Cornified Cell Envelope Precursor Proteins Research Articles

ROS Quenching Potential of the Epidermal Cornified Cell Envelope

The cornified cell envelope (CE) is a specialized structure assembled beneath the plasma membrane of keratinocytes in the outermost layers of the epidermis. It is essential for the physical and permeability properties of the barrier function of the skin. Our skin is continuously exposed to atmospheric oxygen and threatened by reactive oxygen species (ROS). Here, we identify the CE as a first line of antioxidant defense and show that the small proline-rich (SPRR) family of CE precursor proteins have a major role in ROS detoxification. Cysteine residues within these proteins are responsible for ROS quenching, resulting in inter- and intramolecular S-S bond formation, both in isolated proteins and purified CEs. The related keratinocyte proline-rich protein is also oxidized on several cysteine residues within the CE. Differences in antioxidant potential between various SPRR family members are likely determined by structural differences rather than by the amount of cysteine residues per protein. Loricrin, a major component of the CE with a higher cysteine content than SPRRs, is a weak ROS quencher and oxidized on a single cysteine residue within the CE. It is inferred that SPRR proteins provide the outermost layer of our skin with a highly adaptive and protective antioxidant shield.

Disruption of the suprabasal keratin network by mutation M150T in the helix initiation motif of keratin 10 does not affect cornified cell envelope formation in human epidermis.

Keratin 10 (K10) is known to be tightly bound to the cornified cell envelope (CCE) and this binding is thought to play an important role in enhancing the structural integrity of the cornified cells. Bullous congenital ichthyosiform erythroderma (BCIE) is a genetic disorder of keratinization caused by gene mutations in the conserved sequences of keratin 1 (K1) or K10, which leads to abnormal suprabasal keratin network assembly. In BCIE patients' skin, the keratin network abnormalities make the upper spinous and granular keratinocytes fragile and result in blister formation. However, the exact pathomechanism of the hyperkeratosis seen in BCIE is still unknown. The involvement of the CCE in the pathomechanism of hyperkeratosis in BCIE is controversial. Abnormal CCE assembly may cause hyperkeratosis as reported in cases of lamellar ichthyosis. Binding of K10 to CCE is thought to be a vital connection between the suprabasal keratin filament network and CCE. We hypothesize that abnormal suprabasal keratin assembly caused by either K1 or K10 mutations can disrupt CCE formation, resulting in the hyperkeratosis observed in BCIE. To clarify whether K10 and keratin network defects affect CCE formation in vivo, the ultrastructural and immunohistological features of CCE were studied in the epidermis of two Japanese BCIE patients from two independent families carrying an identical missense mutation M150T in the helix initiation motif of K10. Ultrastructurally, a 15-nm-thick, dense, normal-appearing CCE was formed at the cell periphery of the keratinized epidermal cells. Light and electron microscopic immunolabeling revealed that the major CCE precursor proteins, involucrin and loricrin, were normally distributed and restricted to CCE of the epidermis. Immunofluorescent labeling showed that epidermal TGases, TGase 1, TGase 2 and TGase 3, were expressed normally in the epidermis. These findings suggest that a normal CCE is formed during the process of human epidermal keratinization, even if the suprabasal keratin filament network is disrupted as with this particular K10 mutation, M150T in BCIE.

Formation of cornified cell envelope in human hair follicle development.

Cornified cell envelope (CCE) formation is an important step in the final stage of keratinization, in which CCE precursor proteins including involucrin and loricrin are cross-linked by keratinocyte transglutaminases (TGases) to the inner surface of the plasma membrane of cornified cells, while the outer surface is coated with material derived from secreted lamellar granules. Skin samples from human fetuses of a series of estimated gestational age (EGA) (49-163 days) were studied for the prescence of precursor proteins. Methods TGase activity was studied by in situ TGase activity assay, and ultrastructural features of CCE formation were observed at each stage of hair follicle development. We used immunofluorescent labelling to investigate the time and site of expression of CCE precursor proteins involucrin and loricrin, TGases 1, 2 and 3, and a 25-kDa lamellar granule-associated protein (LGP) in developing human hair follicles. In the hair germ (65-84 days EGA) (corresponding to the stages 1-2 of murine hair follicle morphogenesis), only TGase 2 was observed in the entire hair germ, where in situ TGase activity was weakly positive, although thickening of cell membrane was not seen ultrastructurally. In the hair peg (85-104 days EGA) (corresponding to the stage 3 of murine hair follicle morphogenesis), loricrin and TGase 2 were seen in cells of the upper part of the hair peg while TGase 1, 3 and LGP were observed in the inner cells of the hair peg. In situ TGase activity was weakly positive in the upper part and inner cells of the hair peg. In the bulbous hair peg (105-135 days EGA) (corresponding to the stages 4-6 of murine hair follicle morphogenesis) and differentiated lanugo hair follicle (> 135 days EGA) (corresponding to the stages 7-8 of murine hair follicle morphogenesis), immunoreactivities of involucrin, loricrin, TGase 1, 2, 3, in situ TGase activity and LGP were detected in the inner root sheath cells, hair canals and inner cells of the outer root sheath in the region of the isthmus. Ultrastructurally, thickening of cell membrane was already seen in the inner root sheath cells of the bulbous hair peg and electron-dense, thick CCE was observed in the hair cuticle and hair canal of differentiated lanugo hair follicle. These data indicate that, in terms of CCE formation, certain portions of the developing human hair follicle have already been determined in differentiation of the hair canal and cuticle at the hair peg stage.

Periderm Cells Form Cornified Cell Envelope in Their Regression Process During Human Epidermal Development

Terminally differentiated stratified squamous epithelium forms a lining of the plasma membrane called the cornified cell envelope, a thick layer of several covalently cross-linked precursor proteins including involucrin, small proline-rich proteins, and loricrin. Their cross-linking isodipeptide bonds are formed by epidermal transglutaminases 1-3. Material from lamellar granules is attached on the extracellular surface of corneocytes during the keratinization process. The formation of cornified cell envelope and sequential expression of major cornified cell envelope precursor proteins, transglutaminases, and 25 kDa lamellar granule-associated protein were studied in human embryonic and fetal skin. Ultrastructurally, membrane thickening has already started in periderm cells of the two-layered epidermis and an electron-dense, thickened cell envelope similar to cornified cell envelope in adult epidermis is observed in periderm cells at the three-layered and later stages of skin development. In the two-layered epidermis (49-65 d estimated gestational age), immunoreactivities of involucrin, small proline-rich proteins, all the transglutaminases, and lamellar granule-associated protein were present only in the periderm. In the three-layered epidermis and thereafter (66-160 d estimated gestational age), loricrin became positive in the periderm cells, transglutaminases extended to the entire epidermis, and lamellar granule-associated protein was detected in intermediate cells as well as periderm cells. Immunoelectron microscopy demonstrated that both major cornified cell envelope precursor proteins, involucrin and loricrin, were restricted to the cornified cell envelope in periderm cells at this stage of development. After 160 d estimated gestational age, the periderm had disappeared and cornified cell envelope proteins and lamellar granule-associated proteins were expressed in the spinous, granular, and cornified cells and transglutaminases were detected in the entire epidermis. These findings indicate that cornified cell envelope precursor proteins, transglutaminases, and lamellar granule-associated proteins are expressed in coordination in periderm cells during human epidermal development and suggest that periderm cells form cornified cell envelope in the process of regression.

Abnormalities of basal cell keratin in epidermolysis bullosa simplex do not affect the expression patterns of suprabasal keratins and cornified cell envelope proteins

Basal keratins, suprabasal keratins, filaggrin, and cornified cell envelope (CCE) precursor proteins are expressed during the differentiation of epidermal keratinocytes. These molecules are coordinately expressed during epidermal differentiation. The present study investigated the expression patterns of keratins and CCE precursor proteins in 15 patients with epidermolysis bullosa simplex (EBS), which is caused by mutations in the genes that encode for the basal keratins, keratins 5 and 14. The patterns of expression of keratins 5, 14, 1 and 10, filaggrin, and of the three major CCE precursor proteins, involucrin, loricrin and small proline-rich proteins 1 and 2 (SPRs), were studied immunohistochemically and by electron microscopy. In 14 of the 15 patients with EBS, the distribution pattern of keratins was not altered. In one neonate with EBS, basal cell keratins were expressed in the suprabasal layers. Ultrastructurally, numerous clumped tonofilaments were observed in the basal and suprabasal cells. In all cases, findings were positive for filaggrin in the granular cells, with positivity for involucrin in the upper spinous and granular cells. The upper spinous cells and granular cells were positive for SPRs 1 and 2, and loricrin was expressed in granular cells. Ultrastructurally, no marked abnormality was observed in the suprabasal layers such as a decrease in, or agglutination of, keratin filaments, except in one neonate. A CCE about 15 nm thick was formed normally in the cell membrane of cornified cells. The patterns of distributions of basal cell keratins, suprabasal keratins, filaggrin, and CCE precursor proteins, as well as the ultrastructural findings, resembled those of normal skin. Thus, the abnormality in basal cell keratins in patients with EBS did not appear to alter the patterns of expression of the keratins and CCE precursor proteins.

Interdependent transcription control elements regulate the expression of the SPRR2A gene during keratinocyte terminal differentiation.

Expression of the SPRR2A gene, a member of the small proline-rich family of cornified cell envelope precursor proteins, is strictly linked to keratinocyte terminal differentiation both in vivo and in vitro. In this study, we explored the molecular mechanisms underlying this regulation in transiently transfected primary keratinocytes induced to differentiate in vitro. Deletion mapping and site-directed mutagenesis of SPRR2A promoter-chloramphenicol acetyltransferase constructs indicate that four transcription control elements are essential and sufficient for promoter activity. These elements were further characterized by electrophoretic mobility shift and identified as (i) an inverted octamer doublet, bound by the POU domain factor Oct-11 (Skn-1a/i, Epoc-1), (ii) an interferon-stimulated response element recognized by interferon regulatory factors 1 and 2, (iii) an Ets binding site partially overlapping the interferon-stimulated response element, and (iv) a TG box recognized by the Sp1 family of zinc finger transcription factors. Destruction of a single terminal differentiation element is sufficient to completely abolish transcription from the SPRR2A promoter, indicating that these transcription control elements function in concert in an interdependent manner. Apparently, integration of signals transmitted by the above-mentioned transcription factors is necessary and sufficient to promote gene expression during keratinocyte terminal differentiation.

The Small Proline-Rich Proteins Constitute a Multigene Family of Differentially Regulated Cornified Cell Envelope Precursor Proteins

Loricrin, involucrin, small proline-rich protein (SPRR)1, SPRR2, and SPRR3 genes are located within a cluster of 1.5 Mbp on chromosome 1q21 and most likely evolved from a common ancestor. Monospecific polyclonal antibodies and cDNA probes were produced to investigate SPRR transcripts and proteins. SPRR expression was restricted to terminally differentiating squamous cells, preferentially located at the cell periphery, and immunoreactivity was greatly reduced in cells with a mature cornified cell envelope. Furthermore, detectable SPRR2 and SPRR3 levels were strongly increased in differentiating keratinocyte cultures after addition of LTB-2, a specific inhibitor of transglutaminases, suggesting that they are precursor proteins of the cornified cell envelope. In normal epidermis, SPRR1 was restricted to appendageal areas, SPRR2 was expressed coherently, and SPRR3 was completely absent. In the upper digestive tract, SPRR1 was expressed in sublingual and tongue epithelium, SPRR2 was mostly restricted to lingual papillae, and SPRR3 was abundant in oral and esophageal epithelium. In psoriatic epidermis, SPRR1 and SPRR2 were expressed at much higher levels than in normal epidermis. Addition of 10(-7) M retinoic acid to cultured differentiating keratinocytes significantly down-regulated the expression of SPRR2 and SPRR3 transcripts and slightly decreased that of SPRR1. Thus, SPRR1, SPRR2, and SPRR3 are differentially expressed in vivo and in vitro, suggesting that the SPRR multigene family evolved to serve as highly specialized cornified cell envelope precursor proteins in stratified epithelia.