Miri Seiberg

In vitro 3D Full-Thickness Skin-Equivalent Tissue Model Using Silk and Collagen Biomaterials

Current approaches to skin equivalents often only include the epidermis and dermis. Here, a full-thickness skin equivalent is described including epidermis, dermis, and hypodermis, that could serve as an in vitro model for studying skin biology or as a platform for consumer product testing. The construct is easy to handle and is maintained for >14 d while expressing physiological morphologies of the epidermis and dermis, seen by keratin 10, collagens I and IV expression. The skin equivalent produces glycerol and leptin, markers of adipose metabolism. This work serves as a foundation for understanding a few necessary factors needed to develop a stable, functional model of full-thickness skin.

The role of keratinocyte growth factor in melanogenesis: a possible mechanism for the initiation of solar lentigines

Please cite this paper as: The role of keratinocyte growth factor in melanogenesis: a possible mechanism for the initiation of solar lentigines. Experimental Dermatology 2010; 19: 865–872.

Molecules of the cycling hair follicle — a tabulated review

In this review we tabulated molecules which have been experimentally identified to be associated with, or play a role in, hair follicle growth. While compiling these data we were impressed by the fact that this field is only now beginning to be developed in terms of molecular analysis. Ironically, hair was used in some of the earliest molecular approaches to biologic structure (e.g. Astbury and Street, 1931), but the field did not develop from there. From our review we have come to the following conclusions. (1) As indicated by the growing number of reports dealing with follicle-associated molecules in the past 3 years, the field of hair biology has entered a new molecular era. (2) In many reported hair biology studies not enough emphasis has been placed on the fact that the follicle is a dynamic structure. All too often a study is limited to follicles of one particular phase of the cycle or one phase of development. Students in the field have to be more sensitive to the remarkable changes that this deceptively simple structure can undergo during its cycle. (3) Although we have not been able to find any molecules unique to the follicle, some of the structural molecules come close to an ideal tool. It is our impression that even more specific molecule tags will be found. Whether this requires a subtraction library approach or gene mapping of specific mutants is not yet clear. It would appear that the large, diverse family of intermediate filament-associated proteins will prove to be an excellent source of unique follicle-labeling molecules. (4) There is an acute need for molecules which distinguish the phases of the cycle, e.g. telogen from early anagen. Telogen is by far the most difficult phase to identify morphologically since the earliest phase of anagen and the latest phase of catagen may appear structurally like telogen. That these phases are functionally distinguishable must imply a molecular difference. As the number of recognized hair follicle-associated molecules and their interactions increase, it will be essential to assemble libraries of highly specific RNA and antibody probes for localization and mapping studies. We recognize that this review, as written, is imperfect. It is particularly deficient in making any effort towards identifying unifying principles of structure and function. We look forward to returning to this subject within 3 years.(ABSTRACT TRUNCATED AT 400 WORDS)

Trypsin-induced follicular papilla apoptosis results in delayed hair growth and pigmentation

Programmed cell death is a controlled process that leads to the elimination of single cells via apoptosis. Programmed cell death is fundamental to development, morphogenesis, and homeostasis. Proteases play a major role in the death process. We have previously shown that a serine protease, secreted by a keratinocyte cell line, can induce apoptosis in numerous cell lines. Here we show that serine proteases can induce cell death in vivo as well. Using a synchronized hair growth mouse model, we show that topical trypsin treatment following depilation induces cell death at the follicular papilla. This results in delaying hair growth and pigmentation. We speculate that trypsin might affect a receptor‐mediated signaling pathway that leads to follicular papilla cell death. Dev. Dyn. 208:553–564, 1997.

Soymilk reduces hair growth and hair follicle dimensions

Abstract: We have recently shown that soybean‐derived serine protease inhibitors and soybean extracts alter skin pigmentation, suggesting that soymilk could be used as a natural alternative to skin lightening. The present studies were initiated to examine the possible effect of STI, BBI and soymilk on hair pigmentation. Interestingly, these agents were found to affect not only hair pigmentation, but also the rate of hair growth, the dimensions of the hair follicle and hair shaft, and the appearance of the hair. The studies presented here provide first evidence, at the morphological and histological level, that soymilk and the soybean‐derived serine protease inhibitors could be used as effective agents for hair care and management. These agents could reduce the rate of hair growth, decrease hair shaft dimensions and alter the pattern of melanogenic gene expression.

Immuno-histochemical evaluation of solar lentigines: The association of KGF/KGFR and other factors with lesion development

BACKGROUND Solar lentigines (SLs) are macular hyperpigmented lesions associated with sun exposure and age. Histopathologically, SLs are defined by a hyperpigmented basal layer and elongated rete ridges. The molecular mechanisms involved in the formation and the development of SLs are not completely understood. Our earlier data show that keratinocyte growth factor (KGF) induces hyperpigmentary lesions with histological resemblance to SLs. OBJECTIVE To investigate the association of KGF/KGF receptor (KGFR) and other pigmentary genes with the progression of SL development. To better understand the possible role of KGF in the pathology of SLs. METHODS Archived human skin biopsies (24 SLs and 14 healthy skins) were studied using immunohistochemistry for KGF/KGFR, proliferation marker Ki67, stem cell marker keratin-15 (K15), tyrosinase (TYR), stem cell factor (SCF), and protease-activated receptor-2 (PAR-2). RESULTS An increase in TYR-positive cells and expression was found throughout SL progression, as compared to normal skin. The levels of KGF, KGFR, SCF, Ki67 and PAR-2 varied during SL progression. Ki67, K15 and KGF/KGFR were significantly upregulated at early-mid SL stages. The latest-stage SLs expressed the lowest levels of KGF, KGFR, SCF, Ki67 and PAR-2. CONCLUSIONS The upregulation of KGF/KGFR might induce the formation of rete ridges and hyperpigmentation. The reduced levels of all examined proteins (except TYR and K15) suggest a possible inactive status (dormancy or quiescence) of advanced lesions.

Melanocortin-5 receptor and sebogenesis

The melanocortins (α-MSH, β-MSH, γ-MSH, and ACTH) bind to the melanocortin receptors and signal through increases in cyclic adenosine monophosphate to induce biological effects. The melanocortin MC(5) and MC(1) receptors are expressed in human sebaceous glands, which produce sebum, a lipid mixture of squalene, wax esters, triglycerides, cholesterol esters, and free fatty acids that is secreted onto the skin. Excessive sebum production is one of the major factors in the pathogenesis of acne. The expression of melanocortin MC(5) receptor has been associated with sebocyte differentiation and sebum production. Sebaceous lipids are down-regulated in melanocortin MC(5) receptor-deficient mice, consistent with the observation that α-MSH acts as a sebotropic hormone in rodents. These findings, which suggest that melanocortins stimulate sebaceous lipid production through the MC(5) receptor, led to our search for MC(5) receptor antagonists as potential sebum-suppressive agents. As predicted, an antagonist was shown to inhibit sebocyte differentiation in vitro, and to reduce sebum production in human skin transplanted onto immunodeficient mice. The melanocortin MC(5) receptor antagonists may prove to be clinically useful for the treatment of sebaceous disorders with excessive sebum production, such as acne.

LIGR, a protease‐activated receptor‐2‐derived peptide, enhances skin pigmentation without inducing inflammatory processes

The protease‐activated receptor‐2 (PAR‐2) is a seven transmembrane G‐protein‐coupled receptor that could be activated by serine protease cleavage or by synthetic peptide agonists. We showed earlier that activation of PAR‐2 with Ser‐Leu‐Ile‐Gly‐Arg‐Leu‐NH2 (SLIGRL), a known PAR‐2 activating peptide, induces keratinocyte phagocytosis and increases skin pigmentation, indicating that PAR‐2 regulates pigmentation by controlling phagocytosis of melanosomes. Here, we show that Leu‐Ile‐Gly‐Arg‐NH2 (LIGR) can also induce skin pigmentation. Both SLIGRL and LIGR increased melanin deposition in vitro and in vivo, and visibly darkened human skins grafted onto severe combined immuno‐deficient (SCID) mice. Both SLIGRL and LIGR stimulated Rho‐GTP activation resulting in keratinocyte phagocytosis. Interestingly, LIGR activates only a subset of the PAR‐2 signaling pathways, and unlike SLIGRL, it does not induce inflammatory processes. LIGR did not affect many PAR‐2 signaling pathways, including [Ca2+] mobilization, cAMP induction, the induction of cyclooxgenase‐2 (COX‐2) expression and the secretion of prostaglandin E2, interleukin‐6 and ‐8. PAR‐2 siRNA inhibited LIGR‐induced phagocytosis, indicating that LIGR signals via PAR‐2. Our data suggest that LIGR is a more specific regulator of PAR‐2‐induced pigmentation relative to SLIGRL. Therefore, enhancing skin pigmentation by topical applications of LIGR may result in a desired tanned‐like skin color, without enhancing inflammatory processes, and without the need of UV exposure.

Aldo‐keto reductase 1C subfamily genes in skin are UV‐inducible: possible role in keratinocytes survival

Abstract:  Human skin is endowed with the capacity to synthesize and metabolize steroid hormones, a function of importance in skin physiology and pathology. It is the hormone‐regulatory enzymes, including the aldo‐keto reductase 1C subfamily (AKR1Cs) that are largely responsible for the local levels of active steroid hormones. AKR1C1 and AKR1C2 inactivate progesterone and 5α‐dihydrotestosterone, respectively, whereas AKR1C3 activates oestradiol and testosterone. Here, we show that AKR1C1‐3 are expressed in keratinocytes and fibroblasts, with marginal expression in melanocytes. In human primary keratinocytes, AKR1C1 and ‐2 were UVB‐inducible in a dose‐dependent manner, as shown by quantitative PCR and Western blot analyses. The induction of AKR1C1 by UVB was concomitant with the presence of an apoptotic marker, the cleavage product of poly‐ADP ribose polymerase. Similarly, the activation of AKR1C1 and ‐2 upon UVB exposure was demonstrated in swine skin in vivo and in human skin explants. As expected, hydrogen peroxide‐derived reactive oxygen species also induced AKR1C1 and ‐2 mRNA and protein levels in keratinocytes in a dose‐dependent manner. Furthermore, down‐regulation of AKR1Cs by small interfering ribonucleic acid led to significantly reduced cell viability. Based on the combined evidence of the presence of an apoptotic marker in the UVB‐exposed keratinocytes with increased AKR1Cs expression and reduced cell viability in down‐regulated AKR1Cs, we suggest that AKR1C subfamily genes are stress‐inducible and might function as survival factors in keratinocytes.

Extracts from Glycine max (soybean) induce elastin synthesis and inhibit elastase activity

Abstract:  Elastic fibres are essential extracellular matrix components of the skin, contributing to its resilience and elasticity. In the course of skin ageing, elastin synthesis is reduced, and elastase activity is accelerated, resulting in skin sagging and reduced skin elasticity. Our studies show that non‐denatured Glycine max (soybean) extracts induced elastin promoter activity, inhibited elastase activity and protected elastic fibres from degradation by exogenous elastases in vitro. Mouse and swine skins topically treated with soybean extracts showed enhanced elastic fibre network and increased desmosine content. Elastin expression was also augmented in human skin transplanted onto SCID mice in response to soy treatment. These data suggest that non‐denatured soybean extracts may be used as skin care agents to reduce the signs of skin ageing.