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PubMed2001

Signal transduction system for interleukin-6 synthesis stimulated by lipopolysaccharide in human osteoblasts.

Lipopolysaccharide (LPS) is a bacterial cell component that plays multifunctional roles in inflammatory reactions, and one of the roles is as a powerful stimulator of bone resorption. LPS stimulated bone resorption via CD14 in mouse calvaria and was reported to function as a receptor for bacterial LPS complexed with serum proteins. Interleukin-6 (IL-6) is capable of stimulating the differentiation of osteoclasts from their hematopoietic precursors, and LPS elevates IL-6 synthesis in human osteoblastic cells. However, the signaling pathway of LPS-induced IL-6 synthesis in osteoblasts is unknown. In the present study, we could detect the existence of CD14 in human osteoblastic cells by RT-PCR analysis and show that LPS increased IL-6 mRNA and synthesis via CD14 in human osteoblastic cells. In human osteoblasts (SaM-1 cells) treated with 10 microg/ml LPS, increases in IL-6 mRNA and synthesis were inhibited by anti-CD14 antibody (MEM-18), PD98059 (an inhibitor of classic mitogen-activated protein kinase [MAPK]), or SB203580 (an inhibitor of p38 MAPK) but were not inhibited by H-89 (an inhibitor of protein kinase A [PKA]) and calphostin C (an inhibitor of protein kinase C [PKC]). Furthermore, LPS-induced IL-6 synthesis was inhibited by curcumin (an inhibitor of activating protein-1 [AP-1]) but not by pyrrolidine dithiocarbamate (PDTC) (an inhibitor of nuclear factor kappa B [NF-kappaB]). The findings of the present study suggest that the LPS receptor CD14, existent in human osteoblastic cells, and IL-6 synthesis in response to LPS probably occur via CD14, p38 MAPK, and MAP kinase/extracellular-regulated kinase kinase (MEK), leading to the transcriptional activation of AP-1 in human osteoblastic cells.

Adult↗

PubMed2005

Rescue of vasopressin V2 receptor mutants by chemical chaperones: specificity and mechanism.

Because missense mutations in genetic diseases of membrane proteins often result in endoplasmic reticulum (ER) retention of functional proteins, drug-induced rescue of their cell surface expression and understanding the underlying mechanism are of clinical value. To study this, we tested chemical chaperones and sarco(endo)plasmic reticulum Ca2+ ATPase pump inhibitors on Madin-Darby canine kidney cells expressing nine ER-retained vasopressin type-2 receptor (V2R) mutants involved in nephrogenic diabetes insipidus. Of these nine, only V2R-V206D showed improved maturation and plasma membrane rescue with glycerol, dimethyl sulfoxide (DMSO), thapsigargin/curcumin, and ionomycin but not with other osmolytes or growth at 27 degrees C. This revealed that rescue is mutant specific and that this mutant is prone to rescue by multiple compounds. Rescue did not involve changed expression of molecular chaperones calnexin, heat-shock protein (HSP) 70, or HSP90. V2R antagonist SR121463B treatment revealed that V2R-V206D and V2R-S167T were rescued and matured to a greater extent, suggesting that the rescuing activity of a pharmacological versus chemical chaperone is broader and stronger. Calcium measurements showed that rescue of V2R-V206D by thapsigargin, curcumin, and ionomycin was because of increased cytosolic calcium level, rather than decreased endoplasmic reticulum calcium level. The molecular mechanism underlying rescue by DMSO, glycerol, and SR121463B is different, because with these compounds intracellular calcium levels were unaffected.

Animals↗

PubMed2006

Curry consumption and cognitive function in the elderly.

Curcumin, from the curry spice turmeric, has been shown to possess potent antioxidant and antiinflammatory properties and to reduce beta-amyloid and plaque burden in experimental studies, but epidemiologic evidence is lacking. The authors investigated the association between usual curry consumption level and cognitive function in elderly Asians. In a population-based cohort (n = 1,010) of nondemented elderly Asian subjects aged 60-93 years in 2003, the authors compared Mini-Mental State Examination (MMSE) scores for three categories of regular curry consumption, taking into account known sociodemographic, health, and behavioral correlates of MMSE performance. Those who consumed curry "occasionally" and "often or very often" had significantly better MMSE scores than did subjects who "never or rarely" consumed curry. The authors reported tentative evidence of better cognitive performance from curry consumption in nondemented elderly Asians, which should be confirmed in future studies.

Aged↗

PubMed2006

Increased expression of the MGMT repair protein mediated by cysteine prodrugs and chemopreventative natural products in human lymphocytes and tumor cell lines.

O6-methylguanine-DNA methyltransferase (MGMT) is a DNA repair protein which protects the cellular genome and critical oncogenic genes from the mutagenic action of endogenous and exogenous alkylating agents. An expedited elimination of O6-alkylguanines by increasing MGMT activity levels is likely to be a successful chemoprevention strategy. Here, we report for the first time that cysteine/glutathione enhancing drugs and certain plant antioxidants possess the ability to increase human MGMT expression beyond its steady-state levels that may afford protection. The non-toxic cysteine prodrugs, 2-oxothiazolidine-4-carboxylic acid (OTC) and N-acetyl-L-cysteine (NAC), metabolized, respectively by 5-oxoprolinase and acylases, increased the MGMT protein and its repair activity levels in a dose- and time-dependent manner in several cancer cell lines and peripheral blood lymphocytes with a maximum of 3-fold increase by 72 h. The natural antioxidants, namely, curcumin, silymarin, sulforaphane and resveratrol were also effective in raising the MGMT levels to different extents. Among the synthetic agents, oltipraz and N-(4-hydroxyphenyl) retinamide (4-HPR) also increased MGMT expression, albeit to a lesser extent. Augmented mRNA levels accounted at least, in part, for the increased activity of MGMT in this setting. However, evidence from cysteine/methionine deprivation, acivicin treatment, and protein synthesis measurements in OTC-treated cells suggested that an increased cysteine flux also contributed significantly to enhanced MGMT expression. Many of these treatments increased the glutathione S-transferase-P1 (GSTP1) levels as well. These findings raise the possibility of MGMT-targeted chemoprevention strategies through dietary supplementation of OTC and herbal antioxidants. Further, the studies reveal the antioxidant responsiveness of the human MGMT gene.

Acetylcysteine↗

PubMed2004

Localization and functional analyses of the MLC1 protein involved in megalencephalic leukoencephalopathy with subcortical cysts.

Mutations in the MLC1 gene are responsible for one form of the neurological disorder megalencephalic leukoencephalopathy with subcortical cysts (MLC). The disease is a type of vacuolating myelinopathy. The biochemical properties and the function of the MLC1 protein are unknown. To characterize MLC1, we generated polyclonal antibodies. The MLC1 protein was detected in the brain, assembled into higher molecular complexes, as assessed by assembly-dependent trafficking assays. In situ hybridization and immunohistochemistry were used to determine MLC1 localization within the adult mouse brain. MLC1 was expressed in neurons, detected preferentially in particular axonal tracts. This expression pattern correlates with the major phenotype observed in the disease. In addition, it was expressed in some astrocytes, concentrating in Bergmann glia, the astrocyte end-feet membranes adjacent to blood vessels and in astrocyte-astrocyte membrane contact regions. Other neuronal barriers, such as the ependyma and the pia mater, were also positive for MLC1 expression. MLC1 was detected in vivo and in heterologous systems at the plasma membrane. MLC mutations impaired folding, and the defect was corrected in vitro by addition of curcumin, a Ca(2+)-ATPase inhibitor. In summary, this study provides an explanation as to why mutations in MLC1 provoke the disease and points to a possible therapy for some patients.

Animals↗

PubMed2004

Cyclic mechanical stretch augments both interleukin-8 and monocyte chemotactic protein-3 production in the cultured human uterine cervical fibroblast cells.

Intensive local leukocyte infiltration in the uterine cervix is a characteristic feature in the process of cervical ripening. The infiltrated leukocytes include neutrophils, macrophages and monocytes, which are believed to play important roles in cervical ripening by secreting elastase, matrix metalloproteinase and interleukin-1 (IL-1). Interleukin-8 (IL-8) and monocyte chemotactic protein-3 (MCP-3) belong to the CXC and CC chemokine families, and mediate the chemotaxis of neutrophils and monocytes/macrophages respectively. The aim of the present study was to investigate the possible involvement of IL-8 and MCP-3 in leukocyte chemotaxis in cervical ripening. Immunohistochemistry and RT-PCR detected both IL-8 and MCP-3 expression in human pregnant uterine cervices. Labour-like cyclic mechanical stretch for 48 h significantly elevated both IL-8 (555%) and MCP-3 (360%) secretion from cultured human uterine cervical fibroblast (CxF) cells (P<0.05 for both). Cyclic mechanical stretch for 24, 36 and 48 h significantly increased both IL-8 and MCP-3 mRNA expression in CxF cells (P<0.05 for all). The stretch-induced augmentation of both IL-8 and MCP-3 expression was significantly suppressed by an activator protein-1 (AP-1) inhibitor, curcumin. These data suggest that cyclic mechanical stretch of the uterine cervix by the presenting part of the fetus during labour may augment both IL-8 and MCP-3 production in the uterine cervix via AP-1 activation.

Anti-Inflammatory Agents, Non-Steroidal↗

PubMed2004

Evaluation of DNA damage in mice topically exposed to total particulate matter from mainstream and sidestream smoke from cigarettes and bidis.

The genotoxic potential of total particulate matter (TPM) from mainstream smoke (MS) and sidestream smoke (SS) of Indian smoking products, namely cigarettes and bidis, as well as a brand of US cigarettes, was studied by determining the levels of bulky aromatic DNA adducts in mouse tissues. TPM from MS or SS of various smoking products [equal weights (2.5 mg) or the amount derived from equal (0.25) cigarette/bidi] was applied topically to mouse skin once a day for four consecutive days and adduct levels were determined in DNA from skin and lung by (32)P-post-labelling analysis. Relatively higher levels of bulky aromatic DNA adducts were noted in mouse skin treated with MS from a single Indian non-filter (INF) cigarette when compared with MS of a single bidi (with about half the product weight and one-quarter the tobacco compared with a cigarette), while comparable adduct levels were noted with SS from these two products. Considering the differences in the yields of constituents of tobacco smoke from the different products analyzed, the genotoxic potential of INF, Indian filter king (IFK) and American filter (AF) cigarettes as well as bidis was determined by topically applying an equal amount of TPM (rather than equal product-derived TPM). SS-derived TPM from all the products showed relatively higher levels of total polycyclic aromatic hydrocarbons and induced relatively higher levels of bulky aromatic DNA adducts than those derived from MS. The data indicate that TPM (MS + SS) from cigarettes appears to be more genotoxic than that from bidis and the contribution of tendu leaf (a non-tobacco bidi wrapper) to the generation of bulky aromatic DNA adducts appears to be significant, particularly in SS of bidis. Topical pretreatment with curcumin decreased the levels of TPM-derived adducts while pretreatment with dietary turmeric failed to show such protection.

Acetone↗

PubMed2002

Sensitive markers used to identify compounds that trigger apoptosis in cultured hepatocytes.

Apoptosis may be a major event in chemical-induced injury, and therefore the detection of apoptotic effects when developing new drugs is highly relevant in screening for pharmacotoxicological risk assessment. However, as apoptosis in vitro normally degenerates to secondary necrosis, it is possible that it is underestimated, unless sensitive and specific parameters are used. In this present study we have evaluated the usefulness of a set of markers associated with the pivotal steps in the execution phase of apoptosis, in order to detect apoptotic compounds in hepatocytes before significant necrosis takes place. The markers selected include several biochemical parameters (downregulation of the antiapoptotic bclX(L) gene, caspase-3 activation, and cytochrome C release from mitochondria), and flow cytometry determinations (analysis of the size of the nuclei, chromatin complexity, and DNA integrity). The effects of several well-known model apoptotic toxicants (galactosamine, tertiary-butyl-hydroperoxide, etoposide, campothecine, and curcumin) were analyzed in hepatocytes. The aim was to identify early markers of apoptosis using known inducers of apoptosis in hepatocytes, as this battery of markers is designed to identify compounds triggering apoptosis in hepatocytes prior to necrosis. Concentrations of the compounds, as low as possible in order to keep 90% of hepatocyte viability, were selected according to their intracellular lactate dehydrogenase (LDH) leakage, which is well known as an indicator of cell membrane integrity and cell viability. The results demonstrated that (1) the apoptotic effect of 4 out of 5 compounds could be detected in low concentrations of the drugs long before cell necrosis (tertiary-butyl-hydroperoxide-induced apoptosis was only detected at concentrations causing concomitant necrosis) and (2) among the markers evaluated, caspase 3 activation and nucleus and DNA analysis by flow cytometry were used to fulfil the compromise between reliability, sensitivity, and ease of performance, which are critical issues when screening for an apoptotic effect of newly developed drugs.

Animals↗

PubMed2004

A novel homologous recombination system to study 92 kDa type IV collagenase transcription demonstrates that the NF-kappaB motif drives the transition from a repressed to an activated state of gene expression.

The 92-kDa type IV collagenase (MMP-9) contributes to tissue remodeling in both physiology and pathology. Previous studies on the transcriptional regulation of this gene have used transiently transfected promoter-reporter constructs. However, this approach suffers from several limitations including (a) multiple copies of the plasmid and (b) the plasmid is not genomically integrated and consequently poorly chromatinized. We developed a novel system for studying MMP-9 transcription in which a single copy of a MMP-9 promoter-luciferase construct(s) is integrated at an identical genomic locus in HT1080 cells by homologous recombination. We report that the activity of a genomic-integrated 2.2 kb MMP-9 promoter sequence mirrors expression of the endogenous MMP-9 gene in response to both physiological and pharmacological (curcumin) cues. Further, when constrained into chromatin, the integrated NF-kappaB-mutated MMP-9 promoter is repressed by PMA, a situation not apparent using nonintegrated plasmids. Thus, we have developed a novel method for studying MMP-9 expression that overcomes some of the limitations associated with transient transfection approaches and which may be of utility in screening for agents that repress the expression of this gene.

Cell Line↗

PubMed2002

Involvement of the mitogen-activated protein kinase family in tetracaine-induced PC12 cell death.

BACKGROUND: To explore whether cytotoxicity of local anesthetics is related to apoptosis, the authors examined how local anesthetics affect mitogen-activated protein kinase (MAPK) family members, extracellular signal-regulated kinases (ERKs), c-Jun N-terminal kinases (JNKs)-stress-activated protein kinases, and p38 kinase, which are known to play important roles in apoptosis. METHODS: Cell death was evaluated using PC12 cells. Morphologic changes of cells, cellular membrane, and nuclei were observed. DNA fragmentation was electrophoretically assayed. Western blot analysis was performed to analyze phosphorylation of the MAPK family, cleavage of caspase-3 and poly(adenosine diphosphate-ribose) polymerase. Intracellular Ca2+ concentration was measured using a calcium indicator dye. RESULTS: Tetracaine-induced cell death was shown in a time- and concentration-dependent manner and characterized by nuclear condensation or fragmentation, membrane blebbing, and internucleosomal DNA fragmentation. Caspase-3 activation and phosphorylation of ERK, JNK, and p38 occurred in the cell death. PD98059, an inhibitor of ERK, enhanced tetracaine-induced cell death and JNK phosphorylation, whereas ERK phosphorylation was inhibited. Curcumin, an inhibitor of JNK pathway, attenuated the cell death. Increase of intracellular Ca2+ concentration was detected. In addition to the increase of ERK phosphorylation and the decrease of JNK phosphorylation, two Ca2+ chelators protected cells from death. Neither cell death nor phosphorylation of the MAPK family was caused by tetrodotoxin. Nifedipine did not affect tetracaine-induced apoptosis. CONCLUSIONS: Tetracaine induces apoptosis of PC12 cells via the MAPK family. ERK activation protects cells from death, but JNK plays the opposite role. Toxic Ca2+ influx caused by tetracaine seems to be responsible for the cell death, but blocking of Na+ channels or L-type Ca2+ channels is unlikely involved in the tetracaine's action for apoptosis.

Anesthetics, Local↗

PubMed2003

Arrest of apoptosis in auditory neurons: implications for sensorineural preservation in cochlear implantation.

HYPOTHESIS: The JNK/c-Jun cell death pathway is a major pathway responsible for the loss of oxidative stress-damaged auditory neurons. BACKGROUND: Implantation of patients with residual hearing accentuates the need to preserve functioning sensorineural elements. Although some auditory function may survive electrode insertion, the probability of initiating an ongoing loss of auditory neurons and hair cells is unknown. Cochlear implantation can potentially generate oxidative stress, which can initiate the cell death of both auditory neurons and hair cells. METHODS: Dissociated cell cultures of P4 rat auditory neurons identified the apoptotic pathway initiated by oxidative stress insults (e.g., loss of trophic factor support) and characterized this pathway by arresting translation of pathway-specific mRNA with antisense oligonucleotide treatment and with the use of pathway specific inhibitors. The presence or absence of apoptosis-specific protein and changes in the level of neuronal survival measured the efficacy of these interventional strategies. RESULTS: These in vitro studies identified the JNK/c-Jun cascade as a major initiator of apoptosis of auditory neurons in response to oxidative stress. Neurons pretreated with c-jun antisense oligonucleotide and exposed to high levels of oxidative stress were rescued from apoptosis, whereas neurons in treatment control cultures died. Treatment of oxidative-stressed cultures with either curcumin, a MAPKKK pathway inhibitor, or PD-098059, a MEK1 inhibitor, blocked loss of neurons via the JNK/c-Jun apoptotic pathway. CONCLUSION: Blocking the JNK/c-Jun cell death pathway is a feasible approach to treating oxidative stress-induced apoptosis within the cochlea and may have application as an otoprotective strategy during cochlear implantation.

Animals↗

PubMed2002

Microarray-based analysis of anti-angiogenic activity of demethoxycurcumin on human umbilical vein endothelial cells: crucial involvement of the down-regulation of matrix metalloproteinase.

cDNA microarray-based gene expression analysis has been successfully employed to explore the action mechanism and to validate the targets of several drugs. In the present study, we evaluated anti-angiogenic activity of demethoxycurcumin (DC), a structural analog of curcumin, isolated from Curcuma aromatica, and investigated the effect of DC on genetic reprogramming in cultured human umbilical vein endothelial cells (HUVECs) using cDNA microarray analysis. Of 1024 human cancer-focused genes arrayed, 187 genes were up-regulated and 72 genes were down-regulated at least 2-fold by DC. Interestingly, 9 angiogenesis-related genes were down-regulated over 5-fold in response to DC, suggesting that the genetic reprogramming was crucially involved in anti-angiogenesis by the compound. To verify the results obtained from cDNA microarray analysis, matrix metalloproteinase-9 (MMP-9), the product of one of the angiogenesis-related genes down-regulated over 5-fold by DC, was investigated using gelatin zymography. DC potently inhibited the expression of MMP-9, yet showed no direct effect on its activity. These data show that gene expressional change of MMP-9 is a major mediator for angiogenesis inhibition by DC. All genes identified and microarray data are available on the web at http://dasan.sejong.ac.kr/~bioprobe/.

Angiogenesis Inhibitors↗

PubMed2006

Histone modifications in kainate-induced status epilepticus.

To understand the molecular actions of status epilepticus at the chromatin level, we studied the effects of kainate-induced status epilepticus on two different histone modifications at amino terminal tails: histone H3 phosphorylation at serine 10 and histone H4 acetylation. In addition to induction of c-fos and c-jun immediate early genes (IEGs) expression in mouse hippocampus, we also found the upregulation of acetylation and phosphorylation of histones, coupled with status epilepticus after kainate administration. c-fos and c-jun mRNA were sequentially induced in response to kainate, in different hippocampal subpopulations, starting from the dentate gyrus (DG) and spreading to the cornus ammonis regions. Immunohistochemical analysis showed that the spatio-temporal distribution of histone H4 hyperacetylation after kainate treatment was well correlated with the expression of c-fos and c-jun genes. Additionally, there was a transient appearance of phosphorylated histone H3 specifically in the DG region. CREB-binding protein or CBP, a well-known transcriptional co-activator with histone acetyltransferase (HAT) activity, was also induced by kainate and its expression pattern well correlated with histone H4 hyperacetylation in the hippocampus. Chromatin immunoprecipitation analysis showed that both histone modifications were associated with c-fos gene promoter after kainate stimulation, but only histone acetylation with c-jun gene. Pretreatment with curcumin, which has a HAT inhibitory activity specific for CBP/p300, attenuated histone modifications, IEGs expression and also the severity of status epilepticus after kainate treatment. Our findings suggest the involvement of histone modifications induced by kainate not only in IEGs expression but also in the development of epilepsy.

Animals↗

PubMed2005

Sodium phenylbutyrate prolongs survival and regulates expression of anti-apoptotic genes in transgenic amyotrophic lateral sclerosis mice.

Multiple molecular defects trigger cell death in amyotrophic lateral sclerosis (ALS). Among these, altered transcriptional activity may perturb many cellular functions, leading to a cascade of secondary pathological effects. We showed that pharmacological treatment, using the histone deacetylase inhibitor sodium phenylbutyrate, significantly extended survival and improved both the clinical and neuropathological phenotypes in G93A transgenic ALS mice. Phenylbutyrate administration ameliorated histone hypoacetylation observed in G93A mice and induced expression of nuclear factor-kappaB (NF-kappaB) p50, the phosphorylated inhibitory subunit of NF-kappaB (pIkappaB) and beta cell lymphoma 2 (bcl-2), but reduced cytochrome c and caspase expression. Curcumin, an NF-kappaB inhibitor, and mutation of the NF-kappaB responsive element in the bcl-2 promoter, blocked butyrate-induced bcl-2 promoter activity. We provide evidence that the pharmacological induction of NF-kappaB-dependent transcription and bcl-2 gene expression is neuroprotective in ALS mice by inhibiting programmed cell death. Phenylbutyrate acts to phosphorylate IkappaB, translocating NF-kappaB p50 to the nucleus, or to directly acetylate NF-kappaB p50. NF-kappaB p50 transactivates bcl-2 gene expression. Up-regulated bcl-2 blocks cytochrome c release and subsequent caspase activation, slowing motor neuron death. These transcriptional and post-translational pathways ultimately promote motor neuron survival and ameliorate disease progression in ALS mice. Phenylbutyrate may therefore provide a novel therapeutic approach for the treatment of patients with ALS.

Acetylation↗

PubMed2005

Effect of anti-inflammatory and antioxidant drugs on the long-term repair of severely injured mouse skeletal muscle.

Non-steroidal anti-inflammatory drugs are frequently prescribed after skeletal muscle injury. It is not known whether this type of medication can interfere with muscle repair, although inflammatory response is thought to play an important role in this process. Tibialis anterior muscles of mice were injured by myotoxic agent (snake venom) or crushed. Then, animals were treated daily for 10-14 days with different types of non-steroidal anti-inflammatory and antioxidant drugs. The long-term repair was studied 10-42 days after injury by analysing the recovery of in situ muscle force production, size of regenerating muscle cells and expression of myosin heavy chain. Our results show that diclofenac, diferuloylmethane (curcumin), dimethylthiourea or pyrrolidine dithiocarbamate treatment did not significantly affect muscle recovery after myotoxic injury (P > 0.05). Similarly, diferuloylmethane, dimethyl sulphoxide or indomethacin administration did not markedly change muscle repair after crush injury. However, we noted that high doses (> 2 mg kg(-1)) of diferuloylmethane or indomethacin increased lethality and reduced muscle repair after crush injury. In conclusion, non-steroidal anti-inflammatory and antioxidant drugs did not exhibit long-term detrimental effects on muscle recovery after injury, except at lethal doses.

Animals↗

PubMed1996

CD14-mediated signal pathway of Porphyromonas gingivalis lipopolysaccharide in human gingival fibroblasts.

Lipopolysaccharide (LPS) induces expression of inflammatory cytokines in monocytes/macrophages via CD14, one of the LPS receptors, which is expressed predominantly in these cells. It has been demonstrated that Porphyromonas gingivalis LPS (P-LPS) also is able to induce inflammatory cytokines in human gingival fibroblasts. Therefore, it is important to determine whether CD14 is expressed in gingival fibroblasts and to define the P-LPS-mediated signal-transducing mechanism in the cells. In this study, we observed unexpectedly by immunohistochemical, Western blotting (immunoblotting), and Northern (RNA) blotting assays that CD14 is expressed at high density in human gingival fibroblasts. P-LPS-induced expression of the monocyte chemoattractant protein 1 (MCP-1) gene in the cells was inhibited markedly by treatment with anti-human CD14 antibody and was completely inhibited by herbimycin A, a potent inhibitor of tyrosine kinase. The inhibitor also dramatically inhibited monocyte chemotactic activity of and MCP-1 production by the cells. Furthermore, P-LPS-induced expression of the MCP-1 gene in the cells also was blocked by inhibitors of two transcription factors, i.e., curcumin, an inhibitor of AP-1, and pyrolidine dithiocarbamate, an inhibitor of NF-kappaB. Both inhibitors inhibited monocyte chemotactic activity in the culture supernatant of P-LPS-treated cells. Gel shift mobility assay showed stimulation of the AP-1 and NF-kappaB contents in P-LPS-treated cells. This study is the first to demonstrate the expression of CD14 in human gingival fibroblasts and to show that the signal-transducing pathway of P-LPS in the cells is mediated by CD14.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed2005

Hepatitis C virus NS2 protein is phosphorylated by the protein kinase CK2 and targeted for degradation to the proteasome.

Hepatitis C virus (HCV) nonstructural 2 (NS2) protein is a hydrophobic transmembrane protein, described to be involved in different functions, such as apoptosis inhibition and gene transcription modulation. We investigated here NS2 protein turnover and found that NS2 was rapidly degraded by the proteasome in different cell lines, as in primary human hepatocytes. Since posttranslational modifications can influence protein turnover, we looked for potential phosphoacceptor sites in NS2. Computational sequence analysis in combination with screening of NS2 point mutants revealed that serine residue 168 was critical for degradation. In the quest of a protein kinase for NS2, we identified by sequence analysis that the serine residue 168 was part of a consensus casein kinase 2 (CK2) recognition site (S/TXXE). This motif was highly conserved since it could be found in the NS2 primary consensus sequences from all HCV genotypes. To verify whether CK2 is involved in NS2 phosphorylation, we showed by an in vitro kinase assay that CK2 phosphorylated NS2, as far as this CK2 motif was conserved. Interestingly, NS2 became resistant to protein degradation when the CK2 motif was modified by a single point mutation. Furthermore, inhibition of CK2 activity by curcumin decreased NS2 phosphorylation in vitro and stabilized NS2 expression in HepG2 cells. Finally, we showed in Huh-7.5 replicon cells that NS2, expressed in the context of the HCV polyprotein, was also sensitive to both proteasome-mediated degradation and CK2 inhibitor treatment. We suggest that NS2 is a short-lived protein whose degradation by the proteasome is regulated in a phosphorylation-dependent manner through the protein kinase CK2.

Amino Acid Sequence↗

PubMed2002

Mitogen-activated protein kinases mediate stretch-induced c-fos mRNA expression in myometrial smooth muscle cells.

Evidence indicates that stretch of the uterus imposed by the growing fetus contributes to the onset of labor. Previously we have shown that mechanically stretching rat myometrial smooth muscle cells (SMCs) induces c-fos expression. To investigate this stretch-induced signaling, we examined the involvement of the mitogen-activated protein kinase (MAPK) family. We show that stretching rat myometrial SMCs induces a rapid and transient phosphorylation (activation) of MAPKs: extracellular signal-regulated protein kinase (ERK), c-Jun NH2-terminal kinase (JNK), and p38. The use of selective inhibitors for the ERK pathway (PD-98059 and U-0126), p38 (SB-203580), and JNK pathway (curcumin) demonstrated that activation of all three MAPK signaling pathways was necessary for optimal stretch-induced c-fos expression. We also demonstrate that upstream tyrosine kinase activity is involved in the mechanotransduction pathway leading to stretch-induced MAPK activation and c-fos mRNA expression. To further examine the role of MAPKs in vivo, we used a unilaterally pregnant rat model. MAPKs (ERK and p38) are expressed in the pregnant rat myometrium with maximal ERK and p38 phosphorylation occurring in the 24 h immediately preceding labor. Importantly, the rise in MAPK phosphorylation was confined to the gravid horn and was absent in the empty uterine horn, suggesting that mechanical strain imposed by the growing fetus controls MAPK activation in the myometrium. Collectively, this data indicate that mechanical stretch modulates MAPK activity in the myometrium leading to c-fos expression.

Animals↗