Diacylglycerol Signaling

15 Transgenic Mouse Models to Investigate Functional Specificity of Protein Kinase C
for TACE activation and is believed to be mediated by reactive oxygen species
(ROS). Upon its release, TNFa exerts its biological effects by trimerizing and
binding to two distinct receptors, TNFR1 and TNFR2. Binding of TNFa induces
trimerization of each of these receptors, which then recruit several signaling
proteins to the cytoplasmic membrane. With the ability to activate two distinct
receptors and to recruit different receptor signaling complexes, TNFa has the
ability to regulate a vast array of cellular responses including cellular inflammation,
immunity, cell proliferation, differentiation, and apoptosis (Komori et al. 1993;
Wheeler et al. 2003).
Evidence indicates that TNFa is linked to skin tumor promotion by TPA and
UVR (Komori et al. 1993; Wheeler et al. 2003; Starcher 2000; Suganuma et al.
1999; Moore et al. 1999; Arnott et al. 2004). Experiments using tumor promoters
of the okadaic acid class have provided evidence that TNFa is the central mediator
of tumor promotion in the mouse skin. These experiments indicated that TNFa
shed from the initiated cell or various tissues surrounding the initiated lesion can
induce clonal expansion and transformation of initiated cells. This work led to the
development of in vivo mouse models, which have further implicated TNFa as the
key cytokine for tumor promotion in the mouse skin. Using either the 2-stage model
of carcinogenesis or UVR, mice deficient for TNFa or either of its receptors render
the mice resistant to skin tumor formation (Komori et al. 1993; Wheeler et al. 2003;
Starcher 2000; Suganuma et al. 1999; Moore et al. 1999; Arnott et al. 2004).
PKCe transgenic mice elicit elevated both serum and epidermal TNFa levels
during skin tumor promotion either by TPA or UVR and this increase is linked to
the development of SCC (Wheeler et al. 2004, 2005). A single topical application
of TPA (5 nmol) to the skin, as early as 2.5 h after treatment, result in a significant
(p < 0.01) increase (twofold) in epidermal TNFa and more than a sixfold increase
in ectodomain shedding of TNFa into the serum of PKCe transgenic mice relative
to their wild-type littermates. Furthermore, this TPA-stimulated TNFa shedding is
proportional to the level of expression of PKCe in the epidermis. Using the TNF
alpha converting enzyme (TACE) inhibitor, TAPI-1, TPA-stimulated TNFa shedding
can be completely prevented in PKCe transgenic mice and isolated keratinocytes.
These results indicate that PKCe signal transduction pathways to
TPA-stimulated TNFa ectodomain shedding are mediated by TACE, a transmembrane
metalloprotease. Using the superoxide dismutase mimetic CuDIPs and the
glutathione reductase mimetic ebselen, TPA-stimulated TNFa shedding from
PKCe transgenic mice can be completely attenuated, implying the role of reactive
oxygen species (Wheeler et al. 2003).
To determine whether TNFa is a critical intermediate component in PKCe -mediated
squamous cell carcinoma formation, bigenic mice were created by cross breeding
K14-PKCe 215 transgenic mice with TNFa knockout mice. The bigenic mice were
then used for the two-step DMBA-TPA skin tumor promotion protocol. TNFa
deficiency significantly inhibited (~50%) the development of SCC in PKCe
transgenic mice.
TNFa-deficient PKCe transgenic mice were also evaluated for UVR-induced
cutaneous damage. Deletion of TNFa gene in PKCe transgenic mice inhibited
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