M-CSF and prostratin induced Mregs promote immune tolerance in transplanted mice through Arg-1 pathway.

OBJECTIVE: Regulatory macrophages (Mregs) are a group of heterogeneous macrophages. These cells could induce immunosuppressive effects through the expression of immune regulatory molecules and cytokines. METHODS: The differentiation of Mregs was induced by treating bone marrow cells with M-CSF and prostratin in vitro. The cell-phenotypes and immunosuppressive function were determined by flow cytometry. Rt-PCR was employed to assess the mechanisms of Mregs. Skin grafted mouse model was used for in vivo validation. RESULTS: Mregs induced by M-CSF + prostratin had a strong inhibitory effect on T cell proliferation and cytokines production. The phenotype of induced bone marrow cells changed towards Mregs. These Mregs could induce the differentiation of Tregs in vivo. Arg-1 expression in these cells were significantly upregulated. Inhibition of arginase (Arg) or arginine supplement significantly reversed the immunosuppressive function. In mice skin-grafted models, adoptive transfer of these Mregs significantly prolonged allograft survival. In mice models, Arg-1 expression significantly elevated on skin grafts cells and Tregs increased in graft tissues. CONCLUSIONS: We successfully developed a Mregs-inducing protocol with the combination of M-CSF and prostratin in vitro. M-CSF + prostratin induced Mregs prevented mice skin graft rejection through upregulating the expression Arg-1.

Involvement of LPA receptor-5 in the enhancement of cell motile activity by phorbol ester and anticancer drug treatments in melanoma A375 cells.

Lysophosphatidic acid (LPA) signaling through six subtypes of LPA receptors (LPA1 to LPA6) regulates a variety of biological responses in cancer cells. The aim of our study was to evaluate an involvement of LPA receptors in the activation of cell motility by phorbol ester and anticancer drug treatments in melanoma A375 cells. Cells were treated with 12-O-tetradecanoylphorbol- 13-acetate (TPA) and phorbol-12,13-dibutyrate (PDBu) for 3 days. The cell motile activity of TPA treated cells was significantly higher than that of PDBu treated cells, correlating with LPAR5 expression levels. LPA5 knockdown suppressed the high cell motile activity induced by TPA. To assess whether the cell motile activity of A375 cells is stimulated through LPA5 induced by anticancer drugs, the long-term cisplatin (CDDP) and dacarbazine (DTIC) treated cells were generated from A375 cells (A375-CDDP and A375-DTIC cells, respectively). The expression levels of LPA receptor genes were changed in A375-CDDP and A375-DTIC cells. In particular, CDDP and DTIC treatment markedly elevated LPAR5 expressions. The cell motile activities of A375-CDDP and A375-DTIC cells were significantly higher than that of untreated cells. These results suggest that the cell motile activity is regulated through the induction of LPA5 by phorbol ester and anticancer drug treatments in A375 cells.

Resveratrol inhibits phorbol ester-induced membrane translocation of presynaptic Munc13-1.

BACKGROUND: Resveratrol (1) is a naturally occurring polyphenol that has been implicated in neuroprotection. One of resveratrol's several biological targets is Ca2+-sensitive protein kinase C alpha (PKCα). Resveratrol inhibits PKCα by binding to its activator-binding C1 domain. Munc13-1 is a C1 domain-containing Ca2+-sensitive SNARE complex protein essential for vesicle priming and neurotransmitter release. METHODS: To test if resveratrol could also bind and inhibit Munc13-1, we studied the interaction of resveratrol and its derivatives, (E)-1,3-dimethoxy-5-(4-methoxystyryl)benzene, (E)-5,5'-(ethene-1,2-diyl)bis(benzene-1,2,3-triol), (E)-1,2-bis(3,4,5-trimethoxyphenyl)ethane, and (E)-5-(4-(hexadecyloxy)-3,5-dihydroxystyryl)benzene-1,2,3-triol with Munc13-1 by studying its membrane translocation from cytosol to plasma membrane in HT22 cells and primary hippocampal neurons. RESULTS: Resveratrol, but not the derivatives inhibited phorbol ester-induced Munc13-1 translocation from cytosol to membrane in HT22 cells and primary hippocampal neurons, as evidenced by immunoblot analysis and confocal microscopy. Resveratrol did not show any effect on Munc13-1H567K, a mutant which is not sensitive to phorbol ester. Binding studies with Munc13-1 C1 indicated that resveratrol competes with phorbol ester for the binding site. Molecular docking and dynamics studies suggested that hydroxyl groups of resveratrol interact with phorbol-ester binding residues in the binding pocket. CONCLUSIONS AND SIGNIFICANCE: This study characterizes Munc13-1 as a target of resveratrol and highlights the importance of dietary polyphenol in the management of neurodegenerative diseases.

Induction of IL-17 production from human peripheral blood CD4+ cells by asbestos exposure.

We have previously reported that chronic, recurrent and low-dose exposure to asbestos fibers causes a reduction in antitumor immunity. Investigation of natural killer (NK) cells using an in vitro cell line model and comprising in vitro activation using freshly isolated NK cells co-cultured with chrysotile fibers, as well as NK cells derived from asbestos-exposed patients with pleural plaque (PP) or malignant mesothelioma (MM), revealed decreased expression of NK cell activating receptors such as NKG2D, 2B4 and NKp46. An in vitro differentiation and clonal expansion model for CD8+ cytotoxic T lymphocytes (CTLs) showed reduced cytotoxicity with decreased levels of cytotoxic molecules such as granzyme B and perforin, as well as suppressed proliferation of CTLs. Additionally, analysis of T helper cells showed that surface CXCR3, chemokine receptor, and the productive potential of interferon (IFN)γ were reduced following asbestos exposure in an in vitro cell line model and in peripheral CD4+ cells of asbestos-exposed patients. Moreover, experiments revealed that asbestos exposure enhanced regulatory T cell (Treg) function. This study also focused on CXCR3 expression and the Th-17 cell fraction. Following activation with T-cell receptor and co-culture with various concentrations of chrysotile fibers using freshly isolated CD4+ surface CXCR3 positive and negative fractions, the intracellular expression of CXCR3, IFNγ and IL-17 remained unchanged when co-cultured with chrysotile. However, subsequent re-stimulation with phorbol 12-myristate 13-acetate (PMA) and ionomycin resulted in enhanced IL-17 production and expression, particularly in CD4+ surface CXCR3 positive cells. These results indicated that the balance and polarization between Treg and Th-17 fractions play an important role with respect to the immunological effects of asbestos and the associated reduction in antitumor immunity.

HMBA Enhances Prostratin-Induced Activation of Latent HIV-1 via Suppressing the Expression of Negative Feedback Regulator A20/TNFAIP3 in NF-κB Signaling.

In the past decade, much emphasis has been put on the transcriptional activation of HIV-1, which is proposed as a promised strategy for eradicating latent HIV-1 provirus. Two drugs, prostratin and hexamethylene bisacetamide (HMBA), have shown potent effects as inducers for releasing HIV-1 latency when used alone or in combination, although their cellular target(s) are currently not well understood, especially under drug combination. Here, we have shown that HMBA and prostratin synergistically release HIV-1 latency via different mechanisms. While prostratin strongly stimulates HMBA-induced HIV-1 transcription via improved P-TEFb activation, HMBA is capable of boosting NF-κB-dependent transcription initiation by suppressing prostratin-induced expression of the deubiquitinase A20, a negative feedback regulator in the NF-κB signaling pathway. In addition, HMBA was able to increase prostratin-induced phosphorylation and degradation of NF-κB inhibitor IκBα, thereby enhancing and prolonging prostratin-induced nuclear translocation of NF-κB, a prerequisite for stimulation of transcription initiation. Thus, by blocking the negative feedback circuit, HMBA functions as a signaling enhancer of the NF-κB signaling pathway.

Heme oxygenase-1-mediated anti-inflammatory effects of tussilagonone on macrophages and 12-O-tetradecanoylphorbol-13-acetate-induced skin inflammation in mice.

The dried flower buds of Tussilago farfara L. have been used in traditional medicine, mainly as an antitussive in the treatment of cough and other respiratory problems. In the present study, we investigated the anti-inflammatory signaling pathway via the upregulation of heme oxygenase-1 (HO-1) in response to tussilagonone (TGN), a sesquiterpene compound isolated from T. farfara. TGN induced HO-1 expression and nuclear factor-E2-related factor 2 (Nrf2) activation in RAW 264.7 cells. Nuclear translocation of Nrf2 by TGN also increased in a time- and dose-dependent manner, indicating that TGN induced HO-1 via the Nrf2 pathway. Consistent with the notion that HO-1 has anti-inflammatory properties, TGN suppressed inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression and reduced the mRNA expression of proinflammatory cytokines, as well as nitric oxide (NO) and prostaglandin E2 (PGE2) production in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. TGN inhibited the phosphorylation and degradation of inhibitory κB-α (IκB-α) and the nuclear translocation of nuclear factor (NF)-κB. However, a specific inhibitor of HO-1 reversed the TGN-mediated suppression of NO production and knockdown of HO-1 by small interfering RNA abrogated inhibitory effects of TGN on iNOS and COX-2 protein expression and NF-κB nuclear translocation. Furthermore, TGN reduced iNOS and COX-2 expression in a 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced skin inflammation mouse model. Taken together, these findings suggest an important role for TGN-induced HO-1 activation in regulating inflammatory responses. Moreover, TGN is a potent therapeutic candidate for targeting the crosstalk between Nrf2/HO-1 and the NF-κB signaling pathway in the prevention or treatment of inflammation-associated diseases.

Conventional protein kinase Cß-mediated phosphorylation inhibits collapsin response-mediated protein 2 proteolysis and alleviates ischemic injury in cultured cortical neurons and ischemic stroke-induced mice.

We previously reported that conventional protein kinase C (cPKC)ß participated in hypoxic preconditioning-induced neuroprotection against cerebral ischemic injury, and collapsin response-mediated protein 2 (CRMP2) was identified as a cPKCß interacting protein. In this study, we explored the regulation of CRMP2 phosphorylation and proteolysis by cPKCß, and their role in ischemic injury of oxygen-glucose deprivation (OGD)-treated cortical neurons and brains of mice with middle cerebral artery occlusion-induced ischemic stroke. The results demonstrated that cPKCß-mediated CRMP2 phosphorylation via the cPKCß-selective activator 12-deoxyphorbol 13-phenylacetate 20-acetate (DOPPA) and inhibition of calpain-mediated CRMP2 proteolysis by calpeptin and a fusing peptide containing TAT peptide and the calpain cleavage site of CRMP2 (TAT-CRMP2) protected neurons against OGD-induced cell death through inhibiting CRMP2 proteolysis in cultured cortical neurons. The OGD-induced nuclear translocation of the CRMP2 breakdown product was inhibited by DOPPA, calpeptin, and TAT-CRMP2 in cortical neurons. In addition, both cPKCß activation and CRMP2 proteolysis inhibition by hypoxic preconditioning and intracerebroventricular injections of DOPPA, calpeptin, and TAT-CRMP2 improved the neurological deficit in addition to reducing the infarct volume and proportions of cells with pyknotic nuclei in the peri-infact region of mice with ischemic stroke. These results suggested that cPKCß modulates CRMP2 phosphorylation and proteolysis, and cPKCß activation alleviates ischemic injury in the cultured cortical neurons and brains of mice with ischemic stroke through inhibiting CRMP2 proteolysis by phosphorylation. Focal cerebral ischemia induces a large flux of Ca(2+) to activate calpain which cleaves collapsin response mediator (CRMP) 2 into breakdown product (BDP). Inhibition of CRMP2 cleavage by calpeptin and TAT-CRMP2 alleviates ischemic injury. Conventional protein kinase C (cPKC)ß-mediated phosphorylation could inhibit CRMP2 proteolysis and alleviate ischemic injury in cultured cortical neurons and ischemic stroke-induced mice.

Treatment with protein kinase C activator is effective for improvement of male pronucleus formation and further embryonic development of sperm-injected oocytes in pigs.

To assist the process of oocyte activation, which is essential for promotion of fertilization events, i.e., resumption of meiosis, extrusion of the second polar body and formation of the pronucleus (PN), artificial stimuli such as an electrical pulse have been applied to porcine oocytes after injection of sperm. However, the efficiency of fertilization and embryonic development remains low. It is well known that in vertebrates, inactivation of mitogen-activated protein (MAP) kinase is required for oocyte activation. We have hypothesized that even after electrical stimulation of sperm-injected oocytes, MAP kinase may not be inactivated. As it has been reported that MAP kinase activity is regulated by protein kinase C, we examined the effectiveness of phorbol 12-myristate 13-acetate (PMA), a protein kinase C activator, for improvement of fertilization and embryonic development of sperm-injected porcine oocytes. First, we examined the concentrations (0, 0.01, 0.1, 1, and 10 µM) and durations (0, 1, 3, 5 hours) of PMA treatment that were efficient for the extrusion of two polar bodies and formation of two PNs (2PB+2PN) and embryonic development. When the sperm-injected oocytes were treated with 0.01-µM PMA for 3 hours after electrical stimulation, the rates of 2PB+2PN and embryonic development were higher than those in the other treatment groups. We then examined the effect of PMA treatment (0.01 µM, 3 hours) on MAP kinase activity. Unexpectedly, after electrical stimulation, the activity remained low until PN formation, irrespective of whether or not the oocytes had been treated with PMA. On the other hand, transformation of the injected sperm nucleus into the male PN was accelerated after the PMA treatment. Our present results suggest that the low efficiency of fertilization and embryonic development in sperm-injected oocytes is not due to high activity of MAP kinase but due to poor transformation of the injected sperm nucleus into the male PN. Furthermore, a combination of electrical stimulation and PMA is a fairly effective artificial protocol for promoting 2PB+2PN and embryonic development in sperm-injected porcine oocytes.

K-Ras Promotes Tumorigenicity through Suppression of Non-canonical Wnt Signaling.

K-Ras and H-Ras share identical effectors and have similar properties; however, the high degree of tumor-type specificity associated with K-Ras and H-Ras mutations suggests that they have unique roles in oncogenesis. Here, we report that oncogenic K-Ras, but not H-Ras, suppresses non-canonical Wnt/Ca(2+) signaling, an effect that contributes strongly to its tumorigenic properties. K-Ras does this by binding to calmodulin and so reducing CaMKii activity and expression of Fzd8. Restoring Fzd8 in K-Ras mutant pancreatic cells suppresses malignancy, whereas depletion of Fzd8 in H-Ras(V12)-transformed cells enhances their tumor initiating capacity. Interrupting K-Ras-calmodulin binding using genetic means or by treatment with an orally active protein kinase C (PKC)-activator, prostratin, represses tumorigenesis in K-Ras mutant pancreatic cancer cells. These findings provide an alternative way to selectively target this "undruggable" protein.

12-Deoxyphorbol 13-palmitate inhibits the expression of VEGF and HIF-1α in MCF-7 cells by blocking the PI3K/Akt/mTOR signaling pathway.

Vascular endothelial growth factor (VEGF) is an essential component for angiogenesis, and hypoxia-inducible factor-1α (HIF-1α), which controls the switch of glycolytic and oxidative metabolism, activates the transcription of VEGF. 12-Deoxyphorbol 13-palmitate (DP) is a compound isolated from the roots of Euphorbia fischeriana, and has been revealed to possess anticancer activity. In the present study, we found that DP is an effective inhibitor of VEGF and HIF-1α in MCF-7 cells. DP markedly reduced cell viability as determined by MTT assay. ELISA, western blotting and RT-qPCR assays indicated that DP significantly decreased the protein and mRNA expression of VEGF and the protein expression of HIF-1α, while HIF-1α mRNA remained unchanged. In addition, the entrance of HIF-1α into the nucleus was blocked after DP treatment as detected by immunofluorescence analysis. In a further study, we proved that the effects mentioned above were associated with constitutive interference of the phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway. DP effectively inhibited the phosphorylation of PI3K and its downstream factors p-Akt and p-mTOR, oppositely enhanced the expression of TSC1 (hamartin) and TSC2 (tuberin), which could be reversed by the co-treatment with the PI3K inhibitor wortmannin. Moreover, the addition of wortmanin further downregulated the protein levels of VEGF and HIF-1α. The results revealed that DP inhibited the expression of VEGF and HIF-1α through the PI3K/Akt/mTOR signaling pathway, confirming that DP may be a potential therapeutic candidate for breast cancer.

Skin repair properties of d-Limonene and perillyl alcohol in murine models.

The orange-peel derived terpene d-Limonene, probably through its metabolite, perillyl alcohol (POH), has been reported to have tissue-repair properties. Two murine models of respectively 12-O-Tetradecanoylphorbol-13-Acetate (TPA)-induced dermatitis and mechanical skin lesion were used here to assess the efficacy of d-Limonene or POH applied topically. Macroscopic and microscopic evaluation of skin lesions was performed as well as that of P-selectin expression, together with measurements of serum concentrations of IL-1ß, IL-6 and TNF-α in the first model. Healing and angiogenesis around the scar were examined in the second model. Because differences in angiogenesis were noted, the effect of both d-Limonene and POH was further tested on an in vitro model of endothelial microtubules formation. Both d-Limonene and POH reduced the severity and extension of TPA-induced skin lesions with significantly lowered macroscopic and microscopic scores (p<0.04 in both cases). Moreover, the expression of P-selectin induced by TPA was abrogated by POH and significantly lower serum concentrations of IL-6 and TNF-α were observed in d-Limonene- and POH-treated mice (p<0.04 and 0.03). In the second model, tissue regeneration was improved, especially by POH, and was clearly associated with reduced neovascularization. This surprising anti-angiogenic effect was confirmed in the matrigel model of endothelial microtubules formation. These studies show that d-Limonene and POH demonstrate significant anti-inflammatory effects in murine dermal inflammation and wound-healing. The decreased systemic cytokine production as well as a consistent inhibition of endothelial P-selectin expression and neo-vascularization induced by these terpenic compounds contribute to their healing effects on the epidermal barrier.

Repeated topical application of glucocorticoids augments irritant chemical-triggered scratching in mice.

Topical glucocorticoids, widely used for the treatment of a variety of dermatitises, are known to exacerbate atopic dermatitis after long-term or inappropriate use. In some animal models, topical glucocorticoids augment the allergic cutaneous inflammation after repeated application, suggesting a relationship between these and clinical observations. We investigated whether topical glucocorticoids augment itching, rather than inflammation, resulting in the exacerbation of atopic dermatitis. Mice receiving repeated topical application of glucocorticoids, betamethasone valerate or dexamethasone, to the ear for 1 week showed significantly higher scratching frequency after application of an irritant chemical, 2,4-dinitrofluorobenzene (DNFB) or 12-O-tetradecanoilphorbol 13-acetate (TPA) than those receiving either a glucocorticoid or irritant chemical alone. In contrast, the increase in ear thickness induced by application of TPA was significantly suppressed by dexamethasone. Substance P (SP) and nerve growth factor (NGF) levels were higher in the ear receiving betamethasone valerate followed by DNFB application than in that receiving DNFB alone. In addition, histopathological studies revealed an increased density of nerve fibers in the ear receiving betamethasone valerate or dexamethasone followed by DNFB application. Oral administration of betamethasone valerate was not associated with an increase in either scratching frequency or SP or NGF level in the ear. These results suggest that repeated topical application of glucocorticoids may augment irritant chemical-triggered scratching through an increase in SP and NGF levels and nerve fiber density at the application site. These findings might explain the etiology of the exacerbation of atopic dermatitis and other dermatitises, occurring after long-term or inappropriate use of topical glucocorticoids.

Selenoproteins regulate macrophage invasiveness and extracellular matrix-related gene expression.

BACKGROUND: Selenium, a micronutrient whose deficiency in diet causes immune dysfunction and inflammatory disorders, is thought to exert its physiological effects mostly in the form of selenium-containing proteins (selenoproteins). Incorporation of selenium into the amino acid selenocysteine (Sec), and subsequently into selenoproteins is mediated by Sec tRNA([Ser]Sec). RESULTS: To define macrophage-specific selenoprotein functions, we generated mice with the Sec tRNA([Ser]Sec) gene specifically deleted in myeloid cells. These mutant mice were devoid of the "selenoproteome" in macrophages, yet exhibited largely normal inflammatory responses. However, selenoprotein deficiency led to aberrant expression of extracellular matrix-related genes, and diminished migration of macrophages in a protein gel matrix. CONCLUSION: Selenium status may affect immune defense and tissue homeostasis through its effect on selenoprotein expression and the trafficking of tissue macrophages.

Protein kinase C-dependent trafficking of glutamate transporters excitatory amino acid carrier 1 and glutamate transporter 1b in cultured cerebellar granule cells.

Previous data showed that cell surface expression of the glutamate transporters GLT1a and excitatory amino acid carrier 1 (EAAC1), localized in glia and neurons of the CNS, can be regulated by protein kinase C (PKC). Regulation and physiological importance of GLT1b, a splice variant of GLT1a, is not understood. In the present study we used cultured cerebellar granule cells (CGCs) from mice to investigate PKC dependent trafficking of GLT1b in comparison to GLT1a and EAAC1 using immunohistochemistry and subcellular fractionation followed by Western blotting. In neurites of CGCs, GLT1b and EAAC1 were localized to different aggregates of vesicles that were different from vesicle aggregates containing vesicular glutamate transporters. In CGCs cultured with low-potassium medium, stimulation of PKC by phorbol ester enhanced the formation of large varicosities in neurites that exhibited immunoreactivity for GLT1a, GLT1b, and EAAC1. Stimulation of PKC leads to a significant increase of GLT1b and EAAC1 in the plasma membrane whereas GLT1a in the plasma membrane was decreased. Following PKC stimulation, also a significant increase of transporter-mediated glutamate uptake representing sodium dependent glutamate uptake, was observed. Similarly, the fraction of glutamate uptake, that was sensitive to the inhibitor WAY-213613 and represents uptake by GLT1a and GLT1b, was increased after stimulation by PKC. The findings suggest that PKC is similarly involved in regulation of surface trafficking of GLT1b and EAAC1 and that PKC stimulated increase in surface location of GLT1b and EAAC1 in glutamatergic CGCs.

Phorbol ester-induced PKCepsilon down-modulation sensitizes AML cells to TRAIL-induced apoptosis and cell differentiation.

Despite the relevant therapeutic progresses made in these last 2 decades, the prognosis of acute myeloid leukemia (AML) remains poor. Phorbol esters are used at very low concentrations as differentiating agents in the therapy of myeloid leukemias. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), in turn, is a death ligand that spares normal cells and is therefore currently under clinical trials for cancer therapy. Emerging evidence, however, suggests that TRAIL is also involved in nonapoptotic functions, like cell differentiation. PKCepsilon is differentially modulated along normal hematopoiesis, and its levels modulate the response of hematopoietic precursors to TRAIL. Here, we investigated the effects of the combination of phorbol esters (phorbol ester 4-beta-phorbol-12,13-dibutyrate [PDBu]) and TRAIL in the survival/differentiation of AML cells. We demonstrate here that PDBu sensitizes primary AML cells to both the apoptogenic and the differentiative effects of TRAIL via PKCepsilon down-modulation, without affecting TRAIL receptor surface expression. We believe that the use of TRAIL in combination with phorbol esters (or possibly more specific PKCepsilon down-modulators) might represent a significative improvement of our therapeutic arsenal against AML.

Antidipsogenic effects of a TRPV4 agonist, 4alpha-phorbol 12,13-didecanoate, injected into the cerebroventricle.

Transient receptor potential vanilloid 4 (TRPV4) is one member of the TRP superfamily of nonselective cation channels. Recently, the possibility has been raised that TRPV4 is an osmoreceptor, because it is found in the circumventricular organs where osmoreceptors are supposed to be distributed and because it is sensitive to osmotic pressure in in vitro experiments. In addition, TRPV4 knockout mice have abnormal osmosensitivity. In this study, effects of 4alpha-phorbol 12,13-didecanoate (4alpha-PDD), a TRPV4 agonist, on drinking behavior were examined to investigate roles for TRPV4 as an osmoreceptor in vivo in wild-type animals. Intracerebroventricular injections of 4alpha-PDD inhibited water intake under normal conditions in both light and dark periods of the day, after food deprivation, and after administration of angiotensin II. However, this drug did not influence increased water intake after administration of a hypertonic solution or after water deprivation that significantly increased plasma osmolality. Locomotor activity of the 4alpha-PDD-injected group decreased slightly compared with that of the vehicle-injected group; however, sweet taste, food intake, and body temperature were not different between the two groups. The antidipsogenic effects of 4alpha-PDD were blocked by preinjection into the ventricle of TRPV4 antagonists such as ruthenium red or gadolinium. These findings suggest that TRPV4 regulates drinking behavior under certain conditions, and the regulation interacts with the angiotensin II pathway.

Curcumin suppresses phorbol ester-induced matrix metalloproteinase-9 expression by inhibiting the PKC to MAPK signaling pathways in human astroglioma cells.

The aberrant expression of matrix metalloproteinase-9 (MMP-9) is implicated in the invasion and angiogenesis process of brain tumor. This study has investigated the effects of curcumin on MMP-9 expression in human astroglioma cell lines. Curcumin significantly inhibited the MMP-9 enzymatic activity and protein expression that was induced by PMA. The inhibitory effect of curcumin on MMP-9 expression correlates with the decreased MMP-9 mRNA level and the suppression of MMP-9 promoter activity. The curcumin-mediated inhibition of MMP-9 gene expression appears to occur via NF-kappaB and AP-1 because their DNA binding activities were suppressed by curcumin. Furthermore, curcumin strongly repressed the PMA-induced phosphorylation of ERK, JNK, and p38 MAP kinase, which were dependent on the PKC pathway. Therefore, the inhibition of MMP-9 expression by curcumin might have therapeutic potential for controlling the growth and invasiveness of brain tumor.

Inhibition of tumour promotion in mouse skin by extracts of rooibos (Aspalathus linearis) and honeybush (Cyclopia intermedia), unique South African herbal teas.

The modulating effect of ethanol/acetone (E/A) soluble fractions, prepared from methanolic extracts of processed and unprocessed rooibos (Aspalathus linearis) and honeybush (Cyclopia intermedia) as well as green (Camellia sinensis) teas was established in a two-stage mouse skin carcinogenesis assay. Topical application of the tea fractions prior to the tumour promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA), on ICR mouse skin initiated with 7,12-dimethylbenz[a]anthracene (DMBA) suppressed skin tumorigenesis significantly (P<0.001) with the green tea E/A fraction exhibiting a 100% inhibition, unprocessed honeybush 90%, processed honeybush 84.2%, processed rooibos 75% and unprocessed rooibos 60%. The green tea fraction, with the highest flavanol/proanthocyanidin content, also exhibited the highest protective activity (99%) against hepatic microsomal lipid peroxidation, and completely inhibited skin tumour formation. Differences in the flavanol/proanthocyanidin and flavonol/flavone composition and/or non polyphenolic constituents are likely to be important determinants in the inhibition of tumour promotion by the herbal tea E/A fractions in mouse skin.

Activation of activator protein 1 and extracellular signal-regulated kinases in human hepatocellular transformation.

Hepatocellular carcinoma is one of the most common tumors worldwide. Activator protein 1 (AP-1) is a nuclear transcription factor, and its transactivation is required for transformation in several cell lines. However, no direct correlation between AP-1 activity and human hepatocellular transformation has been proved. Here we analyzed the role of AP-1 on the 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced human hepatocellular transformation. TPA promoted the formation of anchorage-independent colonies, induced the AP-1 activity, and enhanced the DNA-binding ability of AP-1 in human hepatocytes. The phosphorylation of extracellular signal-regulated protein kinases (ERKs) was increased by TPA and the TPA-induced AP-1 activity was inhibited by PD98059, indicating that TPA-induced AP-1 activation was via ERK pathway. Moreover, retinoic acid and PD98059, which inhibited the AP-1 activity, abolished the TPA-induced transformation. Our findings indicated that AP-1 and ERKs activations were required for TPA-induced human hepatocellular transformation. These studies suggested that AP-1 could be the target for the development of antihepatocellular transformation agents.

Influence of lead on rat thoracic aorta contraction and relaxation.

Low levels of lead, but not high levels, produce hypertension. This mystery has not yet been resolved. In this study we compared the in vitro vasoresponsiveness in rat thoracic aorta to low dose (10(-8) mol/L) and high dose (10(-5) mol/L and 10(-4) mol/L) lead acetate. In addition to the direct response to lead, we examined reactivity to norepinephrine, acetylcholine, isoproterenol, phorbol ester, and calcium in the presence and absence of lead. Neither low-dose nor high-dose lead directly affected aortic contractile or relaxant responses. However, lead, only at the highest concentration (10(-4) mol/L), increased the contractions to calcium at all submaximal calcium concentrations. We conclude that low-dose lead must increase blood pressure indirectly through a humoral effect. The reasons for the failure of high-dose lead to influence blood pressure remain to be explored.