Anisomycin induces COX-2 mRNA expression through p38(MAPK) and CREB independent of small GTPases in intestinal epithelial cells

Cyclooxygenase (COX)-2 expression in intestinal epithelial cells is associated with colorectal carcinogenesis. COX-2 expression is induced by numerous growth factors and gastrointestinal hormones through multiple protein kinase cascades. Here, the role of mitogen activated protein kinases (MAPKs) and small GTPases in COX-2 expression was investigated. Anisomycin and sorbitol induced COX-2 expression in non-transformed, intestinal epithelial IEC-18 cells. Both anisomycin and sorbitol activated p38(MAPK) followed by phosphorylation of CREB. SB202190 and PD169316 but neither PD98059 nor U0126 blocked COX-2 expression and CREB phosphorylation by anisomycin or sorbitol. Clostridium difficile toxin B inhibition of small GTPases did not affect anisomycin-induced COX-2 mRNA expression or phosphorylation of p38MAPK and CREB but did inhibit sorbitol-dependent COX-2 expression and phosphorylation of p38MAPK and CREB. Angiotensin (Ang) II-dependent induction of COX-2 mRNA and induced phosphorylation of p38MAPK and CREB were inhibited by toxin B. Reduction of CREB protein in cells transfected with CREB siRNAs inhibited anisomycin-induced COX-2 expression. These results indicate that activation of p38MAPK signaling is sufficient for COX-2 expression in IEC-18 cells. Ang II and sorbitol require small GTPase activity for COX-2 expression via p38MAPK while anisomycin-induced COX-2 expression by p38MAPK does not require small GTPases. This places small GTPase activity down-stream of the AT1 receptor and hyperosmotic stress and up-stream of p38MAPK and CREB.     

Expression and characterization of MAP kinases in bacteria

Mitogen-activated protein kinases (MAPKs) are common signal transducers in all eukaryotic organisms. MAPKs are activated by protein kinase cascades consisting of MAPK kinases (MAP2Ks) and MAPK kinase kinases (MAP3Ks). Extracellular-signal regulated kinases 1 and 2 (ERK1/2) are the best characterized MAPKs. Like other MAPKs their activity is regulated by dual phosphorylation as well as dephosphorylation by a host of phosphoprotein phosphatases. The ability to phosphorylate or thiophosphorylate ERK2 in vitro, as described here, is valuable for use in downstream applications designed to investigate MAPK signaling networks.     

Critical role of cAMP-response element-binding protein for angiotensin II-induced hypertrophy of vascular smooth muscle cells

We reported previously an important role of cyclic AMP-response element (CRE) for the induction of interleukin-6 gene expression by angiotensin II (AngII). We examined signaling pathways that are responsible for AngII-induced phosphorylation of CRE-binding protein (CREB) at serine 133 that is a critical marker for the activation in rat vascular smooth muscle cells (VSMC). AngII time dependently induced phosphorylation of CREB with a peak at 5 min. The AngII-induced phosphorylation of CREB was blocked by CV11974, an AngII type I receptor antagonist, suggesting that AngII type I receptor may mediate the phosphorylation of CREB. Inhibition of extracellular signal-regulated protein kinase (ERK) by PD98059 or inhibition of p38 mitogen-activated protein kinase (MAPK) by SB203580 partially inhibited AngII-induced CREB phosphorylation. A protein kinase A inhibitor, H89, also partially suppressed AngII-induced CREB phosphorylation. Inhibition of epidermal growth factor-receptor by AG1478 suppressed the AngII-induced CREB phosphorylation as well as activation of ERK and p38MAPK. Overexpression of the dominant negative form of CREB by an adenovirus vector suppressed AngII-induced c-fos expression and incorporation of [(3)H]leucine to VSMC. These findings suggest that AngII may activate multiple signaling pathways involving two MAPK pathways and protein kinase A, all of which contribute to the activation of CREB. Transactivation of epidermal growth factor-receptor is also critical for AngII-induced CREB phosphorylation. Activation of CREB may be important for the regulation of gene expression and hypertrophy of VSMC induced by AngII.     

OX2R activation induces PKC-mediated ERK and CREB phosphorylation

Deficiencies in brain orexins and components of mitogen activated protein kinase (MAPK) signaling pathway have been reported in either human depression or animal model of depression. Brain administration of orexins affects behaviors toward improvement of depressive symptoms. However, the documentation of endogenous linkage between orexin receptor activation and MAPK signaling pathway remains to be insufficient. In this study, we report the effects of orexin 2 receptor (OX2R) activation on cell signaling in CHO cells over-expressing OX2R and in mouse hypothalamus cell line CLU172. Short-term extracellular signal-regulated kinase (ERK) phosphorylation and long-term cyclic adenosine monophosphate (cAMP) response element binding protein (CREB) phosphorylation were subsequently observed in CHO cells that over-express OX2R while 20 min of ERK phosphorylation was significantly detected in mouse adult hypothalamus neuron cell line CLU172. Orexin A, which can also activate OX2R, mediated ERK phosphorylation was as the same as orexin B in CHO cells. A MAPK inhibitor eliminated ERK phosphorylation but not CREB phosphorylation in CHO cells. Also, ERK and CREB phosphorylation was not mediated by protein kinase A (PKA) or calmodulin kinase (CaMK). However, inhibition of protein kinase C (PKC) by GF 109203X eliminated the phosphorylation of ERK and CREB in CHO cells. A significant decrease in ERK and CREB phosphorylation was observed with 1 μM GF 109203X pre-treatment indicating that the conventional and novel isoforms of PKC are responsible for CREB phosphorylation after OX2R activation. In contrast, ERK phosphorylation induced by orexin B in CLU172 cells cannot be inhibited by 1 μM of protein kinase C inhibitor. From above observation we conclude that OX2R activation by orexin B induces ERK and CREB phosphorylation and orexin A played the same role as orexin B. Several isoforms of PKC may be involved in prolonged CREB phosphorylation. Orexin B induced ERK phosphorylation in mouse hypothalamus neuron cells differs from CHO cell line and cannot be inhibited by PKC inhibitor GF 109203X. And hypothalamus neuron cells may use different downsteam pathway for orexin B induced ERK phosphorylation. This result supports findings that orexins might have anti-depressive roles.     

Cis-4-methylsphingosine phosphate induces apoptosis in neuroblastoma cells by opposite effects on p38 and ERK mitogen-activated protein kinases

Intracellular phosphorylation of cis-4-methylsphingosine was previously shown to result in a metabolically stable compound that accumulates in Swiss 3T3 fibroblasts and mimics the mitogenic effect induced by the short-lived sphingosine metabolite, sphingosine-1-phosphate. In the present study incubation of neuroblastoma B104 cells with cis-4-methylsphingosine (10 microM) also resulted in an intracellular accumulation of its phosphorylated derivative that was, however, associated with the concentration-dependent induction of apoptosis, not observed after treatment with 10 microM of sphingosine-1-phosphate or sphingosine, respectively. In B104 cells, cis-4-methylsphingosine stimulated p38 mitogen-activated protein kinase (p38 MAPK) and simultaneously inhibited extracellular signal-regulated kinase (ERK), whereas sphingosine and sphingosine-1-phosphate only stimulated p38 MAPK without suppression of ERK. Inhibition of cis-4-methylsphingosine phosphorylation reduced both, apoptosis and concurrent regulation of mitogen-activated protein kinases (MAPKs), suggesting that the unusual accumulation of the phosphorylated sphingoid base was responsible for the biological effects. Furthermore, inhibition of p38 MAPK prevented cis-4-methylsphingosine-induced apoptosis, while suppression of the ERK pathway in the presence of sphingosine or sphingosine-1-phosphate resulted in apoptosis, indicating that the simultaneous opposite regulation of the two MAPKs was required for the induction of apoptosis.     

Clustering of cellular prion protein induces ERK1/2 and stathmin phosphorylation in GT1-7 neuronal cells

The physiological role of the prion protein is largely unknown. Here, clustering of prion at the surface of GT1-7 cells was observed upon anti-prion antibody treatments. This clustering was associated with a rapid and transient phosphorylation of the mitogen activated protein kinases (MAPKs) extracellular receptor kinases 1 and 2 (ERK1/2), and also of the microtubule-destabilizing protein stathmin at serine 16. The specificity of this antibody-mediated activation was ascertained by its inhibition by prion small interfering RNA. The phosphorylation of ERK1/2 but not that of stathmin was abolished by the MAPK/ERK kinase 1 inhibitor U0126, whereas both signaling pathways were blocked by the specific inhibitor of the epidermal growth factor receptor AG1478, suggesting the likely recruitment of this receptor upon prion clustering.     

JNK and p38 MAPK are independently involved in tributyltin-mediated cell death in rainbow trout (Oncorhynchus mykiss) RTG-2 cells

Mitogen-activated protein kinases (MAPKs) are a family of Ser/Thr protein kinases that transmit various extracellular signals to the nucleus inducing gene expression, cell proliferation, and apoptosis. Recent studies have revealed that organotin compounds induce apoptosis and MAPK phosphorylation/activation in mammal cells. In this study, we elucidated the cytotoxic mechanism of tributyltin (TBT), a representative organotin compound, in rainbow trout (Oncorhynchus mykiss) RTG-2 cells. TBT treatment resulted in significant caspase activation, characteristic morphological changes, DNA fragmentation, and consequent apoptotic cell death in RTG-2 cells. TBT exposure induced the rapid and sustained accumulation of phosphorylated MAPKs, including extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAP kinase (p38 MAPK). Further analysis using pharmacological inhibitors against caspases and MAPKs showed that TBT also induced cell death in a caspase-independent manner and that p38 MAPK is involved in TBT-induced caspase-independent cell death, whereas JNK is involved in the caspase-dependent apoptotic pathway. Thus, TBT employs at least two independent signaling cascades to mediate cell death in RTG-2 cells. To our knowledge, this is the first study revealing the relationship between MAPK activation and TBT cytotoxicity in RTG-2 cells.     

Activation and Function of the MAPKs and Their Substrates, the MAPK-Activated Protein Kinases

Summary: The mitogen-activated protein kinases (MAPKs) regulate diverse cellular programs by relaying extracellular signals to intracellular responses. In mammals, there are more than a dozen MAPK enzymes that coordinately regulate cell proliferation, differentiation, motility, and survival. The best known are the conventional MAPKs, which include the extracellular signal-regulated kinases 1 and 2 (ERK1/2), c-Jun amino-terminal kinases 1 to 3 (JNK1 to -3), p38 (α, β, γ, and δ), and ERK5 families. There are additional, atypical MAPK enzymes, including ERK3/4, ERK7/8, and Nemo-like kinase (NLK), which have distinct regulation and functions. Together, the MAPKs regulate a large number of substrates, including members of a family of protein Ser/Thr kinases termed MAPK-activated protein kinases (MAPKAPKs). The MAPKAPKs are related enzymes that respond to extracellular stimulation through direct MAPK-dependent activation loop phosphorylation and kinase activation. There are five MAPKAPK subfamilies: the p90 ribosomal S6 kinase (RSK), the mitogen- and stress-activated kinase (MSK), the MAPK-interacting kinase (MNK), the MAPK-activated protein kinase 2/3 (MK2/3), and MK5 (also known as p38-regulated/activated protein kinase [PRAK]). These enzymes have diverse biological functions, including regulation of nucleosome and gene expression, mRNA stability and translation, and cell proliferation and survival. Here we review the mechanisms of MAPKAPK activation by the different MAPKs and discuss their physiological roles based on established substrates and recent discoveries.     

Regulation of MAPK pathways in response to purinergic stimulation of adult rat cardiac myocytes

We investigated the activation of mitogen-activated protein kinases (MAPKs) pathways by purinergic stimulation in cardiac myocytes from adult rat hearts. ATPS increased the phosphorylation (activation) of the extracellular signal regulated kinase 1 and 2 (ERK1/2) and p38 MAPK. ERK1/2 and p38 MAPK activation was differential, ERK1/2 being rapid and transient while that of p38 MAPK slow and sustained. Using selective inhibitors, activation of ERK1/2 was shown to involve protein kinase C and MEK1/2 while that of p38 MAPK was regulated by both protein kinase C and protein kinase A. Furthermore, we show that purinergic stimulation induces the phosphorylation of the MAPK downstream target, mitogen- and stress-activated protein kinase 1 (MSK1), in cardiac myocytes. The time course of MSK1 phosphorylation closely follows that of ERK activation. Inhibitors of the ERK and p38 MAPK pathways were tested on the phosphorylation of MSK1 at two different time points. The results suggest that ERKs initiate the response but both ERKs and p38 MAPK are required for the maintenance of the complete phosphorylation of MSK1. The temporal relationship of MSK1 phosphorylation and cPLA₂ translocation induced by purinergic stimulation, taken together with previous findings, is an indication that cPLA₂ may be a downstream target of MSK1.     

Multiple signals required for cyclic AMP-responsive element binding protein (CREB) binding protein interaction induced by CD3/CD28 costimulation

The optimal activation of cAMP-responsive element binding protein (CREB), similar to the full activation of T lymphocytes, requires the stimulation of both CD3 and CD28. Using a reporter system to detect interaction of CREB and CREB-binding protein (CBP), in this study we found that CREB binds to CBP only by engagement of both CD3 and CD28. CD3/CD28-promoted CREB-CBP interaction was dependent on p38 mitogen-activated protein kinase (MAPK) and calcium/calmodulin-dependent protein kinase (CaMK) IV in addition to the previously identified extracellular signal-regulated kinase pathway. Extracellular signal-regulated kinase, CaMKIV, and p38 MAPK were also the kinases involved in CREB Ser(133) phosphorylation induced by CD3/CD28. A reconstitution experiment illustrated that optimum CREB-CBP interaction and CREB trans-activation were attained when these three kinase pathways were simultaneously activated in T cells. Our results demonstrate that coordinated activation of different kinases leads to full activation of CREB. Notably, CD28 ligation activated p38 MAPK and CaMKIV, the kinases stimulated by CD3 engagement, suggesting that CD28 acts by increasing the activation extent of p38 MAPK and CaMKIV. These results support the model of a minimum activation threshold for CREB-CBP interaction that can be reached only when both CD3 and CD28 are stimulated.     

Mitogen-activated protein kinase dynamics during the meiotic G2/MI transition of mouse spermatocytes

Cellular and genetic approaches were used to investigate the requirements for activation during spermatogenesis of the extracellular signal-regulated protein kinases (ERKs), more commonly known as the mitogen-activated protein kinases (MAPKs). The MAPKS and their activating kinases, the MEKs, are expressed in specific developmental patterns. The MAPKs and MEK2 are expressed in all premeiotic germ cells and spermatocytes, while MEK1 is not expressed abundantly in pachytene spermatocytes. Phosphorylated (active) variants of these kinases are diminished in pachytene spermatocytes. Treatment of pachytene spermatocytes with okadaic acid (OA), to induce transition from meiotic prophase to metaphase I (G2/MI), resulted in phosphorylation and enzymatic activation of ERK1/2. However, U0126, an inhibitor of the ERK-activating kinases, MEK1/2, did not inhibit OA-induced MAPK activation or chromosome condensation. Analysis of spermatocytes lacking MOS, a mitogen-activated protein kinase kinase kinase responsible for MEK and MAPK activation, revealed that MOS is not required for OA-induced activation of the MAPKs. OA-induced MAPK activation was inhibited by butyrolactone I, an inhibitor of cyclin-dependent kinases 1 and 2 (CDK1, CDK2); thus, these kinases may regulate MAPK activity. Additionally, spermatocytes lacking CDC25C condensed bivalent chromosomes and activated both MPF and MAPKs in response to OA treatment; therefore, there is a CDC25C-independent pathway for MPF and MAPK activation. These studies reveal that spermatocytes do not require either MOS or CDC25C for onset of the meiotic division phase or for activation of MPF and the MAPKs, thus implicating a novel pathway for activation of the ERK1/2 MAPKs in spermatocytes.     

The role of progestin receptors and the mitogen-activated protein kinase pathway in delta opioid receptor facilitation of female reproductive behaviors

The present study investigated the role of the progestin receptor (PR) and the mitogen-activated protein kinase (MAPK) pathway in the facilitation of lordosis behavior by the delta opioid receptor agonist [D-Pen(2), D-Pen(5)]-enkephalin (DPDPE). Ovariectomized, estrogen-primed rats were treated with the PR antagonist RU486 or the MAPK inhibitor PD98059 prior to intraventricular (icv) infusion of DPDPE. Both RU486 and PD98059 blocked receptive and proceptive behaviors induced by DPDPE at 60 min, and RU486 continued to inhibit estrous behavior at 90 min. Because delta opioid receptors can activate the p42/44 MAPKs, extracellular signal regulated kinases (ERK), we determined the effects of DPDPE on ERK phosphorylation. Icv infusion of DPDPE increased the levels of phosphorylated ERK in the hypothalamus and preoptic area of female rats, assessed by immunoblotting. These results support the participation of the PR and the MAPK pathway in the facilitation of lordosis behavior by delta opioid receptors.