Both lipopolysaccharide (LPS) and double-stranded RNA (dsRNA) are adjuvants for the adaptive immune response, inducing upregulation of costimulatory molecules (UCM) on antigen-presenting cells. Trif, an adapter protein that transduces signals from Toll-like receptor 4 (TLR4) and TLR3, permits the induction of many cytokines, including interferon-beta, which signals through the type I interferon receptor. We show here that LPS-induced UCM was strictly dependent on the TLR4-->Trif axis, whereas dsRNA-induced UCM was only partly dependent on the TLR3-->Trif axis. But both LPS- and dsRNA-induced UCM were entirely dependent on type I interferon receptor signaling. These findings show that UCM involves an autocrine or paracrine loop, and indicate that an alternative TLR3-independent, Trif-independent pathway contributes to dsRNA-induced UCM.

Since a release of intracellular contents can induce local inflammatory responses, mechanisms that lead to loss of plasma membrane integrity in cell death are important to know. We showed previously that deficiency of the plasma membrane Ca 2؉ ATPase 4 (PMCA4) in L929 cells impaired tumor necrosis factor alpha (TNF-␣)-induced enlargement of lysosomes and reduced cell death. The lysosomal changes can be determined by measuring the total volume of intracellular acidic compartments per cell (VAC), and we show here that inhibition of the increase in VAC due to PMCA4 deficiency not only reduced cell death but also converted TNF-␣-induced cell death from a process involving disruption of the plasma membrane to a cell demise with a nearly intact plasma membrane. The importance of the size of lysosomes in determining plasma membrane integrity during cell death was supported by the observations that chemical inhibitors that reduce VAC also reduced the plasma membrane disruption induced by TNF-␣ in wild-type L929 cells, while increases in VAC due to genetic mutation, senescence, cell culture conditions, and chemical inhibitors all changed the morphology of cell death from one with an originally nearly intact plasma membrane to one with membrane disruption in a number of different cells. Moreover, the ATP depletion-mediated change from apoptosis to necrosis is also associated with the increases of VAC. The increase in lysosomal size may due to intracellular self-digestion of dying cells. Big lysosomes are easy to rupture, and the release of hydrolytic enzymes from ruptured lysosomes can cause plasma membrane disruption.

Abstract-Recent studies suggest that p38 mitogen-activated protein kinase (MAPK) may be involved in ischemic preconditioning (PC). To further test this possibility, the regulation of MAPK-activated protein kinase 2 (MAPKAPK2), a kinase immediately downstream from p38 MAPK, and the activity of c-Jun NH 2 -terminal kinase (JNK), a second MAPK, were examined in preconditioned hearts. Isolated, perfused rabbit hearts were subjected to 20 to 30 minutes of global ischemia. Ventricular biopsies before treatment and after 20 minutes of ischemia were homogenized, and the activities of MAPKAPK2 and JNK were evaluated. For the MAPKAPK2 experiments, 7 groups were studied, as follows: control hearts; preconditioned hearts; hearts treated with 500 nmol/L R(-) N 6 -(2-phenylisopropyl) adenosine (PIA), an A 1 -adenosine receptor agonist; preconditioned hearts pretreated with 100 mol/L 8-(p-sulfophenyl) theophylline (SPT), an adenosine receptor antagonist; preconditioned hearts also treated with SB 203580, a potent inhibitor of p38 MAPK activation; hearts treated with 50 ng/mL anisomycin (a p38 MAPK/JNK activator); and hearts treated with both anisomycin (50 ng/mL) and the tyrosine kinase inhibitor genistein (50 mol/L). MAPKAPK2 activity was not altered in control hearts after 20 minutes of global ischemia. By contrast, there was a 3.8-fold increase in activity during ischemia in preconditioned hearts. Activation of MAPKAPK2 in preconditioned hearts was blocked by both SPT and SB 203580. MAPKAPK2 activity during ischemia increased 3.5-fold and 3.3-fold in hearts pretreated with PIA or anisomycin, respectively. MAPKAPK2 activation during ischemia in hearts pretreated with anisomycin was blocked by genistein. In separate hearts, anisomycin mimicked the anti-infarct effect of PC, and that protection was abolished by genistein. JNK activity was measured in control and preconditioned hearts. There was a comparable, modest decline in activity during 30 minutes of global ischemia in both groups. As a positive control, a third group of hearts was treated with anisomycin before global ischemia, and in these, JNK activity increased by 290% above baseline. These results confirm that the p38 MAPK/MAPKAPK2 pathway is activated during ischemia only if the heart is in a preconditioned state. These data further support p38 MAPK as an important signaling component in ischemic PC. (Circ Res. 2000;86:144-151.)

Protein kinase CK2 has been implicated in the regulation of a wide range of proteins that are important in cell proliferation and differentiation. Here we demonstrate that the stress signaling agents anisomycin, arsenite, and tumor necrosis factor-␣ stimulate the specific enzyme activity of CK2 in the human cervical carcinoma HeLa cells by up to 8-fold, and this could be blocked by the p38 MAP kinase inhibitor SB203580. We show that p38␣ MAP kinase, in a phosphorylation-dependent manner, can directly interact with the ␣ and ␤ subunits of CK2 to activate the holoenzyme through what appears to be an allosteric mechanism. Furthermore, we demonstrate that anisomycin-and tumor necrosis factor-␣-induced phosphorylation of p53 at Ser-392, which is important for the transcriptional activity of this growth suppressor protein, requires p38 MAP kinase and CK2 activities.The signaling pathways that regulated protein kinase CK2 1 (formerly casein kinase II) have been elusive since the discovery of this highly conserved and ubiquitous protein-serine/ threonine kinase over 45 years ago (1-5). Despite the identification of over 160 putative substrates for CK2, the overriding function of this kinase has been evasive. In most cells, the holoenzyme form of CK2 is a constitutively active heterotetramer of two catalytic ␣ and/or ␣Ј) and two regulatory (␤) subunits, but CK2␤-free pools of CK2␣ also exist. Further increases (2-3-fold) in the phosphotransferase activity of CK2 in cell lysates toward synthetic peptide substrates have been reported following treatment of murine and human fibroblastic cell lines, A431 and 3T3-L1 adipocytes with serum (6), insulin (7-9), insulin-like growth factor-18 (10), epidermal growth factor (7, 11), bombesin (12), and tumor necrosis factor (TNF) (13). CK2 phosphotransferase activity and protein levels are also commonly elevated in solid human tumors and transformed cell lines (2-4, 14, 15). Furthermore, CK2␣ overexpression along with c-Myc induces lymphomas in mice (16). While the increased expression of CK2 is linked with neoplastic transformation, little is known concerning acute regulation of this protein kinase. EXPERIMENTAL PROCEDURESCell Culture and Lysates Preparation-HeLa cells were grown at 37°C on 125-cm 2 flasks in M199 medium (Life Technologies, Inc.) supplemented with 10% fetal calf serum. For cell stimulation, HeLa cells were split at 2 ϫ 10 6 cells/10-cm 2 dish 24 h prior to starvation. Cells were starved with 0.5% fetal calf serum for 15-20 h before exposure to stimuli. Starved cells were incubated with 10 M SB203580 (Calbiochem) for 30 min prior to addition of 10 g/ml anisomycin (Sigma) for 30 min, 50 M arsenite (Sigma) for 15 min, or 20 ng/ml TNF␣ for 15 min. Adhering cells were washed twice with ice-cold phosphate-buffered saline, scraped, then lysed in 50 mM Hepes (pH 7.2), 100 mM NaCl, 1 mM EGTA, and 20 mM sodium fluoride in Buffer A (1 mM sodium orthovanadate, 1% aprotinin, 1 mM phenylmethylsulfonyl fluoride, 1 M pepstatin, 0.5 g/ml leupeptin, 10 g/ml soybean trypsin inhibitor, ...