Lipid-bound factor Xa regulates tissue factor activity

The activation of coagulation factor X by tissue factor (TF) and coagulation factor VIIa (VIIa) on a phospholipid surface is thought to be the key step in the initiation of blood coagulation. In this reaction, the product, fXa, is transiently and reversibly bound to the TF-VIIa enzyme complex. This in effect leads to a probabilistic inhibition of subsequent fX activations; a new fX substrate molecule cannot be activated until the old fXa molecule leaves. In this study, we demonstrate that benzamidine and soybean trypsin inhibitor-conjugated Sepharose beads, which bind fXa and sequester it away from the reaction, serve to enhance fX activation by the TF-VIIa complex. Thus, removal of fXa from the reactive zone, by either flow, fXa sequestration, or binding to distant lipid surfaces, can serve to enhance the levels of TF-VIIa activity. Using resonance energy transfer, we found the dissociation constants of fX and fXa for 100 nm diameter phospholipid vesicles to be on the order of 30-60 nM, consistent with previous measurements employing planar lipid surfaces. On the basis of the measurements of binding of fXa to phospholipid surfaces, we demonstrate that the rates of fX activation by the TF-VIIa complex under a variety of experimental conditions depend inversely on the amount of product (fXa) bound to the TF-phospholipid surface. These data support an inhibitory role for the reaction product, fXa, and indicate that models previously employed in understanding this initial coagulation reaction must now be re-evaluated to account for both the product occupancy of the phospholipid surface and the binding of the product to the enzyme. Moreover, the inhibitory properties of fXa can be described on the basis of the estimated surface density of fXa molecules on the TF-phospholipid surface.     

Regulation of polyamine transport by polyamines and polyamine analogs

Regulation of polyamine transport in murine L1210 leukemia cells was characterized in order to better understand its relationship to specific intracellular polyamines and their analogs and to quantitate the sensitivity by which it is controlled. Up-regulation of polyamine uptake was evaluated following a 48-hr treatment with a combination of biosynthetic enzyme inhibitors to deplete intracellular polyamine pools. The latter declined gradually over 48 hr and was accompanied by a steady increase in spermidine (SPD) and spermine (SPM) transport as indicated by rises in V_max to levels ～4.5 times higher than control values. Restoration of individual polyamine pools during a 6-hr period following inhibitor treatment revealed that SPD and SPM uptake could not be selectively affected by specific pool changes. The effectiveness of individual polyamines in reversing inhibitor-induced stimulation of uptake was as follows: putrescine < SPD < SPM = the SPM analog, N¹, N¹²-bis(ethyl)spermine (BESPM). In contrast to stimulation of transport, down-regulation by exogenous polyamines or analogs occurred rapidly and in response to subtle increases in intracellular pools. Following a 1-hr exposure to 10 μM BESPM, V_max values for SPD and SPM fell by 70%, whereas the analog pool increased to only 400–500 pmol/10⁶ cells—about 15–20% of the total polyamine pool (～2.8 nmol/10⁶ cells). SPM produced nearly identical regulatory effects on transport kinetics. Both BESPM and SPM were even more effective at down-regulating transport that had been previously stimulated four to fivefold by polyamine depletion achieved with enzyme inhibitors. A dose response with BESPM at 48 hr revealed a biphasic effect on uptake whereby concentrations of analog < 3 μM produced an increase in SPD and SPM V_max values, whereas concentrations 3 μM and higher produced a marked suppression of these values. Cells treated with 3 μM BESPM for 2 hr and placed in analog-free medium recovered transport capability in only 3 hr. Thus, whereas stimulation of polyamine transport is a relatively insensitive and slowly responsive process that tends to parallel polyamine depletion, down-regulation of polyamine transport by exogenous polyamines and analogs and its reversal are rapidly responsive events that correlate with relatively small (i.e., 15–20%) changes in intracellular polyamine pools.     

The mechanism by which MEK/ERK regulates JNK and p38 activity in polyamine depleted IEC-6 cells during apoptosis

Polyamine-depletion inhibited apoptosis by activating ERK1/2, while, preventing JNK1/2 activation. MKP-1 knockdown by SiRNA increased ERK1/2, JNK1/2, and p38 phosphorylation and apoptosis. Therefore, we predicted that polyamines might regulate MKP1 via MEK/ERK and thereby apoptosis. We examined the role of MEK/ERK in the regulation of MKP1 and JNK, and p38 activities and apoptosis. Inhibition of MKP-1 activity with a pharmacological inhibitor, sanguinarine (SA), increased JNK1/2, p38, and ERK1/2 activities without causing apoptosis. However, pre-activation of these kinases by SA significantly increased camptothecin (CPT)-induced apoptosis suggesting different roles for MAPKs during survival and apoptosis. Inhibition of MEK1 activity prevented the expression of MKP-1 protein and augmented CPT-induced apoptosis, which correlated with increased activities of JNK1/2, caspases, and DNA fragmentation. Polyamine depleted cells had higher levels of MKP-1 protein and decreased JNK1/2 activity and apoptosis. Inhibition of MEK1 prevented MKP-1 expression and increased JNK1/2 and apoptosis. Phospho-JNK1/2, phospho-ERK2, MKP-1, and the catalytic subunit of PP2Ac formed a complex in response to TNF/CPT. Inactivation of PP2Ac had no effect on the association of MKP-1 and JNK1. However, inhibition of MKP-1 activity decreased the formation of the MKP-1, PP2Ac and JNK complex. Following inhibition by SA, MKP-1 localized in the cytoplasm, while basal and CPT-induced MKP-1 remained in the nuclear fraction. These results suggest that nuclear MKP-1 translocates to the cytoplasm, binds phosphorylated JNK and p38 resulting in dephosphorylation and decreased activity. Thus, MEK/ERK activity controls the levels of MKP-1 and, thereby, regulates JNK activity in polyamine-depleted cells.     

Epidermal growth factor: modulator of murine embryonic palate mesenchymal cell proliferation, polyamine biosynthesis, and polyamine transport

Polyamines (putrescine, spermidine, and spermine) are normal cellular constituents able to modulate cellular proliferation and differentiation in a number of tissues and cell types. This investigation explores the response of murine embryonic palate mesenchymal (MEPM) cells to epidermal growth factor (EGF) in terms of biosynthesis of putrescine and its transport across the plasma membrane and tests the hypothesis that polyamine transport can serve as an alternative mechanism (other than biosynthesis) for elevating intracellular polyamines during stimulation of MEPM cellular proliferation. MEPM cells treated with EGF were stimulated to proliferate and showed a dose- and time-dependent stimulation of ornithine decarboxylase (ODC) which was maximal at 4-6 hours. EGF also stimulated the initial rate of putrescine transport in a dose- and time-dependent manner. This stimulation was found to be maximal 3 hours after treatment and specific for the putrescine transport system. The kinetic parameters of putrescine transport shifted from 2.52 microM (Km) and 23.6 nmol/mg protein/15 minutes (Vmax) in nonstimulated cells to 4.48 microM (Km) and 39.8 nmol/mg protein/15 minutes (Vmax) in EGF-treated cells. This kinetic shift did not require de novo protein or RNA synthesis, as cycloheximide (10 micrograms/ml) and actinomycin D (50 micrograms/ml) had little effect on the ability of EGF to stimulate the initial rate of putrescine uptake. The rate of transport, however, was found to be inversely related to cell density. The addition of exogenous putrescine concomitantly with EGF blocked the induction of ODC, while in the presence of difluoromethylornithine (DFMO) (irreversible inhibitor of ODC) the initial rate of putrescine transport remained elevated throughout the time course studied. This stimulation of putrescine uptake caused by polyamine deprivation was reversed by exogenous putrescine and Ca++ while alpha-aminoisobutyric acid (AIB) further stimulated the rate of uptake. EGF's ability to stimulate cellular DNA synthesis was inhibited by DFMO. If DFMO-treated cells were stimulated with EGF in the presence of exogenous putrescine, this stimulatory effect was preserved. These studies indicate that the rate of polyamine transportation is highly responsive to a signal which initiates biosynthesis of polyamines. Further, this transportation system provides a compensatory mechanism allowing the cell to increase intracellular levels of polyamines when environmental conditions inhibit biosynthesis or when polyamines are abundant.     

Taxol induced apoptosis regulates amino acid transport in breast cancer cells

A major outcome from Taxol treatment is induction of tumor cell apoptosis. However, metabolic responses to Taxol-induced apoptosis are poorly understood. In this study, we hypothesize that alterations in specific amino acid transporters may affect the Taxol-induced apoptosis in breast cancer cells. In this case, the activity of the given transporter may serve as a biomarker that could provide a biological assessment of response to drug treatment. We have examined the mechanisms responsible for Taxol-induced neutral amino acid uptake by breast cancer cells, such as MCF-7, BT474, MDAMB231 and T47D. The biochemical and molecular studies include: (1) growth-inhibition (MTT); (2) transport kinetics: (3) substrate-specific inhibition; (4) effect of thiol-modifying agents NEM and NPM; (5) gene expression of amino acid transporters; and (6) apoptotic assays. Our data show that Taxol treatment of MCF-7 cells induced a transient increase in Na⁺-dependent transport of the neutral amino acid transporter B0 at both gene and protein level. This increase was attenuated by blocking the transporter in the presence of high concentrations of the substrate amino acid. Other neutral amino acid transporters such as ATA2 (System A) and ASC were not altered. Amino acid starvation resulted in the expected up-regulation of System A (ATA2) gene, but not for B0 and ASC. B0 was significantly down regulated. Taxol treatment had no significant effect on the uptake of arginine and glutamate as measured by System y⁺ and X⁻ _GC respectively. Tunel assays and FACS cell cycle analysis demonstrated that both Taxol- and doxorubicin-induced upregulation of B0 transporter gene with accompanying increase in cell apoptosis, could be reversed partially by blocking the B0 transporter with high concentration of alanine, and/or by inhibiting the caspase pathway. Both Taxol and doxorubicin treatment caused a significant decrease in S-phase of the cell cycle. However, Taxol-induced an increase primarily in the G2 fraction while doxorubicin caused increase in G1/G0 together with a small increase in G2. In summary, our study showed that Taxol induced apoptosis in several breast cancer cells results in activation of amino acid transporter System B0 at both gene and protein level. Similar response was observed with another chemotherapeutic agent Doxorubicin, suggesting that this increase is in response to apoptosis, and not only due to changes in cell cycle related events. Drs. Wu and Shen contributed equally to this study.     

Smad7 differentially regulates transforming growth factor beta-mediated signaling pathways

Smad7 has been identified as a negative regulator of transforming growth factor beta (TGF-beta) signaling by interfering with the phosphorylation of other Smad proteins by TGF-beta receptor type I (TbetaRI). We established a mink lung epithelial (Mv1Lu) cell line where ectopic expression of Smad7 is tightly controlled by doxycycline using an improved Tet-on system. Once induced by doxycycline, the recombinant Smad7 was localized predominantly in the perinuclear region and in the cytoplasm. However, the type of culture surface alters the subcellular localization of Smad7: on plastic or on fibronectin-coated glass, Smad7 was localized in the cytoplasm; but when the cells were cultured on glass, nuclear localization was observed. TGF-beta stimulation did not alter substantially the cellular distribution of Smad7. Importantly, the expression of recombinant Smad7 differentially inhibited TGF-beta signaling pathways. Consistent with previous studies, Smad7 inhibited TGF-beta-stimulated induction of type 1 plasminogen activator inhibitor as measured by p3TP-Lux reporter. However, expression of Smad7 had little effect on TGF-beta-induced growth inhibition.