Keratin network modifications lead to the mechanical stiffening of the hair follicle fiber.

The complex mechanical properties of biomaterials such as hair, horn, skin, or bone are determined by the architecture of the underlying fibrous bionetworks. Although much is known about the influence of the cytoskeleton on the mechanics of isolated cells, this has been less studied in tridimensional tissues. We used the hair follicle as a model to link changes in the keratin network composition and architecture to the mechanical properties of the nascent hair. We show using atomic force microscopy that the soft keratinocyte matrix at the base of the follicle stiffens by a factor of ∼360, from 30 kPa to 11 MPa along the first millimeter of the follicle. The early mechanical stiffening is concomitant to an increase in diameter of the keratin macrofibrils, their continuous compaction, and increasingly parallel orientation. The related stiffening of the material follows a power law, typical of the mechanics of nonthermal bending-dominated fiber networks. In addition, we used X-ray diffraction to monitor changes in the (supra)molecular organization within the keratin fibers. At later keratinization stages, the inner mechanical properties of the macrofibrils dominate the stiffening due to the progressive setting up of the cystine network. Our findings corroborate existing models on the sequence of biological and structural events during hair keratinization.

Transglutaminase-3 enzyme: a putative actor in human hair shaft scaffolding?

The family of transglutaminases (TGase) is known to be involved in terminal differentiation processes in the epidermis. These enzymes contribute also to the physical resistance and the preservation of the hair follicle structure. Our particular interest in hair fiber keratinization led us to focus on the TGase 3, exclusively expressed in the hair shaft. To date its function is still to be elucidated, thus we have developed a multidisciplinary approach in order to define the localization, activity, and substrates of TGase 3. The hair fiber is characterized by the expression of specific proteins essentially consisting of keratin intermediate filaments and keratin-associated proteins (KAPs), which are essential for the formation of a rigid hair shaft through their extensive disulfide cross-links. Gel electrophoresis combined with mass spectrometry experiments revealed an unexpected protein migration pattern, suggesting the existence of covalent interactions other than disulfide bonds. Western blot and amino-acid analysis revealed the presence of gamma-glutamyl-epsilon-lysine isopeptide linkages that could constitute this second covalent network. Our hypothesis is that TGase 3-driven specific isopeptide bonds between intermediate filaments and KAPs participate to the progressive scaffolding of the hair shaft.

Heparanase 1: a key participant of inner root sheath differentiation program and hair follicle homeostasis.

Heparanase is a heparan sulphate endo-glycosidase which was previously detected in the outer root sheath of murine hair follicles. Heparanase overexpression was reported to improve mouse hair (re)growth. In this study, we investigated its involvement in human hair biology. Immunofluorescence detection was used to explore heparanase distribution in both anagen and catagen hair follicles. Heparanase functionality was assessed in in vitro cultured hair follicles, in the presence of a heparanase activity inhibitor. Our results showed that heparanase expression was (i) primarily located in the inner root sheath (IRS) of human hair follicle, and there (ii) restricted to anagen phase. Furthermore, inhibition of heparanase in in vitro cultured hair follicles induced a catagen-like process. Hair shaft retreat upward was accompanied by a decrease in Ki67-positive cells, the formation of an epithelial strand as evidenced by K14 keratin expression, and the loss of IRS as assessed by transglutaminase 1 and desmoglein labelling. IRS distribution of heparanase and the induction of catagen-like involution of hair follicles when a potent heparanase inhibitor is added suggest that heparanase is a key actor of IRS differentiation and hair homeostasis.

Human hair keratin network and curvature.

BACKGROUND: In human hair, very little is known about the substructures relating to the curl pattern. The interpretation of the macroscopic shape of the fiber at the molecular and cellular scales is still unclear. METHODS: A comparative and multiscale study was carried out on a set of human hair samples, ranging in shape from straight to tightly curled, in order to investigate structural elements that might be related to hair curl pattern. RESULTS: At the macroscopic level, the frequency and amplitude of cross-sectional rotations were found to be crucial for an accurate description of curliness. At the cellular level, transmission electron microscopy experiments made it possible to confirm that macrofibril organization was strongly related to hair curliness. The curly hair follicles exhibited retrocurvature, independent of ethnic origin. A direct comparison of straight hair and curly hair highlighted an intrinsic asymmetry in the proliferative compartment that clearly extended above the Auber line on the convex side of the curvature. This phenomenon caused delayed differentiation of both inner and outer root sheaths. The hair cortex itself was elliptical and asymmetric, as evidenced by hHa8 keratin. In curly hair, this hair keratin accumulated on the concave side of the curvature, whereas, in straight hair, positive cortical cells were evenly distributed throughout the circular fiber. CONCLUSION: The curly shape of the hair shaft seems to result from the asymmetric differentiation of the precortex. Hair fiber can thus be considered as a shape memory material.

Expression of GATA-3 in epidermis and hair follicle: relationship to p63.

The epidermis is a multi-layered stratified epithelium continuously renewed by differentiating keratinocytes that develops by the action of p63, a member of the p53 family. The TP63 contains two promoters, resulting in the expression of different proteins, containing (TAp63) or not (DeltaNp63) an amino-terminal transactivation domain, which contribution in skin formation is not fully understood. We found that p63 binds and transactivate GATA-3 promoter, which in turn transactivate IKKalpha, two pivotal regulators of epithelial development. Indeed, GATA-3 is a regulator of cell lineage in skin and hair follicles formation. To further study the relationship between GATA-3 and p63 isoforms here we investigated their expression during keratinocyte differentiation, in human epidermis and hair follicle.

Absence of TRP-2 in melanogenic melanocytes of human hair.

Skin and hair colour mostly depend on the activity of melanogenic melanocytes. Numerous proteins involved in melanocyte function have been identified including pMel-17, Mitf-M, Sox10, tyrosinase, tyrosinase related proteins-1 (TRP-1) and -2 (TRP-2). In the hair, melanogenic activity occurs only during the anagen phase of the hair cycle. In order to evaluate the implications of some known melanogenic proteins in human hair pigmentation, we performed immunohistochemical studies to reveal the expression of pMel-17, Mitf-M, tyrosinase, TRP-1 and TRP-2 in active bulb melanocytes of eumelanic brown and black anagen hairs of different ethnic origins, e.g. brown Caucasian, black Asian and African hairs. The labelling was compared with that observed in Caucasian and African scalp epidermis (interfollicular epidermis) melanocytes. We found that while pMel-17, TRP-1 and TRP-2 were expressed in epidermal melanocytes irrespective of ethnic origin and melanin content of the scalp epidermis, Mitf-M and tyrosinase expression were clearly evidenced only in pigmented epidermis, e.g. African scalps. Regarding human hair, pMel-17, Mitf-M, tyrosinase and TRP-1 were detected in a similar manner in active bulb melanocytes of brown and black hairs. In contrast and unexpectedly, TRP-2 could not be detected in hair bulb melanocytes, whatever the hair colour and ethnic origin. The lack of TRP-2 was further confirmed by western blot analyses. Reverse transcriptase-polymerase chain reaction (RT-PCR) performed on hair bulb mRNA demonstrated that Mitf-M, tyrosinase and TRP-1 amplimer signals were easily detected, whereas the TRP-2 amplimer signal was barely detectable. Furthermore Sox10 was not detected in hair bulb. Altogether our results suggest that the absence of detectable level of TRP-2 is due to transcriptional control in active melanocytes of human eumelanic hair bulbs. According to the absence of TRP-2 in melanin-producing melanocytes of brown and black hair bulbs, one must consider that eumelanogenesis as well as brown and black colour do not require TRP-2 expression in human hair.