Genetic ablation of cyclooxygenase-2 in keratinocytes produces a cell-autonomous defect in tumor formation.

Using a mouse skin tumor model, we reported previously that cyclooxygenase-2 (COX-2) deficiency reduced papilloma formation. However, this model did not differentiate between the effects of systemic COX-2-deficiency and keratinocyte-specific COX-2 deficiency on tumor formation. To determine whether keratinocyte-specific COX-2 deficiency reduced papilloma formation, v-H-ras-transformed COX-2+/+ and COX-2-/- keratinocytes were grafted onto nude mice and tumor development was compared. Transformed COX-2+/+ and COX-2-/- keratinocytes expressed similar levels of H-ras, epidermal growth factor receptor and phospho-extracellular signal-regulated kinase 1/2 in vitro; and COX-2-deficiency did not reduce uninfected or v-H-ras infected keratinocyte replication. In contrast, tumors arising from grafted transformed COX-2+/+ and COX-2-/- keratinocytes expressed similar levels of H-ras, but COX-2 deficiency reduced phospho-extracellular signal-regulated kinase 1/2 and epidermal growth factor receptor levels 50-60% and tumor volume by 80% at 3 weeks. Two factors appeared to account for the reduced papilloma size. First, papillomas derived from COX-2-/- keratinocytes showed about 70% decreased proliferation, as measured by bromodeoxyuridine incorporation, compared with papillomas derived from COX-2+/+ keratinocytes. Second, keratin 1 immunostaining of papillomas indicated that COX-2-/- keratinocytes prematurely initiated terminal differentiation. Differences in the levels of apoptosis and vascularization did not appear to be contributing factors as their levels were similar in tumors derived from COX-2-/- and COX-2+/+ keratinocytes. Overall, the data are in agreement with our previous observations that decreased papilloma number and size on COX-2-/- mice resulted from reduced keratinocyte proliferation and accelerated keratinocyte differentiation. Furthermore, the data indicate that deficiency/inhibition of COX-2 in the initiated keratinocyte is an important determinant of papilloma forming ability.

Multi-walled carbon nanotubes induce COX-2 and iNOS expression via MAP kinase-dependent and -independent mechanisms in mouse RAW264.7 macrophages.

BACKGROUND: Carbon nanotubes (CNTs) are engineered graphene cylinders with numerous applications in engineering, electronics and medicine. However, CNTs cause inflammation and fibrosis in the rodent lung, suggesting a potential human health risk. We hypothesized that multi-walled CNTs (MWCNTs) induce two key inflammatory enzymes in macrophages, cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS), through activation of extracellular signal-regulated kinases (ERK1,2). METHODS: RAW264.7 macrophages were exposed to MWCNTs or carbon black nanoparticles (CBNPs) over a range of doses and time course. Uptake and subcellular localization of MWCNTs was visualized by transmission electron microscopy (TEM). Protein levels of COX-2, iNOS, and ERK1,2 (total ERK and phosphorylated ERK) were measured by Western blot analysis. Prostaglandin-E(2) (PGE(2)) and nitric oxide (NO) levels in cell supernatants were measured by ELISA and Greiss assay, respectively. RESULTS: MWCNTs, but not CBNPs, induced COX-2 and iNOS in a time- and dose-dependent manner. COX-2 and iNOS induction by MWCNTs correlated with increased PGE(2) and NO production, respectively. MWCNTs caused ERK1,2 activation and inhibition of ERK1,2 (U0126) blocked MWCNT induction of COX-2 and PGE2 production, but did not reduce the induction of iNOS. Inhibition of iNOS (L-NAME) did not affect ERK1,2 activation, nor did L-NAME significantly decrease COX-2 induction by MWCNT. Nickel nanoparticles (NiNPs), which are present in MWCNTs as a residual catalyst, also induced COX-2 via ERK-1,2. However, a comparison of COX-2 induction by MWCNTs containing 4.5 and 1.8% Ni did not show a significant difference in ability to induce COX-2, indicating that characteristics of MWCNTs in addition to Ni content contribute to COX-2 induction. CONCLUSION: This study identifies COX-2 and subsequent PGE(2) production, along with iNOS induction and NO production, as inflammatory mediators involved in the macrophage response to MWCNTs. Furthermore, our work demonstrates that COX-2 induction by MWCNTs in RAW264.7 macrophages is ERK1,2-dependent, while iNOS induction by MWCNTs is ERK1,2-independent. Our data also suggest contributory physicochemical factors other than residual Ni catalyst play a role in COX-2 induction to MWCNT.

The prostaglandin E2 receptor, EP2, stimulates keratinocyte proliferation in mouse skin by G protein-dependent and {beta}-arrestin1-dependent signaling pathways.

The prostaglandin E(2) (PGE(2)) G protein-coupled receptor (GPCR), EP2, plays important roles in mouse skin tumor development (Chun, K. S., Lao, H. C., Trempus, C. S., Okada, M., and Langenbach, R. (2009) Carcinogenesis 30, 1620-1627). Because keratinocyte proliferation is essential for skin tumor development, EP2-mediated signaling pathways that contribute to keratinocyte proliferation were investigated. A single topical application of the EP2 agonist, butaprost, dose-dependently increased keratinocyte replication via activation of epidermal growth factor receptor (EGFR) and PKA signaling. Because GPCR-mediated activation of EGFR can involve the formation of a GPCR-ß-arrestin-Src signaling complex, the possibility of a ß-arrestin1-Src complex contributing to EP2-mediated signaling in keratinocytes was investigated. Butaprost induced ß-arrestin1-Src complex formation and increased both Src and EGFR activation. A role for ß-arrestin1 in EP2-mediated Src and EGFR activation was demonstrated by the observation that ß-arrestin1 deficiency significantly reduced Src and EGFR activation. In agreement with a ß-arrestin1-Src complex contributing to EGFR activation, Src and EGFR inhibition (PP2 and AG1478, respectively) indicated that Src was upstream of EGFR. Butaprost also induced the activation of Akt, ERK1/2, and STAT3, and both ß-arrestin1 deficiency and EGFR inhibition (AG1478 or gefitinib) decreased their activation. In addition to ß-arrestin1-dependent EGFR activation, butaprost increased PKA activation, as measured by phospho-GSK3ß (p-GSK3ß) and p-cAMP-response element-binding protein formation. PKA inhibition (H89 or R(P)-adenosine-3',5'-cyclic monophosphorothioate (R(P)-cAMPS)) decreased butaprost-induced cAMP-response element-binding protein and ERK activation but did not affect EGFR activation, whereas ß-arrestin1 deficiency decreased EGFR activation but did not affect butaprost-induced PKA activation, thus indicating that they were independent EP2-mediated pathways. Therefore, the results indicate that EP2 contributed to mouse keratinocyte proliferation by G protein-independent, ß-arrestin1-dependent activation of EGFR and G protein-dependent activation of PKA.

The prostaglandin receptor EP2 activates multiple signaling pathways and beta-arrestin1 complex formation during mouse skin papilloma development.

Prostaglandin E(2) (PGE(2)) is elevated in many tumor types, but PGE(2)'s contributions to tumor growth are largely unknown. To investigate PGE(2)'s roles, the contributions of one of its receptors, EP2, were studied using the mouse skin initiation/promotion model. Initial studies indicated that protein kinase A (PKA), epidermal growth factor receptor (EGFR) and several effectors-cyclic adenosine 3',5'-monophosphate response element-binding protein (CREB), H-Ras, Src, protein kinase B (AKT) and extracellular signal-regulated kinase (ERK)1/2-were activated in 12-O-tetradecanoylphorbol-13-acetate (TPA)-promoted papillomas and that PKA and EGFR inhibition (H89 and AG1478, respectively) decreased papilloma formation. EP2's contributions to the activation of these pathways and papilloma development were determined by inhibiting endogenous TPA-induced PGE(2) production with indomethacin (Indo) and concomitantly treating with the EP2 agonist, CAY10399 (CAY). CAY treatment restored papilloma formation in TPA/Indo-treated mice and increased cyclic adenosine 3',5'-monophosphate and PKA activation as measured by p-CREB formation. CAY treatment also increased EGFR and Src activation and their inhibition by AG1478 and PP2 indicated that Src was upstream of EGFR. CAY also increased H-Ras, ERK1/2 and AKT activation, and AG1478 decreased their activation indicating EGFR being upstream. Supporting EP2's contribution, EP2-/- mice exhibited 65% fewer papillomas and reduced Src, EGFR, H-Ras, AKT and ERK1/2 activation. G protein-coupled receptor (GPCR) activation of EGFR has been reported to involve Src's activation via a GPCR-beta-arrestin-Src complex. Indeed, immunoprecipitation of beta-arrestin1 or p-Src indicated the presence of an EP2-beta-arrestin1-p-Src complex in papillomas. The data indicated that EP2 contributed to tumor formation via activation of PKA and EGFR and that EP2 formed a complex with beta-arrestin1 and Src that contributed to signaling and/or EP2 desensitization.

Cyclooxygenase-2 deficiency increases epidermal apoptosis and impairs recovery following acute UVB exposure.

The cyclooxygenases, COX-1 and COX-2, are involved in cutaneous responses to both acute and chronic UV exposure. In the present study, wild-type (WT), COX-1-/- and COX-2-/- mice were used to determine the influence of the individual isoform on mouse skin responses to acute UVB treatment. Immunohistochemistry and Western analysis indicated that COX-2, and not COX-1, was induced by UVB (2.5 or 5.0 kJ/m2), but that COX-1 remained the major source of prostaglandin E2 production. UVB exposure significantly increased epidermal apoptosis in all genotypes compared to untreated mice. However, while the number of apoptotic cells in WT and COX-1-/- mice were about equal, the number of apoptotic cells was 2.5-fold greater in COX-2-/- mice. Apoptosis in WT and COX-2-/- mice peaked at 24 h post-exposure. The increased apoptosis and reduced proliferation in COX-2-/- mice resulted in about a 50% decrease in epidermal thickness at 24-48 h post-exposure compared to about a 50% increase in epidermal thickness in WT mice. UVB-induced cell replication, as measured by BrdU labeling, was reduced in COX-2-/- compared to WT mice at 24-96 h. However, by 96 h post-exposure, both WT and COX-2-/- mice showed epidermal hyperplasia. The data indicate that COX-2 induction initially protects against the acute sunburn effects of UVB, but that continuous induction of COX-2 may contribute to skin cancer in chronic UVB exposure.

Genetic deficiency or pharmacological inhibition of cyclooxygenase-1 or -2 induces mouse keratinocyte differentiation in vitro and in vivo.

Previously we demonstrated that genetic deficiency of the cyclooxygenases (COX-1 or COX-2) altered keratinocyte differentiation in mouse skin [Tiano et. al. (2002) Cancer Res. 62, 3395-3401]. In this study, we show that topical application of SC-560 (a COX-1 selective inhibitor) or celecoxib (COX-2 selective) to TPA-treated wild-type skin caused fivefold increases in the number of basal keratinocytes expressing the early differentiation marker keratin 1 (K1). In contrast to skin, COX-2 not COX-1 was the major isoform expressed in cultured primary keratinocytes. COX-1 was predominantly expressed in detached, differentiated cells, whereas COX-2 was found in the attached, proliferating cells. High Ca++ medium induced K1 and COX-1 in wild-type keratinocytes but did not change COX-2 expression. As observed in skin, COX-1-/- and COX-2-/- primary keratinocytes expressed fivefold more K1 than wild-type cells. K1 levels in cultured wild-type keratinocytes were also increased by treatment with celecoxib and indomethacin. However, unlike its in vivo effect, SC-560, possibly due to low COX-1 expression in cultured mouse keratinocytes, did not increase K1 levels. Furthermore, no increases in apoptotic cell numbers were observed in COX-deficient keratinocytes or COX-inhibitor treated wild-type cells. Thus, a major effect of COX inhibitors and COX-deficiency is the induction of keratinocyte differentiation.

Characterization and reduction of formaldehyde emissions from a low-VOC latex paint.

The patterns of formaldehyde emission from a low volatile organic compound (VOC) latex paint applied to gypsum board were measured and analyzed by small environmental chamber tests. It was found that the formaldehyde emissions resulted in a sharp increase of chamber air formaldehyde concentration to a peak followed by transition to a long-term slow decay. A semi-empirical first-order decay in-series model was developed to interpret the chamber data. The model characterized the formaldehyde emissions from the paint in three stages: an initial "puff" of instant release, a fast decay, and a final stage of slow decay controlled by a solid-phase diffusion process that can last for more than a month. The model was also used to estimate the peak concentration and the amount of formaldehyde emitted during each stage. The formaldehyde sources were investigated by comparing emission patterns and modeling outcomes of different paint formulations. The biocide used to preserve the paint was found to be a major source of the formaldehyde. Chamber test results demonstrated that replacing the preservative with a different biocide for the particular paint tested resulted in an approximate reduction of 55% of formaldehyde emissions. But the reduction affected only the third-stage long-term emissions.

Characterization of Curing Emissions from Conversion Varnishes.

Three commercially available conversion varnish coating "systems" (stain, sealer, and topcoat) were selected for an initial scoping study. The total volatile content of the catalyzed varnishes, as determined by U.S. Environmental Protection Agency (EPA) Method 24, ranged from 64 to 73 weight%. Uncombined (free) formaldehyde concentrations, determined by a sodium sulfite titration method, ranged from 0.15 to 0.58 weight% of the uncatalyzed varnishes. Each sealer and topcoat was also analyzed by gas chromatography (EPA Method 311). The primary volatile organic constituents included methyl ethyl ketone (MEK), isobutanol, n-butanol, methyl isobutyl ketone (MIBK), toluene, ethylbenzene, the xylenes, and 1,2,4-trimethylbenzene.