Troglitazone induction of COX-2 expression is dependent on ERK activation in keratinocytes

Cyclooxygenase-2 (COX-2) plays an important role in tumorigenesis of several tissues, including skin. We report here that troglitazone, a thiazolidinedione class of antidiabetic drug, induced COX-2 expression at both the protein and mRNA levels and increased production of prostaglandin E2 (PGE2) in cultured keratinocytes. Troglitazone-induced COX-2 expression in keratinocytes was likely peroxisome proliferator-activated receptor gamma (PPARgamma)-independent. Troglitazone treatment of these cells also resulted in a sustained increase in phosphorylation of ERK. We show that induction of COX-2 by troglitazone was almost completely inhibited by specific inhibitors of ERK activation. These data suggest that troglitazone is capable of inducing COX-2 expression through an ERK-dependent mechanism in mouse skin keratinocytes.     

Protective effects of a Ficus deltoidea (Mas cotek) extract against UVB-induced photoageing in skin cells

Continuous exposure to ultraviolet (UV) irradiation leads to a variety of skin damage, such as sunburn, pigmentation, premature ageing, and photocarcinogenesis. Various phytochemical extracts have been identified to efficiently protect sun exposed skin from UV induced photodamage. A Ficus deltoidea (Mas cotek) water extract has been widely used for women’s health in Malaysia. In a previous study from this lab, the F. deltoidea extract exhibited strong anti-melanogenic effects towards cultured B16F1 melanoma cells. Additional studies were intended to evaluate the effects of the F. deltoidea extract on antiphotoageing activity using cultured human dermal fibroblasts and immortalised human keratinocytes (HaCaT). Both TNF-α and cyclooxygenase (COX-2) play primary roles in the inflammation process upon UV irradiation and are known to be stimulated by UVB irradiation. Treatment with the F. deltoidea extract dramatically inhibited the UVinduced TNF-α, IL-1α, IL-6, and COX-2 expression. The decreased collagen synthesis of fibroblasts as a result of UVB exposure was restored to a normal level after treatment with the F. deltoidea extract. In addition, the enhanced MMP-1 expression upon UVB irradiation was downregulated by the F. deltoidea extract in a dose-dependent manner. The overall findings indicate that the F. deltoidea extract may exert a protective effect against UVB-induced damage in the skin that is useful for anti-photoageing cosmetic products.     

Reciprocal modulation of Toll-like receptor-4 signaling pathways involving MyD88 and phosphatidylinositol 3-kinase/AKT by saturated and polyunsaturated fatty acids

Toll-like receptor-4 (TLR4) can be activated by nonbacterial agonists, including saturated fatty acids. However, downstream signaling pathways activated by nonbacterial agonists are not known. Thus, we determined the downstream signaling pathways derived from saturated fatty acid-induced TLR4 activation. Saturated fatty acid (lauric acid)-induced NFkappaB activation was inhibited by a dominant-negative mutant of TLR4, MyD88, IRAK-1, TRAF6, or IkappaBalpha in macrophages (RAW264.7) and 293T cells transfected with TLR4 and MD2. Lauric acid induced the transient phosphorylation of AKT. LY294002, dominant-negative (DN) phosphatidylinositol 3-kinase (PI3K), or AKT(DN) inhibited NFkappaB activation, p65 transactivation, and cyclooxygenase-2 (COX-2) expression induced by lauric acid or constitutively active (CA) TLR4. AKT(DN) blocked MyD88-induced NFkappaB activation, suggesting that AKT is a MyD88-dependent downstream signaling component of TLR4. AKT(CA) was sufficient to induce NFkappaB activation and COX-2 expression. These results demonstrate that NFkappaB activation and COX-2 expression induced by lauric acid are at least partly mediated through the TLR4/PI3K/AKT signaling pathway. In contrast, docosahexaenoic acid (DHA) inhibited the phosphorylation of AKT induced by lipopolysaccharide or lauric acid. DHA also suppressed NFkappaB activation induced by TLR4(CA), but not MyD88(CA) or AKT(CA), suggesting that the molecular targets of DHA are signaling components upstream of MyD88 and AKT. Together, these results suggest that saturated and polyunsaturated fatty acids reciprocally modulate the activation of TLR4 and its downstream signaling pathways involving MyD88/IRAK/TRAF6 and PI3K/AKT and further suggest the possibility that TLR4-mediated target gene expression and cellular responses are also differentially modulated by saturated and unsaturated fatty acids.     

Involvement of P2Y6 receptor in p38 MAPK-mediated COX-2 expression in response to UVB irradiation of human keratinocytes

Ultraviolet B (UVB) radiation induces inflammation in human skin. Extracellular nucleotides are released from cells in response to various stimuli and act as intercellular signaling molecules through activation of P2 receptors. In this study, we investigated the involvement of extracellular nucleotides and P2 receptors in UVB-radiation-induced inflammation using human keratinocyte-derived HaCaT cells. UVB radiation induced rapid ATP release from HaCaT cells; this was inhibited by pretreatment with anion transporter blockers or maxi-anion channel blockers. In addition, the radiation-induced activation of p38 MAPK was significantly blocked by pretreatment with ecto-nucleotidase (apyrase) or P2Y6 receptor antagonist (MRS2578). Expression of COX-2, mediated by activation of p38 MAPK, was also induced by UVB radiation. Both pretreatment with MRS2578 and knockdown of the P2Y6 receptor by siRNA transfection attenuated the induction of COX-2 in HaCaT cells exposed to UVB radiation. Our results indicate that UVB radiation evokes ATP release from human keratinocytes and also that activation of P2Y6 receptor mediates the UVB-radiation-induced activation of p38 MAPK and expression of COX-2. Thus P2Y6 receptor is a mediator of UVB-radiation-induced inflammatory responses in keratinocytes.     

Ultraviolet light activates NFkappaB through translational inhibition of IkappaBalpha synthesis

UV light induces a delayed and prolonged (3-20 h) activation of NFkappaB when compared with the immediate and acute (10-90 min) activation of NFkappaB in response to tumor necrosis factor alpha treatment. In the early phase (3-12 h) of NFkappaB activation, UV light reduces inhibitor of NFkappaB (IkappaB) through an IkappaB kinase-independent, but polyubiquitin-dependent, pathway. However, the mechanism for the UV light-induced reduction of IkappaB and activation of NFkappaB is not known. In this report, we show that UV light down-regulates the total amount of IkappaB through decreasing IkappaB mRNA translation. Our data show that UV light inhibits translation of IkappaB in wild-type mouse embryo fibroblasts (MEF(S/S)) and that this inhibition is prevented in MEF(A/A) cells in which the phosphorylation site, Ser-51 in the eukaryotic translation initiation factor 2 alpha-subunit, is replaced with a non-phosphorylatable Ala (S51A). Our data also show that UV light-induced NFkappaB activation is delayed in MEF(A/A) cells and in an MCF-7 cell line that is stably transfected with a trans-dominant negative mutant protein kinase-like endoplasmic reticulum kinase (PERK). These results suggest that UV light-induced eukaryotic translation initiation factor 2 alpha-subunit phosphorylation translationally inhibits new IkappaB synthesis. Without a continuous supply of newly synthesized IkappaB, the existing IkappaB is degraded through a polyubiquitin-dependent proteasomal pathway leading to NFkappaB activation. Based upon our results, we propose a novel mechanism by which UV light regulates early phase NFkappaB activation by means of an ER-stress-induced translational inhibition pathway.     

Docosahexaenoic acid reverts resistance to UV-induced apoptosis in human keratinocytes: involvement of COX-2 and HuR

The dramatic increase in the incidence of nonmelanoma skin cancer over the last decades has been related to the augmented exposure to ultraviolet (UV) radiation (UVR). It is known that apoptosis is induced as a protective mechanism after the acute irradiation of keratinocytes, whereas apoptotic resistance and carcinogenesis may follow the chronic exposure to UVR. We found that not all the human keratinocytes lines studied underwent apoptosis following acute exposure to UVR (10-60 mJ/cm²). Whereas UVR induced apoptosis in the HaCaT cells, NCTC 2544 and nr-HaCaT cells showed apoptosis resistance. The cytokeratin pattern of the apoptosis-resistant cells indicated that they possessed a degree of differentiation lower than that of HaCaT cells. They also showed an enhanced expression of cyclooxygenase-2 (COX-2), an early marker of carcinogenesis in various tissues, including skin. n-3 polyunsaturated fatty acids have drawn increasing interest as nutritional factors with the potential to reduce UVR carcinogenesis, and since they are apoptosis inducers and COX-2 inhibitors in cancer cells, we investigated the ability of n-3 polyunsaturated fatty acids to influence the resistance to UVR-induced apoptosis in keratinocytes. We observed that docosahexaenoic acid (DHA) reverted the resistance of nr-HaCaT cells to UVR-induced apoptosis, increasing the Bax/Bcl-2 ratio and caspase-3 activity, and reduced COX-2 levels by inhibiting the expression of the human antigen R (HuR), a known COX-2 mRNA stabilizer in keratinocytes. The transfection of nr-HaCaT cells with HuR siRNA mimicked the proapoptotic effect of DHA. Overall, our findings further support the role of DHA as a suitable anticarcinogenic factor against nonmelanoma skin cancers.     

iNOS signaling interacts with COX-2 pathway in colonic fibroblasts

COX-2 and iNOS are two major inflammatory mediators implicated in colorectal inflammation and cancer. Previously, the role of colorectal fibroblasts involved in regulation of COX-2 and iNOS expression was largely ignored. In addition, the combined interaction of COX-2 and iNOS signalings and their significance in the progression of colorectal inflammation and cancer within the fibroblasts have received little investigation. To address those issues, we investigated the role of colonic fibroblasts in the regulation of COX-2 and iNOS gene expression, and explored possible mechanisms of interaction between COX-2 and iNOS signalings using a colonic CCD-18Co fibroblast line and LPS, a potential stimulator of COX-2 and iNOS. Our results clearly demonstrated that LPS activated COX-2 gene expression and enhanced PGE(2) production, stimulated iNOS gene expression and promoted NO production in the fibroblasts. Interestingly, activation of COX-2 signaling by LPS was not involved in activation of iNOS signaling, while activation of iNOS signaling by LPS contributed in part to activation of COX-2 signaling. Further analysis indicated that PKC plays a major role in the activation and interaction of COX-2 and iNOS signalings induced by LPS in the fibroblasts.     

COX-2 mediates hepatitis B virus X protein abrogation of p53-induced apoptosis

The oncogenic hepatitis B virus X protein (HBx) and cyclooxygenase (COX)-2 are highly co-expressed in chronic hepatitis, cirrhosis and well-differentiated hepatocellular carcinoma (HCC). Although HBx is shown to activate COX-2, the functional consequences of this interaction in hepatocarcinogenesis remain unknown. Using an engineered hepatoma cell system in which the expression of wild-type p53 can be chemically modulated, we show here that COX-2 mediates HBx actions in opposing p53. Enforced expression of HBx sequestrates p53 in the cytoplasm and significantly abolishes p53-induced apoptosis. The anti-apoptotic Mcl-1 protein is suppressed by p53 but reactivated by HBx. The abrogation of apoptosis is completely reversed by specific COX-2 inhibition, suggesting that HBx blocks p53-induced apoptosis via activation of COX-2/PGE₂ pathway. We further show that COX-2 inhibition blocks HBx reactivation of Mcl-1, linking this protein to the anti-apoptotic function of COX-2. These results demonstrate that COX-2 is an important survival factor mediating the oncogenic actions of HBx. Over-expression of HBx and COX-2 may provide a selective clonal advantage for preneoplastic or neoplastic hepatocytes and contribute to the initiation and progression of HCC.     

Resistance of senescent keratinocytes to UV-induced apoptosis.

Irradiation with ultraviolet (UV) triggers programmed cell death (apoptosis) in keratinocytes. This process is believed to protect against skin carcinogenesis since the cells with damaged DNA are selectively removed, limiting the likelihood of the development of a malignant keratinocyte clone. The p53 protein is able to detect mutation-bearing DNA fragments and is thus indispensable for the UV-induced apoptosis in the epidermis. Since age is a risk factor for the development of skin tumors we investigated whether ultraviolet induces apoptosis and p53 activation in senescent keratinocytes. Cultured senescent keratinocytes were irradiated with broad-band ultraviolet, apoptosis was assessed using TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling) technique and the p53 activation pattern was determined with Western blotting and immunofluorescent staining with a panel of anti-p53 antibodies recognising different conformational forms of the protein (PAb 122, PAb 240, DO-7). In senescent keratinocytes arrested in the G1 phase of cell cycle, ultraviolet irradiation (100-2000 J/m2) caused accumulation and nuclear translocation of p53. However, in contrast to young cells where UV induces apoptotic cell death in G1, apoptosis was not detected in senescent cells. There were subtle differences in the p53 activation pattern between senescent keratinocytes and known patterns in young keratinocytes and other cell types. In senescent keratinocytes a constitutional nuclear expression of p53 (conformational form recognized by PAb 240) was present and the p53 induction in response to ultraviolet radiation was rapid. Suppression of apoptosis in senescent keratinocytes may be an important mechanism responsible for enhanced skin carcinogenesis in old age.     

Resveratrol causes COX-2- and p53-dependent apoptosis in head and neck squamous cell cancer cells

Cyclooxygenase-2 (COX-2) content is increased in many types of tumor cells. We have investigated the mechanism by which resveratrol, a stilbene that is pro-apoptotic in many tumor cell lines, causes apoptosis in human head and neck squamous cell carcinoma UMSCC-22B cells by a mechanism involving cellular COX-2. UMSCC-22B cells treated with resveratrol for 24 h, with or without selected inhibitors, were examined: (1) for the presence of nuclear activated ERK1/2, p53 and COX-2, (2) for evidence of apoptosis, and (3) by chromatin immunoprecipitation to demonstrate p53 binding to the p21 promoter. Stilbene-induced apoptosis was concentration-dependent, and associated with ERK1/2 activation, serine-15 p53 phosphorylation and nuclear accumulation of these proteins. These effects were blocked by inhibition of either ERK1/2 or p53 activation. Resveratrol also caused p53 binding to the p21 promoter and increased abundance of COX-2 protein in UMSCC-22B cell nuclei. Resveratrol-induced nuclear COX-2 accumulation was dependent upon ERK1/2 activation, but not p53 activation. Activation of p53 and p53-dependent apoptosis were blocked by the COX-2 inhibitor, NS398, and by transfection of cells with COX-2-siRNA. In UMSCC-22B cells, resveratrol-induced apoptosis and induction of nuclear COX-2 accumulation share dependence on the ERK1/2 signal transduction pathway. Resveratrol-inducible nuclear accumulation of COX-2 is essential for p53 activation and p53-dependent apoptosis in these cancer cells.     

Gq protein mediates UVB-induced cyclooxygenase-2 expression by stimulating HB-EGF secretion from HaCaT human keratinocytes

Ultraviolet (UV) radiation induces cyclooxygenase-2 expression to produce cellular responses including aging and carcinogenesis in skin. We hypothesised that heterotrimeric G proteins mediate UV-induced COX-2 expression by stimulating secretion of soluble HB-EGF (sHB-EGF). In this study, we aimed to elucidate the role and underlying mechanism of the α subunit of Gq protein (Gαq) in UVB-induced HB-EGF secretion and COX-2 induction. We found that expression of constitutively active Gαq (GαqQL) augmented UVB-induced HB-EGF secretion, which was abolished by knockdown of Gαq with shRNA in HaCaT human keratinocytes. Gαq was found to mediate the UVB-induced HB-EGF secretion by sequential activation of phospholipase C (PLC), protein kinase Cδ (PKCδ), and matrix metaloprotease-2 (MMP-2). Moreover, GαqQL mediated UVB-induced COX-2 expression in an HB-EGF-, EGFR-, and p38-dependent manner. From these results, we concluded that Gαq mediates UV-induced COX-2 expression through activation of EGFR by HB-EGF, of which ectodomain shedding was stimulated through sequential activation of PLC, PKCδ and MMP-2 in HaCaT cells.     

Ca2+ stimulates COX-2 expression through calcium-sensing receptor in fibroblasts

Fibroblasts isolated from jaw cysts expressed calcium-sensing receptor (CasR). In the fibroblasts elevated extracellular Ca(2+) ([Ca(2+)](o)) increased fluo-3 fluorescence intensity, and the production of inositol(1,4,5)trisphosphate and active protein kinase C. Phospholipase C inhibitor U-73122 attenuated the Ca(2+)-induced increase in fluo-3 fluorescence intensity. Elevated [Ca(2+)](o) enhanced the expression of cyclooxygenase-2 (COX-2) mRNA and protein, and the secretion of prostaglandin E(2) in the fibroblasts. CasR activator neomycin also increased the expression of COX-2 mRNA, and U-73122 attenuated the Ca(2+)-induced expression of COX-2 mRNA. Elevated [Ca(2+)](o)-induced phosphorylation of extracellular signal-regulated protein kinase-1/2 (ERK1/2), p38 mitogen-activated protein kinase (MAPK), and c-Jun N-terminal kinase (JNK), and U-73122 inhibited the Ca(2+)-induced phosphorylation. The inhibitors for each kinase, PD98059, SB203580, and SP600125, attenuated the Ca(2+)-induced expression of COX-2 mRNA. These results suggest that in jaw cyst fibroblasts elevated extracellular Ca(2+) may enhance COX-2 expression via the activation of ERK1/2, p38 MAPK, and JNK through CasR.     

Myricetin blocks lipoteichoic acid-induced COX-2 expression in human gingival fibroblasts

Periodontitis is an infectious disease caused by microorganisms present in dental bacterial plaque. Lipoteichoic acid (LTA) is a component of the external membrane of Gram-positive bacteria. It causes septic shock. Ingested flavonoids have been reported to directly affect the regulation of cyclooxygenase-2 (COX-2) expression induced by bacterial toxins. In this study, we examined the effects of four flavonoids (luteolin, fisetin, morin and myricetin) on the activation of ERK1/2, p38 and AKT, and on the synthesis of COX-2 in human gingival fibroblasts treated with LTA from Streptococcus sanguinis. We found that luteolin and myricetin blocked AKT and p38 activation and that myricetin blocked LTA-induced COX-2 expression. The results of our study are important for elucidating the mechanism of action of flavonoid regulation of inflammatory responses.     

Signaling pathway requirements for induction of senescence by telomere homolog oligonucleotides

Cellular senescence is a major defense against cancer. In human fibroblasts, suppressing both the p53 and pRb pathways is necessary to bypass replicative senescence as well as senescence induced by ectopic expression of a dominant negative form of the telomere repeat binding factor 2, TRF2(DN). We recently reported that exposure to oligonucleotides homologous to the telomere 3' overhang (T-oligos) activates both the p53 and pRb pathways and leads to senescence in primary human fibroblasts. To further characterize T-oligo-induced senescence, we compared established isogenic fibroblast cell lines lacking functional p53 and/or pRb pathways to the normal parental line. Here, we report that, as in physiologic senescence, inactivation of both the p53 and pRb pathways is necessary to suppress T-oligo-induced senescence. Moreover, T-oligo rapidly induces senescence in a malignant fibroblast-derived cell line, demonstrating the potential of using T-oligo as a novel anticancer therapeutic. Our data support the hypothesis that exposure of the TTAGGG tandem repeat telomere 3' overhang sequence is the event that initiates signaling through DNA damage response pathways after experimental telomere disruption, serial passage, or acute genomic damage of normal cells.     

n-3 and n-6 polyunsaturated fatty acids induce the expression of COX-2 via PPARgamma activation in human keratinocyte HaCaT cells

Polyunsaturated fatty acids (PUFA) n-3 inhibit inflammation, in vivo and in vitro in keratinocytes. We examined in HaCaT keratinocyte cell line whether eicosapentaenoic acid (EPA) a n-3 PUFA, gamma-linoleic acid (GLA) a n-6 PUFA, and arachidic acid a saturated fatty acid, modulate expression of cyclooxygenase-2 (COX-2), an enzyme pivotal to skin inflammation and reparation. We demonstrate that only treatment of HaCaT with GLA and EPA or a PPARgamma ligand (roziglitazone), induced COX-2 expression (protein and mRNA). Moreover stimulation of COX-2 promoter activity was increased by those PUFAs or rosiglitazone. The inhibitory effects of GW9662 and T0070907 (PPARgamma antagonists), on COX-2 expression and on stimulation of COX-2 promoter activity by EPA and GLA suggest that PPARgamma is implicated in COX-2 induction. Finally, PLA2 inhibitor methyl arachidonyl fluorophosphonate blocked the PUFA effects on COX-2 induction, promoter activity and arachidonic acid mobilization suggesting involvement of AA metabolites in PPAR activation. These findings demonstrate that n-3 and n-6 PUFA increased PPARgamma activity is necessary for the COX-2 induction in HaCaT human keratinocyte cells. Given the anti-inflammatory properties of EPA, we suggest that induction of COX-2 in keratinocytes may be important in the anti-inflammatory and protective mechanism of action of PUFAs n-3 or n-6.     

AMP-activated protein kinase contributes to UV- and H2O2-induced apoptosis in human skin keratinocytes

AMP-activated protein kinase or AMPK is an evolutionarily conserved sensor of cellular energy status, activated by a variety of cellular stresses that deplete ATP. However, the possible involvement of AMPK in UV- and H(2)O(2)-induced oxidative stresses that lead to skin aging or skin cancer has not been fully studied. We demonstrated for the first time that UV and H(2)O(2) induce AMPK activation (Thr(172) phosphorylation) in cultured human skin keratinocytes. UV and H(2)O(2) also phosphorylate LKB1, an upstream signal of AMPK, in an epidermal growth factor receptor-dependent manner. Using compound C, a specific inhibitor of AMPK and AMPK-specific small interfering RNA knockdown as well as AMPK activator, we found that AMPK serves as a positive regulator for p38 and p53 (Ser(15)) phosphorylation induced by UV radiation and H(2)O(2) treatment. We also observed that AMPK serves as a negative feedback signal against UV-induced mTOR (mammalian target of rapamycin) activation in a TSC2-dependent manner. Inhibiting mTOR and positively regulating p53 and p38 might contribute to the pro-apoptotic effect of AMPK on UV- or H(2)O(2)-treated cells. Furthermore, activation of AMPK also phosphorylates acetyl-CoA carboxylase or ACC, the pivotal enzyme of fatty acid synthesis, and PFK2, the key protein of glycolysis in UV-radiated cells. Collectively, we conclude that AMPK contributes to UV- and H(2)O(2)-induced apoptosis via multiple mechanisms in human skin keratinocytes and AMPK plays important roles in UV-induced signal transduction ultimately leading to skin photoaging and even skin cancer.     

Inhibition of p38 mitogen-activated protein kinase and phosphatidylinositol 3-kinase decreases UVB-induced activator protein-1 and cyclooxygenase-2 in a SKH-1 hairless mouse model

Activation of activator protein-1 (AP-1) and increased expression of cyclooxygenase-2 (COX-2) have been clearly shown to play a functional role in UVB-induced skin tumor promotion. In this study, we examined UVB-induced signal transduction pathways in SKH-1 mouse epidermis leading to increases in COX-2 expression and AP-1 activity. We observed rapid increases in p38 mitogen-activated protein kinase (MAPK) signaling through activation of p38 MAPK and its downstream target, MAPK activated protein kinase-2. UVB also increased phosphatidylinositol 3-kinase (PI3K) signaling as observed through increases in AKT and GSK-3beta phosphorylation. Activation of the p38 MAPK and PI3K pathways results in the phosphorylation of cyclic AMP-responsive element binding protein, which was also observed in UVB-irradiated SKH-1 mice. Topical treatment with SB202190 (a specific inhibitor of p38 MAPK) or LY294002 (a specific inhibitor of PI3K) significantly decreased UVB-induced AP-1 activation by 84% and 68%, respectively, as well as COX-2 expression. Our data show that in mouse epidermis, UVB activation of the p38 MAPK and PI3K pathways leads to AP-1 activation and COX-2 expression.