Neutral sphingomyelinase-2 mediates growth arrest by retinoic acid through modulation of ribosomal S6 kinase

All-trans-retinoic acid (ATRA) induces growth arrest of many cell types. Previous studies have reported that ATRA can modulate cellular sphingolipids, but the role of sphingolipids in the ATRA response is not clear. Using MCF-7 cells as a model system, we show that ATRA stimulates an increase in ceramide levels followed by G(0)/G(1) growth arrest. Notably, induction of nSMase2 was the primary effect of ATRA on the sphingolipid network and was both time- and dose-dependent. Importantly, pretreatment with nSMase2 siRNA significantly inhibited ATRA effects on ceramide levels and growth arrest. In contrast, nSMase2 overexpression was sufficient to increase ceramide levels and induce G(0)/G(1) growth arrest of asynchronous MCF-7 cells. Surprisingly, neither ATRA stimulation nor nSMase2 overexpression had significant effects on classical cell cycle regulators such as p21/WAF1 or retinoblastoma. In contrast, ATRA suppressed phosphorylation of ribosomal S6 kinase (S6K) and its downstream targets S6 and eIF4B. Importantly, these effects were significantly inhibited by nSMase2 siRNA. Reciprocally, nSMase2 overexpression was sufficient to suppress S6K phosphorylation and signaling. Notably, neither ATRA effects nor nSMase2 effects on S6K phosphorylation required the ceramide-activated protein phosphatase PP2A, previously identified as important for S6K regulation. Finally, nSMase2 overexpression was sufficient to decrease translation as measured by methionine incorporation and analysis of polyribosome profiles. Taken together, these results implicate nSMase2 as a major component of ATRA-induced growth arrest of MCF-7 cells and identify S6K as a novel downstream target of nSMase2.     

EGF receptor exposed to oxidative stress acquires abnormal phosphorylation and aberrant activated conformation that impairs canonical dimerization

Crystallographic studies have offered understanding of how receptor tyrosine kinases from the ErbB family are regulated by their growth factor ligands. A conformational change of the EGFR (ErbB1) was shown to occur upon ligand binding, where a solely ligand-mediated mode of dimerization/activation was documented. However, this dogma of dimerization/activation was revolutionized by the discovery of constitutively active ligand-independent EGFR mutants. In addition, other ligand-independent activation mechanisms may occur. We have shown that oxidative stress (ox-stress), induced by hydrogen peroxide or cigarette smoke, activates EGFR differently than its ligand, EGF, thereby inducing aberrant phosphorylation and impaired trafficking and degradation of EGFR. Here we demonstrate that ox-stress activation of EGFR is ligand-independent, does not induce "classical" receptor dimerization and is not inhibited by the tyrosine kinase inhibitor AG1478. Thus, an unprecedented, apparently activated, state is found for EGFR under ox-stress. Furthermore, this activation mechanism is temperature-dependent, suggesting the simultaneous involvement of membrane structure. We propose that ceramide increase under ox-stress disrupts cholesterol-enriched rafts leading to EGFR re-localization into the rigid, ceramide-enriched rafts. This increase in ceramide also supports EGFR aberrant trafficking to a peri-nuclear region. Therefore, the EGFR unprecedented and activated conformation could be sustained by simultaneous alterations in membrane structure under ox-stress.     

nSMase2 activation and trafficking are modulated by oxidative stress to induce apoptosis

We have previously shown that accumulation of ceramide, triggered by hydrogen peroxide (H(2)O(2)), induces apoptosis of human airway epithelial (HAE) cells. Under oxidant exposure, a lung sphingomyelinase (SMase) is activated and displays continued ceramide generation and pro-apoptotic signaling, thus leading to the pathological apoptosis that causes lung injury. In a search for a specific SMase that is modulated by oxidative stress, we recently cloned nSMase2 from monkey lung tissue and HAE cells. Here, we show that this nSMase2 is up-regulated by an oxidant (H(2)O(2)) and is inhibited by an antioxidant (glutathione (GSH)). Moreover, nSMase2 subcellular localization is governed by oxidant exposure, which leads to its preferential trafficking to the plasma membrane, where it generates ceramide and induces apoptosis. On the other hand, exposure to GSH results in nSMase2 trafficking to the nucleus, where it neither generates ceramide nor induces apoptosis.     

Amyloid beta peptide increases DP5 expression via activation of neutral sphingomyelinase and JNK in oligodendrocytes

There is growing recognition that white matter pathology is a common feature in Alzheimer's disease. We have previously reported that the amyloid beta peptide (Abeta) induces apoptosis in oligodendrocytes (OLG), via activation of neutral sphingomyelinase (nSMase) and resultant generation of ceramide. In the current study, we report that both Abeta and ceramide increased expression of the proapoptotic protein DP5/Hrk (DP5), and release of cytochrome C from mitochondria to cytoplasm in OLGs. We provide evidence that the Jun N-terminal kinase (JNK) signaling pathway mediates Abeta- and ceramide-induced apoptosis: Both Abeta and ceramide activated JNK phosphorylation, and subsequent AP-1 DNA binding activity; JNK siRNA decreased AP-1 DNA binding, DP5 expression and reduced cell death. Furthermore, inhibition of nSMase attenuated Abeta-induced JNK phosphorylation, AP-1 DNA binding activity, DP5 expression, and cytochrome C release. Collectively, these results suggest that Abeta-induced apoptosis involves the sequential activation of nSMase with ceramide generation, JNK activation, AP-1 DNA binding, and DP5 expression.     

Bone Morphogenic Protein (BMP) Signaling Up-regulates Neutral Sphingomyelinase 2 to Suppress Chondrocyte Maturation via the Akt Protein Signaling Pathway as a Negative Feedback Mechanism

Although bone morphogenic protein (BMP) signaling promotes chondrogenesis, it is not clear whether BMP-induced chondrocyte maturation is cell-autonomously terminated. Loss of function of Smpd3 in mice results in an increase in mature hypertrophic chondrocytes. Here, we report that in chondrocytes the Runx2-dependent expression of Smpd3 was increased by BMP-2 stimulation. Neutral sphingomyelinase 2 (nSMase2), encoded by the Smpd3 gene, was detected both in prehypertrophic and hypertrophic chondrocytes of mouse embryo bone cartilage. An siRNA for Smpd3, as well as the nSMase inhibitor GW4869, significantly enhanced BMP-2-induced differentiation and maturation of chondrocytes. Conversely, overexpression of Smpd3 or C₂-ceramide, which mimics the function of nSMase2, inhibited chondrogenesis. Upon induction of Smpd3 siRNA or GW4869, phosphorylation of both Akt and S6 proteins was increased. The accelerated chondrogenesis induced by Smpd3 silencing was negated by application of the Akt inhibitor MK2206 or the mammalian target of rapamycin inhibitor rapamycin. Importantly, in mouse bone culture, GW4869 treatment significantly promoted BMP-2-induced hypertrophic maturation and calcification of chondrocytes, which subsequently was eliminated by C₂-ceramide. Smpd3 knockdown decreased the apoptosis of terminally matured ATDC5 chondrocytes, probably as a result of decreased ceramide production. In addition, we found that expression of hyaluronan synthase 2 (Has2) was elevated by a loss of Smpd3, which was restored by MK2206. Indeed, expression of Has2 protein decreased in nSMase2-positive hypertrophic chondrocytes in the bones of mouse embryos. Our data suggest that the Smpd3/nSMase2-ceramide-Akt signaling axis negatively regulates BMP-induced chondrocyte maturation and Has2 expression to control the rate of endochondral ossification as a negative feedback mechanism.     

Regulation of PTEN stability and activity by Plk3

By studying primary isogenic murine embryonic fibroblasts (MEFs), we have shown that PLK3 null MEFs contain a reduced level of phosphatase and tensin homolog (PTEN) and increased Akt1 activation coupled with decreased GSK3β activation under normoxia and hypoxia. Purified recombinant Plk3, but not a kinase-defective mutant, efficiently phosphorylates PTEN in vitro. Mass spectrometry identifies threonine 366 and serine 370 as two putative residues that are phosphorylated by Plk3. Immunoblotting using a phosphospecific antibody confirms these sites as Plk3 phosphorylation sites. Moreover, treatment of MEFs with LiCl, an inhibitor of GSK3β and CK2, only partially suppresses the phosphorylation, suggesting Plk3 as an additional kinase that phosphorylates these sites in vivo. Plk3-targeting mutants of PTEN are expressed at a reduced level in comparison with the wild-type counterpart, which is associated with an enhanced activity of PDK1, an upstream activator of Akt1. Furthermore, the reduced level of PTEN in PLK3 null MEFs is stabilized by treatment with MG132, a proteosome inhibitor. Combined, our study identifies Plk3 as a new player in the regulation of the PI3K/PDK1/Akt signaling axis by phosphorylation and stabilization of PTEN.     

Neutral sphingomyelinase 2 induces dopamine uptake through regulation of intracellular calcium

Ceramide serves as a second messenger produced from sphingomyelin by the activation of sphingomyelinase (SMase). Here, we suggest that neutral SMase 2 (nSMase2) may regulate dopamine (DA) uptake. nSMase2 siRNA-transfected PC12 cells showed lower levels of nSMase activity and ceramide than scramble siRNA-transfected and control cells. Interestingly, transfection of nSMase2 siRNA or pretreatment with the nSMase2-specific inhibitor GW4869 resulted in decreased DA uptake. Reciprocally, exposure of PC12 cells to cell-permeable C₆-ceramide induced a concentration-dependent increase in DA uptake. Removal of extracellular calcium by EGTA increased DA uptake in scramble-transfected and control cells, but not in nSMase2 siRNA-transfected or GW4869-pretreated cells. Moreover, siRNA-transfected cells showed higher levels of intracellular calcium than scramble cells, while C₆-ceramide treatment resulted in decreased intracellular calcium compared to vehicle treatment alone. Taken together, these data suggest that nSMase2 may increase DA uptake through inducing ceramide production and thereby decreasing intracellular calcium levels.     

Identification of novel anionic phospholipid binding domains in neutral sphingomyelinase 2 with selective binding preference

Sphingolipids such as ceramide are recognized as vital regulators of many biological processes. Neutral sphingomyelinase 2 (nSMase2) is one of the key enzymes regulating ceramide production. It was previously shown that the enzymatic activity of nSMase2 was dependent on anionic phospholipids (APLs). In this study, the structural requirements for APL-selective binding of nSMase2 were determined and characterized. Using lipid-protein overlay assays, nSMase2 interacted specifically and directly with several APLs, including phosphatidylserine and phosphatidic acid. Lipid-protein binding studies of deletion mutants identified two discrete APL binding domains in the N terminus of nSMase2. Further, mutagenesis experiments pinpointed the core sequences and major cationic amino acids in the domains that are necessary for the cooperative activation of nSMase2 by APLs. The first domain included the first amino-terminal hydrophobic segment and Arg-33, which were essential for nSMase2 to interact with APLs. The second binding domain was comprised of the second hydrophobic segment and Arg-92 and Arg-93. Moreover, mutation of one or both domains decreased APL binding and APL-dependent catalytic activity of nSMase2. Further, mutation of both domains in nSMase2 reduced its plasma membrane localization. Finally, these binding domains are also important for the capability of nSMase2 to rescue the defects of yeast lacking the nSMase homologue, ISC1. In conclusion, these data have identified the APL binding domains of nSMase2 for the first time. The analysis of interactions between nSMase2 and APLs will contribute to our understanding of signaling pathways mediated by sphingolipid metabolites.     

Neutral sphingomyelinase 2 is palmitoylated on multiple cysteine residues. Role of palmitoylation in subcellular localization

The neutral sphingomyelinases (nSMases) are considered major candidates for mediating the stress-induced production of ceramide. nSMase2, which has two hydrophobic segments near the NH(2)-terminal region, has been reported to be located at the plasma membrane and play important roles in ceramide-mediated signaling. In this study, we found that nSMase2 is palmitoylated on multiple cysteine residues via thioester bonds. Site-directed mutagenesis of cysteine residues to alanine indicated that two cysteine clusters of the enzyme are multiply palmitoylated; one cluster is located between the two hydrophobic segments, and the second one is located in the middle of the catalytic region of the protein. When overexpressed in the confluent phase of MCF-7 cells, wild-type nSMase2 was strictly localized in the plasma membranes, and the cysteine mutants of each palmitoylated cysteine cluster were seen not only at the plasma membrane but also in some punctate structures. Furthermore, mutation of all potential palmitoylation sites resulted in a dramatic reduction in the plasma membrane distribution and an increase in the punctate structures. The palmitoylation-deficient mutant was directed to lysosomes and rapidly degraded. Palmitoylation had no effect on enzyme activity but affected membrane-association properties of the protein. Finally, the catalytic region of nSMase2 where palmitoylation occurs was found to be localized at the inner leaflet of the plasma membrane. In summary, the results from this study reveal for the first time the palmitoylation of nSMase2 via thioester bonds and its importance in the subcellular localization and stability of this protein.     

Role for furin in tumor necrosis factor alpha-induced activation of the matrix metalloproteinase/sphingolipid mitogenic pathway

Neutral sphingomyelinase (nSMase), the initial enzyme of the sphingolipid signaling pathway, is thought to play a key role in cellular responses to tumor necrosis factor alpha (TNF-alpha), such as inflammation, proliferation, and apoptosis. The mechanism of TNF-alpha-induced nSMase activation is only partly understood. Using biochemical, molecular, and pharmacological approaches, we found that nSMase activation triggered by TNF-alpha is required for TNF-alpha-induced proliferation and in turn requires a proteolytic cascade involving furin, membrane type 1 matrix metalloproteinase (MT1-MMP), and MMP2, and leading finally to extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation and DNA synthesis, in smooth muscle cells (SMC) and fibroblasts. Pharmacological and molecular inhibitors of MMPs (batimastat), furin (alpha1-PDX inhibitor-transfected SMC), MT1-MMP (SMC overexpressing a catalytically inactive MT1-MMP), MMP2 (fibroblasts from MMP2(-/-) mice), and small interfering RNA (siRNA) strategies (siRNAs targeting furin, MT1-MMP, MMP2, and nSMase) resulted in near-complete inhibition of the activation of nSMase, sphingosine kinase-1, and ERK1/2 and of subsequent DNA synthesis. Exogenous MT1-MMP activated nSMase and SMC proliferation in normal but not in MMP2(-/-) fibroblasts, whereas exogenous MMP2 was active on both normal and MMP2(-/-) fibroblasts. Altogether these findings highlight a pivotal role for furin, MT1-MMP, and MMP2 in TNF-alpha-induced sphingolipid signaling, and they identify this system as a possible target to inhibit SMC proliferation in vascular diseases.     

BAD contributes to RAF-mediated proliferation and cooperates with B-RAF-V600E in cancer signaling

BAD (Bcl-2 antagonist of cell death) belongs to the proapoptotic BH3-only subfamily of Bcl-2 proteins. Physiological activity of BAD is highly controlled by phosphorylation. To further analyze the regulation of BAD function, we investigated the role of recently identified phosphorylation sites on BAD-mediated apoptosis. We found that in contrast to the N-terminal phosphorylation sites, the serines 124 and 134 act in an antiapoptotic manner because the replacement by alanine led to enhanced cell death. Our results further indicate that RAF kinases represent, besides PAK1, BAD serine 134 phosphorylating kinases. Importantly, in the presence of wild type BAD, co-expression of survival kinases, such as RAF and PAK1, leads to a strongly increased proliferation, whereas substitution of serine 134 by alanine abolishes this process. Furthermore, we identified BAD serine 134 to be strongly involved in survival signaling of B-RAF-V600E-containing tumor cells and found that phosphorylation of BAD at this residue is critical for efficient proliferation in these cells. Collectively, our findings provide new insights into the regulation of BAD function by phosphorylation and its role in cancer signaling.     

Phosphorylation of human high mobility group N1 protein by protein kinase CK2

High mobility group (HMG) N1 protein, formerly known as HMG 14, is a member of the chromosomal HMG protein family. Protein kinase CK2 was previously reported to be able to phosphorylate bovine HMGN1 in vitro; Ser89 and Ser99, corresponding to Ser88 and Ser98 in human HMGN1, were shown to be major and minor recognition sites, respectively. In this report, we employed mass spectrometry and examined both the extent and the sites of phosphorylation in HMGN1 protein catalyzed by recombinant human protein kinase CK2. We found that five serine residues, i.e., Ser6, Ser7, Ser85, Ser88, and Ser98, in HMGN1 can be phosphorylated by the kinase in vitro. All five sites were previously shown to be phosphorylated in MCF-7 human breast cancer cells in vivo. Among these five sites, Ser6, Ser7, and Ser85 were new sites of phosphorylation induced by protein kinase CK2 in vitro.     

Role for neutral sphingomyelinase-2 in tumor necrosis factor alpha-stimulated expression of vascular cell adhesion molecule-1 (VCAM) and intercellular adhesion molecule-1 (ICAM) in lung epithelial cells: p38 MAPK is an upstream regulator of nSMase2

Neutral sphingomyelinases (N-SMases) are major candidates for stress-induced ceramide production. However, there is little information on the physiological regulation and roles of the cloned N-SMase enzyme, nSMase2. In this study, nSMase2 was found to translocate acutely to the plasma membrane of A549 epithelial cells in response to tumor necrosis factor alpha (TNF-alpha) in a time- and dose-dependent manner. Additionally, TNF-alpha increased N-SMase activity rapidly and transiently both endogenously and in cells overexpressing nSMase2. Furthermore, the translocation of nSMase2 was regulated by p38-alpha MAPK, but not ERK or JNK, and the increase in endogenous N-SMase activity was abrogated by p38 MAPK inhibition. In addition, both p38-alpha MAPK and nSMase2 were implicated in the TNF-alpha-stimulated up-regulation of the adhesion proteins vascular cell adhesion molecule-1 (VCAM) and intercellular adhesion molecule-1 (ICAM), but this was largely independent of NF-kappaB activation. These data reveal p38 MAPK as an upstream regulator of nSMase2 and indicate a role for nSMase2 in pro-inflammatory responses induced by TNF-alpha as a regulator of adhesion proteins.     

p38 MAP Kinase and MAPKAP Kinases MK2/3 Cooperatively Phosphorylate Epithelial Keratins

The MAPK-activated protein kinases (MAPKAP kinases) MK2 and MK3 are directly activated via p38 MAPK phosphorylation, stabilize p38 by complex formation, and contribute to the stress response. The list of substrates of MK2/3 is increasing steadily. We applied a phosphoproteomics approach to compare protein phosphorylation in MK2/3-deficient cells rescued or not by ectopic expression of MK2. In addition to differences in phosphorylation of the known substrates of MK2, HSPB1 and Bag-2, we identified strong differences in phosphorylation of keratin 8 (K8). The phosphorylation of K8-Ser⁷³ is catalyzed directly by p38, which in turn shows MK2-dependent expression. Notably, analysis of small molecule p38 inhibitors on K8-Ser⁷³ phosphorylation also demonstrated reduced phosphorylations of keratins K18-Ser⁵² and K20-Ser¹³ but not of K8-Ser⁴³¹ or K18-Ser³³. Interestingly, K18-Ser⁵² and K20-Ser¹³ are not directly phosphorylated by p38 in vitro, but by MK2. Furthermore, anisomycin-stimulated phosphorylations of K20-Ser¹³ and K18-Ser⁵² are inhibited by small molecule inhibitors of both p38 and MK2. MK2 knockdown in HT29 cells leads to reduced K20-Ser¹³ phosphorylation, which further supports the notion that MK2 is responsible for K20 phosphorylation in vivo. Physiologic relevance of these findings was confirmed by differences of K20-Ser¹³ phosphorylation between the ileum of wild-type and MK2/3-deficient mice and by demonstrating p38- and MK2-dependent mucin secretion of HT29 cells. Therefore, MK2 and p38 MAPK function in concert to phosphorylate K8, K18, and K20 in intestinal epithelia.     

Dopamine transporter trafficking is regulated by neutral sphingomyelinase 2/ceramide kinase

Dopamine (DA) reuptake is the primary mechanism to terminate dopaminergic transmission in the synaptic cleft. The dopamine transporter (DAT) has an important role in the regulation of DA reuptake. This study provides anatomical and physiological evidence that DAT recycling is regulated by ceramide kinase via the sphingomyelin pathway. First, the results show that DAT and neutral sphingomyelinase 2 (nSMase2) were successfully co-precipitated from striatal samples and were colocalized in the mouse striatum or PC12 cells. We also identified a protein-protein interaction between nSMase2 and DAT through in situ proximity ligation assay experiments in the mouse striatum. Second, dopamine (DA) stimulated the formation of ceramide and increased nSMase activity in PC12 cells, while treatment with a cell-permeable ceramide-1-phosphate (C1P) increased DA uptake. Third, we used inhibitors and siRNA to inhibit nSMase2 and ceramide kinase and observed the effects on DAT recycling in PC12 cells. Treatment with ceramide kinase inhibitor K1, or nSMase inhibitor GW4869, decreased DA uptake in PC12 cells, although the application of FB₁, a ceramide synthase inhibitor, did not affect DA uptake. Transfection of nSMase2 and CERK siRNA decreased DAT surface level in PC12 cells. These results suggested that SM-derived C1P affects cell surface levels of DAT.     

Analysis of membrane topology of neutral sphingomyelinase 2

Neutral sphingomyelinase 2 (nSMase2), which has two hydrophobic segments at its NH(2)-terminus, plays an important role in ceramide-mediated cell regulation. Here, we investigated the membrane topology of nSMase2. When a double-tagged nSMase2 at both the NH(2) and COOH termini, was overexpressed in MCF-7 cells, the signals from both tags were detected in the inner leaflet of the plasma membrane. Furthermore, insertion of a tag into the internal sequence and green fluorescent protein-fused deletion mutants revealed that the entire catalytic region of the protein was located on the cytosolic face of the membranes and each hydrophobic segment is integrated into the membranes, but unlikely to span the entire membrane. These results indicate the presence of the enzyme in the inner leaflet of plasma membrane.     

MAO-A-induced mitogenic signaling is mediated by reactive oxygen species, MMP-2, and the sphingolipid pathway

The degradation of biogenic amines by monoamine oxidase A (MAO-A) generates reactive oxygen species (ROS) which participate in serotonin and tyramine signaling. This study aimed to investigate the role of ROS in the mitogenic signaling activated during tyramine and serotonin oxidation by MAO-A in smooth muscle cells (SMC). Incubation of SMC with serotonin or tyramine induced intracellular ROS generation, and a signaling cascade involving metalloproteases and the neutral sphingomyelinase-2 (nSMase2, the initial step of the sphingolipid pathway), ERK1/2 phosphorylation, and DNA synthesis. Silencing MAO-A by siRNA, pharmacological MAO-A inhibitors (pargyline and Ro41-1049), and the antioxidant/ROS scavenger butylated hydroxytoluene (BHT) inhibited the signaling cascade, suggesting that ROS generated during tyramine oxidation by MAO-A are required. The MMP inhibitor Batimastat, MMP2-specific siRNA, and MMP2 deletion (MMP2(-/-) fibroblasts) blocked nSMase activation and SMC proliferation, suggesting a role for MMP2 in this signaling pathway. Silencing nSMase2 by siRNA did not inhibit ROS generation and MMP2 activation, but blocked SMC proliferation induced by tyramine, suggesting that nSMase2 is downstream MMP2. These findings demonstrate that H(2)O(2)-generated during tyramine oxidation by MAO-A triggers a stress-induced mitogenic signaling via the MMP2/sphingolipid pathway, which could participate in excessive remodeling and alteration of the vascular wall.     

The N-terminal region of twitchin binds thick and thin contractile filaments: redundant mechanisms of catch force maintenance

Catch force maintenance in invertebrate smooth muscles is probably mediated by a force-bearing tether other than myosin cross-bridges between thick and thin filaments. The phosphorylation state of the mini-titin twitchin controls catch. The C-terminal phosphorylation site (D2) of twitchin with its flanking Ig domains forms a phosphorylation-sensitive complex with actin and myosin, suggesting that twitchin is the tether (Funabara, D., Osawa, R., Ueda, M., Kanoh, S., Hartshorne, D. J., and Watabe, S. (2009) J. Biol. Chem. 284, 18015-18020). Here we show that a region near the N terminus of twitchin also interacts with thick and thin filaments from Mytilus anterior byssus retractor muscles. Both a recombinant protein, including the D1 and DX phosphorylation sites with flanking 7th and 8th Ig domains, and a protein containing just the linker region bind to thin filaments with about a 1:1 mol ratio to actin and K(d) values of 1 and 15 μM, respectively. Both proteins show a decrease in binding when phosphorylated. The unphosphorylated proteins increase force in partially activated permeabilized muscles, suggesting that they are sufficient to tether thick and thin filaments. There are two sites of thin filament interaction in this region because both a 52-residue peptide surrounding the DX site and a 47-residue peptide surrounding the D1 site show phosphorylation-dependent binding to thin filaments. The peptides relax catch force, confirming the region's central role in the mechanism of catch. The multiple sites of thin filament interaction in the N terminus of twitchin in addition to those in the C terminus provide an especially secure and redundant mechanical link between thick and thin filaments in catch.     

Reactive nitrogen and oxygen species activate different sphingomyelinases to induce apoptosis in airway epithelial cells

Airway epithelial cells are constantly exposed to environmental insults such as air pollution or tobacco smoke that may contain high levels of reactive nitrogen and reactive oxygen species. Previous work from our laboratory demonstrated that the reactive oxygen species (ROS), hydrogen peroxide (H(2)O(2)), specifically activates neutral sphingomyelinase 2 (nSMase2) to generate ceramide and induce apoptosis in airway epithelial cells. In the current study we examine the biological consequence of exposure of human airway epithelial (HAE) cells to reactive nitrogen species (RNS). Similar to ROS, we hypothesized that RNS may modulate ceramide levels in HAE cells and induce apoptosis. We found that nitric oxide (NO) exposure via the NO donor papa-NONOate, failed to induce apoptosis in HAE cells. However, when papa-NONOate was combined with a superoxide anion donor (DMNQ) to generate peroxynitrite (ONOO(-)), apoptosis was observed. Similarly pure ONOO(-)-induced apoptosis, and ONOO(-)-induced apoptosis was associated with an increase in cellular ceramide levels. Pretreatment with the antioxidant glutathione did not prevent ONOO(-)-induced apoptosis, but did prevent H(2)O(2)-induced apoptosis. Analysis of the ceramide generating enzymes revealed a differential response by the oxidants. We confirmed our findings that H(2)O(2) specifically activated a neutral sphingomyelinase (nSMase2). However, ONOO(-) exposure did not affect neutral sphingomyelinase activity; rather, ONOO(-) specifically activated an acidic sphingomyelinase (aSMase). The specificity of each enzyme was confirmed using siRNA to knockdown both nSMase2 and aSMase. Silencing nSMase2 prevented H(2)O(2)-induced apoptosis, but had no effect on ONOO(-)-induced apoptosis. On the other hand, silencing of aSMase markedly impaired ONOO(-)-induced apoptosis, but did not affect H(2)O(2)-induced apoptosis. These findings support our hypothesis that ROS and RNS modulate ceramide levels to induce apoptosis in HAE cells. However, we found that different oxidants modulate different enzymes of the ceramide generating machinery to induce apoptosis in airway epithelial cells. These findings add to the complexity of how oxidative stress promotes lung cell injury.