Apoptosis in the Hair Follicle

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1 PERSPECTIVE Apoptosis in the Hair Follicle Natalia V. Botchkareva 1, Gurpreet Ahluwalia 1 and Douglas Shander 1 Apoptosis plays an important role in many physiological processes, ranging from morphogenetic events to adult tissue homeostasis, and defects in its regulation contribute to many disorders. Here we review molecular mechanisms of apoptosis in the hair follicle (HF), whose cyclical growth pattern is repeatedly interrupted by apoptosis-driven involution (catagen). We review the common mechanisms underlying apoptosis in the HF during catagen, as well as differences in the regulation of apoptosis between distinct HF cell populations. An overview is provided on the expression and function of molecules involved in the control of various phases of the apoptotic process during catagen. Journal of Investigative Dermatology (2006) 126, doi: /sj.jid Apoptosis is a process of eliminating cells that have fulfilled their biological function during development and tissue homeostasis in multicellular organisms. During the last decade, substantial progress was achieved in the delineation of molecular mechanisms involved in the realization of apoptosis. As an energydemanding process, apoptosis is initiated by a variety of stimuli, including a withdrawal of growth factors, loss of cell adhesion, stimulation of proapoptotic receptors, or DNA damage (reviewed by Afford and Randhawa, 2000). The hair follicle (HF) is a cutaneous organ that shows cyclic activity in postnatal life and transits through periods of active hair growth (anagen), apoptosis-driven involution (catagen), hair shedding (exogen), and relative resting (telogen) (Paus and Cotsarelis, 1999; Cotsarelis and Millar, 2001; Stenn and Paus, 2001). During HF growth and hair production, the activity of factors promoting the proliferation, differentiation, and survival predominates, whereas HF regression is characterized by the activation of a variety of signaling pathways that induce apoptosis in HF cells (Lindner et al., 1997; Soma et al., 1998; Stenn and Paus, 2001). This review focuses on the molecular mechanisms of apoptosis in the HF during its physiological involution; mechanisms of apoptosis underlying the distinct hair loss conditions have been reviewed elsewhere (Botchkarev, 2003; Cotsarelis and Millar, 2001; Hendrix et al., 2005). Apoptosis As a Multi-Step Program of Cell Death Morphologically, apoptosis is characterized by cell shrinkage, membrane blebbing, nuclear condensation, and cellular fragmentation, followed by the formation and the phagocytosis of apoptotic bodies. The process of apoptosis can be divided into four stages: initiation, intracellular response, cell fragmentation, and phagocytosis (reviewed by Afford and Randhawa, 2000). Stages of Initiation and Intracellular Response of Apoptosis The extrinsic apoptotic pathway is activated by binding of specific ligands to the members of the death domain family of membrane receptors (Curtin and Cotter, 2003) (Figure 1). All death receptors have an extracellular cysteine-rich domain that is required for ligand binding and an intracellular domain essential for signal transduction (Peter and Krammer, 2003). Signaling via death receptors results in the recruitment of several intracellular adapter molecules specific for each apoptotic receptor (Fas-associated death domain protein, tumor necrosis factor (TNF) receptor 1 associated death domain, receptor-interacting protein 1, and so on; see Figure 1), all of which contain the death effector domains required for the interaction with similar domains of procaspase-8 and procaspase-10 (Curtin and Cotter, 2003; Roux and Barker, 2002). Activation of caspase-8 and caspase-10 results in their recruitment into the death-inducing signaling complex, followed by activation of caspase-3 along the common apoptotic pathway (Peter and Krammer, 2003). Caspase-8 may also activate the cytoplasmic protein Bid, which, after its translocation to the mitochondria, induces release of cytochrome c, thus linking the extrinsic and intrinsic apoptotic pathways (Schultz and Harrington, 2003). The intrinsic pathway of apoptosis is activated by a variety of intracellular events that lead to the release of cytochrome c from the mitochondria. This process is controlled by proteins of the family, which are divided into two groups: inhibitors of apoptosis (for example,, Bcl-x L, and Mcl-1) and promoters of apoptosis (for example, Bax, Bak, Bok, Bcl-x S, Bik, Bim, Bad, and Bid) (van Gurp et al., 2003). Bim, Bad, and Bid may act as sensors for 1 Gillette Technology Center, Needham, Massachusetts, USA Correspondence: Natalia V. Botchkareva or Douglas Shander, Gillette Technology Center, 37 A Street, Needham, Massachusetts 02492, USA. natasha_botchkareva@gillette.com or doug_shander@gillette.com Abbreviations: HF, hair follicle; IAP, inhibitor of apoptosis protein; KC, keratinocyte; TGF-β, transforming growth factor-β; TNF-α, tumor necrosis factor-α Manuscript received 5 July 2005; revised 16 August 2005; accepted for publication 2 September Journal of Investigative Dermatology (2006), Volume The Society for Investigative Dermatology

2 cellular integrity and the growth factor supply. Bim serves as a sensor of cytoskeleton integrity, and its proapoptotic activity is regulated by interaction with the dynein motor complex (Puthalakath et al., 1999). Bad operates as a sensor for growth factor withdrawal, as deprivation of survival growth factors induces Bad dephosphorylation, which results in apoptosis (Chiang et al., 2001). The cytoplasmic protein Bid may be cleaved by caspase-8, granzyme B, and cathepsin into a truncated isoform (tbid), which, after translocation to the mitochondria, promotes cytochrome c release and apoptosis (van Gurp et al., 2003). After its release into the cytoplasm, cytochrome c recruits the caspase adapter molecule Apaf-1 and the apoptosis initiator enzyme procaspase-9, which together form a holoenzyme complex called an apoptosome (van Gurp et al., 2003). Apoptosomes promote the formation of the active form of caspase-3 along the common pathway of apoptosis (Figure 1). Caspase-3 activation may be inhibited by members of the inhibitor of apoptosis protein (IAP) family, which are located in the cytosol and prevent the activation of procaspases. However, during apoptosis the inhibitory activity of IAPs is neutralized by Smac/DIABLO protein, which is released from the mitochondria and is capable of sequestering IAPs (van Gurp et al., 2003; Riedl and Shi, 2004). Stage of Cell Fragmentation and Phagocytosis of Apoptotic Cells Both extrinsic and intrinsic apoptotic pathways result in the activation of caspase-3, which is believed to be the key proteolytic enzyme capable of cleaving a large variety of intracellular substrates. These include structural and signaling proteins, transcription factors, and regulators of DNA repair and RNA metabolism (Shi, 2002). In addition, caspase-3 cleaves caspase-activated deoxyribonuclease, which translates into the nucleus and initiates DNA cleavage (van Gurp et al., 2003). These processes result in the appearance of the degraded chromatin fragments, lysosomes, mitochondria, and other degenerated cell organelles covered by cell membrane, which are morphologically distinguishable as apoptotic bodies (Afford and Randhawa, 2000; Shi, 2002). Initiation Stage Intracellular Response Cell Fragmentation Phagocytosis Apoptotic ligands Neurotrophins TNF-α FasL, DcR3 p75ntr TNFR-1 Fas (CD95/Apo1) NRAGE, NRIF-1/2, NADE TRADD, RIP, TRAF FAF1, DAXX, FADD TL1A DR3 (TRAMP/Apo3) DR4 (TRAILR1/Apo2) TRADD, RIP, TRAF FADD TRAIL Trail DR5 (TRAILR2/TRICK2) DcR1, DcR2 TRAF, FADD? DR6 TRADD, TRAF Growth factor withdrawal Loss of cell adhesion Membrane of the intact cell Death receptors and adapter molecules Lysosomal defects Decrease of Akt kinase activity Phosphatidyl serine residues Cathepsins Bad Mitochondria Caspase-8 activation During the final steps of apoptosis, cellular, and nuclear fragmentation is also accompanied by changes in the cell membrane and by the translocation of phosphatidylserine from the inner to the outer portion of the cell membrane, leading to its recognition by neighboring cells, macrophages, or dendritic cells and then to its phagocytosis (Fadok, 1999; Afford and Randhawa, 2000; Schultz and Harrington, 2003). CD14, CD36, CD68, the class A scavenger receptors and the vitronectin receptor expressed by macrophages are all capable of interaction with several molecular determinants expressed on apoptotic cells, including anionic phospholipids, phosphatidylserine, and ICAM-3 (Afford and Randhawa, 2000). In addition, serum proteins such as β2-glycoprotein and the complement component C1q can bind to apoptotic cells and enhance their uptake by macrophages (Kagan et al., 2003). Bid Loss of cytoskeleton integrity Dynein motor complex - Bim/Bmf tbid Cyt c Endo G AIF Smac Bax Bak Bcl-x L Bim Bmf Jnk c-jun Proteolysis of cytoplasmic substrates Caspase-3 activation Apoptosome Cyt c Apaf-1 Procaspase-9 IAP Smac/ DIABLO Transcription of apoptotic regulators (Bax) Caspaseactivated DNAse Nucleus DNA fragmentation Proteolysis of cytoplasmic substrates Endonuclease G AIF Phagocyte receptors (CD14, CD36, CD68) Phosphatidylserine residues Membrane of apoptotic cell Membrane of phagocyte Figure 1. Molecular mechanisms of the distinct phases of the apoptotic process. Scheme demonstrates integration of the molecules involved in the control of various phases of apoptosis (initiation, intracellular response, cell fragmentation, phagocytosis). AIF, apoptosis-inducing factor; Akt, protein kinase B; Apaf-1, apoptotic peptidase activating factor 1 Cyt c, cytochrome c; Endo G, endonuclease G; Jnk, c-jun N-terminal kinase. Physiological Hair Follicle Regression (Catagen) As a Model of Apoptosis-Driven and Fully Reversible Organ Involution Although catagen is often thought of as a regressive event, it is an exquisitely orchestrated energy-requiring remodeling process, whose progression assures renewal of further generation of HFs. Catagen was first characterized in detail by Kligman and by Straile et al. (Kligman, 1959; Straile et al., 1961). It lasts about 2 3 weeks in humans and 3 days in mice (Kligman, 1959; Parakkal, 1970; Straile et al., 1961). Morphologically and functionally, catagen is divided into eight substages, each characterized by distinct patterns of apoptosis (Müller-Röver et al., 2001) (Figure 2a). During the murine hair cycle, apoptotic cells visualized by TUNEL staining first appear in the melanogenic area of the late-anagen HF (Tobin et al., 1998). In early-catagen HFs, apoptotic cells are seen in the HF matrix; later, TUNEL+ cells are seen in the outer and inner root sheaths (mid- and late catagen) and are then abundant in the epithelial strand (late catagen) (Lindner et al., 1997; Matsuo et al., 1998). In advanced catagen stages, TUNEL+ cells are also present in the club hair and in the bulge (Lindner et al., 1997; Ito et al., 2004). However, apoptotic cells are never seen in the dermal papilla of catagen HFs in humans and mice under physiological conditions (Lindner et al., 1997; 259

3 Matsuo et al., 1998; Soma et al., 1998). Therefore, the spatio-temporal distribution of TUNEL+ cells in the HF during catagen may be considered as a wave starting from the melanogenic area (late anagen), propagating to the hair matrix (early catagen) and then to the proximal/central outer and inner root sheaths and hair shaft (mid- and late catagen). Distinct cell populations in the HF possess differential susceptibility to apoptosis. The most susceptible to apoptosis are the majority of the follicular epithelial cells and melanocytes, whereas dermal papilla fibroblasts, and some of the keratinocytes (KCs) and melanocytes selected for survival, are resistant to apoptosis (Lindner et al., 1997; Seiberg et al., 1995; Tobin et al., 1998). Apoptotic cells in the HF are phagocytosed by macrophages and by neighboring epithelial cells, which fill the space left by apoptotic cells (the apoptotic force hypothesis introduced by K. Stenn (Stenn and Paus, 2001)). Loss of cell adhesion between KCs may also be considered a catagenpromoting factor, as ICAM-1-deficient mice show significant catagen acceleration (Müller-Röver et al., 2000a). This loss of cell adhesion leads to a progressive reduction in the size of the proximal hair bulb (early and midcatagen) and to rapid shortening of the outer and inner root sheaths and hair shaft (mid- and late catagen). Catagen is also characterized by the formation of a specialized structure in the HF, the club hair, that connects the proximal part of the hair shaft with surrounding HF epithelium and anchors hair in the telogen HF (Parakkal, 1970; Pinkus et al., 1981). During catagen, the dermal papilla is transformed into a cluster of quiescent cells closely adjacent to the regressing HF epithelium, which moves from the subcutis to the dermis/subcutis border to contact the distal portion of the HF epithelium, including the secondary hair germ and bulge. In the hairless gene mutation, the disintegration of the dermal papilla and the HF epithelium during catagen leads to the loss of the capacity of the HF to reenter anagen (Ahmad et al., 1998; Panteleyev et al., 1999). Molecular Mechanisms of Apoptosis in the Hair Follicle During Catagen The physiological involution of the HF may be triggered by a variety of stimuli, including signaling via death receptors, and by the withdrawal of growth factors that maintain cell proliferation and differentiation in the anagen HF. Accumulating evidence suggests that apoptosis in every distinct HF compartment is regulated differently (Figure 3). Apoptosis in the follicular melanocytes. Recent data based on monitoring of the fate of melanocytes during catagen in Trp2-LacZ transgenic mice suggest that distinct subsets of melanocytes (located in the bulge, in the outer root sheath, and above the dermal papilla; Botchkareva et al., 2001) show different sensitivities to apoptosis (Sharov et al., 2005). Specifically, apoptosis occurs only in differentiated melanocytes that are located above the dermal papilla and express the Fas receptor, whereas the other subpopulations, which express the c-kit receptor and, most likely survive catagen (Sharov et al., 2005). Thus, Fas signaling may contribute to melanocyte apoptosis during catagen, similar to that seen after exposure of HFs to chemotherapy (Sharov et al., 2003). Stem cell factor/c-kit and are two important candidate molecules that may promote melanocyte survival during catagen. Constitutive stem cell factor/c-kit ablation or administration of c-kit-neutralizing antibody leads to apoptosis and the disappearance of a Anagen Catagen II Catagen V Telogen b c d e f * * * Figure 2. Catagen development in the human HF. (a) Alterations in HF morphology at distinct stages of anagen-catagen-telogen transition. During anagencatagen transition (Catagen II), the size of the hair matrix (arrow) and dermal papilla (arrowhead) is reduced. During the advanced catagen stages (Catagen V), the dermal papilla is condensed and transformed into a cluster of quiescent cells (arrowhead), and the epithelial strand (arrow) and club hair (asterisk) are formed. In telogen (right), HF length is substantially reduced, and the HF contains two specialized structures, the secondary hair germ (arrowhead) and club hair (asterisk). (b), (c) Bax. In anagen, weak Bax expression is seen in the central inner root sheath (b, arrow). In catagen, Bax expression appears in the regressing hair matrix, the inner root sheath, and the connective tissue sheath (c, large arrowhead, arrow, and small arrowhead, respectively). (d), (e) Caspase- 8. In anagen, caspase-8 expression is restricted to the perifollicular connective tissue sheath (d, arrows). In early catagen, in addition to the connective tissue sheath, caspase-8 appears in central and distal parts of the outer root sheath (e, arrow and arrowhead, respectively). (f) TUNEL (green) plus Ki67 (red). Laser treatment promotes catagen development accompanied by massive apoptosis in the HF: TUNEL+ cells are located in the regressing epithelium (arrow), club hair (arrowhead), and dermal papilla (asterisk). 260 Journal of Investigative Dermatology (2006), Volume 126

4 melanoblasts in mouse embryos (Ito et al., 1999). specifically protects the melanocyte stem cells in the HF from apoptosis, and deficiency results in their apoptotic elimination and premature hair graying (Veis et al., 1993; Nishimura et al., 2005). However, cross-talk between the stem cell factor/c-kit pathway and mitochondrial antiapoptotic proteins in the control of melanocyte survival has yet to be further clarified. Apoptosis in the hair matrix keratinocytes. Data obtained from murine and human HFs suggest that apoptosis in hair matrix KCs is controlled by extrinsic and intrinsic pathways. The p55 subunit of the TNF receptor may be involved in the extrinsic apoptotic pathway, as it is expressed in the hair matrix during catagen, and TNF-α treatment results in an increase of TUNEL+ cells in the hair matrix (Lindner et al., 1997; Ruckert et al., 2000). Also, TNF-α promotes anagencatagen transition in human HFs in vitro (Hoffmann et al., 1996). Apoptosis in hair matrix KCs is also accompanied by the activation of intrinsic apoptotic pathways. During catagen in mice, the /Bax ratio in hair matrix KCs is markedly decreased, compared with anagen levels (Figure 2b and c) (Lindner et al., 1997). The Survivin protein, which belongs to the IAP family, is prominently expressed in the proliferating cells of the hair matrix, whereas its expression is progressively decreased during catagen (Botchkareva et al., 2005). The p53 transcription factor also plays an important role in regulation of apoptosis in hair matrix KCs: catagen HFs of p53-null mice contain fewer apoptotic cells and show significantly retarded catagen progression, compared with catagen HFs of wild-type mice (Botchkarev et al., 2001). p53 may promote the intrinsic apoptotic pathway in hair matrix by regulating the expression of the mitochondrial protein Bax, as such expression is increased in the hair matrix during catagen (Lindner et al., 1997; Botchkarev et al., 2001). The hairless transcription factor also serves as an important regulator of apoptosis in the HF: in hairless mice, apoptosis in hair matrix KCs is strongly increased, leading to a premature switch from anagen to catagen and eventually resulting in disintegration of the dermal papilla from the follicular epithelium and permanent hair loss (Ahmad et al., 1998; Panteleyev et al., 1999; Panteleyev et al., 2000). It was recently shown that hairless protein serves as a transcriptional co-repressor for the vitamin D receptor (Hsieh et al., 2003; Potter et al., 2001); this suggests that interactions between hairless protein and vitamin D receptor might contribute to apoptosis in HF KCs. Interestingly, apoptosis in the hair matrix is strongly increased in keratin 17 knockout mice (McGowan et al., 2002); this provides the first evidence that ablation of a structural protein may cause apoptosis in the HF KCs. It remains to be determined, however, whether keratin 17 deletion results in direct activation of the intrinsic apoptotic pathway, employing Bim as a sensor of cytoskeleton integrity (Puthalakath et al., 1999), or whether other mechanisms are involved in this process. Effector mechanisms of apoptosis in the murine and human HFs are most likely mediated by caspase-1, caspase- 3, caspase-4, and caspase-7, which are highly expressed in the hair matrix during catagen and may be involved in the cell fragmentation process (Lindner et al., 1997; Soma et al., 1998). Apoptosis in the outer root sheath. Before or during catagen, outer root sheath KCs produce several important catagen-promoting secreted molecules: fibroblast growth factor-5 short isoform, neurotrophins, transforming growth factor-β1/2 (TGF-β1/2), IGF binding protein 3, and thrombospondin-1 (Botchkarev et al., 2001; Botchkarev et al., 2004; Suzuki et al., 1998; Yano et al., 2003). Overexpression of one of the antiapoptotic mitochondrial proteins and Bcl-x L in the outer root sheath KCs leads to premature termination of anagen and catagen acceleration, whereas fibroblast growth factor-5 deficiency rescues these effects (Pena et al., 1999; Müller-Röver et al., 2000b. This suggests that the elevated levels of and Bcl-x L in the outer root Inner root sheath L-cathepsin hairless GDNF receptors Fas Melanocytes c-kit Fas Outer root sheath Fas p75ntr TGF- β-1/2 receptors p53 Hair matrix c-kit hairless Survivin Bax p53 TNF-α/p55TNFR Dermal papilla Figure 3. Molecular mechanisms of apoptosis control in the distinct HF compartments. Scheme demonstrates the expression pattern of anti- and proapoptotic molecules (shown in brown and black, respectively) in the HF. GDNF, glial cell line derived neurotrophic factor. sheath stimulate survival and/or activity of cells that promote HF anagencatagen transition. Along with the secretion of catagen stimulatory molecules, outer root sheath KCs express apoptotic death receptors such as Fas, p55 TNF receptor, and p75 neurotrophin receptor, as well as TGFβ-1/2 receptors (Lindner et al., 1997; Paus et al., 1997; Foitzik et al., 2000). This suggests that outer root sheath cells are also the targets for active elimination during catagen. The central outer root sheath of human catagen HFs displays high expression of caspase-8 (Figure 2d and 2e), which links death receptor signaling with the activation of caspase-3 (Peter and Krammer, 2003). Outer root sheath KCs expressing p75ntr and TGF-β receptors become TUNEL+ during catagen (Botchkarev et al., 2000; Foitzik et al., 2000). In contrast to corresponding wild-type mice, p75ntr and TGF-β1 knockout mice display significant catagen retardation associated with the decline of apoptotic cells in the regressing outer root sheath (Botchkarev et al., 2000; Foitzik et al., 2000). Thus, by stimulating apoptosis, neurotrophins and TGF-β1/2 may promote shortening of the outer root sheath in catagen HFs. These data are consistent with results that show potent catagen-promoting activities of neurotrophins and TGF-β2 in human HFs (Soma et al., 2002; Tsuji et al., 2003; Peters et al., 2005)

5 Apoptosis in the inner root sheath and the hair shaft. During catagen, inner root sheath KCs show strong upregulation of the Fas/apo receptor (Lindner et al., 1997), implicating its involvement in apoptosis. Proteolytic enzyme L-cathepsin and hairless may also contribute to apoptosis in the inner root sheath, as both L-cathepsindeficient and hairless-deficient mice show retarded inner root sheath shortening and decreased apoptosis during catagen (Panteleyev et al., 2000; Tobin et al., 2002). Antiapoptotic effects in the inner root sheath may be mediated by glial cell line derived neurotrophic factor and neurturin; their corresponding receptors, GFRα1 and GFRα2, are expressed in the inner root sheath during catagen, and GFRα1- and GFRα2- deficient mice show significant catagen acceleration, compared with wild-type controls (Botchkareva et al., 2000). Less is known about the mechanisms that control apoptosis in hair shaft KCs. In catagen, hair shaft KCs do not express apoptotic receptors (Lindner et al., 1997), although single TUNEL+ cells could be detected (Soma et al., 1998). During mid-catagen and late catagen, TUNEL+ cells become abundant in the proximal end of the hair shaft, that is, in the area of trichilemmal keratinization and club hair formation (Lindner et al., 1997; Matsuo et al., 1998). However, as TUNEL staining is not always a marker of apoptotic cells (Magerl et al., 2001), it is difficult to conclude whether apoptosis plays a role in club hair formation. Clearance of the apoptotic cells from the HF during catagen. Little is known about the mechanisms that control clearance of the apoptotic cells from the HF during catagen. As was shown in other models of biological regression (involution of the mammary gland or thymic epithelium), apoptotic cells are eliminated by macrophages or by neighboring epithelial cells (reviewed by Fadok, 1999). Dendritic cells that are present in human anagen HFs in distal HF epithelium and express C1q may be closely involved in the recognition and phagocytosis of apoptotic cells (Christoph et al., 2000). Apoptotic cells may also be eliminated by macrophages that are located in the connective tissue sheath and that express CD68 (Christoph et al., 2000). Interestingly, the molecules that stimulate catagen development (interleukin 1β, TNF-α, TGF-β1) are also known to significantly augment phagocytosis of apoptotic cells by macrophages (Ren and Savill, 1995). However, the precise mechanisms involved in recognition and elimination of apoptotic cells in catagen HFs remain unclear. Hair follicle cell populations that survive catagen. Survival of distinct HF cell populations during catagen is essential for maintenance of HF regeneration in subsequent hair cycles. These cell populations include fibroblasts of the dermal papilla and connective tissue sheath, KC and melanocyte stem cells and some of their daughter (transient amplifying) cells (Ito et al., 2004; Nishimura et al., 2005; Sharov et al., 2005). Dermal papilla fibroblasts show potent resistance to apoptosis associated with high levels of and lack of death receptors during the entire hair cycle in mice and humans (Lindner et al., 1997; Matsuo et al., 1998; Soma et al., 1998). However, they may be susceptible to apoptosis under certain experimental conditions (application of trypsin or staurosporine) or genetically altered conditions (hairless mice) (Ferraris et al., 1997; Seiberg et al., 1997; Panteleyev et al., 1999). Although the majority of KC stem cells survive during catagen, some bulge cells undergo apoptosis (Ito et al., 2004). However, the mechanisms and purpose of this process remain to be further clarified. Perspectives: Modulation of Apoptosis in the Hair Follicle as a Tool for Correction of Hair Growth Abnormalities Defects in the regulation of apoptosis contribute to many diseases, including hair growth disorders (hirsutism and various hair loss conditions; Paus and Cotsarelis, 1999). Recently, new drugs for modulating apoptosis (for example, TNF-α-inhibiting antibodies, /Bclx L antisense oligonucleotides, synthetic p53, and caspase inhibitors) have been identified, and some of them have been successfully tested in clinical practice (Komarov et al., 1999; Reed, 2002; Kiechle and Zhang, 2002). It was demonstrated that HFs in a number of hair loss conditions (androgenetic alopecia, chemotherapy-induced hair loss) show alterations in apoptosis (Sawaya et al., 2002; Botchkarev et al., 2000). Perhaps apoptosis inhibitors can be used to reduce apoptosis activity in the HFs affected by distinct forms of alopecia. Pharmacological suppression of TGFβ2 by plant extract promotes human HF growth in vitro and delays in vivo catagen development in mice (Tsuji et al., 2003); this suggests that a suppressor of TGF-β might also be effective in preventing male pattern baldness. As an alternative to pharmacological apoptosis manipulation, physical methods that are widely used for management of unwanted hair growth (laser- and light-induced hair removal) are also capable of inducing catagen and apoptosis in the HF (Figure 2f) (Shander et al., 1999). These methods are based on the selective absorption of light by the HF chromophores, including melanocytes. It remains to be determined whether these methods may be combined with pharmacological apoptosis modulation for the correction of hair growth abnormalities. Progress in these areas of research will result in the development of novel strategies for therapeutic intervention in the deregulated apoptotic process seen in the disorders of hair growth. 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