Supandi Winoto-Morbach

PAF-mediated pulmonary edema: a new role for acid sphingomyelinase and ceramide

Platelet-activating factor (PAF) induces pulmonary edema and has a key role in acute lung injury (ALI). Here we show that PAF induces pulmonary edema through two mechanisms: acid sphingomyelinase (ASM)-dependent production of ceramide, and activation of the cyclooxygenase pathway. Agents that interfere with PAF-induced ceramide synthesis, such as steroids or the xanthogenate D609, attenuate pulmonary edema formation induced by PAF, endotoxin or acid instillation. Our results identify acid sphingomyelinase and ceramide as possible therapeutic targets in acute lung injury.

Ceramide mediates caspase-independent programmed cell death

Although numerous studies have implicated the sphingolipid ceramide in the induction of cell death, a causative function of ceramide in caspase‐dependent apoptosis remains a highly debated issue. Here, we show that ceramide is a key mediator of a distinct route to programmed cell death (PCD), i.e., caspase‐independent PCD. Under conditions where apoptosis is either not initiated or actively inhibited, TNF induces caspase‐independent PCD in L929 fibrosarcoma cells, NIH3T3 fibroblasts, human leukemic Jurkat T cells, and lung fibroblasts by increasing intracellular ceramide levels prior to the onset of cell death. Survival is significantly enhanced when ceramide accumulation is prevented, as demonstrated in fibroblasts genetically deficient for acid sphingomyelinase, in L929 cells overexpressing acid ceramidase, by pharmacological intervention, or by RNA interference. Jurkat cells deficient for receptor‐interacting protein 1 (RIP1) do not accumulate ceramide and therefore are fully resistant to caspase‐independent PCD whereas Jurkat cells overexpressing the mitochondrial protein Bcl‐2 are partially protected, implicating RIP1 and mitochondria as components of the ceramide death pathway. Our data point to a role of caspases (but not cathepsins) in suppressing the ceramide death pathway under physiological conditions. Moreover, clonogenic survival of tumor cells is clearly reduced by induction of the ceramide death pathway, promising additional options for the development of novel tumor therapies.—Thon, L., Möhlig, H., Mathieu, S., Lange, R., Bulanova, E., Winoto‐Morbach, S., Schütze, S., Bulfone‐Paus, S., Adam, D. Ceramide mediates caspase‐independent programmed cell death. FASEB J. 19, 1945–1956 (2005)

Cathepsin D is involved in the regulation of transglutaminase 1 and epidermal differentiation

We previously demonstrated that the aspartate protease cathepsin D is activated by ceramide derived from acid sphingomyelinase. Increased expression of cathepsin D in the skin has been reported in wound healing, psoriasis and skin tumors. We explored specific functions of cathepsin D during epidermal differentiation. Protein expression and enzymatic activity of cathepsin D increased in differentiated keratinocytes in both stratified organotypic cultures and in mouse skin during epidermal barrier repair. Treatment of cultured keratinocytes with exogenous cathepsin D increased the activity of transglutaminase 1, known to cross-link the cornified envelope proteins involucrin and loricrin during epidermal differentiation. Inhibition of cathepsin D by pepstatin A suppressed the activity of transglutaminase 1. Cathepsin D-deficient mice revealed reduced transglutaminase 1 activity and reduced protein levels of the cornified envelope proteins involucrin and loricrin. Also, amount and distribution of cornified envelope proteins involucrin, loricrin, filaggrin, and of the keratins K1 and K5 were significantly altered in cathepsin D-deficient mice. Stratum corneum morphology in cathepsin D-deficient mice was impaired, with increased numbers of corneocyte layers and faint staining of the cornified envelope only, which is similar to the human skin disease lamellar ichthyosis. Our findings suggest a functional link between cathepsin D activation, transglutaminase 1 activity and protein expression of cornified envelope proteins during epidermal differentiation.

Ceramide as an activator lipid of cathepsin D.

We have identified the aspartic protease cathepsin D as a novel intracellular target protein for the lipid second messenger ceramide. Ceramide specifically binds to and induces CTSD proteolytic activity. A-SMase deficient cells derived from Niemann-Pick patients show decreased CTSD activity that was reconstituted by transfection with A-SMase cDNA. Ceramide accumulation in cells derived from A-ceramidase defective Farber patients correlates with enhanced CTSD activity. These findings suggest that A-SMase-derived ceramide targets endolysosomal CTSD.

Acid and neutral sphingomyelinase, ceramide synthase, and acid ceramidase activities in cutaneous aging.

Abstract:  In aged skin, decreased levels of stratum corneum ceramides have been described. Epidermal ceramides are generated by sphingomyelin hydrolysis or synthesis from sphingosin and fatty acids and are degraded by ceramidase. We recently showed that epidermal acid sphingomyelinase (A‐SMase) generates ceramides with structural function in the stratum corneum lipid bilayers, which provide for the permeability barrier function of the skin. Here, we examined the activities of epidermal A‐SMase, ceramide synthase, and ceramidase in chronologically aged versus young hairless mouse skin. We found reduced A‐SMase and ceramide synthase activities in the epidermis of aged mice. However, studies on enzyme localization revealed unchanged, ongoing high A‐SMase activity in the outer epidermis, which correlated with reported normal barrier function found in aged skin under basal conditions. Reduced A‐SMase and ceramide synthase activity was noted in the inner epidermis, correlating with reduced capacity for permeability barrier repair in aging. Ceramidase activity was not age dependent. In summary, we found reduced activities of ceramide‐generating SMase and ceramide synthase in the inner epidermis of aged skin, explaining its reduced capacity in barrier repair. In contrast, A‐SMase activity in the outer epidermis was unchanged, indicating that this enzyme is crucially involved in basal permeability barrier homeostasis.

Caspase‐8 and caspase‐7 sequentially mediate proteolytic activation of acid sphingomyelinase in TNF‐R1 receptosomes

We previously demonstrated that tumour necrosis factor (TNF)‐induced ceramide production by endosomal acid sphingomyelinase (A‐SMase) couples to apoptosis signalling via activation of cathepsin D and cleavage of Bid, resulting in caspase‐9 and caspase‐3 activation. The mechanism of TNF‐mediated A‐SMase activation within the endolysosomal compartment is poorly defined. Here, we show that TNF‐induced A‐SMase activation depends on functional caspase‐8 and caspase‐7 expression. The active forms of all three enzymes, caspase‐8, caspase‐7 and A‐SMase, but not caspase‐3, colocalize in internalized TNF receptosomes. While caspase‐8 and caspase‐3 are unable to induce activation of purified pro‐A‐SMase, we found that caspase‐7 mediates A‐SMase activation by direct interaction resulting in proteolytic cleavage of the 72‐kDa pro‐A‐SMase zymogen at the non‐canonical cleavage site after aspartate 253, generating an active 57 kDa A‐SMase molecule. Caspase‐7 down modulation revealed the functional link between caspase‐7 and A‐SMase, confirming proteolytic cleavage as one further mode of A‐SMase activation. Our data suggest a signalling cascade within TNF receptosomes involving sequential activation of caspase‐8 and caspase‐7 for induction of A‐SMase activation by proteolytic cleavage of pro‐A‐SMase.

The Polycomb group protein EED couples TNF receptor 1 to neutral sphingomyelinase

The phospholipase neutral sphingomyelinase (N-SMase) has been recognized as a major mediator of processes such as inflammation, development and growth, differentiation and death of cells, as well as in diseases such as Alzheimer’s, atherosclerosis, heart failure, ischemia/reperfusion damage, or combined pituitary hormone deficiency. Although activation of N-SMase by the proinflammatory cytokine TNF was described almost two decades ago, the underlying signaling pathway is unresolved. Here, we identify the Polycomb group protein EED (embryonic ectodermal development) as an interaction partner of nSMase2. In yeast, the N terminus of EED binds to the catalytic domain of nSMase2 as well as to RACK1, a protein that modulates the activation of nSMase2 by TNF in concert with the TNF receptor 1 (TNF-R1)-associated protein FAN. In mammalian cells, TNF causes endogenous EED to translocate from the nucleus and to colocalize and physically interact with both endogenous nSMase2 and RACK1. As a consequence, EED and nSMase2 are recruited to the TNF-R1•FAN•RACK1-complex in a timeframe concurrent with activation of nSMase2. After knockdown of EED by RNA interference, the TNF-dependent activation of nSMase2 is completely abrogated, identifying EED as a protein that both physically and functionally couples TNF-R1 to nSMase2, and which therefore represents the “missing link” that completes one of the last unresolved signaling pathways of TNF-R1.

TRAIL-induced programmed necrosis as a novel approach to eliminate tumor cells

BackgroundThe cytokine TRAIL represents one of the most promising candidates for the apoptotic elimination of tumor cells, either alone or in combination therapies. However, its efficacy is often limited by intrinsic or acquired resistance of tumor cells to apoptosis. Programmed necrosis is an alternative, molecularly distinct mode of programmed cell death that is elicited by TRAIL under conditions when the classical apoptosis machinery fails or is actively inhibited. The potential of TRAIL-induced programmed necrosis in tumor therapy is, however, almost completely uncharacterized. We therefore investigated its impact on a panel of tumor cell lines of wide-ranging origin.MethodsCell death/viability was measured by flow cytometry/determination of intracellular ATP levels/crystal violet staining. Cell surface expression of TRAIL receptors was detected by flow cytometry, expression of proteins by Western blot. Ceramide levels were quantified by high-performance thin layer chromatography and densitometric analysis, clonogenic survival of cells was determined by crystal violet staining or by soft agarose cloning.ResultsTRAIL-induced programmed necrosis killed eight out of 14 tumor cell lines. Clonogenic survival was reduced in all sensitive and even one resistant cell lines tested. TRAIL synergized with chemotherapeutics in killing tumor cell lines by programmed necrosis, enhancing their effect in eight out of 10 tested tumor cell lines and in 41 out of 80 chemotherapeutic/TRAIL combinations. Susceptibility/resistance of the investigated tumor cell lines to programmed necrosis seems to primarily depend on expression of the pro-necrotic kinase RIPK3 rather than the related kinase RIPK1 or cell surface expression of TRAIL receptors. Furthermore, interference with production of the lipid ceramide protected all tested tumor cell lines.ConclusionsOur study provides evidence that TRAIL-induced programmed necrosis represents a feasible approach for the elimination of tumor cells, and that this treatment may represent a promising new option for the future development of combination therapies. Our data also suggest that RIPK3 expression may serve as a potential predictive marker for the sensitivity of tumor cells to programmed necrosis and extend the previously established role of ceramide as a key mediator of death receptor-induced programmed necrosis (and thus as a potential target for future therapies) also to the tumor cell lines examined here.

Adhesion of immunomagnetic particles targeted to antigens and cytokine receptors on tumor cells determined by magnetophoresis

Abstract The adhesion of immunomagnetic particles to surfaces of target cells based on ligand–receptor interactions was evaluated by counting the number of magnetically labeled cells using magnetophoresis and analyzed by the Langmuir adsorption theory. As few as 200 tumor necrosis factor (TNF) receptor molecules per cell could be detected with streptavidin-coated magnetic particles coupled to biotinylated TNF as ligand.

Selective NF-κB inhibition, but not dexamethasone, decreases acute lung injury in a newborn piglet airway inflammation model

Acute respiratory failure in neonates (e.g. ARDS, meconium aspiration pneumonitis, pneumonia) is characterized by an excessive inflammatory response, governing the migration of polymorpho-nuclear leukocytes (PMNLs) into lung tissue and causing consecutive impairment of gas exchange and lung function. Critical to this inflammatory response is the activation of nuclear factor-kappaB (NF-kappaB) that is required for transcription of the genes for many pro-inflammatory mediators. We asked whether the inhibition of NF-kappaB activity using either a selective inhibitor (IKK-NBD peptide) or dexamethasone would be more effective in decreasing NF-kappaB activity and chemokine expression in pulmonary cells. Changes in lung function were repeatedly assessed for 24h following induction of acute respiratory failure and therapeutic intervention. We conducted a randomized, controlled, prospective animal study with mechanically ventilated newborn piglets which underwent repeated airway lavage (20+/-2 [SEM]) to remove surfactant and to induce lung inflammation. Admixed to 100 mg kg(-1) surfactant, piglets then received either IKK-NBD peptide (S+IKK), a selective inhibitor of NF-kappaB activation, its control peptide without intrinsic activity, dexamethasone (S+Dexa), its solvent aqua, or an air bolus only (all groups n=8). After 24h of mechanical ventilation, the following differences were measured: PaO(2)/FiO(2) (S+IKK 230+/-9 mm Hg vs. S+Dexa 188+/-14, p<0.05); ventilation efficiency index (0.18+/-0.01 [3800/(PIP-PEEP)(*)f(*)PaCO(2)] vs. 0.14+/-0.01, p<0.05); extravascular lung water (24+/-1 ml kg(-1) vs. 29+/-2, p<0.05); PMNL in BAL fluid (112+/-21 cells microl(-1) vs. 208+/-34, p<0.05), IL-8 (351+/-117 pg ml(-1) vs. 491+/-144, p=ns) and leukotriene B(4) (23+/-7 pg ml(-1) vs. 71+/-11, p<0.01) in BAL fluid. NF-kappaB activity in the nucleus of pulmonary cells differed by 32+/-5% vs. 55+/-3, p<0.001. Differences between these two intervention groups were more pronounced in the second half of the observation period (hours 12-24). At 24h of mechanical ventilation, inhibition of NF-kappaB activity by IKK-NBD peptide admixed to surfactant as a carrier caused improved gas exchange, lung function and reduced pulmonary inflammation, as evidenced by reduction in PMNL migration into lung tissue due to reduced nuclear NF-kappaB activity. We conclude that IKK-NBD admixture to surfactant in acute neonatal respiratory failure is superior to dexamethasone administration within the first 24h.