Christina Cheng

Calcium regulation of growth and differentiation of mouse epidermal cells in culture

Modification of the ionic calcium concentration in the culture medium markedly alters the pattern of proliferation and differentiation in cultured mouse epidermal cells. When medium calcium is lowered to 0.05--0.1 mM, keratinocytes proliferate rapidly with a high growth fraction and do not stratify, but continue to synthesize keratin. The cells grow as a monolayer for several months and can be subcultured and cloned in low Ca++ medium. Ultrastructural examination of cells cultured under low Ca++ conditions reveals widened intercellular spaces, abundant microvilli and perinuclear organization of tonofilaments and cellular organelles. Desmosomes are absent. Epidermal cells growing as a monolayer in low Ca++ can be induced to terminally differentiate by adding calcium to the level normally found in the culture medium (1.2 mM). Cell-to-cell contact occurs rapidly and desmosomes form within 2 hr. The cells stratify by 1--2 days and terminally differentiate with cell sloughing by 3--4 days. After Ca++ addition, DNA synthesis decreases with a lag of 5--10 hr and is totally inhibited within 34 hr. In contrast, RNA and protein synthesis continue at 40--50% of the low Ca++ level at day 3, a time when many cells are detaching from the culture dish. Keratin synthesis is unaffected by the Ca++ switch.

Identification of a major keratinocyte cell envelope protein, loricrin

During epidermal cell cornification, the deposition of a layer of covalently cross-linked protein on the cytoplasmic face of the plasma membrane forms the cell envelope. We have isolated and characterized cDNA clones encoding a major differentiation product of mouse epidermal cells, which has an amino acid composition similar to that of purified cell envelopes. Transcripts of this gene are restricted to the granular layer and are as abundant as the differentiation-specific keratins, K1 and K10. An antiserum against a C-terminal peptide localizes this protein in discrete granules in the stratum granulosum and subsequently at the periphery of stratum corneum cells. Immunofluorescence and immunoelectron microscopy detect this epitope only on the inner surface of purified cell envelopes. Taken together, these results suggest that it is a major component of cell envelopes. On the basis of its presumed function, this protein is named loricrin.

mtCLIC/CLIC4, an Organellular Chloride Channel Protein, Is Increased by DNA Damage and Participates in the Apoptotic Response to p53

ABSTRACT mtCLIC/CLIC4 (referred to here as mtCLIC) is a p53- and tumor necrosis factor alpha-regulated cytoplasmic and mitochondrial protein that belongs to the CLIC family of intracellular chloride channels. mtCLIC associates with the inner mitochondrial membrane. Dual regulation of mtCLIC by two stress response pathways suggested that this chloride channel protein might contribute to the cellular response to cytotoxic stimuli. DNA damage or overexpression of p53 upregulates mtCLIC and induces apoptosis. Overexpression of mtCLIC by transient transfection reduces mitochondrial membrane potential, releases cytochrome c into the cytoplasm, activates caspases, and induces apoptosis. mtCLIC is additive with Bax in inducing apoptosis without a physical association of the two proteins. Antisense mtCLIC prevents the increase in mtCLIC levels and reduces apoptosis induced by p53 but not apoptosis induced by Bax, suggesting that the two proapoptotic proteins function through independent pathways. Our studies indicate that mtCLIC, like Bax, Noxa, p53AIP1, and PUMA, participates in a stress-induced death pathway converging on mitochondria and should be considered a target for cancer therapy through genetic or pharmacologic approaches.

p53 and Tumor Necrosis Factor α Regulate the Expression of a Mitochondrial Chloride Channel Protein

A novel chloride intracellular channel (CLIC) gene, clone mc3s5/mtCLIC, has been identified from differential display analysis of differentiating mouse keratinocytes from p53+/+ and p53−/− mice. The 4.2-kilobase pair cDNA contains an open reading frame of 762 base pairs encoding a 253-amino acid protein with two putative transmembrane domains. mc3s5/mtCLIC protein shares extensive homology with a family of intracellular organelle chloride channels but is the first shown to be differentially regulated. mc3s5/mtCLIC mRNA is expressed to the greatest extent in vivo in heart, lung, liver, kidney, and skin, with reduced levels in some organs fromp53−/− mice. mc3s5/mtCLIC mRNA and protein are higher in p53+/+ compared withp53−/− basal keratinocytes in culture, and both increase in differentiating keratinocytes independent of genotype. Overexpression of p53 in keratinocytes induces mc3s5/mtCLIC mRNA and protein. Exogenous human recombinant tumor necrosis factor α also up-regulates mc3s5/mtCLIC mRNA and protein in keratinocytes. Subcellular fractionation of keratinocytes indicates that both the green fluorescent protein-mc3s5 fusion protein and the endogenous mc3s5/mtCLIC are localized to the cytoplasm and mitochondria. Similarly, mc3s5/mtCLIC was localized to mitochondria and cytoplasmic fractions of rat liver homogenates. Furthermore, mc3s5/mtCLIC colocalized with cytochrome oxidase in keratinocyte mitochondria by immunofluorescence and was also detected in the cytoplasmic compartment. Sucrose gradient-purified mitochondria from rat liver confirmed this mitochondrial localization. This represents the first report of localization of a CLIC type chloride channel in mitochondria and the first indication that expression of an organellular chloride channel can be regulated by p53 and tumor necrosis factor α.

Cross-talk between epidermal growth factor receptor and protein kinase C during calcium-induced differentiation of keratinocytes

Abstract: The induction of epidermal differentiation by extracellular Ca2+ involves activation of both tyrosine kinase and protein kinase C (PKC) signaling cascades. To determine if the differentiation‐dependent activation of tyrosine kinase signaling can influence the PKC pathway, we examined the tyrosine phosphorylation status of PKC isoforms in primary mouse keratinocytes stimulated to terminally differentiate with Ca2+. Elevation of extracellular Ca2+ induced tyrosine phosphorylation of PKC‐δ, but not the other keratinocyte PKC isoforms (α,ε, η, ζ). We have previously demonstrated that activation of the epidermal growth factor receptor (EGFR) pathway induces PKC‐δ tyrosine phosphorylation in basal keratinocytes (Denning M F, Dlugosz A A, Threadgill D W, Magnuson T, Yuspa S H (1996) J Biol Chem 271: 5325–5331). When basal keratinocytes were stimulated to differentiate by Ca2+, the level of cell‐associated transforming growth factor‐α (TGF‐α) increased 30‐fold, while no increase in secreted TGF‐α was detected. Furthermore, Ca2+‐induced tyrosine phosphorylation of PKC‐δ and phosphotyrosine‐association of the receptor adapter protein Shc was diminished in EGFR −/− keratinocytes, suggesting that EGFR activation may occur during keratinocyte differentiation. Tyrosine phosphorylated PKC‐δ was also detected in mouse epidermis, suggesting that this differentiation‐associated signaling pathway is physiological. These results establish a requirement for the EGFR in Ca2+‐induced tyrosine phosphorylation of PKC‐δ, and document the production of cell‐associated TGF‐α in differentiated keratinocytes which may function independent of its usual mitogenic effects.

CLIC4 mediates and is required for Ca2+-induced keratinocyte differentiation

Keratinocyte differentiation requires integrating signaling among intracellular ionic changes, kinase cascades, sequential gene expression, cell cycle arrest, and programmed cell death. We now show that Cl– intracellular channel 4 (CLIC4) expression is increased in both mouse and human keratinocytes undergoing differentiation induced by Ca2+, serum and the protein kinase C (PKC)-activator, 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Elevation of CLIC4 is associated with signaling by PKCδ, and knockdown of CLIC4 protein by antisense or shRNA prevents Ca2+-induced keratin 1, keratin 10 and filaggrin expression and cell cycle arrest in differentiating keratinocytes. CLIC4 is cytoplasmic in actively proliferating keratinocytes in vitro, but the cytoplasmic CLIC4 translocates to the nucleus in keratinocytes undergoing growth arrest by differentiation, senescence or transforming growth factor β (TGFβ) treatment. Targeting CLIC4 to the nucleus of keratinocytes via adenoviral transduction increases nuclear Cl– content and enhances expression of differentiation markers in the absence of elevated Ca2+. In vivo, CLIC4 is localized to the epidermis in mouse and human skin, where it is predominantly nuclear in quiescent cells. These results suggest that CLIC4 participates in epidermal homeostasis through both alterations in the level of expression and subcellular localization. Nuclear CLIC4, possibly by altering the Cl– and pH of the nucleus, contributes to cell cycle arrest and the specific gene expression program associated with keratinocyte terminal differentiation.

Sequential Changes in Gene Expression during Epidermal Differentiation

Genes encoding the major proteins expressed in mouse epidermis have been isolated and characterized. Using a combination of in situ hybridization with RNA probes, which are specific for individual mRNA’s, and indirect immunofluorescence with specific antisera, which were elicited with synthetic peptides corresponding to unique sequences within each protein, it is possible to show that these genes belong to at least four subsets: those expressed predominantly in the proliferating basal layer of the epidermis (keratins 5 and 14); those expressed predominantly in the differentiated suprabasal spinous layer and to a less extent the granular layer (keratins 1 and 10); those initially expressed in the upper spinous layer but most prominently in the granular layer (the gene encoding the precursor for filaggrin, a protein thought to promote keratin filament aggregation and the gene encoding a major component of the comified envelope) and those only expressed under hyperproliferative conditions (keratin 6). In order to identify sequences regulating the expression of these genes, transgenic mice have been produced which contain one of the human differentiation-associated keratin genes (K1). The human K1 gene is located within a 12 kilobase (Kb) fragment and is flanked by 2 Kb upstream and 3 Kb downstream. This DNA fragment contains sufficient sequence information for tissue-specific, developmental-specific and differentiation-specific expression.

Rocket immunoelectrophoresis in the presence of denaturing agents

Modifications of the Laurel rocket technique for assaying antigen antibody reactions are described. These procedures allow insoluble proteins to be dissolved in a variety of denaturing solvents (e.g., SDS and urea) and subjected to electroimmunoassay without loss of sensitivity or specificity. Methods are also presented for obtaining rockets using gel slices from polyacrylamide gel electrophoresis either in the presence of urea or SDS. Results obtained using keratins, the insoluble proteins of epidermis, hair and nail are summarized.

The In Vitro Analysis of Biochemical Changes Relevant to Skin Carcinogenesis

The phenotypic alterations produced in mouse skin cells during the multistage development of squamous cancer have been well documented. In normal skin, all proliferating cells are confined to the basal cell compartment where less than 10% of the cells are in S phase when pulse-labeled with DNA precursors. Two keratins, K5 (M r 60 000) and K14 (M r 55 000), are transcribed largely in basal cells, although the proteins persist in the upper layers (Roop et al. 1988). The commitment to differentiate is associated with the loss of proliferative potential, the commencement of suprabasal migration, and the expression of two suprabasal keratins, K1 (M r 67 000) and K10 (M r 59 000) in the first spinous cell layer (Roop et al. 1988). Proliferating cells do not express K1 or K10 in normal epidermis. As cells migrate into the granular cell layer, K1 and K10 transcripts diminish and the genes for filaggrin, a M r 27 000 interfilamentous matrix protein, and loricrin, a major component of the cornified envelope, are activated and the proteins synthesized (Mehrel et al. 1990; Roop et al. 1989).