Expression Of Involucrin Research Articles

Significance of Immunohistochemical Expression of p27 and Involucrin as the Marker of Cellular Differentiation of Squamous Cell Carcinoma of the Esophagus

Purpose: p27kip1 belongs to the KIP/CIP family of cyclin-dependent kinase inhibitors and is considered to be a tumor suppressor. Involucrin has been known as a marker of differentiation of squamous cell carcinoma (SCC). The aim of this study was to evaluate the clinicopathologic significance of the expression of p27 and involucrin in esophageal SCC. Methods: Immunohistochemical expression of p27 and involucrin was examined in 70 specimens of esophageal SCC. The correlation of the expression of these proteins and clinicopathologic features was evaluated. Results: Cellular differentiation in esophageal SCC was significantly correlated with the expression of p27 and involucrin (p = 0.010 and p = 0.002, respectively). Among well, moderately and poorly differentiated SCCs, 45.8 ± 21.6, 20.0 ± 15.0 and 10.6 ± 9.1% of carcinoma cells expressed involucrin, respectively (p < 0.0001 for well vs. poorly, p < 0.0001 for well vs. moderately, and p = 0.042 for moderately vs. poorly). There existed a more powerful statistical difference regarding the histological grade between SCCs with the expression of both p27 and involucrin and tumors with other expression patterns (p = 0.0001). Conclusions: Expression of both p27 and involucrin can be a powerful biological marker of cellular differentiation of esophageal SCC.

Novel synthetic ceramide derivatives increase intracellular calcium levels and promote epidermal keratinocyte differentiation

Ceramide is an important constituent of stratum corneum lipids, which act as both physical barriers and signal modulators. We synthesized several ceramide derivatives and investigated their effects on keratinocyte differentiation. RT-PCR and Western blotting showed that the novel synthetic ceramide derivatives K6PC-4 [N-(2,3-dihydroxypropyl)-2-hexyl-3-oxo-decanamide], K6PC-5, [N-(1,3-dihydroxypropyl-2-hexyl-3-oxo-decanamide] and K6PC-9 (N-ethanol-2-hexyl-3-oxo-decanamide) [corrected] These ceramide derivatives elicited a rapid transient increase in intracellular calcium levels, which were measured using laser scanning confocal microscopy. In addition, K6PC-4, K6PC-5, and K6PC-9 stimulated the phosphorylation of p42/44 extracellular signal-regulated kinase and c-Jun N-terminal kinase. In a reconstituted epidermis model, K6PC-4, K6PC-5, and K6PC-9 significantly increased keratin 1 expression in the suprabasal layer. These results indicate that these novel synthetic ceramide derivatives have the potential to promote keratinocyte differentiation, suggesting that the lipid molecules are applicable for treating skin diseases involving abnormal keratinocyte differentiation.

Time-course study of histological and genetic patterns of differentiation in human engineered oral mucosa

The lack of sufficient oral mucosa available for intra-oral grafting is a major surgical problem, and new sources of oral tissues for clinical use are needed. In this regard, some models of engineered oral mucosa have been reported to date, but little is known about the structural and genetic mechanisms that occur during the process of development and maturation of these tissue substitutes. We have carried out a time-course study of the genes and morphological patterns of cell and tissue differentiation that develop in oral mucosa constructs after 3, 7, 11 and 21 days of development. Our electron microscopy and microarray analyses demonstrated that the oral mucosa constructs generated by tissue engineering undergo a progressive process of cell differentiation with the sequential formation and maturation of several layers of epithelium (with expression of stratifin, sciellin, involucrin, trichohyalin and kallikrein 7), intercellular junctions (with expression of plakophilin, desmocollin, desmoglein and cadherins), cytokeratins, a basement membrane (laminins, collagen IV) and the extracellular matrix (biglycan, matrix metalloproteinases). In conclusion, although the level and type of keratinization developed in vitro could be different, the oral mucosa substitutes were very similar to the native tissues.

Simultaneous effect of ursolic acid and oleanolic acid on epidermal permeability barrier function and epidermal keratinocyte differentiation via peroxisome proliferator‐activated receptor‐α

Ursolic acid (UA) and oleanolic acid (ONA) are pentacyclic triterpenoids, which naturally occur in many medicinal herbs and plants. Recent research revealed that several pharmacological effects could be attributed to UA and ONA, such as anti-tumor, anti-inflammatory and anti-microbial activities. To evaluate the effects of UA and ONA on epidermal permeability barrier recovery and normal skin, both flanks of hairless mice were topically treated with either 0.01-0.1 mg/mL UA or 0.1-1.0 mg/mL ONA after tape stripping and transepidermal water loss (TEWL) were assessed, and then hydration and TEWL were measured for 3 weeks with application of UA and ONA (2 mg/mL). We also investigated the morphological changes using light (LM) and electron microscopic (EM) examination. Finally, we observed that UA and ONA stimulated epidermal keratinocyte differentiation via peroxisome proliferator-activated receptor (PPAR)-alpha using Western immunoblotting. The recovery rate of epidermal permeability barrier after tape stripping increased in the UA- and ONA-treated groups (0.1 mg/mL UA and 0.5 mg/mL ONA) at 6 h to more than 20% when compared to the vehicle-treated group (P < 0.05). In both groups, hydration was increased compared to the vehicle group from 1 week without TEWL alteration (P < 0.05). An LM finding showed that epidermal thickening was frequently observed (UA > ONA > vehicle). EM examination revealed an increase in secretion and in the number of lamellar bodies in treated groups and that complete formation of lipid bilayers was also prominent (ONA > UA > vehicle). Protein expression of PPAR-alpha, involucrin, loricrin and filaggrin increased twofold and threefold in HaCaT cells treated for 24 h with either ONA (10 micromol/L) or UA (10 micromol/L), respectively, reflecting that the UA and ONA can improve the recovery of skin barrier function and induce epidermal keratinocyte differentiation via PPAR-alpha. Taken together, these results suggest that UA and ONA will be pertinent candidates for the improvement of epidermal permeability barrier function.

Hypoxia Enhances the Expansion of Human Limbal Epithelial Progenitor Cells In Vitro

To demonstrate the effects of hypoxia on proliferation and differentiation of human limbal epithelial cells in vitro. Primary human limbal epithelial cells were harvested from the rim of donor corneas. Colony-forming efficiency (CFE) and cell proliferation were observed in standard (20% O(2)) or hypoxic (2% O(2)) culture conditions. Cell cycle, forward scatter (FSC) and side scatter (SCC) of cells were analyzed by flow cytometry. Proliferating cells were also observed by pulse labeling (2 hours) with BrdU and Ki67 staining. Apoptosis was detected by TUNEL assay. Isolated colonies were examined by immunohistochemistry against K15, p63, involucrin, and K3. Involucrin expression was also analyzed by Western blot analysis. Both CFE and proliferation of limbal epithelial cells was significantly enhanced in hypoxia. Flow cytometry revealed a higher fraction of hypoxic cells in the G(0)/G(1)-phase and fewer cells in the S-phase, compared with normoxia. However, there was no difference in the uptake of BrdU during a 2-hour pulse, suggesting that hypoxic colonies contained rapidly cycling cells. Apoptotic cells were sparse in both groups, and hypoxic cells showed lower FSC compared with normoxic cells. Although there was no difference in the staining pattern of K15, p63, and Ki67, cells cultivated in normoxia expressed higher levels of the differentiation markers involucrin and K3. Significantly higher involucrin expression was also observed by Western blot. Hypoxic culture (2%) enhances proliferation while inhibiting differentiation of limbal epithelial cells in vitro.

Involucrin Expression Is Decreased in Hailey–Hailey Keratinocytes Owing to Increased Involucrin mRNA Degradation

Hailey-Hailey disease (HHD) (MIM 16960) is an autosomal-dominant blistering skin disease caused by a mutation in the Ca2+-ATPase ATP2C1 (protein SPCA1), responsible for controlling Ca2+ concentrations in the cytoplasm and Golgi in human keratinocytes. Cytosolic Ca2+ concentrations, in turn, play a major role in the regulation of keratinocyte differentiation. To study how ATP2C1 function impacts keratinocyte differentiation, we assessed involucrin expression in HHD keratinocytes. Involucrin is a protein that makes up the cornified envelope of keratinocytes and is expressed in response to increased intracellular Ca2+ concentrations. Even though HHD keratinocytes suffer from abnormally high cytosolic Ca2+, we found that these cells expressed lower involucrin protein levels at both low and high extracellular Ca2+ concentrations when compared with normal control keratinocytes. Decreased involucrin protein levels were caused by lower involucrin mRNA levels in HHD keratinocytes. Decreased involucrin mRNA, in turn, was caused by increased rates of involucrin mRNA degradation. Ca2+-sensitive involucrin AP-1 promotor activity was increased, both in HHD keratinocytes and in an small interfering RNA (siRNA) experimental model, suggesting compensatory promoter upregulation in the face of increased mRNA degradation. This report provides new insights into differentiation defects in HHD and its relationship to Ca2+ signaling.

STAT5a/PPARγ Pathway Regulates Involucrin Expression in Keratinocyte Differentiation

Signal transducers and activators of transcription (STATs) are critical to growth factor-mediated intracellular signal transduction. We observed the rapid expression and activation of STAT5a during keratinocyte differentiation induced by suspension culture. STAT5a expression preceded that of involucrin, an important molecule in the terminal differentiation of keratinocytes. To determine whether STAT5a regulated involucrin expression, we expressed a dominant-negative (dn) STAT5a that blocks the dimerization of STAT5 and inhibits its nuclear translocation. We found that dn-STAT5a inhibited involucrin expression in keratinocytes. Given that STAT5 regulates adipogenesis via activating the peroxisome proliferator-activated receptor (PPAR) gamma signal, we hypothesized that STAT5a regulated involucrin expression in the same manner. To test this hypothesis, we examined the expression and transactivation of PPARgamma in a suspension culture of keratinocytes. Suspension culture induced PPARgamma expression and triggered PPARgamma transactivation rapidly and dn-STAT5a downregulated this induction and suppressed PPARgamma transactivation. Furthermore, preincubation with the PPARgamma/retinoid X-receptor inhibitor HX-531 or the introduction of a dn-PPARgamma prevented the activation of involucrin promoter and inhibited its induction. This report provides early evidence of a major role for STAT5a in the differentiation of keratinocytes, where it contributes to involucrin expression by activating the PPARgamma signal.

Barrier Function, Epidermal Differentiation, and Human β-Defensin 2 Expression in Tinea Corporis

Barrier Function, Epidermal Differentiation, and Human β-Defensin 2 Expression in Tinea Corporis

Culture of Keratinocytes for Transplantation without the Need of Feeder Layer Cells.

Patients with large burn wounds have a limited amount of healthy donor skin. An alternative for the autologous skin graft is transplantation with autologous keratinocytes. Conventionally, the keratinocytes are cultured with mouse feeder layer cells in medium containing fetal calf serum (FCS) to obtain sufficient numbers of cells. These xenobiotic materials can be a potential risk for the patient. The aim of the present study was to investigate if keratinocytes could be expanded in culture without the need of a feeder layer and FCS. Keratinocytes were cultured on tissue culture plastic with or without collagen type IV coating in medium containing Ultroser G (serum substitute) and keratinocyte growth factor (KGF). An in vitro skin equivalent model was used to examine the capacity of these cells to form an epidermis. Keratinocytes in different passages (P2, P4, and P6) and freshly isolated cells were studied. Keratinocytes grown on collagen type IV were able to form an epidermis at higher passage numbers than cells grown in the absence of collagen type IV (P4 and P2, respectively). In both cases the reconstructed epidermis showed an increased expression of Ki-67, SKALP, involucrin, and keratin 17 compared to normal skin. Only 50,000 keratinocytes grown on collagen type IV in P4 were needed to form 1 cm2 epidermis, whereas 150,000 of freshly isolated keratinocytes were necessary. Using this culture technique sufficient numbers of keratinocytes, isolated from 1 cm2 skin, were obtained to cover 400 cm2 of wound surface in 2 weeks. The results show that keratinocytes can be cultured without the need of a fibroblast feeder layer and FCS and that these cells are still able to create a fully differentiated epidermis. This culture technique can be a valuable tool for the treatment of burn wounds and further development of tissue engineered skin.

Subcellular localisation of BAG-1 and its regulation of vitamin D receptor-mediated transactivation and involucrin expression in oral keratinocytes: Implications for oral carcinogenesis

In oral cancers, cytoplasmic BAG-1 overexpression is a marker of poor prognosis. BAG-1 regulates cellular growth, differentiation and survival through interactions with diverse proteins, including the vitamin D receptor (VDR), a key regulator of keratinocyte growth and differentiation. BAG-1 is expressed ubiquitously in human cells as three major isoforms of 50 kDa (BAG-1L), 46 kDa (BAG-1M) and 36 kDa (BAG-1S) from a single mRNA. In oral keratinocytes BAG-1L, but not BAG-1M and BAG-1S, enhanced VDR transactivation in response to 1α,25-dihydroxyvitamin D 3. BAG-1L was nucleoplasmic and nucleolar, whereas BAG-1S and BAG-1M were cytoplasmic and nucleoplasmic in localisation. Having identified the nucleolar localisation sequence in BAG-1L, we showed that mutation of this sequence did not prevent BAG-1L from potentiating VDR activity. BAG-1L also potentiated transactivation of known vitamin-D-responsive gene promoters, osteocalcin and 24-hydroxylase, and enhanced VDR-dependent transcription and protein expression of the keratinocyte differentiation marker, involucrin. These results demonstrate endogenous gene regulation by BAG-1L by potentiating nuclear hormone receptor function and suggest a role for BAG-1L in 24-hydroxylase regulation of vitamin D metabolism and the cellular response of oral keratinocytes to 1α,25-dihydroxyvitamin D 3. By contrast to the cytoplasmic BAG-1 isoforms, BAG-1L may act to suppress tumorigenesis.

State of differentiation defines buccal epithelial cell affinity for cross‐linking to Candida albicans Hwp1

Candida albicans utilizes mammalian cell-associated transglutaminase (TGase) activity to adhere covalently to human buccal epithelial cells (BECs) through Hyphal Wall Protein 1. Little is known about the factors leading to the identity and appearance of Hwp1 binding partners on cells lining the oral cavity. The observation that BECs vary in their ability to attach to C. albicans germ tubes and to bind recombinant Hwp1 (rHwp1) suggested that differentiation may play a role in affinity for germ tube attachment. Individual BECs were characterized for differentiation status and rHwp1 binding. rHwp1 bound to the more terminally differentiated cells displaying SPR3 and keratin 13 but not to less differentiated cells with abundant involucrin. Sequential expression of involucrin followed by SPR3 in oral keratinocytes was demonstrated using stratified organotypic cultures and a feeder layer system with the OKF6/TERT-2 cell line. Increased cross-linking of the lysine analogue 5-(biotinamido)pentylamine to cultured OKF6/TERT-2 cell proteins accompanied this increased expression of SPR3. Western blot analysis demonstrated the presence of rHwp1 cross-links to proteins from BECs or from OKF6/TERT-2 cells that had been mechanically dislodged from culture dishes. Therefore, the differentiation of SPR3 positive from involucrin positive cells is correlated with the acquisition of affinity for cross-linking to rHwp1 and covalent adhesion of germ tubes to BECs.

Matrix Metalloproteinase-19 Expression in Keratinocytes Is Repressed by Transcription Factors Tst-1 and Skn-1a: Implications for Keratinocyte Differentiation

Matrix metalloproteinase-19 (MMP-19), unlike other members of the MMP family, is expressed in basal keratinocytes of intact epidermis whereas keratinocytes in suprabasal and higher epidermal layers express this enzyme only during cutaneous disorders. As the activity of MMP-19 effects proliferation, migration, and adhesion of keratinocytes we examined whether transcription factors involved in keratinocyte differentiation repress the expression of MMP-19. Using luciferase reporter assays, POU transcription factors Tst-1 (Oct-6) and Skn-1a (Oct-11) markedly downregulated the activity of MMP-19 promoter in COS-7 cells and HaCaT keratinocytes. Tst-1 alone was able to inhibit 85% of the promoter activity. Skn-1a exhibited a weak inhibitory effect although it synergistically increased effects of Tst-1. HaCaT cells stably transfected with Tst-1 showed a strong decrease of activity of MMP-19 promoter that correlated with suppression of MMP-19, cytokeratin 14 and 5, decreased cell proliferation, and altered expression of involucrin and loricrin. The expression of MMP-9 was also significantly reduced in Tst-1 expressing keratinocytes. MMP-2 was substantially affected during its activation whereas the expression of MMP-28 was unchanged. Our results suggest that Tst-1 and Skn-1a regulate expression of MMPs in keratinocytes and effect both the expression and activation of these proteolytic enzymes.

The Role of Proline-Rich Protein Tyrosine Kinase 2 in Differentiation-Dependent Signaling in Human Epidermal Keratinocytes

Non-receptor tyrosine kinase proline-rich protein tyrosine kinase 2 (Pyk2) functions as an integrator of multiple signaling pathways involved in the regulation of fundamental cellular processes. Pyk2 expression, regulation, and functions in skin have not been examined. Here we investigated the expression and subcellular localization of Pyk2 in human epidermis and in primary human keratinocytes, and studied the mechanisms of Pyk2 activation by differentiation-inducing stimuli, and the role of Pyk2 as a regulator of keratinocyte differentiation. We demonstrate that Pyk2 is abundantly expressed in skin keratinocytes. Notably, the endogenous Pyk2 protein is predominantly localized in keratinocyte nuclei throughout all layers of healthy human epidermis, and in cultured human keratinocytes. Pyk2 is activated by treatment with keratinocyte-differentiating agents, 12-O-tetradecanoylphorbol-13-acetate and calcium via a mechanism that requires intracellular calcium release and functional protein kinase C (PKC) and Src activities. Particularly, differentiation-promoting PKC delta and PKC eta elicit Pyk2 activation. Our data show that Pyk2 increases promoter activity and endogenous protein levels of involucrin, a marker of keratinocyte terminal differentiation. This regulation is associated with increased expression of Fra-1 and JunD, activator protein-1 transcription factors known to be required for involucrin expression. Altogether, these results provide insights into Pyk2 signaling in epidermis and reveal a novel role for Pyk2 in regulation of keratinocyte differentiation.

Gene expression profiling analysis of solar lentigo in relation to immunohistochemical characteristics

Solar lentigo appears as dark brown spots that occur on sun-exposed areas and is considered to be a hallmark of aged skin. Although considerable knowledge about acute pigmentation has recently been accumulated, little is yet known about the mechanisms underlying chronic- and delayed-type hyperpigmentation, such as solar lentigo. To clarify further the mechanisms underlying the development of solar lentigo, we carried out gene expression analysis in skin biopsy specimens obtained from human solar lentigines using DNA microarray analysis. Two pairs of skin specimens were obtained from solar lentigo and adjacent sun-exposed normal skin, as well as normal skin on the buttocks of 16 volunteers aged 40-55 years. One set of specimens was frozen and RNA was extracted for microarray and the other set was prepared for histological sections and analysed by antibodies and probes. Sixty-five genes were upregulated more than 1.8-fold in solar lentigo compared with adjacent control skin and seven melanocyte-related genes were included. Compared with sun-protected skin, many inflammation-related genes were upregulated in solar lentigo, and compared with sun-exposed control skin, upregulation of genes related to fatty-acid metabolism was apparent in solar lentigo. Moreover, we found downregulation of cornified envelope-related genes, which suggests suppression of cornification in the epidermis in solar lentigo. Immunohistochemically, larger numbers of TRP1-positive cells were found in the basal layer of solar lentigo than in normal skin. Fatty acid-related genes were highly expressed in the epidermis as detected by in situ hybridization, and they were much more prominent in the lesional skin of solar lentigo. However, cycling epidermal cells detectable with Ki67 antibody were fewer in the lesional skin of solar lentigo. Expression of filaggrin and involucrin was decreased in the lesional skin, where the number of cell layers of the stratum corneum was significantly higher than in normal skin. The results of the present microarray analysis of solar lentigo, demonstrating upregulation of genes related to inflammation, fatty-acid metabolism and melanocytes and downregulation of cornified envelope-related genes, suggest that solar lentigo is induced by the mutagenic effect of repeated ultraviolet light exposures in the past, leading to the characteristic enhancement of melanin production, together with decreased proliferation and differentiation of lesional keratinocytes on the background of chronic inflammation.

Endothelin-2/vasoactive Intestinal Contractor via ROCK regulates transglutaminase 1 on differentiation of mouse keratinocytes

We previously found that endothelin-2/vasoactive intestinal contractor (ET-2/VIC) greatly increased in mouse epidermis after birth. In the present study, we evaluated whether ET-2/VIC expression was associated with the calcium-induced differentiation of cultured mouse keratinocytes. The differentiation induction was revealed by morphological change, cornified envelope (CE) formation, and involucrin and transglutaminase 1 (TG 1) expressions. ET-2/VIC gene expression and peptide production subsequently increased in the induction of the differentiation. We also found that Y-27632, a Rho-associated coiled–coil forming protein serine/threonine kinase (ROCK) inhibitor, suppressed up-regulation of ET-2/VIC gene expression, the induction of morphological change, the CE formation, and TG 1 expression, but not involucrin expression. These results indicate new three findings, (1) ET-2/VIC expression increases and has potential as a differentiation marker, (2) ET-2/VIC expression is mediated by ROCK, and (3) the ROCK regulated TG 1 expression, on the calcium-induced differentiation of mouse keratinocytes.

Curcumin Suppresses AP1 Transcription Factor-dependent Differentiation and Activates Apoptosis in Human Epidermal Keratinocytes

The diet-derived cancer preventive agent, curcumin, inhibits skin cancer cell proliferation and tumor formation. However, its effect on normal human keratinocyte differentiation, proliferation, and apoptosis has not been adequately studied. Involucrin (hINV) is a marker of keratinocyte differentiation and a useful model for the study of chemopreventive agent action. We show that curcumin suppresses the differentiation agent-dependent activation of hINV gene expression and that an AP1 transcription factor DNA binding site in the hINV gene is required for this regulation. A protein kinase C, Ras, MEKK1, MEK3 signaling cascade controls hINV expression by regulating AP1 factor level. Curcumin treatment inhibits the novel protein kinase C-, Ras-, and MEKK1-dependent activation of hINV promoter activity and reduces the differentiation agent-dependent increase in AP1 factor level and DNA binding. This reduction requires proteasome function. In addition, curcumin treatment reduces cell number, which is associated with a reduced cyclin and cdk1 levels. Curcumin treatment also suppresses the Bcl-xL level, leading to reduced mitochondrial membrane potential and increased cleavage of procaspases and poly(ADP-ribose) polymerase. These studies provide important insights regarding the mechanism whereby curcumin acts as a chemopreventive agent in normal human epidermis.

Autocrine Extracellular Signal-regulated Kinase Activation in Normal Human Keratinocytes is not Interrupted by Calcium Triggering and is Involved in the Control of Cell Cycle at the Early Stage of Calcium-induced Differentiation

Normal human epidermal keratinocytes (NHEK) respond to the autocrine activated extracellular signal-regulated kinase (ERK) signaling pathway, which contributes to the survival of keratinocytes. However, during the condition of calcium-induced differentiation, how the autocrine ERK signaling is regulated and affected is poorly understood. The purpose of this study was to understand and to obtain clues to the possible function of the autocrine ERK activation during the calcium-induced differentiation of NHEK. We demonstrated that the autocrine activated ERK was not interrupted by calcium triggering and that it was sustained for at least one day after changing the medium. We also found that the autocrine ERK activation was associated with the expression of cyclin D1 and the cell cycle regulation at the early stage of calcium triggering by treating the cells with the mitogen-activated protein kinase inhibitor PD98059. However, the PD98059 treatment did not have a significant influence on the expression of involucrin and loricrin. In addition, we demonstrated that autocrine ERK activation was associated with protein kinase C and p38MAPK signaling. We suggest that the activation of autocrine ERK is not interrupted by calcium triggering and it might participate in cell growth during the early stage of calcium-induced differentiation in NHEK.

Spontaneous trichoepithelioma in a laboratory mouse: gross, microscopic and immunohistochemical findings

A spontaneous trichoepithelioma occurred in a Swiss OF1 outbred, four-month-old, intact, nulliparous female mouse from a breeding colony. At necropsy, the tumour was a single, well-delineated mass measuring 4.2 cm in major diameter, located in the thoracic region and had an intact haired surface. The regional lymph nodes were not enlarged and no other abnormalities were found. Microscopically, it was composed of a random admixture of budding epithelial islands and cystic structures variable in size. The epithelial islands were composed of basaloid cells. The cystic structures were lined by squamous epithelium with or without a granular cell layer and contained lamellar or amorphous keratin, as well as wide areas of matrical keratinization (ghost cells) with or without a peripheral layer of basaloid cells and calcified contents. Mitotic activity of basaloid cells was moderate to high, but nuclear or mitotic atypia were not observed. High and low molecular weight cytokeratins, profilaggrin and involucrin expression were observed in the tumour. The immunohistochemical profile of this rare type of tumour of the skin of mice, which includes a first-time description of involucrin expression, confirms the histological evidence of differentiation towards more than one segment of follicular epithelium.

Effects of methyl paraben on skin keratinocytes

Some ingredients of dermatological formulations result in skin irritation and allergy. In particular, preservatives have been reported extensively as a cause of allergic contact dermatitis. The study focused on parabens which have been used extensively as antimicrobial preservatives in foods, drugs and cosmetics. The aim of this study was to clarify the effects of the daily use of methyl paraben (MP) on human skin. The concentrations of MP in the stratum corneum (SC) of the human forearm were measured using the cup method and GC-MS after daily applications of MP containing formulations. The study also investigated the effects of long-term exposure to MP on keratinocytes in vitro. Normal human keratinocytes and the skin equivalents were cultured in the medium containing MP. The following changes were analysed: proliferating ability, apoptotic cells, morphological changes, mRNA and protein expressions. After 1 month of daily applications of MP containing formulations, MP remained unmetabolized and persisted slightly in the SC. MP decreased the proliferating ability of keratinocytes and changed the cell morphology. MP also decreased the expressions of hyaluronan synthase 1 and 2 mRNAs and type IV collagen. In contrast, it increased the expressions of involucrin and HSP27. Furthermore, MP influenced the epidermal differentiation of the skin equivalent. These results suggest that MP exposure through application of dermatological formulations results in MP persistence and accumulation in the SC, and that MP might influence the aging and differentiation of keratinocytes.

Transglutaminase inhibitors induce hyperproliferation and parakeratosis in tissue-engineered skin

The transglutaminase (TG) family consists of eight distinct isoforms. TG types 1, 3 and 5 play a major role in normal skin development, with TG2 also being elevated during dermal wounding. TG1, 3 and 5 are responsible for the cross-linking of keratin precursors and formation of the cornified envelope during keratinocyte differentiation. TG2 may play a role in keratinocyte basement membrane cross-linking. Abnormal TG expression has been demonstrated in Darier disease, Netherton syndrome, psoriasis and lamellar ichthyosis. During a recent investigation of skin contraction in tissue-engineered skin, transglutaminase inhibitors were found to produce hyperproliferation and parakeratosis. Accordingly, this study was designed to study the effect of pan-transglutaminase inhibition on morphology of tissue-engineered skin and expression of keratinocyte differentiation and proliferation-associated antigens. We used a tissue-engineered model of human skin, based on de-epidermized acellular human dermis, seeded with normal keratinocytes and dermal fibroblasts and cultured at an air-liquid interface. The pan-transglutaminase inhibitors putrescine, NTU283 (1-dimethyl,2-[(oxopropyl)thio]imidazolium) and NTU285 (N-benzyloxycarbonyl-l-glutaminyl-6-dimethylsulfonium-5-oxo-l-norleucine) were added to the culture medium. After 28 days, histology and immunohistochemistry for collagen IV, involucrin and cytokeratins 6, 10 and 16 were performed. Keratinocyte hyperproliferation and parakeratosis were seen in response to transglutaminase inhibition. Inhibition of transglutaminase also resulted in loss of basement membrane collagen IV. Involucrin and cytokeratins 6 and 16 were confined to the basal layers in control composites but expressed throughout the epidermis in response to transglutaminase inhibition. A distinct band of expression of cytokeratin 10 was seen in the upper stratum granulosum of control composites but only patchy expression was seen after transglutaminase expression. Pan-transglutaminase inhibition inhibits terminal differentiation of keratinocytes, leading to a hyperproliferative epidermis with parakeratosis and enhanced expression of involucrin and cytokeratins 6 and 16. Expression of the differentiation-associated cytokeratin, cytokeratin 10, is reduced. Basement membrane integrity is also lost as a result of transglutaminase inhibition.