The Lysosomal Protease Cathepsin L Is an Important Regulator of Keratinocyte and Melanocyte Differentiation During Hair Follicle Morphogenesis and Cycling
2002; Elsevier BV; Volume: 160; Issue: 5 Linguagem: Inglês
10.1016/s0002-9440(10)61127-3
ISSN1525-2191
AutoresDesmond J. Tobin, Kerstin Foitzik, Thomas Reinheckel, Lars Mecklenburg, Vladimir A. Botchkarev, Christoph Peters, Ralf Paus,
Tópico(s)Dermatologic Treatments and Research
ResumoWe have previously shown that the ubiquitously expressed lysosomal cysteine protease, cathepsin L (CTSL), is essential for skin and hair follicle homeostasis. Here we examine the effect of CTSL deficiency on hair follicle development and cycling in ctsl−/− mice by light and electron microscopy, Ki67/terminal dUTP nick-end labeling, and trichohyalin immunofluorescence. Hair follicle morphogenesis in ctsl−/− mice was associated with several abnormalities. Defective terminal differentiation of keratinocytes occurred during the formation of the hair canal, resulting in disruption of hair shaft outgrowth. Both proliferation and apoptosis levels in keratinocytes and melanocytes were higher in ctsl−/− than in ctsl+/+ hair follicles. The development of the hair follicle pigmentary unit was disrupted by vacuolation of differentiating melanocytes. Hair cycling was also abnormal in ctsl−/− mice. Final stages of hair follicle morphogenesis and the induction of hair follicle cycling were retarded. Thereafter, these follicles exhibited a truncated resting phase (telogen) and a premature entry into the first growth phase. Further abnormalities of telogen development included the defective anchoring of club hairs in the skin, which resulted in their abnormal shedding. Melanocyte vacuolation was again apparent during the hair cycle-associated reconstruction of the hair pigmentary unit. A hallmark of these ctsl−/− mice was the severe disruption in the exiting of hair shafts to the skin surface. This was mostly because of a failure of the inner root sheath (keratinocyte layer next to the hair shaft) to fully desquamate. These changes resulted in a massive dilation of the hair canal and the abnormal routing of sebaceous gland products to the skin surface. In summary, this study suggests novel roles for cathepsin proteases in skin, hair, and pigment biology. Principal target tissues that may contain protein substrate(s) for this cysteine protease include the developing hair cone, inner root sheath, anchoring apparatus of the telogen club, and organelles of lysosomal origin (eg, melanosomes). We have previously shown that the ubiquitously expressed lysosomal cysteine protease, cathepsin L (CTSL), is essential for skin and hair follicle homeostasis. Here we examine the effect of CTSL deficiency on hair follicle development and cycling in ctsl−/− mice by light and electron microscopy, Ki67/terminal dUTP nick-end labeling, and trichohyalin immunofluorescence. Hair follicle morphogenesis in ctsl−/− mice was associated with several abnormalities. Defective terminal differentiation of keratinocytes occurred during the formation of the hair canal, resulting in disruption of hair shaft outgrowth. Both proliferation and apoptosis levels in keratinocytes and melanocytes were higher in ctsl−/− than in ctsl+/+ hair follicles. The development of the hair follicle pigmentary unit was disrupted by vacuolation of differentiating melanocytes. Hair cycling was also abnormal in ctsl−/− mice. Final stages of hair follicle morphogenesis and the induction of hair follicle cycling were retarded. Thereafter, these follicles exhibited a truncated resting phase (telogen) and a premature entry into the first growth phase. Further abnormalities of telogen development included the defective anchoring of club hairs in the skin, which resulted in their abnormal shedding. Melanocyte vacuolation was again apparent during the hair cycle-associated reconstruction of the hair pigmentary unit. A hallmark of these ctsl−/− mice was the severe disruption in the exiting of hair shafts to the skin surface. This was mostly because of a failure of the inner root sheath (keratinocyte layer next to the hair shaft) to fully desquamate. These changes resulted in a massive dilation of the hair canal and the abnormal routing of sebaceous gland products to the skin surface. In summary, this study suggests novel roles for cathepsin proteases in skin, hair, and pigment biology. Principal target tissues that may contain protein substrate(s) for this cysteine protease include the developing hair cone, inner root sheath, anchoring apparatus of the telogen club, and organelles of lysosomal origin (eg, melanosomes). The hair follicle (HF) is a unique neuroectodermal-mesodermal interactive organoid that results in the elaboration of at least 15 distinct interacting cell subpopulations, organized into five or six concentric cylinders.1Paus R Cotsarelis G The biology of hair follicles.N Engl J Med. 1999; 341: 491-497Crossref PubMed Scopus (1007) Google Scholar, 2Cotsarelis G Millar SE Towards a molecular understanding of hair loss and its treatment.Trends Mol Med. 2001; 7: 293-301Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar, 3Stenn KS Paus R Controls of hair follicle cycling.Physiol Rev. 2001; 81: 449-494Crossref PubMed Scopus (1157) Google Scholar These together provide a truly exceptional miniorgan that rivals the vertebrate limb bud,4Schaller SA Li S Ngo-Muller V Han MJ Omi M Anderson R Muneoka K Cell biology of limb patterning.Int Rev Cytol. 2001; 203: 483-517Crossref PubMed Scopus (14) Google Scholar and feather and tooth development5Chuong C-M Molecular Basis of Epithelial Appendage Morphogenesis.in: Chuong C-M R.G. Landes Company, Austin1998Google Scholar as models for studies of the genetic regulation of morphogenesis and tissue renewal.2Cotsarelis G Millar SE Towards a molecular understanding of hair loss and its treatment.Trends Mol Med. 2001; 7: 293-301Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar, 6Hardy MH The secret life of the hair follicle.Trends Genet. 1992; 8: 55-61Abstract Full Text PDF PubMed Scopus (820) Google Scholar, 7Philpott MJ Paus R Principles of hair follicle morphogenesis.in: Chuong C-M Molecular Basis of Epithelial Appendage Morphogenesis. R.G. Landes Company, Austin1998: 75-103Google Scholar From its initiation during the perinatal period to its life-long cyclical growth, the HF is unique in the adult mammalian body in experiencing multiple and life-long recapitulations to early stages of its embryogenesis.2Cotsarelis G Millar SE Towards a molecular understanding of hair loss and its treatment.Trends Mol Med. 2001; 7: 293-301Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar, 8Paus R Müller-Röver S Botchkarev VA Chronobiology of the hair follicle: hunting the “hair cycle clock.”.J Invest Dermatol Symp Proc. 1999; 4: 338-345Abstract Full Text PDF PubMed Scopus (77) Google Scholar Critical to the formation of a functional hair fiber and the maintenance of the HF’s cyclical behavior is the highly regulated expression of molecular mediators that form the HF. Although most investigations have focused on the role of classical morphogens, growth factors, and cytokines in the control of HF development and cycling,3Stenn KS Paus R Controls of hair follicle cycling.Physiol Rev. 2001; 81: 449-494Crossref PubMed Scopus (1157) Google Scholar recent evidence has also implicated an important role for protease/anti-protease systems. For example, there are distinct hair cycle-dependent changes in metalloproteases and their inhibitor systems.9Kawabe TT Rea TJ Flenniken AM Williams BR Groppi VE Buhl AE Localization of TIMP in cycling mouse hair.Development. 1991; 111: 877-879PubMed Google Scholar, 10Krejci-Papa NC Paus R A novel in-situ-zymography technique localizes gelatinolytic activity in human skin to mast cells.Exp Dermatol. 1998; 7: 321-326Crossref PubMed Scopus (9) Google Scholar Also, hepatocyte growth factor stimulates HF elongation in organ culture after activation by the serine proteinase, hepatocyte growth factor activator. This elongation can be partially abrogated by the serine proteinase inhibitor, aprotinin.11Yamazaki M Tsuboi R Lee YR Ishidoh K Mitsui S Ogawa H Hair cycle-dependent expression of hepatocyte growth factor (HGF) activator, other proteinases, and proteinase inhibitors correlates with the expression of HGF in rat hair follicles.J Invest Dermatol Symp Proc. 1999; 4: 312-315Abstract Full Text PDF PubMed Scopus (29) Google Scholar, 12Lee YR Yamazaki M Mitsui S Tsuboi R Ogawa H Hepatocyte growth factor (HGF) activator expressed in hair follicles is involved in in vitro HGF-dependent hair follicle elongation.J Dermatol Sci. 2001; 25: 156-163Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar It has also been proposed that among other proteinases/protease inhibitors, gelatinase A,13Karelina TV Bannikov GA Eisen AZ Basement membrane zone remodeling during appendageal development in human fetal skin. The absence of type VII collagen is associated with gelatinase-A (MMP2) activity.J Invest Dermatol. 2000; 114: 371-375Crossref PubMed Scopus (38) Google Scholar nexin-1,14Sonoda T Asada Y Kurata S Takayasu S The mRNA for protease nexin-1 is expressed in human dermal papilla cells and its level is affected by androgen.J Invest Dermatol. 1999; 113: 308-313Crossref PubMed Scopus (30) Google Scholar and stratum corneum chymotryptic enzyme15Ekholm E Egelrud T The expression of stratum corneum chymotryptic enzyme in human anagen hair follicles: further evidence for its involvement in desquamation-like processes.Br J Dermatol. 1998; 139: 585-590Crossref PubMed Scopus (40) Google Scholar have a role in the regulation of hair growth and/or cycling. In this context, the papain-like lysosomal cysteine protease, cathepsin L (CTSL), one of the major lysosomal enzymes that can also be secreted, is of particular interest. CTSL-deficient mice (ctsl−/−) exhibit specific perturbations in both HF morphogenesis and cycling.16Nakagawa T Roth W Wong P Nelson A Farr A Deussing J Villadangos JA Ploegh H Peters C Rudensky AY Cathepsin L: critical role in Ii degradation and CD4 T cell selection in the thymus.Science. 1998; 280: 450-453Crossref PubMed Scopus (596) Google Scholar, 17Roth W Deussing J Botchkarev VA Pauly-Evers M Saftig P Hafner A Schmidt P Schmahl W Scherer J Anton-Lamprecht I Von Figura K Paus R Peters C Cathepsin L deficiency as molecular defect of furless: hyperproliferation of keratinocytes and perturbation of hair follicle cycling.FASEB J. 2000; 14: 2075-2086Crossref PubMed Scopus (284) Google Scholar These, as yet ill-characterized abnormalities, deserve further dissection because they promise novel insights into the full range of functions of CTSL. This multifunctional, ubiquitously expressed, proteinase is involved in the positive thymic selection of CD4+ T cells and the intrathymic degradation of the MHC class II invariant chain.16Nakagawa T Roth W Wong P Nelson A Farr A Deussing J Villadangos JA Ploegh H Peters C Rudensky AY Cathepsin L: critical role in Ii degradation and CD4 T cell selection in the thymus.Science. 1998; 280: 450-453Crossref PubMed Scopus (596) Google Scholar Recent reports also indicate a role for CTSL in bone resorption,18Turk B Turk D Turk V Lysosomal cysteine proteases: more than scavengers.Biochim Biophys Acta. 2000; 7: 98-111Crossref Scopus (703) Google Scholar trophoblast invasion,19Afonso S Romagnano L Babiarz B Expression of cathepsin proteinases by mouse trophoblast in vivo and in vitro.Dev Dyn. 1999; 216: 374-384Crossref PubMed Google Scholar, 20Afonso S Romagnano L Babiarz B The expression and function of cystatin C and cathepsin B and cathepsin L during mouse embryo implantation and placentation.Development. 1997; 124: 3415-3425Crossref PubMed Google Scholar tumor metastasis,21Dohchin A Suzuki JI Seki H Masutani M Shiroto H Kawakami Y Immunostained cathepsins B and L correlate with depth of invasion and different metastatic pathways in early stage gastric carcinoma.Cancer. 2000; 89: 482-487Crossref PubMed Scopus (65) Google Scholar and chronic inflammation.22Iwata Y Mort JS Tateishi H Lee ER Macrophage cathepsin L, a factor in the erosion of subchondral bone in rheumatoid arthritis.Arthritis Rheum. 1997; 40: 499-509Crossref PubMed Scopus (54) Google Scholar In vivo, the level of CTSL mRNA is related to tumor progression/metastatic potential,23Morris VL Tuck AB Wilson SM Percy D Chambers AF Tumor progression and metastasis in murine D2 hyperplastic alveolar nodule mammary tumor cell lines.Clin Exp Metastasis. 1993; 11: 103-112Crossref PubMed Scopus (67) Google Scholar and this is thought to relate to the ability of CTSL to degrade extracellular matrix and basement membranes.24Ohba T Ohba Y Moriyama K Synthesis of mRNAs for cathepsins L and K during development of the rat mandibular condylar cartilage.Cell Tissue Res. 2000; 302: 343-352Crossref PubMed Scopus (10) Google Scholar As part of its extracellular proteolytic activity, CTSL can hydrolyze azocasein, elastin, and collagen.18Turk B Turk D Turk V Lysosomal cysteine proteases: more than scavengers.Biochim Biophys Acta. 2000; 7: 98-111Crossref Scopus (703) Google Scholar, 25Felbor U Dreier L Bryant RA Ploegh HL Olsen BR Mothes W Secreted cathepsin L generates endostatin from collagen XVIII.EMBO J. 2000; 19: 1187-1194Crossref PubMed Scopus (408) Google Scholar Expression of procathepsin L has been reported in normal epidermis, eccrine sweat glands, HFs, and blood vessels.26Thewes M Engst R Jurgens M Borelli S Immunohistochemical analysis of procathepsin L and cathepsin B in cutaneous Kaposi's sarcoma.Int J Dermatol. 1997; 36: 100-103Crossref PubMed Scopus (5) Google Scholar Unlike other members of the mammalian papain family of cysteine proteases, the CTSL gene is activated by various growth factors27Ishidoh K Kominami E Gene regulation and extracellular functions of procathepsin L.Biol Chem. 1998; 379: 131-135PubMed Google Scholar, 28Lemaire R Huet G Zerimech F Grard G Fontaine C Duquesnoy B Flipo RM selective induction of the secretion of cathepsins B and L by cytokines in synovial fibroblast-like cells.Br J Rheumatol. 1997; 36: 735-743Crossref PubMed Scopus (83) Google Scholar and oncogenes.29Janulis M Silberman S Ambegaokar A Gutkind JS Schultz RM Role of mitogen-activated protein kinases and c-Jun/AP-1 trans-activating activity in the regulation of protease mRNAs and the malignant phenotype in NIH 3T3 fibroblasts.J Biol Chem. 1999; 274: 801-813Crossref PubMed Scopus (57) Google Scholar Many of these are also intimately involved in the regulation of hair growth. Notably, the pro-inflammatory cytokine interleukin-6 can up-regulate CTSL, whereas transforming growth factor-β1 suppresses CTSL expression.30Gerber A Wille A Welte T Ansorge S Buhling F Interleukin-6 and transforming growth factor-beta 1 control expression of cathepsins B and L in human lung epithelial cells.J Interferon Cytokine Res. 2001; 21: 11-19Crossref PubMed Scopus (43) Google Scholar Mice expressing the K14-interleukin-6 transgene exhibit retarded hair growth31Turksen K Kupper T Degenstein L Williams I Fuchs E Interleukin 6: insights to its function in skin by overexpression in transgenic mice.Proc Natl Acad Sci USA. 1992; 89: 5068-5072Crossref PubMed Scopus (196) Google Scholar while HF regression (catagen) is regulated in part by transforming growth factor-β1.32Foitzik K Lindner G Mueller-Roever S Maurer M Botchkareva N Botchkarev V Handjiski B Metz M Hibino T Soma T Dotto GP Paus R Control of murine hair follicle regression (catagen) by TGF-beta1 in vivo.FASEB J. 2000; 14: 752-760Crossref PubMed Scopus (280) Google Scholar The level of CTSL mRNA in certain cell types is significantly increased by basic fibroblast growth factor and nerve growth factor.33Liuzzo JP Petanceska SS Devi LA Neurotrophic factors regulate cathepsin S in macrophages and microglia: a role in the degradation of myelin basic protein and amyloid beta peptide.Mol Med. 1999; 5: 334-343PubMed Google Scholar Notably, basic fibroblast growth factor is a hair growth inhibitor in mice,34du Cros DL Fibroblast growth factor influences the development and cycling of murine hair follicles.Dev Biol. 1993; 156: 444-453Crossref PubMed Scopus (52) Google Scholar whereas nerve growth factor promotes murine HF development and the prohormone-responsive neuronal system has been implicated in the regulation of hair growth.35Botchkareva NV Botchkarev VA Albers KM Metz M Paus R Distinct roles for nerve growth factor and brain-derived neurotrophic factor in controlling the rate of hair follicle morphogenesis.J Invest Dermatol. 2000; 114: 314-320Crossref PubMed Scopus (34) Google Scholar CTSL protease activity is also increased by interleukin-1β, interleukin-6, and oncostatin M, and decreased by insulin-like growth factor-1 and growth hormone,36Damiens C Grimaud E Rousselle AV Charrier C Fortun Y Heymann D Padrines M Cysteine protease production by human osteosarcoma cells (MG63, SAOS2) and its modulation by soluble factors.Cytokine. 2000; 12: 539-542Crossref PubMed Scopus (10) Google Scholar ie, bioregulators appreciated as hair growth modulatory agents.3Stenn KS Paus R Controls of hair follicle cycling.Physiol Rev. 2001; 81: 449-494Crossref PubMed Scopus (1157) Google Scholar, 7Philpott MJ Paus R Principles of hair follicle morphogenesis.in: Chuong C-M Molecular Basis of Epithelial Appendage Morphogenesis. R.G. Landes Company, Austin1998: 75-103Google Scholar, 37Stenn KS Parimoo S Prouty S Growth of the hair follicle: a cycling and regenerating biological system.in: Chuong C-M Molecular Basis of Epithelial Appendage Morphogenesis. R. G. Landes, Austin1999: 229-236Google Scholar Furthermore, ras oncogenes (K-ras and N-ras) up-regulate CTSL activities to increase tumorigenic potential,38Kim K Cai J Shuja S Kuo T Murnane MJ Presence of activated ras correlates with increased cysteine proteinase activities in human colorectal carcinomas.Int J Cancer. 1998; 79: 324-333Crossref PubMed Scopus (40) Google Scholar whereas mice overexpressing the ras activator, E2F1, exhibit HF development disrupted via increased apoptosis.39Pierce AM Fisher SM Conti CJ Johnson DG Deregulated expression of E2F1 induces hyperplasia and cooperates with ras in skin tumor development.Oncogene. 1998; 16: 1267-1276Crossref PubMed Scopus (130) Google Scholar Of related interest is the observation that CTSL can generate the angiogenesis inhibitor endostatin, pointing to a CTSL involvement in a regulatory loop of angiogenesis.40Ferreras M Felbor U Lenhard T Olsen BR Delaisse J Generation and degradation of human endostatin proteins by various proteinases.FEBS Lett. 2000; 486: 247-251Abstract Full Text Full Text PDF PubMed Scopus (337) Google Scholar This is of trichological importance, because angiogenesis is fundamental to the HF switch from resting (telogen) to the active growth stage (anagen),41Mecklenburg L Tobin DJ Müller-Röver S Handjiski B Wendt G Peters EM Pohl S Moll I Paus R Active hair growth (anagen) is associated with angiogenesis.J Invest Dermatol. 2000; 114: 909-916Crossref PubMed Scopus (201) Google Scholar whereas HF regression (catagen) is associated with vascular regression.42Mecklenburg L Tobin DJ Paus R Hair follicle involution (catagen) is accompanied by apoptosis-driven regression of cutaneous microvasculature.J Invest Dermatol. 2000; 117: 427AGoogle Scholar In summary, there are numerous biological reasons why lack of functional CTSL may be expected to effect the HF. Indeed, CTSL knockout mice exhibit significant alterations in skin homeostasis and striking defects in hair growth abnormalities. The epidermis of ctsl−/− mice is significantly thicker than that of ctsl+/+ mice, because of increased epidermal proliferation.17Roth W Deussing J Botchkarev VA Pauly-Evers M Saftig P Hafner A Schmidt P Schmahl W Scherer J Anton-Lamprecht I Von Figura K Paus R Peters C Cathepsin L deficiency as molecular defect of furless: hyperproliferation of keratinocytes and perturbation of hair follicle cycling.FASEB J. 2000; 14: 2075-2086Crossref PubMed Scopus (284) Google Scholar Furless (fs) mice exhibit a very similar phenotype to ctsl−/− mice, because of allelism for fs and ctsl with a missense mutation (glycine to arginine substitution, G149R) which results in the loss of CTSL catalytic activity.17Roth W Deussing J Botchkarev VA Pauly-Evers M Saftig P Hafner A Schmidt P Schmahl W Scherer J Anton-Lamprecht I Von Figura K Paus R Peters C Cathepsin L deficiency as molecular defect of furless: hyperproliferation of keratinocytes and perturbation of hair follicle cycling.FASEB J. 2000; 14: 2075-2086Crossref PubMed Scopus (284) Google Scholar HF morphogenesis and the initiation of the first HF regression phase (catagen), which sets off HF cycling,3Stenn KS Paus R Controls of hair follicle cycling.Physiol Rev. 2001; 81: 449-494Crossref PubMed Scopus (1157) Google Scholar, 8Paus R Müller-Röver S Botchkarev VA Chronobiology of the hair follicle: hunting the “hair cycle clock.”.J Invest Dermatol Symp Proc. 1999; 4: 338-345Abstract Full Text PDF PubMed Scopus (77) Google Scholar are significantly delayed in ctsl−/− mice. However, null mice subsequently exhibit a much accelerated growth phase of the first genuine hair cycle (anagen).17Roth W Deussing J Botchkarev VA Pauly-Evers M Saftig P Hafner A Schmidt P Schmahl W Scherer J Anton-Lamprecht I Von Figura K Paus R Peters C Cathepsin L deficiency as molecular defect of furless: hyperproliferation of keratinocytes and perturbation of hair follicle cycling.FASEB J. 2000; 14: 2075-2086Crossref PubMed Scopus (284) Google Scholar Despite this accelerated entry into anagen, ctsl−/− mice are by then macroscopically nude, having shed all their fur during an apparently truncated and abnormal preceding catagen/telogen phase. After hair regrowth during each subsequent anagen phase, it falls out again with entry into catagen. Normally, the club hairs of telogen HFs in mice are not shed, instead being retained in the HF for several cycles until the hair shafts are shed in a separately controlled hair cycle phase (exogen).3Stenn KS Paus R Controls of hair follicle cycling.Physiol Rev. 2001; 81: 449-494Crossref PubMed Scopus (1157) Google Scholar However, hair loss and hair regrowth in later ctsl−/− hair cycles is both incomplete and spatially restricted, so that these mice always remain partially devoid of hair. The shedding of hair shafts has been ascribed to an abnormal formation of the telogen club.17Roth W Deussing J Botchkarev VA Pauly-Evers M Saftig P Hafner A Schmidt P Schmahl W Scherer J Anton-Lamprecht I Von Figura K Paus R Peters C Cathepsin L deficiency as molecular defect of furless: hyperproliferation of keratinocytes and perturbation of hair follicle cycling.FASEB J. 2000; 14: 2075-2086Crossref PubMed Scopus (284) Google Scholar This structure aids the persistent mooring of the hair shaft to its HF.3Stenn KS Paus R Controls of hair follicle cycling.Physiol Rev. 2001; 81: 449-494Crossref PubMed Scopus (1157) Google Scholar, 43Koch PJ Mahoney MG Cotsarelis G Rothenberger K Lavker RM Stanley JR Desmoglein 3 anchors telogen hair in the follicle.J Cell Sci. 1998; 111: 2529-2537PubMed Google Scholar Interestingly, regrowing hair shafts become progressively grayer in ctsl−/− mice.17Roth W Deussing J Botchkarev VA Pauly-Evers M Saftig P Hafner A Schmidt P Schmahl W Scherer J Anton-Lamprecht I Von Figura K Paus R Peters C Cathepsin L deficiency as molecular defect of furless: hyperproliferation of keratinocytes and perturbation of hair follicle cycling.FASEB J. 2000; 14: 2075-2086Crossref PubMed Scopus (284) Google Scholar The current morphological and cytokinetic study was conducted to examine more closely the nature of the defects underlying this intriguing phenotype using light microscopic and ultrastructural techniques. This should facilitate dissecting the functional roles of CTSL in HF biology in particular and epithelial and pigment cell biology in general. The specific questions addressed by this study were how CTSL deficiency affects postnatal HF development and cycling and what components of the developing and cycling HF are primarily targeted. Given that increased epidermal proliferation is a feature of this knockout,17Roth W Deussing J Botchkarev VA Pauly-Evers M Saftig P Hafner A Schmidt P Schmahl W Scherer J Anton-Lamprecht I Von Figura K Paus R Peters C Cathepsin L deficiency as molecular defect of furless: hyperproliferation of keratinocytes and perturbation of hair follicle cycling.FASEB J. 2000; 14: 2075-2086Crossref PubMed Scopus (284) Google Scholar the current study investigated proliferation, apoptosis, and terminal differentiation during HF development and cycling. Such events are critically dependent on spatiotemporally, stringently restricted cell proliferation and death.3Stenn KS Paus R Controls of hair follicle cycling.Physiol Rev. 2001; 81: 449-494Crossref PubMed Scopus (1157) Google Scholar, 44Magerl M Tobin DJ Müller-Röver S Hagen E Lindner G McKay IA Paus R Patterns of proliferation and apoptosis during murine hair follicle morphogenesis.J Invest Dermatol. 2001; 116: 947-955Crossref PubMed Google Scholar, 45Lindner G Botchkarev VA Botchkareva NV Ling G van der Veen C Paus R Analysis of apoptosis during hair follicle regression (catagen).Am J Pathol. 1997; 151: 1601-1617PubMed Google Scholar We examined the involvement of CTSL in HF cycling from the first regression phase (catagen), formation of the telogen resting HF, hair shaft shedding or exogen,37Stenn KS Parimoo S Prouty S Growth of the hair follicle: a cycling and regenerating biological system.in: Chuong C-M Molecular Basis of Epithelial Appendage Morphogenesis. R. G. Landes, Austin1999: 229-236Google Scholar and the subsequent HF regeneration during anagen. The possible involvement of the inner root sheath (IRS) structural protein, trichohyalin, a potential substrate for CTSL, was assessed immunohistochemically by protein expression in ctsl−/− and ctsl+/+ HFs. Finally, the morphological basis for the observed canities (ie, hair graying) in ctsl−/− mice17Roth W Deussing J Botchkarev VA Pauly-Evers M Saftig P Hafner A Schmidt P Schmahl W Scherer J Anton-Lamprecht I Von Figura K Paus R Peters C Cathepsin L deficiency as molecular defect of furless: hyperproliferation of keratinocytes and perturbation of hair follicle cycling.FASEB J. 2000; 14: 2075-2086Crossref PubMed Scopus (284) Google Scholar was examined, as lysosome function (including their biogenic derivatives such as melanosomes,46Orlow SJ Melanosomes are specialized members of the lysosomal lineage of organelles.J Invest Dermatol. 1995; 105: 3-7Crossref PubMed Scopus (239) Google Scholar) is likely to be affected by the absence of this lysosomal enzyme. Mice lacking CTSL expression (ctsl−/−) were generated by insertion of a G418 resistance cassette in exon 3 of CTSL by homologous recombination in embryonic stem cells.16Nakagawa T Roth W Wong P Nelson A Farr A Deussing J Villadangos JA Ploegh H Peters C Rudensky AY Cathepsin L: critical role in Ii degradation and CD4 T cell selection in the thymus.Science. 1998; 280: 450-453Crossref PubMed Scopus (596) Google Scholar, 17Roth W Deussing J Botchkarev VA Pauly-Evers M Saftig P Hafner A Schmidt P Schmahl W Scherer J Anton-Lamprecht I Von Figura K Paus R Peters C Cathepsin L deficiency as molecular defect of furless: hyperproliferation of keratinocytes and perturbation of hair follicle cycling.FASEB J. 2000; 14: 2075-2086Crossref PubMed Scopus (284) Google Scholar Expression of CTSL mRNA, protein, or CTSL activity was completely abolished in ctsl−/− mice.17Roth W Deussing J Botchkarev VA Pauly-Evers M Saftig P Hafner A Schmidt P Schmahl W Scherer J Anton-Lamprecht I Von Figura K Paus R Peters C Cathepsin L deficiency as molecular defect of furless: hyperproliferation of keratinocytes and perturbation of hair follicle cycling.FASEB J. 2000; 14: 2075-2086Crossref PubMed Scopus (284) Google Scholar The mice were housed in a controlled environment with a temperature of 21 ± 1°C and a 12-hour day/night cycle. Mice were kept in macrolon cages and had access to standard food pellets and tap water ad libitum. The microbiological status of the animal facility was checked according to our institutional guidelines. Mice were essentially pathogen-free. Most notably, ectoparasites and dermatophytes were not detectable. For the present study, heterozygous parent mice (ctsl+/−) with hybrid background C57BL/6 × 129 were bred to obtain ctsl−/− and ctsl+/+ littermates. For age determination of the experimental animals, females because of delivery were checked at least three times daily by the authors and by professional animal caretakers. Newborns with obscure time points of birth were omitted from the experiments. Representative tissue samples were harvested from dorsum skin only of ctsl−/− and wild-type (ctsl+/+) mice at various stages during hair morphogenesis.47Müller-Röver S Handjiski B van der Veen C Eichmuller S Foitzik K McKay IA Stenn KS Paus R A comprehensive guide for the accurate classification of murine hair follicles in distinct hair cycle stages.J Invest Dermatol. 2001; 117: 3-15Crossref PubMed Google Scholar These included; day of birth (P0), day 2 (P2), day 6 (P6), day 14 (P14), day 17 (P17), day 20 (P20), and day 28 (P28) postpartum. Per genotype (ctsl−/−, ctsl+/+), two to three mice, each from different litters, were analyzed from each age group. Because reliable sex determination in very young mice is difficult in practice, sex was not determined in newborn, day 2 (P2), or day 6 (P6) mice. For older mice each group contained a mixture of males and females. Again, in the various ctsl−/− strains that we have obtained so far, no differences of hair cycle kinetics between males and females have been observed. There is no evidence to suggest any sex difference is present at least until P25 of age. Tissues were immediately fixed in half-strength Karnovsky’s fixative,48Karnovsky MJ A formaldehyde-glutaraldehyde fixative of high osmolarity for use in electron microscopy.J Cell Biol. 1965; 27: 137a-138aGoogle Scholar postfixed in 2% osmium tetroxide and uranyl acetate, and embedded in resin as previously described.49Tobin DJ Fenton DA Kendall MD Ult
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