Age-induced hair greying - the multiple effects of oxidative stress.

An obvious sign of ageing is hair greying, or the loss of pigment production and deposition within the hair shafts. Numerous mechanisms, acting at different levels and follicular locations, contribute to hair greying, ranging from melanocyte stem cells defects to follicular melanocyte death. One key issue that is in common to these processes is oxidative damage. At the hair follicle stem cells niche, oxidative stress, accelerated by B-cell lymphoma 2 gene (BCL-2) depletion, leads to selective apoptosis and diminution of melanocyte stem cells, reducing the repopulation of newly formed anagen follicles. Melanotic bulbar melanocytes express high levels of BCL-2 to enable survival from melanogenesis- and ultraviolet A (UVA)-induced reactive oxygen species (ROS) attacks. With ageing, the bulbar melanocyte expression of anti-oxidant proteins such as BCL-2, and possibly TRP-2, is reduced, and the dedicated enzymatic anti-oxidant defence system throughout the follicle weakens, resulting in enhanced oxidative stress. A marked reduction in catalase expression and activity results in millimolar accumulation of hydrogen peroxide, contributing to bulbar melanocyte malfunction and death. Interestingly, amelanotic melanocytes at the outer root sheath (ORS) are somewhat less affected by these processes and survive for longer time even within the white, ageing hair follicles. Better understanding of the overtime susceptibility of melanocytes to oxidative stress at the different follicular locations might yield clues to possible therapies for the prevention and reversal of hair greying.

A novel anti-ageing mechanism for retinol: induction of dermal elastin synthesis and elastin fibre formation.

Dermal elastic fibres are extracellular matrix protein complexes produced by fibroblasts and involved in skin elasticity. Elastin fibres decrease with age as a result of reduced synthesis and increased degradation, resulting in skin sagging and reduced skin elasticity. In this study, we show that retinol (ROL), known to enhance dermal collagen production, is also enhancing elastin fibre formation. ROL induced elastin gene expression and elastin fibre formation in cultured human dermal fibroblasts. Topical treatment of cultured human skin explants with a low dose (0.04%) of ROL increased mRNA and protein levels of tropoelastin and of fibrillin-1, an elastin accessory protein, as documented by QPCR and immunohistochemistry staining. Luna staining confirmed the increased elastin fibre network in the ROL-treated skin explants, as compared with untreated controls. These data demonstrate that ROL exerts its anti-ageing benefits not only via enhanced epidermal proliferation and increased collagen production, but also through an increase in elastin production and assembly.

Modulation of microphthalmia-associated transcription factor gene expression alters skin pigmentation.

The microphthalmia-associated transcription factor is implicated in melanocyte development and in the regulation of melanogenesis. Microphthalmia-associated transcription factor is thought to bind to the M-box promoter elements of tyrosinase, tyrosinase-related protein-1 and dopachrome tautomerase/tyrosinase-related protein-2 and transactivate these genes, resulting in increased pigmentation. Using a luciferase reporter construct driven by the microphthalmia-associated transcription factor promoter, we identified agents that modulate microphthalmia-associated transcription factor promoter activity. Changes in endogenous microphthalmia-associated transcription factor expression levels upon treatment with these agents were confirmed using northern and western blots, and their pigmentary modulating activities were demonstrated. Ultraviolet B irradiation and traditional Chinese medicine-1, a natural extract used in traditional Chinese medicine, upregulated microphthalmia-associated transcription factor gene expression and enhanced tyrosinase activity in vitro. Dihydrolipoic acid, lipoic acid, and resveratrol reduced microphthalmia-associated transcription factor and tyrosinase promoter activities. These agents also inhibited the forskolin- and ultraviolet B-stimulated promoter activities of these genes and significantly reduced tyrosinase activity in melanocyte cultures, resulting in depigmentation. Overexpressed microphthalmia-associated transcription factor was capable of rescuing the repressive effects of these compounds on the cotransfected tyrosinase promoter. Dark-skinned Yucatan swine treated with these agents showed visible skin lightening, which was confirmed histologically, whereas ultraviolet B-induced tanning of light-skinned swine was inhibited using these agents. Our findings suggest that modulation of microphthalmia-associated transcription factor expression can alter skin pigmentation and further confirm the central role of microphthalmia-associated transcription factor in melanogenesis.

Soymilk reduces hair growth and hair follicle dimensions.

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.

Keratinocyte-melanocyte interactions during melanosome transfer.

The epidermal-melanin unit is composed of one melanocyte and approximately 36 neighboring keratinocytes, working in synchrony to produce and distribute melanin. Melanin is synthesized in melanosomes, transferred to the dendrite tips, and translocated into keratinocytes, forming caps over the keratinocyte nuclei. The molecular and cellular mechanisms involved in melanosome transfer and the keratinocyte-melanocyte interactions required for this process are not yet completely understood. Suggested mechanisms of melanosome transfer include melanosome release and endocytosis, direct inoculation ('injection'), keratinocyte-melanocyte membrane fusion, and phagocytosis. Studies of the keratinocyte receptor protease-activated receptor-2 (PAR-2) support the phagocytosis theory. PAR-2 controls melanosome ingestion and phagocytosis by keratinocytes and exerts a regulatory role in skin pigmentation. Modulation of PAR-2 activity can enhance or decrease melanosome transfer and affects pigmentation only when there is keratinocyte-melanocyte contact. Moreover, PAR-2 is induced by UV irradiation and inhibition of PAR-2 activation results in the prevention of UVB-induced tanning. The role of PAR-2 in mediating UV-induced responses remains to be elucidated.

An alternative approach to depigmentation by soybean extracts via inhibition of the PAR-2 pathway.

The protease-activated receptor 2, expressed on keratinocytes but not on melanocytes, has been ascribed functional importance in the regulation of pigmentation by phagocytosis of melanosomes. Inhibition of protease-activated receptor 2 activation by synthetic serine protease inhibitors requires keratinocyte-melanocyte contact and results in depigmentation of the dark skinned Yucatan swine, suggesting a new class of depigmenting mechanism and agents. We therefore examined natural agents that could exert their effect via the protease-activated receptor 2 pathway. Here we show that soymilk and the soybean-derived serine protease inhibitors soybean trypsin inhibitor and Bowman-Birk inhibitor inhibit protease-activated receptor 2 cleavage, affect cytoskeletal and cell surface organization, and reduce keratinocyte phagocytosis. The depigmenting activity of these agents and their capability to prevent ultraviolet-induced pigmentation are demonstrated both in vitro and in vivo. These results imply that inhibition of the protease-activated receptor 2 pathway by soymilk may be used as a natural alternative to skin lightening.

Protease-activated receptor 2, a receptor involved in melanosome transfer, is upregulated in human skin by ultraviolet irradiation.

Previous studies have shown that the protease-activated receptor 2 is involved in skin pigmentation through increased phagocytosis of melanosomes by keratinocytes. Ultraviolet irradiation is a potent stimulus for melanosome transfer. We show that protease-activated receptor 2 expression in human skin is upregulated by ultraviolet irradiation. Subjects with skin type I, II, or III were exposed to two or three minimal erythema doses of irradiation from a solar simulator. Biopsies were taken from nonexposed and irradiated skin 24 and 96 h after irradiation and protease-activated receptor 2 expression was detected using immunohistochemical staining. In nonirradiated skin, protease-activated receptor 2 expression was confined to keratinocytes in the lower one-third of the epidermis. After ultraviolet irradiation protease-activated receptor 2 expression was observed in keratinocytes in the upper two-thirds of the epidermis or the entire epidermis at both time points studied. Subjects with skin type I showed delayed upregulation of protease-activated receptor 2 expression, however, compared with subjects with skin types II and III. Irradiated cultured human keratinocytes showed upregulation in protease-activated receptor 2 expression as determined by immunofluorescence microscopy and Western blotting. Cell culture supernatants from irradiated keratinocytes also exhibited a dose-dependent increase in protease-activated receptor-2 cleavage activity. These results suggest an important role for protease-activated receptor-2 in pigmentation in vivo. Differences in protease-activated receptor 2 regulation in type I skin compared with skin types II and III suggest a potential mechanism for differences in tanning in subjects with different skin types.

Inhibition of melanosome transfer results in skin lightening.

The chemical basis of melanogenesis is well documented, but the mechanism of melanosome transfer and the regulation of pigmentation by keratinocyte-melanocyte interactions are not well understood. Therefore we examined the effects of serine protease inhibitors on skin pigmentation and found that the protease-activated receptor 2, expressed on keratinocytes, may regulate pigmentation via keratinocyte-melanocyte interactions. Here we show that modulation of protease-activated receptor 2 activation affects melanosome transfer into keratinocytes, resulting in changes in pigment production and deposition. SLIGRL, the protease-activated receptor 2 activating peptide, enhanced melanosome ingestion by keratinocytes, thus increasing pigment deposition. RWJ-50353, a serine protease inhibitor, led to reduced pigment deposition in melanocytes and depigmentation. Electron microscopy studies illustrated an accumulation of immature melanosomes inside melanocytes and abnormal dendrite dynamics in RWJ-50353-treated epidermal equivalents. RWJ-50353 induced a visible and dose-dependent skin lightening effect in the dark-skinned Yucatan swine. Examinations by electron microscopy indicated that the in vivo transfer of melanosomes from melanocytes to keratinocytes was affected. Our data suggest that modulation of keratinocyte-melanocyte interactions via the protease-activated receptor 2 pathway affects melanosome transfer. The use of RWJ-50353 to modulate protease-activated receptor 2 activation could lead to a new class of depigmenting agents.

The protease-activated receptor-2 upregulates keratinocyte phagocytosis.

The protease-activated receptor-2 (PAR-2) belongs to the family of seven transmembrane domain receptors, which are activated by the specific enzymatic cleavage of their extracellular amino termini. Synthetic peptides corresponding to the tethered ligand domain (SLIGRL in mouse, SLIGKV in human) can activate PAR-2 without the need for receptor cleavage. PAR-2 activation is involved in cell growth, differentiation and inflammatory processes, and was shown to affect melanin and melanosome ingestion by human keratinocytes. Data presented here suggest that PAR-2 activation may regulate human keratinocyte phagocytosis. PAR-2 activation by trypsin, SLIGRL or SLIGKV increased the ability of keratinocytes to ingest fluorescently labeled microspheres or E. coli K-12 bioparticles. This PAR-2 mediated increase in keratinocyte phagocytic capability correlated with an increase in actin polymerization and *-actinin reorganization, cell surface morphological changes and increased soluble protease activity. Moreover, addition of serine protease inhibitors downmodulated both the constitutive and the PAR-2 mediated increases in phagocytosis, suggesting that serine proteases mediate this functional activity in keratinocytes. PAR-2 involvement in keratinocyte phagocytosis is a novel function for this receptor.

The protease-activated receptor 2 regulates pigmentation via keratinocyte-melanocyte interactions.

Close association exists between melanocytes, the pigment melanin-producing cells in the body, and their neighboring keratinocytes. Keratinocytes are the pigment recipients and skin pigmentation is the result of this interaction. While the chemical basis of melanin production (melanogenesis) is well documented, the molecular mechanism of melanosome transfer needs to be elucidated. We are now providing first evidence that the protease-activated receptor 2 (PAR-2) expressed on keratinocytes, but not on melanocytes, is involved in melanosome transfer and therefore may regulate pigmentation. Activation of PAR-2 with trypsin or with the peptide agonist SLIGRL induced pigmentation in both two- and three-dimensional cocultures of keratinocytes and melanocytes, but not in cocultures that were spatially separated, indicating the need for intimate cell-cell contact. Topical application of SLIGRL on human skin transplanted on SCID mice resulted in a visible skin darkening. Histological examination revealed increased deposits of melanin in the keratinocytes. Inhibition of PAR-2 activation by RWJ-50353, a serine protease inhibitor, resulted in depigmentation and changes in expression of melanogenic-specific genes. Keratinocyte-melanocyte contact was essential for this depigmenting effect. Topical application of this inhibitor induced lightening of the dark skin Yucatan swine, which was confirmed by histochemical analysis. The results presented here suggest a novel mechanism for the regulation of pigmentation, mediated by the activation or inhibition of the keratinocyte receptor PAR-2.

The influence of the response of skin equivalent systems to topically applied consumer products by epithelial--mesenchymal interactions.

A number of diverse in vitro model systems have been employed for the prediction of irritation potential of test articles. Monolayer systems have proven to be useful for preliminary screening but are not always capable of distinguishing mild effects or adaptable to fully formulated product. Three-dimensional reconstructed skin equivalents integrate cellular toxicity with the kinetics of exposure and absorption, serving as more realistic models; however, it is not obvious which of the three-dimensional models will give the most predictive response, and which biomarker should be used for an endpoint measurement for different groups of irritants. While evaluating these variables, we have shown that different irritants modulate various cytokine mRNA levels and secretion patterns in a time- and concentration-dependent manner that is unique to each product category. These profiles are also dependent on keratinocyte-fibroblast interactions. The most predictive combinations of model systems and biomarkers for each product category were identified following comparison to preclinical data and human in vivo skin responses. Using a panel of representative consumer products, we identified IL-1alpha, IL-1ra, IL-8 and GM-CSF release from skin equivalents as being the best indicators of irritation.

The effects of trypsin on apoptosis, utriculi size, and skin elasticity in the Rhino mouse.

Acne vulgaris is the result of multifactorial disorders of the pilosebaceous duct. The initial lesion is believed to be hyper-keratinization of the infundibulum. The Rhino mouse has been used as an experimental acne model system for screening anti-keratinizing and comedolytic agents. Using this system we show that trypsin could induce desquamation and utriculi-epidermal differentiation in the absence of irritation. Following five daily trypsin treatments, the biomechanical properties of the mouse skin improved, as demonstrated by cutometer measurements and increased elastin expression. Extensive programmed cell death and apoptosis are demonstrated in the utriculi epithelium of the untreated animals. This cell death is eliminated by the trypsin treatment. We speculate that co-administration of trypsin might increase the therapeutic value of topical acne treatments and improve skin elasticity while reducing irritating effects.

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.

Evidence for the presence of a protease-activated receptor distinct from the thrombin receptor in human keratinocytes.

Thrombin receptor activation was explored in human epidermal keratinocytes and human dermal fibroblasts, cells that are actively involved in skin tissue repair. The effects of thrombin, trypsin, and the receptor agonist peptides SFLLRN and TFRIFD were assessed in inositolphospholipid hydrolysis and calcium mobilization studies. Thrombin and SFLLRN stimulated fibroblasts in both assays to a similar extent, whereas TFRIFD was less potent. Trypsin demonstrated weak efficacy in these assays in comparison with thrombin. Results in fibroblasts were consistent with human platelet thrombin receptor activation. Keratinocytes, however, exhibited a distinct profile, with trypsin being a far better activator of inositolphospholipid hydrolysis and calcium mobilization than thrombin. Furthermore, SFLLRN was more efficacious than thrombin, whereas no response was observed with TFRIFD. Since our data indicated that keratinocytes possess a trypsin-sensitive receptor, we addressed the possibility that these cells express the human homologue of the newly described murine protease-activated receptor, PAR-2 [Nystedt, S., Emilsson, K., Wahlestedt, C. & Sundelin, J. (1994) Proc. Natl. Acad. Sci. USA 91, 9208-9212]. PAR-2 is activated by nanomolar concentrations of trypsin and possesses the tethered ligand sequence SLIGRL. SLIGRL was found to be equipotent with SFLLRN in activating keratinocyte inositolphospholipid hydrolysis and calcium mobilization. Desensitization studies indicated that SFLLRN, SLIGRL, and trypsin activate a common receptor, PAR-2. Northern blot analyses detected a transcript of PAR-2 in total RNA from keratinocytes but not fibroblasts. Levels of thrombin receptor message were equivalent in the two cell types. Our results indicate that human keratinocytes possess PAR-2, suggesting a potential role for this receptor in tissue repair and/or skin-related disorders.

A secreted serine protease can induce apoptosis in Pam212 keratinocytes.

The epidermal keratinocyte cell line Pam212 undergoes spontaneous apoptosis in culture, providing an in vitro model for the early steps of epidermal differentiation. Pam212 cells exhibit characteristics of basal keratinocytes, committed for the transition to the spinous layer of the epidermis. Bcl-2 can regulate the differentiation of these cells by negatively regulating several genes that have been implicated in apoptosis. We show evidence that a serine protease activity, secreted by the Pam212 cells, could induce apoptosis in Pam212 and several other cell lines. This activity might be regulated via the bcl-2 pathway. We suggest that this serine protease could either directly, via binding and/or cleavage of a serine protease-activated receptor, or indirectly, via the cleavage of an unknown protein, activate the signaling for apoptosis in Pam212 cells. Alternatively, this secreted serine protease could reenter the cell and start a proteolytic cascade reaction that leads to cell death. This is based on the induction of apoptosis in several cell lines by the partially purified serine protease activity, and the minimal effect of protein synthesis inhibition on Pam212 apoptosis. We propose that in vivo, a two-step mechanism controls keratinocyte apoptosis and differentiation. The basal cells of the epidermis contain all of the necessary proteins required for apoptosis, as well as the repressor protein Bcl-2. As Bcl-2 levels go down, the cells commit to terminal differentiation. A serine protease, secreted from these cells, then induces the death process. This second step enables the cells to undergo apoptosis and continue the process of terminal differentiation.

Apoptosis in Pam212, an epidermal keratinocyte cell line: a possible role for bcl-2 in epidermal differentiation.

Programmed cell death is a controlled process that leads to the elimination of single cells via apoptosis, a mode of cell death with a characteristic morphology. During epidermal differentiation, keratinocytes migrate outward to become terminally differentiated cornified cells in a process involving programmed cell death pathway(s) and apoptosis. The molecular mechanisms regulating epidermal differentiation and apoptosis have not yet been elucidated. Here we show that a mouse keratinocyte cell line, Pam212, undergoes spontaneous apoptosis in culture. Apoptosis of Pam212 cells is demonstrated by both morphology and DNA oligonucleosomal degradation. The expression of bcl-2, a gene implicated in the negative control of apoptosis, was down-regulated in these cells by transfecting a bcl-2-antisense expression vector. The cells that down-regulate bcl-2 expression exhibit enhanced apoptosis and further progress in the epidermal differentiation pathway. We analyzed the expression patterns of several genes that have been implicated in apoptosis in other systems. We show that the mRNA levels of c-myc, c-myb, c-fos, tumor necrosis factors (TNF) alpha and beta, TNF receptors I and II, interleukin 1 alpha, IFN-gamma, and transforming growth factor beta increase in the antisense-transfected cells. We suggest that bcl-2 influences epidermal differentiation in Pam212 keratinocyte cells, and maybe in vivo, by negatively regulating several genes that are involved in apoptosis.

Clusterin expression within skin correlates with hair growth.

Clusterin/TRPM-2 is a sulfated glycoprotein that is expressed in many tissues. Independently cloned and isolated by several laboratories, it bears many names, and has been shown to be involved in many processes. These include cell-cell adhesion and aggregation, inhibition of complement cytolysis, programmed cell death and apoptosis, tissue remodeling, and terminal differentiation. The hair follicle undergoes cycles of growth, regression, and rest, which involve both tissue remodeling and programmed cell death. To identify whether clusterin expression is involved in hair growth and cycling, we studied the expression of clusterin throughout the hair cycle. We demonstrate that clusterin is expressed during the growth phase of the hair cycle. We found no correlation between clusterin expression and the apoptotic regression of the hair follicle. Using immunohistochemistry we localized clusterin to the inner root sheath of the follicle. This suggests that clusterin might be involved in the morphogenesis and differentiation of the hair follicle. We propose that clusterin has a role in the maintenance of the layered structure of the hair follicle, and in the interactions between the inner root sheath and both the outer root sheath and the

Changes in expression of apoptosis-associated genes in skin mark early catagen.

Programmed cell death is central to hair biology, as the hair follicle undergoes cycles of growth (anagen), regression (catagen), and rest (telogen). During catagen, the hair follicle shortens via a pathway of programmed cell death and apoptosis. The molecular mechanisms involved in this process have not been elucidated yet. Using reverse transcriptase-polymerase chain reaction, we examined in this study the expression in total skin, throughout one hair cycle, of a series of regulatory genes associated with apoptosis. We show that gene expression within skin is hair-cycle-dependent. Transforming growth factor-beta was expressed immediately before catagen; therefore, it might be involved in the early signaling of this process. Tumor necrosis factor-beta was expressed during catagen and might be involved in follicular apoptosis. Several proto-oncogenes and transcription factors have been described in the regulation of apoptosis in other systems. Here we show that the transcript levels of c-myc, c-myb, and c-jun changed immediately before or during early catagen and thus could be involved in the signaling or regulation of catagen. Levels of p53 remained constant throughout anagen and catagen, suggesting that p53 is not involved in the developmentally induced apoptosis of the hair follicle. The variable expression throughout the hair cycle of the genes described demonstrates the dynamic changes of the skin and underscores the importance of studying the complete hair cycle when characterizing any molecule in skin.

1994 William J. Stickel Silver Award. Closing base wedge versus Austin bunionectomies for metatarsus primus adductus.

Hallux abducto valgus with concomitant metatarsus primus adductus was treated by either an isolated Austin bunionectomy or by a combination of a modified McBride bunionectomy, along with a closing base wedge osteotomy, on a total of 73 patients (88 feet) from 1983 to 1993. Both subjective and objective similarities and differences were compared between these two groups of patients. Long-term elevation or depression of the first ray was analyzed by using a technique termed sagittal plane displacement. The prevalent preoperative symptoms were significantly reduced postoperatively in both groups of patients. Initial postoperative elevation of the first ray occurred in approximately one third of the cases in both groups. Long-term elevation of the first ray was greater with the base wedge osteotomy and did not change appreciably with the Austin procedure. The sagittal plane displacement method is a helpful tool in analyzing changes in the position of the first ray perioperatively.

Expression of the bcl-2 protooncogene in the cycling adult mouse hair follicle.

The hair follicle undergoes a cycle of growing, regressing, and resting phases (anagen, catagen, telogen, respectively). As the follicle enters catagen, the cells of the lower, cycling portion undergo a process of controlled cell death (apoptosis). Understanding the mechanism of apoptosis in the follicle should give insight into one of the control steps of hair cycling. In this study we sought the expression of bcl-2, a protooncogene associated with apoptosis control, in the cycling follicle of the adult mouse. Using a monoclonal antibody to the mouse protein we immunolocalized bcl-2 gene product in the cycling pelage follicle of the C57/B6 adult mouse. The protein was expressed in the follicular papilla (a non-cycling portion of the follicle) throughout the cycle-including telogen. The cycling follicular epithelium, however, showed positive antibody staining in anagen, which decreased in catagen and disappeared in telogen. In anagen the cells of the bulb, bulge, and basal layer of the outer root sheath expressed the bcl-2 protein. Understanding the action of this apoptosis-inhibiting molecule should serve to elucidate the dynamics of follicular cycling.