Adam B. Glick

Mice lacking Smad3 show accelerated wound healing and an impaired local inflammatory response.

The generation of animals lacking SMAD proteins, which transduce signals from transforming growth factor-β (TGF-β), has made it possible to explore the contribution of the SMAD proteins to TGF-β activity in vivo. Here we report that, in contrast to predictions made on the basis of the ability of exogenous TGF-β to improve wound healing, Smad3-null (Smad3ex8/ex8) mice paradoxically show accelerated cutaneous wound healing compared with wild-type mice, characterized by an increased rate of re-epithelialization and significantly reduced local infiltration of monocytes. Smad3ex8/ex8 keratinocytes show altered patterns of growth and migration, and Smad3ex8/ex8 monocytes exhibit a selectively blunted chemotactic response to TGF-β. These data are, to our knowledge, the first to implicate Smad3 in specific pathways of tissue repair and in the modulation of keratinocyte and monocyte function in vivo.

Canonical WNT signaling promotes mammary placode development and is essential for initiation of mammary gland morphogenesis

Mammary glands, like other skin appendages such as hair follicles and teeth, develop from the surface epithelium and underlying mesenchyme; however, the molecular controls of embryonic mammary development are largely unknown. We find that activation of the canonical WNT/β-catenin signaling pathway in the embryonic mouse mammary region coincides with initiation of mammary morphogenesis, and that WNT pathway activity subsequently localizes to mammary placodes and buds. Several Wnt genes are broadly expressed in the surface epithelium at the time of mammary initiation, and expression of additional Wnt and WNT pathway genes localizes to the mammary lines and placodes as they develop. Embryos cultured in medium containing WNT3A or the WNT pathway activator lithium chloride (LiCl) display accelerated formation of expanded placodes, and LiCl induces the formation of ectopic placode-like structures that show elevated expression of the placode marker Wnt10b. Conversely, expression of the secreted WNT inhibitor Dickkopf 1 in transgenic embryo surface epithelium in vivo completely blocks mammary placode formation and prevents localized expression of all mammary placode markers tested. These data indicate that WNT signaling promotes placode development and is required for initiation of mammary gland morphogenesis. WNT signals play similar roles in hair follicle formation and thus may be broadly required for induction of skin appendage morphogenesis.

Tubular Overexpression of Transforming Growth Factor-β1 Induces Autophagy and Fibrosis but Not Mesenchymal Transition of Renal Epithelial Cells

We recently showed in a tetracycline-controlled transgenic mouse model that overexpression of transforming growth factor (TGF)-beta1 in renal tubules induces widespread peritubular fibrosis and focal degeneration of nephrons. In the present study we have analyzed the mechanisms underlying these phenomena. The initial response to tubular cell-derived TGF-beta1 consisted of a robust proliferation of peritubular cells and deposition of collagen. On sustained expression, nephrons degenerated in a focal pattern. This process started with tubular dedifferentiation and proceeded to total decomposition of tubular cells by autophagy. The final outcome was empty collapsed remnants of tubular basement membrane embedded into a dense collagenous fibrous tissue. The corresponding glomeruli survived as atubular remnants. Thus, TGF-beta1 driven autophagy may represent a novel mechanism of tubular decomposition. The fibrosis seen in between intact tubules and in areas of tubular decomposition resulted from myofibroblasts that were derived from local fibroblasts. No evidence was found for a transition of tubular cells into myofibroblasts. Neither tracing of injured tubules in electron micrographs nor genetic tagging of tubular epithelial cells revealed cells transgressing the tubular basement membrane. In conclusion, overexpression of TGF-beta1 in renal tubules in vivo induces interstitial proliferation, tubular autophagy, and fibrosis, but not epithelial-to-mesenchymal transition.

Isolation and utilization of epidermal keratinocytes for oncogene research

Publisher Summary This chapter focuses on the isolation and utilization of the epidermal keratinocytes for oncogene research. Keratinocytes have been widely used as target cells for testing the activity of oncogenes in the epithelial neoplasia. Many experimental studies have utilized cultured mouse skin keratinocytes, where in vitro results can be analyzed in the context of a substantial experience in carcinogen-induced mouse skin tumors. Studies of the mouse keratinocytes have also revealed the biological consequences of expressing v-los and derivatives, neu, mutant p53, and transforming growth factor α (TGF-α ). These studies have focused on the participation of oncogenes as mediators of premalignant progression and malignant conversion. The effects of oncogenic DNA viruses have also been tested in keratinocytes and particular emphasis has been directed toward the expression of the simian virus 40 (SV40) and specific subtypes of human or bovine papilloma viruses (HPV, BPV) or their transforming sequences. The chapter also provides details of the techniques employed in the studies testing the influence of oncogenes on keratinocytes and several general conclusions from the results are presented.

Pathological responses to oncogenic Hedgehog signaling in skin are dependent on canonical Wnt/β-catenin signaling

Constitutive Hedgehog (Hh) signaling underlies several human tumors, including basal cell carcinoma (BCC) and basaloid follicular hamartoma in skin. Intriguingly, superficial BCCs arise as de novo epithelial buds resembling embryonic hair germs, collections of epidermal cells whose development is regulated by canonical Wnt/β-catenin signaling. Similar to embryonic hair germs, human BCC buds showed increased levels of cytoplasmic and nuclear β-catenin and expressed early hair follicle lineage markers. We also detected canonical Wnt/β-catenin signaling in epithelial buds and hamartomas from mice expressing an oncogene, M2SMO, leading to constitutive Hh signaling in skin. Conditional overexpression of the Wnt pathway antagonist Dkk1 in M2SMO-expressing mice potently inhibited epithelial bud and hamartoma development without affecting Hh signaling. Our findings uncover a hitherto unknown requirement for ligand-driven, canonical Wnt/β-catenin signaling for Hh pathway-driven tumorigenesis, identify a new pharmacological target for these neoplasms and establish the molecular basis for the well-known similarity between early superficial BCCs and embryonic hair germs.

An efficient and versatile system for acute and chronic modulation of renal tubular function in transgenic mice

We describe a transgenic mouse line, Pax8-rtTA, which, under control of the mouse Pax8 promoter, directs high levels of expression of the reverse tetracycline–dependent transactivator (rtTA) to all proximal and distal tubules and the entire collecting duct system of both embryonic and adult kidneys. Using crosses of Pax8-rtTA mice with tetracycline-responsive c-MYC mice, we established a new, inducible model of polycystic kidney disease that can mimic adult onset and that shows progression to renal malignant disease. When targeting the expression of transforming growth factor-β1 to the kidney, we avoided early lethality by discontinuous treatment and successfully established an inducible model of renal fibrosis. Finally, a conditional knockout of the gene encoding tuberous sclerosis complex-1 was achieved, which resulted in the early outgrowth of giant polycystic kidneys reminiscent of autosomal recessive polycystic kidney disease. These experiments establish Pax8-rtTA mice as a powerful tool for modeling renal diseases in transgenic mice.

Critical Aspects of Initiation, Promotion, and Progression in Multistage Epidermal Carcinogenesis

Abstract Carcinogenesis in mouse skin can be divided into three distinct stages: initiation, promotion, and progression (malignant conversion). Initiation, induced by a single exposure to a genotoxic carcinogen, can result from a mutation in a single critical gene (e.g., rasHa), apparently in only a few epidermal cells. The change is irreversible. Promotion, resulting in the development of numerous benign tumors (papillomas), is accomplished by the repeated application of a nonmutagenic tumor promoter. The effects of single applications of tumor promoters are reversible since papillomas do not develop after insufficient exposure of initiated skin to promoters or when the interval between individual promoter applications is increased sufficiently. The reversibility of promotion suggests an epigenetic mechanism. Promoter treatment provides an environment that allows the selective clonal expansion of foci of initiated cells. The conversion of squamous papillomas to carcinomas (termed progression or malignant conversion) occurs spontaneously at a low frequency. The rate of progression to malignancy can be significantly increased by treatment of papilloma-bearing mice with certain genotoxic agents. These progressor agents or converting agents are likely to act via a second genetic change in papillonias already bearing the initiating mutation. Progression in the skin is characterized by genetic changes that result in several distinct changes in the levels or activity of structural proteins, growth factors, and proteases. The mechanisms involved in progression are being studied in epidermal cell culture. In order to determine the in vivo phenotype of cultured cells, a grafting system was developed in which the cells were transferred from culture to a prepared skin bed in athymic mice. Introduction of an activated v-fos oncogene into initiated cells bearing an activated ras Ha gene produced cells with a carcinoma phenotype, of Initiation i.e., carcinomas formed when the cells were grafted as part of reconstituted skin. Grafted keratinocytes containing the ras Ha gene alone produced papillomas; with v-fos alone, normal skin formed when grafted. The ras Ha/fos carcinomas showed changes in differentiation markers characteristic of chemically induced carcinomas. A cell culture assay utilizing cells initiated by the introduction of an activated ras Ha oncogene was developed to study progression. After exposure of initiated cells to progressor agents under conditions in which the proliferation of the ras Ha-initiated cells was suppressed, proliferating foci developed, with a good correlation of activity in the assay with activity in the progression stage in vivo. The cell culture assay provides a quantitative model to study chemically induced neoplastic progression and may be useful to identify potential progressor agents.

Transgenic expression of interleukin-13 in the skin induces a pruritic dermatitis and skin remodeling

IL-13 has been implicated in the pathogenesis of allergic diseases, including atopic dermatitis (AD). However, a direct role of IL-13 in AD has not been established. We aimed to develop an inducible transgenic model in which IL-13 can be expressed in the skin and to define the resulting dermal phenotype and mechanisms involved. The keratin 5 promoter was used with a tetracycline-inducible system to target IL-13 to the skin. The clinical manifestations, dermal histology, cytokine gene regulation, and systemic immune responses in the transgenic mice were assessed. IL-13 was produced exclusively in the skin and caused a chronic inflammatory phenotype characterized by xerosis and pruritic eczematous lesions; dermal infiltration of CD4+ T cells, mast cells, eosinophils, macrophages, and Langerhans cells; upregulation of chemokine and cytokine genes, including thymic stromal lymphopoietin; and skin remodeling with fibrosis and increased vasculature. The dermal phenotype was accompanied by elevated serum total IgE and IgG1 and increased production of IL-4 and IL-13 by CD4+ cells from lymphoid tissues and peripheral blood mononuclear cells. IL-13 is a potent stimulator of dermal inflammation and remodeling and this transgenic model of AD is a good tool for investigating the underlying mechanisms in the pathogenesis of AD.

Inhibition of Transforming Growth Factor-β1 Signaling Attenuates Ataxia Telangiectasia Mutated Activity in Response to Genotoxic Stress

Ionizing radiation causes DNA damage that elicits a cellular program of damage control coordinated by the kinase activity of ataxia telangiectasia mutated protein (ATM). Transforming growth factor beta (TGFbeta)-1, which is activated by radiation, is a potent and pleiotropic mediator of physiologic and pathologic processes. Here we show that TGFbeta inhibition impedes the canonical cellular DNA damage stress response. Irradiated Tgfbeta1 null murine epithelial cells or human epithelial cells treated with a small-molecule inhibitor of TGFbeta type I receptor kinase exhibit decreased phosphorylation of Chk2, Rad17, and p53; reduced gammaH2AX radiation-induced foci; and increased radiosensitivity compared with TGFbeta competent cells. We determined that loss of TGFbeta signaling in epithelial cells truncated ATM autophosphorylation and significantly reduced its kinase activity, without affecting protein abundance. Addition of TGFbeta restored functional ATM and downstream DNA damage responses. These data reveal a heretofore undetected critical link between the microenvironment and ATM, which directs epithelial cell stress responses, cell fate, and tissue integrity. Thus, Tgfbeta1, in addition to its role in homoeostatic growth control, plays a complex role in regulating responses to genotoxic stress, the failure of which would contribute to the development of cancer; conversely, inhibiting TGFbeta may be used to advantage in cancer therapy.

Defects in TGFβ signaling overcome senescence of mouse keratinocytes expressing v-rasHa

Previous studies have shown that TGFβ1 expression is upregulated in mouse keratinocytes infected with a v-rasHa retrovirus, although the functional significance of this has not been clear. Here we show that v-rasHa retrovirus transduced primary mouse keratinocytes undergo hyperproliferation followed by a TGFβ1 dependent G1 growth arrest and senescence. The growth arrest is accompanied by a 15-fold increase in total TGFβ1 secreted and a fourfold increase in secreted active TGFβ1. When cultured in the presence of a neutralizing antibody to TGFβ1, the senescence response is suppressed. Levels of the TGFβ1 target p15ink4b increase during senescence as does association of this kinase inhibitor with cyclinD/cdk4 complexes. However, p16ink4a, p53 and p19ARF expression also increase during senescence. Genetic analysis shows that TGFβ1 null and dominant negative TβRII expressing v-rasHa keratinocytes resist the G1 growth arrest and do not senescence. This resistance is associated with low expression of p15ink4b and p16ink4a, constitutive Rb phosphorylation and high levels of cdk4 and cdk2 kinase activity. In contrast, inactivation of TGFβ1 secretion or response does not block the induction of p53 and p19ARF, but the level of p21waf1, a p53 target gene, is reduced in cyclin D/cdk4 and cyclin E/cdk2 complexes. Thus, although multiple senescence pathways are activated in response to a ras oncogene, inactivation of TGFβ1 secretion or response is sufficient to block the senescence program. Since v-rasHa transduced TGFβ1−/− keratinocytes form squamous cell carcinomas following skin grafting, these results suggest that in mouse keratinocytes, defects in TGFβ1 signaling accelerate malignant progression by overcoming oncogene induced replicative senescence.