FADD negatively regulates lipopolysaccharide signaling by impairing interleukin-1 receptor-associated kinase 1-MyD88 interaction.

Lipopolysaccharide (LPS) engages Toll-like receptor 4 (TLR4) on various cells to initiate inflammatory and angiogenic pathways. FADD is an adaptor protein involved in death receptor-mediated apoptosis. Here we report a role for FADD in regulation of TLR4 signals in endothelial cells. FADD specifically attenuates LPS-induced activation of c-Jun NH(2)-terminal kinase and phosphatidylinositol 3'-kinase in a death domain-dependent manner. In contrast, FADD-null cells show hyperactivation of these kinases. Examining physical associations of endogenous proteins, we show that FADD interacts with interleukin-1 receptor-associated kinase 1 (IRAK1) and MyD88. LPS stimulation increases IRAK1-FADD interaction and recruitment of the IRAK1-FADD complex to activated MyD88. IRAK1 is required for FADD-MyD88 interaction, as FADD does not associate with MyD88 in IRAK1-null cells. By shuttling FADD to MyD88, IRAK1 provides a mechanism for controlled and limited activation of the TLR4 signaling pathway. Functionally, enforced FADD expression inhibited LPS- but not vascular endothelial growth factor-induced endothelial cell sprouting, while FADD deficiency led to enhanced production of proinflammatory cytokines induced by stimulation of TLR4 and TLR2, but not TLR3. Reconstitution of FADD reversed the enhanced production of proinflammatory cytokines. Thus, FADD is a physiological negative regulator of IRAK1/MyD88-dependent responses in innate immune signaling.

Erythropoietin promotes survival of primary human endothelial cells through PI3K-dependent, NF-kappaB-independent upregulation of Bcl-xL.

Erythropoietin (EPO) regulates the production of red blood cells primarily by preventing apoptosis of erythroid progenitors. More recently, however, EPO has emerged as a major cytoprotective cytokine in several nonhemopoietic tissues in the setting of stress or injury. The underlying mechanisms of the protective responses of EPO have not been fully defined. Here we show that EPO triggers a phosphatidylinositol 3-kinase-(PI3K)-dependent survival pathway that counteracts endothelial cell death. The protection conferred by PI3K relies on the subsequent induction of Bcl-x(L), a prosurvival member of the Bcl-2 protein family. In addition, EPO counteracts the upregulation of the pro-apoptotic BH3-only protein BIM, which is induced by serum withdrawal. EPO also activates extracellular signal-regulated kinase 1 and 2 (ERK1/2), which are involved in a Bcl-x(L)-independent cytoprotective pathway. EPO caused a prolonged activation of nuclear factor (NF)-kappaB, which was blocked by inhibition of PI3K, but not by inhibition of mitogen-activated protein (MAP)/ERK kinase (MEK), suggesting that EPO-activated NF-kappaB requires PI3K activity. However, the activation of the NF-kappaB pathway was not required for the ability of EPO to counteract endothelial apoptosis. Thus EPO promotes survival of endothelial cells through PI3K-dependent Bcl-x(L)-induction and BIM regulation, as well as through a separate mechanism involving the ERK pathway.

Dephosphorylation by default, a potential mechanism for regulation of insulin receptor substrate-1/2, Akt, and ERK1/2.

Protein phosphorylation is an important mechanism that controls many cellular activities. Phosphorylation of a given protein is precisely controlled by two opposing biochemical reactions catalyzed by protein kinases and protein phosphatases. How these two opposing processes are coordinated to achieve regulation of protein phosphorylation is unresolved. We have developed a novel experimental approach to directly study protein dephosphorylation in cells. We determined the kinetics of dephosphorylation of insulin receptor substrate-1/2, Akt, and ERK1/2, phosphoproteins involved in insulin receptor signaling. We found that insulin-induced ERK1/2 and Akt kinase activities were completely abolished 10 min after inhibition of the corresponding upstream kinases with PD98059 and LY294002, respectively. In parallel experiments, insulin-induced phosphorylation of Akt, ERK1/2, and insulin receptor substrate-1/2 was decreased and followed similar kinetics. Our findings suggest that these proteins are dephosphorylated by a default mechanism, presumably via constitutively active phosphatases. However, dephosphorylation of these proteins is overcome by activation of protein kinases following stimulation of the insulin receptor. We propose that, during acute insulin stimulation, the kinetics of protein phosphorylation is determined by the interplay between upstream kinase activity and dephosphorylation by default.

Inhibition of cyclic AMP dependent protein kinase by vanadyl sulfate.

Vanadium salts influence the activities of a number of mammalian enzymes in vitro but the mechanisms by which low concentrations of vanadium ameliorate the effects of diabetes in vivo remain poorly understood. The hypothesis that vanadium compounds act by inhibiting protein tyrosine phosphatases has attracted most support. The studies described here further evaluate the possibility that vanadyl sulfate trihydrate (VS) can also inhibit 3',5'-cyclic adenosine monophosphate (cAMP) dependent protein kinase (PKA). Using conventional assay conditions, VS inhibited PKA only at high concentrations (IC50>400 microM); however, PKA inhibition was seen at dramatically lower concentrations of VS (IC50<10 microM) when sequestration of vanadyl ions was minimized. Vanadyl appears to be the effective PKA inhibitor because sodium orthovanadate did not inhibit PKA and inhibition by vanadyl was abolished by potential chelators such as ethylenediaminetetraacetic acid or glycyl peptides. PKA inhibition by vanadyl appears to be mixed rather than strictly competitive or uncompetitive and may replicate the inhibitory effects of high concentrations of Mg2+. The effect of vanadyl on PKA provides a possible explanation for the effects of vanadium salts on fat tissue lipolysis and perhaps on other aspects of energy metabolism that are controlled by cAMP-dependent mechanisms. Considering the high degree of conservation of the active sites of protein kinases, vanadyl may also influence other members of this large protein family.

Lipopolysaccharide initiates a TRAF6-mediated endothelial survival signal.

Similar to tumor necrosis factor (TNF), bacterial lipopolysaccharide (LPS) elicits parallel apoptotic and antiapoptotic pathways in endothelial cells. The overall result is that there is minimal endothelial cell death in response to LPS without inhibition of the cytoprotective pathway. While the TNF-induced death and survival pathways have been relatively well elucidated, much remains to be learned about LPS signaling events in this regard. It is known that the transcription factor nuclear factor-kappaB (NF-kappaB) provides a critical cell survival signal in response to TNF, but is not an essential component of the LPS-induced survival pathway. The TNF receptor-associated factor 6 (TRAF6) is a major effector of multiple LPS-induced signals, including a c-Jun N-terminal kinase (JNK)-mediated apoptotic response. In this report we demonstrate that following LPS stimulation, TRAF6 also transmits an important endothelial cell survival signal in a situation of complete NF-kappaB blockade. In response to LPS, TRAF6 activates the phosphatidylinositol 3'-kinase (PI3K)/Akt pathway, but not ERK1/2 mitogen-activated protein kinases (MAPKs) in endothelial cells. Activation of PI3K signals a critical antiapoptotic pathway in response to LPS in endothelial cells, whereas ERK1/2 does not. Thus TRAF6 acts as a bifurcation point of the LPS-initiated death and survival signals in endothelial cells.

In vivo phosphorylation of insulin receptor substrate 1 at serine 789 by a novel serine kinase in insulin-resistant rodents.

Insulin resistance is a key pathophysiologic feature of obesity and type 2 diabetes and is associated with other human diseases, including atherosclerosis, hypertension, hyperlipidemia, and polycystic ovarian disease. Yet, the specific cellular defects that cause insulin resistance are not precisely known. Insulin receptor substrate (IRS) proteins are important signaling molecules that mediate insulin action in insulin-sensitive cells. Recently, serine phosphorylation of IRS proteins has been implicated in attenuating insulin signaling and is thought to be a potential mechanism for insulin resistance. However, in vivo increased serine phosphorylation of IRS proteins in insulin-resistant animal models has not been reported before. In the present study, we have confirmed previous findings in both JCR:LA-cp and Zucker fatty rats, two genetically unrelated insulin-resistant rodent models, that an enhanced serine kinase activity in liver is associated with insulin resistance. The enhanced serine kinase specifically phosphorylates the conserved Ser(789) residue in IRS-1, which is in a sequence motif separate from the ones for MAPK, c-Jun N-terminal kinase, glycogen-synthase kinase 3 (GSK-3), Akt, phosphatidylinositol 3'-kinase, or casein kinase. It is similar to the phosphorylation motif for AMP-activated protein kinase, but the serine kinase in the insulin-resistant animals was shown not to be an AMP-activated protein kinase, suggesting a potential novel serine kinase. Using a specific antibody against Ser(P)(789) peptide of IRS-1, we then demonstrated for the first time a striking increase of Ser(789)-phosphorylated IRS-1 in livers of insulin-resistant rodent models, indicating enhanced serine kinase activity in vivo. Taken together, these data strongly suggest that unknown serine kinase activity and Ser(789) phosphorylation of IRS-1 may play an important role in attenuating insulin signaling in insulin-resistant animal models.

Molecular mechanism of insulin-induced degradation of insulin receptor substrate 1.

Insulin receptor substrate 1 (IRS-1) plays an important role in the insulin signaling cascade. In vitro and in vivo studies from many investigators have suggested that lowering of IRS-1 cellular levels may be a mechanism of disordered insulin action (so-called insulin resistance). We previously reported that the protein levels of IRS-1 were selectively regulated by a proteasome degradation pathway in CHO/IR/IRS-1 cells and 3T3-L1 adipocytes during prolonged insulin exposure, whereas IRS-2 was unaffected. We have now studied the signaling events that are involved in activation of the IRS-1 proteasome degradation pathway. Additionally, we have addressed structural elements in IRS-1 versus IRS-2 that are required for its specific proteasome degradation. Using ts20 cells, which express a temperature-sensitive mutant of ubiquitin-activating enzyme E1, ubiquitination of IRS-1 was shown to be a prerequisite for insulin-induced IRS-1 proteasome degradation. Using IRS-1/IRS-2 chimeric proteins, the N-terminal region of IRS-1 including the PH and PTB domains was identified as essential for targeting IRS-1 to the ubiquitin-proteasome degradation pathway. Activation of phosphatidylinositol 3-kinase is necessary but not sufficient for activating and sustaining the IRS-1 ubiquitin-proteasome degradation pathway. In contrast, activation of mTOR is not required for IRS-1 degradation in CHO/IR cells. Thus, our data provide insight into the molecular mechanism of insulin-induced activation of the IRS-1 ubiquitin-proteasome degradation pathway.