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Experiments on the proto-oncogene c-fos of mice with v-H-ras expressing keratinocytes grafted on nude mice reveals that c-fos deficient tumors fail to initiate malignant progression hindering tumorigenesis. The results may be significant for the therapeutic prevention of neoplastic diseases and may point out that the c-fos represents a potential target for pharmacological interventions.

Information regarding the three-day Montagna Symposium 2011 entitled "Advances in Science and Medicine Catalyzed by Pioneering Skin Research," the 60th anniversary meeting is presented. Topics include a timeline denoting the origin and highlights of photoimmunology as a discipline, the milestones in cancer research and discoveries on the skin, and the role of DNA repair defects in skin cancer. The symposium features several speakers including Stuart Yuspa, Andrzej Dlugosz, and Pierre Coulombe.

New born mouse epidermal cells proliferate when cultured in 0.5 mM Ca++ medium and terminally differentiate when the Ca++ is increased to about 1.2 mM, the level found in most cell culture media. We found that hyaluronic acid and proteoglycans were synthesized by isolated cultured newborn mouse epidermal cells and that quantitative and qualitative changes in these macromolecules appeared when proliferating epidermal cultures were induced to differentiate by calcium. A major change that occurred with differentiation was a reduction in synthesis of hyaluronic acid while synthesis of proteoglycans and glycoproteins increased. The proteoglycans synthesized in these cultures were heparan sulfate-proteoglycan (90%) and chondroitin sulfate-proteoglycan (10%), regardless of the calcium level. [ABSTRACT FROM AUTHOR]

Studies performed on mouse skin have indicated that chemical carcinogenesis can be subdivided into two distinct stages, initiation and promotion. Initiation results from exposure to a classical mutagenic carcinogen and is irreversible even after a single exposure. The permanently altered initiated cell and its progeny may never form a tumor or in any way be recognizable in the target tissue, Exposure to tumor promoters permits the expression of the neoplastic change in initiated cells, and tumors develop. In contrast to initiators, promoters must be given repeatedly to be effective; individual exposures are reversible, A similar biology is suggested by epidemiologic studies of certain human cancers, particularly lung, breast, colon, and uterine malignancies. Studies in mouse skin cell culture have provided new insights into the changes associated with initiation and promotion. Initiated cells appear to be resistant to signals for terminal differentiation and can proliferate under conditions where normal epidermal cells are obligated to cease proliferation and begin their maturation program. This change is essential for an epithelial tumor cell since it provides the ability to grow away from a basement- membrane attachment site, In cultured epidermal cells, tumor promoters are capable of selectively stimulating the growth of certain cells, including initiated cells, while simultaneously inducing terminal differentiation in other epidermal cells, The net effect of these responses to promoters is the clonal expansion of cells stimulated to proliferate. In this way, promoters are capable of increasing the clone size of initiated cells. These cell culture data provided a biological framework for under- standing initiation and promotion in terminally differentiating epithelial tissues. [ABSTRACT FROM AUTHOR]

Skin tumors chemically induced in mice have provided an important experimental model for studying carcinogenesis and for bioassaying carcinogenic agents. The information obtained from this model suggests that the events leading to tumor formation can be divided into at least two stages, initiation and promotion. A single small. dose of carcinogen produces initiation which appears to be irreversible. These initiating agents may have to be metabolically activated and can interact with cellular macromolecules. The extent to which they bind to DNA correlates well with their carcinogenicity. Increased DNA replication at the time of or during the first day after these agents have been applied appears to enhance carcinogenesis. Unlike initiation, promotion appears to be reversible and the promoting agents must be applied repeatedly before tumors are formed. Promoters interact with membranes, stimulate and alter genetic expression, and increase the rate of cell proliferation. The knowledge gained from these studies in mouse skin has immeasurably helped the entire field of chemical carcinogenesis. But efforts to determine the cellular and molecular mechanisms involved in the carcinogenic process, particularly in the skin, have been hampered by the difficulties of working on whole animals and by the special problems associated with the biologic and biochemical methods required for this target organ. Such problems, however, can be solved by the use of cell cultures of mouse epidermis which can metabolize and bind carcinogens just as is done in vivo. The fact that epidermal cells in vitro proliferate synchronously should facilitate the study of the relation between the cell cycle and carcinogenesis. These cells repair chemically induced DNA damage by at least two mechanisms, excision repair and base-specific repair. [ABSTRACT FROM AUTHOR]

Mixed cultures of epidermal and dermal cells from term fetuses of Balb/cAn mice were exposed to high concentrations (50 μg/ml) of 7. 12-dimethylbenz(a)anthracene (DMBA) in medium containing Tween-80 or to medium with Tween-80 alone for 45 min. Within 5 weeks the cultures exposed to DMBA began to exhibit accelerated growth in vitro and an epithelioid morphology. These same changes occurred in the Tween-80-treated group starting around 15 weeks in culture. Both sets of cultures remained epithelioid in appearance and rapidly growing for over 9 months. Injections of cells into syngeneic hosts, beginning approximately 21 weeks after treatment. gave rise to undifferentiated tumors. Animals receiving carcinogen-treated cells had more tumors than those receiving vehicle-treated cells. Ultrastructural studies of the transformed cells in vitro suggested that they were of keratinocyte origin, but DMBA + Tween-80-treated cells were more differentiated than cells treated with Tween-80 alone. 80-100 Å cytofilaments, presumably identical to tonofilaments in vivo, were organized into bundles which traversed both the cellular endoplasm and ectoplasm, either terminating on adherens-like junctional structures, or looping back into the cytoplasm after attaching peripherally to such structures. After transplantation into syngeneic hosts, transformed cells from both groups produced tumors which ultrastructurally resembled anaplastic squamous-cell carcinomas. When highly undifferentiated tumors derived from DMBA + Tween-80-treated cells were recultured, more differentiated features reappeared. This observation indicates an important role for the cellular millieu in the determination of phenotypic expression. [ABSTRACT FROM AUTHOR]

Normal human epidermal keratinocytes (NHEK) grown in serum-free medium on a plastic substrate spontaneously differentiate at high cell densities in vitro. Because protein kinase C (PKC) regulates murine keratinocyte differentiation triggered by a variety of stimuli, we examined the role of this signaling pathway in density-dependent activation of NHEK differentiation. Relative to subconfluent cultures, confluent NHEK expressed markedly higher levels of multiple differentiation markers assayed by immunoblotting, including keratin 1, loricrin, filaggrin, involucrin, TGK, and SPR-1. Expression of several of these markers continued to increase for several days after cells reached confluency. The total level of several PKC isoforms was not substantially altered in NHEK harvested at different cell densities, based on immunoblotting; however, subcellular fractionation revealed that PKCα underwent a redistribution to the particulate fraction in confluent and postconfluent NHEK cultures, suggesting that this isozyme was activated under these conditions and may be involved in triggering the terminal differentiation program. Supporting this concept, inhibition of PKC function using bryostatin 1 or GF 109203X blocked the induction of keratinocyte differentiation markers at high cell densities. These data suggest that endogenous activation of PKC is responsible for cell density-mediated stimulation of NHEK differentiation, establishing a critical role for this pathway in regulating human as well as murine keratinocyte differentiation. [ABSTRACT FROM AUTHOR]

The introduction of the techniques of molecular biology as tools to study skin carcinogenesis has provided more precise localization of biochemical pathways that regulate the tumor phenotype. This approach has identified genetic changes that are characteristic of each of the specific stages of squamous cancer pathogenesis: initiation, exogenous promotion, premalignant progression, and malignant conversion. Initiation can result from mutations in a single gene, and the Harvey allele of the ras gene family has been identified as a frequent site for initiating mutations. Heterozygous activating mutations in c-rasHa are dominant, and affected keratinocytes hyperproliferate and are resistant to signals for terminal differentiation. An important pathway impacted by c-rasHa activation is the protein kinase C (PKC) pathway, a major regulator of keratinocyte differentiation. Increased activity of PKCα and suppression of PKCδ by tyrosine phosphorylation contribute to the phenotypic consequences of rasHa gene activation in keratinocytes. Tumor promoters disturb epidermal homeostasis and cause selective clonal expansion of initiated cells to produce multiple benign squamous papillomas. Resistance to differentiation and enhanced growth rate of initiated cells impart a growth advantage when the epidermis is exposed to promoters. The frequency of premalignant progression varies among papillomas, and subpopulations at high risk for progression have been identified. These high-risk papillomas overexpress the α6β4 integrin and are deficient in transforming growth factor β1 and β2 peptides, two changes associated with a very high proliferation rate in this subset of tumors. The introduction of an oncogenic rasHa gene into epidermal cells derived from transgenic mice with a null mutation in the TGFβ1 gene have an accelerated rate of malignant progression when examined in vivo. Thus members of the TGFβ gene family contribute a tumor-suppressor function in carcinogenesis. Accelerated malignant progression is also found with v-rasHa transduced keratinocytes from skin of mice with a null mutation in the p53 gene. The similarities in risk for malignant conversion by initiated keratinocytes from TGFβ and p53 null geneotypes suggest that a common, growth-related pathway may underly the tumor-suppressive functions of these proteins in the skin carcinogenesis model. [ABSTRACT FROM AUTHOR]

During the final stage of epidermal differentiation, activation of keratinocyte transglutaminase results in covalent cross-linking of a variety of proteins to form highly protective cornified cell envelopes. We have studied the regulation of keratinocyte transglutaminase (TGK) gene expression in murine epidermal keratinocytes induced to terminally differentiate in vitro by increasing the level of extracellular Ca++ or treatment with the protein kinase C (PKC) activator 12- O-tetradecanoylphorbol-13-acetate (TPA). Raising extracellular Ca++ induces squamous differentiation of cultured keratinocytes and elicits a concentration-dependent increase in expression of TGK mRNA; keratinocytes grown for 24 h in 0.12 mM Ca++ medium express ∼12 times as much TGK mRNA as basal cells (grown in 0.05 mM Ca++ medium), whereas cultures exposed to 1.4 mM Ca++ express ∼17 times as much. TPA induces squamous differentiation and TGK mRNA even in basal keratinocyte cultures grown in 0.05 mM Ca++ medium, suggesting that expression of this differentiation marker is regulated by the PKC signaling pathway. Induction of TGK mRNA in response to TPA treatment is transient, reaching a peak at 6-8 h and returning to baseline by 24 h. In contrast, elevation of TGK mRNA levels in response to Ca++ persists for at least 24 h. The increased abundance of TGK mRNA reflects increased transcription of the TGK gene, based on nuclear run-on analysis of Ca++- and TPA-treated keratinocytes. Induction of TGK mRNA by either TPA or Ca++ is blocked in the presence of cycloheximide, suggesting that a PKC-dependent protein factor is required for TGK gene expression in response to both stimuli. Furthermore, the accumulation of TGK mRNA in keratinocytes treated with TPA or CA++ is blocked in cells treated with the PKC inhibitor GF 109203X or bryostatin. These results suggest that the induction of TGK gene expression by CA++ is dependent on PKC, providing further support for the hypothesis that PKC plays a central role in regulating the late stages of epidermal differentiation. [ABSTRACT FROM AUTHOR]

During embryonic development presumptive hair follicle cells of epithelial and mesenchymal origin are determined in defined body locations. This is followed by rapid proliferation of epithelial cells and associated penetration into the dermis in response to as yet undetermined signals. A collagen matrix culture system, which maintains the three-dimensional relationships of hair follicle cells to each other, was developed to study the regulation of the enlargement of immature hair follicles and the accompanying remodeling of the dermis. In studies with a heterogeneous dermis-derived preparation of murine hair follicles, ranging in size from the earliest down-growing budding cell mass to hair-forming follicles, we had previously shown that cell proliferation was stimulated by cholera toxin and epidermal growth factor, but only the epidermal growth factor-stimulated proliferation was accompanied by digestion of the collagen matrix due to release of collagenolytic enzymes. Further studies revealed that transforming growth factor-α also stimulated hair follicle cell proliferation and collagenase release. However, although transforming growth factor-β inhibited the transforming growth factor-α-stimulated proliferation, it enhanced the release and activation of collagenases and other gelatin-degrading enzymes detectable by gelatin zymography. Stimulation of collagenolytic activity depended on the three-dimensional hair follicle structure and did not occur in monolayer cultures of hair follicle cells. Comparison of hair follicle buds with more developed dermis-derived hair follicles, plated at the same cell density (based on DNA content), suggested that a greater fraction of cells in the bud-stage follicle responded to the growth factors by release of collagenases. Possibly only the cells in the advancing portion of growing hair follicles that are closest to the dermal papilla cell cluster produce the collagenases in response to growth factors. To examine the participation of dermal papilla cells in collagenase release and activation, several immortalized rat whisker dermal papilla cell liners were co-cultured with mouse hair follicle buds. Co-culture resulted in a marked enlargement of follicles as well as activation of the 92-kDa type IV collagenase, produced by hair follicle buds, that correlated with ability of the dermal papilla cells to stimulate hair formation in grafts of hair follicle buds on nude mice. Dermal papilla cells cultured alone produced the 72-kDa type IV collagenase, which was also activated during co-culture with hair follicle buds. Thus, two activities, both relevant for hair follicle development, namely, cell proliferation and release and activation of collagenases, have been stimulated in immature hair follicle buds by either growth-factor supplementation or interaction with dermal papilla cells. The collagen matrix culture system allows investigation of the mechanism by which these activities are controlled under defined in vitro conditions. [ABSTRACT FROM AUTHOR]

Cellular lipid metabolism can provide a variety of mediators of signal transduction, including diacylglycerols and inositol Phosphates. These factors may be involved in the control of epidermal differentiation and proliferation because they are modulated by extracellular calcium, which also regulates the maturation phenotype of cultured keratinocytes. The effect of non-cytotoxic exposures to ultraviolet light on lipid metabolism was studied in cultured murine keratinocytes. Ultraviolet treatment of cultured murine keratinocytes growing in 0.05 mM Ca++ did not significantly change the total amount of [³H]inositol phosphates at 0.5, 8, or 24 h post-irradiation. Irradiated cells responded to an increase from 0.05 mM Ca++ to 1.4 mM Ca++ medium with increased formation of inositol phosphates suggesting irradiation did not alter the normal inositol lipid turnover in response to the Ca++ signal for terminal differentiation. Irradiation (20- 120 J/m² of UVB) induced a dose-dependent increase in the cellular level of diacylglycerols as measured at 24 h post-irradiation, without changing the turnover of other phospholipids including phosphatidylcholine and phosphatidylethanolamine. The increased cellular levels of diacylglycerols following ultraviolet exposure were accompanied by changes in the activity of diacylglycerol kinase (DAG-kinase). The cytosolic DAG-kinase activity was decreased whereas the DAG-kinase activity in the membrane fraction was increased. These results suggest that ultraviolet irradiation increases the level of diacylglycerols via changes in de novo metabolism through a DAG-kinase pathway. Elevated diacylglycerol may influence signal-transduction pathways mediated by cellular lipids and contribute to some keratinocyte responses to ultraviolet light. [ABSTRACT FROM AUTHOR]

A method for the monolayer culture of fetal mouse skin cells and the behavior of primary cultures have been described. The early cultures consist of discrete colonies of epidermoid cells with individual fibroblasts scattered between these foci. The cell colonies begin to produce a material which may be keratin by the third in vitro day. After one week in vitro the majority of epidermoid foci have degenerated and the cultures consist largely of elongated rapidly multiplying cells. When cells from cultures in vitro five days or less are transplanted back to the panniculus carnosus of F1 hosts, donor skin grows. This skin is hyperplastic but contains normal appendages and produces hair. These grafts have remained recognizable and stable for over four months. [ABSTRACT FROM AUTHOR]

Mouse keratinocytes can he grown at clonal densities in dermal fibroblast conditioned medium with a calcium concentration of 0.02 MM. Colony forming efficiencies of approximately 1-3% can be achieved with primary and secondary cultures and up to 6% with selected subclones. A substrate of frozen-thawed dermal fibroblasts enhances colony formation over plastic. Colony size increases more rapidly in the presence of epidermal growth factor in conditioned medium, Conditioning of medium by fibroblasts is optimum after day 6 of culture and conditioned medium can be stored at -20°C for at least 4 mo. [ABSTRACT FROM AUTHOR]

Presents a letter to the editor on an antibody's ability to recognize soluble keratins in epidermal keratinocyte cultures, published in the November 1997 issue of "The Journal of Investigative Dermatology."

The chloride intracellular channel-4 (CLIC4) is one of the six highly conserved proteins in the CLIC family that share high structural homology with GST-omega in the GST superfamily. While CLIC4 is a multifunctional protein that resides in multiple cellular compartments, the discovery of its enzymatic glutaredoxin-like activity in vitro suggested that it could function as an antioxidant. Here, we found that deleting CLIC4 from murine 6DT1 breast tumor cells using CRISPR enhanced the accumulation of reactive oxygen species (ROS) and sensitized cells to apoptosis in response to H2O2 as a ROS-inducing agent. In intact cells, H2O2 increased the expression of both CLIC4 mRNA and protein. In addition, increased superoxide production in 6DT1 cells lacking CLIC4 was associated with mitochondrial hyperactivity including increased mitochondrial membrane potential and mitochondrial organelle enlargement. In the absence of CLIC4, however, H2O2-induced apoptosis was associated with low expression and degradation of the antiapoptotic mitochondrial protein Bcl2 and the negative regulator of mitochondrial ROS, UCP2. Furthermore, transcriptomic profiling of H2O2-treated control and CLIC4-null cells revealed upregulation of genes associated with ROS-induced apoptosis and downregulation of genes that sustain mitochondrial functions. Accordingly, tumors that formed from transplantation of CLIC4-deficient 6DT1 cells were highly necrotic. These results highlight a critical role for CLIC4 in maintaining redox-homeostasis and mitochondrial functions in 6DT1 cells. Our findings also raise the possibility of targeting CLIC4 to increase cancer cell sensitivity to chemotherapeutic drugs that are based on elevating ROS in cancer cells. [ABSTRACT FROM AUTHOR]

Epithelial cancers are the most common malignancies and the greatest cause of cancer mortality worldwide. The incidence of keratinocyte-derived (non-melanoma) skin cancers is increasing rapidly. Despite access to abundant tumor tissue and ease of observation, acceptance of non-melanoma skin cancers as model carcinomas has been hindered by the lack of a reliable xenograft model. Herein we describe conditions that allow routine xeno-engraftment of primary human squamous cell carcinoma (SCCa) cells. Tumor development required creation of an appropriate stromal bed before xenografting tumor tissue onto the backs of athymic nude mice. We also demonstrate that the stromal bed must be 'humanized' if primary human SCCa is to be propagated from cell suspensions. SCCa xenografts recapitulated the histological grade and phenotype of the original tumors with considerable fidelity, even after serial passage, irrespective of the histological grade of the primary human SCCa. This model, which to our knowledge is previously unreported, can be used for drug testing, as well as for studies that are relevant to the biology of primary human SCCa and other epithelial cancers. [ABSTRACT FROM AUTHOR]

Cutaneous cancers represent a major public health concern due to the very high incidence, associated medical costs, substantial mortality, and cosmetic deformities associated with treatment. Considerable progress in basic research has provided new insights into the underlying genetic basis of the major human cutaneous cancers, malignant melanoma, basal cell carcinoma, and squamous cell carcinoma. In turn, these genetic insights have illuminated biochemical pathways that promise to provide new approaches to the prevention and treatment of cutaneous neoplasms. This review will detail the evolving genetic information and indicate how this information is being used to refine experimental models that serve to both define the biochemistry of cancer pathogenesis and test novel approaches to cancer therapy. Combined with preventive measures to reduce exposure to sunlight, these advances are likely to reduce this major public health burden in the coming decade. [ABSTRACT FROM AUTHOR]

Primary murine keratinocytes can be maintained in culture for extended periods in a proliferative, basal cell state under conditions of reduced extracellular Ca2+ In response to increased Ca2++ concentrations, the cells undergo a well-defined program of terminal differentiation, thus serving as a convenient model in which to study the genes involved in regulating this and possibly other differentiation cascades by DNA- mediated gene transfer. However, because of their sensitivity to increased Ca2++ concentrations, the introduction of exogenous genomic DNA into primary keratinocytes by conventional methods is problematic. We have optimized the calcium phosphate DNA transfection procedure by introducing conditions that reduce the potency of Ca2+ as a differentiation signal. Primary epidermal cells were transfected with pSV2CAT, a plasmid that codes for the enzyme chloramphenicol acetyltransferase CAT. Enzyme activity was measured in cell extracts under varying transfection conditions. When the K+ concentration of the medium used for transfection by calcium phosphate precipitation is reduced from 6.5 to 0.01 mM, CAT activity following transfection increases 2-3 times. Exposure to the DNA precipitate for 2 - 4 h is optimal. By the use of fibroblast conditioned medium following transfection, enzyme activity can be detected in cell extracts for at least 21 d, suggesting that the exogenous gene is integrated. The low K++/Ca2+ transfection method is more effective than SrCl2 used as an alternative for CaC12 in Ca2+ sensitive cells. Low K+ medium enhances cell survival for Ca2+ mediated transfection but also appears to have a beneficial effect on DNA uptake or expression. [ABSTRACT FROM AUTHOR]

Epidermal cells cultured in low-calcium medium (0.02- 0.1 mM) grow as a monolayer, in contrast to the stratified pattern of growth in medium with standard calcium levels (1.2-1.8 mM). These low-calcium cells lack desmosomes and maintain a high proliferation rate. Raising the extracellular calcium to >0.1 mM induces rapid desmosome formation followed by stratification, inhibition of proliferation, formation of cornified envelopes, and sloughing of the cells from the culture dish. This calcium-induced terminal differentiation program is characterized by an increase in the intracellular levels of sodium and potassium at 12 to 24 hours and is not blocked by inhibitors of calcium or sodium flux. Of 40 to 50 agents tested as inhibitors of calcium-induced epidermal differentiation, only ouabain, harmaline, A23187, and 8(diethylamino)octyl-3,4,5-trimethoxybenzoate hydrochloride (TMB-8) were effective. These agents did not block the earliest calcium-induced effect (desmosome formation), but they did inhibit later stages in the program of terminal differentiation. Their detailed mechanism of action is unclear, although ouabain inhibits the sodium pump (Na+K+ATPase), lowering potassium and elevating sodium in the cells. The other inhibitors also prevented the calcium-induced elevation of intracellular potassium with no common effect on intracellular sodium. Reduction of potassium in the medium from the usual level of 6.5 mM to 0.1 mM lowers intracellular potassium by 60 to 70 percent and prevents calcium-induced differentiation, This result, along with the inhibitor studies, suggests that potassium plays an important role in epidermal terminal differentiation. [ABSTRACT FROM AUTHOR]

Modulating extracellular Ca2+ (Cao) and suspension culture are two frequently used methods to induce maturation of cultured human and mouse keratinocytes. To determine if the two methods share a common mechanism, changes in Ca2+ metabolism were studied in suspension cultures of mouse keratinocytes. Spontaneously detached and suspensioncultured keratinocytes in 0.05 mM Ca2+ medium express markers of suprabasal differentiation, while 0.05 mM Ca2+ is not permissive for marker expression by attached keratinocytes. Intracellular free Ca2+ (Cai increased rapidly after placing keratinocytes in suspension in 0.05 mM Ca2+, reaching levels up to 3to 4-fold higher than Cai in attached cells after 4-5 h. In suspended cells, the increase in Cai was associated with a 2- to 6-fold increase in Ca2+ transport across plasma membrane as well as depletion of intracellular Ca2+-stores. Differentiation marker expression and terminal differentiation were inhibited in suspension-cultured keratinocytes by preventing the rise of Cai using either 1,2-bis(o-aminophenoxy)-ethaneN,N,N',N'-tetraacetic acid to chelate intracellular Ca2+ or ethyleneglycol-bis(β-aminoethyl ether)N,N,N',N'-tetraacetic acid to reduce Cao. Together, these results indicate that a rise in Cai is a common mechanism controlling differentiation in cultured mouse keratinocytes, and suspension of keratinocytes enhances Ca2+ transport and alters intracellular Ca2+ sequestration producing a rise in Cai. [ABSTRACT FROM AUTHOR]

Histologic and immunocytochemical analyses were performed on cutaneous biopsies from 10 patients treated with retinoic acid under occlusion for 4 d compared to biopsies from 19 patients treated nightly for 16 weeks. Acute application of RA caused epidermal thickening (9 of 10 samples), stratum granulosum thickening (7 of 10), parakeratosis (4 of 10). a marked increase in the number of cell layers expressing epidermal transglutaminase (7 of 10), and focal expression of two non-epidermal keratins. K6 (8 of I0) and K13 (2 of I0), changes also observed with chronic treatment, Involucrin, filaggrin, and Ioricrin were also altered in samples from both acute and chronic treatment. An increased number of cell layers expressed both involucrin and filaggrin from both the acute (7 of I0) and chronic (14 of 19) treatment groups. In the acute group, loricrin expression was significantly reduced or absent in some regions of the epidermis (5 of 10). whereas most chronic samples showed an increased number of cell layers expressing loricrin (12 of 19). The pattern of expression of three major epidermal differentiation products, keratins K 1, K10, and K14, was not significantly altered in any of the acute or chronic samples. although there was a slight reduction in the detection of K10 in two of the acute samples. Thus, acute topical RA treatment under occlusion caused substantial changes in the epidermis, and reproduced most, but not all of the effects of Chronic treatment. [ABSTRACT FROM AUTHOR]

This article discusses a technique for isolating mouse pelage hair-follicle preparations that differ in their potential to form hair in nude mouse grafts. Major aims of hair follicle (HF) research include the elucidation of signals that control the initiation and duration of the hair-cycle phases as well as signals that control differentiation of HF matrix cells to form the various layers of the inner root sheath and of the hair itself. The challenge to the cell biologist is the creation at in vitro environment in which the relevant cells continue to perform their in vivo function and can be manipulated in a conotrollable manner.

Highlights: [•] MyD88 contributes to carcinogenesis in experimental models. [•] MyD88 promotes tumor formation through its role in cancer-associated inflammation. [•] Through its involvement in tissue repair, MyD88 can also protect against tumor formation. [•] MyD88-dependent adaptive immune responses protect against oncogenic pathogens. [ABSTRACT FROM AUTHOR]

Highlights: [•] Cytoplasmic psoriasin and koebnerisin may protect the tumor cell and block oncogenic pathways. [•] Nuclear psoriasin and likely koebnerisin promote tumor cell growth, survival and spreading. [•] Secreted psoriasin mediates oncogenic inflammation and tumor cell migration. [•] Nuclear psoriasin, koebnerisin and S100 protein secretion are associated with poor prognosis. [•] Psoriasin (S100A7) and koebnerisin (S100A15) are considered epithelial tumor progression markers. [ABSTRACT FROM AUTHOR]

Selenoproteins are essential molecules for the mammalian antioxidant network. We previously demonstrated that targeted loss of all selenoproteins in mouse epidermis disrupted skin and hair development, and caused premature death. In the current study, we targeted specific selenoproteins for epidermal deletion to determine whether similar phenotypes developed. Keratinocyte-specific knockout mice lacking either the glutathione peroxidase 4 (GPx4) or thioredoxin reductase 1 (TR1) gene were generated by cre-lox technology using K14-cre. TR1 knockout mice had a normal phenotype in resting skin, whereas GPx4 loss in the epidermis caused epidermal hyperplasia, dermal inflammatory infiltrate, dysmorphic hair follicles, and alopecia in perinatal mice. Unlike epidermal ablation of all selenoproteins, mice ablated for GPx4 recovered after 5 weeks and had a normal life span. GPx1 and TR1 were upregulated in the skin and keratinocytes of GPx4-knockout mice. GPx4 deletion reduces keratinocyte adhesion in culture and increases lipid peroxidation and cyclooxygenase-2 (COX-2) levels in cultured keratinocytes and whole skin. Feeding a COX-2 inhibitor to nursing mothers partially prevents development of the abnormal skin phenotype in knockout pups. These data link the activity of cutaneous GPx4 to the regulation of COX-2 and hair follicle morphogenesis, and provide insight into the function of individual selenoprotein activity in maintaining cutaneous homeostasis. [ABSTRACT FROM AUTHOR]

Retinoic acid (RA) is essential during embryogenesis and for tissue homeostasis, whereas excess RA is well known as a teratogen. In humans, excess RA is associated with hair loss. In the present study, we demonstrate that specific levels of RA, regulated by Cyp26b1, one of the RA-degrading enzymes, are required for hair follicle (hf) morphogenesis. Mice with embryonic ablation of Cyp26b1 (Cyp26b1-/-) have excessive endogenous RA, resulting in arrest of hf growth at the hair germ stage. The altered hf development is rescued by grafting the mutant skin on immunodeficient mice. Our results show that normalization of RA levels is associated with reinitiation of hf development. Conditional deficiency of Cyp26b1 in the dermis (En1Cre; Cyp26b1f/—) results in decreased hair follicle density and specific effect on hair type, indicating that RA levels also influence regulators of hair bending. Our results support the model of RA-dependent dermal signals regulating hf downgrowth and bending. To elucidate target gene pathways of RA, we performed microarray and RNA-Seq profiling of genes differentially expressed in Cyp26b1-/- skin and En1Cre;Cyp26b1f/— tissues. We show specific effects on the Wnt-catenin pathway and on members of the Runx, Fox, and Sox transcription factor families, indicating that RA modulates pathways and factors implicated in hf downgrowth and bending. Our results establish that proper RA distribution is essential for morphogenesis, development, and differentiation of hfs. [ABSTRACT FROM AUTHOR]

Unraveling the signaling pathways that transmit information from the cell surface to the nucleus has been a major accomplishment of modern cell and molecular biology. The benefit to humans is seen in the multitude of new therapeutics based on the illumination of these pathways. Although considerable insight has been gained in understanding homeostatic and pathological signaling in the epidermis and other skin compartments, the translation into therapy has been lacking. This review will outline advances made in understanding fundamental signaling in several of the most prominent pathways that control cutaneous development, cell-fate decisions, and keratinocyte growth and differentiation with the anticipation that this insight will contribute to new treatments for troubling skin diseases. [ABSTRACT FROM AUTHOR]

Primary human squamous cell carcinomas (SCCas) are heterogeneous invasive tumors with proliferating outer layers and inner differentiating cell masses. To determine if tumor-initiating cells (TICs) are present in SCCas, we utilized newly developed reliable in vitro and in vivo xenograft assays that propagate human SCCas, and demonstrated that a small subset of SCCa cells (∼1%) expressing Prominin-1 (CD133) in the outer layers of SCCas were highly enriched for TICs (∼1/400) compared with unsorted SCCa cells (TICs ∼1/106). Xenografts of CD133+ SCCas recreated the original SCCa tumor histology and organizational hierarchy, whereas CD133− cells did not, and only CD133+ cells demonstrated the capacity for self-renewal in serial transplantation studies. We present a model of human SCCas in which tumor projections expand with outer leading edges that contain CD133+ TICs. Successful cancer treatment will likely require that the TICs identified in cancers be targeted therapeutically. The demonstration that TICs are present in SCCas and are enriched in a CD133− expressing subpopulation has not been, to our knowledge, previously reported. [ABSTRACT FROM AUTHOR]

Nuclear translocation of chloride intracellular channel protein CLIC4 is essential for its role in Ca2+-induced differentiation, stress-induced apoptosis, and modulating TGF-β signaling in mouse epidermal keratinocytes. However, post-translational modifications on CLIC4 that govern nuclear translocation and thus these activities remain to be elucidated. The structure of CLIC4 is dependent on the redox environment, in vitro, and translocation may depend on reactive oxygen and nitrogen species in the cell. Here we show that NO directly induces nuclear translocation of CLIC4 that is independent of the NO-cGMP pathway. Indeed, CLIC4 is directly modified by NO through S-nitrosylation of a cysteine residue, as measured by the biotin switch assay. NO enhances association of CLIC4 with the nuclear import proteins importin α and Ran. This is likely a result of the conformational change induced by S-nitrosylated CLIC4 that leads to unfolding of the protein, as exhibited by CD spectra analysis and trypsinolysis of the modified protein. Cysteine mutants of CLIC4 exhibit altered nitrosylation, nuclear residence, and stability, compared with the wild type protein likely as a consequence of altered tertiary structure. Moreover, tumor necrosis factor a-induced nuclear translocation of CLIC4 is dependent on nitric-oxide synthase activity, inhibition of nitric-oxide synthase activity inhibits tumor necrosis factor a-induced nitrosylation and association with importin a and Ran and ablates CLIC4 nuclear translocation. These results suggest that S-nitrosylation governs CLIC4 structure, its association with protein partners, and thus its intracellular distribution. [ABSTRACT FROM AUTHOR]

Recent advances in the knowledge of the EGFR pathway have revealed its contribution to distinct immune/inflammatory functions of the epidermis. The purpose of our study was to evaluate the role of EGFR in the regulation of keratinocyte GM-CSF expression. In cultured human keratinocytes, proinflammatory cytokines synergized with TGF-α to induce GM-CSF expression. Accordingly, high epidermal levels of EGFR activation are associated with enhanced expression of GM-CSF in lesional skin of patients with psoriasis or allergic contact dermatitis. In cultured keratinocytes, pharmacological inhibition of EGFR activity reduced GM-CSF promoter transactivation, whereas genetic inhibition of AP-1 reduced expression of GM-CSF. Furthermore, EGFR activation enhanced TNF-α-induced c-Jun phosphorylation and DNA binding, whereas c-Jun silencing reduced GM-CSF expression. Using two different mouse models, we showed that the lack of a functional EGFR pathway was associated with reduced cytokine-induced phosphorylation of ERK1/2, JNK1/2, c-Jun and reduced keratinocyte-derived GM-CSF expression both in vitro and in vivo. Finally, the analysis of GM-CSF expression in the skin of cancer patients treated with anti EGFR drugs showed an association between ERK activity, c-Jun phosphorylation, and epidermal GM-CSF expression. These data demonstrate that the EGFR pathway is critical for the upregulation of keratinocyte GM-CSF expression under conditions of cytokine stimulation. [ABSTRACT FROM AUTHOR]

Abstract: Human S100A7 (psoriasin) is considered a marker for specific stages of breast cancer. hS100A15 is almost identical to hS100A7 and difficult to discriminate. We developed specific probes to distinguish hS100A7 and hS100A15, and demonstrate their differential distribution in normal breast tissue. Further, hS100A7 and S100A15 transcripts are elevated in ER/PR negative breast cancers, but hS100A15 protein is detected in all cancer specimens while hS100A7 protein is sporadically expressed. The differential regulation, expression and distribution of hS100A7 and hS100A15 and their reported distinct functions are compelling reasons to discriminate among these proteins in normal breast and breast cancers. [Copyright &y& Elsevier]

The calcium-binding proteins of the human S100A7/A15 (hS100A7/A15) subfamily are differentially expressed in normal and pathological epidermis. The hS100A7 (psoriasin) and S100A15 reside in a chromosomal cluster of highly similar paralogs. To exploit the power of mouse models for determining functions of gene products, the corresponding S100A7/A15 ortholog was cloned and examined in murine skin. The single mouse S100A15 (mS100A15) gene encodes a protein of 104 amino acids with a predicted molecular weight of 12,870 Da and two EF-hand calcium binding sites. Using gene-specific primers and specific antibodies, expression of mS100A15 in both skin and isolated keratinocytes is confined to differentiating granular and cornified epidermal cells. Immunoblotting of epidermal extracts revealed a series of high molecular weight bands that are also recognized by an antibody for transglutaminase-mediated protein crosslinks. mS100A15 expression is upregulated in cultured keratinocytes induced to differentiate by calcium or phorbol esters. Maximal induction occurs concordantly with expression of late differentiation markers. Induction is enhanced in keratinocytes overexpressing protein kinase Cα and is dependent on activator protein-1 transcription factors. The regulation, expression pattern and crosslinking of mS100A15 are consistent with the characteristics of the human orthologs, providing a valid surrogate model to study changes in these proteins associated with cutaneous pathologies.Journal of Investigative Dermatology (2006) 126, 1600–1608. doi:10.1038/sj.jid.5700210; published online 9 March 2006 [ABSTRACT FROM AUTHOR]

Skin keratinocytes are subject to frequent chemical and physical injury and have developed elaborate cell survival mechanisms to compensate. Among these, the Akt/protein kinase B (PKB) pathway protects keratinocytes from the toxic effects of ultraviolet light (UV). In contrast, the protein kinase C (PKC) family is involved in several keratinocyte death pathways. During an examination of potential interactions among these two pathways, we found that the insulin-like growth factor (IGF-1) activates both the PKC and the Akt signaling pathways in cultured primary mouse keratinocytes as indicated by increased phospho-PKC and phospho-Ser-473-Akt. IGF-1 also selectively induced translocation of PKCδ and PKC∈ from soluble to particulate fractions in mouse keratinocytes. Furthermore, the PKC-specific inhibitor, GF109203X, increased IGF-1-induced phospho-Ser-473-Akt and Akt kinase activity and enhanced IGF-1 protection from UVC-induced apoptosis. Selective activation of PKC by 12-O-tetradecanoylphorbol-13-acetate (TPA) reduced phospho-Ser-473-Akt, suggesting that activation of PKC inhibits Akt activity. TPA also attenuated IGF-1 and epidermal growth factor-induced phospho-Ser-473-Akt, reduced Akt kinase activity, and blocked IGF-1 protection from UVC-induced apoptosis. The inhibition of Akt activity by TPA was reduced by inhibitors of protein phosphatase 2A, and TPA stimulated the association of phosphatase 2A with Akt. Individual PKC isoforms were overexpressed in cultured keratinocytes by transduction with adenoviral vectors or inhibited with PKC-selective inhibitors. These studies indicated that PKCδ and PKC∈ were selectively potent at causing dephosphorylation of Akt and modifying cell survival, whereas PKCα enhanced phosphorylation of Akt on Ser-473. Our results suggested that activation of PKCδ and PKC∈ provide a negative regulation for Akt phosphorylation and kinase activity in mouse keratinocytes and serve as modulators of cell survival pathways in response to external stimuli. [ABSTRACT FROM AUTHOR]

Myc is a key regulatory protein in higher eukaryotes controlling important cellular functions such as proliferation, differentiation, and apoptosis. Myc is profoundly involved in the genesis of many human and animal cancers, and the abrogation of Myc-induced apoptosis is a critical event in cancer progression. Because the mechanisms that mediate Myc-induced apoptosis are largely unknown, we analyzed protein expression during Myc-induced apoptosis using an isotope-coded affinity tag quantitative proteomics approach and identified that a proapoptotic mitochondrial chloride ion channel, mtCLIC/CLIC4, is induced by Myc. Myc binds to the mtCLIC gene promoter and activates its transcription. Suppression of mtCLIC expression by RNA interference inhibited Myc-induced apoptosis in response to different stress conditions and abolished the cooperative induction of apoptosis by Myc and Bax. We also found that Myc reduces the expression of Bcl-2 and Bcl-xL and that the apoptosis-inducing stimuli up-regulate Bax expression. These results suggest that up-regulation of mtCLIC, together with a reduction in Bci-2 and Bcl-xL, sensitizes Myc-expressing cells to the proapoptotic action of Bax. [ABSTRACT FROM AUTHOR]

Chloride intracellular channel (CLIC)4 is a p53- and tumor necrosis factor α (TNFα)-regulated chloride channel protein that is localized to the mitochondria and cytoplasm of mouse and human keratinocytes. CLIC4 protein increases in differentiating keratinocytes and in keratinocytes exposed to DNA-damaging agents and metabolic inhibitors. Increasing CLIC4 levels by transduction of recombinant CLIC4 causes apoptosis. CLIC4 translocates to the nucleus under a variety of conditions of cell stress, and nuclear CLIC4 is associated with cell cycle arrest and accelerated apoptosis. Reduction of CLIC4 and several other CLIC family members by expressing a doxycycline-regulated CLIC4 antisense also causes apoptosis in squamous cancer cell lines. Expressing antisense CLIC4 in tumors derived from transplanting these cells into nude mice inhibits tumor growth, increases tumor apoptosis, and reduces tumor cell proliferation. Co-administration of TNFα intraperitoneally enhances the tumor-inhibitory influence of CLIC4 antisense expression. Together, these results suggest that CLIC4 is important for keratinocyte viability and may be a novel target for anti-cancer therapy. [ABSTRACT FROM AUTHOR]

The epidermal growth factor receptor (EGFR) has been implicated in the regulation of wound healing. In order to directly evaluate the role of endogenous EGFR in cutaneous incisional wound healing, we examined EGFR null- and wild-type skin after injury. By 5 d after wounding, re-epithelialization was complete in all EGFR wild-type wounds, but in only 40% of EGFR null wounds. Delayed wound closure in EGFR null skin was accompanied by an increase in edema, longer lasting and more prominent eschar, and increased distance between apposing wound edges. EGFR altered neutrophil and mast cell infiltration, and enhanced angiogenesis. EGFR enhanced epithelial proliferation during the first 3 d following injury, although proliferation was greater in EGFR null wounds at 5 d. Although migration was decreased in EGFR null keratinocytes cultured with standard medium or in medium supplemented with transforming growth factor-α when compared with controls, the addition of the wound-associated motogen keratinocyte growth factor eliminated the differences between genotypes. Epithelial migration into the wound was decreased in EGFR null skin, suggesting that both EGFR-dependent and -independent mechanisms regulate migration during wound healing. These data demonstrate that EGFR regulates multiple facets of cutaneous wound healing, including inflammation, wound contraction, proliferation, migration, and angiogenesis. [ABSTRACT FROM AUTHOR]

CLIC4/mtCLIC, a chloride intracellular channel protein, localizes to the mitochondria and cytoplasm of keratinocytes and participates in the apoptotic response to stress. We now show that multiple stress inducers cause the translocation of cytoplasmic CLIC4 to the nucleus. Immunogold electron microscopy and confocal analyses indicate that nuclear CLIC4 is detected prior to the apoptotic phenotype. CLIC4 associates with the Ran, NTF2, and Importin-α nuclear import complexes in immunoprecipitates of lysates from cells treated with apoptotic/stress-inducing agents. Deletion or mutation of the nuclear localization signal in the C terminus of CLIC4 eliminates nuclear translocation, whereas N terminus deletion enhances nuclear localization. Targeting CLIC4 to the nucleus via adenoviral transduction accelerates apoptosis when compared with cytoplasmic CLIC4, and only nuclear-targeted CLIC4 causes apoptosis in Apaf null mouse fibroblasts or in Bcl-2-overexpressing keratinocytes. These results indicate that CLIC4 nuclear translocation is an integral part of the cellular response to stress and may contribute to the initiation of nuclear alterations that are associated with apoptosis. [ABSTRACT FROM AUTHOR]

The cytokine interferon-β is a regulator of cell replication and function, including invasion and induction of angiogenesis. The goal of this study was to determine whether the expression of interferon-β by cells in the epidermis correlated with terminal differentiation. In situ hybridization analysis and immunohistochemical staining of formalin-fixed, paraffin-embedded specimens of normal human and murine epidermis and human and murine skin tumors of epithelial origin revealed that only differentiated, nondividing cells of the epidermis expressed interferon-β protein. Keratinocyte cultures established from the epidermis of 3 d old mice were maintained under conditions permitting continuous cell division or induction of differentiation. Continuously dividing cells did not produce interferon-β whereas nondividing differentiated cells expressing keratin 1 did. Growth-arrested, undifferentiated keratinocytes also expressed interferon-β protein. Neutralizing interferon-β in the culture medium inhibited differentiation, but the addition of exogenous interferon-β did not stimulate differentiation. These data indicate that interferon-β is produced by growth-arrested, terminally differentiated keratinocytes. [ABSTRACT FROM AUTHOR]

Techniques are described for the isolation and cultivation of functionally intact mouse hair follicles. Follicles were isolated by collagenase digestion of dermis from 5-day-old mice and purified by differential centrifugation and filtration. Purified follicles were cultured in a Type 1 collagen matrix using Medium 199 and 8% fetal calf serum as the basic nutrient. Viability of follicles was maintained in culture Since the cultures incorporated thymidine into DNA and methionine into proteins for at least 7 days. Further- more, follicles isolated from the collagen matrix after 7 days could reattach to a plastic culture substrate or be further cultivated in a fresh collagen matrix. Functional integrity of cultured follicles was maintained since some follicle-specific cytoskeletal proteins were synthesized in vitro, and follicles isolated from the collagen matrix after 7 days formed a haired skin when recombined with dermal fibroblasts and grafted to a skin site on nude mice. Only a minority of follicles appeared to produce a mature hair shaft in vitro by morphologic criteria, however, and synthesis of the total complement of hair proteins was not observed. Cholera toxin was a strong mitogen for cultured follicles, whereas epidermal growth factor was slightly mitogenic. Epidermal growth factor stimulated the release of a Type 1 collagenase by follicle cells, however. This model system provides an opportunity for the systematic analysis of factors required for the induction of hair growth and the underlying physiology of hair follicle development. This model should also be useful for studying the role of the hair follicle in skin carcinogenesis. [ABSTRACT FROM AUTHOR]

A large number of cross-linked envelopes form spontaneously when cell lines derived from chemically induced mouse skin papillomas are cultured in medium containing 1.2 mM calcium. This phenomenon is associated with high activity of the cross-linking enzyme, epidermal transglutamjnase (TGase). The influence of retinoic acid (RA) on envelope formation was studied in detail in a papilloma cell line, PE. Retinoic acid (3 μM) completely blocked cornified envelope (CE) production but reduced TGase activity only 50%. A rabbit antiserum was produced against sonicated CEs isolated from newborn mouse skin. On Western blots of epidermal extracts, diffuse staining was observed for particulate proteins of suprabasal, but not basal, cells and similar immunoreactive material was absent from the cytosolic fraction of both cell layers. The antibody also recognized particulate proteins from PE cells induced to differentiate by calcium, but not from cells grown in the presence of high calcium and RA. The antiserum appears to recognize partially cross-linked CE precursor proteins judging by the diffuse staining, the molecular weight range of the proteins stained, and their origin in the particulate cellular fraction. Cross-linked envelopes could be induced in RA-treated PE cells by permeabilization with 0.75 M NaCI or 50 μg/ml A23187. However, this treatment failed to cause the appearance of proteins recognized by the antiserum. Preincubation of the antiserum with purified fragments of CEs from newborn mouse epidermis, but not with cross-linked envelopes from permeabilized, RA-treated PE cells, removed immunoreactivity. These results indicate that the cross-linked envelopes formed in RA-treated cells after permeabilization lack a set of proteins contained in CEs from stratum corneum and may even be composed of different proteins. Retinoic acid appears to prevent CE formation in part by inhibiting activation of epidermal TGase but in addition by influencing the synthesis of precursor proteins. [ABSTRACT FROM AUTHOR]

Primary cultures of mouse epidermal cells are induced to terminally differentiate when extracellular calcium levels are increased to more than 0.1 mM After carcinogen treatment, cellular foci can be selected that resist this calcium signal to terminally differentiate Calcium causes these foci to stratify, however, in contrast to normal epidermis, DNA- synthesizing cells in these foci are found in the suprabasal cell layers as well as in basal cells Cell lines derived from these foci may be considered to be putative initiated cells Three of these cell lines, designated 308, D, and F, have been characterized for their response to calcium and phorbol ester tumor promoters. The formation of cornified cells and the activity of epidermal transglutaminase were utilized as markers of epidermal differentiation. Neither calcium nor the tumor promoter 12-O-tetradecanoylphorbol-13- acetate (TPA) increased transglutaminase activity or cornification of any of the 3 lines Proliferation was estimated by the [³H]thymidine labeling index, by incorporation of [³H]thymidine into DNA, and by a clonal growth assay. Unlike primary normal cultures, rising the calcium level of the medium did not markedly reduce the rate of proliferation of any of the 3 cell lines. in 2 of the lines, line 308 and line D, proliferation increased in response to TPA exposure. in line F, [³H]thymidine incorporation in confluent cultures was inhibited by TRA, while in cells plated at clonal densities, TPA was cytotoxic at doses of 5 ng/ml or higher. It these calcium-resistant epidermal cell lines correspond to initiated cells, their lack of sensitivity to the induction of terminal differentiation by TPA could account for their growth relative to normal cells. Those lines that also respond to stimulation of proliferation by TPA to a greater extent than normal cells would have a further growth advantage. [ABSTRACT FROM AUTHOR]

Skin extracts contain an epidermal mitosis inhibitor that recently has been purified and identified as a pentapeptide. To develop an in vitro assay system for further biologic characterization, primary mouse epidermal cells and an established mouse epidermal cell line (line 308) were used for testing of the purified pentapeptide. In primary cell cultures the mitotic activity, as estimated by means of vinblastine, was reversibly inhibited by 44% at a peptide concentration of 10-8 M in high-calcium (1.2 mM Ca++), and by 27-38% at peptide concentrations of 10-10 and 10-8 M in low-calcium (0.02 mM Ca++) medium. The 308 cells were inhibited by 46% at a peptide concentration of 10-6 M but only after the cells had reached near-confluence and had a moderate rate of proliferation. A low concentration of adrenalin (0.18 μg/ml) in the medium rendered the primary cultures more sensitive to the peptide. After repeated peptide treatments over 24 h, the number of cornified envelopes (a marker of terminal differentiation) was increased both in primary cultures and in the 308 cells. The epidermal pentapeptide thus seems to influence both proliferation and terminal differentiation in cultured mouse epidermal cells. [ABSTRACT FROM AUTHOR]

Proteins from mouse epidermis cytosol extracts react on immunoblots with a polyclonal rabbit antiserum raised against rat skin calcium-binding protein (SCaBP), a parvalbumin of the panniculus carnosus. Three mouse epidermal proteins with molecular weights between 10-12K, which are distinct from SCaBP, are recognized by the antiserum. The synthesis of these proteins in keratinocyte culture is modulated by Ca++, as is the differentiation of the keratinocytes. Proliferating mouse keratinocytes in medium containing 0.07 mM Ca++ (low CaCa++) undergo terminal differentiation when the Ca++ concentration is elevated to 1.8 mM (high Ca++). Synthesis of the 3 antigens can be demonstrated when soluble extracts of keratinocytes labeled with [35S]methionine in low Ca++ medium are immunoprecipitated with anti-SCaBP serum. These antigens are not synthesized in cultures of dermal fibroblasts. When keratinocytes are switched to high Ca++ medium, synthesis of these antigens is greatly diminished over the course of 48-72 h. However, the antigens persist in differentiating cells. When proliferating keratinocytes in low Ca++ medium are exposed to the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), differentiation is induced in a subpopulation of cells, and specific antigen synthesis is transiently inhibited. The inhibition correlates with the time when many cells are differentiating in response to TPA. When proliferating keratinocytes are pulse-labeled with 32PO4, the 11 K antigen is phosphorylated and the phosphorylation is not enhanced by TPA exposure. All 3 antigens are synthesized in a reticulocyte lysate preparation with added newborn mouse epidermis messenger RNA or mRNA from keratinocytes cultured in low Ca++ medium. Thus, these antigens are likely to represent unique proteins rather than processed or degraded ones. The coordinately regulated expression of these antigens associated with the differentiation state of the keratinocyte proliferation and differentiation. [ABSTRACT FROM AUTHOR]

Skin calcium-binding protein (SCaBP) is a calcium binding protein purified from whole rat skin. It has a molecular weight of approximately 12,000 daltons but migrates at Mr 13,000 on sodium dodecyl sulfate (SDS)-polyacrylamide gels. On nitrocellulose blots of SDS-polyacrylamide gels, 6 different antisera to SCaBP reacted equally well with SCaBP and parvalbumin (PV), an 11,500-dalton calcium-binding protein purified from rat skeletal muscle, which also migrates at Mr 13,000 on SDS-polyacrylamide gels. Rabbit antiserum to muscle PV also recognized both PV and SCaBP, and either protein absorbed specific antibodies against either antigen from both types of antisera. Soluble protein extracts from whole adult rat and mouse skin contained a Mr 13,000 protein which was recognized on nitrocellulose blots of SDS gels by both antisera. Blots of extracts from epidermis, dermis, whole skin, and skin scraped on the dermal side to remove hypodermal tissue revealed that the Mr 13,000 PV/SCaBP cross-reacting antigen was restricted to the hypodermal tissue removed by scraping. Immunofluorescent staining of Bouin-fixed skin sections with these antisera confirmed the localization of PV/SCaBP to the panniculus carnosus, a hypodermal muscle layer. Newborn mouse skin does not contain this antigen. Additional polypeptides of Mr 10,500 and 12,000 on SDS gels of extracts from the epidermis of newborn and adult rats and mice were found to be immunoreactive with anti-SCaBp serum. These polypeptides were not recognized by the PV antiserum, and the reactivity of anti-SCaBP for these antigens was not absorbed by purified PV or SCaBP. Our results indicate that SCaBP is antigenically indistinguishable from PV and is localized in the adult rodent panniculus carnosus, and that antisera to SCaBP are poly-specific, recognizing epidermal proteins in addition to SCaBP/PV. [ABSTRACT FROM AUTHOR]