The cDNA-deduced amino acid sequence for trichohyalin, a differentiation marker in the hair follicle, contains a 23 amino acid repeat

Trichohyalin is a highly expressed protein within the inner root sheath of hair follicles and is similar, or identical, to a protein present in the hair medulla. In situ hybridization studies have shown that trichohyalin is a very early differentiation marker in both tissues and that in each case the trichohyalin mRNA is expressed from the same single copy gene. A partial cDNA clone for sheep trichohyalin has been isolated and represents approximately 40% of the full-length trichohyalin mRNA. The carboxy-terminal 458 amino acids of trichohyalin are encoded, and the first 429 amino acids consist of full- or partial-length tandem repeats of a 23 amino acid sequence. These repeats are characterized by a high proportion of charged amino acids. Secondary structure analyses predict that the majority of the encoded protein could form alpha-helical structures that might form filamentous aggregates of intermediate filament dimensions, even though the heptad motif obligatory for the intermediate filament structure itself is absent. The alternative structural role of trichohyalin could be as an intermediate filament-associated protein, as proposed from other evidence.     

The origin of citrulline-containing proteins in the hair follicle and the chemical nature of trichohyalin, an intracellular precursor.

The present studies have demonstrated that the medulla and inner root sheath cells develop within their cytoplasm a protein that is unique in composition and is present in the trichohyalin granules. The protein is rich in arginine residues, some of which undergo a side-chain conversion in situ into citrulline residues. An unusual Ca2+-dependent enzyme activity distinguishable from cross-linking transamidase has been detected in the hair follicle and will act in vitro on trichohyalin protein as the natural substrate. The conversion in vivo must occur during the time that the medullary and inner root sheath cells move up the follicle and their cytoplasm fills with cross-linked protein containing citrulline. The function of citrulline in these proteins is not understood but its formation is a major process during hair growth.     

Fine structure of marsupial hairs, with emphasis on trichohyalin and the structure of the inner root sheath

The fine structure and cornification of marsupial hairs are unknown. The distribution of keratins, trichohyalin, and transglutaminase in marsupial hairs was studied here for the first time by electron microscopy and immunocytochemistry. The localization of acidic and basic keratins in marsupial hairs is similar to that of hairs in placental mammals, and the keratins are mainly localized in the outer root sheath and surrounding epidermis. Marsupial trichohyalin in both medulla and inner root sheath (IRS) cross-reacts with a trichohyalin antibody that recognizes trichohyalin across placental species, indicating a common epitope(s) among mammalian trichohyalin. Roundish to irregular trichohyalin granules are composed of a network of immunolabeled 10-15-nm-thick coarse filaments within an amorphous matrix in which a weak labeling for transglutaminases is present. This suggests that the enzyme, and its substrate trichohyalin, are associated in mature granules. Transglutaminase labeling mainly occurs in condensing chromatin of mature cells of the outer and inner root sheaths, suggesting formation of the nuclear envelope connected with terminal differentiation of these cells. In mature Huxley or Henle layers the filaments lose the immunolabeling for trichohyalin when they are reoriented into parallel rows linked by short bridges, thus suggesting that the filaments with their reactive epitopes are chemically modified during cornification, as seen in the IRS of hairs of placental mammals. The Huxley layer probably acts as a cushion, absorbing the tensions connected with the distalward movement of the growing hair fiber. Variations in stratification of the Huxley layer are probably related to the diameter of the hair shaft. The cytoplasmic and junctional connections between cells of the Huxley layer and the companion layer and the outer root sheath enhance the grip of the IRS and hair fiber within the follicle. The role of cells of the IRS in sculpturing the fiber cuticle and in the mechanism of shedding that allows the exit of hair on the epidermal surface in mammals are discussed.     

Trichohyalin mechanically strengthens the hair follicle: multiple cross-bridging roles in the inner root shealth

Trichohyalin is expressed in specialized epithelia that are unusually mechanically strong, such as the inner root sheath cells of the hair follicle. We have previously shown that trichohyalin is sequentially subjected to post-synthetic modifications by peptidylarginine deaminases, which convert many of its arginines to citrullines, and by transglutaminases, which introduce intra- and interprotein chain cross-links. Here we have characterized in detail the proteins to which it becomes cross-linked in vivo in the inner root sheath of the mouse hair follicle. We suggest that it has three principal roles. First, it serves as an interfilamentous matrix protein by becoming cross-linked both to itself and to the head and tail end domains of the inner root sheath keratin intermediate filament chains. A new antibody reveals that arginines of the tail domains of the keratins are modified to citrullines before cross-linking, which clarifies previous studies. Second, trichohyalin serves as a cross-bridging reinforcement protein of the cornified cell envelope of the inner root sheath cells by becoming cross-linked to several known or novel barrier proteins, including involucrin, small proline-rich proteins, repetin, and epiplakin. Third, it coordinates linkage between the keratin filaments and cell envelope to form a seamless continuum. Together, our new data document that trichohyalin is a multi-functional cross-bridging protein that functions in the inner root sheath and perhaps in other specialized epithelial tissues by conferring to and coordinating mechanical strength between their peripheral cell envelope barrier structures and their cytoplasmic keratin filament networks.     

Fine structure and immunocytochemistry of monotreme hairs, with emphasis on the inner root sheath and trichohyalin-based cornification during hair evolution

The fine structure of hairs in the most ancient extant mammals, the monotremes, is not known. The present study analyzes the ultrastructure and immunocytochemistry for keratins, trichohyalin, and transglutaminase in monotreme hairs and compares their distribution with that present in hairs of the other mammals. The overall ultrastructure of the hair and the distribution of keratins is similar to that of marsupial and placental hairs. Acidic and basic keratins mostly localize in the outer root sheath. The inner root sheath (IRS) comprises 4-8 cell layers in most hairs and forms a tile-like sheath around the hair shaft. No cytological distinction between the Henle and Huxley layers is seen as cells become cornified about at the same time. Externally to the last cornified IRS cells (homologous to the Henle layer), the companion layer contains numerous bundles of keratin. Occasionally, some granules in the companion layer show immunoreactivity for the trichohyalin antibody. This further suggests that the IRS in monotremes is ill-defined, as the companion layer of placental hairs studied so far does not express trichohyalin. A cross-reactivity with an antibody against sheep trichohyalin is present in the IRS of monotremes, suggesting conserved epitopes across mammalian trichohyalin. Trichohyalin granules in the IRS consist of a framework of immunolabeled coarse filaments of 10-12 nm. The latter assume a parallel orientation and lose the immunoreactivity in fully cornified cells. Transglutaminase immunolabeling is diffuse among trichohyalin granules and among the parallel 10-12 nm filaments of maturing inner root cells. Transglutaminase is present where its substrate, trichohyalin, is modified as matrix protein. Cornification of IRS is different from that of hair fiber cuticle and from that of the cornified layer of the epidermis above the follicle. The different consistency among cuticle, IRS, and corneous layer of the epidermis determines separation between hair fiber, IRS, and epidermis. This allows the hair to exit on the epidermal surface after shedding from the IRS and epidermis. Based on comparative studies of reptilian and mammalian skin, a speculative hypothesis on the evolution of the IRS and hairs from the skin of synapsid reptiles is presented.     

Involucrin, but not filaggrin and Kdap mRNA, expression is downregulated in 3-D cultures of intact rat hair bulbs after calcium stimulation

The hair follicle consists of several distinctive epidermal cell layers. The hair root, which undergoes keratinization, is surrounded by two sheaths: the inner root sheath (IRS) and the outer root sheath (ORS). The ORS is continuous with the basal layer of the epidermis. Its cells do not keratinize in situ, unlike IRS. We have previously demonstrated that keratinization of the ORS was prevented by contact with the IRS in hair follicle mid-segments (i.e. fragments dissected from skin at the level above the hair bulb and below the opening of the sebaceous gland duct) cultured on agarose layer. The purpose of this study was to determine whether the same applies to the hair bulb. After isolation, intact bulbs or hair bulb-derived cells were incubated in suspension in a low or high calcium medium. The level of mRNA for differentiation markers: involucrin, filaggrin, keratinocyte differentiation associated protein and trichohyalin, was studied by RealTime PCR. We observed increased Ca(2+) upregulated expression of involucrin, filaggrin, trichohyalin and Kdap in cultures of bulb-derived cells, but in hair bulbs downregulation of involucrin and trichohyalin was observed. We concluded that the inner root sheath exerts an inhibitory effect on the expression of involucrin and trichohyalin already in the hair bulbs. The observation that downregulation of involucrin expression under Ca(2+) influence occurs both in hair bulb and midsegments could simplify future experiments, since their separation does not seem to be necessary.     

Trichohyalin, an intermediate filament-associated protein of the hair follicle

A precursor protein associated with the formation of the citrulline-containing intermediate filaments of the hair follicle has been isolated and characterized. The protein, with a molecular weight of 190,000, was isolated from sheep wool follicles and purified until it yielded a single band on a SDS polyacrylamide gel. The Mr 190,000 protein has a high content of lysine and glutamic acid/glutamine residues and is rich in arginine residues, some of which, it is postulated, undergo a side chain conversion in situ into citrulline residues. Polyclonal antibodies were raised to the purified protein, and these cross-react with similar proteins from extracts of guinea pig and human follicles and rat vibrissae inner root sheaths. Tissue immunochemical methods have localized the Mr 190,000 protein to the trichohyalin granules of the developing inner root sheath of the wool follicle. We propose that the old term trichohyalin be retained to describe this Mr 190,000 protein. Immunoelectron microscopy has located the Mr 190,000 protein to the trichohyalin granules but not to the newly synthesized filaments. This technique has revealed that trichohyalin becomes associated with the filaments at later stages of development. These results indicate a possible matrix role for trichohyalin.     

Characterization of the proteins of guinea-pig hair and hair-follicle tissue

This work forms a part of a study of the mechanism and control of protein synthesis in the hair follicle and concerns the characterization of the proteins of hair-follicle tissue and for comparative reasons those of the hair itself. 1. Five different groups of reduced carboxymethylated proteins were delineated from both tissues; these were: group 1A proteins, which appeared to be aggregates of the group 2 proteins; group 1B proteins, soluble at pH4.4, which were thought to originate from the medulla and inner-rootsheath layers; group 2 proteins, which were defined as the main low-sulphur keratin proteins insoluble at pH4.4; group 3 proteins, the precise origin of which is not known; and the group 4 proteins, which were defined as the main high-sulphur keratin proteins soluble at pH4.4. 2. With the single exception of the group 1B proteins, the types and properties of all hair and hair-follicle proteins were identical as far as could be determined by use of such criteria as multiplicity of components, molecular charge, molecular weight and amino acid composition. 3. Two significant quantitative differences were noted: in follicle extracts there were more group 2 proteins but less group 3 and group 4 proteins than in hair extracts; and secondly, in the follicle group 4 proteins, there were more proteins of lowest molecular weight and S-carboxymethylcysteine content, but fewer proteins of the highest molecular weight and S-carboxymethylcysteine conent than in the hair group 4 proteins. 4. These quantitative differences are discussed in terms of the mechanism of synthesis of the keratin proteins. 5. Follicle group 1B proteins are postulated to have arisen from the trichohyalin droplets of the developing medulla and inner-root-sheath layers of the follicle and may be precursors of the proteins of the mature medulla and inner root sheath.     

Perspectives on hair evolution based on some comparative studies on vertebrate cornification

Hair evolution contributed to the biological success of mammals. Hair origin from synapsid scales is speculative and requires extensive modifications of the morphogenetic process transforming lens-shaped dermis of scales into small dermal papillae in hair. Hair evolution from glands is hypothetical but is supported from studies on the signaling control of hair vs. glandular morphogenesis. Based on immunocytochemical and comparative studies, it is hypothesized that the onion-like organization of hair derived from glandular pegs which central part produced lipids and some keratin. In a following stage, involucrin, trichohyalin, and keratins were produced in the central cells of the gland and formed a solid medulla surrounded by keratinocytes of the inner root sheath. The origin of this protohair was possibly related to increased concentration of beta-catenin and other signaling molecules in epithelial cells following the evolution of a dermal papilla. The latter activated the keratogenic genes, already utilized in cells of the claws, in concentric layers of cells of the glandular peg. Lipidogenic genes were depressed. As new genes evolved in the genome of synapsids, new circular layers of keratinocytes containing specialized hard keratins and keratin-associated proteins were formed around medullary cells. The new keratinocytes probably originated the cortex separating medulla from the external cells that became the inner root sheath. The hypothesis indicates that in a following stage, the medulla was obliterated or replaced by cortical cells while the external part of the cortex formed a cuticular surface due to the different growth rate with inner root sheath cells.     

Epidermal and hair follicle trans glutaminases and crosslinking in skin

Summary Epidermal and hair follicle transglutaminases crosslink structural proteins in the skin by epsilon-(gamma-glutamyl)-lysine bonds. This crosslinking produces protein polymers that are extremely insoluble and, until recently, difficult to characterize.Epidermal transglutaminase is localized to the granular layer of the epidermis. It catalyzes the crosslinking of a soluble cytoplasmic precursor to form the cornified envelope that lines the inner membrane of the mature keratinocyte in the stratum corneum.Hair follicle transglutaminase is localized to the inner root sheath and medulla of the hair follicle. It crosslinks a poorly characterized citrulline-rich protein.The enzymes and their substrates have been shown to be important markers of normal differentiation. Regulation of these processes is currently under investigation.     

Segmental Igfbp5 expression is specifically associated with the bent structure of zigzag hairs

The murine hair coat consists of four different hair types that are characterised by hair length, the number of medulla columns, and the presence and number of bends. The molecular mechanisms underlying the establishment and maintenance of distinct hair follicle fates are unknown. We identify Igfbp5 as the first molecular marker that distinguishes among different hair follicle types. High-resolution expression analysis revealed that its expression in the medulla of hair shafts is associated with the bend-forming zones of zigzag hairs. To directly examine the functional importance of segmental gene expression in the hair follicle, we have generated transgenic mice expressing Igfbp5 in differentiating keratinocytes of the medulla and inner root sheath. Ectopic expression of Igfbp5 resulted in the appearance of remarkable curvatures and thinning of hair shafts, two hallmarks of hair bends. Both effects and the natural bending process are under negative control of IGF signalling. Thus, our data identify Igfbp5 as a central regulator of hair shaft differentiation and hair type determination.     

Expression of nucleosomal protein HMGN1 in the cycling mouse hair follicle

Here we examine the expression pattern of HMGN1, a nucleosome binding protein that affects chromatin structure and activity, in the hair follicle and test whether loss of HMGN1 affects the development or cycling of the follicle. We find that at the onset of hair follicle development, HMGN1 protein is expressed in the epidermal placode and in aggregated dermal fibroblasts. In the adult hair follicle, HMGN1 is specifically expressed in the basal layer of epidermis, in the outer root sheath, in the hair bulb, but not in the inner root sheath and hair shaft. The expression pattern of HMGN1 is very similar to p63, suggesting a role for HMGN1 in the transiently amplifying cells. We also find HMGN1 expression in some, but not all hair follicle stem cells as detected by its colocalization with Nestin and with BrdU label-retaining cells. The appearance of the skin and hair follicle of Hmgn1^?/? mice was indistinguishable from that of their Hmgn1^+/+ littermates. We found that in the hair follicle the expression of HMGN2 is very similar to HMGN1 suggesting functional redundancy between these closely related HMGN variants.     

常见毛囊细胞角蛋白在毛囊周期中的表达研究

目的探讨常见毛囊细胞角蛋白在毛囊周期中的表达特征。 方法取毛囊发育期、生长期启动、生长期、退化期和静止期的小鼠皮肤,石蜡切片后通过免疫荧光的方法,检测细胞角蛋白Krt5、Krt6、Krt10、Krt14、Krt15和Krt19的表达情况。 结果Krt5在静止期和生长期启动表达于所有毛囊上皮细胞,在其他时期表达不一致;Krt6表达于所有时期的外根鞘细胞和内根鞘细胞;Krt10表达于生长期和退化期的毛母质和内根鞘细胞,在其他时期表达不一致;Krt14在生长期和退化期表达于所有毛囊上皮细胞,在其他时期表达不一致;Krt15和Krt19表达于毛囊发育期、生长期启动和静止期的毛囊隆突区细胞,在生长期和退化期表达不一致。 结论角蛋白作为毛囊结构或毛囊干细胞标记物仅适用于特定的毛囊周期。研究者在使用毛囊角蛋白作为标记物时,应首先明确其在毛囊周期中的表达情况。     

The electron histochemical demonstration of cystine-containing proteins in the guinea pig hair follicle

Summary A previously described electron histochemical method for detecting cystine in fully keratinised human hair has been modified for the examination of this amino acid residue amongst the cellular components of anagen guinea pig hair follicles. Using the technique the progressive incorporation of cystine into the hair cuticle and cortex has been observed. Although cystine was absent from the hair medulla and outer root sheath cells at all stages of development, a narrow layer of cystine-containing material was found adjacent to the cell membranes of hardened inner root sheath cells.     

Common Variants in the Trichohyalin Gene Are Associated with Straight Hair in Europeans

Hair morphology is highly differentiated between populations and among people of European ancestry. Whereas hair morphology in East Asian populations has been studied extensively, relatively little is known about the genetics of this trait in Europeans. We performed a genome-wide association scan for hair morphology (straight, wavy, curly) in three Australian samples of European descent. All three samples showed evidence of association implicating the Trichohyalin gene (TCHH), which is expressed in the developing inner root sheath of the hair follicle, and explaining ∼6% of variance (p = 1.5 × 10⁻³¹). These variants are at their highest frequency in Northern Europeans, paralleling the distribution of the straight-hair EDAR variant in Asian populations.     

Purinergic receptors are part of a signalling system for proliferation and differentiation in distinct cell lineages in human anagen hair follicles

We investigated the expression of P2X₅, P2X₇, P2Y₁ and P2Y₂ receptor subtypes in adult human anagen hair follicles and in relation to markers of proliferation [proliferating cell nuclear antigen (PCNA) and Ki-67], keratinocyte differentiation (involucrin) and apoptosis (anticaspase-3). Using immunohistochemistry, we showed that P2X₅, P2Y₁ and P2Y₂ receptors were expressed in spatially distinct zones of the anagen hair follicle: P2Y₁ receptors in the outer root sheath and bulb, P2X₅ receptors in the inner and outer root sheaths and medulla and P2Y₂ receptors in living cells at the edge of the cortex/medulla. P2X₇ receptors were not expressed. Colocalisation experiments suggested different functional roles for these receptors: P2Y₁ receptors were associated with bulb and outer root sheath keratinocyte proliferation, P2X₅ receptors were associated with differentiation of cells of the medulla and inner root sheaths and P2Y₂ receptors were associated with early differentiated cells in the cortex/medulla that contribute to the formation of the hair shaft. The therapeutic potential of purinergic agonists and antagonists for controlling hair growth is discussed.     

Cellular dynamics of the outer layers of the hair follicle of fine-wool sheep during the phase of stable hair growth

The structure, origin, and migration of outer sheath cells of the hair follicles of domestic sheep were studied by electron microscopic, autoradiographic, and histochemical (glycogen) methods in order to understand the role of this layer in hair morphogenesis. We demonstrated that the cells of the outer layers of the outer sheath interpose into the inner “companion” layer of the outer sheath. Although this process takes place all along the hair follicle from the lower bulb up to the sebaceous glands orifices, it mainly takes place over the bulb. Labeled cells interposed into the companion layer reach sebaceous glands orifices more than 24 h faster than labeled cells of the inner sheath and hair, because these cells included the label not in the bulb cambium (as hair and inner sheath) but over the bulb, and from this point they start movement. Interposition of cells into the companion layer must cause increase of its volume and additional volume supposed to be led away into the pillar canal around the hair near the sebaceous glands orifices. This can provide the mechanism of the hair and inner sheath promotion to sebaceous gland orifices.     

BMPR1A signaling is necessary for hair follicle cycling and hair shaft differentiation in mice

Interactions between ectodermal and mesenchymal extracellular signaling pathways regulate hair follicle (HF) morphogenesis and hair cycling. Bone morphogenetic proteins (BMPs) are known to be important in hair follicle development by affecting the local cell fate modulation. To study the role of BMP signaling in the HF, we disrupted Bmpr1a, which encodes the BMP receptor type IA (BMPR1A) in an HF cell-specific manner, using the Cre/loxP system. We found that the differentiation of inner root sheath, but not outer root sheath, was severely impaired in mutant mice. The number of HFs was reduced in the dermis and subcutaneous tissue, and cycling epithelial cells were reduced in mutant mice HFs. Our results strongly suggest that BMPR1A signaling is essential for inner root sheath differentiation and is indispensable for HF renewal in adult skin.     

The biology of hair follicle

The human hair follicle is a unique appendage which results from epithelio-mesenchymal interactions initiated around the 3rd month of development. This appendage has a very complex structure, with more than 20 different cell types distributed into 6 main compartments, namely the connective tissue sheath, the dermal papilla, the outer root sheath, the inner root sheath, the shaft and the sebaceous gland. The pigmentation unit, responsible for hair color, is made of fully active melanocytes located on top of the dermal papilla. This complex appendage has a unique behavior in mammals since, after a hair production phase, it involutes in situ before entering a resting phase after which it renews in a cyclical but stochastic fashion, out of a double reservoir of pluripotent stem cells also to able regenerate epidermis. The pigmentation unit also renews in a cyclical fashion, out of a melanocyte progenitor reservoir which progressively declines with time, provoking the hair whitening process. Finally, the shape of the hair shaft is programmed from the bulb. The hair follicle thus behaves as a fully autonomous skin appendage with its own hormonal control, its own autocrine and paracrine network, its own cycle, appearing as an incredibly complex and stable structure which summarizes the main rules of tissue homeostasis.