Positive modulation of the TMEM16B mediated currents by TRPV4 antagonist

Calcium-activated chloride channels (CaCCs) play important roles in many physiological processes and their malfunction is implicated in diverse pathologies such as cancer, asthma, and hypertension. TMEM16A and TMEM16B proteins are the structural components of the CaCCs. Recent studies in cell cultures and animal models have demonstrated that pharmacological inhibition of CaCCs could be helpful in the treatment of some diseases, however, there are few specific modulators of these channels. CaCCs and Transient Receptor Potential Vanilloid-4 (TRPV4) channels are co-expressed in some tissues where they functionally interact. TRPV4 is activated by different stimuli and forms a calcium permeable channel that is activated by GSK1016790A and antagonized by GSK2193874. Here we report that GSK2193874 enhances the chloride currents mediated by TMEM16B expressed in HEK cells at nanomolar concentrations and that GSK1016790A enhances native CaCCs of Xenopus oocytes. Thus, these compounds may be used as a tool for the study of CaCCs, TRPV4 and their interactions.     

Effects of calcium on transendothelial albumin transfer and electrical resistance

Nicolaysen, and more recently Kern and Malik, reported that chelation of calcium increased microvascular hydraulic conductivity and albumin permeability in isolated perfused lungs. To begin to understand how calcium affects endothelial function we examined the effect of calcium chelation on an in vitro endothelium. Chelation of calcium with ethyleneglycol-bis(beta-aminoethylether)-N,N'-tetraacetic acid increased the rate of transendothelial albumin transfer by 125%. Reincubation of the endothelium in calcium-repleted medium restored the rate of transfer to its original value. Chelation of extracellular calcium abolished transendothelial electrical resistance. The transendothelial electrical resistance was also restored to normal by reincubation of the endothelium in calcium-repleted medium. Chelation of extracellular calcium caused adjacent endothelial cells to retract from one another, and normal apposition of adjacent cells was restored after reincubation in calcium-repleted medium. Chelation of extracellular calcium produced a centripetal retraction of the peripheral band of actin in individual endothelial cells, and the actin band resumed its normal location after reincubation in calcium-repleted medium. Calcium is an important determinant of endothelial integrity and alterations in calcium produce dynamic changes in endothelial barrier properties and in endothelial-cell shape.     

Capsaicin-induced apoptosis of glioma cells is mediated by TRPV1 vanilloid receptor and requires p38 MAPK activation

We provide evidence on the expression of the transient receptor potential vanilloid type-1 (TRPV1) by glioma cells, and its involvement in capsaicin (CPS)-induced apoptosis. TRPV1 mRNA was identified by quantitative RT-PCR in U373, U87, FC1 and FLS glioma cells, with U373 cells showing higher, and U87, FC1 and FLS cells lower TRPV1 expression as compared with normal human astrocytes. By flow cytometry we found that a substantial portion of both normal human astrocytes, and U87 and U373 glioma cells express TRPV1 protein. Moreover, we analyzed the expression of TRPV1 at mRNA and protein levels of glioma tissues with different grades. We found that TRPV1 gene and protein expression inversely correlated with glioma grading, with marked loss of TRPV1 expression in the majority of grade IV glioblastoma multiforme. We also described that CPS trigger apoptosis of U373, but not U87 cells. CPS-induced apoptosis involved Ca(2+) influx, p38 but not extracellular signal-regulated mitogen-activated protein kinase activation, phosphatidylserine exposure, mitochondrial permeability transmembrane pore opening and mitochondrial transmembrane potential dissipation, caspase 3 activation and oligonucleosomal DNA fragmentation. TRPV1 was functionally implicated in these events as they were markedly inhibited by the TRPV1 antagonist, capsazepine. Finally, p38 but not extracellular signal-regulated protein kinase activation was required for TRPV1-mediated CPS-induced apoptosis of glioma cells.     

Stimulus-specific modulation of the cation channel TRPV4 by PACSIN 3

TRPV4, a member of the vanilloid subfamily of the transient receptor potential (TRP) channels, is activated by a variety of stimuli, including cell swelling, moderate heat, and chemical compounds such as synthetic 4alpha-phorbol esters. TRPV4 displays a widespread expression in various cells and tissues and has been implicated in diverse physiological processes, including osmotic homeostasis, thermo- and mechanosensation, vasorelaxation, tuning of neuronal excitability, and bladder voiding. The mechanisms that regulate TRPV4 in these different physiological settings are currently poorly understood. We have recently shown that the relative amount of TRPV4 in the plasma membrane is enhanced by interaction with the SH3 domain of PACSIN 3, a member of the PACSIN family of proteins involved in synaptic vesicular membrane trafficking and endocytosis. Here we demonstrate that PACSIN 3 strongly inhibits the basal activity of TRPV4 and its activation by cell swelling and heat, while leaving channel gating induced by the synthetic ligand 4alpha-phorbol 12,13-didecanoate unaffected. A single proline mutation in the SH3 domain of PACSIN 3 abolishes its inhibitory effect on TRPV4, indicating that PACSIN 3 must bind to the channel to modulate its function. In line herewith, mutations at specific proline residues in the N terminus of TRPV4 abolish binding of PACSIN 3 and render the channel insensitive to PACSIN 3-induced inhibition. Taken together, these data suggest that PACSIN 3 acts as an auxiliary protein of TRPV4 channel that not only affects the channel's subcellular localization but also modulates its function in a stimulus-specific manner.     

Eosinophil resistance to glucocorticoid-induced apoptosis is mediated by the transcription factor NFIL3

The mainstay of asthma therapy, glucocorticoids (GCs) exert their therapeutic effects through the inhibition of inflammatory signaling and induction of eosinophil apoptosis. However, laboratory and clinical observations of GC-resistant asthma suggest that GCs’ effects on eosinophil viability may depend on the state of eosinophil activation. In the present study we demonstrate that eosinophils stimulated with IL-5 show impaired pro-apoptotic response to GCs. We sought to determine the contribution of GC-mediated transactivating (TA) and transrepressing (TR) pathways in modulation of activated eosinophils’ response to GC by comparing their response to the selective GC receptor (GR) agonist Compound A (CpdA) devoid of TA activity to that upon treatment with Dexamethasone (Dex). IL-5-activated eosinophils showed contrasting responses to CpdA and Dex, as IL-5-treated eosinophils showed no increase in apoptosis compared to cells treated with Dex alone, while CpdA elicited an apoptotic response regardless of IL-5 stimulation. Proteomic analysis revealed that both Nuclear Factor IL-3 (NFIL3) and Map Kinase Phosphatase 1 (MKP1) were inducible by IL-5 and enhanced by Dex; however, CpdA had no effect on NFIL3 and MKP1 expression. We found that inhibiting NFIL3 with specific siRNA or by blocking the IL-5-inducible Pim-1 kinase abrogated the protective effect of IL-5 on Dex-induced apoptosis, indicating crosstalk between IL-5 anti-apoptotic pathways and GR-mediated TA signaling occurring via the NFIL3 molecule. Collectively, these results indicate that (1) GCs’ TA pathway may support eosinophil viability in IL-5-stimulated cells through synergistic upregulation of NFIL3; and (2) functional inhibition of IL-5 signaling (anti-Pim1) or the use of selective GR agonists that don’t upregulate NFIL3 may be effective strategies for the restoring pro-apoptotic effect of GCs on IL-5-activated eosinophils.     

Apoptosis resistance in epithelial tumors is mediated by tumor-cell-derived interleukin-4

We investigated the mechanisms involved in the resistance to cell death observed in epithelial cancers. Here, we identify that primary epithelial cancer cells from colon, breast and lung carcinomas express high levels of the antiapoptotic proteins PED, cFLIP, Bcl-xL and Bcl-2. These cancer cells produced interleukin-4 (IL-4), which amplified the expression levels of these antiapoptotic proteins and prevented cell death induced upon exposure to TRAIL or other drug agents. IL-4 blockade resulted in a significant decrease in the growth rate of epithelial cancer cells and sensitized them, both in vitro and in vivo, to apoptosis induction by TRAIL and chemotherapy via downregulation of the antiapoptotic factors PED, cFLIP, Bcl-xL and Bcl-2. Furthermore, we provide evidence that exogenous IL-4 was able to upregulate the expression levels of these antiapoptotic proteins and potently stabilized the growth of normal epithelial cells rendering them apoptosis resistant. In conclusion, IL-4 acts as an autocrine survival factor in epithelial cells. Our results indicate that inhibition of IL-4/IL-4R signaling may serve as a novel treatment for epithelial cancers.     

Oxidative stress-induced apoptosis is mediated by ERK1/2 phosphorylation

Oxidative stress is known to induce apoptosis in a wide variety of cell types, apparently by modulating intracellular signaling pathways. High concentrations of H2O2 have been found to induce apoptosis in L929 mouse fibroblast cells. To elucidate the mechanisms of H2O2-mediated apoptosis, ERK1/2, p38-MAPK, and JNK1/2 phosphorylation was examined, and ERK1/2 and JNK1/2 were found to be activated by H2O2. Inhibition of ERK1/2 activation by treatment of L929 cells with PD98059 or dominant-negative ERK2 transfection blocked H2O2-induced apoptosis, while inhibition of JNK1/2 by dominant-negative JNK1 or JNK2 or MKK4 or MKK7 transfection did not affect H2O2-mediated apoptosis. H2O2-mediated ERK1/2 activation was not only Ras-Raf dependent, but also both tyrosine kinase (PDGFbeta receptor and Src) and PKCdelta dependent. H2O2-mediated PKCdelta-dependent and tyrosine kinase-dependent ERK1/2 activations were independent from each other. Based on the above results, we suggest for the first time that oxidative damage-induced apoptosis is mediated by ERK1/2 phosphorylation which is not only Ras-Raf dependent, but also both tyrosine kinase and PKCdelta dependent.     

Nitric-oxide-induced necrosis and apoptosis in PC12 cells mediated by mitochondria

Nitric oxide (NO) can trigger either necrotic or apoptotic cell death. We have used PC12 cells to investigate the extent to which NO-induced cell death is mediated by mitochondria. Addition of NO donors, 1 mM S-nitroso-N-acetyl-DL-penicillamine (SNAP) or 1 mM diethylenetriamine-NO adduct (NOC-18), to PC12 cells resulted in a steady-state level of 1-3 microM: NO, rapid and almost complete inhibition of cellular respiration (within 1 min), and a rapid decrease in mitochondrial membrane potential within the cells. A 24-h incubation of PC12 cells with NO donors (SNAP or NOC-18) or specific inhibitors of mitochondrial respiration (myxothiazol, rotenone, or azide), in the absence of glucose, caused total ATP depletion and resulted in 80-100% necrosis. The presence of glucose almost completely prevented the decrease in ATP level and the increase in necrosis induced by the NO donors or mitochondrial inhibitors, suggesting that the NO-induced necrosis in the absence of glucose was due to the inhibition of mitochondrial respiration and subsequent ATP depletion. However, in the presence of glucose, NO donors and mitochondrial inhibitors induced apoptosis of PC12 cells as determined by nuclear morphology. The presence of apoptotic cells was prevented completely by benzyloxycarbonyl-Val-Ala-fluoromethyl ketone (a nonspecific caspase inhibitor), indicating that apoptosis was mediated by caspase activation. Indeed, both NO donors and mitochondrial inhibitors in PC12 cells caused the activation of caspase-3- and caspase-3-processing-like proteases. Caspase-1 activity was not activated. Cyclosporin A (an inhibitor of the mitochondrial permeability transition pore) decreased the activity of caspase-3- and caspase-3-processing-like proteases after treatment with NO donors, but was not effective in the case of the mitochondrial inhibitors. The activation of caspases was accompanied by the release of cytochrome c from mitochondria into the cytosol, which was partially prevented by cyclosporin A in the case of NO donors. These results indicate that NO donors (SNAP or NOC-18) may trigger apoptosis in PC12 cells partially mediated by opening the mitochondrial permeability transition pores, release of cytochrome c, and subsequent caspase activation. NO-induced apoptosis is blocked completely in the absence of glucose, probably due to the lack of ATP. Our findings suggest that mitochondria may be involved in both types of cell death induced by NO donors: necrosis by respiratory inhibition and apoptosis by opening the permeability transition pore. Further, our results indicate that the mode of cell death (necrosis versus apoptosis) induced by either NO or mitochondrial inhibitors depends critically on the glycolytic capacity of the cell.     

Differential modulation of Beta-adrenergic receptor signaling by trace amine-associated receptor 1 agonists

Trace amine-associated receptors (TAAR) are rhodopsin-like G-protein-coupled receptors (GPCR). TAAR are involved in modulation of neuronal, cardiac and vascular functions and they are potentially linked with neurological disorders like schizophrenia and Parkinson's disease. Subtype TAAR1, the best characterized TAAR so far, is promiscuous for a wide set of ligands and is activated by trace amines tyramine (TYR), phenylethylamine (PEA), octopamine (OA), but also by thyronamines, dopamine, and psycho-active drugs. Unfortunately, effects of trace amines on signaling of the two homologous β-adrenergic receptors 1 (ADRB1) and 2 (ADRB2) have not been clarified yet in detail. We, therefore, tested TAAR1 agonists TYR, PEA and OA regarding their effects on ADRB1/2 signaling by co-stimulation studies. Surprisingly, trace amines TYR and PEA are partial allosteric antagonists at ADRB1/2, whereas OA is a partial orthosteric ADRB2-antagonist and ADRB1-agonist. To specify molecular reasons for TAAR1 ligand promiscuity and for observed differences in signaling effects on particular aminergic receptors we compared TAAR, tyramine (TAR) octopamine (OAR), ADRB1/2 and dopamine receptors at the structural level. We found especially for TAAR1 that the remarkable ligand promiscuity is likely based on high amino acid similarity in the ligand-binding region compared with further aminergic receptors. On the other hand few TAAR specific properties in the ligand-binding site might determine differences in ligand-induced effects compared to ADRB1/2. Taken together, this study points to molecular details of TAAR1-ligand promiscuity and identified specific trace amines as allosteric or orthosteric ligands of particular β-adrenergic receptor subtypes.     

Differential activation of phospholipases during necrosis or apoptosis: A comparative study using tumor necrosis factor and anti-Fas antibodies

Phospholipases generate important secondary messengers in several cellular processes, including cell death. Tumor necrosis factor (TNF) can induce two distinct modes of cell death, viz. necrosis and apoptosis. Here we demonstrate that phospholipase D (PLD) and cytosolic phospholipase A₂ (cPLA₂) are differentially activated during TNF-induced necrosis or apoptosis. Moreover, a comparative study using TNF and anti-Fas antibodies as cell death stimuli showed that PLD and cPLA₂ are specifically activated by TNF. These results indicate that both the mode of cell death and the type of death stimulus determine the potential role of phospholipases as generators of secondary messengers. J. Cell. Biochem. 71:392–399, 1998. © 1998 Wiley-Liss, Inc.     

Differential modulation of cell cycle, apoptosis and PPARgamma2 gene expression by PPARgamma agonists ciglitazone and 9-hydroxyoctadecadienoic acid in monocytic cells

We sought to compare the effects of the thiazolidinedione ciglitazone with the endogenous fatty acid PPARgamma agonists 9- and 13-hydroxyoctadecadienoic acid (9- and 13-HODE), in U937 monocytic cells. Ciglitazone and 9-HODE inhibited cell proliferation and all three agonists increased cellular content of C18:0 fatty acids. Ciglitazone and 13-HODE resulted in an increased percentage of cells in S phase and ciglitazone reduced the percentage of cells in G2/M phase of cell cycle, whilst 9-HODE increased the percentage of cells in G0/1 and reduced the fraction in S and G2/M phases. 9-HODE selectively induced apoptosis in U937 cells, and increased PPARgamma2 gene expression. Induction of apoptosis by 9-HODE was not abrogated by the presence of the PPARgamma antagonist GW9662. Synthetic (TZD) and endogenous fatty acid ligands for PPARgamma, ciglitazone and 9- and 13-HODE, possess differential, ligand specific actions in monocytic cells to regulate cell cycle progression, apoptosis and PPARgamma2 gene expression.     

Monocytic cell necrosis is mediated by potassium depletion and caspase-like proteases

Apoptosis is aphysiological cell death that culminates in mitochondrial permeabilitytransition and the activation of caspases, a family of cysteineproteases. Necrosis, in contrast, is a pathological cell deathcharacterized by swelling of the cytoplasm and mitochondria and rapidplasma membrane disruption. Necrotic cell death has long been opposedto apoptosis, but it now appears that both pathways involvemitochondrial permeability transition, raising the question of whatmediates necrotic cell death. In this study, we investigated mechanismsthat promote necrosis induced by various stimuli(Clostridium difficile toxins,Staphylococcus aureus alpha toxin,ouabain, nigericin) in THP-1 cells, a human monocytic cell line, and in monocytes. All stimuli induced typical features of necrosis and triggered protease-mediated release of interleukin-1 (IL-1) andCD14 in both cell types. K⁺depletion was actively implicated in necrosis because substituting K⁺ forNa⁺ in the extracellular mediumprevented morphological features of necrosis and IL-1 release.N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone, a broad-spectrum caspase inhibitor, prevented morphological features of necrosis, plasma membrane destruction, loss ofmitochondrial membrane potential, IL-1 release, and CD14 sheddinginduced by all stimuli. Thus, in monocytic cells, necrosis is a celldeath pathway mediated by passiveK⁺ efflux and activation ofcaspase-like proteases.     

FK506 (tacrolimus) inhibits extravasation of lymphoid cells by abrogating VLA-4/VCAM-1 mediated transendothelial migration

Extravasation is a critical process for the physiological lymphocyte traffic as well as the hematogenous spread of malignant hemopoietic cells. Here we report that abrogation of calcineurin activity leads to in vitro transendothelial migration and in vivo infiltration of human lymphoma Nalm-6 cells, which are associated with the abrogation of the VLA-4/VCAM-1 mediated pathway. Rapamycin, which can antagonize FK506 but not CsA to inhibit calcineurin, abrogates FK-506 mediated but not CsA mediated inhibition of in vitro transendothelial migration. FK506 may exert its potent immunosuppressive action partly by inhibiting VLA-4/VCAM-1 mediated transendothelial migration or insinuation of lymphoid cells to tissues.     

Ca2+-dependent potentiation of the nonselective cation channel TRPV4 is mediated by a C-terminal calmodulin binding site

Most Ca2+-permeable ion channels are inhibited by increases in the intracellular Ca2+ concentration ([Ca2+]i), thus preventing potentially deleterious rises in [Ca2+]i. In this study, we demonstrate that currents through the osmo-, heat- and phorbol ester-sensitive, Ca2+-permeable nonselective cation channel TRPV4 are potentiated by intracellular Ca2+. Spontaneous TRPV4 currents and currents stimulated by hypotonic solutions or phorbol esters were reduced strongly at all potentials in the absence of extracellular Ca2+. The other permeant divalent cations Ba2+ and Sr2+ were less effective than Ca2+ in supporting channel activity. An intracellular site of Ca2+ action was supported by the parallel decrease in spontaneous currents and [Ca2+]i on removal of extracellular Ca2+ and the ability of Ca2+ release from intracellular stores to restore TRPV4 activity in the absence of extracellular Ca2+. During TRPV4 activation by hypotonic solutions or phorbol esters, Ca2+ entry through the channel increased the rate and extent of channel activation. Currents were also potentiated by ionomycin in the presence of extracellular Ca2+. Ca2+-dependent potentiation of TRPV4 was often followed by inhibition. By mutagenesis, we localized the structural determinant of Ca2+-dependent potentiation to an intracellular, C-terminal calmodulin binding domain. This domain binds calmodulin in a Ca2+-dependent manner. TRPV4 mutants that did not bind calmodulin lacked Ca2+-dependent potentiation. We conclude that TRPV4 activity is tightly controlled by intracellular Ca2+. Ca2+ entry increases both the rate and extent of channel activation by a calmodulin-dependent mechanism. Excessive increases in [Ca2+]i via TRPV4 are prevented by a Ca2+-dependent negative feedback mechanism.     

MEKK1-induced apoptosis is mediated by Smac/Diablo release from the mitochondria

During apoptotic stimulation, the serine threonine kinase, MEKK1, is cleaved into an activated 91 kDa kinase fragment. This cleavage is mediated by caspase 3 and leads to further caspase 3 activation and apoptosis. Forced expression of the 91 kDa kinase fragment induces apoptosis through changes in membrane potential of the mitochondria mediated by permeability transition pore opening. MEKK1 activation, however, fails to release cytochrome c from the mitochondria. Herein, we determined that overexpression of MEKK1 causes mitochondrial Smac/Diablo release correlating with MEKK1-induced apoptosis. Furthermore, using siRNA that lowers Smac/Diablo expression, MEKK1-induced apoptosis was significantly reduced. Mouse embryonic fibroblast cells lacking MEKK1 expression are also resistant to etoposide-induced mitochondrial Smac/Diablo release. In contrast, etoposide-induced mitochondrial cytochrome c release was not inhibited. MEKK1 also activates the MAP kinase JNK, but MEKK1-induced mitochondrial Smac/Diablo release and apoptosis are independent of MEKK1 mediated JNK activation. Taken together, release of Smac/Diablo from the mitochondria plays a role in MEKK1-induced apoptosis.     

Halogenation of 4-hydroxy/amino-3-methoxyphenyl acetamide TRPV1 agonists showed enhanced antagonism to capsaicin

As an extension of our analysis of the effect of halogenation on thiourea TRPV1 agonists, we have now modified selected 4-hydroxy(or 4-amino)-3-methoxyphenyl acetamide TRPV1 agonists by 5- or 6-halogenation on the aromatic A-region and evaluated them for potency for TRPV1 binding and regulation and for their pattern of agonism/antagonism (efficacy). Halogenation shifted the functional activity at TRPV1 toward antagonism with a greater extent of antagonism as the size of the halogen increased (I>Br>Cl), as previously observed for the thiourea series. The extent of antagonism was greater for halogenation at the 5-position than at the 6-position, in contrast to SAR for the thiourea series. In this series, compounds 55 and 75 showed the most potent antagonism, with K(i) (ant)=2.77 and 2.19nM, respectively, on rTRPV1 expressed in Chinese hamster ovary cells. The compounds were thus ca. 40-60-fold more potent than 6'-iodononivamide.     

Differential contribution of necrosis and apoptosis in myocardial ischemia-reperfusion injury

Necrosis and apoptosis differentially contribute to myocardial injury. Determination of the contribution of these processes in ischemia-reperfusion injury would allow for the preservation of myocardial tissue. Necrosis and apoptosis were investigated in Langendorff-perfused rabbit hearts (n = 47) subjected to 0 (Control group), 5 (GI-5), 10 (GI-10), 15 (GI-15), 20 (GI-20), 25 (GI-25), and 30 min (GI-30) of global ischemia (GI) and 120 min of reperfusion. Myocardial injury was determined by triphenyltetrazolium chloride (TTC) staining, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL), bax, bcl2, poly(ADP)ribose polymerase (PARP) cleavage, caspase-3, -8, and -9 cleavage and activity, Fas ligand (FasL), and Fas-activated death domain (FADD). The contribution of apoptosis was determined separately (n = 42) using irreversible caspase-3, -8, and -9 inhibitors. Left ventricular peak developed pressure (LVPDP) and systolic shortening (SS) were significantly decreased and infarct size and TUNEL-positive cells were significantly increased (P < 0.05 vs. Control group) at GI-20, GI-25, and GI-30. Proapoptotic bax, PARP cleavage, and caspase-3 and -9 cleavage and activity were apparent at GI-5 to GI-30. Fas, FADD, and caspase-8 cleavage and activity were unaltered. Irreversible inhibition of caspase-3 and -9 activity significantly decreased (P < 0.05) infarct size at GI-25 and GI-30 but had no effect on LVPDP or SS. Myocardial injury results from a significant increase in both necrosis and apoptosis (P < 0.05 vs. Control group) evident by TUNEL, TTC staining, and caspase activity at GI-20. Intrinsic proapoptotic activation is evident early during ischemia but does not significantly contribute to infarct size before GI-25. The contribution of necrosis to infarct size at GI-20, GI-25, and GI-30 is significantly greater than that of apoptosis. Apoptosis is significantly decreased by caspase inhibition during early reperfusion, but this protection does not improve immediate postischemic functional recovery.     

12-O-tetradecanoylphorbol-13-acetate-induced apoptosis is mediated by tumor necrosis factor alpha in human monocytic U937 cells.

12-O-tetradecanoylphorbol-13-acetate (TPA), a phorbol ester that is known as a tumor promoter, induces differentiation of myeloid cells and suppresses their proliferation. We studied the regulation of apoptosis by TPA in human monocytic cell line U937 cells that lack p53. Untreated U937 cells constitutively underwent apoptosis, and TPA enhanced apoptosis in these cells. Further studies showed that TPA increased production of tumor necrosis factor-alpha (TNFalpha) in U937 cells, and exogenously added TNFalpha induced apoptosis. Moreover, the induction of apoptosis by TPA was blocked by anti-TNFalpha antibody. Similar results were obtained in the myeloblastic cell line KY821 cells. We also found that the induction of apoptosis by TPA was increased in cells overexpressed with TNF receptor 1 but not in control cells. Furthermore, TPA failed to induce the production of TNFalpha and apoptosis in cells with either their protein kinase C or mitogen-activated protein kinase pathway blocked. Our results indicate that TPA induces apoptosis, at least in part, through a pathway that requires endogenous production of TNFalpha in U937 cells. Our data also suggest that the induction of apoptosis by TPA occurs through activation of protein kinase C and mitogen-activated protein kinase and TNFalpha is an autocrine-stimulating factor for the induction of apoptosis in these cells.     

Execution of macrophage apoptosis by PE_PGRS33 of Mycobacterium tuberculosis is mediated by Toll-like receptor 2-dependent release of tumor necrosis factor-alpha

Combating tuberculosis requires a detailed understanding of how mycobacterial effectors modulate the host immune response. The role of the multigene PE family of proteins unique to mycobacteria in the pathogenesis of tuberculosis is still poorly understood, although certain PE_PGRS genes have been linked to virulence. Tumor necrosis factor-alpha (TNF-alpha) is essential for successfully combating tuberculosis. In this study we provide evidence that PE_PGRS33, a surface exposed protein, elicits TNF-alpha release from macrophages in a TLR2 (Toll-like receptor 2)-dependent manner. ASK1 (apoptosis signal-regulating kinase 1) is activated downstream of TLR2. ASK1 activates the MAPKs p38 and JNK. PE_PGRS33-induced signaling leads to enhanced expression of TNF-alpha and TNF receptor I (TNFRI) genes. Mycobacterium smegmatis expressing PE_ PGRS33 elicits the same effects as purified PE_PGRS33. TNF-alpha release occurs even when internalization of the bacteria is blocked by cytochalasin D, suggesting that interaction of PE_ PGRS33 with TLR2 is sufficient to trigger the effects described. Release of TNF-alpha plays the determining role in triggering apoptosis in macrophages challenged with PE_PGRS33. The death receptor-dependent signals are amplified through classical caspase 8-dependent mitochondrial release of cytochrome c, leading to the activation of caspases 9 and 3. An important aspect of our findings is that deletions within the PGRS domain (simulating those occurring in clinical strains) attenuate the TNF-alpha-inducing ability of PE_PGRS33. These results provide the first evidence that variations in the polymorphic repeats of the PGRS domain modulate the innate immune response.     

Apoptosis induction and modulation of P-glycoprotein mediated multidrug resistance by new macrocyclic lathyrane-type diterpenoids

The macrocyclic lathyrane diterpenes, latilagascenes D-F (1-3) and jolkinol B (4), were isolated from the methanol extract of Euphorbia lagascae, and evaluated for multidrug resistance reversing activity on mouse lymphoma cells. All compounds displayed very strong activity compared with that of the positive control, verapamil. The structure-activity relationship is discussed. The evaluation of compounds 1 and 4, and of latigascenes A-C (5-7), isolated from the same species, as apoptosis-inducers was also carried out. Compound 1 was the most active. Furthermore, in the model of combination chemotherapy, the interaction between the doxorubicine and latilagascene B (6) was studied in vitro, on human MDR1 gene transfected mouse lymphoma cells, showing that the type of interaction was synergistic. Latilagascenes D-F (1-3) are new compounds whose structures were established on the basis of spectroscopic methods, including 2D NMR experiments (COSY, HMQC, HMBC and NOESY).