AP1 (jun/fos) transcription factors (c-jun, junB, junD, c-fos, FosB, Fra-1, and Fra-2) are key regulators of epidermal keratinocyte survival and differentiation and important drivers of cancer development. Understanding the role of these factors in epidermis is complicated by the fact that each protein is expressed, at different levels, in multiple cells layers in differentiating epidermis, and because AP1 transcription factors regulate competing processes (i.e., proliferation, apoptosis, and differentiation). Various in vivo genetic approaches have been used to study these proteins including targeted and conditional knockdown, overexpression, and expression of dominant-negative inactivating AP1 transcription factors in epidermis. Taken together, these studies suggest that individual AP1 transcription factors have different functions in the epidermis and in cancer development and that altering AP1 transcription factor function in the basal versus suprabasal layers differentially influences the epidermal differentiation response and disease and cancer development.
Cholesterol has been recently suggested to regulate the early steps of keratinocyte differentiation through lipid rafts. In many cell types, depletion of cholesterol activates signaling proteins like epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), or extracellular signal-regulated kinase (ERK) known to affect cell differentiation. In this study, we explored the effects of cholesterol depletion on the phenotype of cultured keratinocytes, using a treatment with methyl-beta-cyclodextrin (MbetaCD) to extract cholesterol and a treatment with lovastatin to inhibit cholesterol neosynthesis. Analysis of the expression of differentiation marker genes in early differentiating confluent cultures reveals that cholesterol depletion induces downregulation of keratin 14 (K14) and keratin 10 (K10) and upregulation of involucrin. MbetaCD treatment induces phosphorylation of EGFR, HER2, and ERK, but not HER3. Inhibition of EGFR with PD153035 impairs the MbetaCD-induced phosphorylation of EGFR, HER2, and ERK, but does not impair the alteration of K14, K10, or involucrin gene expression, indicating that other signaling proteins regulate this phenomenon. p38 has been suggested to regulate the expression of involucrin during keratinocyte differentiation. We found that MbetaCD treatment induces a prolonged phosphorylation of p38 in general and p38alpha in particular. An inhibition of p38 with PD169316 impairs the upregulation of involucrin mRNAs by a treatment with MbetaCD, but not by a p38delta-activating TPA treatment, which might suggest that cholesterol depletion alters involucrin gene expression through activation of p38alpha/beta.
Tazarotene-induced gene 3 (TIG3) regulates keratinocyte terminal differentiation by activating type I transglutaminase (TG1). TIG3 consists of an amino-terminal (N-terminal) segment, that encodes several conserved motifs, and a carboxy-terminal (C-terminal) membrane-anchoring domain. By producing a series of truncation mutants that remove segments of the N-terminal region, and monitoring the ability of each mutant to co-precipitate TG1, function as a TG1 substrate, or functionally localize with TG1 in cells, we show that the TIG3 domain that interacts with TG1 is located within a TIG3 segment spanning amino acids 112-164. Although they bind TG1, TIG3 mutants lacking the conserved N-terminal region drive apoptosis-like cell death characterized by cell rounding, membrane blebbing, cytochrome c release, procaspase-3 and poly(ADP-ribose)polymerase (PARP) cleavage, and reduced p53 and p21 levels. Compared with TIG3, these truncated mutants have an increased tendency to associate with membranes. A mutant lacking the C-terminal membrane-anchoring domain is inactive. These findings suggest that TIG3 interaction with TG1 does not require the N-terminal conserved domains, that the TIG3 N-terminal region is required for TIG3-dependent keratinocyte differentiation, that its removal converts TIG3 into a proapoptotic protein, and that this change in action of TIG3 is associated with an intracellular redistribution.
AP1 (jun/fos) factors comprise a family of transcriptional regulators (c-jun, junB, junD, c-fos, FosB, Fra-1 and Fra-2) that are key controllers of epidermal keratinocyte survival and differentiation, and are important drivers of cancer development. Understanding the role of these factors in epidermis is complicated by the fact that each member is expressed in defined cell layers during epidermal differentiation, and because AP1 factors regulate competing processes (i.e., proliferation, apoptosis and differentiation). We have proposed that AP1 factors function differently in basal versus suprabasal epidermis. To test this, we inactivated suprabasal AP1 factor function in mouse epidermis by targeted expression of dominant-negative c-jun (TAM67) which inactivates function of all AP1 factors. This produces increased basal keratinocyte proliferation, delayed differentiation, and extensive hyperkeratosis. These findings contrast with previous studies showing that basal layer AP1 factor inactivation does not perturb resting epidermis. It is interesting that in spite of extensive keratinocyte hyperproliferation, susceptibility to carcinogen-dependent tumor induction is markedly attenuated. These novel observations strongly suggest that AP1 factors have distinct roles in the basal versus suprabasal epidermis, confirm that AP1 factor function is required for normal terminal differentiation, and suggest that AP1 factors play a different role in normal epidermis versus in cancer progression.
TIG3 is a tumor suppressor protein that limits keratinocyte survival during normal differentiation. It is also important in cancer, as TIG3 level is reduced in tumors and in skin cancer cell lines, suggesting that loss of expression may be required for cancer cell survival. An important goal is identifying how TIG3 limits cell survival. In the present study we show that TIG3 expression in epidermal squamous cell carcinoma SCC-13 cells reduces cell proliferation and promotes morphological and biochemical apoptosis. To identify the mechanism that drives these changes, we demonstrate that TIG3 localizes near the centrosome and that pericentrosomal accumulation of TIG3 alters microtubule and microfilament organization and organelle distribution. Organelle accumulation at the centrosome is a hallmark of apoptosis and we demonstrate that TIG3 promotes pericentrosomal organelle accumulation. These changes are associated with reduced cyclin D1, cyclin E and cyclin A, and increased p21 level. In addition, Bax level is increased and Bcl-XL level is reduced, and cleavage of procaspase 3, procaspase 9 and PARP is enhanced. We propose that pericentrosomal localization of TIG3 is a key event that results in microtubule and microfilament redistribution and pericentrosomal organelle clustering and that leads to cancer cell apoptosis.
Lysophosphatidic acid (LPA) enhances cell migration and promotes wound healing in vivo, but the intracellular signaling pathways regulating these processes remain incompletely understood. Here we investigated the involvement of agonist-induced Ca2+ entry and STIM1 and Orai1 proteins in regulating nuclear factor of activated T cell (NFAT) signaling and LPA-induced keratinocyte cell motility. As monitored by Fluo-4 imaging, stimulation with 10 μℳ LPA in 60 μℳ Ca2+o evoked Ca2+i transients owing to store release, whereas addition of LPA in physiological 1.2 mℳ Ca2+o triggered store release coupled to extracellular Ca2+ entry. Store-operated Ca2+ entry (SOCE) was blocked by the SOCE inhibitor diethylstilbestrol (DES), STIM1 silencing using RNA interference (RNAi), and expression of dominant/negative Orai1R91W. LPA induced significant NFAT activation as monitored by nuclear translocation of green fluorescent protein-tagged NFAT2 and a luciferase reporter assay, which was impaired by DES, expression of Orai1R91W, and inhibition of calcineurin using cyclosporin A (CsA). By using chemotactic migration assays, LPA-induced cell motility was significantly impaired by STIM1, CsA, and NFAT2 knockdown using RNAi. These data indicate that in conditions relevant to epidermal wound healing, LPA induces SOCE and NFAT activation through Orai1 channels and promotes cell migration through a calcineurin/NFAT2-dependent pathway.
Keratinocytes undergo a process of terminal cell differentiation that results in the construction of a multilayered epithelium designed to produce a structure that functions to protect the body from dehydration, abrasion and infection. These protective properties are due to the production of a crosslinked layer of protein called the cornified envelope. Type I transglutaminase (TG1), an enzyme that catalyzes the formation of ε-(γ-glutamyl)lysine bonds, is the key protein responsible for generation of the crosslinks. The mechanisms that lead to activation of transglutaminase during terminal differentiation are not well understood. We have identified a protein that interacts with TG1 and regulates its activity. This protein, tazarotene-induced gene 3 (TIG3), is expressed in the differentiated layers of the epidermis and its expression is associated with transglutaminase activation and cornified envelope formation. We describe a novel mechanism whereby TIG3 regulates TG1 activity.
Lysosomes and their components are suspected to be involved in epidermal differentiation. In this study, lysosomal enzyme activities, expression of the lysosome-associated membrane protein 1 (Lamp-1) and expression of the epidermal galectins-1, -3 and -7 were investigated in human keratinocytes cultured at different cell densities (subconfluence, confluence and postconfluence) in order to induce differentiation. Detected by Western blot and immunofluorescence, Lamp-1 expression is transiently upregulated at culture confluence, but reduced at postconfluence. Northern blot analyses performed on subconfluent, confluent and post-confluent cultures of keratinocytes show that Lamp-1 mRNA expression is also upregulated at culture confluence, but downregulated at postconfluence. Measurements of lysosomal enzyme activities indicate a transient upregulation at culture confluence, whereas cathepsins B, C and L are particularly downregulated at postconfluence. Cell density and differentiation of epidermal cells also differentially regulates galectin expression in autocrine cultures. As the expression of galectin-1 mRNA is high in subconfluent cells, it is assumed to be associated with their proliferative state. On the other hand, as the mRNA levels for galectins-3 and -7 are notably upregulated at culture confluence (galectin-7) or at postconfluence (galectin-3), their expression is thought to be related to the differentiated state of keratinocytes. However, we collected evidence by confocal microscopy that galectin-3 and Lamp-1 do not colocalize in vitro in keratinocytes. Altogether, our results suggest that the upregulated Lamp-1 expression at confluence could be involved in keratinocyte differentiation, but apparently not through interaction with galectin-3.
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