N-p-Tosyl-L-phenylalanine chloromethyl ketone (TPCK) inhibits HIV-1 by suppressing the activity of viral protease.

Human Immunodeficiency Virus (HIV), the etiological agent for Acquired Immunodeficiency Syndrome (AIDS), continues to kill humans despite stupendous advances in antiviral research. With the presently available combination antiretroviral therapeutic arsenal, AIDS is now a manageable disease but with no cure available till date. The development of novel antivirals consumes an extensive amount of time and resources. Hence, repurposing of the established gold standard molecules for their anti-HIV application is enormously advantageous. In this study, we report that N-p-Tosyl-L-phenylalanine chloromethyl ketone (TPCK) inhibits HIV-1 replication in a highly-conserved manner. Further, TPCK inhibits HIV-1 replication at the late stages of its life cycle by impeding viral protease (PR) enzyme activity. Additionally, our results demonstrate that the combination of TPCK with established HIV-1 PR inhibitors shows significant synergistic inhibitory potential, suggesting the potential use of TPCK in cART regimen. Collectively, we report the anti-HIV activity of TPCK, which should be further characterized for its translational applications.

MG132 exerts anti-viral activity against HSV-1 by overcoming virus-mediated suppression of the ERK signaling pathway.

Herpes simplex virus 1 (HSV-1) causes a number of clinical manifestations including cold sores, keratitis, meningitis and encephalitis. Although current drugs are available to treat HSV-1 infection, they can cause side effects such as nephrotoxicity. Moreover, owing to the emergence of drug-resistant HSV-1 strains, new anti-HSV-1 compounds are needed. Because many viruses exploit cellular host proteases and encode their own viral proteases for survival, we investigated the inhibitory effects of a panel of protease inhibitors (TLCK, TPCK, E64, bortezomib, or MG132) on HSV-1 replication and several host cell signaling pathways. We found that HSV-1 infection suppressed c-Raf-MEK1/2-ERK1/2-p90RSK signaling in host cells, which facilitated viral replication. The mechanism by which HSV-1 inhibited ERK signaling was mediated through the polyubiquitination and proteasomal degradation of Ras-guanine nucleotide-releasing factor 2 (Ras-GRF2). Importantly, the proteasome inhibitor MG132 inhibited HSV-1 replication by reversing ERK suppression in infected cells, inhibiting lytic genes (ICP5, ICP27 and UL42) expression, and overcoming the downregulation of Ras-GRF2. These results indicate that the suppression of ERK signaling via proteasomal degradation of Ras-GRF2 is necessary for HSV-1 infection and replication. Given that ERK activation by MG132 exhibits anti-HSV-1 activity, these results suggest that the proteasome inhibitor could serve as a novel therapeutic agent against HSV-1 infection.

The serine peptidase inhibitor TPCK induces several morphophysiological changes in the opportunistic fungal pathogen Candida parapsilosis sensu stricto.

Candida parapsilosis sensu stricto (C. parapsilosis) has emerged as the second/third commonest Candida species isolated from hospitals worldwide. Candida spp. possess numerous virulence attributes, including peptidases that play multiple roles in both physiological and pathological events. So, fungal peptidases are valid targets for new drugs development. With this premise in mind, we have evaluated the effect of serine peptidase inhibitors (SPIs) on both cell biology and virulence aspects of C. parapsilosis. First, five different SPIs, phenylmethylsulfonyl fluoride, benzamidine, 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride, N-α-tosyl-L-lysine chloromethyl ketone hydrochloride, and N-tosyl-L-phenylalanine chloromethyl ketone (TPCK) were tested, and TPCK showed the best efficacy to arrest fungal growth. Subsequently, the ability of TPCK to modulate physiopathological processes was investigated. Overall, TPCK was able to (i) inhibit the cell-associated serine peptidase activities, (ii) promote morphometric and ultrastructural alterations, (iii) induce an increase in the intracellular oxidation level, which culminates in a vigorous lipid peroxidation and accumulation of neutral lipids in cytoplasmic inclusions, (iv) modulate the expression/exposition of surface structures, such as mannose/glucose-rich glycoconjugates, N-acetylglucosamine-containing molecules, chitin, polypeptides and surface aspartic peptidases, (v) reduce the adhesion to either polystyrene or glass surfaces as well as to partially disarticulate the mature biofilm, (vi) block the fungal interaction with macrophages, and (vii) protect Galleria mellonella from fungal infection, enhancing larvae survivability. Altogether, these results demonstrated that TPCK induced several changes over fungal biology besides the interference with aspects associated to C. parapsilosis virulence and pathogenesis, which indicates that SPIs could be novel promising therapeutic agents in dealing with candidiasis.

Pterostilbene inhibits amyloid-ß-induced neuroinflammation in a microglia cell line by inactivating the NLRP3/caspase-1 inflammasome pathway.

Neuroinflammation has been known as an important pathogenetic contributor of Alzheimer's disease (AD). Pterostilbene is a natural compound which has neuroprotective activity. However, the effect of pterostilbene on amyloid-ß (Aß)-induced neuroinflammation has not been clarified. The aim of the present study was to investigate the effect of pterostilbene on Aß-induced neuroinflammation in microglia. The results indicated that pterostilbene attenuated Aß1-42 -induced cytotoxicity of BV-2 cells. Aß1-42 induced NO production and iNOS mRNA and protein expression, while pterostilbene inhibited the induction. The expression and secretion levels of IL-6, IL-1ß, and TNF-α were enhanced by Aß1-42 treatment, whereas pterostilbene decreased them. Aß1-42 activated NLRP3/caspase-1 inflammasome, which was inactivated by pterostilbene. In addition, the inhibitor of caspase-1 Z-YVAD-FMK attenuated the Aß1-42 -induced neuroinflammation in BV-2 cells. In conclusion, pterostilbene attenuated the neuroinflammatory response induced by Aß1-42 in microglia through inhibiting the NLRP3/caspase-1 inflammasome pathway, indicating that pterostilbene might be an effective therapy for AD.

Laquinimod decreases Bax expression and reduces caspase-6 activation in neurons.

Laquinimod is an immunomodulatory compound that has shown neuroprotective benefits in clinical trials for multiple sclerosis. Laquinimod ameliorates both white and gray matter damage in human patients, and prevents axonal degeneration in animal models of multiple sclerosis. Axonal damage and white matter loss are a common feature shared between different neurodegenerative diseases. Caspase-6 activation plays an important role in axonal degeneration on the molecular level. Increased activity of caspase-6 has been demonstrated in brain tissue from presymptomatic Huntington disease mutation carriers, and it is an early marker of axonal dysfunction. Since laquinimod is currently undergoing a clinical trial in Huntington disease (LEGATO-HD, clinicaltrials.gov ID: NCT02215616), we set out to evaluate its impact on neuronal caspase-6 activation. We find that laquinimod ameliorates DNA-damage induced activation of caspase-6 in primary neuronal cultures. This is an indirect effect that is not mediated by direct inhibition of the enzyme. The investigation of potential caspase-6 activating mechanisms revealed that laquinimod reduces the expression of Bax, a pro-apoptotic molecule that causes mitochondrial cytochrome c release and caspase activation. Bax expression is furthermore increased in striatal tissues from the YAC128 mouse model of HD in an age-dependent manner. Our results demonstrate that laquinimod can directly downregulate neuronal apoptosis pathways relevant for axonal degeneration in addition to its known effects on astrocytes and microglia in the CNS. It targets a pathway that is relevant for the pathogenesis of HD, supporting the hypothesis that laquinimod may provide clinical benefit.

Nicotine-Mediated Ca(2+)-Influx Induces IL-8 Secretion in Oral Squamous Cell Carcinoma Cell.

Cigarette smoking is one of the most important risk factors for the development of various diseases. Nicotine is the most extensively investigated component of cigarette smoke, and a comprehensive analysis of the genes induced by nicotine stimulation revealed that interleukin-8 (IL-8) was induced in oral squamous cell carcinoma cell (OSCC). Based on this background, the signaling mechanisms of nicotine-mediated IL-8 induction in OSCC was investigated. Augmented IL-8 secretion by Ca9-22 cells was blocked by the NF-κB inhibitor L-1-4'-tosylamino-phenylethyl-chloromethyl ketone (TPCK) and the nicotinic acetylcholine receptor (nAChR)-specific inhibitor α-bungarotoxin (αBtx). The downstream signaling pathway was further examined by pre-incubating the cells with inhibitors against mitogen-activated protein kinase (MEK), protein kinase C (PKC), and Ca(2+)/calmodulin-dependent kinase II (CaMK II). Only the CaMK II inhibitor was found to exert an inhibitory effect on nicotine-mediated IL-8 secretion. Pre-treatment of the Ca9-22 cells with the Ca(2+) chelator BAPTA-AM drastically inhibited IL-8 secretion. Although nicotine stimulation induced the phosphorylation of the NF-κB p65 subunit, pre-treatment with BAPTA-AM was found to inhibit this activity significantly. CaMK II-dependent p65 phosphorylation was confirmed by pre-incubation of the cells with CaMK II inhibitor. The results from this study indicate that the binding of nicotine to nAChR induces Ca(2+) influx, which results in the activation and phosphorylation of CaMK II and NF-κB p65, respectively. Nicotine-mediated IL-8 induction should be a trigger for the initiation of various diseases.

Heterogeneity of subordination of the IL-18/IFN-γ axis to caspase-1 among patients with Crohn's disease.

In Crohn's disease (CD), hierarchical architecture of the inflammatory network, including subordination of IL-18, an IFN-γ-inducing cytokine, to the inflammasome, have remained undeciphered. Heterogeneity among patients of such a subordination cannot be evaluated by animal models, monofactorial in their etiology and homogenous in disease progression. To address these issues, we set up an ex vivo model of inflamed mucosa explant cultures from patients with active long-standing CD. Th1 cytokine production, especially IFN-γ and IL-18, was assessed in relation with inflammation intensity. Subordination of the Th1 response to caspase-1, effector of the inflammasome, was determined in explant cultures subjected to pharmacological inhibition of caspase-1 by YVAD. We showed a correlation between secreted IFN-γ/IL-18 levels, and caspase-1 activation, with inflammation intensity of intestinal CD mucosa explants. Inhibition of caspase-1 activation using the specific inhibitor YVAD identified a homogenous non responder group featuring a caspase-1-independent IL-18/IFN-γ response, and a heterogenous responder group, in which both IL-18 and IFN-γ responses were caspase-1-dependent, with a 40-70% range of inhibition by YVAD. These findings bring out the concept of heterogeneity of subordination of the Th1 response to inflammasome activation among CD patients. This ex vivo model should have therapeutic relevance in allowing to determine eligibility of CD patients for new targeted therapies.

A critical assessment of the effect of serine protease inhibitors on porcine fertilization and quality parameters of porcine spermatozoa in vitro.

Proteases play an important role during mammalian fertilization. Their function is frequently investigated using specific inhibitors. We analyzed four serine protease inhibitors [4-(2-aminoethyl) benzene sulfonyl fluoride hydrochloride (AEBSF), soybean trypsin inhibitor from glycine max (STI), Nα-tosyl-L-lysine-chloromethyl ketone hydrochloride (TLCK) and N(p)-tosyl-L-phenylalanine-chloromethyl ketone (TPCK)] for their in vitro effect on fertilization and sperm quality in pigs. Inhibitor concentrations were chosen based on the reduction of fertilization rate during preliminary dose-response experiments with cryopreserved epididymal spermatozoa. The inhibitor effects on in vitro fertilization (IVF) and sperm parameters (membrane and acrosomal integrity, motility and mitochondrial membrane potential - MMP) were evaluated using diluted fresh semen. AEBSF (100 µM), TLCK (100 µM) and TPCK (100 µM) decreased total fertilization and polyspermy rates by at least 50%. STI (5 µM) lowered total fertilization rates but not the level of polyspermy. AEBSF and TPCK reduced fertilization parameters to a similar degree using cryopreserved epididymal spermatozoa (dose-response experiment) or diluted fresh semen. Inhibition by STI was more pronounced using cryopreserved epididymal spermatozoa, whereas TLCK inhibited IVF only with diluted fresh semen. AEBSF and STI had no effect on sperm parameters, and TLCK significantly reduced motility. TPCK diminished MMP and motility and affected membrane and acrosomal integrity in a negative way. In summary, serine protease inhibitors differed in the way they reduce the fertilization rate. These results emphasize the necessity of inhibitor testing before they can be applied in fertilization studies. AEBSF and STI can be used in the future IVF studies without compromising sperm quality.

Fine-tuning of NFκB by glycogen synthase kinase 3ß directs the fate of glomerular podocytes upon injury.

Nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) is regulated by a myriad of signaling cascades including glycogen synthase kinase (GSK) 3ß and plays a Janus role in podocyte injury. In vitro, lipopolysaccharide (LPS) or adriamycin (ADR) elicited podocyte injury and cytoskeletal disruption, associated with NFκB activation and induced expression of NFκB target molecules, including pro-survival Bcl-xL and podocytopathic mediators like MCP-1, cathepsin L, and B7-1. Broad-range inhibition of NFκB diminished the expression of all NFκB target genes, restored cytoskeleton integrity, but potentiated apoptosis. In contrast, blockade of GSK3ß by lithium or 4-benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione (TDZD-8) mitigated the expression of podocytopathic mediators, ameliorated podocyte injury, but barely affected Bcl-xL expression or sensitized apoptosis. Mechanistically, GSK3ß was sufficient and essential for RelA/p65 phosphorylation, specifically at serine 467, which specifies the expression of selective NFκB target molecules, including podocytopathic mediators, but not Bcl-xL. In vivo, lithium or TDZD-8 therapy improved podocyte injury and proteinuria in mice treated with LPS or ADR, concomitant with the suppression of podocytopathic mediators, but retained Bcl-xL in glomerulus. Broad-range inhibition of NFκB conferred similar but much weakened antiproteinuric and podoprotective effects accompanied with a blunted glomerular expression of Bcl-xL and marked podocyte apoptosis. Thus, the GSK3ß-dictated fine-tuning of NFκB may serve as a novel therapeutic target for podocytopathy.

Serine protease inhibitors interact with IFN-γ through up-regulation of FasR; a novel therapeutic strategy against cancer.

Among the many immunomodulatory and anti-tumor activities, IFN-γ up-regulates tumor cell death mediated by Fas receptor (FasR). Our and several other studies have demonstrated the involvement of trypsin-like proteases (TLPs) in the mode of action of IFN-γ. In the present study, we tried to unravel the role of serine proteases in IFN-γ induced Fas-mediated cell death. Our present results show that both tosyl-l-Lysine chloromethylketone (TLCK), a trypsin like protease inhibitor and tosyl-l-phenylalanine chloromethylketone (TPCK) - a chymotrypsin like protease (CLP) inhibitor, sensitize HeLa cells to Fas-mediated cell death. The combined effect of these protease inhibitors with anti-Fas was stronger than additive. In contrast, elastase inhibitor III (EI), which also contains the chloromethyl ketone moiety, was not active. Furthermore, co-addition of TLCK or TPCK with IFN-γ markedly enhanced Fas-induced cell death. IFN-γ led to up-regulation of FasR on its own, which was further enhanced by the co-addition of TLCK or TPCK. This was evident both by increased expression of Fas receptor on cell surface and by elevated Fas mRNA level. This study may provide the basis for the design of a novel combinatory therapeutic strategy that could enhance the eradication of tumors.

The serine protease inhibitor TLCK attenuates intrinsic death pathways in neurons upstream of mitochondrial demise.

It is well-established that activation of proteases, such as caspases, calpains and cathepsins are essential components in signaling pathways of programmed cell death (PCD). Although these proteases have also been linked to mechanisms of neuronal cell death, they are dispensable in paradigms of intrinsic death pathways, e.g. induced by oxidative stress. However, emerging evidence implicated a particular role for serine proteases in mechanisms of PCD in neurons. Here, we investigated the role of trypsin-like serine proteases in a model of glutamate toxicity in HT-22 cells. In these cells glutamate induces oxytosis, a form of caspase-independent cell death that involves activation of the pro-apoptotic protein BH3 interacting-domain death agonist (Bid), leading to mitochondrial demise and ensuing cell death. In this model system, the trypsin-like serine protease inhibitor Nα-tosyl-l-lysine chloromethyl ketone hydrochloride (TLCK) inhibited mitochondrial damage and cell death. Mitochondrial morphology alterations, the impairment of the mitochondrial membrane potential and ATP depletion were prevented and, moreover, lipid peroxidation induced by glutamate was completely abolished. Strikingly, truncated Bid-induced cell death was not affected by TLCK, suggesting a detrimental activity of serine proteases upstream of Bid activation and mitochondrial demise. In summary, this study demonstrates the protective effect of serine protease inhibition by TLCK against oxytosis-induced mitochondrial damage and cell death. These findings indicate that TLCK-sensitive serine proteases play a crucial role in cell death mechanisms upstream of mitochondrial demise and thus, may serve as therapeutic targets in diseases, where oxidative stress and intrinsic pathways of PCD mediate neuronal cell death.

Epinephrine evokes renalase secretion via α-adrenoceptor/NF-κB pathways in renal proximal tubular epithelial cells.

BACKGROUND/AIMS: Renalase is a recently discovered, kidney-specific monoamine oxidase that metabolizes circulating catecholamines. These findings present new insights into hypertension and chronic kidney diseases. Previous data demonstrated that renalase was mainly secreted from proximal tubules which could be evoked by catecholamines. The purpose of this study is to investigate whether renalase expression is induced by epinephrine via α-adrenoceptor/NFκB pathways. METHODS: HK2 cells were utilized to explore renalase expression in response to epinephrine in vitro. Phentolamine, an α-adrenoceptor antagonist, and Tosyl Phenylalanyl Chloromethyl Ketone (TPCK) were used to block α-adrenoceptor and to knock down the transcription factor NFκB, respectively. Renalase expression was analyzed using Western blot and quantitative PCR. RESULTS: Both protein and mRNA levels of renalase in HK2 cells increased in response to epinephrine (P<0.05). Epinephrine-evoked renalase expression was attenuated by phentolamine and TPCK separately (P<0.05). CONCLUSION: Epinephrine evokes renalase secretion via α-adrenoceptor/NF-κB pathways in renal proximal tubular epithelial cells.

Regulation of autophagy by α1-antitrypsin: "a foe of a foe is a friend".

Autophagy is involved in both the cell protective and the cell death process but its mechanism is largely unknown. The present work unravels a novel intracellular mechanism by which the serpin α1-antitrypsin (AAT) acts as a novel negative regulator of autophagic cell death. For the first time, the role of intracellularly synthesized AAT, other than in liver cells, is demonstrated. Autophagic cell death was induced by N-α-tosyl-L-phenylalanine chloromethyl ketone (TPCK) and tamoxifen. By utilizing a fluorescently tagged TPCK analog, AAT was "fished out" (pulled out) as a TPCK intracellular protein target. The interaction was further verified by competition binding experiments. Both inducers caused downregulation of AAT expression associated with activation of trypsin-like proteases. Furthermore, silencing AAT by siRNA induced autophagic cell death. Moreover, AAT administration to cultured cells prevented autophagic cell death. This new mechanism could have implications in the treatment of diseases by the regulation of AAT levels in which autophagy has a detrimental function. Furthermore, the results imply that the high synthesis of endogenous AAT by cancer cells could provide a novel resistance mechanism of cancer against autophagic cell death.

Protease in sturgeon sperm and the effects of protease inhibitors on sperm motility and velocity.

In mammals, proteases are present in sperm acrosome and play key role in fertilization. Sturgeon sperm has an acrosome, but its physiology, biochemistry, and potential role in fertilization are unknown. In the present study, we have observed high protease activity in acidic extract of intact sperm compared to that of seminal plasma in sterlet (Acipenser ruthenus). The protease activity was decreased and increased in acidic extract of motility-activated sperm and in the activation medium, respectively. Molecular analysis revealed total protease and serine (acrosin) protease activities in sperm acidic extract which was accumulated in a protein band with relative molecular mass of 35 kDa. Immunoelectron microscopy using an affinity-purified polyclonal antibody for boar acrosin localized the protease at the acrosome region. Moreover, initiation of sperm motility was inhibited after activation in the presence of inhibitors for both trypsin-like and chymotrypsin-like proteases, while the effects of protease inhibitors on sperm velocity were uncertain. Our results indicate similarities in physiology and biochemistry of acrosome between sturgeon and mammals and suggest potential role of protease in the initiation of sperm motility in sturgeon.

The cellular inhibitor of apoptosis protein-2 is a possible target of novel treatment for endometriosis.

PROBLEM: How is the tumor necrosis factor (TNF) α-induced inhibitor of apoptosis (IAP) protein expression in endometriotic stromal cells (ESCs) involved in cell viability and signaling pathways? METHOD OF STUDY: Endometriotic stromal cells were isolated from ovarian chocolate cysts in 20 patients who underwent laparoscopic surgery. IAP protein expression and IκB phosphorylation were evaluated by Western blot analysis. Interleukin (IL)-8 protein expression and cell proliferation were assessed by ELISA. RESULTS: Cellular IAP (cIAP)-2 protein expression in endometriotic tissue was higher than that of endometrium. TNFα markedly enhanced cIAP-2 protein expression in ESCs. Pretreatment with a nuclear factor (NF)-κB inhibitor attenuated TNFα-induced cIAP-2 expression. An antagonist of IAPs abrogated TNFα-induced cIAP-2 protein expression and showed a decrease in TNFα-induced IL-8 protein expression and BrdU incorporation in ESCs. CONCLUSIONS: TNFα and its downstream NFκB pathway have proven to be critical regulators of highly expressed cIAP-2 in ESCs. cIAP-2 may be a novel therapeutic target for endometriosis.

Platelets mediate increased endothelium permeability in dengue through NLRP3-inflammasome activation.

Dengue is the most frequent hemorrhagic viral disease and re-emergent infection in the world. Although thrombocytopenia is characteristically observed in mild and severe forms of dengue, the role of platelet activation in dengue pathogenesis has not been fully elucidated. We hypothesize that platelets have major roles in inflammatory amplification and increased vascular permeability during severe forms of dengue. Here we investigate interleukin (IL)-1ß synthesis, processing, and secretion in platelets during dengue virus (DV) infection and potential contribution of these events to endothelial permeability during infection. We observed increased expression of IL-1ß in platelets and platelet-derived microparticles from patients with dengue or after platelet exposure to DV in vitro. We demonstrated that DV infection leads to assembly of nucleotide-binding domain leucine rich repeat containing protein (NLRP3) inflammasomes, activation of caspase-1, and caspase-1-dependent IL-1ß secretion. Our findings also indicate that platelet-derived IL-1ß is chiefly released in microparticles through mechanisms dependent on mitochondrial reactive oxygen species-triggered NLRP3 inflammasomes. Inflammasome activation and platelet shedding of IL-1ß-rich microparticles correlated with signs of increased vascular permeability. Moreover, microparticles from DV-stimulated platelets induced enhanced permeability in vitro in an IL-1-dependent manner. Our findings provide new evidence that platelets contribute to increased vascular permeability in DV infection by inflammasome-dependent release of IL-1ß.

A novel chemical screening strategy in zebrafish identifies common pathways in embryogenesis and rhabdomyosarcoma development.

The zebrafish is a powerful genetic model that has only recently been used to dissect developmental pathways involved in oncogenesis. We hypothesized that operative pathways during embryogenesis would also be used for oncogenesis. In an effort to define RAS target genes during embryogenesis, gene expression was evaluated in Tg(hsp70-HRAS(G12V)) zebrafish embryos subjected to heat shock. dusp6 was activated by RAS, and this was used as the basis for a chemical genetic screen to identify small molecules that interfere with RAS signaling during embryogenesis. A KRAS(G12D)-induced zebrafish embryonal rhabdomyosarcoma was then used to assess the therapeutic effects of the small molecules. Two of these inhibitors, PD98059 and TPCK, had anti-tumor activity as single agents in both zebrafish embryonal rhabdomyosarcoma and a human cell line of rhabdomyosarcoma that harbored activated mutations in NRAS. PD98059 inhibited MEK1 whereas TPCK suppressed S6K1 activity; however, the combined treatment completely suppressed eIF4B phosphorylation and decreased translation initiation. Our work demonstrates that the activated pathways in RAS induction during embryogenesis are also important in oncogenesis and that inhibition of these pathways suppresses tumor growth.

Angiogenic changes in co-cultures of mast cells and myocardial microvascular endothelial cells under hyperglycemic conditions.

The aim of the present study was to determine the correlation between angiogenesis and the differential expression of growth factors and their receptors when myocardial microvascular endothelial cells (MMVECs) were co-cultured with mast cell granules (MCGs) under hyperglycemic conditions. MMVECs and mast cells (MCs) were isolated from Wistar rats. An in vitro angiogenesis assay was used to observe any differences when MMVECs were co-cultured with MCGs in normal or hyperglycemic medium. The mRNA and protein expression of growth factors and their receptors were analyzed by real-time reverse transcription (RT)-PCR and western blot analysis. Real-time RT-PCR analysis demonstrated the upregulated mRNA and protein expression of vascular endothelial growth factor (VEGF) in the MMVECs; however, the expression of its receptor, fms-like tyrosine kinase-1 (Flt-1) and fetal liver kinase-1 (Flk­1), decreased significantly, and the angiogenic ability of the MMVECs decreased under hyperglycemic conditions. The angiogenic ability of the MMVECs cultured under hyperglycemic conditions (even after the addition of MCGs) was inferior to that of the MMVECs cultured under normal glucose conditions. The specific inhibitor of tryptase, N-tosyl-L-lysine chloromethyl ketone (TLCK), suppressed angiogenesis regardless of the glucose concentration, and the specific inhibitor of chymase, N-tosyl-L-phenylalanyl chloromethyl ketone (TPCK), was not as effective as TLCK, which was mainly detected under hyperglycemic conditions. High glucose levels have a profound effect on angiogenesis; this effect may be more pronounced than the effects of MCGs on angiogenesis.

CpG ODN-induced matrix metalloproteinase-13 expression is mediated via activation of the ERK and NF-κB signalling pathways in odontoblast cells.

AIM: To investigate the effects of CpG ODN (CpG oligodeoxynucleotides) to model the action of bacterial challenge on pulpal matrix metalloproteinase-13 (MMP-13) expression and elucidate the associated intracellular signalling pathways. METHODOLOGY: Real-time PCR was used to detect the effects of CpG ODN on MMP-13 mRNA expression levels in a murine odontoblast-lineage cell line (OLCs). The possible involvement of TLR9/MyD88, NF-κB or MAPK pathways involved in the CpG ODN-induced MMP-13 expression was examined by real-time PCR, transient transfection, luciferase activity assay and ELISA. Western blotting was performed to assay the phosphorylation of ERK at a range of time points. RESULTS: MMP-13 was constitutively expressed in OLCs, and their exposure to CpG ODN significantly increased MMP-13 expression. Pre-treatment of OLCs with the inhibitory peptide MyD88, or chloroquine, attenuated the CpG ODN-induced expression of MMP-13. Treatment of the OLCs with CpG ODN increased NF-κB-luciferase activity. This activity was decreased by the over-expression of a nondegrading mutant of IκBα (IκBαSR), although enhanced by the over-expression of NF-κB p65. MMP-13 expression induced by CpG ODN was markedly suppressed by NF-κB inhibitors (pyrrolidine dithiocarbamate, PDTC), IκBα phosphorylation inhibitors (Bay 117082) or IκB protease inhibitor (L-1-tosylamido-2-phenylethyl chloromethyl ketone, TPCK). The inhibitor of ERK1/2, U0126, but not inhibitors of p38 MAPK and JNK, SB203580 and SP600125, decreased CpG ODN-mediated MMP-13 expression. CONCLUSION: The CpG ODN-induced MMP-13 expression in OLCs is mediated through TLR9, NF-κB and the ERK pathway indicating that potentially the recognition of CpG ODN by TLR9 on odontoblasts may regulate the remodelling of injured dental pulp and hard tissues by inducing MMP-13 expression.

TPCK inhibits AGC kinases by direct activation loop adduction at phenylalanine-directed cysteine residues.

N-alpha-tosyl-L-phenylalanyl chloromethyl ketone (TPCK) has anti-tumorigenic properties, but its direct cellular targets are unknown. Previously, we showed TPCK inhibited the PDKl-dependent AGC kinases RSK, Akt and S6K1 without inhibiting PKA, ERK1/2, PI3K, and PDK1 itself. Here we show TPCK-inhibition of the RSK-related kinases MSK1 and 2, which can be activated independently of PDK1. Mass spectrometry analysis of RSK1, Aktl, S6K1 and MSK1 immunopurified from TPCK-treated cells identified TPCK adducts on cysteines located in conserved activation loop Phenylalanine-Cysteine (Phe-Cys) motifs. Mutational analysis of the Phe-Cys residues conferred partial TPCK resistance. These studies elucidate a primary mechanism by which TPCK inhibits several AGC kinases, inviting consideration of TPCK-like compounds in chemotherapy given their potential for broad control of cellular growth, proliferation and survival.