Mite and cockroach allergens activate protease-activated receptor 2 and delay epidermal permeability barrier recovery

Protease-activated receptor-2 (PAR-2) is known to be involved in epidermal permeability barrier function homeostasis. PAR-2 activation occurs after barrier disruption and further activation of PAR-2 by activating peptide significantly delays barrier recovery rate. Cockroach and house dust mite allergens, both known to be associated with the development of asthma, allergic rhinitis, and atopic dermatitis, have protease activity, which can activate PAR-2. In this study, we investigated the effects of both allergens on the epidermal barrier function as well as on the epidermal calcium gradient. Both allergens, when topically applied on the barrier-disrupted site, increased protease activities in the epidermis and delayed barrier recovery and lamellar body secretion in murine skin. The topical application of PAR-2-specific antagonist or protease inhibitors normalized the barrier recovery. Cockroach allergens induced intracellular calcium oscillations in cultured human keratinocytes through PAR-2-involved pathway, which was confirmed by desensitization protocol. Abnormal calcium ion distribution after barrier disruption was also observed in allergens-applied skin. These results suggest that allergens with protease activity can influence the epidermal permeability barrier homeostasis through PAR-2 activation and consequent modulation of the calcium ions in skin.     

Topical hesperidin improves epidermal permeability barrier function and epidermal differentiation in normal murine skin

Orange peel extract appears to exhibit beneficial effects on skin whitening, inflammation, UVB protection, as well as keratinocyte proliferation. In the present study, we determine whether topical hesperidin influences epidermal permeability barrier function and its underlying mechanisms. Hairless mice were treated topically with 2% hesperidin or 70% ethanol alone twice daily for 6 days. At the end of treatment, basal transepidermal water loss (TEWL) was measured 2 and 4 h post barrier disruption. Epidermal proliferation and differentiation were evaluated by immunohistochemical staining and Western blot analysis. Additionally, lamellar body density and secretion were assessed by electron microscopy. Although there were no significant differences in basal barrier function, in comparison with control animals, topical hesperidin significantly accelerated barrier recovery at both 2 and 4 h after acute barrier abrogation. Enhanced barrier function in hesperidin-treated skin correlated with stimulation of both epidermal proliferation and differentiation, as well as enhanced lamellar body secretion. These results indicate that topical hesperidin enhances epidermal permeability barrier homeostasis at least in part due to stimulation of epidermal proliferation, differentiation, as well as lamellar body secretion.     

Actin dynamics regulate immediate PAR-2-dependent responses to acute epidermal permeability barrier abrogation

Background

Lamellar body (LB) secretion and terminal differentiation of stratum granulosum (SG) cells are signaled by both protease activated receptor-2 (PAR-2) and caveolin-1 (cav-1).

Objective

To address the early dynamics of LB secretion, we examined cytoskeletal remodeling of keratinocytes in 3 mouse models following acute barrier abrogation: hairless mice, PAR-2 knockout (−/−) and cav-1 −/−.

Methods and results

Under basal conditions, globular (G)-actin accumulates in SG cells cytosol, while filamentous (F)-actin is restricted to peri-membrane domains. Barrier abrogation induces the apical movement of F-actin and the retreat of the SG-G-actin front, paralleled by upstream cytoskeletal kinases activation. This phenomenon was both enhanced by PAR-2 agonist, and inhibited by cytochalasin-D and in PAR-2 knockout mice. We found that plasma membrane conformational changes causing LB secretion are controlled by PAR-2-dependent cytoskeletal rearrangements. We next addressed the interaction dynamics between cytoskeleton and plasma membrane following PAR-2-induced actin stress fiber formation in both cav-1 −/− and wildtype cells. Actin stress fiber formation is increased in cav-1 −/− cells prior to and following PAR-2 agonist peptide-treatment, while absence of cav-1 inhibits E-cadherin-mediated cell-to-cell adhesion.

Conclusion

PAR-2 drives cytoskeletal/plasma membrane dynamics that regulate early LB secretion following barrier abrogation, stress fiber formation and keratinocyte adhesion.     

Mechanisms by which psychologic stress alters cutaneous permeability barrier homeostasis and stratum corneum integrity

Although many skin disorders, including psoriasis and atopic dermatitis, are adversely affected by psychologic stress (PS), the pathophysiologic link between PS and disease expression remains unclear. Recent studies demonstrated PS-induced alterations in permeability barrier homeostasis, mediated by increased endogenous glucocorticoids. Here, we assessed the mechanisms by which PS alters stratum corneum (SC) function. Insomniac psychologic stress (IPS) altered both barrier homeostasis and SC integrity. IPS decreased epidermal cell proliferation, impaired epidermal differentiation, and decreased the density and size of corneodesmosomes (CD), which was linked to degradation of CD proteins (e.g., desmoglein1). Barrier compromise was linked to decreased production and secretion of lamellar bodies (LB), which in turn could be attributed to a decrease in de novo synthesis of epidermal lipids. Topical physiologic lipids (equimolar cholesterol, ceramides, and free fatty acids) normalized both barrier homeostasis and SC integrity in IPS mice, further evidence that lipid deficiency accounted for these functional abnormalities. Thus, PS inhibition of epidermal lipid synthesis results in decreased LB formation and secretion, as well as decreased CD, compromising both permeability barrier homeostasis and SC integrity. These studies suggest that topical treatment with epidermal physiologic lipids could be beneficial in stress-induced, barrier-associated dermatoses, such as psoriasis and atopic dermatitis.     

Epidermal proteases in the pathogenesis of rosacea

A number of different proteases and their inhibitors have a role in skin physiology and in the pathophysiology of inflammatory skin diseases. Proteases are important in the desquamation process and orderly regulation of the skin's barrier function. On the basis of the catalytic domain, proteases are classified into aspartate-, cysteine-, glutamate-, metallo-, serine-, and threonine proteases. Particularly, serine proteases (SPs) contribute to epidermal permeability barrier homeostasis, as acute barrier disruption increases SP activity in skin and inhibition by topical SP inhibitors accelerated recovery of barrier function after acute abrogation. In rosacea, increased levels of the vasoactive and inflammatory host-defense peptide cathelicidin LL-37 and its proteolytic peptide fragments were found, which were explained by an abnormal production of tryptic activity originating from kallikrein-related peptidase (KLK) 5. It is therefore possible that also other proteases, even from microbial or parasite origin, have a role in rosacea by forming alternate angiogenic and proinflammatory cathelicidin peptides. Further, the regulation of protease activity, in particular KLK-5 activity, might have a role in rosacea. This review briefly summarizes our current knowledge about keratinocyte-derived proteases and protease inhibitors, which might have a role in the pathophysiology of rosacea.     

Basis for improved permeability barrier homeostasis induced by PPAR and LXR activators: liposensors stimulate lipid synthesis, lamellar body secretion, and post-secretory lipid processing

Previously, we demonstrated that topical applications of peroxisome proliferator-activated receptors (PPARs) and liver X receptor (LXR) activators improve permeability barrier homeostasis. We showed further that stimulation of epidermal differentiation provides one mechanism that could account for such improvement. Here, we studied the effects of these agents on the lipid matrix of the stratum corneum. Hairless mice were treated topically with activators of PPARalpha (WY14643), PPARdelta (GW1514), PPARgamma (ciglitazone), and LXR (22(R)-cholesterol or TO901317) or vehicle twice daily for 3 days. All activators significantly increased epidermal cholesterol, fatty acid, and sphingolipid synthesis, including the production of barrier-specific ceramide species. In addition, lamellar body (LB) formation, secretion, and post-secretory processing accelerated significantly following acute barrier disruption in PPAR/LXR-activator-treated animals. Finally, the activity of epidermal beta-glucocerebrosidase, a key lipid-processing enzyme, increased in PPAR/LXR-activator-treated animals. Thus, topical PPAR and LXR activators stimulate epidermal lipid synthesis, increase LB secretion, and accelerate extracellular lipid processing, providing additional mechanisms that further account for their ability to improve epidermal permeability barrier homeostasis. Since the liposensors are activated by endogenous lipid metabolites, they may serve as unique regulators of barrier homeostasis.     

Testosterone perturbs epidermal permeability barrier homeostasis

Although there are no known gender-related differences in permeability barrier function in adults, estrogens accelerate whereas testosterone retards barrier development in fetal skin, and male fetuses demonstrate slower barrier development than female littermates. Moreover, prenatal administration of the androgen receptor antagonist, flutamide, equalizes developmental rates in male and female fetuses. Therefore, we evaluated the effects of changes in testosterone on barrier homeostasis in adult murine and human skin. Hypogonadal mice (whether by castration or by treatment with systemic flutamide) displayed significantly faster barrier recovery at 3, 6, and 12 h than did controls, and testosterone replacement slowed barrier recovery in castrated mice. Moreover, testosterone directly effects the skin, as topical flutamide also accelerated barrier recovery in normal male mice. These findings appear to be of physiologic significance, since prepubertal male mice (age 5 wk) displayed accelerated barrier recovery in comparison with adult postpubertal (11 wk) males. These studies also appear to be relevant for humans, as a hypopituitary human subject demonstrated repeated changes in barrier recovery in parallel with peaks and nadirs in serum testosterone levels during intermittent testosterone replacement. Mechanistic studies showed that differences in epidermal lipid synthesis do not account for the testosterone-induced functional alterations. Instead, epidermal lamellar body (LB) formation and secretion both decrease, resulting in decreased extracellular lamellar bilayers in testosterone-replete animals. These studies demonstrate that fluctuations in testosterone modulate barrier function, and that testosterone repletion can have negative consequences for permeability barrier homeostasis.     

Cannabinoid receptors 1 and 2 oppositely regulate epidermal permeability barrier status and differentiation

Cannabinoid receptors (CBR) 1 and 2 have been implicated in keratinocyte differentiation/proliferation. How CB receptors affect epidermal permeability barrier and stratum corneum structure and function remains unclear. Permeability barrier abrogation was induced by sequential tape-stripping of the SC and assessed in both CB1R and CB2R knockout (-/-) mice in comparison with wild-type (+/+) littermates. Absence of CB1R delays permeability barrier recovery, while the latter was found to be accelerated in CB2R -/- mice. While increased lamellar body (LB) secretion is observed in CB2R -/- mice accounting for the enhanced recovery, CB1R -/- animals display strong alterations in lipid bilayer structures. Markers for epidermal differentiation (i.e. filaggrin, loricrin and involucrin) and terminal differentiation (i.e. TUNEL assay and caspase-14 activation) were respectively decreased and increased in CB1R and CB2R -/- mice. Surprisingly, CB1R agonist treatment of human cultured keratinocytes increases mRNA of p21 and cytokeratin 1 and 10 and decreases cyclin D1 but protein levels remained unchanged. Such paradox could partially be explained by the increase in non-phosphorylated-4E-BP1, an inhibitor of mRNA translation, following CB1R agonist treatment. Altogether, these observations put forward the importance and the complexity of cannabinoid signalling for the regulation of permeability barrier and epidermal differentiation.     

Potassium channel openers accelerate epidermal barrier recovery

BACKGROUND: Maintenance of a competent permeability barrier in the face of external and internal stressors requires signals between the stratum corneum interface and the metabolic machinery in the underlying nucleated layers. For example, reductions in the ion gradients for Ca2+ after acute barrier disruption stimulate lamellar body (LB) secretion, a response required to restore barrier homeostasis. Although alterations in external K+ levels also regulate barrier recovery after acute insults, the mechanisms whereby K+ regulates barrier function remain unknown. OBJECTIVES: To evaluate effects of regulators of K+ channels on barrier homeostasis in hairless mice. METHODS: We tested a number of chemically different drugs that alter intracellular K+ levels. Results Single applications of either K+ channel openers (i.e. 1-EBIO, minoxidil, diazoxide) or the K+ ionophore, valinomycin, accelerated barrier recovery after acute insults to murine skin, paralleled by a reduction in intracellular K+ levels in cultured human keratinocytes. In contrast, applications of K+ channel blockers (i.e. gilbenclamide, dequalinium) delayed barrier recovery. Alterations in intracellular K+ regulated barrier homeostasis by either stimulating (reduced K+) or inhibiting (elevated K+) LB secretion. Finally, development of epidermal hyperplasia, a downstream consequence of barrier disruption, was also inhibited by agents that reduce intracellular K+ levels. CONCLUSIONS: These results demonstrate that changes in K+ levels that can be presumed to occur after barrier disruption signal metabolic responses, i.e. LB secretion, which accelerates normalization of barrier function.     

Structural and biochemical basis for the UVB-induced alterations in epidermal barrier function

Ultraviolet light (UVR) induces a myriad of cutaneous changes, including delayed disruption of the permeability barrier with higher doses. To investigate the basis for the UVB-induced barrier alteration, we assessed the epidermal lamellar body secretory system at various time points before and after barrier disruption with a single high dose of UVB (7.5 MED) to murine epidermis. Morphological data were correlated with changes in epidermal proliferation and lipid synthesis, indicative of lamellar body generation. Twenty-four hours following UVB, the stratum corneum (SC) is normal, but a layer of abnormal, vacuolated, and lamellar body (LB)-deficient cells is present, immediately beneath the stratum granulosum (SG)/SC interface. Immediately subjacent to this band of damaged cells, normal keratinocytes that contain intact LBs are present. By 72 h, concomitant with the appearance of a barrier abnormality, extensively damaged cells persist at the SC/SG interface, and abnormal lamellar membrane structures appear in the lower SC. Upper stratum spinosum (SS) and lower SG cells appear normal, with increased numbers of LBs. A barrier abnormality is still present at 96 h, in association with membrane abnormalities in the lower SC interstices, but up to four normal-appearing, subjacent SG cell layers are present. By 120 h, accelerated LB formation and precocious LB extrusion occur throughout the thickened SG; normal lamellar membranes are present in the lower SC; and barrier recovery is almost complete. Whereas, epidermal synthesis of the major barrier lipid species (i.e., cholesterol, fatty acids, and ceramides, including acylceramides) is reduced or unchanged at 24 and 48 h, it increases significantly 72 h after exposure to UVB. Therefore, the delayed disruption of the permeability barrier following acute UVB exposure results from the arrival of a band of lamellar body-incompetent (i.e., damaged) cells at the SG/SC interface. The subsequent, rapid recovery of the barrier, in turn, results from compensatory hyperplasia of subjacent, undamaged SS/SG cells, generating increased numbers and contents of LB. These results underscore the critical role of the stratum compactum in mediating barrier function, and suggest that beneficial therapeutic effects of UV exposure may be due to enhanced lipid production and barrier regeneration.     

Permeability barrier disorder in Niemann-Pick disease: sphingomyelin-ceramide processing required for normal barrier homeostasis

Prior studies have established the requirement for enzymatic hydrolysis of glucosylceramides to ceramide for epidermal barrier homeostasis. In this study, we asked whether sphingomyelin-derived ceramide, resulting from acid-sphingomyelinase activity, is also required for normal barrier function. We showed first, that a subset of Niemann-Pick patients with severe acid-sphingomyelinase deficiency (i.e., <2% residual activity) demonstrate abnormal permeability barrier homeostasis, i.e., delayed recovery kinetics following acute barrier disruption by cellophane tape-stripping. To obtain further mechanistic insights into the potential requirement for sphingomyelin-to-ceramide processing for the barrier, we next studied the role of acid-sphingomyelinase in hairless mouse skin. Murine epidermis contains abundant acid-sphingomyelinase activity (optimal pH 5.1-5.6). Two hours following acute barrier disruption by tape-stripping, acid-sphingomyelinase activity increases 1. 44-fold (p<0.008 versus vehicle-treated controls), an increase that is blocked by a single topical application of the acid-sphingomyelinase inhibitor, palmitoyldihydrosphingosine. Furthermore, both palmitoyldihydrosphingosine and desipramine, a chemically and mechanically unrelated acid-sphingomyelinase inhibitor, significantly delay barrier recovery both 2 and 4 h after acute barrier abrogation. Inhibitor application also causes both an increase in sphingomyelin content, and a reduction of normal extracellular lamellar membrane structures, in the stratum corneum. Both of the inhibitor-induced delays in barrier recovery can be overridden by co-applications of topical ceramide, demonstrating that an alteration of the ceramide-sphingomyelin ratio, rather than sphingomyelin accumulation, is likely responsible for the barrier abnormalities that occur with acid-sphingomyelinase deficiency. These studies demonstrate an important role for enzymatic processing of sphingomyelin-to-ceramide by acid-sphingomyelinase as a mechanism for generating a portion of the stratum corneum ceramides for permeability barrier homeostasis in mammalian skin.     

Sustained serine proteases activity by prolonged increase in pH leads to degradation of lipid processing enzymes and profound alterations of barrier function and stratum corneum integrity

We showed recently that short-term increases in stratum corneum (SC) pH are accompanied by minor alterations in permeability barrier homeostasis and SC integrity/cohesion. Since prolonged SC neutralization more closely mirrors clinical situations (i.e., neonatal skin, occupational dermatitis conditions), we assessed here whether sustained elevations of SC pH by long-term application of 1,1,3,3-tetramethylguanidine superbase provoke profound alterations in SC function. Sustained SC neutralization altered not only barrier recovery kinetics but also basal permeability barrier function. These abnormalities were attributable to a decrease in beta-glucocerebrosidase (beta-GlcCer'ase) and acidic sphingomyelinase (aSMase) catalytic activity and enzyme degradation consequent to a pH-induced sustained serine protease (SP) activity. The role of SP in this process was shown by the normalization of enzyme activities/content by co-applied SP inhibitors (SPI). To address whether lipid-processing enzymes are potential substrates for the stratum corneum chymotryptic enzyme (SCCE), protein extracts from human SC were treated for 2 h at 37 degrees C with recombinant active SCCE at pH 7.2. Recombinant SCCE induced a significant decrease in the immunoblotting of both beta-GlcCer'ase or aSMase compared with control experiments performed in the absence of the active SCCE. Finally, with sustained SC neutralization, SC integrity/cohesion deteriorated, attributable to SP-mediated degradation of corneodesmosomes (CD) as well as CD constituent proteins, desmoglein 1. These abnormalities were again reversed by co-applied SPI. In conclusion, prolonged SC neutralization provokes profound abnormalities in SC function, due to pH-induced high SP activity that, in turn, degrades lipid processing enzymes and CD proteins.     

Proteolytically active allergens cause barrier breakdown

Two major allergens--the house dust mite Dermatophagoides pteronyssinus (Der p 1) and cockroach allergens--are proteolytically active and stimulate the protease-activated receptor 2 (PAR-2). Jeong et al. (2008, this issue) exposed mouse and human epidermis to both allergens and correlated the observed delay in permeability barrier recovery to PAR-2 activation/signaling. This article exposes the secretive boundaries between barrier homeostasis and immunity.     

Co-regulation and interdependence of the mammalian epidermal permeability and antimicrobial barriers

Human epidermis elaborates two small cationic, highly hydrophobic antimicrobial peptides (AMP), beta-defensin 2 (hBD2), and the carboxypeptide cleavage product of human cathelicidin (hCAP18), LL-37, which are co-packaged along with lipids within epidermal lamellar bodies (LBs) before their secretion. Because of their colocalization, we hypothesized that AMP and barrier lipid production could be coregulated by altered permeability barrier requirements. mRNA and immunostainable protein levels for mBD3 and cathelin-related antimicrobial peptide (CRAMP) (murine homologues of hBD2 and LL-37, respectively) increase 1-8 hours after acute permeability barrier disruption and normalize by 24 hours, kinetics that mirror the lipid metabolic response to permeability barrier disruption. Artificial permeability barrier restoration, which inhibits the lipid-synthetic response leading to barrier recovery, blocks the increase in AMP mRNA/protein expression, further evidence that AMP expression is linked to permeability barrier function. Conversely, LB-derived AMPs are also important for permeability barrier homeostasis. Despite an apparent increase in mBD3 protein, CRAMP-/- mice delayed permeability barrier recovery, attributable to defective LB contents and abnormalities in the structure of the lamellar membranes that regulate permeability barrier function. These studies demonstrate that (1) the permeability and antimicrobial barriers are coordinately regulated by permeability barrier requirements and (2) CRAMP is required for permeability barrier homeostasis.     

Expression of voltage-gated calcium channel subunit alpha1C in epidermal keratinocytes and effects of agonist and antagonists of the channel on skin barrier homeostasis

Recent studies demonstrated that skin surface electric conditions affect epidermal permeability barrier homeostasis. These results suggest the existence of voltage sensor on the keratinocytes of the epidermis. On the contrary, specific blockers of the voltage-gated calcium channel (VGCC) also affect epidermal barrier homeostasis, but the existence and function of the channel has not been determined. We demonstrated here immunohistochemically the expression of the main subunit of the L-type VGCC, alpha1C, which alone has a calcium channel function, in mouse and human epidermis. Immunostaining, RT-PCR, and Western blotting were carried out to detect the channel protein. Messenger RNA of alpha1C was also detected in mouse epidermis and human keratinocyte culture by RT-PCR. We also evaluated the function of the channel in the cultured human keratinocytes. Previously, we demonstrated that influx of calcium ion into epidermal keratinocytes delayed the barrier recovery after barrier disruption and topical application of calcium channel blocker accelerated the barrier recovery. In this study, topical application of nifedipine and R-(+)-BAY K8644 after tape stripping of hairless mice accelerated the barrier repair rate while application of S-(-)-BAY K8644 delayed the barrier recovery. These results suggest that the VGCC exists on epidermal keratinocytes and plays an important role in skin barrier homeostasis.     

Topical peroxisome proliferator activated receptor activators accelerate postnatal stratum corneum acidification

Previous studies have shown that pH declines from between 6 and 7 at birth to adult levels (pH 5.0-5.5) over 5-6 days in neonatal rat stratum corneum (SC). As a result, at birth, neonatal epidermis displays decreased permeability barrier homeostasis and SC integrity, improving days 5-6. We determined here whether peroxisome proliferator-activated receptor (PPAR) activators accelerate postnatal SC acidification. Topical treatment with two different PPARalpha activators, clofibrate and WY14643, accelerated the postnatal decline in SC surface pH, whereas treatment with PPARgamma activators did not and a PPARbeta/delta activator had only a modest effect. Treatment with clofibrate significantly accelerated normalization of barrier function. The morphological basis for the improvement in barrier function in PPARalpha-treated animals includes accelerated secretion of lamellar bodies and enhanced, postsecretory processing of secreted lamellar body contents into mature lamellar membranes. Activity of beta-glucocerebrosidase increased after PPARalpha-activator treatment. PPARalpha activator also improved SC integrity, which correlated with an increase in corneodesmosome density and increased desmoglein-1 content, with a decline in serine protease activity. Topical treatment of newborn animals with a PPARalpha activator increased secretory phospholipase A2 activity, which likely accounts for accelerated SC acidification. Thus, PPARalpha activators accelerate neonatal SC acidification, in parallel with improved permeability homeostasis and SC integrity/cohesion. Hence, PPARalpha activators might be useful to prevent or treat certain common neonatal dermatoses.     

Deficiency of PPARbeta/delta in the epidermis results in defective cutaneous permeability barrier homeostasis and increased inflammation

In cultured human keratinocytes or murine epidermis, peroxisome proliferator-activated receptor beta/delta (PPARbeta/delta) (NR1C2) activators (1) stimulate keratinocyte differentiation; (2) decrease keratinocyte proliferation; (3) accelerate permeability barrier repair; (4) increase epidermal lipid synthesis; and (5) reduce cutaneous inflammation. Since these results suggest that PPARbeta/delta could play an important role in cutaneous homeostasis, we assessed here the skin phenotype of mice deficient in PPARbeta/delta. Gross cutaneous abnormalities were not evident, and both stratum corneum (SC) skin hydration and surface pH were normal. However, the epidermis was thickened and proliferating cell nuclear antigen (PCNA) staining was increased, indicating increased cell proliferation. No change in apoptosis was observed but the expression of differentiation markers, such as filaggrin, involucrin, and loricrin, was slightly increased in PPARbeta/delta(-/-) mice. Although basal permeability barrier function was normal, PPARbeta/delta knockout (KO) mice show a significant delay in barrier recovery rates following acute barrier disruption by either acetone treatment or tape-stripping. Delayed barrier recovery correlated with decreased production and secretion of lamellar bodies (LBs), and with reduced numbers of extracellular lamellar membranes in the SC. Finally, PPARbeta/delta KO mice displayed increased inflammation in response to 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment. Together, these results further demonstrate that PPARbeta/delta in the epidermis: (1) is required for permeability barrier homeostasis; (2) regulates keratinocyte proliferation; and (3) modulates cutaneous inflammation.     

Ablation of the calcium-sensing receptor in keratinocytes impairs epidermal differentiation and barrier function

The calcium-sensing receptor (CaR) has an essential role in mediating Ca(2+)-induced keratinocyte differentiation in vitro. In this study, we generated keratinocyte-specific CaR knockout ((Epid)CaR(-/-)) mice to investigate the function of the CaR in epidermal development in vivo. (Epid)CaR(-/-) mice exhibited a delay in permeability barrier formation during embryonic development. Ion capture cytochemistry detected the loss of the epidermal Ca(2+) gradient in the (Epid)CaR(-/-) mice. The expression of terminal differentiation markers and key enzymes mediating epidermal sphingolipid transport and processing in the (Epid)CaR(-/-) epidermis was significantly reduced. The (Epid)CaR(-/-) epidermis displayed a marked decrease in the number of lamellar bodies (LBs) and LB secretion, thinner lipid-bound cornified envelopes, and a defective permeability barrier. Consistent with in vivo results, epidermal keratinocytes cultured from (Epid)CaR(-/-) mice demonstrated abnormal Ca(2+)(i) handling and diminished differentiation. The impairment in epidermal differentiation and permeability barrier in (Epid)CaR(-/-) mice maintained on a low calcium (0.02%) diet is more profound and persistent with age than in (Epid)CaR(-/-) mice maintained on a normal calcium (1.3%) diet. Deleting CaR perturbs the epidermal Ca(2+) gradient and impairs keratinocyte differentiation and permeability barrier homeostasis, indicating a key role for the CaR in normal epidermal development.     

Barrier dysfunction and pathogenesis of neutral lipid storage disease with ichthyosis (Chanarin-Dorfman syndrome)

Neutral lipid storage disease with ichthyosis (NLSDI; Chanarin-Dorfman syndrome) is an ichthyosiform syndrome, often associated with mutations in a lipid hydrolase, CGI-58. The presence of oil red O-positive, neutral lipid droplets in tissue biopsies, and/or in leukocytes on blood smears, coupled with a constellation of multisystem abnormalities and a pruritic ichthyosiform erythroderma, are together diagnostic of NLSDI. We investigated the pathogenesis of the ichthyosiform erythroderma in patients from three unrelated kindreds with a clinical diagnosis of NLSDI. Basal permeability barrier function and stratum corneum (SC) integrity were abnormal, but barrier recovery rates were faster than normal, as in atopic dermatitis. The basal barrier abnormality was linked to the secretion of lipid micro-inclusions, first segregated within lamellar bodies (LB), which then form a non-lamellar phase within the SC interstices, shown by combined ruthenium tetroxide post-fixation and lipid-retaining resin-white embedding. With colloidal lanthanum nitrate perfusion, excess water/solute movement was restricted to the SC interstices, and further localized to non-lamellar domains. Phase separation of excess stored lipid provides a unifying pathogenic mechanism not only for NLSDI, but also in several other inherited ichthyosiform disorders of lipid metabolism, such as recessive X-linked ichthyosis and type 2 Gaucher's disease.     

Hyaluronan-CD44 interaction stimulates keratinocyte differentiation, lamellar body formation/secretion, and permeability barrier homeostasis

In this study we investigated whether hyaluronan (HA)-CD44 interaction influences epidermal structure and function. Our data show that CD44 deficiency is accompanied by reduction in HA staining in CD44 knockout (k/o) mouse skin leading to a marked thinning of epidermis versus wild-type mouse skin. A significant delay in the early barrier recovery (following acute barrier disruption) occurs in CD44 k/o versus wild-type mouse skin. To assess the basis for these alterations in CD44 k/o mouse epidermis, we determined that differentiation markers are greatly reduced in the epidermis of CD44 k/o versus wild-type mice, while conversely HA binding to CD44 triggers differentiation in cultured human keratinocytes. CD44 downregulation (using CD44 small interfering RNAs) also inhibits HA-mediated keratinocyte differentiation. Slower barrier recovery in CD44 k/o mice could be further attributed to reduced lamellar body formation, loss of apical polarization of LB secretion, and downregulation of cholesterol synthesis. Accordingly, HA-CD44 binding stimulates both LB formation and secretion. Together, these observations demonstrate new roles for HA-CD44 interaction in regulating both epidermal differentiation and lipid synthesis/secretion, which in turn influence permeability barrier homeostasis. HA-CD44 signaling could comprise a novel approach to treat skin disorders characterized by abnormalities in differentiation, lipid synthesis, and/or barrier function.