GTPase-deficient G alpha 16 and G alpha q induce PC12 cell differentiation and persistent activation of cJun NH2-terminal kinases.

Persistent stimulation of specific protein kinase pathways has been proposed as a key feature of receptor tyrosine kinases and intracellular oncoproteins that signal neuronal differentiation of rat pheochromocytoma (PC12) cells. Among the protein serine/threonine kinases identified to date, the p42/44 mitogen-activated protein (MAP) kinases have been highlighted for their potential role in signalling PC12 cell differentiation. We report here that retrovirus-mediated expression of GTPase-deficient, constitutively active forms of the heterotrimeric Gq family members, G alpha qQ209L and G alpha 16Q212L, in PC12 cells induces neuronal differentiation as indicated by neurite outgrowth and the increased expression of voltage-dependent sodium channels. Differentiation was not observed after cellular expression of GTPase-deficient forms of alpha i2 or alpha 0, indicating selectivity for the Gq family of G proteins. As predicted, overexpression of alpha qQ209L and alpha 16Q212L constitutively elevated basal phospholipase C activity approximately 10-fold in PC12 cells. Significantly, little or no p42/44 MAP kinase activity was detected in PC12 cells differentiated with alpha 16Q212L or alpha qQ209L, although these proteins were strongly activated following expression of constitutively active cRaf-1. Rather, a persistent threefold activation of the cJun NH2-terminal kinases (JNKs) was observed in PC12 cells expressing alpha qQ209L and alpha 16Q212L. This level of JNK activation was similar to that achieved with nerve growth factor, a strong inducer of PC12 cell differentiation. Supportive of a role for JNK activation in PC12 cell differentiation, retrovirus-mediated overexpression of cJun, a JNK target, in PC12 cells induced neurite outgrowth. The results define a p42/44 MAP kinase-independent mechanism for differentiation of PC12 cells and suggest that persistent activation of the JNK members of the proline-directed protein kinase family by GTPase-deficient G alpha q and G alpha 16 subunits is sufficient to induce differentiation of PC12 cells.     

BCR/ABL activates Rap1 and B-Raf to stimulate the MEK/Erk signaling pathway in hematopoietic cells

The BCR/ABL fusion tyrosine kinase activates various intracellular signaling pathways, thus causing chronic myeloid leukemia (CML). Here we demonstrate that the inducible expression of BCR/ABL in a murine hematopoietic cell line, TonB210, leads to the activation of the Ras family small GTPase Rap1, which is inhibited by the ABL kinase inhibitor imatinib. The Rap1 activity in a CML cell line, K562, was also inhibited by imatinib. Inhibition of Rap1 activation by a dominant negative mutant of Rap1, Rap1-N17, or SPA-1 inhibited the BCR/ABL-induced activation of Elk-1. BCR/ABL also activated in a kinase activity-dependent manner the B-Raf kinase, which is an effector molecule of Rap1 and a potent activator of the MEK/Erk/Elk-1 signaling pathway. Together, these data suggest that, in addition to the well-established Ras/Raf-1 pathway, BCR/ABL activates the alternative signaling pathway involving Rap1 and B-Raf to activate Erk, which may play important roles in leukemogenesis.     

Ephrin-B1 reverse signaling activates JNK through a novel mechanism that is independent of tyrosine phosphorylation

Eph receptors and their cognate ligand ephrins play important roles in various biological processes such as cell migration, axon guidance, and synaptic plasticity. One characteristic feature of the Eph-ephrin signal transduction is that, upon interaction with the receptor, the transmembrane B-class ephrins become tyrosine-phosphorylated and transduce intracellular signals that lead to reorganization of the cytoskeleton. Although in vitro and genetic studies have demonstrated unequivocally the significance of this reverse signaling, the underlying mechanism remains unclear. We report here that transfection of ephrin-B1 into 293 cells resulted in robust increase in JNK activity, whereas expression of truncated ephrin-B1 lacking the cytoplasmic domain had a negligible effect, indicating that the induction of JNK activity was attributed mainly to the reverse signaling. The ephrin-B1-mediated JNK activation was reduced significantly by dominant-negative TAK1, MKK4, or MKK7. Ephrin-B1 over-expressing 293 cells became rounded in morphology. Surprisingly, ephrin-B1 that lacked all six intracellular tyrosine residues still triggered JNK activation and rounding morphology of the transfected cells. Consistent with these observations, activation of JNK and the resulting morphological changes mediated by ephrin-B1 could be abolished by the JNK inhibitor SP600125 but not the Src inhibitor PP2. Taken together, our findings have identified a novel reverse signaling pathway transduced by ephrin-B1, which is independent of tyrosine phosphorylation but involves the activation of JNK through TAK1 and MKK4/MKK7 and leads to changes in cell morphology.     

Phospholipase D1 is an Important Regulator of bFGF-Induced Neurotrophin-3 Expression and Neurite Outgrowth in H19-7 Cells

The purpose of this study was to examine the role of phospholipase D1 (PLD1) in basic fibroblast growth factor (bFGF)-induced neurotrophin-3 (NT-3) expression and neurite outgrowth in H19-7 rat hippocampal neuronal progenitor cells. Overexpression of PLD1 increased bFGF-induced NT-3 expression, and dominant-negative-PLD1 or PLD1 siRNA abolished bFGF-induced NT-3 expression and neurite outgrowth. Treatment with bFGF activated the RhoA/Rho-associated kinase (ROCK)/c-jun N-terminal kinase (JNK) pathway, and bFGF-induced NT-3 expression was blocked by a dominant-negative RhoA as well as by a specific Rho-kinase inhibitor (Y27632) and a SAPK/JNK inhibitor (SP600125). Furthermore, bFGF-induced JNK activation was also blocked by Y27632. These results indicate that the RhoA/ROCK/JNK pathway acts as an upstream signaling pathway in bFGF-induced NT-3 expression. Also, phosphatidic acid, the product of PLD, increased NT-3 expression. We found that PLD regulated the RhoA/ROCK/JNK pathway, which then led to Elk-1 transactivation. When Elk-1 activity was blocked by Elk-1 siRNA, bFGF-induced NT-3 expression and neurite outgrowth decreased. NT-3 overexpression increased neurite outgrowth, indicating that NT-3 is important for neurite outgrowth. Taken together, these results suggest that PLD1 is an important regulator of bFGF-induced NT-3 expression and neurite outgrowth, which are mediated by the RhoA/ROCK/JNK pathway via Elk-1 in H19-7 cells.     

Adrenomedullin promotes cell cycle transit and up-regulates cyclin D1 protein level in human glioblastoma cells through the activation of c-Jun/JNK/AP-1 signal transduction pathway

Adrenomedullin is a secreted peptide hormone with multiple functions. Although a number of reports have indicated that adrenomedullin may be involved in tumor progression, its mechanism of action remains obscure. In this study, we have analysed the signal transduction pathway activated by adrenomedullin in human glioma cells. Our results revealed that adrenomedullin induced the phosphorylation of both c-Jun and JNK in glioblastoma cells. Silencing JNK expression with siRNA reversed the phosphorylation of c-Jun induced by adrenomedullin, indicating that JNK is responsible of c-Jun activation. In addition, electrophoretic mobility-shift assays showed that the increase in phosphorylation of c-Jun was associated with increased AP-1 DNA binding activity. Supershift assays and co-immunoprecipitation demonstrated that c-Jun and JunD are part of the AP-1 complex, indicating that activated c-Jun is dimerized with JunD in response to adrenomedullin. Furthermore, adrenomedullin was shown to promote cell transit beyond cell cycle phases with a concomittant increase in cyclin D1 protein level, suggesting that adrenomedullin effect cell proliferation through up-regulation of cyclin D1. The inhibition of JNK activation or the suppression of c-Jun or JunD expression with siRNA impaired the effects of adrenomedullin on cell proliferation and on cyclin D1. Taken together, these data demonstrate that activation of cJun/JNK pathway is involved in the growth regulatory activity of adrenomedullin in glioblastoma cells.     

M-Ras induces Ral and JNK activation to regulate MEK/ERK-independent gene expression in MCF-7 breast cancer cells

Constitutive activation of M-Ras has previously been reported to cause morphologic and growth transformation of murine cells, suggesting that M-Ras plays a role in tumorigenesis. Cell transformation by M-Ras correlated with weak activation of the Raf/MEK/ERK pathway, although contributions from other downstream effectors were suggested. Recent studies indicate that signaling events distinct from the Raf/MEK/ERK cascade are critical for human tumorigenesis. However, it is unknown what signaling events M-Ras triggers in human cells. Using constitutively active M-Ras (Q71L) containing additional mutations within its effector-binding loop, we found that M-Ras induces MEK/ERK-dependent and -independent Elk1 activation as well as phosphatidylinositol 3 kinase (PI3K)/Akt and JNK/cJun activation in human MCF-7 breast cancer cells. Among several human cell lines examined, M-Ras-induced MEK/ERK-independent Elk1 activation was only detected in MCF-7 cells, and correlated with Rlf/M-Ras interaction and Ral/JNK activation. Supporting a role for M-Ras signaling in breast cancer, EGF activated M-Ras and promoted its interaction with endogenous Rlf. In addition, constitutive activation of M-Ras induced estrogen-independent growth of MCF-7 cells that was dependent on PI3K/Akt, MEK/ERK, and JNK activation. Thus, our studies demonstrate that M-Ras signaling activity differs between human cells, highlighting the importance of defining Ras protein signaling within each cell type, especially when designing treatments for Ras-induced cancer. These findings also demonstrate that M-Ras activity may be important for progression of EGFR-dependent tumors.     

Selective upregulated expression of the alpha2-macroglobulin signaling receptor in highly metastatic 1-LN prostate carcinoma cells

Cellular binding of receptor-recognized forms of alpha2-macroglobulin (alpha2M*) is mediated by the low-density lipoprotein receptor related protein (LRP) and the alpha2M signaling receptor (alpha2MSR). In nonmalignant cells, ligation of alpha2MSR promotes DNA synthesis and cellular proliferation. Here, we report that insulin treatment of highly metastatic 1-LN human prostate carcinoma selectively increases alpha2MSR expression and binding of alpha2M* to 1-LN cells. alpha2M* induces transient increases in intracellular calcium and inositol 1,4,5-trisphosphate in insulin-treated 1-LN cells, consistent with activation of alpha2MSR. Inhibition of signaling cascades activated by insulin blocks upregulation of alpha2MSR. By contrast, alpha2M* does not bind to nor induce intracellular signaling in PC-3 cells, even though 1-LN cells were subcloned from PC-3 cells. We suggest that alpha2M* behaves like a growth factor in these highly malignant cells. The 1-LN metastatic phenotype may result, in part, from aberrant expression of alpha2MSR, indicating the possible involvement of alpha2M* in tumor progression.     

A novel mechanism for Wnt activation of canonical signaling through the LRP6 receptor

LDL receptor-related protein 6 (LRP6) is a Wnt coreceptor in the canonical signaling pathway, which plays essential roles in embryonic development. We demonstrate here that wild-type LRP6 forms an inactive dimer through interactions mediated by epidermal growth factor repeat regions within the extracellular domain. A truncated LRP6 comprising its transmembrane and cytoplasmic domains is expressed as a constitutively active monomer whose signaling ability is inhibited by forced dimerization. Conversely, Wnts are shown to activate canonical signaling through LRP6 by inducing an intracellular conformational switch which relieves allosteric inhibition imposed on the intracellular domains. Thus, Wnt canonical signaling through LRP6 establishes a novel mechanism for receptor activation which is opposite to the general paradigm of ligand-induced receptor oligomerization.     

Effect of the alpha3beta1 integrin on the IL-1 stimulated activation of c-Jun N-terminal kinase (JNK) in CACO-2 cells

Intestinal epithelial cells (IEC) are capable of responding to IL-1 stimulation by producing a variety of pro-inflammatory cytokines. Recently, we have found that binding of the alpha3beta1 integrin may have a regulatory effect on IL-1 responses and intracellular signaling by suppressing cytokine secretion, mRNA expression and the downstream intracellular signaling events from IKK to NF-kappaB activation. In this study, we extend these findings by showing that treatment of the Caco-2 epithelial cells with a cross-linking anti-alpha3 integrin antibody resulted in a suppression in the levels of IL-1 induced AP-1 binding activity in nuclear extracts. Furthermore, suppressed levels of IL-1 induced c-Jun N-terminal kinase (JNK) phosphorylation and kinase activity were seen with the antibody treated cells. Cells cultured on purified laminin-5, the ligand for the alpha3beta1 integrin, did not show significantly elevated levels of JNK phosphorylation after IL-1 stimulation while cells cultured on fibronectin yielded significantly elevated levels of IL-1 induced JNK phosphorylation. These results indicate that binding of the alpha3beta1 integrin results in a suppression in the activation of the IL-1 induced intracellular signaling pathway from JNK to AP-1. This novel regulatory effect may be a potentially important mechanism to regulate IL-1 mediated responses by IEC.     

CD28 signaling augments Elk-1-dependent transcription at the c-fos gene during antigen stimulation.

Untransformed CD4(+) Th1 cells stimulated with Ag and APC demonstrated a dependence on B7- and CD28-mediated costimulatory signals for the expression and function of AP-1 proteins. The induction of transactivation by the c-fos gene regulator Elk-1 mirrored this requirement for TCR and CD28 signal integration. c-Jun N-terminal kinase (JNK) (but not extracellular signal-regulated kinase or p38) protein kinase activity was similarly inhibited by neutralizing anti-B7 mAbs. Blockade of JNK protein kinase activity with SB 202190 prevented both Elk-1 transactivation and c-Fos induction. These results identify a unique role for B7 costimulatory molecules and CD28 in the activation of JNK during Ag stimulation in Th1 cells, and suggest that JNK regulates Elk-1 transactivation at the c-fos gene to promote the formation of AP-1 complexes important to IL-2 gene expression.     

Multiple mechanisms for Wnt11-mediated repression of the canonical Wnt signaling pathway

The effect of a noncanonical Wnt, Wnt11, on canonical Wnt signaling stimulated by Wnt1 and activated forms of LRP5 (low density lipoprotein receptor-related protein-5), Dishevelled1 (Dvl1), and beta-catenin was examined in NIH3T3 cells and P19 embryonic carcinoma cells. Wnt11 repressed Wnt1-mediated activation of LEF-1 reporter activity in both cell lines. However, Wnt11 was unable to inhibit canonical signaling activated by LRP5, Dvl1, or beta-catenin in NIH3T3 cells, although it could in P19 cells. In addition, Wnt11-mediated inhibition of canonical signaling in NIH3T3 cells is ligand-specific; Wnt11 could effectively repress canonical signaling activated by Wnt1, Wnt3, or Wnt3a but not by Wnt7a or Wnt7b. Co-culture experiments with NIH3T3 cells showed that the co-expression of Wnt11 with Wnt1 was not an essential requirement for the inhibition, suggesting receptor competition as a possible mechanism. Moreover, in both cell types, elevation of intracellular Ca(2+) levels, which can result from Wnt11 treatment, led to the inhibition of canonical signaling. This result suggests that Wnt11 might not be able to signal in NIH3T3. Furthermore, P19 cells were found to express both endogenous canonical Wnts and Wnt11. Knockdown of Wnt11 expression using siRNA resulted in increased LEF-1 reporter activity, thus indicating that Wnt11-mediated suppression of canonical signaling exists in vivo.     

Insulin induces the low density lipoprotein receptor‐related protein 1 (LRP1) degradation by the proteasomal system in J774 macrophage‐derived cells

Low‐density lipoprotein receptor‐related protein 1 (LRP1) is an endocytic receptor, which binds and internalizes diverse ligands such as activated α₂‐macroglobulin (α₂M*). LRP1 promotes intracellular signaling, which downstream mediates cellular proliferation and migration of different types of cells, including macrophages. Unlike the LDL receptor, LRP1 expression is not sensitive to cellular cholesterol levels but appears to be responsive to insulin. It has been previously demonstrated that insulin increases the cell surface presentation of LRP1 in adipocytes and hepatocytes, which is mediated by the intracellular PI₃K/Akt signaling activation. The LRP1 protein distribution is similar to other insulin‐regulated cell surface proteins, including transferring receptor (Tfr). However, in macrophages, the insulin effect on the LRP1 distribution and expression is not well characterized. Considering that macrophages play a central role in the pathogenesis of atherosclerosis, herein we evaluate the effect of insulin on the cellular expression of LRP1 in J774 macrophages‐derived cells using Western blot and immunofluorescence microscopy. Our data demonstrate that insulin induces a significant decrease in the LRP1 protein content, without changing the specific mRNA level of this receptor. Moreover, insulin specifically affected the protein expression of LRP1 but not Tfr. The insulin‐induced protein degradation of LRP1 in J774 cells was mediated by the activation of the PI₃K/Akt pathway and proteasomal system by an enhanced ubiquitin–receptor conjugation. The decreased content of LRP1 induced by insulin affected the cellular internalization of α₂M*. Thus, we propose that the protein degradation of LRP‐1 induced by insulin in macrophages could have important effects on the pathogenesis of atherosclerosis. J. Cell. Biochem. 106: 372–380, 2009. © 2008 Wiley‐Liss, Inc.     

The G alpha(o/i)-coupled cannabinoid receptor-mediated neurite outgrowth involves Rap regulation of Src and Stat3

The study of the signaling pathways regulating neurite outgrowth in culture is important because of their potential role in neuronal differentiation in vivo. We have previously shown that the G alpha(o/i)-coupled CB1 cannabinoid receptor (CB1R) activates Rap1 to induce neurite outgrowth. G alpha(o/i) also activates the Src-Stat3 pathway. Here, we studied the relationship between the G alpha(o/i)-Rap1 and Src-Stat3 pathways and the role of these signaling pathways in CB1R-mediated neurite outgrowth in Neuro-2A cells. The CB1 agonist HU-210 induced pertussis toxin-sensitive Src and Stat3 phosphorylation. Dominant negative (DN) mutants of Src and Stat3 blocked CB1R-induced neurite outgrowth. Constitutively active Rap 1B and Ral-activated Src and CB1R-induced Src phosphorylation was inhibited by Rap1-DN and Ral-DN, indicating that both Rap1 and Ral mediate downstream signaling from G alpha(o/i) for Src activation. Rap1-activated Ral and Ral-DN blocked Rap-induced Src phosphorylation. G alpha(o)-induced Stat3 activation was blocked by Ral-DN, whereas v-Src-induced Stat3 activation was not inhibited by Ral-DN, indicating that the CB1R, through G alpha(o), mediates the sequential activation of Rap1 to Ral to Src to Stat3 in Neuro-2A cells. Downstream of Src, the CB1R also activated Rac1 and JNK, which enhanced CBR1-mediated Stat3 activation. Rac-DN blocked CB1R-induced activation of JNK. Pharmacological inhibition of JNK blocked Src and CB1R activation of Stat3, indicating that Rac and JNK are also involved in CB1R-mediated neurite outgrowth. Overall, this study demonstrated that G alpha(o/i)-coupled CB1R triggers neurite outgrowth in Neuro-2A through the activation of a signaling network containing two pathways that bifurcate at Src and converge at Stat3.