Cultured Mouse Epidermal Cells Research Articles

Reversal of Murine Epidermal Atrophy by Topical Modulation of Calcium Signaling

Cytosolic Ca(2+) signals are performed by Ca(2+) releases from the endoplasmic reticulum and Ca(2+) influx from the extracellular medium. Releases rely on the refilling of the intracellular Ca(2+) stores by the Ca(2+) influx "Store-Operated Calcium Entry" (SOCE) via the channel Orai1. Here we show that Orai1 expression, SOCE amplitude, and epidermal proliferation are decreased in the epidermis of patients with skin fragility when compared with aged nonatrophic skin. Epidermal atrophy was induced in mice by the inhibition of Orai1 with small interfering RNA and the topical application of a SOCE blocker BTP2. The inhibition of Orai1 impaired the heparin-binding epidermal growth factor (HB-EGF)-induced Ca(2+) influxes and fully prevented the mitogen effect of HB-EGF in primary human keratinocytes. Importantly, epidermal proliferation correlated with Orai1 expression in mice. Conversely, the topical application of an Orai1 activator, the benzohydroquinone (BHQ), increased the epidermal thickness and proliferation, whereas the pro-proliferative effect of BHQ was prevented by the inhibition of Orai1. Finally, the topical application of BHQ reversed the epidermal atrophy induced by corticosteroids in mice. The topical modulation of Ca(2+) signals may thus be a promising therapeutic strategy in dermatology.

Abstract 5665: Zerumbone inhibits phorbol ester-induced intracellular ROS accumulation and neoplastic transformation by inducing Nrf2-driven heme oxygenase-1 expression in mouse epidermal cells

Abstract Zerumbone, derived from tropical ginger (Zingibers zerumbet, Zingiberaceae), was found to suppress chemically-induced skin tumor formation in mice. However, the molecular mechanisms underlying the chemopreventive effects of zerumbone on mouse skin carcinogenesis are poorly understood. Here, we report that topical application of zerumbone onto dorsal skin of HR-1 hairless mice induced expression of heme oxygenase-1 (HO-1) through activation of Nrf2, a major transcription factor that plays a pivotal role in regulating expression of several antioxidant/detoxifying enzymes. To verify the possible involvement of Nrf2 in zerumbone-derived induction of HO-1, we compared HO-1 protein levels in the zerumbone-treated skin of Nrf2 wild-type (+/+) and Nrf2 knock out (−/−) mice. Nrf2-deficient mice expressed HO-1 in the skin to a much lesser extent than did the wild-type animals following topical application of zerumbone (10 μmol). Moreover, zerumbone (10 μM) inhibited anchorage-independent growth as well as ROS production in mouse epidermal JB6 cells stimulated with 12-0-tetradecanoylphorbol-13-acetate (TPA; 10 ng/ml). Likewise, treatment of JB6 cells with zerumbone resulted in a markedly increased nuclear translocation of Nrf2 as well as the promoter activity of HO-1. In addition, siRNA knock down of nrf2 in JB6 cells diminished the zerumbone-induced expression of HO-1. Using a chromatin immunoprecipitation (ChIP) assay, we confirmed that zerumbone induced the direct binding of Nrf2 to antioxidant response element (ARE). We hypothesized that the electrophilic α,β-unsaturated carbonyl moiety present in zerumbone could modify critical cysteine thiols of Keap1, a cytosolic repressor of Nrf2, thereby facilitating its dissociation from Keap1 for nuclear translocation. α-Humulene and 8-hydroxy-α-humulene, structural derivatives of zerumbone that lack the α,β-unsaturated carbonyl group, were unable to activate Nrf2 and induce HO-1 expression in both mouse skin in vivo and cultured mouse epidermal cells. Unlike zerumbone, these non-electrophilic analogs failed to suppress TPA-induced JB6 cell transformation and ROS accumulation. Interestingly, treatment of JB6 cells with 50 μM carbon monoxide-releasing molecule (CORM) markedly reduced ROS accumulation indicating that carbon monoxide formed as a consequence of zerumbone-induced HO-1 expression has an important role in abolishing TPA-induced ROS generation. Taken together, the above findings suggest that up-regulation of HO-1 by zerumbone is mediated via the Nrf2 signaling pathway, which provides a mechanistic basis for the anti-tumor promoting effects of this sesquiterpene on mouse skin carcinogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5665.

Differential Inhibition of UV-Induced Activation of NFκ B and AP-1 by Extracts From Black Raspberries, Strawberries, and Blueberries

Recent studies from our laboratory have shown that the transactivation of nuclear factor κ B (NFκ B) and activator protein-1 (AP-1) plays an important mechanistic role in ultraviolet (UV)-induced skin carcinogenesis in mice. We also demonstrated that a methanol extract (ME) fraction from black raspberries (Rubus occidentalis) (RO; RO-ME) inhibits benzo[a]pyrene-7,8-diol-9,10-epoxide [B(a)PDE]–induced activation of NFκ B and AP-1 in cultured mouse epidermal cells. In the present study, we determined if RO-ME might also inhibit the induction of NFκ B and AP-1 in mouse epidermal cells exposed to mid UV radiation (UVB) and short UV radiation (UVC) and whether methanol fractions from strawberries and blueberries would also be effective. Our results showed that RO-ME inhibited UVB-induced activation of NFκ B in mouse epidermal cells in a time- and dose-dependent manner; however, the methanol fractions from strawberries and blueberries were ineffective. Interestingly, none of the fractions from all 3 berry types inhibited UVB- or UVC-induced activation of AP-1, suggesting that inhibition of UV-induced signaling pathways is specific for black raspberries and NFκ B. Cyanidin-3-rutinoside, an anthocyanin found in abundance in black raspberries and not in strawberries or high-bush blueberries, was found to contribute to the inhibition of UVB-induced activation of NFκ B. These results suggest that berries differ in their ability to influence signaling pathways leading to activation of NFκ B and AP-1 when using UV light as the inducer.

Effect of tick salivary gland extract on the cytokine production by mouse epidermal cells

Previous studies have demonstrated that both tick saliva and Borrelia burgdorferi sensu lato antigens modulate the cytokine response of the host. In this paper, the effect of salivary gland extract (SGE) from Ixodes ricinus (L., 1758) ticks on cytokine production by primary cultures of mouse epidermal cells stimulated with Borrelia afzelii Canica, Nato, du Merle, Mazie, Baranton et Postic, 1993 spirochetes was analysed. Epidermal cells were derived from C3H/HeN mice, susceptible to Lyme disease, and BALB/c mice, which are resistant. In cultures from C3H/HeN mice, SGE down regulated production of tumour necrosis factor alpha (TNF-alpha) and up regulated Th2 cytokine, interleukin 4 (IL-4). Cultures from BALB/c mice produced higher basal levels of monitored cytokines, but their production was affected by SGE a different way. While Th2 cytokines IL-6 and IL-10 were down regulated, the effect on TNF-alpha and IL-4 was ambiguous. These results indicate that the effect of tick saliva on the epidermal cells of Lyme disease-susceptible C3H/HeN mice mirrors its effect on other cells of the immune system.

Inhibitory effects of [6]-gingerol on PMA-induced COX-2 expression and activation of NF-kappaB and p38 MAPK in mouse skin.

[6]-Gingerol, a major pungent ingredient of ginger (Zingiber officinale Roscoe, Zingiberaceae), has a wide array of pharmacologic effects. Previous studies have demonstrated that [6]-gingerol inhibits mouse skin tumor promotion and anchorage-independent growth of cultured mouse epidermal cells stimulated with epidermal growth factor. Cyclooxygenase-2 (COX-2), a key enzyme in the prostaglandin biosynthesis, has been recognized as a molecular target for many anti-inflammatory as well as chemopreventive agents. Topical application of [6]-gingerol inhibited phorbol 12-myristate 13-acetate -induced COX-2 expression. One of the essential transcription factors responsible for COX-2 induction is NF-kappaB. [6]-Gingerol suppressed NF-kappaB DNA binding activity in mouse skin. In addition, [6]-gingerol inhibited the phoshorylation of p38 mitogen-activated protein kinase which may account for its inactivation of NF-kappaB and suppression of COX-2 expression.

A highly efficient ligand-regulated Cre recombinase mouse line shows that LoxP recombination is position dependent.

Conditional gene inactivation using the Cre/loxP system is widely used, but the difficulty in properly regulating Cre expression remains one of the bottlenecks. One approach to regulate Cre activity utilizes a mutant estrogen hormone-binding domain (ERT) to keep Cre inactive unless the non-steroidal estrogen analog 4-hydroxytamoxifen (OHT) is present. Here we describe a mouse strain expressing Cre-ERT from the ubiquitously expressed ROSA26 (R26) locus. We demonstrate efficient temporal and spatial regulation of Cre recombination in vivo and in primary cells derived from these mice. We show the existence of marked differences in recombination frequencies between different substrates within the same cell. This has important consequences when concurrent switching of multiple alleles within the same cell is needed, and highlights one of the difficulties that may be encountered when using reporter mice as indicator strains.

Induction of protein kinase Cζ-related protein kinase by growth suppression in carcinogen-initiated epidermal cell-line WYF31 cells

In primary cultured mouse epidermal cells, protein kinase C isozyme ζ (PKCζ) consists of multiple forms, for example, low-salt eluted PKCζ (l-PKCζ; 79 and 85 kDa) and high-salt eluted PKCζ (h-PKCζ; 79 and 85 kDa) on anion-exchange column chromatography. In this study, biochemical and biophysical differences between l-PKCζ and h-PKCζ were examined by using carcinogen-initiated mouse epidermal cell-line WYF31 cells, whose growth is stimulated by tumour promoter phorbol 12-myristate 13-acetate (PMA). The binding efficiency of h-PKCζ to anti-PKCζ antibody-affinity column was 10 times higher than that of l-PKCζ. T7-tagged rat PKCζ overexpressed in WYF31 cells was recovered only in the high-salt eluted area on the anion-exchange column. Furthermore, when rat PKCζ was stably overexpressed in WYF31 cells, the content of h-PKCζ increased 4 to 5 times compared to that of parental cells, but the content of l-PKCζ was not altered. All of these results indicate that h-PKCζ is the product of the PKCζ gene (referred to as PKCζ) and that l-PKCζ is closely related but different from PKCζ (referred to as PKCζ-related kinase). Interestingly, serum starvation of WYF31 cells caused a marked increase of the content of PKCζ-related kinase with a concomitant decrease of PKCζ content. These changes were reversed by stimulating the cell growth with 10% foetal calf serum. Prolonged treatment of starved cells with PMA, which induces the proliferation of WYF31 cells, also caused the downregulation of PKCζ-related kinase. These results suggest that the expression levels of PKCζ-related kinase and PKCζ are differently regulated, and that the increased expression of PKCζ-related kinase might play a significant role in the growth-suppression processes of WYF31 cells.

Activation of Phorbol Ester Responsive Form of Protein Kinase Cζ in Association with Ca2+-Induced Differentiation of Primary Cultured Mouse Epidermal Cells

In primary cultured mouse epidermal cells, protein kinase C (PKC) ζ consists of multiple forms: a low salt-eluted PKCζ (1-PKCζ, 79 and 85 kDa) and a high salt-eluted PKCζ (h-PKCζ, 79 and 85 kDa) by anion-exchange column chromatography (K. Nishikawa et al., Cell. Signal. 7, 491-504, 1995). In the present study, PKC isozyme-specific responses during terminal differentiation of epidermal cells, which were induced by the increase of Ca2+-concentration in culture medium, were examined. After 24 hr-treatment with 1.8 mM Ca2+, 79-kDa 1-PKCζ in the particulate fraction was apparently shifted to the 85-kDa form. The phosphatidylserine-dependent kinase activity of this 1-PKCζ was increased in association with the shift. These results suggest the pivotal role of 1-PKCζ in the particulate fraction in the Ca2+-induced epidermal cell differentiation processes.

Activation of phorbol ester responsive form of protein kinase C zeta in association with Ca(2+)-induced differentiation of primary cultured mouse epidermal cells.

In primary cultured mouse epidermal cells, protein kinase C (PKC) zeta consists of multiple forms: a low salt-eluted PKC zeta (1-PKC zeta, 79 and 85 kDa) and a high salt-eluted PKC zeta (h-PKC zeta, 79 and 85 kDa) by anion-exchange column chromatography (K. Nishikawa et al., Cell. Signal. 7, 491-504, 1995). In the present study, PKC isozyme-specific responses during terminal differentiation of epidermal cells, which were induced by the increase of Ca(2+)-concentration in culture medium, were examined. After 24 hr-treatment with 1.8 mM Ca2+, 79-kDa 1-PKC zeta in the particulate fraction was apparently shifted to the 85-kDa form. The phosphatidylserine-dependent kinase activity of this 1-PKC zeta was increased in association with the shift. These results suggest the pivotal role of 1-PKC zeta in the particulate fraction in the Ca(2+)-induced epidermal cell differentiation processes.

Inhibition of anthralin-caused skin tumor promotion and interleukin-1 alpha production by potent immunosuppressant FK506.

The effect of FK506, a potent immunosuppressive agent, on 7,12-dimethylbenz[alpha]anthracene-initiated and anthralin-promoted skin tumor formation was examined in CD-1 mice. A topical application of 0.1 mumol FK506 to mouse skin 15 min prior to each anthralin treatment markedly inhibited skin tumor formation. Anthralin stimulated IL-1 alpha production in primary cultured mouse epidermal cells, and the peak IL-1 alpha level was observed at 6 h after the stimulation. Anthralin also stimulated IL-1 alpha release into culture medium. Both production and release of Il-1 alpha were markedly inhibited by FK506 (0.1 or 1 microM). FK506 (1 microM) alone neither affected IL-1 alpha production nor its release. It may be possible that the inhibition of IL-1 alpha production by FK506 is related to its anti-tumor-promoting action.

KP-10, a novel protein kinase C substrate in intact mouse epidermal cells, is phosphorylated by novel protein kinase Cη and/or ξ

Recently this group found an endogenous substrate protein for Ca(2+)-independent novel protein kinase C (nPKC), i.e. KP-10 (pI 4.7/25,500 M(r)), in primary cultured mouse epidermal cells [Nishikawa, K. et al. (1992) Cell. Signal. 4, 757-776]. In the present study, the nPKC isozymes which phosphorylate KP-10 in these cells were determined. Western blot analysis revealed that PKC alpha, eta and zeta were present in epidermal cell 105,000 g supernatants and that the content of PKC zeta was much higher than those of PKC alpha and eta. Neither PKC beta, delta nor epsilon was detected in the 105,000 g supernatants. Phosphatidylserine and phorbol 12-myristate 13-acetate (PMA)-dependent KP-10 phosphorylating activity was immunoprecipitated by anti-PKC eta and zeta antibodies, but not by antiPKC alpha antibody. These results suggest that PKC eta and/or zeta phosphorylate KP-10 and play pivotal roles in intracellular signal pathways in intact epidermal cells.

Modulation of phorbol ester-associated events in epidermal cells by linoleate and arachidonate.

To elucidate the events elicited by the skin tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), which are modulated by linoleic acid (LA) and arachidonic acid (AA), the activity of these fatty acids in cultured mouse epidermal cells was compared. Approximately 94% of either exogenous radiolabelled fatty acid was incorporated into the total phospholipid pool over 15 h. The relative distribution among the phospholipid classes differed, however, such that approximately 70% of phospholipid-associated [14C]-LA was found in phosphatidylcholine, compared to approximately 30% for [14C]AA. Phosphatidylethanolamine and phosphatidylinositol/phosphatidylserine contained 17 and 13% of the phospholipid [14C]LA, and 34 and 30% of [14C]AA, respectively. Prostaglandin (PG) E2 production was low but similar in unstimulated cultures prelabelled with either [14C]LA or [14C]AA. However, in cultures treated with TPA (1.6 microM), [14C]AA-prelabelling resulted in approximately three times the amount of [14C]PGE2 compared with cultures prelabelled with [14C]LA. Cultured cells were found to contain significant delta 6 desaturase activity, which may enable conversion of LA to AA, and thus may account for the observed PGE2 production from [14C]LA treated cells. AA-Supplemented (1.6 microM) cultures supported approximately twice the induction of ornithine decarboxylase activity by TPA compared with cultures treated with 1.8 microM LA. Activation of partially purified protein kinase C was similar for either fatty acid tested over a 10-300 microM dose range. Overall, the results suggest that LA does not have the same biological activity as AA with regard to several TPA-associated events known to be important in skin tumor promotion.(ABSTRACT TRUNCATED AT 250 WORDS)

Suppressive Effect of Transforming Growth Factor-β on the Phosphorylation of Endogenous Substrates by Conventional and Novel Protein Kinase C in Primary Cultured Mouse Epidermal Cells

The effect of transforming growth factor-β (TGF-β) on the endogenous protein phosphorylation caused by phorbol 12-myristate 13-acetate (PMA), a potent activator of protein kinase C (PKC), was examined in primary cultured mouse epidermal cells. PMA markedly stimulates phosphorylation of endogenous proteins, i.e. KP-1 and KP-2, through Ca 2+-dependent conventional PKC (cPKC), and KP-10 through Ca 2+-independent novel PKC (nPKC) in intact epidermal cells. TGF-β strongly suppressed the PMA-stimulated phosphorylation of these three proteins. Rate of dephosphorylation of these phosphorylated proteins was not affected by TGF-β. Treatment of epidermal cells with TGF-β decreased cPKC activity both in cytosolic and particulate fractions, but not nPKC activity. These results indicate that TGF-β suppresses cPKC- and nPKC-mediated endogenous protein phosphorylation in intact epidermal cells, but the mechanisms of suppression are different.

Characterization of endogenous substrates for novel-type protein kinase C as well as conventional-type protein kinase C in primary cultured mouse epidermal cells

In primary cultured mouse epidermal cells, phorbol 12-myristate 13-acetate (PMA), which activates protein kinase C (PKC), induced changes in the phosphorylation levels of 10 proteins, termed KP-1 to 10, in two-dimensional PAGE. Seven of these proteins were phosphorylated and three were dephosphorylated. Similar changes were induced by other PKC activators, but not by inactive phorbol ester. Among these substrate proteins, phosphorylation of three proteins, i.e. KP-1 (pI 4.7/23,000 M r ), KP-2 (pI 4.7/20,700 M r ) and KP-10 (pI 4.7/25,000 M r was markedly enhanced by PMA and inhibited by a potent PKC inhibitor staurosporine. In vitro phosphorylation studies and phosphoamino acid analysis, using these proteins as substrate and PKC preparations obtained from epidermal cell lysate, revealed that KP-1 and -2 were directly phosphorylated by Ca 2+-, phospholipid-dependent protein kinase (conventional-type PKC; cPKC), but not by Ca 2+-independent, phospholipid-dependent protein kinase (novel-type PKC; nPKC). On the other hand, KP-10 was mainly phosphorylated by nPKC in intact epidermal cells. These results indicate that cPKC and nPKC in epidermal cells have different substrate specificity for endogenous proteins and may induce different signal transduction.

Protein kinase C-dependent and -independent actions of a potent protein kinase C inhibitor, staurosporine

12-O-Tetradecanoylphorbol 13-acetate (TPA), an activator of protein kinase C (PKC), induced ornithine decarboxylase (ODC) in primary cultured mouse epidermal cells. Staurosporine, a potent protein kinase C inhibitor, also induced ODC activity. Both TPA- and staurosporine-caused ODC inductions were markedly suppressed in the PKC-down-regulated cells. Another PKC inhibitor, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7), inhibited both TPA- and staurosporine-caused ODC inductions. H-7 by itself never induced ODC activity. Under our experimental conditions, staurosporine induced no detectable phosphorylation of endogenous proteins. TPA induced a translocation of PKC from cytosol to membrane whereas an optimal concentration of staurosporine to induce ODC did not induce an obvious translocation of PKC. Indomethacin, a cyclooxygenase inhibitor, inhibited staurosporine-caused ODC induction, but not TPA-caused ODC induction. Staurosporine induced specific morphological changes of epidermal cells both in normal and in PKC-down-regulated cells. These results indicate that staurosporine induces ODC activity in a PKC-dependent manner and morphological changes possibly through a PKC-independent mechanism. The mechanism of ODC induction caused by staurosporine may be in some way different from that caused by TPA.

Anthralin, a non-TPA type tumor promoter, synergistically enhances phorbol ester-caused prostaglandin E2 release from primary cultured mouse epidermal cells.

Primary cultures of mouse epidermal cells (i.e., target cells of skin tumor promotion) stimulated by 12-O-tetradecanoylphorbol-13-acetate (TPA) release prostaglandin E2 within 30 min. Anthralin, a non-TPA type tumor promoter, also stimulated PGE2 release; however, no release was detectable at least up to 4 hr after the addition of anthralin. When the cells were incubated with TPA plus anthralin, both PGE2 and arachidonic acid release were synergistically enhanced. Other non-TPA type tumor promoters, i.e., chrysarobin, 7-bromomethylbenz[a]anthracene, benzoylperoxide, okadaic acid and palytoxin, did not potentiate the TPA-caused PGE2 release. In protein kinase C-down regulated cells, the synergistic stimulation of PGE2 and arachidonic acid release by TPA plus anthralin were not detected. Anthralin plus TPA did not alter the incorporation of arachidonic acid into cellular phospholipids. Cellular cyclooxygenase activity was increased 2 hr after TPA stimulation. Anthralin-caused increase in cyclooxygenase activity was detected at 6 hr after the addition of anthralin. Cyclooxygenase activity was synergistically increased by treating the cells with TPA plus anthralin. Cycloheximide and actinomycin D inhibited the increase in cyclooxygenase activity caused by anthralin or TPA plus anthralin. These results indicate that anthralin synergistically stimulates TPA-caused PGE2 release by synergistically increasing arachidonic acid release and cellular cyclooxygenase activity.

Anthralin, a Non-TPA Type Tumor Promoter, Synergistically Enhances Phorbol Ester-Caused Prostaglandin E2 Release from Primary Cultured Mouse Epidermal Cells

Primary cultures of mouse epidermal cells (i.e., target cells of skin tumor promotion) stimulated by 12-O-tetradecanoylphorbol-13-acetate (TPA) released prostaglandin E2 within 30 min. Anthralin, a non-TPA type tumor promoter, also stimulated PGE2 release; however, no release was detectable at least up to 4 hr after the addition of anthralin. When the cells were incubated with TP A plus anthralin, both PGE2 and arachidonic acid release were synergistically enhanced. Other non-TPA type tumor promoters, i.e., chrysarobin, 7-bromomethylbenz[a]anthracene, benzoylperoxide, okadaic acid and palytoxin, did not potentiate the TPA-caused PGE2 release. In protein kinase C-down regulated cells, the synergistic stimulation of PGE2 and arachidonic acid release by TPA plus anthralin were not detected. Anthralin plus TPA did not alter the incorporation of arachidonic acid into cellular phospholipids. Cellular cyclooxygenase activity was increased 2 hr after TPA stimulation. Anthralin-caused increase in cyclooxygenase activity was detected at 6 hr after the addition of anthralin. Cyclooxygenase activity was synergistically increased by treating the cells with TPA plus anthralin. Cycloheximide and actinomycin D inhibited the increase in cyclooxygenase activity caused by anthralin or TPA plus anthralin. These results indicate that anthralin synergistically stimulates TPA-caused PGE2 release by synergistically increasing arachidonic acid release and cellular cyclooxygenase activity.

Analysis of point mutations in murine c-Ha-ras of skin tumors initiated with dibenz[a,j]anthracene and derivatives.

This study was designed to evaluate the point mutations in the murine c-Ha-ras gene of skin papillomas induced by initiation with dibenz[a,j]anthracene (DB[a,j]A), its bay-region anti-diol epoxide ((+/-)anti-DB[a,j]A-DE), and a 7,14-dimethyl analogue (7,14-diMeDB[a,j]A). Recent studies (Nair RV, et al., Chem Res Toxicol 4:115-122, 1991) in our laboratory have revealed both deoxyguanosine (dGuo) and deoxyadenosine (dAdo) adducts formed from the anti- and syn-diol epoxides of DB[a,j]A in cultured mouse epidermal cells after exposure to this hydrocarbon. Using PCR amplification and direct sequencing, we found specific A182----T transversion mutations (eight of 10 tumors) in codon 61 of c-Ha-ras in papillomas induced by initiation with DB[a,j]A. Analysis of papillomas generated by initiation with the more biologically potent analogue 7,14-diMeDB[a,j]A revealed that five of five tumors exhibited A182----T transversions in codon 61. The nature of the changes in the two DB[a,j]A tumors not showing codon 61 mutations in Ha-ras is currently not known since these tumor DNAs also did not possess c-Ha-ras mutations at codons 12, 13, or 59. Interestingly, papillomas produced by initiation with (+/-)anti-DB[a,j]A-DE also possessed A182----T transversion mutations in codon 61 of c-Ha-ras (five of five tumors). These data suggest that dAdo adducts derived from both parent hydrocarbons may play an important role in their tumor-initiating activity and possibly implicate a specific diol epoxide-dAdo adduct in this process.

12-O-tetradecanoylphorbol-13-acetate--induced levels of AP-1 proteins: a 46-kDa protein immunoprecipitated by anti-fra-1 and induced in promotion-resistant but not promotion-sensitive JB6 cells.

Neoplastic transformation and transcriptional activation by activator protein-1 (AP-1) complex are stimulated by tumor-promoting agents in promotion-sensitive (P+) but not promotion-resistant (P-) mouse epidermal JB6 cells in culture. This implicates AP-1 as a specific regulator of signal transduction pathways in the promotion phase of neoplastic transformation. We therefore hypothesized that the defective P- responsiveness may be due to limiting levels of AP-1 protein components in those cells. In this investigation, steady-state levels of AP-1 protein components were measured by immunoprecipitating proteins from 12-O-tetradecanoylphorbol-13-acetate (TPA)-treated P+ and P- cells to discern what may limit the AP-1 response. Whereas the AP-1 proteins junB, junD, and fosB did not show differential basal or TPA-inducible levels in P+ and P- cells, a 46-kDa species precipitated by anti-fra-1 antibody was TPA-inducible in P- cells but not in P+ cells, and c-jun protein was present at higher levels in TPA-treated and untreated P+ cells than in P- cells. These data raise the possibility that the 46-kDa fra-1-related protein may be a negative modulator of AP-1 activity and suggest that elevated levels of this 46-kDa species and limiting levels of c-jun may significantly impair AP-1 function or transformation response in P- cells or both.

Regulation of connexin 43-mediated gap junctional intercellular communication by Ca2+ in mouse epidermal cells is controlled by E-cadherin.

Gap junctional intercellular communication (GJIC) of cultured mouse epidermal cells is mediated by a gap junction protein, connexin 43, and is dependent on the calcium concentration in the medium, with higher GJIC in a high-calcium (1.2 mM) medium. In several mouse epidermal cell lines, we found a good correlation between the level of GJIC and that of immunohistochemical staining of E-cadherin, a calcium-dependent cell adhesion molecule, at cell-cell contact areas. The variant cell line P3/22 showed both low GJIC and E-cadherin protein expression in low- and high-Ca2+ media. P3/22 cells showed very low E-cadherin mRNA expression. To test directly whether E-cadherin is involved in the Ca(2+)-dependent regulation of GJIC, we transfected the E-cadherin expression vector into P3/22 cells and obtained several stable clones which expressed high levels of E-cadherin mRNA. All transfectants expressed E-cadherin molecules at cell-cell contact areas in a calcium-dependent manner. GJIC was also observed in these transfectants and was calcium dependent. These results suggest that Ca(2+)-dependent regulation of GJIC in mouse epidermal cells is directly controlled by a calcium-dependent cell adhesion molecule, E-cadherin. Furthermore, several lines of evidence suggest that GJIC control by E-cadherin involves posttranslational regulation (assembly and/or function) of the gap junction protein connexin 43.