Inhibitory effects of Sanguisorba officinalis root extract on HYBID (KIAA1199)-mediated hyaluronan degradation and skin wrinkling.

OBJECTIVES: Hyaluronan (HA), an important constituent of extracellular matrix in the skin, has many biological activities such as hydration that contributes to firmness and bounciness of the skin. We have reported that reduction in HA in the papillary dermis and over-expression of HYBID (HYaluronan Binding protein Involved in hyaluronan Depolymerization, alias KIAA1199 or CEMIP), a key molecule for HA degradation in skin fibroblasts, are implicated in facial skin wrinkling in Japanese and Caucasian women. However, little or no information is available for substances which inhibit the HYBID-mediated HA degradation. METHODS: Inhibition of Sanguisorba officinalis root extract and ziyuglycoside I, one of the components of Sanguisorba officinalis root extract, to the HYBID-mediated HA degradation was assessed by size-exclusion chromatography of HA depolymerized by stable transfectants of HYBID in HEK293 cells (HYBID/HEK293 cells) or normal human skin fibroblasts (Detroit 551 cells and NHDF-Ad cells). The HYBID mRNA and protein expression was examined by quantitative real-time PCR and immunoblotting in the skin fibroblasts treated with Sanguisorba officinalis root extract, and size distribution of newly produced HA was evaluated by preparing metabolically radiolabelled HA. A double-blind, randomized and placebo-controlled study was carried out in the 21 healthy Japanese women, who were topically treated with the formulation containing Sanguisorba officinalis root extract or the placebo on each side of the face including crow's foot area. RESULTS: Sanguisorba officinalis root extract, but not ziyuglycoside I, abolished HYBID-mediated HA degradation by HYBID/HEK293 cells. Sanguisorba officinalis root extract also inhibited HYBID-mediated HA degradation in skin fibroblasts by down-regulating HYBID mRNA and protein expression. Although control untreated skin fibroblasts produced polydispersed HA, the cells treated with Sanguisorba officinalis root extract produced only high-molecular-weight HA. Treatment with Sanguisorba officinalis root extract-formulated lotion significantly improved skin elasticity, and reduced skin wrinkling scores at the outer eye corner compared with the placebo formulation. CONCLUSION: Sanguisorba officinalis root extract showed an anti-HYBID-mediated HA degradation activity and anti-wrinkle activity on human facial skin, which is accompanied by the improvement in elasticity. Our study provides the possibility of a new strategy to inhibit HYBID-mediated HA degradation for anti-wrinkle care.

OBJECTIFS: l'acide hyaluronique (AH), un composant important de la matrice extracellulaire de la peau, assure de nombreuses activités biologiques, telles que l'hydratation qui contribue à la fermeté et l'élasticité de la peau. Nous avons rapporté que la réduction d'AH dans le derme papillaire et une surexpression de la protéine de liaison de l'AH impliquée dans la dépolymérisation de l'AH (HYBID, alias KIAA1199 ou CEMIP), une molécule clé de la dégradation de l'AH des fibroblastes cutanés, sont impliquées dans la formation des rides au niveau de la peau du visage chez les femmes d'origine japonaise et caucasienne. Cependant, peu ou aucune information n'est disponible concernant les substances qui inhibent la dégradation de l'AH provoquée par la protéine HYBID. MÉTHODES: l'inhibition de l'extrait de racine de la pimprenelle (Sanguisorba officinalis) et du ziyuglycoside I, l'un des composants de l'extrait de racine de Sanguisorba officinalis, sur la dégradation de l'AH provoquée par la protéine HYBID a été évaluée à l'aide d'une chromatographie par exclusion stérique de l'AH dépolymérisé par des transfectants stables de la protéine HYBID dans les cellules HEK293 (cellules HYBID/HEK293) ou les fibroblastes cutanés humains normaux (lignée cellulaire Detroit 551 et cellules des fibroblastes du derme humain chez l'adulte). L'expression de l'ARNm et de la protéine HYBID a été examinée par PCR quantitative en temps réel et par immuno-empreinte des fibroblastes cutanés traités avec de l'extrait de racine de Sanguisorba officinalis, et l'attribution des tailles des nouveaux échantillons produits de l'AH a été évaluée par préparation d'AH radiomarqué métaboliquement. Une étude en double aveugle, randomisée et contrôlée par placebo a été menée auprès des 21 femmes japonaises en bonne santé, qui ont été traitées localement avec la formulation élaborée à partir d'extraits de racine de Sanguisorba officinalis ou un placebo, sur chaque côté du visage, notamment sur la zone à pattes d'oie. RÉSULTATS: l'extrait de racine de Sanguisorba officinalis a permis d'arrêter la dégradation de l'AH provoquée par la protéine HYBID par les cellules HYBID/HEK293, mais ce n'était pas le cas du ziyuglycoside I. L'extrait de racine de Sanguisorba officinalis a également inhibé la dégradation de l'AH provoquée par la protéine HYBID des fibroblastes cutanés en diminuant l'expression de l'ARNm et des protéines HYBID. Bien que les fibroblastes cutanés témoins non traités aient produit de l'AH polydispersé, les cellules traitées aux extraits de racine de Sanguisorba officinalis ont produit uniquement de l'AH de haut poids moléculaire. Le traitement par lotion formulée à partir d'extraits de racine de Sanguisorba officinalis a amélioré de manière significative l'élasticité de la peau et réduit les scores de vieillissement du coin extérieur de la peau autour des yeux, par rapport à la formulation placebo. CONCLUSION: l'extrait de racine de Sanguisorba officinalis a démontré une action anti-dégradation de l'AH provoquée par la protéine HYBID et une activité antirides au niveau de la peau du visage humain, s'accompagnant d'une amélioration de l'élasticité. Notre étude fournit la possibilité d'une nouvelle stratégie pour inhiber la dégradation de l'AH provoquée par la protéine HYBID dans le cadre des soins antirides.

Relationship of hyaluronan and HYBID (KIAA1199) expression with roughness parameters of photoaged skin in Caucasian women.

BACKGROUND: Hyaluronan (HA) is an important constituent of extracellular matrix (ECM) in the skin, and HA degradation mediated by HYBID (KIAA1199) is suggested to be implicated in facial skin wrinkling in Japanese women. Ethnic difference in skin wrinkle formation is known between Caucasian and Japanese women, but no information is available for the relations of HA and HYBID expression levels with skin wrinkling in Caucasian women. METHODS: The skin surface roughness at the eye corner of the Caucasian female subjects was measured, and the skin specimens biopsied from the same areas were subjected to microarray gene analysis, HA staining, and immunohistochemistry for HYBID. RESULTS: Among the ECM genes and those related to ECM metabolism, only HYBID expression levels positively correlated with the skin roughness parameters. When the skin sample groups with high expression of HYBID or low expression of HYBID were compared, the HA staining intensity and the ratio of HYBID-immunoreactive cells to total cells in the superficial dermis were significantly reduced and increased in the high-HYBID-expression group compared with the low-HYBID-expression group, respectively. CONCLUSION: Our data suggest that like Japanese women, HYBID-mediated reduction of HA in the superficial dermis is involved in the formation of wrinkles in Caucasian women.

Reduction of hyaluronan and increased expression of HYBID (alias CEMIP and KIAA1199) correlate with clinical symptoms in photoaged skin.

BACKGROUND: Hyaluronan (HA) metabolism in skin fibroblasts is mediated by HYBID (hyaluronan binding protein involved in hyaluronan depolymerization, alias CEMIP and KIAA1199) and the HA synthases HAS1 and HAS2. However, photoageing-dependent changes in HA and their molecular mechanisms, and the relationship between HA metabolism and clinical symptoms in photoaged skin remain elusive. OBJECTIVES: We examined the amount, size and tissue distribution of HA and expression levels of HYBID, HAS1 and HAS2 in photoaged skin, and analysed their relationship with the degree of photoageing. METHODS: Photoageing-dependent changes of HA were investigated by studying skin biopsies isolated from photoprotected and photoexposed areas of the same donors, and the relationships between HA and photoageing symptoms such as skin wrinkling and sagging were examined. RESULTS: Skin biopsy specimens showed that the amount and size of HA are decreased in photoexposed skin compared with photoprotected skin, and this was accompanied by increased expression of HYBID and decreased expression of HAS1 and HAS2. Histologically, HA staining in the papillary dermis was decreased in photoexposed skin, showing reverse correlation with HYBID expression. HYBID expression in the photoexposed skin directly correlated with skin roughness and sagging parameters, and the reduced HA staining in the papillary dermis in the photoexposed skin positively correlated with these symptoms. CONCLUSIONS: These data demonstrate that imbalance between HYBID-mediated HA degradation and HAS-mediated HA synthesis may contribute to enhanced HA catabolism in photoaged skin, and suggest that HYBID-mediated HA reduction in the papillary dermis is related to skin wrinkling and sagging of photoaged skin.

Synergistic effect of N-acetylglucosamine and retinoids on hyaluronan production in human keratinocytes.

Hyaluronan (HA) is well known to reside in the extracellular matrix as a water-sorbed macromolecule. The aims of this study were twofold: to investigate the regulation of HA synthesis in keratinocytes, and to develop a method to modulate this regulatory process. We found that N-acetylglucosamine (NAG) increased the production of HA by cultured keratinocytes dose dependently, but had no effect on the production by skin fibroblasts. The effect of NAG in keratinocytes was found to be specific for HA production, as there was no change in sulfated glycosaminoglycan formation. The copresence of NAG with either of two retinoids, retinoic acid (RA) or retinol, exerted a synergistic effect on HA production. To investigate whether human HA synthase (HAS) genes were regulated by NAG or retinoids, total RNA extracted from cells treated with these agents was subjected to Northern blot analysis. We observed that RA and retinol markedly induced the expression of HA synthase-3 (HAS3) mRNA. Moreover, beta-carotene, a provitamin A, influenced HA production and HAS3 gene expression in a manner similar to the retinoids. Conversely, NAG had no effect on the expression of HAS3 transcripts. Pretreatment of cells with RA stimulated the activity of membrane-associated HAS, whereas pretreatment with NAG did not. These results suggest that HA production is regulated by at least two pathways: one involving the regulation of HAS gene expression, and the other independent of such a regulatory effect. Taken together, our findings suggest that NAG is a new modulator of HA synthesis.

Hyaluronan exists in the normal stratum corneum.

Hyaluronan is well known to exist as a water-sorbed macromolecule in the extracellular matrix. We here examined whether hyaluronan exists in the normal stratum corneum. High performance liquid chromatography was used to quantify hyaluronan content in the stratum corneum, epidermis (including stratum corneum), and dermis of mice, with the resulting dry weights being 22.3 +/- 2.9, 15.1 +/- 1.5, and 738.6 +/- 31.6 microg per g, respectively. Normal mouse skin was then labeled with [3H]-glucosamine in an organ culture, and accumulation of [3H]-labeled hyaluronan and its molecular mass were determined separately for the stratum corneum, epidermis, and dermis. In the stratum corneum, [3H]-labeled hyaluronan was accumulated linearly over the 3-d culture period. After the 3-d culture period, the epidermis synthesized twice the amount (expressed as dpm per mg dry weight) of [3H]-labeled hyaluronan as the dermis, whereas the stratum corneum and dermis showed nearly the same content of [3H]-labeled hyaluronan. The molecular mass of [3H]-labeled hyaluronan was highest (>1.0 x 106) in the dermis and clearly lower (<6.0 x 104) in the stratum corneum. Based on these results, we here confirm that hyaluronan is supplied from keratinocytes beneath the stratum corneum layer, and is present in the normal stratum corneum. We speculate that hyaluronan may play a role in moisturizing the stratum corneum and/or regulating its mechanical properties.

N-Methyl-L-serine stimulates hyaluronan production in human skin fibroblasts.

We examined the effects of N-methyl-L-serine (NMS), an amino acid derivative, on hyaluronan (HA) synthesis in human skin fibroblasts. NMS (1-10 mM), but not L-serine, stimulated the incorporation of [(3)H]glucosamine into HA dose-dependently, with a maximum stimulation of 1.5-fold compared to the control. The effect of NMS was specific for HA production, because there was no change in sulfated glycosaminoglycan formation. Neither the N-methyl derivatives of L-glycine or L-alanine, nor N-methyl-D-serine, could stimulate HA synthesis, indicating that the beta-hydroxyl group and the L-configuration were essential for the activity. Gel filtration of the products showed that NMS stimulated the production of high-molecular-mass HA (>10(6) D) without affecting the production of low-molecular-mass HA. NMS required 24 h to stimulate HA production, and when fibroblasts were pretreated for 10-24 h with NMS (1-10 mM), membrane-associated HA synthase activity was increased dose-dependently. Thus, a second messenger is likely to be involved in the stimulation of HA production by NMS.

Putative hyaluronan synthase mRNA are expressed in mouse skin and TGF-beta upregulates their expression in cultured human skin cells.

We examined in situ expression of putative hyaluronan synthase genes, Has1 and Has2, and effects of transforming growth factor-beta on their expression. In situ mRNA hybridization showed that mouse skin expressed both Has1 and Has2 mRNA in dermis and epidermis. In dermis, the number of cells expressing the Has1 mRNA was less than that of the Has2 mRNA, and in epidermis, some strong signals from both mRNA were seen in stratum granulosum. Northern blot analysis showed that cultured human skin fibroblasts expressed Has1 mRNA of 2.4 kb and Has2 mRNA of 3.2 and 4.8 kb, whereas human keratinocytes expressed Has1 mRNA of 4.8 but not 2.4 kb and a trace of Has2 mRNA. When the cultures were stimulated with transforming growth factor-beta, both Has1 and Has2 mRNA were upregulated in fibroblasts, and only Has1 mRNA of 2.4 but not 4.8 kb was induced in keratinocytes. The maximal amount of the upregulated Has1 mRNA in keratinocytes at 2 h after stimulation decreased time-dependently to the nonstimulated level at 18 h, although the stimulation for 18 h of fibroblasts was effective on the expression of both Has mRNA. Differences in expression pattern of Has and Has2 mRNA in mouse skin and a higher response of fibroblasts to transforming growth factor-beta suggest that Has1 and Has2 genes are regulated independently and synthesized hyaluronan may have a different function in epidermis and dermis.