Apoptosis as the mechanism of the involution of hair follicles in catagen transformation

Electron microscopy was performed on mouse skin to study the mechanism of the spontaneous involution of hair follicles during catagen. The reduction in follicles size appears to result from cell deletion by apoptosis, a distinct type of cell death with importance in tissue kinetics. These ultrastructural changes have been misinterpreted in the past as autophagic vacuole formation because of the prominent phagocytosis of the apoptotic fragments by adjacent, surviving cells of the hair follicle.     

Cell degeneration in alopecia areata. An ultrastructural study

The morphology and prevalence of different forms of cell degeneration in hair follicles in acute alopecia areata were investigated. In addition to apoptosis and necrosis, a third morphological pattern of cell degeneration, dark-cell transformation, was evident. Fifteen patients with untreated acute alopecia areata and three normal adults without hair loss were studied. Electron microscopy revealed that although apoptosis of outer root sheath keratinocytes produces normal hair follicle involution (catagen), increased levels of apoptosis, necrosis, and dark-cell formation appear to be related to the pathology of alopecia areata. Although cell degeneration was generally restricted to keratinocytes of the lower follicle, melanocytes, Langerhans' and dermal papilla cells were also affected. Keratinocytic degeneration may affect layers of matrix cells in alopecia areata, unlike the apoptosis of scattered outer root sheath cells in normal catagen. The extent of cell death suggests a pathological rather than a physiological event in alopecia areata.     

Apoptosis in murine hair follicles during catagen regression

Catagen hair follicle involution has been reported to involve apoptosis, although the precise mechanism has not been satisfactorily resolved. Previous studies have involved solely morphological or electron microscopical methods. We report here studies on murine hair follicles during the first postnatal hair cycle conducted using the terminal deoxy-nucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) method. Electrophoresis of DNA isolated from the hair follicles of the same animals was carried out in order to confirm the systematic fragmentation of DNA that typifies apoptosis. On day 10, when all the follicles were growing, there was no evidence of staining with TUNEL in the hair bulbs. Electrophoresis similarly did not show characteristic DNA ladders. By day 15, a few positive cells were observed in the hair bulbs and the numbers had increased by day 17 when many positive cells were seen, especially in the lower portions of the follicles. Electrophoresis demonstrated DNA ladders on days 15, 16 and 17, although the DNA ladder on day 15 was less prominent than that on day 17. These studies confirmed that apoptosis, as identified by techniques that measure DNA fragmentation, occurs in the lower regions of hair follicles towards the end of catagen. Received: 19 March 1997     

Morphological analysis of in vitro human hair growth

The histological and ultrastructural aspect of normal human hair follicles maintained ex vivo for 12 days was evaluated. Anagen hair follicles, dissected free of contaminating connective tissue, were maintained for up to 12 days in a serum-free medium. Macroscopic observations revealed continued viability for 12 days, at which time some follicles involuted in a manner morphologically similar to catagen. Increased growth of maintained follicles was measured from the abrupt ending of the connective tissue sheath (CTS), as no increase in this component was observed from initiation of culture. In general follicles maintained up to 8 days exhibited little divergence from normal in vivo morphologies including the persistence of functional hair bulb melanocytes — a marker of anagen. After this time melanin granules were present in dermal papilla cells, as occurs during impending involution in vivo. Heterotypic cell contact occurred in the middle to upper follicle between outer root sheath (ORS) keratinocytes and disorganized CTS. Herniation of some ORS cells away from the follicle and the occurrence of loose desmosomal junctions between ORS keratinocytes reflected loss of normal follicular cell interactions in upper follicles maintained after 8 days. Continued follicle growth correlated with the presence of mitotic matrix keratinocytes even at 12 days. After 12 days in culture most follicles involuted displaying apoptotic-like keratinocytes and hair bulb melanocytes and the presence of highly keratinized hair club structures. While most follicles exhibited this orderly sequence of events, a few follicles involuted after 24 h with synchronous degeneration of all cells. Two follicles exhibited upregulated cortical cell differentiation at the level of the dermal papilla (DP). Normal cell cytoplasmic constituents were replaced with ribosomes and keratin bundles. This study reveals for the first time the histology and ultrastructure of normal hair follicles in culture for up to 12 days in serum-free medium. Although involuted hair follicles may exhibit some features of catagen, it is possible that the mechanisms involved are entirely different.This study was supported in part by a grant to D. J. Tobin from Upjohn (UK)     

Apoptosis in the hair follicle

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.     

何首乌、女贞子等中药煎剂对体外培养的猪毛囊毛发生长的影响

目的 探讨何首乌、女贞子等中药混合煎剂对体外培养的猪毛囊毛发生长的影响。方法 将离体培养的猪毛囊分为对照组(Williams E培养基)和中药组(Williams E培养基+中药煎剂),显微镜下观察各组毛囊毛发生长和毛球部形态变化,并用末端脱氧核苷酸转移酶介导dUTP缺口末端标记法(TUNEL)检测各组毛囊中的凋亡细胞。结果 培养第7天,对照组毛囊毛发生长长度低于中药组(P<0.001);对照组毛囊TUNEL阳性细胞数明显高于中药组(P<0.001);毛球形态观察,对照组毛囊出现退行性变化,而中药组毛囊仍维持生长期毛囊形态。结论 何首乌、女贞子等中药煎剂能在一定程度上抑制猪毛囊细胞内凋亡,延缓生长期毛囊进入退行期。     

Syndecan-1 is strongly expressed in the anagen hair follicle outer root sheath and in the dermal papilla but expression diminishes with involution of the hair follicle

Syndecan-1 is the prototypic member of a family of heparan sulfate-bearing cell surface proteoglycans that function in adhesion, cell-extracellular matrix interactions, migration, and proliferation. During embryogenesis, syndecan-1 expression in the epithelium is downregulated when the epithelium gives rise to motile mesenchymal cells, whereas mesenchymal syndecan-1 expression is upregulated during organ formation. In aggressive basal cell carcinomas, syndecan-1 expression is evident in the stroma. Some neoplastic cells induce stroma to meet needs for growth, and it may be the mesenchymal cells that produce and shed syndecan-1 into the stroma. The physiologic mechanism by which the hair follicle undergoes its cyclic process of involution and formation of a new active hair follicle is not well understood. Sixty scalp biopsies and a large scalp resection were evaluated for syndecan-1 expression within hair follicles in the growing (anagen), involuting (catagen), and resting (telogen) phases. Strong syndecan-1 immunoreactivity was evident in the outer root sheath (ORS) of the anagen hair follicle, but this expression diminished in intensity with the involution and resting stages in the hair follicle cycle. The diminution of syndecan-1 immunoreactivity in the ORS of involuting and resting hair follicles may be a result of terminal keratinocyte differentiation. Syndecan-1 was also present in the dermal papilla of the anagen hair follicle, where it may promote growth factor-mediated cell signaling that induces and maintains growth of the hair shaft and the inner root sheath.     

Transforming growth factor-beta receptor II is preferentially expressed in the companion layer of the human anagen hair follicle

BACKGROUND: Transforming growth factor (TGF)-beta is a multifunctional growth factor with multiple roles in skin including hair follicle development and cycling, where it regulates cell proliferation, differentiation and apoptosis, as well as in wound healing. While TGF-beta receptor I (TGF-beta RI) and receptor II (TGF-beta RII) expression helps define early human hair follicle morphogenesis, expression in the adult human hair follicle remains to be established. OBJECTIVES: To assess TGF-beta receptor expression in human scalp anagen hair follicles. METHODS: Immunohistochemical and double immunofluorescence analysis of TGF-beta RI and RII was conducted on frozen sections of haired human scalp obtained from 10 healthy individuals. RESULTS: TGF-beta RI expression was detected in the outer root sheath of anagen hair follicles while TGF-beta RII was expressed almost exclusively in the companion layer of inner root sheath and less so in premedulla keratinocytes. Both receptors were colocalized in the companion layer of the proximal and mid follicle. CONCLUSIONS: The well-described role of TGF-beta in keratinocyte apoptosis during catagen is likely to involve anagen-specific hair follicle components including the companion layer, as this layer provides the slippage plane supporting the inner root sheath and hair shaft as they ascend to the skin surface. Results of this study suggest that the colocalization of TGF-beta RI/RII complexes at the companion layer would facilitate TGF-beta signalling at this site to regulate apoptosis of the companion layer keratinocytes, facilitating shrinkage/contraction of this cell layer during hair follicle regression/catagen.     

Insulin-like growth factor 1 and hair growth

Insulin-like growth factor 1 (IGF-1) has been identified as an important growth factor in many biological systems.[1] It shares considerable structural homology with insulin and exerts insulin-like effects on food intake and glucose metabolism. Recently it has been suggested to play a role in regulating cellular proliferation and migration during the development of hair follicles. [2,3] To exert its biological effects, the IGF-1 is required to activate cells by binding to specific cell-surface receptors. The type I IGF receptor (IGF-1R) is the only IGF receptor to have IGF-mediated signaling functions.[1] In circulation, this growth factor mediates endocrine action of growth hormone (GH) on somatic growth and is bound to specific binding proteins (BPs). The latter control IGF transport, efflux from vascular compartments and association with cell surface receptors.[4] In tissues, IGF-1 is produced by mesenchymal type cells and acts in a paracrine and autocrine fashion by binding to the IGF-1R. This binding activates the receptor tyrosine kinase (RTK) that triggers the downstream responses and finally stimulates cell division.[5] IGF-1 may therefore be able to stimulate the proliferation of hair follicle cells through cellular signaling pathways of its receptors. Local infusion of IGF-1 into sheep has been reported to be capable of stimulating protein synthesis in the skin.[6] It may also increase the production of wool keratin. Recently, transgenic mice overexpressing IGF-1 in the skin have been shown to have earlier hair follicle development than controls.[7] In addition, this growth factor plays an important role in many cell types as a survival factor to prevent cell death.[8] This anti-apoptotic function of IGF-1 may be important to the development of follicle cells as follicles undergo a growth cycle where the regressive, catagen phase is apoptosis driven. In this review, the effects of IGF-1 on follicle cell proliferation and differentiation are discussed. In particular, the paracrine versus endocrine action of IGF-1 on hair growth and the targeting of expression of the growth factor to the follicles of transgenic animals will be emphasized. The anti-apoptotic role of IGF-1 in hair follicles is also reviewed. Prospects for future studies on hair and fiber growth by IGF-1 are discussed.     

Identification of differentially expressed genes during a wool follicle growth cycle induced by prolactin

The wool follicles of New Zealand Wiltshire sheep can be induced to undergo growth cycles by manipulating circulating prolactin levels. Altered patterns of gene expression through this cycle were examined using differential display, and nine sequence tags for differentially expressed genes were isolated. Four of these tags were identified as fragments of known genes, encoding a wool keratin, KRTAP3.2, a desmosome component, desmoglein 1, an epithelial cell marker, stratifin, and a protein kinase, Clk3. All four genes were shown to be downregulated in telogen skin compared with anagen. In situ hybridization showed that all had localization patterns which included cells that are absent in telogen. The stratifin tag was used to clone a cDNA that incorporated a complete open-reading frame for ovine stratifin. Ovine stratifin is similar to the human form, showing only six single residue differences in the predicted amino acid sequence. Stratifin probably acts as a regulator of other proteins involved in trichocyte cell cycling and differentiation. Clk3 is involved in regulating RNA splicing. KRTAP3.2 and Dsg1 both play structural roles in hair follicles. The other five tags, including two representing genes that were upregulated during catagen, could not be identified by homology. Differential display is an effective means of identifying genes involved in follicle function and, potentially, of genes controlling the growth cycle.     

Interleukin-4 induces apoptosis in cultured human follicular keratinocytes,but not in dermal papilla cells

The importance of apoptosis in hair follicle cycling is still not clearly understood, however, its regulation in follicular keratinocytes (FK) during bulb regression (catagen) may be essential for hair regrowth. So far, the control of FK apoptosis remains unknown. In this study, the anti-inflammatory cytokine IL-4 was found to induce apoptosis dose and time dependently in cultured human FK, in contrast to other agents known to inhibit hair growth such as IL-1alpha, IL-1beta, TNFalpha and TGFbeta, as shown by DNA fragmentation. On the other hand, cytokines reported to be involved in hair follicle cycling including IL-4 were not able to induce apoptosis in dermal papilla cells (DPC), in contrast to staurosporine. This PKC inhibitor revealed dose-dependent apoptotic signals not only for DPC but also for FK in vitro. In further experiments the expression of apoptosis regulating proteins, possibly involved in catagen formation, was analyzed in FK and DPC. However, no striking difference in RNA expression was seen in either cell population under culture conditions and after incubation with IL-4. We conclude, therefore, that IL-4 mediated apoptosis may participate in regulating catagen formation in the hair follicle, acting selectively on cultured FK and being independent of bcl-2 and bax expression.     

Evaluation of radiation-induced hair follicle apoptosis in mice and the preventive effects of fibroblast growth factor-1

Abstract:  Radiation-induced hair loss is a clinically important, but under-researched topic. The aim of the study was to develop an in vivo assay system for radiation-induced apoptosis in hair follicles to promote hair research and exploit new radioprotectors. BALB/c mice received total body irradiation (TBI) with γ-rays at doses in the range from 8 to 16 Gy at 6 days after depilation. Pathological changes were detected progressively in the hair follicles over the time course after TBI and the dystrophy was evaluated on the basis of stage-specific parameters reported previously, which were found to be well-suited for classification of the radiation-induced hair follicle dystrophy. As a result, regression from anagen to catagen was determined in these follicles after irradiation. In addition, radiation-induced apoptosis was a good early dystrophic parameter. In this system, it was found that fibroblast growth factor-1 effectively prevented hair follicle apoptosis in mice.     

双氢睾酮对人毛囊生长影响的初步研究

目的:探讨不同浓度双氢睾酮(DHT)对体外培养人毛囊生长的影响。方法:将分离的人毛囊培养于浓度为10~(-5)mol/L、10~(-6)mol/L、10~(-7)mol/L、10~(-8)mol/L、10~(-9)mol/L的DHT中,设置空白对照组,每日测量毛囊生长长度;运用免疫荧光技术评估各组毛囊毛母质细胞增殖程度。结果:不同浓度DHT对体外培养的毛囊生长产生不同影响:DHT培养浓度为10~(-5)mol/L、10~(-6)mol/L时抑制毛囊生长(正常对照组与DHT 10~(-5)mol/L、10~(-6)mol/L实验组毛囊每日平均生长长度相比,P值均0.05),毛母质细胞增殖活性减弱,毛囊较早进入退行期;DHT培养浓度为10~(-7)mol/L时促进毛囊生长(正常对照组与DHT10~(-7)mol/L实验组毛囊每日平均生长长度相比,P0.05),毛母质细胞增殖活性强,毛囊生长期(anagen)延长;DHT培养浓度为10~(-8)mol/L、10~(-9)mol/L时对毛囊生长的作用与对照组相比无明显差异(正常对照组与DHT 10~(-8)mol/L、10~(-9)mol/L实验组毛囊每日平均生长长度相比,P值均0.05)。结论:不同浓度DHT对体外培养毛囊生长的影响不同。     

Osteopontin gene is expressed in the dermal papilla of pelage follicles in a hair-cycle-dependent manner

Hair follicle formation and maintenance involve intimate interactions between follicular epithelial cells and a group of specialized mesenchymal cells known as the dermal papilla. Using the random primer polymerase chain reaction, we have identified an approximately 1.4 kb osteopontin mRNA that is present in large quantities in cultured rat vibrissa dermal papilla cells but undetectable in cultured rat skin fibroblasts. In situ hybridization showed that the osteopontin gene is expressed in dermal papilla cells of pelage follicles during catagen but not in anagen or telogen. As an acidic glycosylated RGD-containing extracellular matrix protein, osteopontin can function both as a cell attachment protein and as a soluble cytokine playing roles in signaling, cell migration, tissue survival, anti-inflammation, and T-cell-mediated cellular immunity. Our results indicate that the comparison of the mRNA of cultured dermal papilla cells and fibroblasts can lead to the identification of not only anagen-specific genes (e.g., nexin 1), but also a catagen-specific gene. We have thus provided evidence that specific genes are turned on during catagen, which is therefore not simply a passive "degenerative" phase. The functional role of osteopontin in catagen is unclear but it may promote the formation of a tightly aggregated dermal papilla, and/or protect the dermal papilla cells from apoptosis induced by cytokines or hypoxia during catagen.     

Defolliculated (dfl): a dominant mouse mutation leading to poor sebaceous gland differentiation and total elimination of pelage follicles

Defolliculated is a novel spontaneous mouse mutation that maps to chromosome 11 close to the type I keratin locus. Histology shows abnormal differentiation of the sebaceous gland, with the sebocytes producing little or no sebum and undergoing abnormal cornification. The hair follicles fail to regress during catagen leading to abnormally long follicles. In contrast the hair shafts are shorter than normal, suggesting altered differentiation or proliferation of matrix cells during anagen. The shafts emerge from the follicle with cornified material still attached. The dermis contains increased numbers of immune cells, including T cells (CD4-positive), macrophages, and mast cells, at all time points examined. Complete elimination of all pelage and tail follicles occurs after two to three hair cycles, apparently by necrosis. Defolliculated may be a useful model for determining further functions of the sebaceous gland, and for understanding the regulation of catagen and hair follicle immunology.     

Proteoglycan expression patterns in human hair follicle

BACKGROUND: Proteoglycans (PGs) are known to play key roles in many cellular signalling pathways involved in hair follicle biology. Although some PG core proteins have previously been described in adult human hair follicles, their glycosaminoglycan (GAG) moieties have been less studied. OBJECTIVES: To add knowledge about PG core protein and GAG distributions in human anagen hair follicle and, for selected follicles, during catagen. METHODS: We used immunohistochemistry and immunohistofluorescence to revisit the expression pattern of GAG chains and core proteins in human hair follicle. The studied epitopes included CD44v3, syndecan-1, perlecan, versican, aggrecan, biglycan, heparan sulphate (HS), chondroitin sulphate (CS), dermatan sulphate (DS) and keratan sulphate (KS). RESULTS: The membrane PGs syndecan-1 and CD44v3 were respectively detected in the epithelial part of whole hair and in the outer root sheath basal layer. The dermal part of the hair follicle contained high amounts of extracellular PGs such as perlecan, versican, aggrecan, biglycan and their saccharidic moieties, namely HS, CS, DS and KS. We also observed a variable distribution of these components along the hair follicle. Especially, we noted a PG impoverishment at the very bottom of the anagen bulb. Moreover, while type D chondroitin expression remained unaffected, 4C3-CS and PG4-CS/DS epitopes respectively decreased in the dermal papilla and the connective tissue sheath, at the onset of catagen. CONCLUSIONS: GAG and PG expression along the human anagen hair follicle was characterized by (i) discontinuities mainly affecting the basement membrane and (ii) disappearance of some epitopes at catagen onset. These results are discussed in term of functionalities in nutrient diffusion, cell proliferation and differentiation, and hair protection.     

Immunohistochemical analysis of tissue remodelling during the anagen—catagen transition of the human hair follicle

The transition from the growth phase (anagen) to the involution phase (catagen) involves profound morphological changes in the human hair follicle. Club hair and epithelial column formation, for example, are key features of the catagen phase, which result in the disruption of physical interaction between the bulb and the dermal papilla. However, the dynamics and tissue remodelling that occur during this involution process remain largely unknown. Using monoclonal antibodies directed against K14 keratin, trichohyalin, transglutaminase I, desmoglein and Ki67 antigen, we followed the movements of each of the main hair follicle compartments during the onset of catagen. Our results indicate that the inner root sheath is an early target in this process, suggesting a key role for this compartment in the maintenance of hair follicle homeostasis.     

CD10 and CD34 in fetal and adult human hair follicles: dynamic changes in their immunohistochemical expression during embryogenesis and hair cycling

Background  CD10 and CD34 have been detected in both epithelial and mesenchymal components of anagen human hair follicles.
Objectives  To analyse the expression of CD10 and CD34 in human hair follicle development as well as in different phases of the hair cycle.
Methods  Fetal and adult hair follicles at different stages of the hair cycle were examined by immunohistochemistry for CD10 and CD34.
Results  In fetal follicles, CD10 is expressed by the cells of the placodes, and CD34 by the mesenchymal cells of the dermal condensate. As the follicle matures, CD10 can be seen in the matrix cells, inner root sheath and dermal sheath. In adult follicles, the expression of CD10 in the follicular epithelium is present in anagen follicles, but tends to disappear in catagen, and is not detected in telogen. The CD10 positivity of the dermal sheath is more intense in catagen than in anagen follicles. CD34 immunostaining of the external root sheath was seen in adult anagen follicles but not in fetal follicles. This staining of the anagen outer sheath tends to disappear in catagen and is not detected in telogen.
Conclusions  CD10 and CD34 are not proteins constantly present in a specific cell type of the hair follicle, but are proteins that can be expressed by both epithelial and mesenchymal cells depending on the stage of development and hair cycle. The distribution of the immunoreactivity to CD10 in the placode and CD34 in the dermal condensate suggests a role of these proteins in initial stages of hair formation.     

A possible role for Langerhans cells in the removal of melanin from early catagen hair follicles

Hair pigmentation is coupled to the hair follicle growth cycle. A common feature of catagen is the translocation of melanin from the matrix to the dermal papilla of the hair follicle. However, the mechanism whereby this pigment, not incorporated into the hair shaft, is removed from the hair bulb during early catagen is poorly understood. Routine ultrastructural examination of four normal scalp specimens revealed a rare hair follicle in early catagen. Close study of the hair bulb of this catagen follicle revealed a Langerhans cell in the process of transferring pigment from the matrix to the dermal papilla. This cell also contained numerous characteristic Langerhans granules (LG) (also known as Birbeck granules). Interestingly, these granules were intimately associated with melanosomes: so intimate, in fact, that melanosomes appeared to have been endocytosed by LG. This unique demonstration of removal of hair follicle melanin by Langerhans cells during early catagen and of pigment uptake by Langerhans cells by endocytosis into LG, suggests one way by which 'unused' pigment can be removed from the hair follicle during catagen.