Signal transduction pathways involved in rheumatoid arthritis synovial fibroblast interleukin-18-induced vascular cell adhesion molecule-1 expression

Vascular cell adhesion molecule (VCAM)-1 has been implicated in interactions between leukocytes and connective tissue, including rheumatoid arthritis (RA) synovial tissue fibroblasts. Such interactions within the synovium contribute to RA inflammation. Using phosphoinositide 3-kinase (PI3-kinase) inhibitor LY294002 and Src inhibitor PP2, we show that interleukin (IL)-18-induced ERK1/2 activation is Src kinase-dependent. Antisense (AS) c-Src oligonucleotide (ODN) treatment reduced IL-18-induced ERK1/2 expression by 32% compared with control, suggesting an upstream role of Src in ERK1/2 activation. AS c-Src ODN treatment also inhibited Akt expression by 74% compared with sense control. PI3-kinase inhibitor LY294002 or AS PI3-kinase ODN inhibited Akt expression. AS c-Src ODN inhibited Akt phosphorylation, confirming Src is upstream of PI3-kinase in IL-18-induced RA synovial fibroblast signaling. IL-18 induced a time-dependent activation of c-Src, Ras, and Raf-1, suggesting this signaling cascade plays a role in ERK activation. IL-18 directly activated Src kinase by more than 4-fold over basal levels by enzymatic assay. Electrophoretic mobility shift assay showed that activator protein-1 (AP-1) is activated by IL-18 through ERK and Src but not through PI3-kinase. In an alternate pathway, inhibition of IL-1 receptor-associated kinase-1 (IRAK) with AS ODN to IRAK reduced IL-18-induced expression of nuclear factor kappaB (NFkappaB). Finally, IL-18-induced cell surface VCAM-1 expression was inhibited by treatment with AS ODNs to c-Src, IRAK, PI3-kinase, and ERK1/2 by 57, 43, 41, and 32% compared with control sense ODN treatment, respectively. These data support a role for IL-18 activation of three distinct pathways during RA synovial fibroblast stimulation: two Src-dependent pathways and the IRAK/NFkappaB pathway. Targeting VCAM-1 signaling mechanisms may represent therapeutic approaches to inflammatory and angiogenic diseases characterized by adhesion molecule up-regulation.     

The non-provitamin A carotenoid, lutein, inhibits NF-kappaB-dependent gene expression through redox-based regulation of the phosphatidylinositol 3-kinase/PTEN/Akt and NF-kappaB-inducing kinase pathways: role of H(2)O(2) in NF-kappaB activation

Reactive oxygen species (ROS) have been implicated in the regulation of NF-kappaB activation, which plays an important role in inflammation and cell survival. However, the molecular mechanisms of ROS in NF-kappaB activation remain poorly defined. We found that the non-provitamin A carotenoid, lutein, decreased intracellular H(2)O(2) accumulation by scavenging superoxide and H(2)O(2) and the NF-kappaB-regulated inflammatory genes, iNOS, TNF-alpha, IL-1beta, and cyclooxygenase-2, in lipopolysaccharide (LPS)-stimulated macrophages. Lutein inhibited LPS-induced NF-kappaB activation, which highly correlated with its inhibitory effect on LPS-induced IkappaB kinase (IKK) activation, IkappaB degradation, nuclear translocation of NF-kappaB, and binding of NF-kappaB to the kappaB motif of the iNOS promoter. This compound inhibited LPS- and H(2)O(2)-induced increases in phosphatidylinositol 3-kinase (PI3K) activity, PTEN inactivation, NF-kappaB-inducing kinase (NIK), and Akt phosphorylation, which are all upstream of IKK activation, but did not affect the interaction between Toll-like receptor 4 and MyD88 and the activation of mitogen-activated protein kinases. The NADPH oxidase inhibitor apocynin and gp91(phox) deletion reduced the LPS-induced NF-kappaB signaling pathway as lutein did. Moreover, lutein treatment and gp91(phox) deletion decreased the expressional levels of the inflammatory genes in vivo and protected mice from LPS-induced lethality. Our data suggest that H(2)O(2) modulates IKK-dependent NF-kappaB activation by promoting the redox-sensitive activation of the PI3K/PTEN/Akt and NIK/IKK pathways. These findings further provide new insights into the pathophysiological role of intracellular H(2)O(2) in the NF-kappaB signal pathway and inflammatory process.     

PTEN/MMAC1/TEP1 in signal transduction and tumorigenesis.

The level of phosphorylation within cells is tightly regulated by the concerted action of protein kinases and protein phosphatases [Hunter, T. (1995) Cell 80, 225-236]. Disregulation in the activity of either of these players can lead to cellular transformation. Many protein tyrosine kinases are proto-oncogenes and it has been postulated that some protein phosphatases may act as tumor suppressors. Herein we will review the recent findings addressing the roles the candidate tumor suppressor PTEN/MMAC1/TEP1 (PTEN, phosphatase and tensin homologue deleted from chromosome 10; MMAC 1, mutated in multiple advanced cancers 1; TEP1, TGF beta regulated and epithelial cell enriched phosphatase 1) plays in signal transduction and tumorigenesis. PTEN is a dual specificity protein phosphatase (towards phospho-Ser/Thr and phospho-Tyr) and, unexpectedly, also has a phosphoinositide 3-phosphatase activity. PTEN plays an important role in the modulation of the 1-phosphatidylinositol 3-kinase (PtdIns 3-kinase) pathway, by catalyzing the degradation of the PtdIns(3,4,5)P3 generated by PtdIns 3-kinase; this inhibits the downstream functions mediated by the PtdIns 3-kinase pathway, such as activation of protein kinase B (PKB, also known as Akt), cell survival and cell proliferation. Furthermore, PTEN modulates cell migration and invasion by negatively regulating the signals generated at the focal adhesions, through the direct dephosphorylation and inhibition of focal adhesion kinase (FAK). Growth factor receptor signaling is also negatively regulated by PTEN, through the inhibition of the adaptor protein Shc. While some of the functions of PTEN have been elucidated, it is clear that there is much more to discover about the roles of this unique protein.     

The mechanism of action of the tumour suppressor gene PTEN

Intracellular levels of phosphorylation are regulated by the coordinated action of protein kinases and phosphatases. Disregulation of this balance can lead to cellular transformation. Here we review knowledge of the mechanisms of one protein phosphatase, the tumour suppressor PTEN/MMAC/TEP 1 apropos its role in tumorigenesis and signal transduction. PTEN plays an important role in the phosphatidyl-inositol-3-kinase (PI3-K) pathway by catalyzing degradation of phosphatidylinositol-(3,4,5)-triphosphate generated by PI3-K. This inhibits downstream targets mainly protein kinase B (PKB/Akt), cell survival and proliferation. PTEN contributes to cell cycle regulation by blockade of cells entering the S phase of the cell cycle, and by upregulation of p27(Kip1) which is recruited into the cyclin E/cdk2 complex. PTEN also modulates cell migration and motility by regulation of the extracellular signal-related kinase - mitogen activated protein kinase (ERK-MAPK) pathway and by dephosphorylation of focal adhesion kinase (FAK). We also emphasize the increasingly important role that PTEN has from an evolutionary point of view. A number of PTEN functions have been elucidated but more information is needed for utilization in clinical application and potential cancer therapy.     

Phosphatase PTEN is inactivated in bovine aortic endothelial cells exposed to cyclic strain

Hemodynamic forces, including cyclic strain (CS) and shear stress (SS), have been recognized as important modulators of vascular cell morphology and function. PTEN (also known as MMAC1/TEP1) is a lipid phosphatase that leads to a decrease in intracellular phosphatidylinositol 3,4,5 trisphosphate (PIP3) and therefore can modulate the stimulating effect of phosphatidylinositol 3-kinase (PI3K). In this study, we focused on the upstream regulators of the PI3K-Akt pathway by assessing Akt, PTEN, casein kinase 2 (CK2) (a kinase that catalyzes phosphorylation of PTEN), and PI3K activity in endothelial cells (EC) exposed to CS. The activity of phospho-PTEN (n = 4) and phospho-CK2 (n = 4) increased in a time-dependent fashion, reaching maximal activity by 10 min of CS stimulation. The peak of phospho-Akt activity (n = 4) occurred later, at 60 min. Akt activity was altered by transfection of EC with dominant negative PTEN plasmids. Furthermore, CS increased PIP3 immunoreactivity in a time-dependent manner, reaching maximal activity after 60 min of CS stimulation, and these effects were affected by transfection of EC with dominant negative PTEN plasmids. Inhibition of PTEN activity had no effect on CS-mediated cell proliferation but inhibited CS-mediated suppression of apoptosis.     

Osteopontin stimulates cell motility and nuclear factor kappaB-mediated secretion of urokinase type plasminogen activator through phosphatidylinositol 3-kinase/Akt signaling pathways in breast cancer cells

We have recently reported that osteopontin (OPN) induces nuclear factor kappaB (NFkappaB)-mediated promatrix metalloproteinase-2 activation through IkappaBalpha/IKK signaling pathways and that curcumin (diferulolylmethane) down-regulates these pathways (Philip, S., and Kundu, G. C. (2003) J. Biol. Chem. 278, 14487-14497). However, the molecular mechanism by which upstream kinases regulate the OPN-induced NFkappaB activation and urokinase type plasminogen activator (uPA) secretion in human breast cancer cells is not well defined. Here we report that OPN induces the phosphatidylinositol 3'-kinase (PI 3'-kinase) activity and phosphorylation of Akt in highly invasive MDA-MB-231 and low invasive MCF-7 cells. The OPN-induced Akt phosphorylation was inhibited when cells were transfected with a dominant negative mutant of the p85 domain of PI 3-kinase (Deltap85) and enhanced when cells were transfected with an activated form of PI 3-kinase (p110CAAX), indicating that PI 3'-kinase is involved in Akt phosphorylation. OPN enhances the interaction between IkappaBalpha kinase (IKK) and phosphorylated Akt. OPN also induces NFkappaB activation through phosphorylation and degradation of IkappaBalpha by inducing the IKK activity. However, both pharmacological (wortmannin and LY294002) and genetic (Deltap85) inhibitors of PI 3'-kinase inhibited OPN-induced Akt phosphorylation, IKK activity, and NFkappaB activation through phosphorylation and degradation of IkappaBalpha. OPN also enhances uPA secretion, cell motility, and extracellular matrix invasion. Furthermore, cells transfected with Deltap85 or the super-repressor form of IkappaBalpha suppressed the OPN-induced uPA secretion and cell motility, whereas cells transfected with p110CAAX enhanced these effects. Pretreatment of cells with PI 3-kinase inhibitors or NFkappaB inhibitory peptide (SN-50) reduced the OPN-induced uPA secretion, cell motility, and invasion. To our knowledge, this is first report that OPN induces NFkappaB activity and uPA secretion by activating PI 3'-kinase/Akt/IKK-mediated signaling pathways and further demonstrates a functional molecular link between OPN-induced PI 3'-kinase-dependent Akt phosphorylation and NFkappaB-mediated uPA secretion, and all of these ultimately control the motility of breast cancer cells.     

Interleukin-6 impairs the insulin signaling pathway, promoting production of nitric oxide in human umbilical vein endothelial cells

Interleukin 6 (IL-6) is an independent predictor of type 2 diabetes and cardiovascular disease and is correlated with insulin resistance. Insulin stimulates nitric oxide (NO) production through the IRS-1/PI3-kinase/Akt/eNOS pathway (where IRS-1 is insulin receptor substrate 1, PI3-kinase is phosphatidylinositol 3-kinase, and eNOS is endothelial NO synthase). We asked if IL-6 affects insulin vasodilator action both in human umbilical vein endothelial cells (HUVEC) and in the aortas of C57BL/6J mice and whether this inhibitory effect was caused by increased Ser phosphorylation of IRS-1. We observed that IL-6 increased IRS-1 phosphorylation at Ser(312) and Ser(616); these effects were paralleled by increased Jun N-terminal protein kinase (JNK) and extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation and reversed by JNK and ERK1/2 inhibition. In addition, IL-6 treatment resulted in impaired IRS-1 phosphorylation at Tyr(612), a site essential for engaging PI3-kinase. Furthermore, IL-6 treatment reduced insulin-stimulated phosphorylation of eNOS at the stimulatory Ser(1177) site and impaired insulin-stimulated eNOS dephosphorylation at the inhibitory Thr(495) site. Insulin-stimulated eNOS activation and NO production were also inhibited by IL-6; these effects were reversed by inhibition of JNK and ERK1/2. Treatment of C57BL/6J mice with IL-6 resulted in impaired insulin-dependent activation of the Akt/eNOS pathway in the aorta as a result of JNK and ERK1/2 activation. Our data suggest that IL-6 impairs the vasodilator effects of insulin that are mediated by the IRS-1/PI3-kinase/Akt/eNOS pathway through activation of JNK and ERK1/2.     

The IL-1 receptor accessory protein is essential for PI 3-kinase recruitment and activation

Interleukin-1 (IL-1) binds to its type I receptors (IL-1R), which in complex with IL-1R accessory protein (IL-1R AcP) induces various intracellular signaling events. We report here that IL-1 triggers the recruitment of phosphoinositide 3-kinase (PI 3-kinase) to a signaling complex and induces its lipid kinase activity in a biphasic manner. This IL-1-induced complex consists of IL-1R, IL-1R AcP, PI 3-kinase, and the IL-1-receptor-associated kinase (IRAK). Deletion of the C-terminus 27 amino acids of IL-1R AcP resulted in a mutant, CDelta27, that could not recruit PI 3-kinase to the signalsome nor stimulate PI3-kinase activity. Moreover, CDelta27 functioned as a dominant-negative mutant that inhibited IL-1-induced PI 3-kinase and NFkappaB activation. CDelta27, however, had no effect on IL-1-dependent activation of the Jun N-terminal kinase (JNK), indicating that distinct regions of IL-1R AcP mediate the activation of PI 3-kinase and JNK. Thus, our results identified a functional region in the IL-1R AcP required for the recruitment and activation of PI 3-kinase.     

Evidence that phosphatidylinositol 3-kinase- and mitogen-activated protein kinase kinase-4/c-Jun NH2-terminal kinase-dependent Pathways cooperate to maintain lung cancer cell survival

Cancer cells in which the PTEN lipid phosphatase gene is deleted have constitutively activated phosphatidylinositol 3-kinase (PI3K)-dependent signaling and require activation of this pathway for survival. In non-small cell lung cancer (NSCLC) cells, PI3K-dependent signaling is typically activated through mechanisms other than PTEN gene loss. The role of PI3K in the survival of cancer cells that express wild-type PTEN has not been defined. Here we provide evidence that H1299 NSCLC cells, which express wild-type PTEN, underwent proliferative arrest following treatment with an inhibitor of all isoforms of class I PI3K catalytic activity (LY294002) or overexpression of the PTEN lipid phosphatase. In contrast, overexpression of a dominant-negative mutant of the p85alpha regulatory subunit of PI3K (Deltap85) induced apoptosis. Whereas PTEN and Delta85 both inhibited activation of AKT/protein kinase B, only Deltap85 inhibited c-Jun NH2-terminal kinase (JNK) activity. Cotransfection of the constitutively active mutant Rac-1 (Val12), an upstream activator of JNK, abrogated Deltap85-induced lung cancer cell death, whereas constitutively active mutant mitogen-activated protein kinase kinase (MKK)-1 (R4F) did not. Furthermore, LY294002 induced apoptosis of MKK4-null but not wild-type mouse embryo fibroblasts. Therefore, we propose that, in the setting of wild-type PTEN, PI3K- and MKK4/JNK-dependent pathways cooperate to maintain cell survival.     

Ultraviolet light activates NFkappaB through translational inhibition of IkappaBalpha synthesis

UV light induces a delayed and prolonged (3-20 h) activation of NFkappaB when compared with the immediate and acute (10-90 min) activation of NFkappaB in response to tumor necrosis factor alpha treatment. In the early phase (3-12 h) of NFkappaB activation, UV light reduces inhibitor of NFkappaB (IkappaB) through an IkappaB kinase-independent, but polyubiquitin-dependent, pathway. However, the mechanism for the UV light-induced reduction of IkappaB and activation of NFkappaB is not known. In this report, we show that UV light down-regulates the total amount of IkappaB through decreasing IkappaB mRNA translation. Our data show that UV light inhibits translation of IkappaB in wild-type mouse embryo fibroblasts (MEF(S/S)) and that this inhibition is prevented in MEF(A/A) cells in which the phosphorylation site, Ser-51 in the eukaryotic translation initiation factor 2 alpha-subunit, is replaced with a non-phosphorylatable Ala (S51A). Our data also show that UV light-induced NFkappaB activation is delayed in MEF(A/A) cells and in an MCF-7 cell line that is stably transfected with a trans-dominant negative mutant protein kinase-like endoplasmic reticulum kinase (PERK). These results suggest that UV light-induced eukaryotic translation initiation factor 2 alpha-subunit phosphorylation translationally inhibits new IkappaB synthesis. Without a continuous supply of newly synthesized IkappaB, the existing IkappaB is degraded through a polyubiquitin-dependent proteasomal pathway leading to NFkappaB activation. Based upon our results, we propose a novel mechanism by which UV light regulates early phase NFkappaB activation by means of an ER-stress-induced translational inhibition pathway.     

PD-1 Increases PTEN Phosphatase Activity While Decreasing PTEN Protein Stability by Inhibiting Casein Kinase 2

Programmed death 1 (PD-1) is a potent inhibitor of T cell responses. PD-1 abrogates activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, but the mechanism remains unclear. We determined that during T cell receptor (TCR)/CD3- and CD28-mediated stimulation, PTEN is phosphorylated by casein kinase 2 (CK2) in the Ser380-Thr382-Thr383 cluster within the C-terminal regulatory domain, which stabilizes PTEN, resulting in increased protein abundance but suppressed PTEN phosphatase activity. PD-1 inhibited the stabilizing phosphorylation of the Ser380-Thr382-Thr383 cluster within the C-terminal domain of PTEN, thereby resulting in ubiquitin-dependent degradation and diminished abundance of PTEN protein but increased PTEN phosphatase activity. These effects on PTEN were secondary to PD-1-mediated inhibition of CK2 and were recapitulated by pharmacologic inhibition of CK2 during TCR/CD3- and CD28-mediated stimulation without PD-1. Furthermore, PD-1-mediated diminished abundance of PTEN was reversed by inhibition of ubiquitin-dependent proteasomal degradation. Our results identify CK2 as a new target of PD-1 and reveal an unexpected mechanism by which PD-1 decreases PTEN protein expression while increasing PTEN activity, thereby inhibiting the PI3K/Akt signaling axis.     

Activation of Phosphatidylinositol 3-Kinase in Response to Interleukin-1 Leads to Phosphorylation and Activation of the NF-κB p65/RelA Subunit

The work of Reddy et al. (S. A. Reddy, J. A. Huang, and W. S. Liao, J. Biol. Chem. 272:29167-29173, 1997) reveals that phosphatidylinositol 3-kinase (PI3K) plays a role in transducing a signal from the occupied interleukin-1 (IL-1) receptor to nuclear factor kappaB (NF-kappaB), but the underlying mechanism remains to be determined. We have found that IL-1 stimulates interaction of the IL-1 receptor accessory protein with the p85 regulatory subunit of PI3K, leading to the activation of the p110 catalytic subunit. Specific PI3K inhibitors strongly inhibit both PI3K activation and NF-kappaB-dependent gene expression but have no effect on the IL-1-stimulated degradation of IkappaBalpha, the nuclear translocation of NF-kappaB, or the ability of NF-kappaB to bind to DNA. In contrast, PI3K inhibitors block the IL-1-stimulated phosphorylation of NF-kappaB itself, especially the p65/RelA subunit. Furthermore, by using a fusion protein containing the p65/RelA transactivation domain, we found that overexpression of the p110 catalytic subunit of PI3K induces p65/RelA-mediated transactivation and that the specific PI3K inhibitor LY294,002 represses this process. Additionally, the expression of a constitutively activated form of either p110 or the PI3K-activated protein kinase Akt also induces p65/RelA-mediated transactivation. Therefore, IL-1 stimulates the PI3K-dependent phosphorylation and transactivation of NF-kappaB, a process quite distinct from the liberation of NF-kappaB from its cytoplasmic inhibitor IkappaB.