Keratinization of outer root sheath cells is prevented by contact with inner root sheath of rat hair follicles

The purpose of the present study was to elucidate why keratinocytes of the outer root sheath (ORS) do not keratinize in situ. Two possibilities were considered—inhibition of keratinization is caused by contact of ORS with inner root sheath (IRS) or insufficient supply of keratinization promoting factors from the surrounding tissues to the ORS. In order to distinguish between these possibilities mid-segments of hair follicles were liberated from the dermis by dissection followed by collagenase digestion. ORS cells were then either allowed to migrate from the mid-segments or were kept on the agarose layer which prevented cell spreading and preserved three dimensional structure of hair root. Cultures were stimulated with calcium or EGF, and studied morphologically at the light and transmission electron microscope level. The level of mRNA for differentiation cell markers was also studied by RealTime PCR. ORS cells growing in a medium with low Ca²⁺ content formed monolayers, which after elevation of Ca²⁺ produced multilayers with cells containing keratohyalin-like granules. Ca²⁺ or EGF treatment upregulated expression of involucrin, filaggrin and keratinocyte differentiation associated protein (Kdap). Culture of mid-segments of hair follicles in low calcium culture medium kept on agarose increased expression of filaggrin and Kdap, but downregulated expression of involucrin. Stimulation by Ca²⁺ further increased expression of filaggrin and Kdap, but had no effect on the level of involucrin expression. EGF stimulated expression of filaggrin only. It is concluded that IRS exerted an inhibitory effect on the expression of involucrin, an essential component of the cornified envelope, thus preventing keratinization of ORS cells in situ. On the other hand, improved access of nutrients or promoting factors of keratinization to the mid-segment of hair follicles augmented expression of filaggrin and Kdap, proteins engaged in the differentiation of keratinocytes but not involved in its terminal phase.     

A novel type II cytokeratin, mK6irs, is expressed in the Huxley and Henle layers of the mouse inner root sheath

Hair follicle differentiation involves the expression of both epithelial-type keratins or cytokeratins and hair keratins as well as hair keratin-associated proteins. In this study, a cDNA clone encoding a cytokeratin family member was isolated using RNA differential display techniques. The predicted amino acid sequence derived from this clone, revealed a homology with a number of cytokeratins, not only in the central alpha-helical regions but also in the conserved portions of the amino and carboxy terminal domains, indicating that this protein represents a new member of the mouse type II cytokeratin family. Northern blot analysis showed expression in mouse skin, but not in other tissues, including tongue, esophagus, and forestomach. One- and two-dimensional western blot analysis showed that this new cytokeratin was 57 kDa in size and ran slightly below the area of cytokeratin 5, which corresponded to that of the cytokeratin 6 family members. Both RNA in situ hybridization and immunohistochemical studies of mouse anagen hair follicles demonstrated expression of this cytokeratin in the inner root sheath hair cone during anagen III and in the Henle and Huxley layers of the inner root sheath during anagen VI. The expression of the new cytokeratin began in the hair bulb and progressed up to the height of the keratogenous zone. Taken together the sum of the data analyzed, we have termed this novel cytokeratin mK6irs (mouse gene nomenclature k2-6g) to indicate both its similar mobility with K6 in two-dimensional gels and its specific expression in the inner root sheath of the hair follicle.     

Placental glutathione-S-transferase-pi mRNA is abundantly expressed in human skin

Four overlapping cDNA clones were isolated from a lambda gt11 human placenta cDNA library using purified human IgG antibody, from a patient with bullous pemphigoid. The sequence was homologous to human placenta glutathione-S-transferase-pi (GST-pi). Using the placenta clone, epidermal cDNA clones were isolated from a human keratinocyte library. Expression of GST-pi mRNA in human skin, cultured keratinocytes and fibroblasts, and disorders of squamous hyperplasia was demonstrated by Northern blotting and in situ hybridization. Human epidermal and placental cDNA clones hybridized to the same genomic DNA fragments. Hybridization of placental cDNA to interspecific somatic cell hybrids showed retention of chromosome 11, confirming the assignment of GST 3 to the long arm of chromosome 11 by molecular means. Anti-GST-pi antibody did not give a basement membrane zone pattern, although some normal and BP sera contained antibodies to GST-pi. Human skin expresses glutathione-S-transferase-pi, which belongs to an enzyme family important for detoxification and carcinogenesis.     

Investigation of the hair follicle inner root sheath in scarring and non-scarring alopecia

BACKGROUND: Premature desquamation of the inner root sheath (IRS) is described as a defining histologic feature of follicular degeneration syndrome/central centrifugal cicatricial alopecia (CCCA). However, IRS abnormalities have been noted in other types of alopecia. DESIGN: We evaluated the IRS in terminal hair follicles with transverse sections stained with hematoxylin-eosin and Ziehl-Neelsen stains in 22 non-scarring (7 areata and 15 androgenetic) and 21 scarring (13 CCCA, 2 lichen planopilaris, 2 lupus erythematosus, 1 folliculitis decalvans and 3 end stage) alopecia cases. In addition, we evaluated 15 normal controls with longitudinal sections to establish the level of IRS desquamation. RESULTS: The IRS was present in 99.5 +/- 0.01% (mean +/- standard error) of normal follicles at the level of the arrector pili muscle/sebaceous gland/sweat gland coil (L2) and variably present at higher levels. The IRS was present at L2 in 97.9 +/- 1.5% of alopecia areata, 87.4 +/- 5.3% of androgenetic alopecia, 59.3 +/- 7.0% of CCCA and 49.4 +/- 11.3% of other scarring alopecia follicles. CONCLUSIONS: Premature desquamation of the IRS was identified in CCCA; however, it was also noted in other scarring alopecia cases. IRS premature desquamation is a non-specific histologic feature in scarring alopecia and cannot be used alone as a defining feature of CCCA.     

Functional analysis of keratin components in the mouse hair follicle inner root sheath

BACKGROUND: Recently, a family of novel type I keratins of the inner root sheath of the hair follicle were discovered, increasing the number of keratins known to be expressed in the hair follicle. The mouse database shows three keratins that are possible orthologues of these inner root sheath keratins. The sequences of these keratins include rather unusual changes to a highly conserved motif at the end of the alpha-helical rod domain of the proteins, thought to be important in filament assembly. OBJECTIVES: To investigate whether these keratins are expressed in the inner root sheath and to determine whether they assemble normally. METHODS: To investigate this, polyclonal antibodies were raised for immunolocalization of the keratins and their cDNAs were cloned for transfection into cultured cells. RESULTS: At least two of these keratins were expressed in the inner root sheath but the timing of expression of the different keratins was variable. Transfection of the relevant cDNAs into cells in culture indicated that these keratins were capable of integrating into existing keratin networks without disruption, but that de novo filament assembly with the type II inner root sheath keratin, mK6irs, was poor. CONCLUSIONS: These results provide further evidence of the complexity of keratin expression in the three concentric layers of the inner root sheath.     

In vitro degradation of the inner root sheath in human hair follicles lacking sebaceous glands

BACKGROUND: Cultured hair follicles lacking sebaceous glands do not appear to degrade the inner root sheath (IRS), suggesting that the gland may be involved in this process. OBJECTIVES: To examine this supposition in cultured hair follicles. METHODS: Pilosebaceous units were isolated from hair follicles cultured in vitro, and IRS degradation was studied by histology. RESULTS: When grown in culture, the fibres of follicles lacking sebaceous glands were encased in a layer of translucent tissue. During hair growth in vitro this tissue remained intact at the distal end of the follicle but disappeared further down towards the bulb and then reappeared towards the proximal end. Transection within the region lacking this tissue resulted in the release of a naked hair fibre and the production of hair with no attached tissue upon subsequent hair growth. The translucent tissue represented the IRS, thereby demonstrating that this tissue is indeed degraded in vitro. Histological comparison with freshly isolated pilosebaceous units indicated that IRS degradation in vitro strongly resembled the process that occurs in vivo. CONCLUSIONS: These data suggest that the sebaceous gland does not itself participate in IRS degradation. Indeed, this phenomenon appears to be a function of the follicle itself and is probably intimately linked with the processes of cellular proliferation, differentiation and death that occur during hair biogenesis.     

An unusual hybrid cyst in Gardner's syndrome with partial differentiation toward the inner root sheath

A 26-year-old man with Gardner's syndrome had numerous subcutaneous nodules over almost his entire body. Histologically, one of them revealed an unusual so-called hybrid cyst composed of epidermal type keratinization and inner root sheath type keratinization. The histologic findings and the possibility of cutaneous cysts based on hair matrix differentiation, more specifically partial differentiation toward the inner root sheath, are discussed.     

Pili trianguli et canaliculi is a defect of inner root sheath keratinization: ultrastructural observations of anomalous tonofilament organization in a case

A 4-year-old Caucasian girl with pili trianguli et canaliculi had distinctive findings. These included unique ultrastructural alterations consisting of tonofilament-desmosomal detachment and tonofilament clumping within inner root sheath cells. We believe that the hair anomaly in this condition may be due to a compromised cytoskeleton with subsequent configurational changes of the inner root sheath. Despite these configurational changes keratinization of the inner root sheath occurs. As inner root sheath keratinization typically precedes keratinization of the hair shaft, the abnormally configured inner root sheath determines the surface characteristics of the hair shaft in pili trianguli et canaliculi.     

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.     

Cytochemistry of trichohyalin granules: a possible role for cornification of inner root sheath cell in the hair follicle

We studied ultracytochemically how trichohyalin granules (TGs) were involved in the cornification of the inner root sheath (IRS) cells in the hair follicle. After storing unfixed mouse skin samples in glycerol and then low ionic strength salt solution (G-LISS), the TGs reduced electron-dense amorphous material (EDM) to various degrees, with the appearance of filamentous material in routine staining. As the IRS cells were differentiating, these changes in the TGs became successively prominent; Some TGs consisted of the filamentous material and residual granular EDM, showing a "filamentogranular structure". In the cells in transition to cornified cells, or transitional cells, the EDM of the TGs disappeared and consisted of only filamentous material which was intertwined with keratin filaments (KFs). The internal substructure of the TGs induced by the G-LISS treatment stained well with ethanolic phosphotungstic acid, which reacts with basic proteins, and the KFs associated with the TGs also stained well. In skin samples not treated with G-LISS, the TGs of the transitional cells often exhibited fibrogranular structure after PTA staining to detect tissue basic proteins, suggesting a release of PTA stainable proteins(s) by the TGs in living transitional cells. The KFs of the cornified cells were more intensively stained and thicker (about 20 nm wide) than those of the differentiating IRS cells. These findings suggest that the TGs may contain at least two kinds of basic proteins; One is G-LISS-soluble, and the other is a very insoluble filamentous protein. These protein materials may be added to or incorporated into the KFs in the cornified cells with the disappearance of the TGs, since the KFs are markedly thick with PTA staining.