BBA - Molecular Cell Research (v.1813, #6)

Biphasic chromatin binding of histone chaperone FACT during eukaryotic chromatin DNA replication by Lena R. Kundu; Masayuki Seki; Nanae Watanabe; Hiromu Murofushi; Asako Furukohri; Shou Waga; Alan J. Score; J. Julian Blow; Masami Horikoshi; Takemi Enomoto; Shusuke Tada (1129-1136).
The facilitates chromatin transcription (FACT) complex affects nuclear DNA transactions in a chromatin context. Though the involvement of FACT in eukaryotic DNA replication has been revealed, a clear understanding of its biochemical behavior during DNA replication still remains elusive. Here, we analyzed the chromatin-binding dynamics of FACT using Xenopus egg extract cell-free system. We found that FACT has at least two distinct chromatin-binding phases: (1) a rapid chromatin-binding phase at the onset of DNA replication that did not involve origin licensing and (2) a second phase of chromatin binding that initiated after origin licensing. Intriguingly, early-binding FACT dissociated from chromatin when DNA replication was blocked by the addition of Cdc6 in the licensed state before origin firing. Cdc6-induced removal of FACT was blocked by the inhibition of origin licensing with geminin, but not by suppressing the activity of DNA polymerases, CDK, or Cdc7. Furthermore, chromatin transfer experiments revealed that impairing the later binding of FACT severely compromises DNA replication activity. Taken together, we propose that even though FACT has rapid chromatin-binding activity, the binding pattern of FACT on chromatin changes after origin licensing, which may contribute to the establishment of its functional link to the DNA replication machinery.► Histone chaperone FACT has at least two distinct chromatin-binding modes during DNA replication. ► An initial rapid binding of FACT independent of DNA replication events and a latter binding initiating after origin licensing essential for DNA replication. ► Cdc6 can inhibit the latter binding of FACT. ► This biphasic behavior of FACT may thus be a critical basis in elucidating the cross-talk between DNA replication and chromatin-modifying factors.
Keywords: FACT; Spt16; SSRP1; DUF; DNA replication; Origin licensing;

The role and therapeutic potential of prohibitin in disease by Arianne L. Theiss; Shanthi V. Sitaraman (1137-1143).
Prohibitin 1 (PHB1), a pleiotropic protein in the cell, has been implicated in the regulation of proliferation, apoptosis, transcription, mitochondrial protein folding, and as a cell-surface receptor. This diverse array of functions of PHB1 is attributed to the cell type studied and its subcellular localization. This review discusses recent data that indicate a diverse role of PHB1 in disease pathogenesis and suggest that targeting PHB1 may be a potential therapeutic option for treatment of diseases including cancer, inflammatory bowel disease, insulin resistance/type 2 diabetes, and obesity. These diseases are associated with increased oxidative stress and mitochondrial dysfunction and therefore, the role of PHB1 in both responses will also be discussed.► We examine the role of prohibitin 1 in the pathogenesis of cancer, obesity, diabetes, and colitis. ► Oxidative stress and mitochondrial dysfunction are associated with each of these diseases. ► Prohibitin 1 modulates oxidative stress and mitochondrial dysfunction. ► Therapeutic potential of targeting prohibitin 1 is discussed for cancer, obesity, diabetes, and colitis.
Keywords: Prohibitin; Therapeutic; Cancer; Inflammation; Obesity; Diabetes;

Changes in mitochondrial redox state, membrane potential and calcium precede mitochondrial dysfunction in doxorubicin-induced cell death by Andrey V. Kuznetsov; Raimund Margreiter; Albert Amberger; Valdur Saks; Michael Grimm (1144-1152).
Mitochondria play central roles in cell life as a source of energy and in cell death by inducing apoptosis. Many important functions of mitochondria change in cancer, and these organelles can be a target of chemotherapy. The widely used anticancer drug doxorubicin (DOX) causes cell death, inhibition of cell cycle/proliferation and mitochondrial impairment. However, the mechanism of such impairment is not completely understood. In our study we used confocal and two-photon fluorescence imaging together with enzymatic and respirometric analysis to study short- and long-term effects of doxorubicin on mitochondria in various human carcinoma cells. We show that short-term (< 30 min) effects include i) rapid changes in mitochondrial redox potentials towards a more oxidized state (flavoproteins and NADH), ii) mitochondrial depolarization, iii) elevated matrix calcium levels, and iv) mitochondrial ROS production, demonstrating a complex pattern of mitochondrial alterations. Significant inhibition of mitochondrial endogenous and uncoupled respiration, ATP depletion and changes in the activities of marker enzymes were observed after 48 h of DOX treatment (long-term effects) associated with cell cycle arrest and death.► Apoptotic cell death is associated with mitochondrial redox changes. ► Short- and long-term effects of doxorubicin on mitochondria are clearly distinguished. ► Changes in redox state, membrane potentials and calcium precede mitochondrial injury. ► Monitoring mitochondrial alterations is useful for detecting therapy responses in vivo. ► This may provide better understanding of the role of mitochondria in chemotherapy.
Keywords: Confocal imaging; Doxorubicin; Mitochondria; Mitochondrial function; Redox state; Reactive oxygen species, ROS;

The EGF-like proteins DLK1 and DLK2 function as inhibitory non-canonical ligands of NOTCH1 receptor that modulate each other's activities by Beatriz Sánchez-Solana; María Luisa Nueda; María Desamparados Ruvira; María José Ruiz-Hidalgo; Eva María Monsalve; Samuel Rivero; José Javier García-Ramírez; María José M. Díaz-Guerra; Victoriano Baladrón; Jorge Laborda (1153-1164).
The protein DLK2, highly homologous to DLK1, belongs to the EGF-like family of membrane proteins, which includes NOTCH receptors and their DSL-ligands. The molecular mechanisms by which DLK proteins regulate cell differentiation and proliferation processes are not fully established yet. In previous reports, we demonstrated that DLK1 interacts with itself and with specific EGF-like repeats of the NOTCH1 extracellular region involved in the binding to NOTCH1 canonical ligands. Moreover, the interaction of DLK1 with NOTCH1 caused an inhibition of basal NOTCH signaling in preadipocytes and mesenchymal multipotent cells. In this work, we demonstrate, for the first time, that DLK2 interacts with itself, with DLK1, and with the same NOTCH1 receptor region as DLK1 does. We demonstrate also that the interaction of DLK2 with NOTCH1 similarly results in an inhibition of NOTCH signaling in preadipocytes and Mouse Embryo fibloblasts. In addition, we demonstrate that a membrane DLK1 variant, lacking the sequence recognized by the protease TACE, also inhibits NOTCH signaling. Furthermore, both DLK1 and DLK2 are able to decrease NOTCH activity also when triggered by specific NOTCH ligands. However, the decrease in NOTCH signaling induced by overexpression of Dlk2 is reversed by the overexpression of Dlk1, and viceversa. We conclude that DLK1 and DLK2 act as inhibitory non-canonical protein ligands for the NOTCH1 receptor that modulate NOTCH signaling.► EGF-like protein DLK2 interacts with DLK1/Pref-1 and the EGF 10–15 region of NOTCH1 receptor. ► DLK2 protein inhibits NOTCH activity. ► DLK proteins compete with JAGGED1 and DLL4 canonical ligands for NOTCH receptor binding. ► The co-expresion of DLK1 and DLK2 results in higher levels of NOTCH activity.
Keywords: DLK1; DLK2; NOTCH signaling; NOTCH canonical ligand; Protein–protein interaction;

ERp29 regulates response to doxorubicin by a PERK-mediated mechanism by E. Farmaki; S. Mkrtchian; I. Papazian; A.G. Papavassiliou; H. Kiaris (1165-1171).
ERp29 is an endoplasmic reticulum (ER) luminal protein with a putative secretion factor/escort chaperone function. Accumulated evidence has implicated ERp29 in the thyroglobulin secretion, polyoma virus transport and recently in carcinogenesis. ERp29 levels were elevated in the tumors of various origins and under the conditions of genotoxic stress, such as ionizing radiation. Here we report the induction of ERp29 during the treatment of cells with doxorubicin, a commonly used antineoplastic agent. Experiments in the p53 −/− cells and p53 knockout mouse revealed that doxorubicin effect on ERp29 is p53 dependent. The increase of ERp29 level appears to activate a negative feedback loop where the elevated amounts of ERp29 augment cell viability as shown by a clonogenic cell survival assay. To elucidate the mechanisms behind the doxorubicin effects we have studied the impact of ERp29 on the interaction with the ER stress-activated eukaryotic translation initiation factor 2-alpha kinase 3 (PERK) that was shown to facilitate tumor cells' resistance to drug toxicity. Co-immunoprecipitation demonstrated physical interaction of ERp29 with PERK and moreover, overexpression of ERp29 enhanced endogenous levels of PERK. Our results identify ERp29 as a novel regulator of PERK and provide evidence for the role of ER resident factors in the regulation of chemotherapeutic efficacy. These findings show that PERK may represent a nodal point between ER stress and chemotherapeutic response.► Doxorubicin induces the levels of ERp29. ► ERp29 interacts physically with PERK. ► PERK levels are modulated by ERp29 expression. ► ERp29 modulates sensitivity to doxorubicin by a PERK-dependent manner.
Keywords: PERK; ERp29; ER stress; Chemotherapy; Adriamycin; UPR;

Role of somatostatin receptor 1 and 5 on epidermal growth factor receptor mediated signaling by Geetanjali Kharmate; Padmesh S. Rajput; Heather L. Watt; Rishi K. Somvanshi; Nicole Chaudhari; Xiaofan Qiu; Ujendra Kumar (1172-1189).
Epidermal growth factor (EGF) regulates normal and tumor cell proliferation via epidermal growth factor receptor (EGFR) phosphorylation, homo- or heterodimerization and activation of mitogen-activated protein kinases (MAPKs) and PI3K/AKT cell survival pathways. In contrast, SST via activation of five different receptor subtypes inhibits cell proliferation and has been potential target in tumor treatment. To gain further insight for the effect of SSTRs on EGFR activated signaling, we determine the role of SSTR1 and SSTR1/5 in human embryonic kidney (HEK) 293 cells. We here demonstrate that cells transfected with SSTR1 or SSTR1/5 negatively regulates EGF mediated effects attributed to the inhibition of EGFR phosphorylation, MAPKs as well as the cell survival signaling. Furthermore, SSTR effects were significantly enhanced in cells when EGFR was knock down using siRNA or treated with selective antagonist (AG1478). Most importantly, the presence of SSTR in addition to modulating signaling pathways leads to the dissociation of the constitutive and EGF induced heteromeric complex of EGFR/ErbB2. Furthermore, cells cotransfected with SSTR1/5 display pronounced effect of SST on the signaling and dissociation of the EGFR/ErbB2 heteromeric complex than the cells expressing SSTR1 alone. Taken together this study provides the first evidence that the presence of SSTR controls EGF mediated cell survival pathway via dissociation of ErbB heteromeric complex. We propose that the activation of SSTR and blockade of EGFR might serve novel therapeutic approach in inhibition of tumor proliferation.► In this study we demonstrate the activation of SSTRs inhibits epidermal growth factor mediated EGFR phosphorylation and mitogen-activated protein kinases. ► The presence of SSTRs blocks the epidermal growth factor mediated PI3K/AKT cell survival pathway. ► SSTRs in receptor-specific manner impede the epidermal growth factor stimulated heterodimerization between EGFR and ErbB2.
Keywords: Epidermal growth factor receptor; Mitogen-activated protein kinases; Photobleaching fluorescence resonance energy transfer; Somatostatin; Somatostatin receptor; Heterodimerization;

The focus of this research was to investigate the role of protein kinase C-iota (PKC-ι) in regulation of Bad, a pro-apoptotic BH3-only molecule of the Bcl-2 family in glioblastoma. Robust expression of PKC-ι is a hallmark of human glioma and benign and malignant meningiomas. The results were obtained from the two human glial tumor derived cell lines, T98G and U87MG. In these cells, PKC-ι co-localized and directly associated with Bad, as shown by immunofluorescence, immunoprecipitation, and Western blotting. Furthermore, in-vitro kinase activity assay showed that PKC-ι directly phosphorylated Bad at phospho specific residues, Ser-112, Ser-136 and Ser-155 which in turn induced inactivation of Bad and disruption of Bad/Bcl-XL dimer. Knockdown of PKC-ι by siRNA exhibited a corresponding reduction in Bad phosphorylation suggesting that PKC-ι may be a Bad kinase. PKC-ι knockdown also induced apoptosis in both the cell lines. Since, PKC-ι is an essential downstream mediator of the PI (3)-kinase, we hypothesize that glioma cell survival is mediated via a PI (3)-kinase/PDK1/PKC-ι/Bad pathway. Treatment with PI (3)-kinase inhibitors Wortmannin and LY294002, as well as PDK1 siRNA, inhibited PKC-ι activity and subsequent phosphorylation of Bad suggesting that PKC-ι regulates the activity of Bad in a PI (3)-kinase dependent manner. Thus, our data suggest that glioma cell survival occurs through a novel PI (3)-kinase/PDK1/PKC-ι/BAD mediated pathway.Display Omitted► PKC-ι co-localizes and directly associates with Bad in glioma cells. ► PKC-ι directly phosphorylates Bad at Ser-112, Ser-136 and Ser-155. ► Knockdown of PKC-ι leads to reduction in Bad phosphorylation. ► PI (3)-kinase and PDK1 inhibition blocks PKC-ι activity. ► PI (3)-kinase/PDK1/PKC-ι/Bad cell survival pathway in glioblastoma
Keywords: PKC-ι; Bad; PI (3)-kinase; Glioma; Cell survival;

Rin-like, a novel regulator of endocytosis, acts as guanine nucleotide exchange factor for Rab5a and Rab22 by Barbara Woller; Susan Luiskandl; Milica Popovic; Barbara E.M. Prieler; Gloria Ikonge; Michaela Mutzl; Holger Rehmann; Ruth Herbst (1198-1210).
RIN proteins serve as guanine nucleotide exchange factors for Rab5a. They are characterized by the presence of a RIN homology domain and a C-terminal Vps9 domain. Currently three family members have been described and analyzed. Here we report the identification of a novel RIN family member, Rin-like (Rinl), that represents a new interaction partner of the receptor tyrosine kinase MuSK, which is an essential key regulator of neuromuscular synapse development. Rinl is localized to neuromuscular synapses but shows the highest expression in thymus and spleen. Rinl preferentially binds to nucleotide-free Rab5a and catalyzes the exchange of GDP for GTP. Moreover, Rinl also binds GDP-bound Rab22 and increases the GDP/GTP exchange implicating Rinl in endocytotic processes regulated by Rab5a and Rab22. Interestingly, Rinl shows a higher catalytic rate for Rab22 compared to Rab5a. Rinl is closely associated with the cytoskeleton and thus contributes to the spatial control of Rab5a and Rab22 signaling at actin-positive compartments. Most importantly, overexpression of Rinl affects fluid-phase as well as EGFR endocytosis.► Rinl is a novel member of the RIN protein family and specifically binds MuSK. ► Rinl catalyzes the nucleotide exchange on Rab5a and Rab22. ► Rinl shows a higher catalytic activity for Rab22 than Rab5a. ► Rinl regulates the localization of Rab5a and Rab22 to actin-positive membrane ruffles. ► Overexpression of Rinl increases both fluid-phase and receptor-mediated endocytosis.
Keywords: Endocytosis; Rab5; GEF; MuSK; Receptor tyrosine kinase;

Hyperglycemia induces apoptosis of pancreatic islet endothelial cells via reactive nitrogen species-mediated Jun N-terminal kinase activation by Lei Gong; Fu-qiang Liu; Juan Wang; Xu-ping Wang; Xin-guo Hou; Yu Sun; Wei-dong Qin; Shu-jian Wei; Yun Zhang; Li Chen; Ming-Xiang Zhang (1211-1219).
Hyperglycemia significantly stimulates pancreatic islet endothelial cell apoptosis; however, the precise mechanisms are not fully understood. In the present study, treating pancreatic islet endothelial (MS-1) cells with high glucose (30 mmol/l) but not mannitol significantly increased the number of apoptotic cells as compared with a physiological glucose concentration (5.5 mmol/l). Hyperglycemia significantly stimulated the expression of inducible nitric oxide synthase (iNOS) and production of NO and peroxynitrite (ONOO), relevant to MS-1 cell apoptosis. Moreover, induced reactive nitrogen species (RNS) significantly increased the expression of bax, cleaved caspase-3 and poly adenosine diphosphate (ADP)-ribose polymerase (PARP) via JNK activation, but the expression of bcl-2 was not altered. Furthermore, SP600125 (a specific inhibitor of JNK) and 1400W (a specific inhibitor of iNOS) significantly attenuated cell apoptosis induced by high glucose. Therefore, hyperglycemia triggers MS-1 cell apoptosis by activating an intrinsic-dependent apoptotic pathway via RNS-mediated JNK activation.► Hyperglycemia-induced apoptosis in islet ECs is triggered by RNS. ► Apoptosis via an intrinsic-dependent apoptotic pathway. ► RNS regulate apoptosis via activation of JNK.
Keywords: Apoptosis; Hyperglycemia; Pancreatic islet endothelial cells; Reactive nitrogen species; JNK;

FTIR spectral signatures of mouse antral oocytes: Molecular markers of oocyte maturation and developmental competence by Diletta Ami; Paolo Mereghetti; Antonino Natalello; Silvia Maria Doglia; Mario Zanoni; Carlo Alberto Redi; Manuela Monti (1220-1229).
Mammalian antral oocytes with a Hoescht-positive DNA ring around the nucleolus (SN) are able to resume meiosis and to fully support the embryonic development, while oocytes with a non-surrounded nucleolus (NSN) cannot. Here, we applied FTIR microspectroscopy to characterize single SN and NSN mouse oocytes in order to try to elucidate some aspects of the mechanisms behind the different chromatin organization that impairs the full development of NSN oocyte-derived embryos. To this aim, oocytes were measured at three different stages of their maturation: just after isolation and classification as SN and NSN oocytes (time 0); after 10 h of in vitro maturation, i.e. at the completion of the metaphase I (time 1); and after 20 h of in vitro maturation, i.e. at the completion of the metaphase II (time 2). Significant spectral differences in the lipid (3050–2800 cm− 1) and protein (1700–1600 cm− 1) absorption regions were found between the two types of oocytes and among the different stages of maturation within the same oocyte type. Moreover, dramatic changes in nucleic acid content, concerning mainly the extent of transcription and polyadenylation, were detected in particular between 1000 and 800 cm− 1. The use of the multivariate principal component–linear discriminant analysis (PCA–LDA) enabled us to identify the maturation stage in which the separation between the two types of oocytes took place, finding as the most discriminating wavenumbers those associated to transcriptional activity and polyadenylation, in agreement with the visual analysis of the spectral data.► FTIR microspectroscopy has been applied to the study of single Surrounded Nucleolus (SN) and Not Surrounded Nucleolus (NSN) murine oocytes at three stages of their maturation. ► The two types of oocytes displayed differences in the lipid, protein, and in the nucleic absorption regions. ► The most significant differences concerned the IR bands reflecting transcriptional activity and polyadenylation. ► The multivariate statistical analysis allowed to identify the maturation stage where the separation between the two types of oocytes takes place.
Keywords: FTIR microspectroscopy; SN oocytes; NSN oocytes; Oocyte maturation; Developmental competence; Polyadenylation;

Glucose deprivation is associated with Chk1 degradation through the ubiquitin–proteasome pathway and effective checkpoint response to replication blocks by Ae Jeong Kim; Hyun-Ju Kim; Hye Jin Jee; Naree Song; Minjee Kim; Yoe-Sik Bae; Jay H. Chung; Jeanho Yun (1230-1238).
Chk1 plays a key role in the DNA replication checkpoint and in preserving genomic integrity. Previous studies have shown that reduced Chk1 function leads to defects in the checkpoint response and is closely associated with tumorigenesis. Here, we report that glucose deprivation caused the degradation of Chk1 protein without perturbing cell cycle progression. The induction of Chk1 degradation in response to glucose deprivation was observed in various cancer cell lines and in normal human fibroblasts. Therefore, it appears to be a universal phenomenon in mammalian cells. A specific proteasome inhibitor blocked glucose deprivation-induced Chk1 degradation. Ubiquitination of Chk1 was detected, indicating that the proteasome–ubiquitin pathway mediates Chk1 degradation upon glucose deprivation. Mechanistic studies have demonstrated that ATR-dependent phosphorylation of Chk1 at the Ser317 and Ser345 sites is not required, suggesting that the molecular mechanism for Chk1 degradation upon glucose deprivation is distinct from genotoxic stress-induced degradation. Under conditions of glucose deprivation, the cells manifested a defective checkpoint response to replication stress, camptothecin or hydroxyurea. The forced expression of Myc-Chk1 partially rescued the defective response to the replication block upon glucose deprivation. Taken together, our results indicate that glucose deprivation induces ubiquitin-mediated Chk1 degradation and defective checkpoint responses, implying its potential role in genomic instability and tumor development.► Chk1 protein levels are regulated differently depending on glucose concentrations. ► Chk1 is degraded through the ubiquitin-proteasome pathway upon glucose deprivation. ► The molecular mechanism is distinct from genotoxic stress-induced degradation. ► Glucose deprivation induces defective checkpoint response to replication blocks.
Keywords: Chk1; Glucose deprivation; Protein stability; Ubiquitin–proteasome pathway; Genomic instability;

Growth factor activates mitogen-activated protein kinase kinases to promote cell growth. Mitochondrial biogenesis is an integral part of cell growth. How growth factor regulates mitochondrial biogenesis is not fully understood. In this study, we found that mitochondrial mass was specifically reduced upon serum starvation and induced upon re-feeding with serum. Using mitogen-activated protein kinase kinases inhibitor U0126, we found that the mRNA expression levels of ATP synthase, cytochrome-C, mitochondrial transcription factor A, and mitofusin 2 were reduced. Since the transcriptional levels of these genes are under the control of peroxisome proliferator-activated receptor γ coactivator-1α and -1β (PGC-1α and PGC-1β), we examined and found that only the mRNA and protein levels of PGC-1β were suppressed. Importantly, over-expression of PGC-1β partially reversed the reduction of mitochondrial mass upon U0126 treatment. Thus, we conclude that mitogen-activated protein kinase kinases direct mitochondrial biogenesis through selectively inducing PGC-1β expression.► Mitochondrial mass is reduced by withdrawing serum and MEK1/2 inhibitor U0126. ► MEK1 or MEK2 over-expression induces mitochondrial biogenesis. ► U0126 specifically suppresses mitochondrial regulator PGC-1β expression. ► PGC-1β over-expression blunts the U0126-mediated mitochondrial mass reduction. ► MEK–ERK cascade directs mitochondrial biogenesis by inducing PGC-1β expression.
Keywords: MEK1/2; PGC-1β; Mitochondrial biogenesis;

TRIM29 negatively regulates p53 via inhibition of Tip60 by Takuya Sho; Tadasuke Tsukiyama; Tomonobu Sato; Takeshi Kondo; Jun Cheng; Takashi Saku; Masahiro Asaka; Shigetsugu Hatakeyama (1245-1253).
Ataxia-telangiectasia (AT) is an autosomal recessive genetic disease characterized by immunological deficiencies, neurological degeneration, developmental abnormalities and an increased risk of cancer. Ataxia-telangiectasia group D (ATDC) was initially described as a gene related to AT. Ataxia-telangiectasia group D, also known as TRIM29, is structurally a member of the tripartite motif (TRIM) family of proteins, some of which have been reported to be highly expressed in some human carcinomas, but the involvement of TRIM29 in carcinogenesis has not been fully elucidated. In this study, we found by using yeast two-hybrid screening that TRIM29 binds to Tip60, which has been reported as a cellular acetyltransferase protein. Overexpression of TRIM29 promoted degradation and changed localization of Tip60 and reduced acetylation of p53 at lysine 120 by Tip60, resulting in enhancement of cell growth and transforming activity. In addition, we found that TRIM29 suppresses apoptosis induced by UV irradiation in HCT116 cell lines. These findings suggest that TRIM29 functions as an oncogene that promotes tumor growth.► TRIM29 binds to Tip60, which functions as a cellular acetyltransferase. ► Overexpression of TRIM29 promotes degradation and changes localization of Tip60. ► TRIM29 reduces acetylation of p53 at lysine 120 by Tip60. ► TRIM29 suppresses apoptosis induced by UV irradiation in HCT116 cell lines.
Keywords: TRIM29; p53; Tip60; Ubiquitin; Ataxia-telangiectasia;

Tissue factor pathway inhibitor 2 is induced by thrombin in human macrophages by Jordi Pou; Alba Rebollo; Lídia Piera; Manuel Merlos; Núria Roglans; Juan C. Laguna; Marta Alegret (1254-1260).
Tissue factor pathway inhibitor 2 (TFPI2) is a serine protease inhibitor critical for the regulation of extracellular matrix remodeling and atherosclerotic plaque stability. Previously, we demonstrated that TFPI2 expression is increased in monocytes from patients with familial combined hyperlipidemia (FCH). To gain insight into the molecular mechanisms responsible for this upregulation, we examined TFPI2 expression in THP-1 macrophages exposed to lipoproteins and thrombin. Our results showed that TFPI2 expression was not affected by treatment with very low density lipoproteins (VLDL), but was induced by thrombin (10 U/ml) in THP-1 (1.9-fold increase, p  < 0.001) and human monocyte-derived macrophages (2.3-fold increase, p  < 0.005). The specificity of the inductive effect was demonstrated by preincubation with the thrombin inhibitors hirudin and PPACK, which ablated thrombin effects. TFPI2 induction was prevented by pre-incubation with MEK1/2 and JNK inhibitors, but not by the EGF receptor antagonist AG1478. In the presence of parthenolide, an inhibitor of NFκB, but not of SR-11302, a selective AP-1 inhibitor, thrombin-mediated TFPI2 induction was blunted. Our results also show that thrombin treatment increased ERK1/2, JNK and IκBα phosphorylation. Finally, we ruled out the possibility that TFPI2 induction by thrombin was mediated by COX-2, as preincubation with a selective COX-2 inhibitor did not prevent the inductive effect. In conclusion, thrombin induces TFPI2 expression by a mechanism involving ERK1/2 and JNK phosphorylation, leading finally to NFkB activation. In the context of atherosclerosis, thrombin-induced macrophage TFPI2 expression could represent a means of avoiding excessive activation of matrix metalloproteases at sites of inflammation.► TFPI2 expression is upregulated in human macrophages by thrombin exposure. ► Thrombin induced TFPI2 expression involves cAMP, ERK1/2 and JNK pathways, leading finally to NFkB activation. ► TFPI2 induction by thrombin is independent of EGFR and COX-2, and does not involve AP-1 activation. ► In the context of atherosclerosis TFPI2 induction could represent a regulatory mechanism to avoid excessive MMP activation.
Keywords: Human macrophage; Lipoprotein; Thrombin; Tissue factor pathway inhibitor 2;