclic stretch activates ERK1/2 via G proteins and EGFR, in Na(+) and Ca(2+) influxes and Grb2-SOS-, Ras-, and Raf-1-independent pathways.     

Signal transduction of MEK/ERK and PI3K/Akt activation by hypoxia/reoxygenation in renal epithelial cells

The extracellular signal-regulated kinase (ERK) and Akt have been reported to be activated by ischemia/reperfusion in vivo. However, the signaling pathways involved in activation of these kinases and their potential roles were not fully understood in the postischemic kidney. In the present study, we observed that these kinases are activated by hypoxia/reoxygenation (H/R), an in vitro model of ischemia/reperfusion, in opossum kidney (OK) cells and elucidated the signaling pathways of these kinases. ERK and Akt were transiently activated during the early phase of reoxygenation following 4-12h of hypoxia. The ERK activation was inhibited by U0126, a specific inhibitor of ERK upstream MAPK/ERK kinase (MEK), but not by LY294002, a specific inhibitor of phosphoinositide 3-kinase (PI3K), whereas Akt activation was blocked by LY294002, but not by U0126. Inhibitors of epidermal growth factor receptor (EGFR) (AG 1478), Ras and Raf, as well as antioxidants inhibited activation of ERK and Akt, while the Src inhibitor PP2 had no effect. PI3K/Akt activation was shown to be associated with up-regulation of X chromosome-linked inhibitor of apoptosis (XIAP), but not survivin. Reoxygenation following 4-h hypoxia-stimulated cell proliferation, which was dependent on ERK and Akt activation and was also inhibited by antioxidants and AG 1478. Taken together, these results suggest that H/R induces activation of MEK/ERK and PI3K/Akt/XIAP survival signaling pathways through the reactive oxygen species-dependent EGFR/Ras/Raf cascade. Activation of these kinases may be involved in the repair process during ischemia/reperfusion.     

Angiotensin II stimulates ERK via two pathways in epithelial cells: protein kinase C suppresses a G-protein coupled receptor-EGF receptor transactivation pathway.

In GN4 rat liver epithelial cells, angiotensin II (Ang II) produces intracellular calcium and protein kinase C (PKC) signals and stimulates ERK and JNK activity. JNK activation appears to be mediated by a calcium-dependent tyrosine kinase (CADTK). To define the ERK pathway, we established GN4 cells expressing an inhibitory Ras(N17). Induction of Ras(N17) blocked EGF- but not Ang II- or phorbol ester (TPA)-dependent ERK activation. In control cells, Ang II and TPA produced minimal increases in Ras-GTP level and Raf kinase activity. PKC depletion by chronic TPA exposure abolished TPA-dependent ERK activation but failed to diminish the effect of Ang II. In PKC-depleted cells, Ang II increased Ras-GTP level and activated Raf and ERK in a Ras-dependent manner. In PKC depleted cells, Ang II stimulated Shc and Cbl tyrosine phosphorylation, suggesting that without PKC, Ang II activates another tyrosine kinase. PKC-depletion did not alter Ang II-dependent tyrosine phosphorylation or activity of p125(FAK), CADTK, Fyn or Src, but PKC depletion or incubation with GF109203X resulted in Ang II-dependent EGF receptor tyrosine phosphorylation. In PKC-depleted cells, EGF receptor-specific tyrosine kinase inhibitors blocked Ang II-dependent EGF receptor and Cbl tyrosine phosphorylation, and ERK activation. In summary, Ang II can activate ERK via two pathways; the latent EGF receptor, Ras-dependent pathway is equipotent to the Ras-independent pathway, but is masked by PKC action. The prominence of this G-protein coupled receptor to EGF receptor pathway may vary between cell types depending upon modifiers such as PKC.     

Mammalian Sprouty4 suppresses Ras-independent ERK activation by binding to Raf1

The signalling cascade including Raf, mitogen-activated protein kinase (MAPK) kinase and extracellular-signal-regulated kinase (ERK) is important in many facets of cellular regulation. Raf is activated through both Ras-dependent and Ras-independent mechanisms, but the regulatory mechanisms of Raf activation remain unclear. Two families of membrane-bound molecules, Sprouty and Sprouty-related EVH1-domain-containing protein (Spred) have been identified and characterized as negative regulators of growth-factor-induced ERK activation. But the molecular functions of mammalian Sproutys have not been clarified. Here we show that mammalian Sprouty4 suppresses vascular epithelial growth factor (VEGF)-induced, Ras-independent activation of Raf1 but does not affect epidermal growth factor (EGF)-induced, Ras-dependent activation of Raf1. Sprouty4 binds to Raf1 through its carboxy-terminal cysteine-rich domain, and this binding is necessary for the inhibitory activity of Sprouty4. In addition, Sprouty4 mutants of the amino-terminal region containing the conserved tyrosine residue, which is necessary for suppressing fibroblast growth factor signalling, still inhibit the VEGF-induced ERK pathway. Our results show that receptor tyrosine kinases use distinct pathways for Raf and ERK activation and that Sprouty4 differentially regulates these pathways.     

HIV-1 Tat activates dual Nox pathways leading to independent activation of ERK and JNK MAP kinases

Human immunodeficiency virus, type 1 Tat is known to exert pleiotropic effects on the vascular endothelium through mitogen-activated protein (MAP) kinases, although the signaling pathways leading to MAP kinase activation are incompletely understood. We focused on proximal pathways potentially governing downstream MAP kinase activity by Tat. Within 2 min, Tat activated both Ras and Rho GTPases in endothelial cells, leading to ERK phosphorylation by 10 min. Notably, Rac1 was necessary for downstream activation of RhoA and both Rac1 and RhoA acted upstream of the Ras/ERK cassette. Antioxidants and the oxidase inhibitor diphenylene iodonium blocked ERK phosphorylation, but specific interference with the canonical Nox2 oxidase had no effect on ERK. Instead, knock down of the novel oxidase Nox4 completely suppressed Tat-dependent Ras and ERK activation downstream of Rac1 and RhoA. Conversely, interference with Rac1, PAK1, and Nox2 blocked JNK phosphorylation, whereas RhoA(N19) and Nox4 knock down did not. Further, knock down of Nox2, but not Nox4, blocked Tat-induced cytoskeletal rearrangement, whereas knock down of Nox4, but not Nox2, blocked Tat-dependent proliferation. Rac1, therefore, bifurcates Tat signaling, leading to concurrent but separate Nox4-dependent Ras/ERK activation, and Nox2-dependent JNK activation. Tat signaling, therefore, provides an example of Nox-specific differential control of MAP kinase pathways.     

D2 Dopamine Receptors Stimulate Mitogenesis Through Pertussis Toxin-Sensitive G Proteins and Ras-Involved ERK and SAP/JNK Pathways in Rat C6-D2L Glioma Cells

Abstract: Dopamine D2 receptors are members of the G protein-coupled receptor superfamily and are expressed on both neurons and astrocytes. Using rat C6 glioma cells stably expressing the rat D2L receptor, we show here that dopamine (DA) can activate both the extracellular signal-regulated kinase (ERK) and c-Jun NH₂-terminal kinase (JNK) pathways through a mechanism involving D2 receptor-G protein complexes and the Ras GTP-binding protein. Agonist binding to D2 receptors rapidly activated both kinases within 5 min, reached a maximum between 10 and 15 min, and then gradually decreased by 60 min. Maximal activation of both kinases occurred with 100 nM DA, which produced a ninefold enhancement of ERK activity and a threefold enhancement of JNK activity. DA-induced kinase activation was prevented by either (+)-butaclamol, a selective D2 receptor antagonist, or pertussis toxin, an uncoupler of G proteins from receptors, but not by (?)-butaclamol, the inactive isomer of (+)-butaclamol. Cotransfection of RasN17, a dominant negative Ras mutant, prevented DA-induced activation of both ERK and JNK. PD098059, a specific MEK1 inhibitor, also blocked ERK activation by DA. Transfection of SEK1(K → R) vector, a dominant negative SEK1 mutant, specifically prevented DA-induced JNK activation and subsequent c-Jun phosphorylation without effect on ERK activation. Furthermore, stimulation of D2 receptors promoted [³H]thymidine incorporation with a pattern similar to that for kinase activation. DA mitogenesis was tightly linked to Ras-dependent mitogen-activated protein kinase (MAPK) and JNK pathways. Transfection with RasN17 and application of PD098059 blocked DA-induced DNA synthesis. Transfection with FlagΔ169, a dominant negative c-Jun mutant, also prevented stimulation of [³H]thymidine incorporation by DA. The demonstration of D2 receptor-stimulated MAPK pathways may help to understand dopaminergic physiological functions in the CNS.     

Role of Ras in metal-induced EGF receptor signaling and NF-kappaB activation in human airway epithelial cells

We showed previously that epithelial growth factor (EGF) receptor (EGFR) signaling is triggered by metallic compounds associated with ambient air particles. Specifically, we demonstrated that As, Zn, and V activated the EGFR tyrosine kinase and the downstream kinases MEK1/2 and ERK1/2. In this study, we examined the role of Ras in EGFR signaling and the nuclear factor-kappaB (NF-kappaB) activation pathway and the possible interaction between these two signaling pathways in a human airway epithelial cell line (BEAS-2B) exposed to As, V, or Zn ions. Each metal significantly increased Ras activity, and this effect was inhibited by the EGFR tyrosine kinase activity inhibitor PD-153035. Adenoviral-mediated overexpression of a dominant-negative mutant form of Ras(N17) significantly blocked MEK1/2 or ERK1/2 phosphorylation in As-, Zn-, or V-exposed BEAS-2B cells but caused little inhibition of V-, Zn- or EGF-induced EGFR tyrosine phosphorylation. This confirmed Ras as an important intermediate effector in EGFR signaling. Interestingly, V, but not As, Zn, or EGF, induced IkappaBalpha serine phosphorylation, IkappaBalpha breakdown, and NF-kappaB DNA binding. Moreover, PD-153035 and overexpression of Ras(N17) each significantly blocked V-induced IkappaBalpha breakdown and NF-kappaB activation, while inhibition of MEK activity with PD-98059 failed to do so. In summary, exposure to As, Zn, and V initiated EGFR signaling and Ras-dependent activation of MEK1/2 and ERK1/2, but only V induced Ras-dependent NF-kappaB nuclear translocation. EGFR signaling appears to cross talk with NF-kappaB signaling at the level of Ras, but additional signals appear necessary for NF-kappaB activation. Together, these data suggest that, in V-treated BEAS-2B cells, Ras-dependent signaling is essential, but not sufficient, for activation of NF-kappaB.     

Stretch-Induced Stress Fiber Remodeling and the Activations of JNK and ERK Depend on Mechanical Strain Rate,but Not FAK

Background

Cells within tissues are subjected to mechanical forces caused by extracellular matrix deformation. Cells sense and dynamically respond to stretching of the matrix by reorienting their actin stress fibers and by activating intracellular signaling proteins, including focal adhesion kinase (FAK) and the mitogen-activated proteins kinases (MAPKs). Theoretical analyses predict that stress fibers can relax perturbations in tension depending on the rate of matrix strain. Thus, we hypothesized stress fiber organization and MAPK activities are altered to an extent dependent on stretch frequency.

Principal Findings

Bovine aortic endothelial cells and human osteosarcoma cells expressing GFP-actin were cultured on elastic membranes and subjected to various patterns of stretch. Cyclic stretching resulted in strain rate-dependent increases in stress fiber alignment, cell retraction, and the phosphorylation of the MAPKs JNK, ERK and p38. Transient step changes in strain rate caused proportional transient changes in the levels of JNK and ERK phosphorylations without affecting stress fiber organization. Disrupting stress fiber contractile function with cytochalasin D or Y27632 decreased the levels of JNK and ERK phosphorylation. Previous studies indicate that FAK is required for stretch-induced cell alignment and MAPK activations. However, cyclic uniaxial stretching induced stress fiber alignment and the phosphorylation of JNK, ERK and p38 to comparable levels in FAK-null and FAK-expressing mouse embryonic fibroblasts.

Conclusions

These results indicate that cyclic stretch-induced stress fiber alignment, cell retraction, and MAPK activations occur as a consequence of perturbations in fiber strain. These findings thus shed new light into the roles of stress fiber relaxation and reorganization in maintenance of tensional homeostasis in a dynamic mechanical environment.     

Retinoic acid selectively activates the ERK2 but not JNK/SAPK or P38 map kinases when inducing myeloid differentiation

Summary Among the three major mitogen-activated protein kinase (MAPK) cascades—the extracellular signal regulated kinase (ERK) pathway, the c-JUN N-terminal/stress-activated protein kinase (JNK/SAPK) pathway, and the reactivating kinase (p38) pathway—retinoic acid selectively utilizes ERK but not JNK/SAPK or p38 when inducing myeloid differentiation of HL-60 human myeloblastic leukemia cells. Retinoic acid is known to active ERK2. The present data show that the activation is selective for this MAPK pathway. JNK/SAPK or p38 are not activated by retinoic acid. Presumably because it activates relevant signaling pathways including MAPK, the polyoma middle T antigen, as well as certain transformation defective mutants thereof, is known to promote retinoic acid-induced differentiation, although the mechanism of action is not well understood. The present results show that consistent with the selective involvement of ERK2, ectopic expression of either the polyoma middle T antigen or its dl23 mutant, which is defective for PLCγ and PI-3 kinase activation, or the Δ205 mutant, which in addition is also weakened for activation of src-like kinases, caused no enhanced JNK/SAPK or p38 kinase activity that promoted the effects of retinoic acid. However, all three of these polyoma antigens are known to enhance ERK2 activation and promote differentiation induced by retinoic acid. Polyoma-activated MAPK signaling relevant to retinoic acid-induced differentiation is thus restricted to ERK2 and does not involve JNK/SAPK or p38. Taken together, the data indicate that among the three parallel MAPK pathways, retinoic acid-induced HL-60 myeloid differentiation selectively depends on activating ERK but not the other two MAPK pathways, JNK/SAPK or p38, with no apparent cross talk between pathways. Furthermore, the striking ability of polyoma middle T antigens to promote retinoic acid-induced differentiation appears to utilize ERK, but not JNK/SPK or p38 signaling.     

Acute Mechanical Stretch Promotes eNOS Activation in Venous Endothelial Cells Mainly via PKA and Akt Pathways

In the vasculature, physiological levels of nitric oxide (NO) protect against various stressors, including mechanical stretch. While endothelial NO production in response to various stimuli has been studied extensively, the precise mechanism underlying stretch-induced NO production in venous endothelial cells remains incompletely understood. Using a model of continuous cellular stretch, we found that stretch promoted phosphorylation of endothelial NO synthase (eNOS) at Ser¹¹⁷⁷, Ser⁶³³ and Ser⁶¹⁵ and NO production in human umbilical vein endothelial cells. Although stretch activated the kinases AMPKα, PKA, Akt, and ERK1/2, stretch-induced eNOS activation was only inhibited by kinase-specific inhibitors of PKA and PI3K/Akt, but not of AMPKα and Erk1/2. Similar results were obtained with knockdown by shRNAs targeting the PKA and Akt genes. Furthermore, inhibition of PKA preferentially attenuated eNOS activation in the early phase, while inhibition of the PI3K/Akt pathway reduced eNOS activation in the late phase, suggesting that the PKA and PI3K/Akt pathways play distinct roles in a time-dependent manner. Finally, we investigated the role of these pathways in stretch-induced endothelial exocytosis and leukocyte adhesion. Interestingly, we found that inhibition of the PI3K/Akt pathway increased stretch-induced Weibel-Palade body exocytosis and leukocyte adhesion, while inhibition of the PKA pathway had the opposite effects, suggesting that the exocytosis-promoting effect of PKA overwhelms the inhibitory effect of PKA-mediated NO production. Taken together, the results suggest that PKA and Akt are important regulators of eNOS activation in venous endothelial cells under mechanical stretch, while playing different roles in the regulation of stretch-induced endothelial exocytosis and leukocyte adhesion.     

PKC mediates cyclic stretch-induced cardiac hypertrophy through Rho family GTPases and mitogen-activated protein kinases in cardiomyocytes

Signaling events, including Rho GTPases and protein kinase C (PKC), are involved in cardiac hypertrophy. However, the mechanisms by which these pathways cooperate during the hypertrophic process remain unclear. Using an in vitro cyclic stretch model with neonatal rat cardiomyocytes, we demonstrated that stretch-induced activation of RhoA, Rac1/Cdc42, and phosphorylation of Rho-guanine nucleotide dissociation inhibitor (GDI) were prevented by inhibition or depletion of PKC, using chelerythrine and phorbol 12-myristate 13-acetate, indicating that phorbol ester-sensitive PKC isozymes may be upstream regulators of Rho GTPases. Using adenoviral-mediated gene transfer of wild-type (WT) and dominant-negative (DN) mutants of PKCalpha and delta, we found that stretch-induced activation of Rho GTPases and phosphorylation of Rho-GDI were mainly regulated by PKCalpha. PKCdelta was involved in regulation of the activation of Rac1. Stretch-induced increases in [(3)H]-leucine incorporation, myofibrillar reorganization and cell size, were blocked by inhibition of Rho GTPases, or overexpression of DN PKCalpha and delta, suggesting that PKCalpha and delta are both required in stretch-induced hypertrophy, through Rho GTPases-mediated signaling pathways. The mechanism, whereby PKC and Rho GTPases regulate hypertrophy, was associated with mitogen-activated protein (MAP) kinases. Stretch-stimulated phosphorylation of MEK1/ERK1/2 and MKK4/JNK was inhibited by overexpression of DN PKCalpha and delta, and that of MKK3/p38 inhibited by DN PKCdelta. The phosphorylation of ERK and JNK induced by overexpression of WT PKCalpha, and the phosphorylation of p38 induced by WT PKCdelta, were regulated by Rho GTPases. This study represents the first evidence that PKCalpha and delta are important regulators in mediating activation of Rho GTPases and MAP kinases, in the cyclic stretch-induced hypertrophic process.     

Analysis of Ras-dependent signals that prevent caspase-3 activation and apoptosis induced by cytokine deprivation in hematopoietic cells

In hematopoietic cells, Ras has been implicated in signaling pathways that prevent apoptosis triggered by deprivation of cytokines, such as interleukin-3 (IL-3). However, the mechanism whereby Ras suppresses cell death remains incompletely understood. We have investigated the role of Ras in IL-3 signal transduction by using the cytokine-dependent BaF3 cell line. Herein, we show that the activation of the pro-apoptotic protease caspase-3 upon IL-3 removal is suppressed by expression of activated Ras, which eventually prevents cell death. For caspase-3 suppression, the Raf/extracellular signal-regulated kinase (ERK)- or phosphatidylinositol 3-kinase (PI3-K)/Akt-mediated signaling pathway downstream of Ras was required. However, inhibition of both pathways did not block activated Ras-dependent suppression of cell death-associated phenotypes, such as nuclear DNA fragmentation. Thus, a pathway that is independent of both Raf/ERK and PI3-K/Akt pathways may function downstream of Ras, preventing activated caspase-3-initiated apoptotic processes. Conditional activation of c-Raf-1 also suppressed caspase-3 activation and subsequent cell death without affecting Akt activity, providing further evidence for a PI3-K/Akt-independent mechanism.     

Static strain stimulates expression of matrix metalloproteinase-2 and VEGF in microvascular endothelium via JNK- and ERK-dependent pathways

VEGF and MMP protein production are both required for exercise-induced capillary growth in skeletal muscle. The underlying process by which muscle activity initiates an angiogenic response is not established, but it is known that mechanical forces such as muscle stretch are involved. We hypothesized that stretch of skeletal muscle microvascular endothelial cells induces production of MMP-2 and VEGF through a common signal pathway. Endothelial cells were grown on Bioflex plates and exposed to 10% static stretch for up to 24 h. MMP-2 protein level was measured by gelatin zymography and VEGF, MMP-2, and MT1-MMP mRNA levels were quantified by real-time quantitative PCR. ERK1/2 and JNK phosphorylation and VEGF protein levels were assessed by Western blotting. Effects of mitogen-activated protein kinases (MAPKs) (ERK1/2, JNK) and reactive oxygen species (ROS) on stretch-induced expression of MMP-2 and VEGF were tested using pharmacological inhibitors. Stretching of endothelial cells for 24 h caused significant increases in MMP-2 protein and mRNA level, but no change in MT1-MMP mRNA. While MMP-2 protein production was enhanced by H(2)O(2) in unstretched cells, ROS inhibition during stretch did not diminish MMP-2 mRNA or protein production. Inhibition of JNK suppressed stretch-induced MMP-2 protein and mRNA, but inhibition of ERK had no effect. In contrast, inhibition of ERK but not JNK attenuated the stretch-induced increase in VEGF mRNA. Our results demonstrate that differential regulation of MMP-2 and VEGF by MAPK signal pathways contribute to stretch-induced activation of microvascular endothelial cells.     

Teratogen-induced activation of ERK, JNK, and p38 MAP kinases in early postimplantation murine embryos

BACKGROUND: Although many teratogens are known to activate apoptotic pathways culminating in abnormal development, little is known about how the embryo transduces a teratogenic exposure into specific responses. Signal reception and transduction are regulated by a number of signal transduction pathways, including the extracellular signal-regulated protein kinases (ERKs), c-Jun N-terminal kinases (JNKs) and the stress-activated protein kinase, p38. METHODS: To analyze the effects of teratogens on MAP kinases, we used whole embryo culture, Western blot analyses, and antibodies recognizing inactive or active MAP kinases, or both. RESULTS: We show that heat shock (HS) induces a rapid, strong, but transient activation of ERK, JNK, and p38 with maximal activation occurring within 30 min of the heat shock. By contrast, cyclophosphamide (CP) and staurosporine (ST) failed to activate ERK or JNK during the time period studied (7. 5 hr). ST and CP did induce a low but reproducible activation of p38 beginning at around 3 hr and 5 hr, respectively, after the initiation of exposure. Previous work has shown that heat shock induces elevated cell death in the embryo, primarily in the developing neuroepithelium, but not in the embryonic heart. Thus, we also compared the activation of these three MAP kinase pathways in heads, hearts, and trunks isolated from day 9 embryos exposed to 43 degrees C for 15 min. The results show that ERK, JNK, and p38 are activated in heads, hearts, and trunks. CONCLUSIONS: Our results show that day 9 embryos do activate MAP kinase signaling pathways in response to teratogenic exposures; however, activation of a particular pathway does not appear to be required for teratogen-induced apoptosis.     

Deregulated MAPK activity prevents adipocyte differentiation of fibroblasts lacking the retinoblastoma protein

A functional retinoblastoma protein (pRB) is required for adipose conversion of preadipocyte cell lines and primary mouse embryo fibroblasts (MEFs) in response to treatment with standard adipogenic inducers. Interestingly, lack of functional pRB in MEFs was recently linked to elevated Ras activity. Ras-dependent signaling plays a significant, although incompletely understood, role in adipocyte differentiation, because activated Ras has been reported to either promote or inhibit adipogenesis depending on the cellular context. In various cell types activation of Ras leads to activation of the mitogen-activated protein kinases (MAPKs), extracellular signal-regulated kinase 1/2 (ERK1/2), and protein kinase B (PKB)/Akt, which exert opposing effects on adipogenesis, with ERK1/2 inhibiting and PKB/Akt promoting terminal differentiation. Here we report that the levels of activated ERK1/2 and PKB/Akt are significantly increased in pRB-deficient MEFs both before and after the addition of adipogenic inducers. Consistently, we detected higher levels of activated Ras in MEFs lacking pRB. Suppression of ERK1/2 activation by the MEK inhibitor UO126 restored the ability of pRB-deficient MEFs to undergo adipocyte differentiation, as manifested by expression of adipocyte marker genes and lipid accumulation. Furthermore and reflecting the elevated levels of activated PKB/Akt in the pRB-deficient MEFs, differentiation proceeded in an insulin-independent manner. In conclusion, we suggest that pRB plays a pivotal role in adipogenesis by suppressing MAPK activity.     

Sphingosine-1-phosphate promotes mouse melanocyte survival via ERK and Akt activation

In this study, we investigated the signalling pathways induced by ultraviolet B (UVB) and the effects of sphingosine-1-phosphate on UVB-induced apoptosis of mouse melanocytes, Mel-Ab, and observed the cytoprotective effects of sphingosine-1-phosphate on UVB-induced apoptosis. Since sphingosine-1-phosphate is a well-known mitogenic agent, we thought it possible that the mitogenic effect of sphingosine-1-phosphate might contribute to cell survival. However, we found that sphingosine-1-phosphate significantly inhibits DNA synthesis. We next examined the regulation of the three major subfamilies of mitogen-activated protein (MAP) kinases and of the Akt pathway by sphingosine-1-phosphate against UVB-induced apoptosis. UVB irradiation resulted in the remarkable and sustained activation of c-Jun N-terminal kinase (JNK), while p38 MAP kinase was only transiently activated. The basal level of extracellular signal-regulated protein kinase (ERK) phosphorylation decreased 30 min after UVB irradiation, whereas the basal level of Akt phosphorylation was unaffected by UVB. We also found that sphingosine-1-phosphate potently stimulates the phosphorylation of both ERK and Akt, which are involved in the cell survival-signalling cascade. Furthermore, the specific inhibition of the ERK and Akt pathways by PD98059 and LY294002, respectively, restored the cytoprotective effect induced by sphingosine-1-phosphate. On the other hand, the p38 inhibitor SB203580 additively enhanced the cytoprotective effect on sphingosine-1-phosphate. Based on these results, we conclude that the activation of p38 MAP kinase plays an important role in UVB-induced apoptosis, and that sphingosine-1-phosphate probably exert its cytoprotective effect in Mel-Ab cells through ERK and Akt activation.     

Lose and gain: impacts of ERK5 and JNK cascades on each other

Kinase cascades in ERK5 (Extracellular signal-regulated kinases) and JNK (c-Jun N-terminal kinases) signaling pathways mediate the sensing and processing of stimuli. Cross-talks between signaling cascades is a likely phenomenon that can cause apparently different biological responses from a single pathway, on its activation. Feedback loops have the potential to greatly alter the properties of a pathway and its response to stimuli. Based on enzyme kinetic reactions, mathematical models have been developed to predict and analyze the impacts of cross-talks and feedback loops in ERK5 and JNK cascades. It has been observed that, there is no significant impact on neither ERK5 activation nor JNKs’ activation due to cross-talks between them. But it is due to cross-talks and feedback loops in ERK5 and JNK cascade, ERK5 gets activated in a transient manner in the absence of input signals. Planning to obtain the parameter values from the experimentalist and the result should be validated by experimental verification.