Hiroyuki Kanoh

Molecular cloning of a second phospholipase B gene, caPLB2 from Candida albicans

Accumulating evidence suggests that phospholipase B, secreted by pathogenic fungi such as Candida albicans, Cryptococcus neoformans and Aspergillus fumigatus, functions as one of the virulence factors. In the present study, we have attempted to clone phospholipase B gene from C. albicans. By RT-PCR analysis with degenerate primers based on conserved regions of phospholipase B from Saccharomyces cerevisiae, Penicillium notatum and Torulaspora delbrueckii two similar but different cDNA fragments were obtained. One corresponded to the partial sequence of caPLB1, recently cloned phospholipase B gene from C. albicans by a different approach (Leidich et al.: J Biol Chem 1998; 273: 26078-86). The other fragments contained sequences similar to the corresponding sequences of phospholipase B from other fungi. The presence of two related genes was confirmed by Southern and Northern blot analyses. The full length of the second C. albicans phospholipase B gene (caPLB2) encoded a putative protein with 608 amino acids and contained a potential signal peptide sequence and a putative catalytic region, which are found in phospholipase B from other fungi. Consistent with the findings of caPLB1, caPLB2 also lacks a cluster of hydrophobic amino acids at the COOH-terminal, which may function as a signal of glycosylphosphatidylinositol anchor.

Interferon-γ Decreases Ceramides with Long-Chain Fatty Acids: Possible Involvement in Atopic Dermatitis and Psoriasis

Ceramide (CER) with long-chain fatty acids (FAs) in the human stratum corneum (SC) is important for the skin barrier functions. Changes in the CER profile have been associated with abnormal permeability of dermatoses such as atopic dermatitis (AD) and psoriasis. In addition, interferon-γ (IFN-γ) has been known to be abundant in both AD and psoriatic skin lesions. In this study, we aimed to identify the mechanism underlying the alteration of FA chain length of CERs in these diseases. Mass spectrometry analysis of CERs in the SC showed that the proportion of CERs with long-chain FAs was significantly lower in AD and psoriasis patients than in healthy controls, and this reduction was more pronounced in psoriasis than in AD. Using cultured human keratinocytes and epidermal sheets, we found that only IFN-γ among various cytokines decreased the mRNA expression of elongase of long-chain fatty acids (ELOVL) and ceramide synthase (CerS), enzymes involved in FA chain elongation. Furthermore, quantitative analysis showed that IFN-γ decreased the levels of CERs with long-chain FAs. These results suggest that IFN-γ decreases CERs with long-chain FAs through the downregulation of ELOVL and CerS and that this mechanism may be involved in the CER profile alteration observed in psoriasis and AD.

Activation of phospholipase D in rabbit neutrophils by fMet-Leu-Phe is mediated by a pertussis toxin-sensitive GTP-binding protein that may be distinct from a phospholipase C-regulating protein

Stimulation by N‐formyl‐Met‐Leu‐Phe (fMLP) of rabbit peritoneal neutrophils, in which phosphatidylcholine was preferentially labeled with l‐O‐[3H]octadecyl lyso platelet‐activating factor, activated phospholipase D, resulting in the formation of [3H]PA from [3H]PC. A direct activator of GTP‐binding proteins (G‐proteins), NaF, also stimulated [3H]PA formation. fMLP‐stimulated [3H]PA formation was inhibited by pertussis toxin (IAP) in a time‐ and dose‐dependent manner. IAP also inhibited fMLP‐stimulated IP3 formation, but the inhibition of IP3 formation was significantly greater than that of [3H]PA formation. These results indicate that activation of phospholipase D by fMLP in rabbit neutrophils is mediated by an IAP‐sensitive G‐protein that may be distinct from a phospholipase C‐regulating protein.

Activation and solubilization by triton X-100 of membrane-bound phospholipase D of rat brain

In the present study, we examined the ability of detergents to stimulate and solubilize phospholipase D (PLD) of a particulate fraction of rat brain. PLD activity was assayed by measuring the [3H]choline produced from the exogenous substrate dipalmitoyl phosphatidyl[3H]choline (dipalmitoyl [3H]PC). In the absence of detergents, PLD activity was not detectable. Of the detergents examined, Triton X-100 was found to markedly enhance PLD activity, whereas other detergents including sodium doexycholate, sodium cholate, CHAPS and Lubrol-PX caused only a small, if any increase in activity. Enhancement by Triton X-100 was maximal at 0.1–0.2% (w/v) and decreased at higher concentrations. The optimal pH was 7.1–7.3. Both Ca2+ and Mg2+ inhibited enzyme activity stimulated by Triton X-100 in a concentration-dependent manner. Triton X-100 effectively solubilized PLD from the particulate fraction of rat brain; more than 70% of the activity of the particulate fraction was extracted by 0.5–1.0% (w/v) Triton X-100. Furthermore, when the PLD activities in brains of three different species (rat, rabbit and bovine) were measured under optimal conditions, the activities were found to differ greatly. PLD activity was highest in rabbit brain, followed by rat and bovine brains; the activity in bovine brain was extremely low compared to the activities in rat and rabbit brains. We conclude that Triton X-100 is potentially useful for the purification of PLD and that rabbit and rat brains are the preferred sources.

Possible role of a septin, SEPT1, in spreading in squamous cell carcinoma DJM-1 cells.

Abstract We performed biochemical, histochemical and cell biological characterization of septins by focusing on SEPT1 in human skin tissues and a squamous cell carcinoma (SCC) cell line DJM-1. In immunoblotting, SEPT1, together with other septins, was detected in normal human epidermis, SCC and DJM-1. In immunohistochemical analyses, SEPT1 was detected diffusely in the cytoplasm of human epidermal cells and eccrine gland epithelial cells, and the protein level was increased in some skin tumors. In DJM-1 cells, SEPT1 together with other members of SEPT2-subgroup, SEPT4 and SEPT5, was enriched in lamellipodia and the localization was dependent on the cortical actin structure. SEPT1 distribution at lamellipodia was also observed in melanoma B16 cells. SEPT9, SEPT11 and SEPT14, in contrast, were localized along with microtubules in DJM-1 cells. In immunoprecipitation assays, SEPT1 and SEPT5 were found to form a complex in DJM-1 cells, whereas SEPT9, SEPT11 and SEPT14 formed a distinct complex with other septins including SEPT7, SEPT8 and SEPT10, in which SEPT5 was not included. When SEPT1 was silenced in DJM-1 cells, cell spreading was inhibited. These results suggest that SEPT1 may participate in cell-cell and/or cell-substrate interaction in DJM-1 and exert its function in a coordinated manner with other septins.

Pertussis toxin-insensitive G protein mediates carbachol activation of phospholipase D in rat pheochromocytoma PC12 cells.

Abstract: In the present study, an activation mechanism for phospholipase D (PLD) in [3H]palmitic acid‐labeled pheochromocytoma PC12 cells in response to carbachol (CCh) was investigated. PLD activity was assessed by measuring the formation of [3H]phosphatidylethanol ([3H]PEt), the specific marker of PLD activity, in the presence of 0.5% (vol/vol) ethanol. CCh caused a rapid accumulation of [3H]PEt, which reached a plateau within 1 min, in a concentration‐dependent manner. The [3H]PEt formation by CCh was completely antagonized by atropine, demonstrating that the CCh effect was mediated by the muscarinic acetylcholine receptor (mAChR). A tumor promoter, phorbol 12‐myristate 13‐acetate (PMA), also caused an increase in [3H]PEt content, which reached a plateau at 30–60 min after exposure, but an inactive phorbol ester, 4a‐phorbol 12,13‐didecanoate, did not. Although a protein kinase C (PKC) inhibitor, staurosporine (5 μM), blocked PMA‐induced [3H]PEt formation by 77%, it had no effect on the CCh‐induced formation. These results suggest that mAChR‐induced PLD activation is independent of PKC, whereas PLD activation by PMA is mediated by PKC. NaF, a common GTP‐binding protein (G protein) activator, and a stable analogue of GTP, guanosine 5′‐O‐(3‐thiotriphosphate) (OTPGmS), also stimulated [3H]PEt formation in intact and digitonin‐permeabilized cells, respectively. GTP, UTP, and CTP were without effect. Furthermore, guanosine 5′‐O‐(2‐thiodiphosphate) significantly inhibited CCh‐ and GTPΓS‐ induced [3H]PEt formation in permeabilized cells but did not inhibit the formation by PMA, and staurosporine (5 μM) had no effect on [3H]PEt formation by GTPγS. Pretreatment of cells with pertussis toxin (10–200 ng/ml) for 15 h failed to suppress CCh‐induced [3H]PEt formation, although the pertussis toxin‐sensitive G protein(s) in membranes was completely ADP‐ribosylated under the same conditions. From these results, we conclude that the mechanisms of PMA‐ and CCh‐stimulated PLD activation are different from each other and that CCh‐induced PLD activation is independent of PKC and mediated, at least in part, via a pertussis toxin‐insensitive G protein.

Molecular cloning of a gene encoding phospholipase D from the pathogenic and dimorphic fungus, Candida albicans.

A phospholipase D gene (CaPLD) has been cloned from the Candida albicans genomic DNA library. The CaPLD is a member of a highly conserved gene family of PLD and has the highest homology to Saccharomyces cerevisiae PLD (SPO14) with an overall homology of 42%. Phylogenetic analysis indicated that fungus PLDs including CaPLD composed one of the three clusters of PLD genes.

FTY720 and cisplatin synergistically induce the death of cisplatin-resistant melanoma cells through the downregulation of the PI3K pathway and the decrease in epidermal growth factor receptor expression

Sphingosine kinase (SK), a key enzyme in sphingosine-1-phosphate (S1P) synthesis, is known to be overexpressed in various types of cancer cells. The effects of anticancer agents on SK1/S1P signaling have not yet been fully assessed in melanoma cells. In the present study, we investigated the effects of the combination of FTY720, an S1P receptor antagonist, and cisplatin, a DNA-damaging agent, on the induction of the death of human melanoma cells, as well as the molecular mechanisms involved. The viability of various human melanoma cell lines was examined following treatment with anticancer drugs. The cisplatin-resistant SK-Mel-28 and cisplatin-sensitive A375 cell lines were selected for this analysis. Protein expression and apoptotic rates were evaluated by western blot analysis following treatment with cisplatin and/or FTY720. Following treatment with a combination of FTY720 and cisplatin, cell viability significantly decreased and the expression of apoptosis-associated cleaved poly(ADP-ribose) polymerase (PARP) was significantly higher in comparison to treatment with cisplatin alone in the SK-Mel-28 cells. In addition, the combination of FTY720 and cisplatin reduced the protein expression of SK1 and the phosphorylation levels of phosphoinositide 3-kinase (PI3K), Akt and mTOR in the SK-Mel-28 cells; the expression of epidermal growth factor receptor (EGFR) was also markedly reduced. These findings suggest that FTY720 and cisplatin synergistically induce cell death through the downregulation of the PI3K/Akt/mTOR pathway and the decrease in EGFR expression in SK-Mel-28 cells. Thus, the combination of FTY720 and cisplatin may have therapeutic potential for chemotherapy-resistant melanoma, and the effects are likely exerted through the downregulation of S1P signaling.

Implication of Ca2+‐Dependent Protein Tyrosine Phosphorylation in Carbachol‐Induced Phospholipase D Activation in Rat Pheochromocytoma PC12 Cells

Abstract: The mechanism for carbachol (CCh)‐induced phospholipase D (PLD) activation was investigated in [3H]palmitic acid‐labeled pheochromocytoma PC12 cells with respect to the involvement of protein tyrosine phosphorylation and Ca2+. PLD activity was assessed by measuring the formation of [3H]phosphatidylbutanol in the presence of 0.3% butanol. Pretreatment of cells with the tyrosine kinase inhibitors herbimycin A, genistein, and tyrphostin inhibited PLD activation by CCh. Western blot analysis revealed several apparent tyrosine‐phosphorylated protein bands (111, 91, 84, 74, 65–70, 44, and 42 kDa) in PC12 cells treated with CCh. Phosphorylation of the 111‐, 91‐, 84‐, and 65–70‐kDa proteins peaked within 1 min, and their time‐dependent changes seemingly correlated with that of PLD activation. Others (74, 44MAPK, and 42MAPK kDa) were phosphorylated rather slowly, and maximal tyrosine phosphorylation was observed at 2 min. Herbimycin A inhibited PLD activity and tyrosine phosphorylation of four proteins (111, 91, 84, and 65–70 kDa) in a preincubation time‐ and concentration‐dependent fashion. In Ca2+‐free buffer, CCh‐induced [3H]phosphatidylbutanol formation and protein tyrosine phosphorylation were abolished. A Ca2+ ionophore, A23187, caused PLD activation and tyrosine phosphorylation of four proteins of 111, 91, 84, and 65–70 kDa only in the presence of extracellular Ca2+. Extracellular Ca2+ dependency for CCh‐induced PLD activation was well correlated with that for tyrosine phosphorylation of the four proteins listed above, especially the 111‐kDa protein. These results suggest that Ca2+‐dependent protein tyrosine phosphorylation is closely implicated in CCh‐induced PLD activation in PC12 cells.

Cell biological characterization of a multidomain adaptor protein, ArgBP2, in epithelial NMuMG cells, and identification of a novel short isoform

ArgBP2 is a member of the SoHo (sorbin-homology) family of adaptor proteins believed to play roles in cell adhesion, cytoskeletal organization, and signaling. We show here a novel splicing isoform of ArgBP2, i.e., ArgBP2™, composed of only three SH3 (src-homology 3) domains and structurally similar to vinexinß. We then characterized the biochemical and cell biological properties of ArgBP2 to compare these with vinexin. Similar to vinexin, ArgBP2 was enriched at focal adhesions in REF52 fibroblast cells and induced anchorage-dependent extracellular signal-regulated kinase activation in NIH3T3 fibroblast cells. In epithelial NMuMG cells, immunofluorescence analyses revealed localization of ArgBP2 at tight junctions (TJs), whereas vinexin was distributed in cytoplasm as well as cell-cell boundaries. During TJ formation, recruitment of ZO-1 to TJs was followed by ArgBP2. Based on mutation analyses, a second SH3 domain was found to be important for ArgBP2 localization to the cell-cell contact sites. These data suggest some role of ArgBP2 in NMuMG cells at TJs that may be distinct from the function of vinexin.