José Luis Cascallana

Deregulated Activity of Akt in Epithelial Basal Cells Induces Spontaneous Tumors and Heightened Sensitivity to Skin Carcinogenesis

Aberrant activation of the phosphoinositide-3-kinase (PI3K)/PTEN/Akt pathway, leading to increased proliferation and decreased apoptosis, has been implicated in several human pathologies including cancer. Our previous data have shown that Akt-mediated signaling is an essential mediator in the mouse skin carcinogenesis system during both the tumor promotion and progression stages. In addition, overexpression of Akt is also able to transform keratinocytes through transcriptional and posttranscriptional processes. Here, we report the consequences of the increased expression of Akt1 (wtAkt) or constitutively active Akt1 (myrAkt) in the basal layer of stratified epithelia using the bovine keratin K5 promoter. These mice display alterations in epidermal proliferation and differentiation. In addition, transgenic mice with the highest levels of Akt expression developed spontaneous epithelial tumors in multiple organs with age. Furthermore, both wtAkt and myrAkt transgenic lines displayed heightened sensitivity to the epidermal proliferative effects of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) and heightened sensitivity to two-stage skin carcinogenesis. Finally, enhanced susceptibility to two-stage carcinogenesis correlated with a more sustained proliferative response following treatment with TPA as well as sustained alterations in Akt downstream signaling pathways and elevations in cell cycle regulatory proteins. Collectively, the data provide direct support for an important role for Akt signaling in epithelial carcinogenesis in vivo, especially during the tumor promotion stage.

IKKβ Leads to an Inflammatory Skin Disease Resembling Interface Dermatitis

IKKbeta is a subunit of the IkappaB kinase (IKK) complex required for NF-kappaB activation in response to pro-inflammatory signals. NF-kappaB regulates the expression of many genes involved in inflammation, immunity, and apoptosis, and also controls cell proliferation and differentiation in different tissues; however, its function in skin physiopathology remains controversial. In this study we report the alterations caused by increased IKKbeta activity in skin basal cells of transgenic mice. These animals suffered chronic inflammation with abundant macrophages and other CD45(+) infiltrating cells in the skin, which resulted in epidermal basal cell injury and degeneration of hair follicles. They showed histological features characteristic of interface dermatitis (ID). This phenotype is accompanied by an increased production of inflammatory cytokines by transgenic keratinocytes. Accordingly, transcriptome studies show upregulation of genes associated with inflammatory responses. The inflammatory phenotype observed as a consequence of IKKbeta overexpression is independent of T and B lymphocytes, as it also arises in mice lacking these cell types. In summary, our data indicate the importance of IKKbeta in the development of ID and in the homeostasis of stratified epithelia. Our results also support the idea that IKKbeta might be a valid therapeutic target for the treatment of skin inflammatory diseases.

IKKβ Overexpression Leads to Pathologic Lesions in Stratified Epithelia and Exocrine Glands and to Tumoral Transformation of Oral Epithelia

Alterations in nuclear factor kappaB (NFκB) signaling have been related with several diseases and importantly also with cancer. Different animal models with increased or diminished NFκB signaling have shown that NFκB subunits and their regulators are relevant to the pathophysiology of different organs and tissues. In particular, both the deletion of the regulatory subunit β of the kinase of the inhibitor of NFκB (IKKβ) and its overexpression in epidermis lead to the development of skin inflammatory diseases not associated with tumoral lesions. In this work, we have studied the consequences of IKKβ overexpression in other organs and tissues. We found that elevated IKKβ levels led to altered development and functionality of exocrine glands (i.e., mammary glands) in transgenic female mice. In oral epithelia, increased IKKβ expression produced lichenoid inflammation with abundant granulocytes, macrophages, and B cells, among other inflammatory cells. This inflammatory phenotype was associated with high incidence of tumoral lesions in oral epithelia, contrary to what was found in skin. Moreover, IKKβ also increased the malignant progression of both spontaneous and experimentally induced oral tumors. These results highlight the importance of IKKβ in epithelial and glandular homeostasis as well as in oral tumorigenesis and open the possibility that IKKβ activity might be implicated in the development of oral cancer in humans. Mol Cancer Res; 9(10); 1329–38. ©2011 AACR.

Transrepression Function of the Glucocorticoid Receptor Regulates Eyelid Development and Keratinocyte Proliferation but Is Not Sufficient to Prevent Skin Chronic Inflammation

Glucocorticoids (GCs) play a key role in skin homeostasis and stress responses acting through the GC receptor (GR), which modulates gene expression by DNA binding-dependent (transactivation) and -independent (transrepression) mechanisms. To delineate which mechanisms underlie the beneficial and adverse effects mediated by GR in epidermis and other epithelia, we have generated transgenic mice that express a mutant GR (P493R, A494S), which is defective for transactivation but retains transrepression activity, under control of the keratin 5 promoter (K5-GR-TR mice). K5-GR-TR embryos exhibited eyelid opening at birth and corneal defects that resulted in corneal opacity in the adulthood. Transgenic embryos developed normal skin, although epidermal atrophy and focal alopecia was detected in adult mice. GR-mediated transrepression was sufficient to inhibit keratinocyte proliferation induced by acute and chronic phorbol 12-myristate 13-acetate exposure, as demonstrated by morphometric analyses, bromodeoxyuridine incorporation, and repression of keratin 6, a marker of hyperproliferative epidermis. These antiproliferative effects were mediated through negative interference of GR with MAPK/activator protein-1 and nuclear factor-kappaB activities, although these interactions occurred with different kinetics. However, phorbol 12-myristate 13-acetate-induced inflammation was only partially inhibited by GR-TR, which efficiently repressed IL-1beta and MMP-3 genes while weakly repressing IL-6 and TNF-alpha. Our data highlight the relevance of deciphering the mechanisms underlying GR actions on epithelial morphogenesis as well as for its therapeutic use to identify more restricted targets of GC administration.

Differential distribution of cAMP-dependent protein kinase isoforms in various tissues of the bivalve mollusc Mytilus galloprovincialis.

The cAMP signalling pathway is involved in the regulation of basic physiological processes in bivalve molluscs. We had previously identified and characterized two isoforms of cAMP-dependent protein kinase (PKA) from the sea mussel Mytilus galloprovincialis that differ at their regulatory (R) subunit, namely, R(myt1) or R(myt2). Here we investigated the immunohistochemical expression of both PKA isoforms in various mussel tissues. R(myt1) and R(myt2) displayed a complementary subcellular localization. In general, R(myt1) was found to be uniformly distributed in the cytoplasm of most cell types, whereas R(myt2) appears to be localized only in the cell periphery and associated with certain cellular structures, such as the cilia of labial palps and gill filaments. Thus, both PKA isoforms appear to be non-redundant, but they have specific functions. R(myt1) was the main isoform present in catch muscle fibers, which suggests that PKA(myt1) may be the isoform involved in the regulation of the catch state. Conversely, R(myt2) was the only isoform detected in the cilia of gill filaments, indicating that PKA(myt2) could mediate the effects of cAMP on the ciliary beat frequency.