Retention of corneodesmosomes and increased expression of protease inhibitors in dandruff.

BACKGROUND: Dandruff is a common, relapsing and uncomfortable scalp condition affecting a large proportion of the global population. The appearance of flakes on the scalp and in the hair line, and associated itch are thought to be consequences of a damaged skin barrier, altered corneocyte cohesion and abnormal desquamation in dandruff. The balance between skin proteases and protease inhibitors is essential for driving the key events, including corneodesmosome degradation, in the desquamation process and to maintain stratum corneum (SC) barrier integrity. OBJECTIVES: To investigate the distribution of corneodesmosomes, the key component of the SC cohesivity and barrier function, and the protease inhibitors lympho-epithelial Kazal-type-related inhibitor (LEKTI-1) and squamous cell carcinoma antigen (SCCA1) in the scalp of dandruff-affected participants. METHODS: The methods utilized were immunohistochemistry, scanning immunoelectron microscopy, phase-contrast microscopy, Western blotting and serine protease activity assay on tape-stripped SC or scalp skin biopsies. RESULTS: In SC samples from healthy subjects, corneodesmosomes were peripherally located in the corneocytes. In samples of dandruff lesions, corneodesmosomes were located both peripherally and on the entire surface area of the corneocytes. LEKTI-1 and SCCA1 protein levels and parakeratosis were found to be highly elevated in the lesional samples. CONCLUSIONS: The persistence of nonperipheral corneodesmosomes is a characteristic feature of the perturbed desquamation seen in dandruff. The increased expression levels of LEKTI-1 and SCCA1 are consistent with the view that the dandruff condition is characterized by an imbalance in protease-protease inhibitor interaction in the SC.

Filaggrin - revisited.

Profilaggrin (proFLG) and its processing products are critical to the health and appearance of skin. The recent identification of loss-of-function filaggrin (FLG) mutations as a predisposing factor in ichthyosis vulgaris and atopic dermatitis has lead to a resurgent interest in this enigmatic protein. Here, we review the literature on the structure and many functions of proFLG, from its role as a filament-aggregating protein and a source of natural moisturizing factor (NMF), to the more recent discoveries of its role in epidermal barrier formation and its more speculative functions as an antimicrobial and sunscreen. Finally, we discuss the relationship of proFLG with dry skin, the influence of moisturizers on NMF generation and speculate on next generation of FLG research.

Stratum corneum dysfunction in dandruff.

Dandruff is characterized by a flaky, pruritic scalp and affects up to half the world's population post-puberty. The aetiology of dandruff is multifactorial, influenced by Malassezia, sebum production and individual susceptibility. The commensal yeast Malassezia is a strong contributory factor to dandruff formation, but the presence of Malassezia on healthy scalps indicates that Malassezia alone is not a sufficient cause. A healthy stratum corneum (SC) forms a protective barrier to prevent water loss and maintain hydration of the scalp. It also protects against external insults such as microorganisms, including Malassezia, and toxic materials. Severe or chronic barrier damage can impair proper hydration, leading to atypical epidermal proliferation, keratinocyte differentiation and SC maturation, which may underlie some dandruff symptoms. The depleted and disorganized structural lipids of the dandruff SC are consistent with the weakened barrier indicated by elevated transepidermal water loss. Further evidence of a weakened barrier in dandruff includes subclinical inflammation and higher susceptibility to topical irritants. We are proposing that disruption of the SC of the scalp may facilitate dandruff generation, in part by affecting susceptibility to metabolites from Malassezia. Treatment of dandruff with cosmetic products to directly improve SC integrity while providing effective antifungal activity may thus be beneficial.

Studies on the synthesis and degradation of a high molecular weight, histidine-rich phosphoprotein from mammalian epidermis.

The synthesis and subsequent fate of the histidine-rich proteins, which form a major component of keratohyalin granules in mammalian epidermis, have been studied in the guinea-pig and new-born rat. In both species the protein first synthesised is of very high molecular weight, approximately 340 000. It is short-lived and breaks down to lower molecular weight proteins 1-2 days after its synthesis. These smaller proteins differ in the two species. In the guinea-pig, the high molecular weight protein breaks down to proteins of molecular weight 250 000 and 200 000, which are themselves unstable and break down to low molecular weight species, probably amino acids. The initial breakdown of the high molecular weight protein coincides with the dispersion of the keratohyalin granules and the transition of the granular cell into the stratum corneum. This high molecular weight histidine-rich protein has been purified to homogeneity, despite its instability to several treatments during purification. The protein is highly phosphorylated, containing 6 mol% of phosphoserine, but is otherwise very basic. The possibility that dephosphorylation of the protein produces highly basic matrix proteins in the stratum corneum is discussed.

Histidine-rich protein of the keratohyalin granules. Source of the free amino acids, urocanic acid and pyrrolidone carboxylic acid in the stratum corneum.

The pool of free amino acids, urocanic acid and pyrrolidone carboxylic acid in mammalian stratum corneum has been shown to be derived principally or totally from the histidine-rich protein of the keratohyalin granules. The time course of appearance of free amino acids and breakdown of the histidine-rich protein are similar, as are the analyses of the free amino acids and the histidine-rich protein. Quantitative studies show that between 70 and 100% of the total stratum corneum-free amino acids are derived from the histidine-rich protein.

Histidine-rich proteins (filaggrins): structural and functional heterogeneity during epidermal differentiation.

The urea-soluble protein profiles of guinea pig, rat, mouse and human epidermis have been compared by non-equilibrium pH gradient/sodium dodecyl sulphate two-dimensional gel electrophoresis. The histidine-rich proteins (filaggrins) were identified firstly by their characteristic specificity and kinetics of labelling with [3H]histidine and [32P]phosphate, and secondly by their ability in vitro to aggregate keratin filaments specifically into bundles. In all species the phosphorylated filaggrin precursor, profilaggrin, is resolved as a single or doublet band with an apparent molecular weight greater than 300,000 and a neutral or slightly acidic iso-electric point. In striking contrast, the strongly basic filaggrins produced from similar profilaggrins form molecular weight families that are clearly species specific. In rat and man there is a single, principal molecular weight form of filaggrin (Mr 45,000 and 38,000, respectively), while mouse and guinea pig have heterogeneous families, including high molecular weight variants (Mr greater than 200,000). Even filaggrins of a particular molecular weight are not homogeneous proteins, but consist of a number of iso-electric variants, some of which are considerably less basic than the bulk of the filaggrins. Incorporation studies using [3H]arginine and [32P]phosphate indicate that the iso-electric variance is not due to residual phosphate, following profilaggrin breakdown, but rather to a conversion of basic arginine residues into neutral citrulline residues. Filaggrins of all the molecular weights from all the species studied share the ability to aggregate keratin filaments into large, insoluble macrofibrils. However, the more acidic iso-electric variants have lower affinities for keratin, particularly in man and guinea pig where the most acidic filaggrins have completely lost the ability to aggregate keratins. We discuss the possibility that a loss of keratin binding ability, resulting in a loosening of the keratin fibre/filaggrin matrix is necessary before the normal complete proteolysis of the filaggrins can occur.

Markers of epidermal differentiation expressed by rat keratinocytes cultured by a modified feeder layer technique.

A broad range of analytical methods has been used to investigate the expression of key differentiation markers in keratinocytes cultured by a modified feeder layer technique. Cultures were stratified and showed many of the features characteristic of epidermal differentiation in vivo including tonofilaments, desmosomes, loss of organelles and thickening of the plasma membrane to form the cornified envelope. Profilaggrin synthesis was detected by 32P-incorporation and the presence of filaggrin suggested that it was broken down by the normal route. Staining with the lectin from Ulex europeus revealed the presence of a fucose-containing cell-surface glycoprotein. Keratin synthesis was shown by 3H-leucine incorporation and keratins were analysed by two-dimensional gel electrophoresis in comparison with those from different levels of the epidermis. Quantitative and qualitative differences were found between in vivo and in vitro epidermal differentiation. In particular, cornified envelope numbers were low, in keeping with the observation by electron microscopy of only one layer of cells with this structure. The absence of a true stratum corneum in vitro was also indicated by the virtual absence of histidase activity and stratum corneum keratins. The keratin species present in vitro most closely resembled those of the basal cells of the epidermis, although even in this case differences were observed. The evidence as a whole is consistent with the belief that epidermal cells do synthesise in vitro many of the important proteins involved in differentiation, but that they nevertheless do not develop a true keratinised stratum corneum.

The occurrence of profilaggrin and its processing in cultured keratinocytes.

An affinity-purified antibody to rat filaggrin detects filaggrin and profilaggrin in extracts of newborn rat epidermis, and a monoclonal antibody to human filaggrin, HF-1, detects the two proteins in extracts of human epidermis. Immunohistologic studies show that HF-1 reacts with keratohyaline granules of human epidermis and those seen in cultured human keratinocytes. Immunoblotting studies have demonstrated that profilaggrin is synthesized in both cultured human keratinocytes and in a long-lived line of cultured rat keratinocytes, but only in the latter is the protein processed to a product of the molecular weight of filaggrin.

Dandruff: a condition characterized by decreased levels of intercellular lipids in scalp stratum corneum and impaired barrier function.

Dandruff is a major problem, yet little is known about the underlying mechanism and subsequent biochemical changes occurring in the scalp skin that lead to its manifestation. The characteristic flaking and scaling of the scalp experienced by dandruff sufferers suggests, similar to the changes classically seen in xerosis, that the desquamation process is impaired. We initiated studies to quantify the biochemical nature of the stratum corneum in the scalp of healthy individuals and dandruff sufferers. Total amounts and relative ratios of stratum corneum lipids species were analysed in scalp stratum corneum samples collected during studies conducted in the UK and Thailand in order to examine ethnic differences. In both populations, dandruff was associated with a dramatic decrease in free lipid levels, with significant decreases in ceramides, fatty acids, and cholesterol. Detailed sub-analysis of the major ceramide species within the total ceramide fraction revealed a decrease in ceramide 1 and increased proportions of ceramide 6i and 6ii. In a separate study, we demonstrated that dandruff sufferers show both an elevated blood flow and an increased reported incidence of itch in response to histamine topically applied to the scalp compared with no-dandruff controls. Taken together these two studies indicate that the quality and resilience of the epidermal water barrier is impaired in the scalp of dandruff sufferers. We propose that the perturbed barrier leaves dandruff sufferers more prone to the adverse effects of microbial and fungal toxins, and environmental pollutants, thus perpetuating their impaired barrier.

Dry skin, moisturization and corneodesmolysis.

The process leading to the loss of corneocytes form the skin surface is termed desquamation. In healthy skin it is an orderly and essentially invisible process whereby individual or small groups of corneocytes detach from neighbouring cells to be lost to the environment and replaced by younger cells from the deeper layers. Desquamation is carefully controlled to ensure that corneum cohesion and integrity, and hence tissue thickness, is maintained. The most important components of the corneocytes contributing towards intercellular cohesion are the corneodesmosomes and lipids. Corneodesmosomes are proteinaceous complexes which effectively rivet corneocytes together. The intercellular lipids, primarily responsible for the water barrier, also provide part of the extracellular cement. In addition, the shape of the corneocyte itself plays a role in stratum corneum cohesion. Through interdigitation along their peripheral edges, adjacent corneocytes become physically locked together, a process which reinforces the integrity of the tissue. For effective desquamation to occur corneodesmosomes must be degraded: a process catalysed by serine proteases present within the intercellular space and facilitated by subtle changes in lipid composition and phase behaviour. Ultimately, it is the availability of free water which controls corneodesmolysis. In healthy skin this proteolytic process leaves relatively few corneodesmosomes intact in the most superficial layers. By contrast, in chronic and acute dry skin conditions, corneodesmosomal degradation and hence the final stages of desquamation are perturbed, leading to the characteristic formation of visible, powdery flakes on the skin surface. The inability to degrade these structures ultimately reflects a decreased hydrolytic activity of the desquamatory enzymes, either through reduced synthesis of the enzymes, inherent loss of activity, leaching from the surface layers of the corneum or changes in the surrounding lipid-rich microenvironment, which may indirectly reduce enzyme functionality. Increased understanding of the desquamation process is providing new insights into the mode of action of current moisturizing ingredients and is offering opportunities to develop novel therapies for preventing and correcting dry skin.

The cornified cell envelope: an important marker of stratum corneum maturation in healthy and dry skin.

The cornified cell envelope (CE) formed by transglutaminase-mediated epsilon-(gamma-glutamyl)lysine cross-linking of specialized corneocyte proteins is the most insoluble component of the terminally differentiated keratinocyte. Under normal Nomarski optics, two types of CE are readily distinguishable: an irregularly shaped, readily deformed 'fragile' envelope (CEf), which predominates in the deepest layers of the stratum corneum, and a polygonal "resilient" or 'rigid' envelope (CEr), which represents over 80% of the CE population in the superficial layers. This distinct spatial distribution indicates a maturation of the CE from the fragile to the resilient morphology during stratum corneum maturation. In this study, we have examined morphological and physical changes occurring in the CE during the terminal differentiation. The proportion of CEf and CEr present in superficial samples of stratum corneum were readily distinguishable following staining with Tetrarhodamine isothiocyanate (TRITC) and showed significant body site variation. The percentage of CEf was highest on samples recovered from exposed body sites (back of hand > cheek > inner arm [bicep region]) suggesting innate body site differences or that photodamage and other environmental trauma can reduce or delay normal CE maturation. Soap-induced dryness resulted in a significant decrease in CE maturation coincidental with reduced corneodesmosomal hydrolysis. Effective moisturization of winter-induced dry skin enhanced CE maturation (33% increase in TRITC fluorescence, n = 14 following 4-week treatment). Using a novel micromanipulation instrument, the force required (microN) to maximally deform individual CEf and CEr was compared. CEf recovered from deep stratum corneum were significantly softer and weaker than CEr recovered from superficial layers. These studies indicate that the normal process of CE maturation is associated with an actual strengthening of this insoluble protective structure and that the impairment of this process is associated with poor quality of the stratum corneum.

Fluorography--limitations on its use for the quantitative detection of 3H- and 14C-labeled proteins in polyacrylamide gels.

The suitability of fluorography for the detection of 3H- and 14C-labeled proteins on polyacrylamide gradient gels has been investigated. It was found that the absorbance of the fluorographic film image produced by a given level of radioactivity decreased as the acrylamide concentration in the gel increased. The use of Coomassie brilliant blue protein dyes to stain the gel prior to fluorography reduced the absorbance of the fluorographic image. It is concluded that quantitative fluorography can only be applied to unstained gels of a uniform acrylamide concentration.

Moisturization and skin barrier function.

Over the past decade, great progress has been made toward elucidating the structure and function of the stratum corneum (SC), the outermost layer of the epidermis. SC cells (corneocytes) protect against desiccation and environmental challenge by regulating water flux and retention. Maintenance of an optimal level of hydration by the SC is largely dependent on several factors. First, intercellular lamellar lipids, organized predominantly in an orthorhombic gel phase, provide an effective barrier to the passage of water through the tissue. Secondly, the diffusion path length also retards water loss, since water must traverse the tortuous path created by the SC layers and corneocyte envelopes. Thirdly, and equally important, is natural moisturizing factor (NMF), a complex mixture of low-molecular-weight, water-soluble compounds first formed within the corneocytes by degradation of the histidine-rich protein known as filaggrin. Each maturation step leading to the formation of an effective moisture barrier--including corneocyte strengthening, lipid processing, and NMF generation--is influenced by the level of SC hydration. These processes, as well as the final step of corneodesmolysis that mediates exfoliation, are often disturbed upon environmental challenge, resulting in dry, flaky skin conditions. The present paper reviews our current understanding of the biology of the SC, particularly its homeostatic mechanisms of hydration.

Skeletal maturity in myelodysplasia.

Incongruent skeletal age in 76 myelomeningocele patients was assessed by a single hand and wrist bone-age film. 28 per cent of patients were found to have a bone age of more than two standard deviations from their chronological age. 22 per cent had advanced skeletal age. Below eight years of age, only 11 per cent were advanced, but over this age 32 per cent showed skeletal maturity exceeding chronological age. Gender, neurological level, presence of hydrocephalus and number of shunt revisions did not correlate with the prevalence of advanced bone age.

Filaggrin breakdown to water binding compounds during development of the rat stratum corneum is controlled by the water activity of the environment.

Filaggrin is a specific epidermal protein which is the precursor of the free amino acids, urocanic acid and pyrrolidone carboxylic acid which are largely responsible for the ability of the stratum corneum of the skin to remain hydrated at low environmental humidity. The distribution of filaggrin shown by immunofluorescence in the stratum corneum of the rat changed dramatically during the first hours of postnatal life. During late foetal development, filaggrin accumulated through the entire thickness of the stratum corneum, indicating that there was a block on the subsequent processing of the protein which normally would convert it to free amino acids. Immediately after birth this block was lifted and normal proteolysis of the filaggrin took place in the outer part of the stratum corneum, leaving the normal adult pattern of a thin zone of cells containing filaggrin at the bottom of the stratum corneum. This activation of filaggrin proteolysis was dependent on the drop in external water activity caused by the transition from an aqueous environment in utero to a dryer environment after birth and it could be blocked by maintaining a 100% humidity atmosphere around the newborn rat after birth. In isolated stratum corneum in vitro, filaggrin proteolysis took place only between 80 and 95% relative humidity, both higher and lower relative humidity blocked the proteolysis. Application of occlusive patches to adult rats prevented the normal proteolysis of filaggrin, indicating that this mechanism controls not only the massive filaggrin proteolysis occurring after birth but also the proteolysis occurring during normal stratum corneum maturation. The stratum corneum therefore has the ability to respond to changes in external humidity by altering the level of the stratum corneum where it converts its reserves of filaggrin into water binding amino acids, such that under humid conditions water binding components will be produced in only the most superficial stratum corneum, or even not produced at all.

Seasonal influences on stratum corneum ceramide 1 fatty acids and the influence of topical essential fatty acids.

Synopsis Ceramide 1 is the main repository of stratum corneum linoleic acid, and changes in the levels of ceramide 1 linoleate are associated with cutaneous abnormalities: essential fatty acid deficiency, atopic dermatitis and acne. We have previously reported seasonal variation in stratum corneum lipids where lipids, particularly ceramides, are very much reduced in winter which probably influences the appearance of skin xerosis in this season. However, investigations into the seasonal variation in ceramide 1 fatty acid levels and the effects of topical essential fatty acids on the composition of these lipid species have not been conducted in healthy subjects. We determined the composition of stratum corneum ceramide 1 esterified fatty acids in leg skin from Caucasian women in winter and summer and also investigated the effects of topical triglycerides rich in linoleic acid on stratum corneum ceramide 1 fatty acid levels in winter. A dramatic seasonal variation was observed. Whereas decreased levels of linoleic acid and some saturated fatty acids (C-15 and C-24) were observed in the winter months, increased levels of oleic acid were found. Nevertheless, it was possible to normalize the levels of stratum corneum ceramide 1 linoleate following topical application of formulations containing linoleic acid-rich triglycerides for 4 weeks. The reduction in ceramide 1 linoleate may lead to impaired stratum corneum function and increased susceptibility to skin xerosis in winter. However, by using formulations containing linoleic acid esters it is possible to correct these changes and possibly improve barrier resilience.

Evidence for filaggrin as a component of the cell envelope of the newborn rat.

A substrate of transglutaminase, specific to the epidermis, was identified, by fluorescent and radioactive labelling with the lysine analogues dansylcadaverine and [14C]putrescine respectively, in newborn-rat epidermal homogenates and whole-skin organ cultures. The labelled analogues were preferentially incorporated into the stratum-corneum protein filaggrin in a Ca2+-dependent manner in both 'in vitro' systems. When filaggrin was labelled in vivo with [3H]histidine and then incubated with rat epidermal preparations, the label was rendered SDS/thiol-insoluble. Incorporation of [3H]filaggrin into the insoluble envelope fraction was Ca2+-dependent and inhibited by EDTA and exogenous amines. Antisera to newborn-rat filaggrin cross-reacted with purified newborn-rat cell envelopes, and this reaction was blocked by adsorbing the antiserum with purified filaggrin. Quantification of the 'envelope-bound' filaggrin showed it to be a significant component, accounting for approx. 10% of the cell-envelope protein.