Birgit Haarhaus

Expression and Functional Activity of the Bitter Taste Receptors TAS2R1 and TAS2R38 in Human Keratinocytes

Recent studies have shown that human bitter taste receptors (TAS2Rs) are not only expressed in mucous epithelial cells of the tongue, but also in epithelial cells of the colon, stomach and upper respiratory tract. These cell types come in close contact with external bitter compounds by ingestion or breathing. In the present work we addressed the question whether bitter taste receptors might also be expressed in cornified epithelial cells of the skin. Here, we show for the first time the expression of TAS2R1 and TAS2R38 in human skin. Double staining of HaCaT cells and primary keratinocytes demonstrated the colocalization of TAS2R1 and TAS2R38 with the adaptor protein α-gustducin that is essential for signal transduction upon ligand binding. To test if TAS2Rs in keratinocytes are functional, we stimulated HaCaT cells with diphenidol, a clinically used bitter-tasting antiemetic, or amarogentin, the bitterest plant substance, that binds TAS2Rs, including TAS2R1 and TAS2R38. Diphenidol and amarogentin induced calcium influx. Furthermore, in keratinocytes diphenidol and amarogentin stimulated the expression of the differentiation markers keratin 10, involucrin and transglutaminase. Therefore, apart from the known role in mucous membranes of the gastrointestinal tract, TAS2Rs are expressed in the epidermis and might play a role in keratinocyte differentiation.

The Photoprotective and Antioxidative Properties of Luteolin are Synergistically Augmented by Tocopherol and Ubiquinone

Ultraviolet radiation induces DNA damage and oxidative stress which can result in skin inflammation, photoaging, and photocarcinogenesis. The flavonoid luteolin that is present in high amounts in the dyers weld, Reseda luteola, is one of the most potent antioxidative plant metabolites and also has ultraviolet-absorbing properties.The aim of this study was to determine whether tocopherol and ubiquinone add synergistic antioxidative values to luteolin. None of the substances showed cytotoxic effects in concentrations from 0.25 to 4 µg/mL. The photoprotective and antioxidant effect of equivalent concentrations of luteolin, tocopherol, and ubiquinone and their combination in a ratio of 4 : 4 : 1 were studied in solar simulator irradiated human skin fibroblasts. Luteolin had a half-maximal radical scavenging concentration of 2 µg/mL, whereas tocopherol and ubiquinone were only effective at higher concentrations. None of the substances showed a phototoxic effect, and only luteolin had a moderate photoprotective effect at 2 µg/mL. The combination of luteolin, tocopherol, and ubiquinone exerted a synergistic radical scavenging effect already at a concentration of 0.25 µg/mL and a complete photoprotection at 2 µg/mL.In summary, our findings suggest that the potent antioxidant and photoprotective effect of flavonoids like luteolin may be further increased by the addition of low concentrations of other antioxidants such as tocopherol and ubiquinone.

Expression and Functional Activity of the Human Bitter Taste Receptor TAS2R38 in Human Placental Tissues and JEG-3 Cells

Bitter taste receptors (TAS2Rs) are expressed in mucous epithelial cells of the tongue but also outside the gustatory system in epithelial cells of the colon, stomach and bladder, in the upper respiratory tract, in the cornified squamous epithelium of the skin as well as in airway smooth muscle cells, in the testis and in the brain. In the present work we addressed the question if bitter taste receptors might also be expressed in other epithelial tissues as well. By staining a tissue microarray with 45 tissue spots from healthy human donors with an antibody directed against the best characterized bitter taste receptor TAS2R38, we observed an unexpected strong TAS2R38 expression in the amniotic epithelium, syncytiotrophoblast and decidua cells of the human placenta. To analyze the functionality we first determined the TAS2R38 expression in the placental cell line JEG-3. Stimulation of these cells with diphenidol, a clinically used antiemetic agent that binds TAS2Rs including TAS2R38, demonstrated the functionality of the TAS2Rs by inducing calcium influx. Restriction enzyme based detection of the TAS2R38 gene allele identified JEG-3 cells as PTC (phenylthiocarbamide)-taster cell line. Calcium influx induced by PTC in JEG-3 cells could be inhibited with the recently described TAS2R38 inhibitor probenecid and proved the specificity of the TAS2R38 activation. The expression of TAS2R38 in human placental tissues points to further new functions and hitherto unknown endogenous ligands of TAS2Rs far beyond bitter tasting.

Luteolin Prevents Solar Radiation-Induced Matrix Metalloproteinase-1 Activation in Human Fibroblasts: A Role for p38 Mitogen-Activated Protein Kinase and Interleukin-20 Released from Keratinocytes

Human skin is continuously exposed to solar radiation, which can result in photoaging, a process involving both dermal and, to a lesser extent, epidermal structures. Previously, we have shown that the flavonoid luteolin protects the epidermis from ultraviolet (UV)-induced damage by a combination of UV-absorbing, antioxidant, and antiinflammatory properties. The aim of the present study was to determine direct and indirect effects of luteolin on dermal fibroblasts as major targets of photoaging. Stimulation of fibroblasts with UVA light or the proinflammatory cytokine interleukin-20 (IL-20) is associated with wrinkled skin, increased IL-6 secretion, matrix metalloproteinase (MMP-1) expression, and hyaluronidase activity. All of these targets were inhibited by luteolin via interference with the p38 mitogen-activated protein kinase (MAPK) pathway. Next, we assessed the role of conditioned supernatants from keratinocytes irradiated with solar-simulated radiation (SSR) on nonirradiated dermal fibroblasts. In keratinocytes, luteolin inhibited SSR-induced production of IL-20, also via interference with the p38 MAPK pathway. Similarly, keratinocyte supernatant-induced IL-6 and MMP-1 expression in fibroblasts was reduced by pretreatment of keratinocytes with luteolin. Finally, these results were confirmed ex vivo on skin explants treated with luteolin before UV irradiation. Our results suggest that SSR-mediated production of soluble factors in keratinocytes is modulated by luteolin and may attenuate photoaging in dermal fibroblasts.

Anti-Inflammatory Effects of Agrimoniin-Enriched Fractions of Potentilla erecta.

Potentilla erecta (PE) is a small herbaceous plant with four yellow petals belonging to the Rosaceae family. The rhizome of PE has traditionally been used as an antidiarrheal, hemostatic and antihemorrhoidal remedy. PE contains up to 20% tannins and 5% ellagitannins, mainly agrimoniin. Agrimoniin is a hydrolyzable tannin that is a potent radical scavenger. In this study we tested the anti-inflammatory effect of four PE fractions with increasing amounts of agrimoniin obtained by Sephadex column separation. First, we analyzed in HaCaT keratinocytes the expression of cyclooxygenase-2 (COX-2) induced by ultraviolet-B (UVB) irradiation. As COX-2 catalyzes the metabolism of arachidonic acid to prostanoids such as PGE2, we also measured the PGE2 concentration in cell culture supernatants. PE inhibited UVB-induced COX-2 expression in HaCaT cells and dose-dependently reduced PGE2. The PE fraction with the highest agrimoniin amount (PE4) was the most effective in this experiment, whereas fraction PE1 containing mainly sugars had no effect. PE4 also dose dependently inhibited the phosphorylation of the epidermal growth factor receptor (EGFR) which plays a crucial role in UVB-mediated COX-2 upregulation. A placebo-controlled UV-erythema study with increasing concentrations of PE4 demonstrated a dose dependent inhibition of UVB-induced inflammation in vivo. Similarly, PE4 significantly reduced UVB-induced PGE2 production in suction blister fluid in vivo. In summary, PE fractions with a high agrimoniin content display anti-inflammatory effects in vitro and in vivo in models of UVB-induced inflammation.

Salicin from Willow Bark can Modulate Neurite Outgrowth in Human Neuroblastoma SH‐SY5Y Cells

Salicin from willow bark has been used throughout centuries in China and Europe for the treatment of pain, headache, and inflammatory conditions. Recently, it could be demonstrated that salicin binds and activates the bitter taste receptor TAS2R16. Studies on rodent tissues showed the general expression of bitter taste receptors (TAS2Rs) in rodent brain. Here, we demonstrate the expression of hTAS2R16 in human neuronal tissues and the neuroblastoma cell line SH‐SY5Y. The functionality was analyzed in the neuroblastoma cell line SH‐SY5Y after stimulation with salicin, a known TAS2R16 agonist. In this setting salicin induced in SH‐SY5Y cells phosphorylation of ERK and CREB, the key transcription factor of neuronal differentiation. PD98059, an inhibitor of the ERK pathway, as well as probenecid, a TAS2R16 antagonist, inhibited receptor phosphorylation as well as neurite outgrowth. These data show that salicin might modulate neurite outgrowth by bitter taste receptor activation. Copyright

Amarogentin Displays Immunomodulatory Effects in Human Mast Cells and Keratinocytes

Keratinocytes express the bitter taste receptors TAS2R1 and TAS2R38. Amarogentin as an agonist for TAS2R1 and other TAS2Rs promotes keratinocyte differentiation. Similarly, mast cells are known to express bitter taste receptors. The aim of this study was to assess whether bitter compounds display immunomodulatory effects on these immunocompetent cells in the skin, so that they might be a target in chronic inflammatory diseases such as atopic dermatitis and psoriasis. Here, we investigated the impact of amarogentin on substance P-induced release of histamine and TNF-α from the human mast cell line LAD-2. Furthermore, the effect of amarogentin on HaCaT keratinocytes costimulated with TNF-α and histamine was investigated. Amarogentin inhibited in LAD-2 cells substance P-induced production of newly synthesized TNF-α, but the degranulation and release of stored histamine were not affected. In HaCaT keratinocytes histamine and TNF-α induced IL-8 and MMP-1 expression was reduced by amarogentin to a similar extent as with azelastine. In conclusion amarogentin displays immunomodulatory effects in the skin by interacting with mast cells and keratinocytes.

The Herbal Bitter Drug Gentiana lutea Modulates Lipid Synthesis in Human Keratinocytes In Vitro and In Vivo

Gentiana lutea is a herbal bitter drug that is used to enhance gastrointestinal motility and secretion. Recently we have shown that amarogentin, a characteristic bitter compound of Gentiana lutea extract (GE), binds to the bitter taste receptors TAS2R1 and TAS2R38 in human keratinocytes, and stimulates the synthesis of epidermal barrier proteins. Here, we wondered if GE also modulates lipid synthesis in human keratinocytes. To address this issue, human primary keratinocytes were incubated for 6 days with GE. Nile Red labeling revealed that GE significantly increased lipid synthesis in keratinocytes. Similarly, gas chromatography with flame ionization detector indicated that GE increases the amount of triglycerides in keratinocytes. GE induced the expression of epidermal ceramide synthase 3, but not sphingomyelinase. Lipid synthesis, as well as ceramide synthase 3 expression, could be specifically blocked by inhibitors of the p38 MAPK and PPARγ signaling pathway. To assess if GE also modulates lipid synthesis in vivo, we performed a proof of concept half side comparison on the volar forearms of 33 volunteers. In comparison to placebo, GE significantly increased the lipid content of the treated skin areas, as measured with a sebumeter. Thus, GE enhances lipid synthesis in human keratinocytes that is essential for building an intact epidermal barrier. Therefore, GE might be used to improve skin disorders with an impaired epidermal barrier, e.g., very dry skin and atopic eczema.

Anti-inflammatory and vasoconstrictive properties of Potentilla erecta – A traditional medicinal plant from the northern hemisphere

ETHNOPHARMACOLOGICAL RELEVANCE Potentilla erecta (L.) Raeusch is a medicinal plant of the Northern hemisphere belonging to the plant family of roses (Rosaceae). It has traditionally been used to treat inflammatory disorders of the skin and mucous membranes as well as chronic diarrhea. AIM OF THE STUDY In the present study we analyzed the anti-inflammatory and vasoconstrictive effect of a Potentilla erecta extract (PE) and questioned if PE is similar effective as mild corticosteroids. Then we analyzed if PE acts in the skin via a similar mode of action as corticosteroids. MATERIAL AND METHODS The anti-inflammatory effect of PE was analyzed in irradiated HaCaT keratinocytes by measuring the formation of IL-6 and PGE2. Additionally the effect of PE on TNF-α induced NF-κB activation was determined. As the anti-inflammatory effect of corticosteroids correlates with their vasoconstrictive properties we tested if PE displays also vasoconstriction. Therefore we performed an occlusive patch test and a collagen contraction assay. Furthermore the binding of PE to the glucocorticoid receptor was determined with stainings and reporter assays. The interaction of PE on the nitric oxide (NO) content was examined with radical scavenging and endothelial NO synthase (eNOS) reporter assays. RESULTS In irradiated or TNF-α stimulated HaCaT cells the formation of IL-6 and PGE2 or NF-κB activation was strongly reduced by PE. Furthermore PE showed a blanching effect comparable to hydrocortisone. However, in contrast to glucocorticoids, PE did not cause nuclear translocation of the glucocorticoid receptor in HaCaT cells. The blanching effect of PE was at least partly attributable to a scavenging effect of NO and inhibition of eNOS. CONCLUSIONS PE displays anti-inflammatory and vasoconstrictive effects and might therefore be beneficial for the topical treatment of inflammatory skin disorders.