Eicosapentaenoic acid inhibits TNF-alpha-induced matrix metalloproteinase-9 expression in human keratinocytes, HaCaT cells

Eicosapentaenoic acid (EPA) is an omega-3 (ω-3) polyunsaturated fatty acid (PUFA), which has anti-inflammatory and anti-cancer properties. Some reports have demonstrated that EPA inhibits NF-κB activation induced by tumor necrosis factor (TNF)-α or lipopolysaccharide (LPS) in various cells. However, its detailed mode of action is unclear. In this report, we investigated whether EPA inhibits the expression of TNF-α-induced matrix metalloproteinases (MMP)-9 in human immortalized keratinocytes (HaCaT). TNF-α induced MMP-9 expression by NF-κB-dependent pathway. Pretreatment of EPA inhibited TNF-α-induced MMP-9 expression and p65 phosphorylation. However, EPA could not affect IκB-α phosphorylation, nuclear translocation of p65, and DNA binding activity of NF-κB. EPA inhibited TNF-α-induced p65 phosphorylation through p38 and Akt inhibition and this inhibition was IKKα-dependent event. Taken together, we demonstrate that EPA inhibits TNF-α-induced MMP-9 expression through inhibition of p38 and Akt activation.     

Cholesterol inhibits MMP-9 expression in human epidermal keratinocytes and HaCaT cells

Cholesterol is a major component of skin lipids and acts as a regulator of vesicular trafficking and signal transduction. However, the function of cholesterol on matrix metalloproteinases (MMPs) expression of human skin is not fully understood. Here, we investigated the effects of cholesterol on MMP-9 expression in normal human keratinocytes (NHK) and HaCaT cells. Basal level of MMP-9 expression was decreased by cholesterol in NHK. On the other hand, MMP-9 expression was increased by the cholesterol depletion agent, methyl-beta-cyclodextrin (MbetaCD), while it was inhibited by cholesterol repletion in HaCaT cells. MbetaCD induced ERK and JNK phosphorylation were prevented by cholesterol repletion. The inhibition of ERK and JNK decreased MbetaCD-induced MMP-9 expression. Therefore, our results suggest that cholesterol regulates MMP-9 expression through ERK and JNK-dependent pathways.     

Photoprotective and anti-skin-aging effects of eicosapentaenoic acid in human skin in vivo

Skin aging can be attributed to photoaging (extrinsic) and chronological (intrinsic) aging. Photoaging and intrinsic aging are induced by damage to human skin attributable to repeated exposure to ultraviolet (UV) irradiation and to the passage of time, respectively. In our previous report, eicosapentaenoic acid (EPA) was found to inhibit UV-induced matrix metalloproteinase-1 (MMP-1) expression in human dermal fibroblasts. Therefore, we investigated the effects of EPA on UV-induced skin damage and intrinsic aging by applying EPA topically to young and aged human skin, respectively. By immunohistochemical analysis and Western blotting, we found that topical application of EPA reduced UV-induced epidermal thickening and inhibited collagen decrease induced by UV light. It was also found that EPA attenuated UV-induced MMP-1 and MMP-9 expression by inhibiting UV-induced c-Jun phosphorylation, which is closely related to UV-induced activator protein-1 activation, and by inhibiting JNK and p38 activation. EPA also inhibited UV-induced cyclooxygenase-2 (COX-2) expression without altering COX-1 expression. Moreover, it was found that EPA increased collagen and elastic fibers (tropoelastin and fibrillin-1) expression by increasing transformin growth factor-beta expression in aged human skin. Together, these results demonstrate that topical EPA has potential as an anti-skin-aging agent.     

Eicosapentaenoic acid and docosahexaenoic acid modulate MAP kinase enzyme activity in human T-cells

In order to investigate the implication of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) in T signalling, we assessed their effects on the activation of two mitogen activated protein (MAP) kinases, i.e. extracellularly-regulated kinases 1 and 2 (ERK1/ERK2) in Jurkat T-cells. The n-3 polyunsaturated fatty acids (PUFAs) alone failed to induce MAP kinase (MAPK) enzyme activity. To elucidate whether DHA and EPA act via protein kinase C (PKC) dependent and independent pathways, we employed their respective activators, i.e. phorbol 12-myristate 13-acetate (PMA) and antiCD3 antibodies. We observed that U0126, an inhibitor of MAPK kinase-ERK kinase 1/2 (MEK1/2), abolished the actions of these two agents on MAPK activation, suggesting that they act upstream of MEK1/2. Further EPA and DHA diminished both the PMA- and antiCD3 antibodies-induced enzyme activity of ERK1/ERK2 in Jurkat T-cells. Interestingly, okadaic acid (OA), a phosphatase inhibitor seems to act downstream of MEK1/2 as U0126 failed to inhibit the OA-induced MAPK activation. It is noteworthy that EPA and DHA not only failed to curtail the OA-induced MAPK activity but also these n-3 PUFAs at 20 M potentiated the action of OA. Therefore, EPA and DHA seem to modulate MAPK activation upstream and downstream of MEK1/2. On the hand, arachidonic acid, an n-6 PUFA potentiated the MAPK enzyme activity. In conclusion, our study shows that EPA and DHA may regulate T-cells functions by modulating MAPK enzyme activity.     

10t,12c-conjugated linoleic acid inhibits lipopolysaccharide-induced cyclooxygenase expression in vitro and in vivo

Previous data demonstrated that conjugated linoleic acid (CLA) reduced eicosanoid release from select organs. We hypothesized that one active CLA isomer was responsible for the reduced prostaglandin release and that the mechanism was through the inhibition of inducible cyclooxygenase-2 (COX-2). Here, we examined the effects of 10t,12c-CLA and 9c,11t-CLA on COX-2 protein/mRNA expression, prostaglandin E(2) (PGE(2)) production, and the mechanism by which CLA affects COX-2 expression and prostaglandin release. The COX-2 protein expression level was inhibited 80% by 10t, 12c-CLA and 26% by 9c,11t-CLA at 100 microM in vitro. PGE(2) production was decreased from 5.39 to 1.12 ng/2 x 10(6) cells by 10t,12c-CLA and from 5.7 to 4.5 ng/2 x 10(6) cells by 9c,11t-CLA at 100 microM. Mice fed 10t,12c-CLA but not 9c,11t-CLA were found to have a 34% decrease in COX-2 protein and a 43% reduction of PGE(2) release in the lung. 10t,12c-CLA reduced COX-2 mRNA expression level by 30% at 100 microM in vitro and by 30% in mouse lung in vivo. Reduced COX-2 mRNA was attributable to an inhibition of the nuclear factor kappaB (NF-kappaB) pathway by 10t,12c-CLA. These data suggested that the inhibition of NF-kappaB was one of the mechanisms for the reduced COX-2 expression and PGE(2) release by 10t,12c-CLA.     

Profibrillin-1 maturation by human dermal fibroblasts: proteolytic processing and molecular chaperones

Fibrillin-1 is synthesized as a proprotein that undergoes proteolytic processing in the unique C-terminal domain by a member of the PACE/furin family of endoproteases. This family of endoproteases is active in the trans-Golgi network (TGN), but metabolic labeling studies have been controversial as to whether profibrillin-1 is processed intracellularly or after secretion. This report provides evidence that profibrillin-1 processing is not an intracellular event. Bafilomycin A(1) and incubation of dermal fibroblasts at 22 degrees C were used to block secretion in the TGN to confirm that profibrillin-1 processing did not occur in this compartment. Profibrillin-1 immunoprecipitation studies revealed that two endoplasmic reticulum-resident molecular chaperones, BiP and GRP94, interacted with profibrillin-1. To determine the proprotein convertase responsible for processing profibrillin-1, a specific inhibitor of furin, alpha-1-antitrypsin, Portland variant, was both expressed in the cells and added to cells exogenously. In both cases, the inhibitor blocked the processing of profibrillin-1, providing evidence that furin is the enzyme responsible for profibrillin-1 processing. These studies delineate the secretion and proteolytic processing of profibrillin-1, and identify the proteins that interact with profibrillin-1 in the secretory pathway.     

Eicosapentaenoic acid decreases expression of anandamide synthesis enzyme and cannabinoid receptor 2 in osteoblast-like cells

Anandamide (AEA) is an endogenous agonist for the cannabinoid receptor 2 (CB2) which is expressed in osteoblasts. Arachidonic acid (AA) is the precursor for AEA and dietary n-3 polyunsaturated fatty acids (PUFA) are known to reduce the concentrations of AA in tissues and cells. Therefore, we hypothesized that n-3 PUFA, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), which reduce AA in cells, could lower AEA in osteoblasts by altering enzyme expression of the endocannabinoid (EC) system. MC3T3-E1 osteoblast-like cells were grown for 6, 10, 15, 20, 25 or 30 days in osteogenic medium. Osteoblasts were treated with 10 μM of AA, EPA, DHA, oleic acid (OA) or EPA+DHA (5 μM each) for 72 h prior to their collection for measurement of mRNA and alkaline phosphatase (ALP) activity. Compared to vehicle control, osteoblasts treated with AA had higher levels of AA and n-6 PUFA while those treated with EPA and DHA had lower n-6 but higher n-3 PUFA. Independent of the fatty acid treatments, osteoblasts matured normally as evidenced by ALP activity. N-acyl phosphatidylethanolamine-selective phospholipase D (NAPE-PLD), fatty acid amide hydrolase (FAAH) and CB2 mRNA expression were higher at 20 days compared to 10 days. NAPE-PLD and CB2 mRNA was lower in osteoblasts treated with EPA compared to all other groups. Thus, mRNA expression for NAPE-PLD, FAAH, and CB2 increased during osteoblast maturation and EPA reduced mRNA for NAPE-PLD and CB2 receptor. In conclusion, EPA lowered mRNA levels for proteins of the EC system and mRNA for AEA synthesis/degradation is reported in osteoblasts.     

Berberine inhibits TPA-induced MMP-9 and IL-6 expression in normal human keratinocytes

Berberine is a plant ingredient that has anti-inflammatory and anti-oxidative effects. Matrix metalloproteinase-9 (MMP-9) and interleukin-6 (IL-6) are known to be highly induced by ultraviolet (UV) light and may play important roles in UV-induced skin inflammation and the skin aging process. In this study, we investigated the effects of berberine on MMP-9 and IL-6 expression in normal human keratinocytes (NHK). Our results demonstrated that berberine dose-dependently inhibited basal and TPA-induced expression and activity of MMP-9, and also suppressed TPA-induced IL-6 expression. Berberine prevented TPA-induced ERK activation and AP-1 DNA binding activity. Therefore, berberine may be used as an effective ingredient for anti-skin aging products, which can prevent skin inflammation and the degradation of extracellular matrix proteins, including collagen, by MMPs.     

Eicosapentaenoic and docosahexaenoic acid ethyl esters differentially enhance B-cell activity in murine obesity

EPA and DHA are not biologically equivalent; however, their individual activity on B cells is unknown. We previously reported fish oil enhanced murine B-cell activity in obesity. To distinguish between the effects of EPA and DHA, we studied the ethyl esters of EPA and DHA on murine B-cell function as a function of time. We first demonstrate that EPA and DHA maintained the obese phenotype, with no improvements in fat mass, adipose inflammatory cytokines, fasting insulin, or glucose clearance. We then tested the hypothesis that EPA and DHA would increase the frequency of splenic B cells. EPA and DHA differentially enhanced the frequency and/or percentage of select B-cell subsets, correlating with increased natural serum IgM and cecal IgA. We next determined the activities of EPA and DHA on ex vivo production of cytokines upon lipopolysaccharide stimulation of B cells. EPA and DHA, in a time-dependent manner, enhanced B-cell cytokines with DHA notably increasing IL-10. At the molecular level, EPA and DHA differentially enhanced the formation of ordered microdomains but had no effect on Toll-like receptor 4 mobility. Overall, the results establish differential effects of EPA and DHA in a time-dependent manner on B-cell activity in obesity, which has implications for future clinical studies.     

Late G1 amino acid restriction point in human dermal fibroblasts

Human dermal fibroblasts arrested in G0 by maintenance in medium supplemented with 0.1% serum were not restimulated to divide when fresh medium containing 10% dialyzed serum but lacking group B amino acids (cystine, isoleucine, lysine, phenylalanine and tyrosine) was added. Unlike rodent cells, the addition of fresh serum-supplemented medium lacking only isoleucine did not cause a growth arrest. The amino acid sensitive growth arrest in human fibroblasts was dependent both on presynchronization in G0 as well as a prestarvation for amino acids prior to stimulation with high serum. When cells were restimulated in the absence of amino acids, they arrested predominantly in G1, although a small percentage of cells entered early S phase. When medium containing a complete complement of amino acids was then added, cells initiated DNA synthesis following a minimum lag of 2-3 hr. Growth arrested cells initiated DNA synthesis even when complete unsupplemented medium was added, although the addition of high concentrations of insulin or 10% serum increased the rate of entry.     

Inhibition of MMP-3 or -9 suppresses lipopolysaccharide-induced expression of proinflammatory cytokines and iNOS in microglia

Recently, matrix metalloproteinases (MMPs) are emerging as important molecules in neuroinflammation as well as neuronal cell death. However, the role of MMPs in activated microglia remains unclear. In the present study, we found that expressions of MMP-1, -3, -8 and -9 were significantly induced by single or combined treatment of immunostimulants lipopolysaccharide (LPS) or phorbol myristate acetate (PMA) in primary cultured microglia and BV2 microglial cells. Inhibition of MMP-3 or -9 significantly suppressed the expression of iNOS and pro-inflammatory cytokines and the activities of NF-κB, AP-1, and MAPK in LPS-stimulated microglia. The results suggest that MMP-3 and -9 both mediate LPS-induced inflammatory reactions. Inhibition of reactive oxygen species (ROS) by N-acetyl-cysteine or diphenylene iodonium significantly suppressed the expression of MMP-3, MMP-9, NO and TNF-α in LPS-stimulated microglia, suggesting that ROS is an early signaling inducer in LPS-stimulated microglial cells. MMP inhibitors also suppressed ROS production, suggesting a cross-talk between ROS and MMPs. Collectively, the present study demonstrates that MMP-3 and MMP-9 play a role as inflammatory mediators in activated microglia. Pharmacological intervention of MMPs especially MMP-3 and -9 would be a therapeutic strategy for the treatment of inflammatory diseases in the CNS caused by over-activation of microglial cells.     

Up-regulated type I collagen expression by the inhibition of Rac1 signaling pathway in human dermal fibroblasts

Tissue remodeling is known to play important roles in wound healing. Although Rac1 is reported to be one of the key signaling molecules in cutaneous wound healing process, the exact mechanisms of Rac1-mediated tissue remodeling is still unknown. This study investigated the role of Rac1 in the regulation of extracellular matrix in cultured human dermal fibroblasts obtained by skin biopsy from three healthy donors. Protein levels of type I collagen in cultured human fibroblasts were increased by the treatment with Rac1 inhibitor NSC23766 in a dose-dependent manner. However, the mRNA levels of α2(I) collagen was not altered by the inhibitor. On the other hand, by the addition of inhibitor, half-lives of type I collagen protein were increased and MMP1 levels were reduced. These data suggest that blockade of Rac1 signaling results in accumulation of type I collagen due to decreased collagenase activity. This study also suggests that controlling Rac1 signaling is a new therapeutic approach to chronic/untreatable ulcer.     

Free fatty acid release from human breast cancer tissue inhibits cytotoxic T-lymphocyte-mediated killing

Immune-mediated antitumor activities confront a variety of tumor-mediated defense mechanisms. Here, we describe a new mechanism involving FFA that may allow breast cancer to evade immune clearance. We determined the IC50 at which unbound free fatty acids (FFA(u)) inhibit murine cytotoxic T-lymphocyte (CTL)-mediated killing to assess the physiologic relevance of this phenomenon. We found that the IC50 for unbound oleate is 125 +/- 30 nM, approximately 200-fold greater than normal plasma levels. FFA inhibition, however, may play an important role in breast cancer because we found that large quantities of FFAs are released constitutively into the media surrounding samples of human breast cancer but not normal or benign tissue. FFA(u) concentration ([FFA(u)]) increased to at least 25 nM in 20 of 22 cancer tissue samples and exceeded 100 nM in 11 patients. Media from these samples inhibited CTL-mediated killing. Extrapolation from our in vitro conditions suggests that for tumor interstitial fluid, in vivo [FFA(u)] may be 300-fold greater than we observed in vitro. Although breast cancer release of FFA may suppress effector cell antitumor activity, strategies that reduce interstitial [FFA(u)] may significantly improve antitumor immune therapies.