Indications of Endocrine Disruptor Effects of JP-5 Jet Fuel Using a Rat-Model Reproductive Study and an In Vitro Human Hormone Receptor Assay.

Recent events concerning jet fuel contamination of drinking water have shown that we need a better understanding of the effects of ingested jet fuel. To this end, a reproductive study with ingested jet fuel in rats was undertaken with relatively high concentrations of Jet Propellant (JP)-5 along with a human estrogen receptor activation in vitro assay using JP-5, JP-8, and an alternative jet fuel derived from the camelina plant referred to as HydroRenewable Jet (HRJ) fuel, to help evaluate potential effects of ingested jet fuel. The results of the in vivo study provide evidence that JP-5 can act as an endocrine disruptor, with specific observations including altered hormone levels with JP-5 exposure (significantly lower estradiol levels in male rats and significantly increased Dehydroepiandrosterone levels in females), and a decreased male/female offspring ratio. The in vitro hormone receptor activation assay indicated that JP-5 and JP-8 are capable of upregulating human estrogen receptor (ER) activity, while HRJ was not active in the ER assay. The jet fuels were not able to activate androgen or glucocorticoid receptors in further in vitro assays. These results infer potential endocrine disruption associated with JP-5, with activation of the estrogen receptor as one potential mechanism of action.

Phospholipase D2 (PLD2) shortens the time required for myeloid leukemic cell differentiation: mechanism of action.

Cell differentiation is compromised in acute leukemias. We report that mammalian target of rapamycin (mTOR) and S6 kinase (S6K) are highly expressed in the undifferentiated promyelomonocytic leukemic HL-60 cell line, whereas PLD2 expression is minimal. The expression ratio of PLD2 to mTOR (or to S6K) is gradually inverted upon in vitro induction of differentiation toward the neutrophilic phenotype. We present three ways that profoundly affect the kinetics of differentiation as follows: (i) simultaneous overexpression of mTOR (or S6K), (ii) silencing of mTOR via dsRNA-mediated interference or inhibition with rapamycin, and (iii) PLD2 overexpression. The last two methods shortened the time required for differentiation. By determining how PLD2 participates in cell differentiation, we found that PLD2 interacts with and activates the oncogene Fes/Fps, a protein-tyrosine kinase known to be involved in myeloid cell development. Fes activity is elevated with PLD2 overexpression, phosphatidic acid or phosphatidylinositol bisphosphate. Co-immunoprecipitation indicates a close PLD2-Fes physical interaction that is negated by a Fes-R483K mutant that incapacitates its Src homology 2 domain. All these suggest for the first time the following mechanism: mTOR/S6K down-regulation→PLD2 overexpression→PLD2/Fes association→phosphatidic acid-led activation of Fes kinase→granulocytic differentiation. Differentiation shortening could have a clinical impact on reducing the time of return to normalcy of the white cell counts after chemotherapy in patients with acute promyelocytic leukemia.

Accessibility of the coxsackievirus and adenovirus receptor and its importance in adenovirus gene transduction efficiency.

Viruses are commonly investigated as vector systems for gene therapy. To be effective, virus-mediated gene-delivery systems require the presence of specific virus receptors to enter the target cell. One example is adenovirus and its primary receptor is the coxsackievirus and adenovirus receptor (CAR). Madin-Darby canine kidney (MDCK) cells have become a choice model system for studying CAR and adenovirus infection due to their ability to polarize rapidly into an epithelium with high transepithelial resistance. We show here that, whilst MDCK cells are resistant to adenovirus infection and hence appear functionally CAR-deficient, polarized MDCK cells express significant levels of CAR sequestered on the basolateral surface, where it is inaccessible for virus infection. Thus, although a cell type may be resistant to adenovirus infection, it is impossible to know whether it is due to a deficiency, as both CAR absence and inaccessibility are barriers to adenovirus-mediated gene transfer.

Phosphatidic acid is a leukocyte chemoattractant that acts through S6 kinase signaling.

Phosphatidic acid (PA) is a pleiotropic lipid second messenger in mammalian cells. We report here that extracellular PA acts as a leukocyte chemoattractant, as membrane-soluble dioleoyl-PA (DOPA) elicits actin polymerization and chemotaxis of human neutrophils and differentiated proleukemic HL-60 cells. We show that the mechanism for this involves the S6 kinase (S6K) signaling enzyme. Chemotaxis was inhibited >90% by the S6K inhibitors rapamycin and bisindolylmaleimide and by S6K1 silencing using double-stranded RNA. However, it was only moderately ( approximately 30%) inhibited by mTOR siRNA, indicating the presence of an mTOR-independent mechanism for S6K. Exogenous PA led to robust time- and dose-dependent increases in S6K enzymatic activity and Thr(421)/Ser(424) phosphorylation, further supporting a PA/S6K connection. We also investigated whether intracellular PA production affects cell migration. Overexpression of phospholipase D2 (PLD2) and, to a lesser extent, PLD1, resulted in elevation of both S6K activity and chemokinesis, whereas PLD silencing was inhibitory. Because the lipase-inactive PLD2 mutants K444R and K758R neither activated S6K nor induced chemotaxis, intracellular PA is needed for this form of cell migration. Lastly, we demonstrated a connection between extracellular and intracellular PA. Using an enhanced green fluorescent protein-derived PA sensor (pEGFP-Spo20PABD), we showed that exogenous PA or PA generated in situ by bacterial (Streptomyces chromofuscus) PLD enters the cell and accumulates in vesicle-like cytoplasmic structures. In summary, we report the discovery of PA as a leukocyte chemoattractant via cell entry and activation of S6K to mediate the cytoskeletal actin polymerization and leukocyte chemotaxis required for the immune function of these cells.

Mutation of Y179 on phospholipase D2 (PLD2) upregulates DNA synthesis in a PI3K-and Akt-dependent manner.

Phospholipase D2 (PLD2), one of the two mammalian members of the PLD family, has been implicated in cell proliferation, transformation, tumor progression and survival. However, as precise mechanistic details are still unknown, we investigated here if the PLD2 isoform would signal through the PI3K/AKT pathway. Transient expression of PLD2 in COS7 cells with either the WT or with a Y179F mutant, resulted in an increased basal phosphorylation of AKT in residues T308 and S473, in a PI3K-dependent manner. Transfection of PLD2-Y179F (but not the wild type) caused an increased (>2-fold) DNA synthesis even in the absence of extracellular stimuli. Other signaling mechanisms downstream such PLD/PI3K dependence (that might lead to DNA synthesis regulation) were further studied. PLD2-Y179F caused an increase in phosphorylation of p42/p44 ERK and in the expression of G0/G1 phase transition markers (p21 CIP, PCNA), and these effects, too, were dependent on PI3K. Interestingly, Akt, once activated induced the phosphorylation of PLD2 on residue T175, an effect that was inhibited by LY296004. Lastly, if PLD2-Y179F is further mutated in residue K758 (PLD2 Y179F-K758R), which renders inactive a catalytic site, DNA synthesis is then abrogated, indicating that the activity of the enzyme (i.e. synthesis of PA) is necessary for the observed effects. In conclusion, the unavailability of residue Y179 on PLD2 to become phosphorylated leads to an augmentation of DNA synthesis concomitantly with MEK and AKT phosphorylation, in a process that is dependent on PI3K and independent of any extracellular stimuli. This might be critical for the maintenance of the PLD2-regulated proliferative status.

The Grb2/PLD2 interaction is essential for lipase activity, intracellular localization and signaling in response to EGF.

The adaptor protein Grb2 associates with phospholipase D2 (PLD2), but it is not known if this interaction is necessary for the functionality of the lipase in vivo. We demonstrate that stable short hairpin RNA (shRNA)-based silencing of Grb2, a critical signal transducer of the epidermal growth factor receptor (EGFR) and linker to the Ras/Erk pathway, resulted in the reduction of PLD2 activity in COS7 cells. Transfection of a Grb2 construct refractory to shGrb2 silencing (XGrb2(SiL)) into the Grb2-knockdown cells (COS7(shGrb2)), resulted in the nearly full rescue of PLD2 activity. However, Grb2-R86K, an SH2-deficient mutant of Grb2 that is incapable of binding to PLD2, failed to induce an enhancement of the impaired PLD2 activity in COS7(shGrb2) cells. Grb2 and PLD2 are directly associated and Grb2 is brought down with anti-myc antibodies irrespective of the presence or absence of EGFR activation. Immunofluorescence microscopy showed that co-transfected PLD2 and Grb2 re-localize to Golgi-like structures after EGF stimulation. Since this was not observed in cotransfection experiments with Grb2 and PLD2-Y169/179F, a lipase mutant that does not bind to Grb2, we inferred that Grb2 serves to hijack PLD2 to the perinuclear Golgi region through its SH2 domain. Supporting this is the finding that the primary cell line HUVEC expresses PLD2 diffusely in the cytoplasm and in the perinuclear Golgi region, where PLD2 and Grb2 colocalize. Such colocalization in primary cells increased after stimulation with EGF. These results demonstrate for the first time that the presence of Grb2 and its interaction with localized intracellular structures is essential for PLD2 activity and signaling in vivo.

Phospholipase D2-derived phosphatidic acid binds to and activates ribosomal p70 S6 kinase independently of mTOR.

The product of phospholipase D (PLD) enzymatic action in cell membranes, phosphatidic acid (PA), regulates kinases implicated in NADPH oxidase activation, as well as the mammalian target of rapamycin (mTOR) kinase. However, other protein targets for this lipid second messenger must exist in order to explain other key PA-mediated cellular functions. In this study, PA was found to specifically and saturably bind to and activate recombinant and immunoprecipitated endogenous ribosomal S6 kinase (S6K) with a stoichiometry of 94:1 lipid/protein. Polyphosphoinositides PI4-P and PI4,5P2 and cardiolipin could also bind to and activate S6K, albeit with different kinetics. Conversely, PA with at least one acyl side chain saturated (10:0) was ineffective in binding or activating the enzyme. Transfection of COS-7 cells with a wild-type myc-(pcDNA)-PLD2 construct resulted in high PLD activity, concomitantly with an increase in ribosomal p70S6K enzyme activity and phosphorylation in T389 and T421/S424 as well as phosphorylation of p70S6K's natural substrate S6 protein in S235/S236. Overexpression of a lipase inactive mutant (K758R), however, failed to induce an increase in both PLD and S6K activity or phosphorylation, indicating that the enzymatic activity of PLD2 (i.e., synthesis of PA) must be present to affect S6K. Neither inhibiting mTOR kinase activity with rapamycin nor silencing mTOR gene expression altered the augmentative effect of PLD2 exerted on p70S6K activity. This finding indicates that PA binds to and activates p70S6K, even in the absence of mTOR. Lastly, COS-7 transfection with PLD2 changed the pattern of subcellular expression, and a colocalization of S6K and PLD2 was observed by immunofluorescence microscopy. These results show for the first time a direct (mTOR-independent) participation of PLD in the p70S6K pathway and implicate PA as a nexus that brings together cell phospholipases and kinases.

IL-8-induced neutrophil chemotaxis is mediated by Janus kinase 3 (JAK3).

Janus kinase 3 (JAK3) is a non-receptor tyrosine kinase vital to the regulation of T-cells. We report that JAK3 is a mediator of interleukin-8 (IL-8) stimulation of a different class of hematopoietic relevant cells: human neutrophils. IL-8 induced a time- and concentration-dependent activation of JAK3 activity in neutrophils and differentiated HL-60 leukemic cells. JAK3 was more robustly activated by IL-8 than other kinases: p70S6K, mTOR, MAPK or PKC. JAK3 silencing severely inhibited IL-8-mediated chemotaxis. Thus, IL-8 stimulates chemotaxis through a mechanism mediated by JAK3. Further, JAK3 activity and chemotaxis were inhibited by the flavonoid apigenin (4',5,7-trihydroxyflavone) at ∼5nM IC(50). These new findings lay the basis for understanding the molecular mechanism of cell migration as it relates to neutrophil-mediated chronic inflammatory processes.

A comprehensive model that explains the regulation of phospholipase D2 activity by phosphorylation-dephosphorylation.

We report here that the enzymatic activity of phospholipase D2 (PLD2) is regulated by phosphorylation-dephosphorylation. Phosphatase treatment of PLD2-overexpressing cells showed a biphasic nature of changes in activity that indicated the existence of "activator" and "inhibitory" sites. We identified three kinases capable of phosphorylating PLD2 in vitro-epidermal growth factor receptor (EGFR), JAK3, and Src (with JAK3 reported for the first time in this study)-that phosphorylate an inhibitory, an activator, and an ambivalent (one that can yield either effect) site, respectively. Mass spectrometry analyses indicated the target of each of these kinases as Y(296) for EGFR, Y(415) for JAK3, and Y(511) for Src. The extent to which each site is activated or inhibited depends on the cell type considered. In COS-7, cells that show the highest level of PLD2 activity, the Y(415) is a prominent site, and JAK3 compensates the negative modulation by EGFR on Y(296). In MCF-7, cells that show the lowest level of PLD2 activity, the converse is the case, with Y(296) unable to compensate the positive modulation by Y(415). MTLn3, with medium to low levels of lipase activity, show an intermediate pattern of regulation but closer to MCF-7 than to COS-7 cells. The negative effect of EGFR on the two cancer cell lines MTLn3 and MCF-7 is further proven by RNA silencing experiments that yield COS-7 showing lower PLD2 activity, and MTLn3 and MCF-7 cells showing an elevated activity. MCF-7 is a cancer cell line derived from a low-aggressive/invasive form of breast cancer that has relatively low levels of PLD activity. We propose that PLD2 activity is low in the breast cancer cell line MCF-7 because it is kept downregulated by tyrosyl phosphorylation of Y(296) by EGFR kinase. Thus, phosphorylation of PLD2-Y(296) could be the signal for lowering the level of PLD2 activity in transformed cells with low invasive capabilities.

The molecular basis of phospholipase D2-induced chemotaxis: elucidation of differential pathways in macrophages and fibroblasts.

We report the molecular mechanisms that underlie chemotaxis of macrophages and cell migration of fibroblasts, cells that are essential during the body's innate immune response and during wound repair, respectively. Silencing of phospholipase D1 (PLD1) and PLD2 reduced cell migration (both chemokinesis and chemotaxis) by approximately 60% and >80%, respectively; this migration was restored by cell transfection with PLD2 constructs refractory to small interfering RNA (siRNA). Cells overexpressing active phospholipase D1 (PLD1) but, mostly, active PLD2 exhibited cell migration capabilities that were elevated over those elicited by chemoattractants alone. The mechanism for this enhancement is complex. It involves two pathways: one that is dependent on the activity of the lipase (and signals through its product, phosphatidic acid [PA]) and another that involves protein-protein interactions. The first is evidenced by partial abrogation of chemotaxis with lipase activity-defective constructs (PLD2-K758R) and by n-butanol treatment of cells. The second is evidenced by PLD association with the growth factor receptor-bound protein 2 (Grb2) through residue Y(169), located within a Src homology 2 (SH2) consensus site. The association Grb2-PLD2 could be visualized by fluorescence microscopy in RAW/LR5 macrophages concentrated in actin-rich membrane ruffles, making possible that Grb2 serves as a docking or intermediary protein. The Grb2/PLD2-mediated chemotaxis process also depends on Grb2's ability to recognize other motility proteins, like the Wiskott-Aldrich syndrome protein (WASP). Cell transfection with WASP, PLD2, and Grb2 constructs yields the highest levels of cell migration response, particularly in a macrophage cell line (RAW/LR5) and only modestly in the fibroblast cell line COS-7. Further, RAW/LR5 macrophages utilize for cell migration an additional pathway that involves S6 kinase (S6K) through PLD2-Y(296), known to be phosphorylated by epidermal growth factor receptor (EGFR) kinase. Thus, both fibroblasts and macrophages use activity-dependent and activity-independent signaling mechanisms. However, highly mobile cells like macrophages use all signaling machinery available to them to accomplish their required function in rapid immune response, which sets them apart from fibroblasts, cells normally nonmobile that are only briefly involved in wound healing.