27-hydroxycholesterol is an endogenous ligand for liver X receptor in cholesterol-loaded cells

The nuclear receptors liver X receptor alpha (LXRalpha) (NR1H3) and LXRbeta (NR1H2) are important regulators of genes involved in lipid metabolism, including ABCA1, ABCG1, and sterol regulatory element-binding protein-1c (SREBP-1c). Although it has been demonstrated that oxysterols are LXR ligands, little is known about the identity of the physiological activators of these receptors. Here we confirm earlier studies demonstrating a dose-dependent induction of ABCA1 and ABCG1 in human monocyte-derived macrophages by cholesterol loading. In addition, we show that formation of 27-hydroxycholesterol and cholestenoic acid, products of CYP27 action on cholesterol, is dependent on the dose of cholesterol used to load the cells. Other proposed LXR ligands, including 20(S)-hydroxycholesterol, 22(R)-hydroxycholesterol, and 24(S),25-epoxycholesterol, could not be detected under these conditions. A role for CYP27 in regulation of cholesterol-induced genes was demonstrated by the following findings. 1) Introduction of CYP27 into HEK-293 cells conferred an induction of ABCG1 and SREBP-1c; 2) upon cholesterol loading, CYP27-expressing cells induce these genes to a greater extent than in control cells; 3) in CYP27-deficient human skin fibroblasts, the induction of ABCA1 in response to cholesterol loading was ablated; and 4) in a coactivator association assay, 27-hydroxycholesterol functionally activated LXR. We conclude that 27-hydroxylation of cholesterol is an important pathway for LXR activation in response to cholesterol overload.     

Expression of a constitutively active calcineurin encoded by an intron-retaining mRNA in follicular keratinocytes

Hair growth is a highly regulated cyclical process. Immunosuppressive immunophilin ligands such as cyclosporin A (CsA) and FK506 are known as potent hair growth modulatory agents in rodents and humans that induce active hair growth and inhibit hair follicle regression. The immunosuppressive effectiveness of these drugs has been generally attributed to inhibition of T cell activation through well-characterized pathways. Specifically, CsA and FK506 bind to intracellular proteins, principally cyclophilin A and FKBP12, respectively, and thereby inhibit the phosphatase calcineurin (Cn). The calcineurin (Cn)/NFAT pathway has an important, but poorly understood, role in the regulation of hair follicle development. Here we show that a novel-splicing variant of calcineurin Aß CnAß-FK, which is encoded by an intron-retaining mRNA and is deficient in the autoinhibitory domain, is predominantly expressed in mature follicular keratinocytes but not in the proliferating keratinocytes of rodents. CnAß-FK was weakly sensitive to Ca²⁺ and dephosphorylated NFATc2 under low Ca²⁺ levels in keratinocytes. Inhibition of Cn/NFAT induced hair growth in nude mice. Cyclin G2 was identified as a novel target of the Cn/NFATc2 pathway and its expression in follicular keratinocytes was reduced by inhibition of Cn/NFAT. Overexpression of cyclin G2 arrested the cell cycle in follicular keratinocytes in vitro and the Cn inhibitor, cyclosporin A, inhibited nuclear localization of NFATc2, resulting in decreased cyclin G2 expression in follicular keratinocytes of rats in vivo. We therefore suggest that the calcineurin/NFAT pathway has a unique regulatory role in hair follicle development.     

The roles of ABCA12 in epidermal lipid barrier formation and keratinocyte differentiation

ATP-binding cassette (ABC) transporters form a large superfamily of transporters that bind and hydrolyze ATP to transport various molecules across limiting membranes or into vesicles. The ABCA subfamily members are thought to transport lipid materials. ABCA12 is a keratinocyte transmembrane lipid transporter protein associated with the transport of lipids via lamellar granules. ABCA12 is considered to transport lipids including ceramides to form extracellular lipid layers in the stratum corneum of the epidermis, which is essential for skin barrier function. ABCA12 mutations are known to underlie the three major types of autosomal recessive congenital ichthyoses: harlequin ichthyosis, lamellar ichthyosis and congenital ichthyosiform erythroderma. ABCA12 mutations result in defective lipid transport via lamellar granules in the keratinocytes, leading to ichthyosis phenotypes from malformation of the stratum corneum lipid barrier. Studies on ABCA12-deficient bioengineered models have revealed that lipid transport by ABCA12 is required for keratinocyte differentiation and epidermal morphogenesis. Defective lipid transport due to loss of ABCA12 function leads to the accumulation of intracellular lipids, including glucosylceramides and gangliosides, in the epidermal keratinocytes. The accumulation of gangliosides seems to result in the apoptosis of Abca12^−/− keratinocytes. It was reported that AKT activation occurs in Abca12^−/− granular-layer keratinocytes, which suggests that AKT activation serves to prevent the cell death of Abca12^−/− keratinocytes. This article is part of a Special Issue entitled The Important Role of Lipids in the Epidermis and their Role in the Formation and Maintenance of the Cutaneous Barrier. Guest Editors: Kenneth R. Feingold and Peter Elias.     

ABCA1 mediates high-affinity uptake of 25-hydroxycholesterol by membrane vesicles and rapid efflux of oxysterol by intact cells

ATP Binding Cassette (ABC) transporter, ABCA1, plays a pivotal role in reverse cholesterol transport by mediating the cellular efflux of phospholipid and cholesterol. Studies using intact cells strongly suggest that ABCA1 acts as a phospholipid floppase, but there has been no direct demonstration that the protein is a primary active sterol transporter. Using membrane vesicles from insect Sf21 cells, we found that ABCA1 mediated ATP-dependent uptake of [³H]25-hydroxycholesterol with an apparent K_m of 0.7 µM. Consistent with this high apparent affinity, expression of ABCA1 in human embryonic kidney cells both increased rapid efflux of 25-hydroxcholesterol and prevented oxysterol-mediated repression of low-density lipoprotein (LDL) receptor and 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase mRNAs. Comparison of wild-type and ABCA1^–/– murine fibroblasts indicates that 25-hydroxycholesterol is effluxed 5-fold more rapidly by wild-type cells. In addition, the rate of efflux from the wild-type but not the ABCA1^–/– fibroblasts is increased a further twofold by inducers of ABCA1 expression. Thus under the experimental conditions employed, endogenous ABCA1 is a major contributor to 25-hydroxycholesterol efflux from wild-type fibroblasts. Evidence from in vitro studies indicates that oxysterols are potent inducers of genes involved in cellular cholesterol efflux and metabolism, including the ABCA1 gene, and repressors of genes involved in cholesterol synthesis or uptake. Our observations raise the possibility that efflux of oxysterols by ABCA1 could contribute to a homeostatic mechanism, which both attenuates oxysterol-induced expression of its cognate gene and alleviates repression of genes encoding proteins, such as HMG-CoA reductase and LDL receptor. active transport; cholesterol homeostasis     

The role of cathepsin E in terminal differentiation of keratinocytes

Cathepsin E (CatE) is predominantly expressed in the rapidly regenerating gastric mucosal cells and epidermal keratinocytes, in addition to the immune system cells. However, the role of CatE in these cells remains unclear. Here we report a crucial role of CatE in keratinocyte terminal differentiation. CatE deficiency in mice induces abnormal keratinocyte differentiation in the epidermis and hair follicle, characterized by the significant expansion of corium and the reduction of subcutaneous tissue and hair follicle. In a model of skin papillomas formed in three different genotypes of syngeneic mice, CatE deficiency results in significantly reduced expression and altered localization of the keratinocyte differentiation induced proteins, keratin 1 and loricrin. Involvement of CatE in the regulation of the expression of epidermal differentiation specific proteins was corroborated by in vitro studies with primary cultures of keratinocytes from the three different genotypes of mice. In wild-type keratinocytes after differentiation inducing stimuli, the CatE expression profile was compatible to those of the terminal differentiation marker genes tested. Overexpression of CatE in mice enhances the keratinocyte terminal differentiation process, whereas CatE deficiency results in delayed differentiation accompanying the reduced expression or the ectopic localization of the differentiation markers. Our findings suggest that in keratinocytes CatE is functionally linked to the expression of terminal differentiation markers, thereby regulating epidermis formation and homeostasis.     

22(R)-hydroxycholesterol and pioglitazone synergistically decrease cholesterol ester via the PPARγ–LXRα–ABCA1 pathway in cholesterosis of the gallbladder

Cholesterosis is a disease of cholesterol metabolism characterized by the presence of excessive lipid droplets in the cytoplasm. These lipid droplets are mainly composed of cholesterol esters derived from free cholesterol. The removal of excess cholesterol from gallbladder epithelial cells (GBECs) is very important for the maintenance of intracellular cholesterol homeostasis and the preservation of gallbladder function. Several lines of evidence have indicated that the activation of either peroxisome proliferator-activated receptor gamma (PPARγ) or liver X receptor α (LXRα) relates to cholesterol efflux. While pioglitazone can regulate the activation of PPARγ, 22(R)-hydroxycholesterol can activate LXRα and is a metabolic intermediate in the biosynthesis of steroid hormones. However, the effect of 22(R)-hydroxycholesterol in combination with pioglitazone on cholesterosis of the gallbladder is unclear. GBECs were treated with pioglitazone, 22(R)-hydroxycholesterol or PPARγ siRNA followed by Western blot analysis for ATP-binding cassette transporter A1 (ABCA1), PPARγ and LXRα. Cholesterol efflux to apoA-I was determined, and Oil Red O staining was performed to monitor variations in lipid levels in treated GBECs. Our data showed that 22(R)-hydroxycholesterol can modestly up-regulate LXRα while simultaneously increasing ABCA1 by 56%. The combination of 22(R)-hydroxycholesterol and pioglitazone resulted in a 3.64-fold increase in ABCA1 expression and a high rate of cholesterol efflux. Oil Red O staining showed an obvious reduction in the lipid droplets associated with cholesterosis in GBECs. In conclusion, the present findings indicate that the anti-lipid deposition action of 22(R)-hydroxycholesterol combined with pioglitazone involves the activation of the PPARγ–LXRα–ABCA1 pathway, increased ABCA1 expression and the efflux of cholesterol from GBECs. Thus, 22(R)-hydroxycholesterol synergistically combined with pioglitazone to produce a remarkable effect on lipid deposition in cholesterosis GBECs.     

Wnt5a/Ror2 pathway contributes to the regulation of cholesterol homeostasis and inflammatory response in atherosclerosis

Atherosclerosis (AS) is characterized by lipids metabolism disorder and inflammatory response. Accumulating evidence has demonstrated that Wingless type 5a (Wnt5a) is implicated in cardiovascular diseases through non-canonical Wnt cascades. However, its precise role during the pathogenesis of AS is still unclear. Therefore, the present study aims to investigate the role and the underlying mechanism of Wnt5a/receptor tyrosine kinase-like orphan receptor 2 (Ror2) pathways in the promotion of AS process through affecting lipid accumulation and inflammation. In atherosclerotic clinical samples, Wnt5a levels were measured by using enzyme-linked immunosorbent assay (ELISA) assay. In vivo experiments were conducted by using apolipoprotein E knockout (apoE^−/−) mice model. Vascular smooth muscle cells (VSMCs) were applied for in vitro studies. Wnt5a was highly expressed in both of atherosclerotic clinical samples and apoE^−/− mice. The knockdown of Wnt5a significantly inhibited cholesterol accumulation and inflammatory response. Additionally, the lipopolysaccharide (LPS)-induced inflammation aggravated the cholesterol accumulation and decreased adenosine triphosphate (ATP)-binding cassette transporter A1 (ABCA1) expression in VSMCs. Depletion of intracellular cholesterol by β-cyclodextrin (β-CD) led to the upregulation of ABCA1 and the inhibition of inflammation. Conversely, the overexpression of Wnt5a inhibited ABCA1 expression, facilitated cholesterol accumulation, impared cholesterol efflux, promoted NF-κB nuclear translocation and the inflammatory cytokines secretion. Moreover, the knockdown of Ror2 increased ABCA1 expression and reduced Wnt5a-induced cholesterol accumulation and inflammatory responses. Furthermore, the knockdown of ABCA1 enhanced cholesterol accumulation and inflammatory response. Therefore, Wnt5a/Ror2 pathway was critical in regulating cholesterol homeostasis and inflammatory response, which might be a promising therapeutic target for AS therapy.     

Impaired hair follicle morphogenesis and polarized keratinocyte movement upon conditional inactivation of integrin-linked kinase in the epidermis

Integrin-linked kinase (ILK) is key for cell survival, migration, and adhesion, but little is known about its role in epidermal development and homeostasis in vivo. We generated mice with conditional inactivation of the Ilk gene in squamous epithelia. These mice die perinatally and exhibit skin blistering and severe defects in hair follicle morphogenesis, including greatly reduced follicle numbers, failure to progress beyond very early developmental stages, and pronounced defects in follicular keratinocyte proliferation. ILK-deficient epidermis shows abnormalities in adhesion to the basement membrane and in differentiation. ILK-deficient cultured keratinocytes fail to attach and spread efficiently and exhibit multiple abnormalities in actin cytoskeletal organization. Ilk gene inactivation in cultured keratinocytes causes impaired ability to form stable lamellipodia, to directionally migrate, and to polarize. These defects are accompanied by abnormal distribution of active Cdc42 to cell protrusions, as well as reduced activation of Rac1 upon induction of cell migration in scraped keratinocyte monolayers. Significantly, alterations in cell spreading and forward movement in single cells can be rescued by expression of constitutively active Rac1 or RhoG. Our studies underscore a central and distinct role for ILK in hair follicle development and in polarized cell movements, two key aspects of epithelial morphogenesis and function.     

Lysosomal acid lipase deficiency impairs regulation of ABCA1 gene and formation of high density lipoproteins in cholesteryl ester storage disease

ATP-binding cassette transporter A1 (ABCA1) mediates the rate-limiting step in high density lipoprotein (HDL) particle formation, and its expression is regulated primarily by oxysterol-dependent activation of liver X receptors. We previously reported that ABCA1 expression and HDL formation are impaired in the lysosomal cholesterol storage disorder Niemann-Pick disease type C1 and that plasma HDL-C is low in the majority of Niemann-Pick disease type C patients. Here, we show that ABCA1 regulation and activity are also impaired in cholesteryl ester storage disease (CESD), caused by mutations in the LIPA gene that result in less than 5% of normal lysosomal acid lipase (LAL) activity. Fibroblasts from patients with CESD showed impaired up-regulation of ABCA1 in response to low density lipoprotein (LDL) loading, reduced phospholipid and cholesterol efflux to apolipoprotein A-I, and reduced α-HDL particle formation. Treatment of normal fibroblasts with chloroquine to inhibit LAL activity reduced ABCA1 expression and activity, similar to that of CESD cells. Liver X receptor agonist treatment of CESD cells corrected ABCA1 expression but failed to correct LDL cholesteryl ester hydrolysis and cholesterol efflux to apoA-I. LDL-induced production of 27-hydroxycholesterol was reduced in CESD compared with normal fibroblasts. Treatment with conditioned medium containing LAL from normal fibroblasts or with recombinant human LAL rescued ABCA1 expression, apoA-I-mediated cholesterol efflux, HDL particle formation, and production of 27-hydroxycholesterol by CESD cells. These results provide further evidence that the rate of release of cholesterol from late endosomes/lysosomes is a critical regulator of ABCA1 expression and activity, and an explanation for the hypoalphalipoproteinemia seen in CESD patients.     

Induction of the cholesterol transporter ABCA1 in central nervous system cells by liver X receptor agonists increases secreted Abeta levels

The expression, function, and regulation of the cholesterol efflux molecule, ABCA1, has been extensively examined in peripheral tissues but only poorly studied in the brain. Brain cholesterol metabolism is of interest because several lines of evidence suggest that elevated cholesterol increases the risk of Alzheimer's disease. We found a largely neuronal expression of ABCA1 in normal rat brain by in situ hybridization. ABCA1 message was dramatically up-regulated in neurons and glia in areas of damage by hippocampal AMPA lesion after 3-7 days. Immunoblot analysis demonstrated ABCA1 protein in cultured neuronal and glial cells, and expression was induced by ligands of the nuclear hormone receptors of the retinoid X receptor and liver X receptor family. ABCA1 was induced by treatment with retinoic acid and several oxysterols, including 22(R)-hydroxycholesterol and 24-hydroxycholesterol. Expression of an ABCA1-green fluorescent protein construct in neuroblastoma cells demonstrated fluorescence in perinuclear compartments and on the plasma membrane. Because the Abeta peptide is important in Alzheimer's disease pathogenesis, we examined whether ABCA1 induction altered Abeta levels. Treatment of neuroblastoma cells with retinoic acid and 22(R)-hydroxycholesterol caused significant increases in secreted Abeta40 (29%) and Abeta42 (65%). Treatment with a nonsteroidal liver X receptor ligand, TO-901317, similarly increased levels of secreted Abeta40 (25%) and Abeta42 (126%). The increase in secreted Abeta levels was reduced by RNAi blocking of ABCA1 expression. These data suggest that the cholesterol efflux molecule ABCA1 may also be involved in the secretion of the membrane-associated molecule, Abeta.     

Rapid Genetic Analysis of Epithelial-Mesenchymal Signaling During Hair Regeneration

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an attractive model system to study organ development and tissue-specific signaling. Although hair follicle development is genetically tractable, fast and reproducible analysis of factors essential for this process remains a challenge. Here we describe a procedure to generate targeted overexpression or shRNA-mediated knockdown of factors using lentivirus in a tissue-specific manner. Using a modified version of a hair regeneration model ^{5, 6, 11}, we can achieve robust gain- or loss-of-function analysis in primary mouse keratinocytes or dermal cells to facilitate study of epithelial-mesenchymal signaling pathways that lead to hair follicle morphogenesis. We describe how to isolate fresh primary mouse keratinocytes and dermal cells, which contain dermal papilla cells and their precursors, deliver lentivirus containing either shRNA or cDNA to one of the cell populations, and combine the cells to generate fully formed hair follicles on the backs of nude mice. This approach allows analysis of tissue-specific factors required to generate hair follicles within three weeks and provides a fast and convenient companion to existing genetic models.     

Inflammatory Mediator TAK1 Regulates Hair Follicle Morphogenesis and Anagen Induction Shown by Using Keratinocyte-Specific TAK1-Deficient Mice

Transforming growth factor-β-activated kinase 1 (TAK1) is a member of the NF-κB pathway and regulates inflammatory responses. We previously showed that TAK1 also regulates keratinocyte growth, differentiation, and apoptosis. However, it is unknown whether TAK1 has any role in epithelial–mesenchymal interactions. To examine this possibility, we studied the role of TAK1 in mouse hair follicle development and cycling as an instructive model system. By comparing keratinocyte-specific TAK1-deficient mice (Map3k7 ^fl/flK5-Cre) with control mice, we found that the number of hair germs (hair follicles precursors) in Map3k7 ^fl/flK5-Cre mice was significantly reduced at E15.5, and that subsequent hair follicle morphogenesis was retarded. Next, we analyzed the role of TAK1 in the cyclic remodeling in follicles by analyzing hair cycle progression in mice with a tamoxifen-inducible keratinocyte-specific TAK1 deficiency (Map3k7 ^fl/flK14-Cre-ER^T2). After active hair growth (anagen) was induced by depilation, TAK1 was deleted by topical tamoxifen application. This resulted in significantly retarded anagen development in TAK1-deficient mice. Deletion of TAK1 in hair follicles that were already in anagen induced premature, apoptosis-driven hair follicle regression, along with hair follicle damage. These studies provide the first evidence that the inflammatory mediator TAK1 regulates hair follicle induction and morphogenesis, and is required for anagen induction and anagen maintenance.     

RXR-alpha ablation in skin keratinocytes results in alopecia and epidermal alterations

RXR-alpha is the most abundant of the three retinoid X receptors (RXRs) in the epidermis. In this study, we have used Cre-mediated recombination to selectively disrupt the mouse gene for RXR-alpha in epidermal and hair follicle keratinocytes. We show that RXR-alpha is apparently dispensable for prenatal epidermal development, while it is involved in postnatal skin maturation. After the first hair pelage, mutant mice develop a progressive alopecia, histologically characterised by the destruction of hair follicle architecture and the formation of utriculi and dermal cysts in adult mice. Our results demonstrate that RXR-alpha plays a key role in anagen initiation during the hair follicle cycle. In addition, RXR-alpha ablation results in epidermal interfollicular hyperplasia with keratinocyte hyperproliferation and aberrant terminal differentiation, accompanied by an inflammatory reaction of the skin. Our data not only provide genetic evidence that RXR-alpha/VDR heterodimers play a major role in controlling hair cycling, but also suggest that additional signalling pathways mediated by RXR-alpha heterodimerised with other nuclear receptors are involved in postnatal hair follicle growth, and homeostasis of proliferation/differentiation of epidermal keratinocytes and of the skin's immune system.     

The multipotency of adult vibrissa follicle stem cells

Several studies focused on the characterization of bulge keratinocytes have proved that they are multipotent stem cells, being recruited not only to regenerate the hair follicle itself, but also the sebaceous gland and the epidermis. However, due to the difficulty in preparing transplantable cell sheets harvested with conventional enzymatic digestion, there is still no direct evidence of the bulge stem cells’ multipotency. Whether they can respond to adult dermal papilla (DP) signals in recombination experiments also remains unclear. In this study, we addressed this problem by culturing and detaching intact bulge keratinocyte sheets from thermo-responsive culture dishes, only by reducing its temperature. When sheets of mass cultured bulge keratinocytes isolated from rat vibrissa follicles were recombined with fresh adult DPs and sole skin dermis in vivo, regeneration of epidermis and sebaceous gland-like structures, and formation of hair bulb with differentiating inner root sheath and hair cuticle were observed within 3 weeks. However, regardless the expression of stem cells markers like CD34, SA1004 and SA1006, no structures were observed when cloned bulge keratinocytes were used to prepare cell sheets and recombinants, revealing the possible existence of monoclonal stem cells within the bulge region. This report is the first to succeed in harvesting adult bulge keratinocyte sheets. Using these sheets it is demonstrated that bulge stem cells directly respond to adult DP signals to induce hair bulb formation in vivo.     

LXR/RXR activation enhances basolateral efflux of cholesterol in CaCo-2 cells

Regulation of gene expression of ATP-binding cassette transporter (ABC)A1 and ABCG1 by liver X receptor/retinoid X receptor (LXR/RXR) ligands was investigated in the human intestinal cell line CaCo-2. Neither the RXR ligand, 9-cis retinoic acid, nor the natural LXR ligand 22-hydroxycholesterol alone altered ABCA1 mRNA levels. When added together, ABCA1 and ABCG1 mRNA levels were increased 3- and 7-fold, respectively. T0901317, a synthetic non-sterol LXR agonist, increased ABCA1 and ABCG1 gene expression 11- and 6-fold, respectively. ABCA1 mass was increased by LXR/RXR activation. T0901317 or 9-cis retinoic acid and 22-hydroxycholesterol increased cholesterol efflux from basolateral but not apical membranes. Cholesterol efflux was increased by the LXR/RXR ligands to apolipoprotein (apo)A-I or HDL but not to taurocholate/phosphatidylcholine micelles. Actinomycin D prevented the increase in ABCA1 and ABCG1 mRNA levels and the increase in cholesterol efflux induced by the ligands. Glyburide, an inhibitor of ABCA1 activity, attenuated the increase in basolateral cholesterol efflux induced by T0901317. LXR/RXR activation decreased the esterification and secretion of cholesterol esters derived from plasma membranes. Thus, in CaCo-2 cells, LXR/RXR activation increases gene expression of ABCA1 and ABCG1 and the basolateral efflux of cholesterol, suggesting that ABCA1 plays an important role in intestinal HDL production and cholesterol absorption.     

Cyclosporin A-induced hair growth in mice is associated with inhibition of calcineurin-dependent activation of NFAT in follicular keratinocytes

One of the most common side effects of treatment with cyclosporin A (CsA) is hypertrichosis. This study shows that calcineurin activity is associated with hair keratinocyte differentiation in vivo, affecting nuclear factor of activated T cells (NFAT1) activity in these cells. Treatment of nude or C57BL/6 depilated normal mice with CsA inhibited the expression of keratinocyte terminal differentiation markers associated with catagen, along with the inhibition of calcineurin and NFAT1 nuclear translocation. This was associated with induction of hair growth in nude mice and retardation of spontaneous catagen induction in depilated normal mice. Furthermore, calcineurin inhibition blocked the expression of p21(waf/cip1) and p27(kip1), which are usually induced with differentiation. This was also associated with an increase in interleukin-1alpha expression (nude mice), a decrease in transforming growth factor-beta (nude and normal mice), and no change in keratinocyte growth factor expression in the skin. Retardation of catagen in CsA-treated mice was accompanied by significant alterations in apoptosis-related gene product expression in hair follicle keratinocytes. The ratio of the anti-apoptotic Bcl-2 to proapoptotic Bax expression increased, and expression of p53 and interleukin-1beta converting enzyme activity decreased. These data provide the first evidence that calcineurin is functionally active in follicular keratinocytes and that inhibition of the calcineurin-NFAT1 pathway in these cells in vivo by CsA enhances hair growth.