Mei-Zhen Cui

Activation of protein kinase C zeta by peroxynitrite regulates LKB1-dependent AMP-activated protein kinase in cultured endothelial cells.

We previously reported the phosphoinositide 3-kinase-dependent activation of the 5′-AMP-activated kinase (AMPK) by peroxynitrite (ONOO-) and hypoxia-reoxygenation in cultured endothelial cells. Here we show the molecular mechanism of activation of this pathway. Exposure of bovine aortic endothelial cells to ONOO- significantly increased the phosphorylation of both Thr172 of AMPK and Ser1179 of endothelial nitric-oxide synthase, a known downstream enzyme of AMPK. In addition, activation of AMPK by ONOO- was accompanied by increased phosphorylation of protein kinase Cζ (PKCζ) (Thr410/403) and translocation of cytosolic PKCζ into the membrane. Further, inhibition of PKCζ abrogated ONOO--induced AMPK-Thr172 phosphorylation as that of endothelial nitric-oxide synthase. Furthermore, overexpression of a constitutively active PKCζ mutant enhanced the phosphorylation of AMPK-Thr172, suggesting that PKCζ is upstream of AMPK activation. In contrast, ONOO- activated PKCζ in LKB1-deficient HeLa-S3 but affected neither AMPK-Thr172 nor AMPK activity. These data suggest that LKB1 is required for PKCζ-enhanced AMPK activation. In vitro, recombinant PKCζ phosphorylated LKB1 at Ser428, resulting in phosphorylation of AMPK at Thr172. Further, direct mutation of Ser428 of LKB1 into alanine, like the kinase-inactive LKB1 mutant, abolished ONOO--induced AMPK activation. In several cell types originating from human, rat, and mouse, inhibition of PKCζ significantly attenuated the phosphorylation of both LKB1-Ser428 and AMPK-Thr172 that were enhanced by ONOO-. Taken together, we conclude that PKCζ can regulate AMPK activity by increasing the Ser428 phosphorylation of LKB1, resulting in association of LKB1 with AMPK and consequent AMPK Thr172 phosphorylation by LKB1.

Transcriptional Regulation of the Tissue Factor Gene in Human Epithelial Cells Is Mediated by Sp1 and EGR-1

Tissue factor (TF) gene expression is rapidly induced in epithelial cells by phorbol 12-myristate 13-acetate and serum. We have shown that this induction is mediated by a novel serum response region (SRR) (−111 to +14 bp) within the human TF promoter. In this study, we characterized cis-acting genetic elements within the SRR that regulated basal and inducible expression of the TF gene in HeLa cells. Gel mobility shift assays using oligonucleotides spanning the entire SRR identified three 12-base pair (bp) motifs within subregions 1, 2, and 3 that bound constitutively expressed Sp1 and inducibly expressed EGR-1. Analysis of protein binding to these 12-bp motifs by competition with Sp1 and EGR-1 sites, mutation, and antibody supershift experiments indicated that they each contained distinct EGR-1 and Sp1 sites that overlapped by 6 bp. Functional studies using HeLa cells transfected with plasmids containing the wild-type TF promoter (−111 to +14 bp) or derivatives containing mutations in the three Sp1 and/or EGR-1 sites examined basal and inducible expression. The Sp1 sites mediated basal promoter activity, and both Sp1 and EGR-1 sites were required for maximal induction of the TF promoter by phorbol 12-myristate 13-acetate or serum. These data indicated that TF gene expression in HeLa cells was regulated by both Sp1 and EGR-1.

Identification of a New Presenilin-dependent ζ-Cleavage Site within the Transmembrane Domain of Amyloid Precursor Protein

γ-Secretase cleavage of β-amyloid precursor protein (APP) is crucial in the pathogenesis of Alzheimer disease, because it is the decisive step in the formation of the C terminus of β-amyloid protein (Aβ). To better understand the molecular events involved in γ-secretase cleavage of APP, in this study we report the identification of a new intracellular long Aβ species containing residues 1–46 (Aβ46), which led to the identification of a novel ζ-cleavage site between the known γ- and ϵ-cleavage sites within the transmembrane domain of APP. Our data clearly demonstrate that the new ζ-cleavage is a presenilin-dependent event. It is also noted that the new ζ-cleavage site at Aβ46 is the APP717 mutation site. Furthermore, we show that the new ζ-cleavage is inhibited by γ-secretase inhibitors known as transition state analogs but less affected by inhibitors known as non-transition state γ-secretase inhibitors. Thus, the identification of Aβ46 establishes a system to determine the specificity or the preference of the known γ-secretase inhibitors by examining their effects on the formation or turnover of Aβ46.

γ-Cleavage Is Dependent on ζ-Cleavage during the Proteolytic Processing of Amyloid Precursor Protein within Its Transmembrane Domain

β-Amyloid precursor protein apparently undergoes at least three major cleavages, γ-, ϵ-, and the newly identified ζ-cleavage, within its transmembrane domain to produce secreted β-amyloid protein (Aβ). However, the roles of ϵ- and ζ-cleavages in the formation of secreted Aβ and the relationship among these three cleavages, namely ϵ-, ζ-, and γ-cleavages, remain elusive. We investigated these issues by attempting to determine the formation and turnover of the intermediate products generated by these cleavages, in the presence or absence of known γ-secretase inhibitors. By using a differential inhibition strategy, our data demonstrate that Aβ46 is an intermediate precursor of secreted Aβ. Our co-immunoprecipitation data also reveal that, as an intermediate, Aβ46 is tightly associated with presenilin in intact cells. Furthermore, we identified a long Aβ species that is most likely the long sought after intermediate product, Aβ49, generated by ϵ-cleavage, and this Aβ49 is further processed by ζ- and γ-cleavages to generate Aβ46 and ultimately the secreted Aβ40/42. More interestingly, our data demonstrate that γ-cleavage not only occurs last but also depends on ζ-cleavage occurring prior to it, indicating that ζ-cleavage is crucial for the formation of secreted Aβ. Thus, we conclude that the C terminus of secreted Aβ is most likely generated by a series of sequential cleavages, namely first ϵ-cleavage which is then followed by ζ- and γ-cleavages, and that Aβ46 produced by ζ-cleavage is the precursor of secreted Aβ40/42.

Native and Oxidized Low Density Lipoprotein Induction of Tissue Factor Gene Expression in Smooth Muscle Cells Is Mediated by Both Egr-1 and Sp1

Tissue factor, in association with factor VIIa, initiates the coagulation cascade. We studied the influences of two pathophysiological stimuli, native (unmodified) and oxidized low density lipoprotein, on tissue factor gene expression in a cell important in vascular remodeling and vascular diseases, the smooth muscle cell. Our results demonstrated that both lipoproteins significantly induced tissue factor gene expression in rat aortic smooth muscle cells; oxidized low density lipoprotein was slightly more potent. Both lipoproteins increased tissue factor mRNA in a concentration- and time-dependent manner. Results from nuclear run-on assays and mRNA stability experiments indicated that increased tissue factor mRNA accumulation in response to the lipoproteins was principally controlled at the transcriptional level. By using lipid extracts of low density lipoprotein or methylation of the intact lipoprotein to block receptor recognition, we showed that this lipoprotein induced tissue factor mRNA via both receptor-independent and receptor-augmented pathways. Transfection studies using a series of deleted tissue factor promoters revealed that a −143- to +106-base pair region of the rat tissue factor promoter contained regulatory elements required for lipoprotein-mediated induction. Electrophoretic mobility shift assays showed that the binding activities of the transcription factor Egr-1, but not Sp1, were markedly elevated in response to these lipoproteins. Transfection of site-directed mutants of the tissue factor (TF) promoter demonstrated that not only Egr-1 but also Sp1 cis-acting elements in the TF (−143) promoter construct were necessary for optimal TF gene induction. Our data show for the first time that both low density lipoprotein and oxidized low density lipoprotein induce tissue factor gene expression in smooth muscle cells and that this tissue factor gene expression is mediated by both Egr-1 and Sp1 transcription factors.

LDL increases inactive tissue factor on vascular smooth muscle cell surfaces: hydrogen peroxide activates latent cell surface tissue factor.

BACKGROUND Tissue factor, which is required for the initiation of the extrinsic coagulation cascade, is known to be upregulated in cells within atherosclerotic lesions, including smooth muscle cells. Tissue factor expression on the smooth muscle cell surface could be of pathological significance as a contributor to plaque growth, thrombus formation, and the acute coronary syndrome after plaque rupture. METHODS AND RESULTS In this study, we show that LDL increased tissue factor mRNA and cell surface protein in smooth muscle cells without a marked increase in surface tissue factor activity. Hydrogen peroxide activated tissue factor on the cell surface but did not increase tissue factor mRNA or cell surface protein. Sequentially added LDL and hydrogen peroxide increased mRNA, cell surface protein, and activity; surface activity was greater than that observed with hydrogen peroxide alone. The action of hydrogen peroxide did not involve a regulatory mechanism associated with the cytoplasmic tail of tissue factor because a truncated tissue factor lacking the cytoplasmic tail was activated by hydrogen peroxide. CONCLUSIONS These results suggest a novel 2-step pathway for increased tissue factor activity on smooth muscle cell surfaces in which lipoproteins regulate synthesis of a latent tissue factor and oxidants activate the protein complex.

Thrombin Rapidly Induces Protein Kinase D Phosphorylation, and Protein Kinase C δ Mediates the Activation

Thrombin plays a critical role in hemostasis, thrombosis, and inflammation. However, the responsible intracellular signaling pathways triggered by thrombin are still not well defined. We report here that thrombin rapidly and transiently induces activation of protein kinase D (PKD) in aortic smooth muscle cells. Our data demonstrate that protein kinase C (PKC) inhibitors completely block thrombin-induced PKD activation, suggesting that thrombin induces PKD activation via a PKC-dependent pathway. Furthermore, our results show that thrombin rapidly induces PKCδ phosphorylation and that the PKCδ-specific inhibitor rottlerin blocks thrombin-induced PKD activation, suggesting that PKCδ mediates the thrombin-induced PKD activation. Using dominant negative approaches, we demonstrated that expression of a dominant negative PKCδ inhibits the phosphorylation and activation of PKD induced by thrombin, whereas neither PKCε nor PKCζ affects thrombin-induced PKD activation. In addition, our results of co-immunoprecipitation assays showed that PKD forms a complex with PKCδ in smooth muscle cells. Taken together, the findings of the present study demonstrate that thrombin induces activation of PKD and reveal a novel role of PKCδ in mediating thrombin-induced PKD activation in vascular smooth muscle cells.

The Novel Presenilin-1-associated Protein Is a Proapoptotic Mitochondrial Protein

Recent studies have suggested a possible role for presenilin proteins in apoptotic cell death observed in Alzheimers disease. The mechanism by which presenilin proteins regulate apoptotic cell death is not well understood. Using the yeast two-hybrid system, we previously isolated a novel protein, presenilin-associated protein (PSAP) that specifically interacts with the C terminus of presenilin 1 (PS1), but not presenilin 2 (PS2). Here we report that PSAP is a mitochondrial resident protein sharing homology with mitochondrial carrier protein. PSAP was detected in a mitochondria-enriched fraction, and PSAP immunofluorescence was present in a punctate pattern that colocalized with a mitochondrial marker. More interestingly, overexpression of PSAP caused apoptotic death. PSAP-induced apoptosis was documented using multiple independent approaches, including membrane blebbing, chromosome condensation and fragmentation, DNA laddering, cleavage of the death substrate poly(ADP-ribose) polymerase, and flow cytometry. PSAP-induced cell death was accompanied by cytochrome c release from mitochondria and caspase-3 activation. Moreover, the general caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone, which blocked cell death, did not block the release of cytochrome c from mitochondria caused by overexpression of PSAP, indicating that PSAP-induced cytochrome c release was independent of caspase activity. The mitochondrial localization and proapoptotic activity of PSAP suggest that it is an important regulator of apoptosis.

Identification of a Novel PSD-95/Dlg/ZO-1 (PDZ)-like Protein Interacting with the C Terminus of Presenilin-1

Presenilin-1 (PS-1) is the most causative Alzheimer gene product, and its function is not well understood. In an attempt to elucidate the function of PS-1, we screened a human brain cDNA library for PS-1-interacting proteins using the yeast two-hybrid system and isolated a novel protein containing a PSD-95/Dlg/ZO-1 (PDZ)-like domain. This novel PS-1-associated protein (PSAP) shares a significant similarity with a Caenorhabditis elegans protein of unknown function. Northern blot analysis revealed that PSAP is predominantly expressed in the brain. Deletion of the first four C-terminal amino acid residues of PS-1, which contain the PDZ domain-binding motif (Gln-Phe-Tyr-Ile), reduced the binding activity of PS-1 toward PSAP 4-fold. These data suggest that PS-1 may associate with a PDZ-like domain-containing protein in vivoand thus may participate in receptor or channel clustering and intracellular signaling events in the brain.

Lysophosphatidic Acid Induction of Tissue Factor Expression in Aortic Smooth Muscle Cells

Objective—Tissue factor (TF), the initiator of the coagulation cascade, is expressed by cells in atherosclerotic lesions. Lysophosphatidic acid (LPA) is a component of oxidized lipoproteins and an agent released by activated platelets. The present study investigated whether and how TF expression is regulated by LPA. Methods and Results—Northern blotting, Western blotting, and TF activity assays demonstrated that LPA markedly induced TF mRNA, protein, and activity in vascular smooth muscle cells. LPA-induced TF expression is primarily controlled at the transcriptional level. Phosphorylation of mitogen-activated protein kinase kinase (MEK) and extracellular signaling–regulated kinases (ERK1/2) was rapidly and markedly induced by LPA. MEK inhibitors U0126 and PD98059 blocked both ERK activation and the increase in TF mRNA. In contrast, the specific p38 MAP kinase inhibitor SB203580 had no effect on LPA-induced TF mRNA increase. The G&agr;i protein inhibitor, pertussis toxin, abolished LPA-induced phosphorylation of MEKs and ERKs, as well as the induction of TF mRNA. Conclusions—Our data demonstrate that a G&agr;i protein and activation of MEKs and ERKs mediate LPA-induced TF expression. Our data suggest that elevated LPA could be a thrombogenic risk factor by upregulating TF expression. These results may have important implications in vascular remodeling and vascular diseases.