LPA1-mediated PKD2 activation promotes LPA-induced tissue factor expression via the p38α and JNK2 MAPK pathways in smooth muscle cells.

Tissue factor (TF) is the principal initiator of blood coagulation and is necessary for thrombosis. We previously reported that lysophosphatidic acid (LPA), a potent bioactive lipid, highly induces TF expression at the transcriptional level in vascular smooth muscle cells. To date, however, the specific role of the LPA receptor is unknown, and the intracellular signaling pathways that lead to LPA induction of TF have been largely undetermined. In the current study, we found that LPA markedly induced protein kinase D (PKD) activation in mouse aortic smooth muscle cells (MASMCs). Small-interfering RNA-mediated knockdown of PKD2 blocked LPA-induced TF expression and activity, indicating that PKD2 is the key intracellular mediator of LPA signaling leading to the expression and cell surface activity of TF. Furthermore, our data reveal a novel finding that PKD2 mediates LPA-induced TF expression via the p38α and JNK2 MAPK signaling pathways, which are accompanied by the PKD-independent MEK1/2-ERK-JNK pathway. To identify the LPA receptor(s) responsible for LPA-induced TF expression, we isolated MASMCs from LPA receptor-knockout mice. Our results demonstrated that SMCs isolated from LPA receptor 1 (LPA1)-deficient mice completely lost responsiveness to LPA stimulation, which mediates induction of TF expression and activation of PKD and p38/JNK MAPK, indicating that LPA1 is responsible for PKD2-mediated activation of JNK2 and p38α. Taken together, our data reveal a new signaling mechanism in which the LPA1-PKD2 axis mediates LPA-induced TF expression via the p38α and JNK2 pathways. This finding provides new insights into LPA signaling, the PKD2 pathway, and the mechanisms of coagulation/atherothrombosis.

Proapoptotic Mitochondrial Carrier Homolog Protein PSAP Mediates Death Receptor 6 Induced Apoptosis.

Presenilin-associated protein (PSAP) was originally identified as a mitochondrial proapoptotic protein. To further explore the apoptotic pathway that involves PSAP, our yeast two-hybrid screen revealed that PSAP interacts with a death receptor, DR6. DR6 is a relatively less common member of the death receptor family and has been shown to mediate the neurotoxicity of amyloid-ß, mutant SOD1, and prion proteins and has also been implicated in the regulation of immune cell proliferation and differentiation. Our previous study showed that DR6 induces apoptosis via a unique mitochondria-dependent pathway different from the conventional death receptor-mediated extrinsic apoptotic pathways. Thus, the interaction of DR6 with PSAP established a direct molecular link between DR6 and mitochondrial apoptotic pathway. We investigated the possible role of PSAP in DR6-induced apoptosis. Interestingly, it was discovered that knockdown of PSAP strongly inhibited DR6-induced apoptosis. To further elucidate the mechanism by which PSAP mediates DR6-induced mitochondria-dependent apoptosis, our data demonstrated that knockdown of PSAP blocked DR6-induced Bax translocation and cytochrome c release from the mitochondria. Moreover, it was found that both PSAP and DR6 form complexes with Bax, but at different subcellular locations. The DR6-Bax complex was detected in the cytosolic fraction while the PSAP-Bax complex was detected in the mitochondrial fraction. The observation that knockdown of DR6 significantly reduced the amount of PSAP-Bax complex detected in mitochondria suggests a possibility that DR6-bound Bax is transferred to PSAP upon interaction with PSAP at the mitochondria, leading to cytochrome c release and eventually apoptosis.

Platelet CD40 Mediates Leukocyte Recruitment and Neointima Formation after Arterial Denudation Injury in Atherosclerosis-Prone Mice.

The role of platelets in the development of thrombosis and abrupt closure after angioplasty is well recognized. However, the direct impact of platelets on neointima formation after arterial injury remains undetermined. Herein, we show that neointima formation after carotid artery wire injury reduces markedly in CD40-/- apolipoprotein E-deficient (apoE-/-) mice but only slightly in CD40 ligand-/-apoE-/- mice, compared with apoE-/- mice. Wild-type and CD40-deficient platelets were isolated from blood of apoE-/- and CD40-/-apoE-/- mice, respectively. The i.v. injection of thrombin-activated platelets into CD40-/-apoE-/- mice was performed every 5 days, starting at 2 days before wire injury. Injection of wild-type platelets promoted neointima formation, which was associated with increased inflammation by stimulating leukocyte recruitment via up-regulation of circulating platelet surface P-selectin expression and the formation of platelet-leukocyte aggregates. It was also associated with further promoting the luminal deposition of platelet-derived regulated on activation normal T cell expressed and secreted/chemokine (C-C motif) ligand 5 and expression of monocyte chemoattractant protein-1 and vascular cell adhesion molecule 1 in wire-injured carotid arteries. Remarkably, all these inflammatory actions by activated platelets were abrogated by lack of CD40 on injected platelets. Moreover, injection of wild-type platelets inhibited endothelial recovery in wire-injured carotid arteries, but this effect was also abrogated by lack of CD40 on injected platelets. Results suggest that platelet CD40 plays a pivotal role in neointima formation after arterial injury and might represent an attractive target to prevent restenosis after vascular interventions.

CD14 is a key mediator of both lysophosphatidic acid and lipopolysaccharide induction of foam cell formation.

Macrophage uptake of oxidized low-density lipoprotein (oxLDL) plays an important role in foam cell formation and the pathogenesis of atherosclerosis. We report here that lysophosphatidic acid (LPA) enhances lipopolysaccharide (LPS)-induced oxLDL uptake in macrophages. Our data revealed that both LPA and LPS highly induce the CD14 expression at messenger RNA and protein levels in macrophages. The role of CD14, one component of the LPS receptor cluster, in LPA-induced biological functions has been unknown. We took several steps to examine the role of CD14 in LPA signaling pathways. Knockdown of CD14 expression nearly completely blocked LPA/LPS-induced oxLDL uptake in macrophages, demonstrating for the first time that CD14 is a key mediator responsible for both LPA- and LPS-induced oxLDL uptake/foam cell formation. To determine the molecular mechanism mediating CD14 function, we demonstrated that both LPA and LPS significantly induce the expression of scavenger receptor class A type I (SR-AI), which has been implicated in lipid uptake process, and depletion of CD14 levels blocked LPA/LPS-induced SR-AI expression. We further showed that the SR-AI-specific antibody, which quenches SR-AI function, blocked LPA- and LPS-induced foam cell formation. Thus, SR-AI is the downstream mediator of CD14 in regulating LPA-, LPS-, and LPA/LPS-induced foam cell formation. Taken together, our results provide the first experimental evidence that CD14 is a novel connecting molecule linking both LPA and LPS pathways and is a key mediator responsible for LPA/LPS-induced foam cell formation. The LPA/LPS-CD14-SR-AI nexus might be the new convergent pathway, contributing to the worsening of atherosclerosis.

Lysophosphatidic Acid Triggers Apoptosis in HeLa Cells through the Upregulation of Tumor Necrosis Factor Receptor Superfamily Member 21.

Lysophosphatidic acid (LPA), a naturally occurring bioactive phospholipid, activates G protein-coupled receptors (GPCRs), leading to regulation of diverse cellular events including cell survival and apoptosis. Despite extensive studies of the signaling pathways that mediate LPA-regulated cell growth and survival, the mechanisms underlying the apoptotic effect of LPA remain largely unclear. In this study, we investigated this issue in HeLa cells. Our data demonstrate that LPA induces apoptosis in HeLa cells at pathologic concentrations with a concomitant upregulation of the expression of TNFRSF21 (tumor necrosis factor receptor superfamily member 21), also known as death receptor number 6 (DR6) involved in inflammation. Moreover, treatment of cells with LPA receptor (LPAR) antagonist abolished the DR6 upregulation by LPA. LPA-induced DR6 expression was also abrogated by pertussis toxin (PTX), an inhibitor of GPCRs, and by inhibitors of PI3K, PKC, MEK, and ERK. Intriguingly, LPA-induced DR6 expression was specifically blocked by dominant-negative form of PKCδ (PKCδ-DN). LPA-induced DR6 expression was also dramatically inhibited by knockdown of ERK or CREB. These results suggest that activation of the MEK/ERK pathway and the transcription factor CREB mediate LPA-induced DR6 expression. More interestingly, knockdown of DR6 using siRNA approach remarkably attenuated LPA-induced apoptosis. In conclusion, our results suggest that LPA-induced apoptosis in HeLa cells is mediated by the upregulation of DR6 expression.

Pen-2 and Presenilin are Sufficient to Catalyze Notch Processing.

Presenilin-1 (PS1) or presenilin-2 (PS2), nicastrin (NCT), anterior pharynx-defective 1 (Aph-1), and presenilin enhancer-2 (Pen-2) have been considered the minimal essential subunits required to form an active γ-secretase complex. Besides PS, which has been widely believed to function as the catalytic subunit of the complex, the functional roles of the other subunits in the γ-secretase complex remain debatable. In the current study, we set out to determine the role of Pen-2 in γ-secretase activity. To this end, using knockout cells in combination with siRNA and immunoprecipitation approaches, our results revealed that Pen-2 together with presenilin are sufficient to form a functionally active enzyme to process Notch. Specifically, our data demonstrated that Pen-2 plays a crucial role in substrate binding, a mechanism by which Pen-2 contributes directly to the catalytic mechanism of γ-secretase activity. Our data also suggested that there may be different requirements for components to process AßPP and Notch. This information would be important for therapeutic strategy aimed at inhibition or modulation of γ-secretase activity.

CRE and SRE mediate LPA-induced CCN1 transcription in mouse aortic smooth muscle cells.

Lysophosphatidic acid (LPA), one component of oxidized low-density lipoprotein (ox-LDL), is a potent bioactive phospholipid. Our recent data reveal that LPA induces matricellular protein CCN1 (also known as Cyr61) expression in aortic smooth muscle cells (SMCs) and that CCN1 bridges LPA and integrin signaling pathways leading to SMC migration. Whether and how LPA regulates the transcriptional machinery of the CCN1 gene are unknown. In this study, we found that LPA markedly induces CCN1 mRNA expression in SMCs. Using deleting mutation and reporter gene strategies, we demonstrated regions from -2038 to -1787 and from -101 to +63 of the CCN1 promoter contain the essential regulatory elements. The serum response element (SRE) and cyclic AMP-response element (CRE) are located in these regions. LPA induced time-dependent phosphorylation of serum response factor (SRF) and CRE-binding protein (CREB) in mouse SMCs. Luciferase assays of a series of deleted, mutated CCN1 promoter-reporter gene constructs and dominant negative construct revealed the distal SRE and the proximal CRE in the CCN1 promoter are required for LPA-induced CCN1 gene expression. Our results imply that elevated LPA levels may trigger SMC migration and exacerbate restenosis and atherosclerotic lesions through the induced CCN1, which communicates with a set of plasma membrane proteins and intracellular kinases.

JNK1 Mediates Lipopolysaccharide-Induced CD14 and SR-AI Expression and Macrophage Foam Cell Formation.

Foam cell formation is the key process in the development of atherosclerosis. The uptake of oxidized low-density lipoprotein (oxLDL) converts macrophages into foam cells. We recently reported that lipopolysaccharide (LPS)-induced foam cell formation is regulated by CD14 and scavenger receptor AI (SR-AI). In this study, we employed pharmaceutical and gene knockdown approaches to determine the upstream molecular mediators, which control LPS-induced foam cell formation. Our results demonstrated that the specific c-Jun N-terminal kinase (JNK) pathway inhibitor, SP600125, but neither the specific inhibitor of extracellular signaling-regulated kinase (ERK) kinase MEK1/2, U0126, nor the specific inhibitor of p38 MAPK, SB203580, significantly blocks LPS-induced oxLDL uptake, suggesting that the JNK pathway is the upstream mediator of LPS-induced oxLDL uptake/foam cell formation. To address whether JNK pathway mediates LPS-induced oxLDL uptake is due to JNK pathway-regulated CD14 and SR-AI expression, we assessed whether the pharmaceutical inhibitor of JNK influences LPS-induced expression of CD14 and SR-AI. Our results indicate that JNK pathway mediates LPS-induced CD14 and SR-AI expression. To conclusively address the isoform role of JNK family, we depleted JNK isoforms using the JNK isoform-specific siRNA. Our data showed that the depletion of JNK1, but not JNK2 blocked LPS-induced CD14/SR-AI expression and foam cell formation. Taken together, our results reveal for the first time that JNK1 is the key mediator of LPS-induced CD14 and SR-AI expression in macrophages, leading to LPS-induced oxLDL uptake/foam cell formation. We conclude that the novel JNK1/CD14/SR-AI pathway controls macrophage oxLDL uptake/foam cell formation.

Lysophosphatidic acid-induced vascular neointimal formation in mouse carotid arteries is mediated by the matricellular protein CCN1/Cyr61.

Vascular smooth muscle cell (SMC) migration is an essential step involved in neointimal formation in restenosis and atherosclerosis. Lysophosphatidic acid (LPA) is a bioactive component of oxidized low-density lipoprotein and is produced by activated platelets, implying that LPA influences vascular remodeling. Our previous study revealed that matricellular protein CCN1, a prominent extracellular matrix (ECM) protein, mediates LPA-induced SMC migration in vitro. Here we examined the role of CCN1 in LPA-induced neointimal formation. By using LPA infusion of carotid artery in a mouse model, we demonstrated that LPA highly induced CCN1 expression (approximately six- to sevenfold) in neointimal lesions. Downregulation of CCN1 expression with the specific CCN1 siRNA in carotid arteries blocked LPA-induced neointimal formation, indicating that CCN1 is essential in LPA-induced neointimal formation. We then used LPA receptor knockout (LPA1-/-, LPA2-/-, and LPA3-/-) mice to examine LPA receptor function in CCN1 expression in vivo and in LPA-induced neointimal formation. Our data reveal that LPA1 deficiency, but not LPA2 or LPA3 deficiency, prevents LPA-induced CCN1 expression in vivo in mouse carotid arteries. We also observed that LPA1 deficiency blunted LPA infusion-induced neointimal formation, indicating that LPA1 is the major mediator for LPA-induced vascular remodeling. Our in vivo model of LPA-induced neointimal formation established a key role of the ECM protein CCN1 in mediating LPA-induced neointimal formation. Our data support the notion that the LPA1-CCN1 axis may be the central control for SMC migration and vascular remodeling. CCN1 may serve as an important vascular disease marker and potential target for vascular therapeutic intervention.

Nanospherical arabinogalactan proteins are a key component of the high-strength adhesive secreted by English ivy.

Over 130 y have passed since Charles Darwin first discovered that the adventitious roots of English ivy (Hedera helix) exude a yellowish mucilage that promotes the capacity of this plant to climb vertical surfaces. Unfortunately, little progress has been made in elucidating the adhesion mechanisms underlying this high-strength adhesive. In the previous studies, spherical nanoparticles were observed in the viscous exudate. Here we show that these nanoparticles are predominantly composed of arabinogalactan proteins (AGPs), a superfamily of hydroxyproline-rich glycoproteins present in the extracellular spaces of plant cells. The spheroidal shape of the AGP-rich ivy nanoparticles results in a low viscosity of the ivy adhesive, and thus a favorable wetting behavior on the surface of substrates. Meanwhile, calcium-driven electrostatic interactions among carboxyl groups of the AGPs and the pectic acids give rise to the cross-linking of the exuded adhesive substances, favor subsequent curing (hardening) via formation of an adhesive film, and eventually promote the generation of mechanical interlocking between the adventitious roots of English ivy and the surface of substrates. Inspired by these molecular events, a reconstructed ivy-mimetic adhesive composite was developed by integrating purified AGP-rich ivy nanoparticles with pectic polysaccharides and calcium ions. Information gained from the subsequent tensile tests, in turn, substantiated the proposed adhesion mechanisms underlying the ivy-derived adhesive. Given that AGPs and pectic polysaccharides are also observed in bioadhesives exuded by other climbing plants, the adhesion mechanisms revealed by English ivy may forward the progress toward understanding the general principles underlying diverse botanic adhesives.

Nicastrin is required for amyloid precursor protein (APP) but not Notch processing, while anterior pharynx-defective 1 is dispensable for processing of both APP and Notch.

The γ-secretase complex is composed of at least four components: presenilin 1 or presenilin-2, nicastrin (NCT), anterior pharynx-defective 1 (Aph-1), and presenilin enhancer 2. In this study, using knockout cell lines, our data demonstrated that knockout of NCT, as well as knockout of presenilin enhancer 2, completely blocked γ-secretase-catalyzed processing of C-terminal fragment (CTF)α and CTFß, the C-terminal fragments of ß-amyloid precursor protein (APP) produced by α-secretase and ß-secretase cleavages, respectively. Interestingly, in Aph-1-knockout cells, CTFα and CTFß were still processed by γ-secretase, indicating Aph-1 is dispensable for APP processing. Furthermore, our results indicate that Aph-1 as well as NCT is not absolutely required for Notch processing, suggesting that NCT is differentially required for APP and Notch processing. In addition, our data revealed that components of the γ-secretase complex are also important for proteasome- and lysosome-dependent degradation of APP and that endogenous APP is mostly degraded by lysosome while exogenous APP is mainly degraded by proteasome. There are unanswered questions regarding the roles of each component of the γ-secretase complex in amyloid precursor protein (APP) and Notch processing. The most relevant, novel finding of this study is that nicastrin (NCT) is required for APP but not Notch processing, while Aph-1 is not essential for processing of both APP and Notch, suggesting NCT as a therapeutic target to restrict Aß formation without impairing Notch signaling.

Cellular FLICE-like Inhibitory Protein (c-FLIP) and PS1-associated Protein (PSAP) Mediate Presenilin 1-induced γ-Secretase-dependent and -independent Apoptosis, Respectively.

Presenilin 1 (PS1) has been implicated in apoptosis; however, its mechanism remains elusive. We report that PS1-induced apoptosis was associated with cellular FLICE-like inhibitory protein (c-FLIP) turnover and that γ-secretase inhibitor blocked c-FLIP turnover and also partially blocked PS1-induced apoptosis. A complete inhibition of PS1-induced apoptosis was achieved by knockdown of PS1-associated protein (PSAP), a mitochondrial proapoptotic protein that forms a complex with Bax upon induction of apoptosis, in the presence of γ-secretase inhibitor. PS1-induced apoptosis was partially inhibited by knockdown of caspase-8, Fas-associated protein with death domain (FADD), or Bid. However, knockdown of Bax or overexpression of Bcl-2 resulted in complete inhibition of PS1-induced apoptosis. These data suggest that PS1 induces apoptosis through two pathways: the γ-secretase-dependent pathway mediated by turnover of c-FLIP and the γ-secretase-independent pathway mediated by PSAP-Bax complex formation. These two pathways converge on Bax to activate mitochondria-dependent apoptosis. These findings provide new insight into the mechanisms by which PS1 is involved in apoptosis and the mechanism by which PS1 exerts its pathogenic effects. In addition, our results suggest that PS2 induces apoptosis through a pathway that is different from that of PS1.

The matricellular protein Cyr61 is a key mediator of platelet-derived growth factor-induced cell migration.

Platelet-derived growth factor (PDGF), a potent chemoattractant, induces cell migration via the MAPK and PI3K/Akt pathways. However, the downstream mediators are still elusive. In particular, the role of extracellular mediators is largely unknown. In this study, we identified the matricellular protein Cyr61, which is de novo synthesized in response to PDGF stimulation, as the key downstream mediator of the ERK and JNK pathways, independent of the p38 MAPK and AKT pathways, and, thereby, it mediates PDGF-induced smooth muscle cell migration but not proliferation. Our results revealed that, when Cyr61 was newly synthesized by PDGF, it was promptly translocated to the extracellular matrix and physically interacted with the plasma membrane integrins α6ß1 and αvß3. We further demonstrate that Cyr61 and integrins are integral components of the PDGF signaling pathway via an "outside-in" signaling route to activate intracellular focal adhesion kinase (FAK), leading to cell migration. Therefore, this study provides the first evidence that the PDGF-induced endogenous extracellular matrix component Cyr61 is a key mediator in modulating cell migration by connecting intracellular PDGF-ERK and JNK signals with integrin/FAK signaling. Therefore, extracellular Cyr61 convergence with growth factor signaling and integrin/FAK signaling is a new concept of growth factor-induced cell migration. The discovered signaling pathway may represent an important therapeutic target in growth factor-mediated cell migration/invasion-related vascular diseases and tumorigenesis.

Inhibition of Egr1 expression underlies the anti-mitogenic effects of cAMP in vascular smooth muscle cells.

AIMS: Cyclic AMP inhibits vascular smooth muscle cell (VSMC) proliferation which is important in the aetiology of numerous vascular diseases. The anti-mitogenic properties of cAMP in VSMC are dependent on activation of protein kinase A (PKA) and exchange protein activated by cAMP (EPAC), but the mechanisms are unclear. METHODS AND RESULTS: Selective agonists of PKA and EPAC synergistically inhibited Egr1 expression, which was essential for VSMC proliferation. Forskolin, adenosine, A2B receptor agonist BAY60-6583 and Cicaprost also inhibited Egr1 expression in VSMC but not in endothelial cells. Inhibition of Egr1 by cAMP was independent of cAMP response element binding protein (CREB) activity but dependent on inhibition of serum response element (SRE) activity. SRF binding to the Egr1 promoter was not modulated by cAMP stimulation. However, Egr1 expression was dependent on the SRF co-factors Elk1 and 4 but independent of MAL. Inhibition of SRE-dependent Egr1 expression was due to synergistic inhibition of Rac1 activity by PKA and EPAC, resulting in rapid cytoskeleton remodelling and nuclear export of ERK1/2. This was associated with de-phosphorylation of the SRF co-factor Elk1. CONCLUSION: cAMP inhibits VSMC proliferation by rapidly inhibiting Egr1 expression. This occurs, at least in part, via inhibition of Rac1 activity leading to rapid actin-cytoskeleton remodelling, nuclear export of ERK1/2, impaired Elk1-phosphorylation and inhibition of SRE activity. This identifies one of the earliest mechanisms underlying the anti-mitogenic effects of cAMP in VSMC but not in endothelial cells, making it an attractive target for selective inhibition of VSMC proliferation.

Matricellular protein Cyr61 bridges lysophosphatidic acid and integrin pathways leading to cell migration.

Lysophosphatidic acid (LPA), a potent bioactive lipid found in atherosclerotic lesions, markedly induces smooth muscle cell (SMC) migration, which is an important process in atherogenesis. Therefore, understanding the mechanism of LPA-induced SMC migration is important. Several microarray databases suggest that the matricellular protein Cyr61 is highly induced by LPA. We hypothesized that Cyr61 mediates LPA-induced cell migration. Our data show that LPA induced temporal and spatial expression of Cyr61, which promptly accumulated in the cellular Golgi apparatus and then translocated to the extracellular matrix. Cyr61 antibody blockade and siRNA inhibition both diminished LPA-induced SMC migration, indicating a novel regulatory role of Cyr61. SMCs derived from LPA receptor 1 (LPA1) knock-out mice lack the ability of Cyr61 induction and cell migration, supporting the concept that LPA1 is required for Cyr61 expression and migration. By contrast, PPARγ was not found to be involved in LPA-mediated effects. Furthermore, focal adhesion kinase (FAK), a nonreceptor tyrosine kinase important for regulating cell migration, was activated by LPA at a late time frame coinciding with Cyr61 accumulation. Interestingly, knockdown of Cyr61 blocked LPA-induced FAK activation, indicating that an LPA-Cyr61-FAK axis leads to SMC migration. Our results further demonstrate that plasma membrane integrins α6ß1 and ανß3 transduced the LPA-Cyr61 signal toward FAK activation and migration. Taken together, these data reveal that de novo Cyr61 in the extracellular matrix bridges LPA and integrin pathways, which in turn, activate FAK, leading to cell migration. The current study provides new insights into mechanisms underlying cell migration-related disorders, including atherosclerosis, restenosis, and cancers.

Lysophosphatidic acid induces increased BACE1 expression and Aß formation.

The abnormal production and accumulation of ß-amyloid peptide (Aß), which is produced from amyloid precursor protein (APP) by the sequential actions of ß-secretase and γ-secretase, are thought to be the initial causative events in the development of Alzheimer's disease (AD). Accumulating evidence suggests that vascular factors play an important role in the pathogenesis of AD. Specifically, studies have suggested that one vascular factor in particular, oxidized low density lipoprotein (oxLDL), may play an important role in regulating Aß formation in AD. However, the mechanism by which oxLDL modulates Aß formation remains elusive. In this study, we report several new findings that provide biochemical evidence suggesting that the cardiovascular risk factor oxLDL may contribute to Alzheimer's disease by increasing Aß production. First, we found that lysophosphatidic acid (LPA), the most bioactive component of oxLDL induces increased production of Aß. Second, our data strongly indicate that LPA induces increased Aß production via upregulating ß-secretase expression. Third, our data strongly support the notion that different isoforms of protein kinase C (PKC) may play different roles in regulating APP processing. Specifically, most PKC members, such as PKCα, PKCß, and PKCε, are implicated in regulating α-secretase-mediated APP processing; however, PKCδ, a member of the novel PKC subfamily, is involved in LPA-induced upregulation of ß-secretase expression and Aß production. These findings may contribute to a better understanding of the mechanisms by which the cardiovascular risk factor oxLDL is involved in Alzheimer's disease.

PSAP induces a unique Apaf-1 and Smac-dependent mitochondrial apoptotic pathway independent of Bcl-2 family proteins.

Presenilin-associated protein (PSAP) has been identified as a mitochondrial proapoptotic protein. However, the mechanism by which PSAP induces apoptosis remains unknown. To this end, we have established an inducible expression system. Using this system, we have examined the roles of B-cell lymphoma 2 (Bcl-2) family proteins, cytochrome c, Smac (Smac/Diablo, second mitochondria-derived activator of caspases/direct IAP binding protein with low PI), and Apaf-1 (apoptotic protease-activating factor) in PSAP-induced apoptosis. Our results demonstrate that knockdown of Apaf-1 abolished PSAP-induced caspase activation and poly(ADP ribose) polymerase (PARP) cleavage, indicating that the apoptosome formation triggered by cytochrome c is crucial for PSAP-induced apoptosis. Our data also demonstrate that knockdown of Smac abolished PSAP-induced caspase activation and PARP cleavage, indicating that, in addition to Apaf-1 or apoptosome formation, Smac is also essential for PSAP-induced apoptosis. However, interestingly, our data demonstrate that overexpression of Bcl-2 and Bcl-xL did not protect cells from PSAP-induced apoptosis, and that knockdown of Bid, Bax, and Bak had no effect on PSAP-induced cytochrome c and Smac release, indicating that PSAP-induced apoptosis is not regulated by Bcl-2 family proteins. These results strongly suggest that PSAP evokes mitochondrial apoptotic cascades via a novel mechanism that is not regulated by Bcl-2 family proteins, but that both the formation of cytochrome c-Apaf-1 apoptosome and the presence of Smac are absolutely required for PSAP-induced apoptosis.

Histamine induces activation of protein kinase D that mediates tissue factor expression and activity in human aortic smooth muscle cells.

Histamine, an inflammatory mediator, has been shown to influence the pathogenesis of vascular wall cells. However, the molecular basis of its influence is not well understood. Our data reveal that histamine markedly induces protein kinase D (PKD) activation in human aortic smooth muscle cells. PKD belongs to a family of serine/threonine protein kinases, and its function in vascular disease is largely unknown. Our data show that histamine-induced PKD phosphorylation is dependent on the activation of histamine receptor 1 and protein kinase C (PKC). To determine the role of PKD in the histamine pathway, we employed a small-interfering RNA approach to downregulate PKD expression and found that PKD1 and PKD2 are key mediators for expression of tissue factor (TF), which is the key initiator of blood coagulation and is important for thrombosis. Our results show that PKD2 predominantly mediates histamine-induced TF expression via the p38 mitogen-activated protein kinase (MAPK) pathway, whereas PKD1 mediates histamine-induced TF expression through a p38 MAPK-independent pathway. We demonstrate that histamine induces TF expression via the PKC-dependent PKD activation. Our data provide the first evidence that PKD is a new component in histamine signaling in live cells and that PKD has a novel function in the histamine signaling pathway leading to gene expression, as evidenced by TF expression. Importantly, our data reveal a regulatory link from histamine to PKD and TF, providing new insights into the mechanisms of coagulation and the development of atherothrombosis.

Pen-2 is dispensable for endoproteolysis of presenilin 1, and nicastrin-Aph subcomplex is important for both γ-secretase assembly and substrate recruitment.

γ-secretase is a protease complex with at least four components: presenilin, nicastrin (NCT), anterior pharynx-defective 1 (Aph-1), and presenilin enhancer 2 (Pen-2). In this study, using knockout cell lines and small interfering RNA technology, our data demonstrated that the disappeared presenilin 1 C-terminal fragment (PS1C) caused by knockdown of pen-2 or knockout of NCT or Aph-1 was recovered by the addition of proteasome inhibitors, indicating that Pen-2, as well as NCT and Aph-1α, is dispensable for presenilin endoproteolysis. Our data also demonstrate that the formation of the nicastrin-Aph-1 subcomplex plays not only an important role in γ-secretase complex assembly but also in recruiting substrate C-terminal fragment of amyloid precursor protein generated by ß-cleavage. Ablating any one component resulted in the instability of other components of the γ-secretase complex, and the presence of all three of the other components is required for full maturation of NCT.