Encyclopedia of Signaling Molecules

2018 Edition
| Editors: Sangdun Choi


  • Niharika Swain
  • Rashmi Maruti Hosalkar
  • Samapika Routray
Reference work entry
DOI: https://doi.org/10.1007/978-3-319-67199-4_102003


Historical Background

Keratinocytes undergo progressive terminal differentiation to form a stratified squamous epithelium. During the process of keratinization, certain numbers of genes are expressed in suprabasal layer which include a class of genes whose products are cross-linked through action of transglutamase reaction, thus helping in the formation of upper cornified strata. Of the number of proteins said to play a significant role as precursor in the formation of this strata, involucrin (IVL) is one (Eckert et al. 1993). The term “involucrin” is derived from Latin word “involucrum” meaning envelope and was first observed in 1979 in crude extracts of cultured keratinocyctes (Rice and Green 1979). Studies have suggested IVL to assume a rod like shape having glutamate and glutamine residues which promote and serve as major glutamyl donors in transglutaminase catalyzed cross-linking reactions (Eckert et al. 1993).

Gene Transcription and Structure

IVL gene is assigned to chromosome 1q21-q22 with a 6 kb segment of DNA encoding it and 2.5 kb of DNA upstream of the transcription start site helping in its appropriate expression in epidermis (Crish et al. 1993) (Fig. 1). It consists of a 5′ exon (43 bp) and a 3′ exon (2107 bp) separated by an intron (1188 bp) in 5′ untranslated sequence of upstream DNA (Lopez-Bayghen 1996). In transient transfection of cultured human keratinocytes, a 2456-nt noncoding sequence located at 5′ end of the first IVL exon has transcriptional regulatory elements that control its transcriptional activity and is termed as upstream regulatory region. This region is divided functionally into three regulatory domains: the distal enhancer/regulatory region, transcriptional silencer, and proximal enhancer/promoter region. The distal enhancer region consists of an activator protein (AP1, AP1-5) and special protein (Sp1) site, while the proximal enhancer region consists of CCAAT enhancer binding protein (C/EBP) and activator protein (AP1) sites. The transcriptional silencer is located immediately 3′ of the AP1-5/Sp1 site (Azuara Liceaga et al. 2004). Based on in vivo studies, it was proposed that the distal enhancer/regulatory region consists of three key regulatory binding sites: AP1-5 which functions as on/off switch and absolutely required for expression, Sp1 site that maximizes the function, and ISE site which is required for expression in immediate suprabasal cell layer. AP1-5 site and transcription factors play a role in maintaining IVL gene expression in a range of surface epithelial tissues while Sp1 facilitates the loading of AP1 factor at AP1-5 site (Banks et al. 1998; Crish et al. 2006). The C/EBP site on proximal enhancer/promoter region also plays a role in the continuous expression of IVL in all tissues. Based on all findings, it was proposed that JunB, JunD, and Fra-1 at AP1-5 site, Sp1 at Sp1 site, and unknown differentiation regulatory factors at ISE forms a transcriptional factor complex on distal enhancer, while C/EBP factors included complex forms on proximal region and together activate differentiation-appropriate IVL gene expression (Crish et al. 2006). The transcriptional silencer region helps in the regulation of IVL expression through a mechanism that includes AP1 related activators and transcriptional repressors targeting the silencer region (Alvarez-Salas et al. 2005).
Involucrin, Fig. 1

Structure of involucrin gene. The gene is comprised of exons 1 and 2 (blue boxes) separated by an intron (black box) and a noncoding sequence is located at 5′ noncoding region of exon 1. The noncoding region is composed of three regulatory regions: distal enhancer/regulatory region (DER/DRR), transcriptional silencer (pink box), proximal enchancer/promoter region (PER/PPR) with activator protein (AP)1 (green box), AP1-5 (red box), and special protein (Sp1) (purple box) located on the DER/DRR segment and CCAAT enhancer binding protein (C/EBP) (light blue box) and AP1 sites located on PER/PPR segment. The exon 2 codes for involucrin gene and is comprised of ancestral and modern segment (central segment) (red box).

The second exon consists of coding region and comprises of ancestral and modern segment (central segment). The central segment is comprised of 39 highly conserved repeats of a ten amino acid sequence having the consensus sequence (GLU-GLN-GLN-GLY-LEU-LYS-HIS-LEU) with an addition of 19 repeats of lesser similarity of which 15 are present at the amino terminus and four at the carboxyl terminus (Yaffe et al. 1992). The repeating structure of the central segment is conserved in IVL from all higher primates, although the number of tandem repeats varies (Tseng and Green 1988). The glutamic acid and glutamine residues are spaced at regular intervals along the entire molecule such that they are pointing out away from the axis of helical structure, thus contributing to reactivity and accessibility of the gene for reaction (Eckert and Rorke 1989). Also, the intron comprises of interesting features including a consensus “Blessing” sequence (bp 618–625 of intron) and a stem-loop structure (bp 712–732 of intron) that have been found to be important in cotranscriptional regulation of various genes. Studies have also shown that both intron and distal/proximal regions are essential for high levels of tissue-specific expression, though the mechanism behind has yet to be understood. Interplay between the negative and positive components of the intron with sequences upstream in the distal region may result in the strata-specific pattern of IVL expression observed in keratinocytes (Carroll and Taichman 1992).

Structure and Distribution

Based on computer modeling, circular dichorism, and electron microscopy, IVL structure was proposed. It appears as an elongated flexible rod with a central segment along with the C- and N- terminal . The entire central segment and adjacent regions of the amino and carboxy terminal flanking segments are composed of long stretches of α-helical structures separated by short random coil segments in vicinity of proline residues. The amino and carboxy terminal ends are predicted to contain three and four β-sheet domains, respectively, with each β-sheet segment separated again by a series of short random coil segments. The central α-helical structure is hydrophilic with highly similar ten amino acid repeats with each repeat containing at least three glutamine residues, while the two terminals are hydrophobic The axial ratio (i.e., length:width) of the gene is calculated to be 30:1 (Rorke and Eckert 1991; Yaffe et al. 1992).

IVL is present in cells of the outer half of stratum spinosum and granulosum of human epidermis. Human stratified epithelia from conjunctiva, cornea, tongue, esophagus, and vagina also showed its distributions in the same stratum. Even human stratified epithelium lacking stratum corneum has demonstrated IVL expression. The inner or scaffold layer of cornified epithelium is composed of various proteins, of which IVL forms a major component. In addition to acting as a scaffolding protein, it also acts as a scaffold to which lipids (predominantly ceramides, but also cholesterol esters and free fatty acids) are covalently attached (Presland and Dale 2000). Cultured human keratinocytes of any origin also demonstrated it. Though involucrin is not expressed in basal layer, studies have demonstrated its expression several layers beyond (suprabasally), thus suggesting onset of its synthesis to be after cessation of cell division but before occurrence of cross-linking (Banks Schlegel and Green 1981). Murphy et al. in their study demonstrated that cells of upper third dermis exhibit a homogeneous cytoplasmic structure of the IVL gene. It was also exhibited in the distal portion of the eccrine ducts and acrosyringium, cells forming inner root sheath of the hair follicle, the lining of apocrine ducts, follicular infundibula, and isthmi. Normal orthokeratinized epithelia showed intracytoplasmic or pericellular staining in the suprabasal epithelial layers in a pattern similar to that of the normal epidermis. Parakeratinized and nonkeratinized epithelia were less stained (Murphy et al. 1984).

Involucrin in Health

The human keratinocytes leave the proliferation pool (basal cell layer) to enter the differentiation pool by undergoing certain changes like increase in size and incorporation of some specialized molecules like IVL, a soluble protein precursor of the cross-linked envelope. The envelope is a highly insoluble structure (10–16 nm thick) present beneath the plasma membrane, which is formed during terminal differentiation of keratinocytes. The stabilization of the envelope occurs via the cross-linking of various proteins, including IVL, loricrin, and small proline-rich proteins (cornifin or pancomulin) by formation of γ-glutamyl-ε-lysine isopeptide bonds mediated by calcium-dependent transglutaminase. The whole process of assembly of the envelope occurs in two steps as proposed by Eckert et al. (1993). In the first step, deposition of soluble precursors including IVL and small proline-rich proteins occurs which is dependent on only the membrane-bound transglutaminase, whereas in the second step, deposition of insoluble precursors such as loricrin occurs which depends on both the membrane-bound and soluble transglutaminases. So, IVL immunoreactivity is mainly restricted to those cells beneath the stratum corneum (stratum granulosum and upper layer of stratum spinosum) that are about to undergo terminal differentiation (Ishida Yamamoto and Iizuka 1995).

Involucrin in Disease

Apart from its physiological cytoplasmic expression in keratinocytes, alterations in involucrin expression also reflect a lack of normal cellular coordination in preterminal differentiation in hyperplastic, benign, and malignant proliferations of squamous epithelium/epidermis (Fig. 2).
Involucrin, Fig. 2

Physiological and pathological expression of IVL. In epidermis and orthokeratinized epithelium, IVL expression is normally restricted to spinous and granulosum layers but mild expression is observed in parakeratinized and nonkeratinized epithelium. The pathological expression of IVL is noticed in various diseases of skin, mucous membrane, and tumors of odontogenic and salivary gland origin.

In Skin

Among major clinical studies, Kanitakis et al. conducted an extensive study on IVL expression in various genetic skin disorders. They observed varied expression of IVL, based on which the diseases were divided into two groups: disorders like collodion baby, Darier’s disease, Flegel’s disease, erythrokeratoderma variabilis, epidermal nevus with epidermolytic hyperkeratosis, and congenital bullous and nonbullous ichthyosiform erythroderma showed intense increase in IVL expression, whereas diseases like ichthyosis vulgaris, X-linked ichthyosis, confluent and reticulate papillomatosis, and simple epidermal nevus showed no to mild increase in its expression. Altered IVL expression in some of the keratinization disorders, not all, threw lights on etiopathogenesis of these skin disorders other than disturbances due to only keratin metabolism (Kanitakis et al. 1987). Premature expression (lower epidermal layer) and altered intracellular staining (both cytoplasmic and membranous) of IVL was noticed in Darier’s disease, an autosomal dominant skin disorder characterized by acantholysis and abnormal keratinization (Kassar et al. 2008).

In context of IVL expression in other skin lesion or tumors, Murphy et al. conducted an exclusive study which showed similar observation in normal skin but epidermal hyperplasia and benign lesions such as lichen planus and keratoacanthoma showed similar or little variation in expression pattern to that of normal skin. In contrast, lesions with hyperplasia of basaloid keratinocytes such as seborrheic keratosis and basal cell carcinoma showed negative staining for IVL. IVL was readily detectable with more uniform and diffuse patterns of reactivity in benign lesions characterized by proliferation of squamoid epithelium, whereas abnormal patterns (patchy immunoreactivity) of IVL reactivity were emphasized in malignant squamoid lesion i.e. squamous cell carcinoma. These alterations in IVL expression reflect uncoordinated preterminal differentiation in benign and malignant proliferations of squamous epithelium (Murphy et al. 1984). Said et al. in their studies demonstrated expression of IVL extended to adjacent normal basal layer areas of squamous cell carcinoma, probably suggestive of early dysplastic or preneoplastic stage (Said et al. 1984).

In Mucosa

Expression of IVL has also been observed in various preneoplastic and neoplastic lesions of mucous membrane of cervix, pharynx, and oral cavity. In cervix, Sassoon et al. reported diminished immunohistochemical expression as the degree of cell differentiation/squamous differentiation decreased in intraepithelial neoplasia. In both microinvasive and infiltrative squamous cell carcinoma, they noticed strong immunoreactivity of IVL, especially in differentiated area. So they proposed loss of IVL staining could be used as a criterion for assessing neoplastic transformation in cervical epithelium (Sassoon et al. 1985). In oral cavity, leukoplakia showed intense staining whereas lesser intensity but a distinct staining pattern (homogeneous pericellular staining) was observed in lichen planus. Itioz et al. also observed irregular but marked staining pattern in verrucous carcinoma, whereas distribution pattern of IVL was well correlated to the degree of cell differentiation ranging from carcinoma in situ to invasive and infiltrative squamous cell carcinoma (Itioz et al. 1986). Furthermore, Eisenberg et al. observed the importance of irregular IVL expression in dysplastic lichenoid lesions despite squamoid differentiation as it could support disturbances in terminal differentiation (Eisenberg et al. 1987).

In addition, few authors also studied the IVL expression in odontogenic cyst and tumors and salivary gland lesions. IVL positivity was observed in acanthomatous or follicular ameloblastoma. Strong immunoreactivity of IVL was observed in squamous odontogenic tumors, whereas adenomatoid odontogenic tumors gave a negative staining reaction. Among odontogenic cysts, radicular cysts showed a very irregular distribution of IVL where stratification of squamous epithelium was found to be a critical factor for its immunopositivity (Yamada et al. 1989). Sumimoto et al. conducted a study on IVL reactivity in salivary gland and associated lesion. In normal glandular tissues and obstructive sialadenitis, IVL staining was negative. In pleomorphic adenomas, the luminal surface of tubular, ductal, duct-like, and cystic structures was positive for IVL, whereas myoepithelial cells surrounding the duct-like structure were found to be immunonegative for IVL staining. They observed the potential of IVL as a specific marker for detecting squamous metaplasia or keratinizing change in the epithelia of salivary gland tumors (Sumitomo et al. 1986).


IVL is strictly expressed in upper spinous and granular layers of epithelium/epidermis. Decreased and irregular expression of IVL in various keratin disorders, immune-related, preneoplastic, and neoplastic disorders indicates about its potential as a marker for abnormal squamous cell differentiation and maturation. It can also be very useful as an adjunct to routine histopathological diagnosis of keratinizing lesions of both skin and mucosa.


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© Springer International Publishing AG 2018

Authors and Affiliations

  • Niharika Swain
    • 1
  • Rashmi Maruti Hosalkar
    • 2
    • 4
  • Samapika Routray
    • 3
  1. 1.MGM Dental College and HospitalNavi MumbaiIndia
  2. 2.Indian Association of Oral and Maxillofacial PathologistsMumbaiIndia
  3. 3.Department of Dental SurgeryAll India Institute of Medical SciencesBhubaneswarIndia
  4. 4.Maharashtra State Dental CouncilMumbai, MaharashtraIndia