Ultrastructural immunolocalization of involucrin in the medulla and inner root sheath of the human hair

Summary

The participation of involucrin in the cornification of the human hair has been studied by light and electron microscopy immunohistochemistry. Immunoreactivity for involucrin is absent in keratinized cuticle and cortical cells although some immunolabeling is observed in the corneous membrane of internal cortical cells surrounding the hair medulla. Conversely, immunolabeling for involucrin is present in the cytoplasm of keratinizing cells of the medulla and inner root sheath. During the maturation and final cornification of medullary and inner root sheath cells the immunolabeling for involucrin tends to concentrate in the peripheral cytoplasm and along the cornified cell plasma membrane in both medullary and inner root sheath cells, a pattern similar to that known for corneocytes of the epidermis. This observation suggests that in the hair involucrin mainly participates in the formation of the corneous material of the medulla and inner root sheath in conjunction with trichohyalin, probably by the formation of isopeptide-bonds. Therefore, together with trichohyalin, the cross-linking due to involucrin is also responsible for the mechanical resistance of the corneous trabeculae present among the empty spaces of the medulla of the human hair.

Introduction

During epidermal differentiation in mammalian skin, the protein involucrin is synthesized in the upper layers of the spinosus layer and accumulates in the granular and transitional cells (Ishida-Yamamoto et al., 1996, Kalinin et al., 2002, Alibardi and Toni, 2004). The protein is mainly cross-linked by epidermal transglutaminase to the corneous cell envelope of corneocytes forming the corneal layer of stratified epithelium.

Beyond the epidermis, some immunocytochemical and in situ hybridization studies have also indicated that involucrin is present in developing and growing hairs (Piao et al., 2002, Rogers and Koike, 2009), especially in the inner root sheath (IRS) and in the medulla (De Viragh et al., 1994, Fig. 1). Despite these initial data, details on the localization of involucrin in hair are not known and the presence of this protein in the different compartments of the hair remains to be demonstrated at the ultrastructural level (Rogers, 2004). While in cuticle and cortical cells numerous hard keratins and matrix proteins rich in tyrosine or in cysteine explain the high mechanical and chemical resistance of the hair shaft, the protein composition of medullary cells is less known (Rogers et al., 2006, Langbein et al., 2009).

The differentiation of medullary cells occurs with a peculiar process of granule accumulation, granule fusion and eventually margination that results in the formation of corneous trabeculae among hollow spaces (Matoltsy, 1953, Parakkal and Matoltsy, 1964, Roth and Helwig, 1964, Orwin, 1979, Morioka, 2005, De Cassia-Comis Wagner et al., 2007). Aside from trichohyalin (Rothnagel and Rogers, 1984), other proteins capable of mechanically sustaining the corneous trabeculae among medullary spaces are likely present. It is known that the medulla in mammalian hairs contains transglutaminase (especially Transglutaminase 3) and that iso-peptide bonds are formed during medulla maturation (Harding and Rogers, 1972, Peterson et al., 1983, Taresa et al., 1997, Piao et al., 2002; Alibardi, unpublished observations). A major substrate for this enzyme is represented by trichohyalin which is specifically present in the medulla and IRS (Rothnagel and Rogers, 1984). However a sole cornification of medullary cells only using trichohyalin probably cannot produce the mechanically and chemically resistant corneous material of the medulla. In fact, in the other trichohyalin-rich hair compartment, the IRS, trichohyalin is degraded before the hair exits from the hair canal, indicating that trichohyalin-based corneous material is susceptible to broad proteolysis (Rogers, 1964, Rogers, 2004). The cornified medulla is highly resistant to various extractive treatments (Rice et al., 1994) and this suggests that cross-linked trichohyalin is not sufficient to mechanically sustain the trabeculae delimiting the large spaces within the medullary tissue. Therefore specific keratins (Langbein and Schweizer, 2005, Langbein et al., 2009) and keratin-associated proteins capable of cross-linking by transglutaminase are likely present in the hair medulla.

Ultrastructural studies have shown that the membrane of cuticle and cortical cells undergoes a progressive transformation into a pentalaminar structure known as “cell membrane complex” (Rogers, 1959, Orwin and Thomson, 1972, Orwin et al., 1973). Studies on medullary cell differentiation (Parakkal and Matoltsy, 1964, Roth and Helwig, 1964, Orwin, 1979, De Cassia-Comis Wagner et al., 2007) have shown the formation of a hard material that remains within the trabeculae of fully cornified medullary cells. This condition suggests that mechanically resistant corneous proteins help to prevent the collapse of cells of the hair medulla as they form empty spaces at maturity. Besides, after complete cornification, the medulla presents a strong chemical resistance (Matoltsy, 1953, Rice et al., 1994), suggesting that resistant chemical bonds are formed in addition to that derived from trichohyalin.

The present immunocytochemical study on the maturing and fully cornified human hair has been conducted in order to evaluate whether involucrin is present and may participate in the formation of the hard and chemically resistant corneous material of the human hair medulla.