RhoA Activation Sensitizes Cells to Proteotoxic Stimuli by Abrogating the HSF1-Dependent Heat Shock Response.

BACKGROUND: The heat shock response (HSR) is an ancient and highly conserved program of stress-induced gene expression, aimed at reestablishing protein homeostasis to preserve cellular fitness. Cells that fail to activate or maintain this protective response are hypersensitive to proteotoxic stress. The HSR is mediated by the heat shock transcription factor 1 (HSF1), which binds to conserved heat shock elements (HSE) in the promoter region of heat shock genes, resulting in the expression of heat shock proteins (HSP). Recently, we observed that hyperactivation of RhoA conditions cardiomyocytes for the cardiac arrhythmia atrial fibrillation. Also, the HSR is annihilated in atrial fibrillation, and induction of HSR mitigates sensitization of cells to this disease. Therefore, we hypothesized active RhoA to suppress the HSR resulting in sensitization of cells for proteotoxic stimuli. METHODS AND RESULTS: Stimulation of RhoA activity significantly suppressed the proteotoxic stress-induced HSR in HL-1 atrial cardiomyocytes as determined with a luciferase reporter construct driven by the HSF1 regulated human HSP70 (HSPA1A) promoter and HSP protein expression by Western Blot analysis. Inversely, RhoA inhibition boosted the proteotoxic stress-induced HSR. While active RhoA did not preclude HSF1 nuclear accumulation, phosphorylation, acetylation, or sumoylation, it did impair binding of HSF1 to the hsp genes promoter element HSE. Impaired binding results in suppression of HSP expression and sensitized cells to proteotoxic stress. CONCLUSION: These results reveal that active RhoA negatively regulates the HSR via attenuation of the HSF1-HSE binding and thus may play a role in sensitizing cells to proteotoxic stimuli.

Extensive sphingolipid depletion does not affect lipid raft integrity or lipid raft localization and efflux function of the ABC transporter MRP1.

We show that highly efficient depletion of sphingolipids in two different cell lines does not abrogate the ability to isolate Lubrol-based DRMs (detergent-resistant membranes) or detergent-free lipid rafts from these cells. Compared with control, DRM/detergent-free lipid raft fractions contain equal amounts of protein, cholesterol and phospholipid, whereas the classical DRM/lipid raft markers Src, caveolin-1 and flotillin display the same gradient distribution. DRMs/detergent-free lipid rafts themselves are severely depleted of sphingolipids. The fatty acid profile of the remaining sphingolipids as well as that of the glycerophospholipids shows several differences compared with control, most prominently an increase in highly saturated C(16) species. The glycerophospholipid headgroup composition is unchanged in sphingolipid-depleted cells and cell-derived detergent-free lipid rafts. Sphingolipid depletion does not alter the localization of MRP1 (multidrug-resistance-related protein 1) in DRMs/detergent-free lipid rafts or MRP1-mediated efflux of carboxyfluorescein. We conclude that extensive sphingolipid depletion does not affect lipid raft integrity in two cell lines and does not affect the function of the lipid-raft-associated protein MRP1.

Calpain mediates cardiac troponin degradation and contractile dysfunction in atrial fibrillation.

The self-perpetuation of atrial fibrillation (AF) is associated with atrial remodeling, including the degradation of the myofibril structure (myolysis). Myolysis is related to AF-induced activation of cysteine proteases and underlies loss of contractile function. In this study, we investigated which proteases are involved in the degradation of myofibrillar proteins during AF and whether their inhibition leads to preservation of contractile function after AF. In tachypaced HL-1 cardiomyocytes and atrial tissue from AF and control patients, degradation of myofibrillar proteins troponin (cTn) T, I, C, human cTnT and actin was investigated by Western blotting, and contractile function was analyzed by cell-shortening measurements. The role of major proteases was determined by applying specific inhibitors. Tachypacing of HL-1 cardiomyocytes induced a gradual and significant degradation of cTns but not actin, and caused contractile dysfunction. Both were prevented by inhibition of calpain but not by inhibition of caspases or the proteasome. In patients with persistent AF, a significant degradation of cTnT, cTnI and cTnC was found compared to sinus rhythm or paroxysmal AF, which correlated significantly with both calpain activity and the amount of myolysis. Additionally, by utilizing tachypaced human cTnT-transfected HL-1 cardiomyocytes, we directly showed that the degradation of human cTnT was mediated by calpain and not by caspases or proteasome. Our results suggest that calpain inhibition may therefore represent a key target in combating AF-related structural and functional remodeling.

Heat shock proteins as molecular targets for intervention in atrial fibrillation.

Atrial fibrillation (AF) is the most common sustained clinical tachyarrhythmia. AF is a progressive condition as demonstrated by the finding that maintenance of normal rhythm and contractile function becomes more difficult the longer AF exists. AF causes cellular stress, which induces atrial remodelling, involving reduction in the expression of L-type Ca(2+) channels and structural changes (myolysis), finally resulting in contractile dysfunction. Heat shock proteins (HSPs) comprise a family of proteins involved in the protection against different forms of cellular stress. Their classical function is the prevention of toxic protein aggregation by binding to (partially) unfolded proteins. Recent investigations reveal that HSPs prevent atrial remodelling and attenuate the promotion of AF in both cellular and animal experimental models. Furthermore, studies in humans suggest a protective role for HSPs against progression from paroxysmal AF to chronic, persistent AF. Therefore, manipulation of the HSP system may offer novel therapeutic approaches for the prevention of atrial remodelling. Such approaches may contribute to the maintenance or restoration of tissue integrity and contractile function. Ultimately, this concept may offer an additional treatment strategy to delay progression towards chronic AF and/or improve the outcome of cardioversion.

Gangliosides do not affect ABC transporter function in human neuroblastoma cells.

Previous studies have indicated a role for glucosylceramide synthase (GCS) in multidrug resistance (MDR), either related to turnover of ceramide (Cer) or generation of gangliosides, which modulate apoptosis and/or the activity of ABC transporters. This study challenges the hypothesis that gangliosides modulate the activity of ABC transporters and was performed in two human neuroblastoma cell lines, expressing either functional P-glycoprotein (Pgp) or multidrug resistance-related protein 1 (MRP1). Two inhibitors of GCS, D,L-threo-1-phenyl-2-hexadecanoylamino-3-pyrrolidino-1-propanol (t-PPPP) and N-butyldeoxynojirimycin (NB-dNJ), very efficiently depleted ganglioside content in two human neuroblastoma cell lines. This was established by three different assays: equilibrium radiolabeling, cholera toxin binding, and mass analysis. Fluorescence-activated cell sorting (FACS) analysis showed that ganglioside depletion only slightly and in the opposite direction affected Pgp- and MRP1-mediated efflux activity. Moreover, both effects were marginal compared with those of well-established inhibitors of either MRP1 (i.e., MK571) or Pgp (i.e., GF120918). t-PPPP slightly enhanced cellular sensitivity to vincristine, as determined by 3-[4,5-dimethylthiazol-2-yl]2,5-diphenyl tetrazolium bromide analysis, in both neuroblastoma cell lines, whereas NB-dNJ was without effect. MRP1 expression and its localization in detergent-resistant membranes were not affected by ganglioside depletion. Together, these results show that gangliosides are not relevant to ABC transporter-mediated MDR in neuroblastoma cells.

PDMP sensitizes neuroblastoma to paclitaxel by inducing aberrant cell cycle progression leading to hyperploidy.

The sphingolipid ceramide has been recognized as an important mediator in the apoptotic machinery, and its efficient conversion to glucosylceramide has been associated with multidrug resistance. Therefore, inhibitors of glucosylceramide synthase are explored as tools for treatment of cancer. In this study, we used D,L-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol to sensitize Neuro-2a murine neuroblastoma cells to the microtubule-stabilizing agent paclitaxel. This treatment resulted in a synergistic inhibition of viable cell number increase, which was based on a novel mechanism: (a) After a transient mitotic arrest, cells proceeded through an aberrant cell cycle resulting in hyperploidy. Apoptosis also occurred but to a very limited extent. (b) Hyperploidy was not abrogated by blocking de novo sphingolipid biosynthesis using ISP-1, ruling out involvement of ceramide as a mediator. (c) Cyclin-dependent kinase 1 and 2 activities were synergistically decreased on treatment. In conclusion, instead of inducing apoptosis through ceramide accumulation, D,L-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol by itself affects cell cycle-related proteins in paclitaxel-arrested Neuro-2a cells resulting in aberrant cell cycle progression leading to hyperploidy.

Differential expression of sphingolipids in P-glycoprotein or multidrug resistance-related protein 1 expressing human neuroblastoma cell lines.

The sphingolipid composition and multidrug resistance status of three human neuroblastoma cell lines were established. SK-N-FI cells displayed high expression and functional (efflux) activity of P-glycoprotein, while multidrug resistance-related protein 1 was relatively abundant and most active in SK-N-AS cells. These two cell lines exhibited higher sphingolipid levels, compared to SK-N-DZ, which had the lowest activity of either ATP-binding cassette transporter protein. SK-N-DZ cells also differed in ganglioside composition with predominant expression of b-series gangliosides. In conclusion, these three neuroblastoma cell lines offer a good model system to study sphingolipid metabolism in relation to ATP-binding cassette transporter protein function.

Interaction between nitric oxide and subsets of human T lymphocytes with differences in glutathione metabolism.

Nitric oxide (NO) modulates human T-lymphocyte responses through several mechanisms. In the current study we show that interactions between NO and glutathione (GSH) metabolism are related to the selective persistent inhibition of interferon-gamma (IFN-gamma) production by NO, which we previously identified. T cells were exposed to NO using the NO-donor compound Spermine-nonoate (Sper) and activated using anti-CD3 plus anti-CD28 monoclonal antibodies. Persistent inhibition of IFN-gamma by Sper was prevented by addition of the GSH precursor l-cysteine, which inhibits Sper induced GSH depletion. Subsets of cells were either susceptible (GSH(low)) or resistant (GSH(high)) to NO-induced GSH depletion. The GSH(low) subset was characterized by enhanced numbers of CD4+ cells, reduced numbers of activated cells as characterized by CD25 and CD69, and reduced numbers of memory (CD45RO+) cells relative to the GSH(high) population. Rather than directly affecting susceptibility to NO, these surface markers reflected different expression patterns. Particularly, the GSHlow subset was further characterized by decreased activity of the GSH synthesis related enzymes multi-drug resistance related protein (MRP)-1 and gamma-glutamyltranspeptidase (gamma-GT). Blocking gamma-GT, using acivicin was shown to exacerbate NO-induced GSH depletion and NO-induced apoptosis. Since NO induced apoptosis selectively affects IFN-gamma production these findings implicate GSH metabolism in the modulation and maintenance of the T helper (Th)1/Th2 balance.

Sphingolipids in neuroblastoma: their role in drug resistance mechanisms.

Disseminated neuroblastoma usually calls for chemotherapy as the primary approach for treatment. Treatment failure is often attributable to drug resistance. This involves a variety of cellular mechanisms, including increased drug efflux through expression of ATP-binding cassette transporters (e.g., P-glycoprotein) and the inability of tumor cells to activate or propagate the apoptotic response. In recent years it has become apparent that sphingolipid metabolism and the generation of sphingolipid species, such as ceramide, also play a role in drug resistance. This may involve an autonomous mechanism, related to direct effects of sphingolipids on the apoptotic response, but also a subtle interplay between sphingolipids and ATP-binding cassette transporters. Here, we present an overview of the current understanding of the multiple levels at which sphingolipids function in drug resistance, with an emphasis on sphingolipid function in neuroblastoma and how modulation of sphingolipid metabolism may be used as a novel treatment paradigm.

The role of hydrophobic active-site residues in substrate specificity and acyl transfer activity of penicillin acylase.

Penicillin acylase of Escherichia coli catalyses the hydrolysis and synthesis of beta-lactam antibiotics. To study the role of hydrophobic residues in these reactions, we have mutated three active-site phenylalanines. Mutation of alphaF146, betaF24 and betaF57 to Tyr, Trp, Ala or Leu yielded mutants that were still capable of hydrolysing the chromogenic substrate 2-nitro-5-[(phenylacetyl)amino]-benzoic acid. Mutations on positions alphaF146 and betaF24 influenced both the hydrolytic and acyl transfer activity. This caused changes in the transferase/hydrolase ratios, ranging from a 40-fold decrease for alphaF146Y and alphaF146W to a threefold increase for alphaF146L and betaF24A, using 6-aminopenicillanic acid as the nucleophile. Further analysis of the betaF24A mutant showed that it had specificity constants (kcat/Km) for p-hydroxyphenylglycine methyl ester and phenylglycine methyl ester that were similar to the wild-type values, whereas the specificity constants for p-hydroxyphenylglycine amide and phenylglycine amide had decreased 10-fold, due to a decreased kcat value. A low amidase activity was also observed for the semisynthetic penicillins amoxicillin and ampicillin and the cephalosporins cefadroxil and cephalexin, for which the kcat values were fivefold to 10-fold lower than the wild-type values. The reduced specificity for the product and the high initial transferase/hydrolase ratio of betaF24A resulted in high yields in acyl transfer reactions.