HPV16-E6 Oncoprotein Activates TGF-ß and Wnt/ß-Catenin Pathways in the Epithelium-Mesenchymal Transition of Cataracts in a Transgenic Mouse Model.

OBJECTIVE: This work aimed to determine if cataractous changes associated with EMT occurring in the K14E6 mice lenses are associated with TGF-ß and Wnt/ß-catenin signaling activation. MATERIALS AND METHODS: Cataracts of K14E6 mice were analysed histologically; and components of TGF-ß and Wnt/ß-catenin signaling were evaluated by Western blot, RT-qPCR, in situ RT-PCR, IHC, or IF technics. Metalloproteinases involved in EMT were also assayed using zymography. The endogenous stabilisation of Smad7 protein was also assessed using an HDAC inhibitor. RESULTS: The K14E6 mice, which displayed binocular cataracts in 100% of the animals, exhibited loss of tissue organisation, cortical liquefaction, and an increase in the number of hyperproliferative-nucleated cells with mesenchymal-like characteristics in the lenses. Changes in lenses' cell morphology were due to actin filaments reorganisation, activation of TGF-ß and Wnt/ß-catenin pathways, and the accumulation of MTA1 protein. Finally, the stabilisation of Smad7 protein diminishes cell proliferation, as well as MTA1 protein levels. CONCLUSION: The HPV16-E6 oncoprotein induces EMT in transgenic mice cataracts. The molecular mechanism may involve TGF-ß and Wnt/ß-catenin pathways, suggesting that the K14E6 transgenic mouse could be a useful model for the study or treatment of EMT-induced cataracts.

Tissue-specific conditional PKCε knockout mice: a model to precisely reveal PKCε functional role in initiation, promotion and progression of cancer.

PKCε is a transforming oncogene and a predictive biomarker of various human cancers. However, a precise in vivo link of PKCε to cancer induction, progression and metastasis remain undefined. To achieve these goals, we generated tissue specific conditional PKCε knockout mice (PKCε-CKO) using cre-lox technology. Homozygous PKCε(LoxP/LoxP) mice have normal body weight and phenotype. To determine what effect loss of PKCε would have on the prostate, the PKCε(LoxP/LoxP) mice were bred to probasin cre (PB-Cre4+) mice which express cre specifically in the prostate epithelium of postnatal mice. Western blot and immunohistochemical analyses showed reduced levels of PKCε specifically in the prostate of PKCε-CKO mice. Histopathological analyses of prostate from both PKCε(LoxP/LoxP) and prostate PKCε-CKO mice showed normal pathology. To determine the functional impact of prostate specific deletion of PKCε on prostate tumor growth, we performed an orthotopic xenograft study. Transgenic adenocarcinoma of the mouse prostate (TRAMP) cells (TRAMPC1, 2×106) were implanted in the prostate of PKCε-CKO mice. Mice were sacrificed at 6th week post-implantation. Results demonstrated a significant (P<0.05) decrease in the growth of TRAMPC1 cells-derived xenograft tumors in PKCε-CKO mice compared to wild type. To determine a link of PKCε to ultraviolet radiation (UVR) exposure-induced epidermal Stat3 phosphorylation, PKCε(LoxP/LoxP) mice were bred to tamoxifen-inducible K14 Cre mice. PKCε deletion in the epidermis resulted in inhibition of UVR-induced Stat3 phosphorylation. In summary, our novel PKCε(LoxP/LoxP) mice will be useful for defining the link of PKCε to various cancers in specific organ, tissue, or cells.

Scrib is required for epithelial cell identity and prevents epithelial to mesenchymal transition in the mouse.

The integrity and function of epithelial tissues depend on the establishment and maintenance of defining characteristics of epithelial cells, cell-cell adhesion and cell polarity. Disruption of these characteristics can lead to the loss of epithelial identity through a process called epithelial to mesenchymal transition (EMT), which can contribute to pathological conditions such as tissue fibrosis and invasive cancer. In invertebrates, the epithelial polarity gene scrib plays a critical role in establishing and maintaining cell adhesion and polarity. In this study we asked if the mouse homolog, Scrib, is required for establishment and/or maintenance of epithelial identity in vivo. To do so, we conditionally deleted Scrib in the head ectoderm tissue that gives rise to both the ocular lens and the corneal epithelium. Deletion of Scrib in the lens resulted in a change in epithelial cell shape from cuboidal to flattened and elongated. Early in the process, the cell adhesion protein, E-cadherin, and apical polarity protein, ZO-1, were downregulated and the myofibroblast protein, αSMA, was upregulated, suggesting EMT was occurring in the Scrib deficient lenses. Correlating temporally with the upregulation of αSMA, Smad3 and Smad4, TGFß signaling intermediates, accumulated in the nucleus and Snail, a TGFß target and transcriptional repressor of the gene encoding E-cadherin, was upregulated. Pax6, a lens epithelial transcription factor required to maintain lens epithelial cell identity also was downregulated. Loss of Scrib in the corneal epithelium also led to molecular changes consistent with EMT, suggesting that the effect of Scrib deficiency was not unique to the lens. Together, these data indicate that mammalian Scrib is required to maintain epithelial identity and that loss of Scrib can culminate in EMT, mediated, at least in part, through TGFß signaling.

Cell-autonomous requirements for Dlg-1 for lens epithelial cell structure and fiber cell morphogenesis.

Cell polarity and adhesion are thought to be key determinants in organismal development. In Drosophila, discs large (dlg) has emerged as an important regulator of epithelial cell proliferation, adhesion, and polarity. Herein, we investigated the role of the mouse homolog of dlg (Dlg-1) in the development of the mouse ocular lens. Tissue-specific ablation of Dlg-1 throughout the lens early in lens development led to an expansion and disorganization of the epithelium that correlated with changes in the distribution of adhesion and polarity factors. In the fiber cells, differentiation defects were observed. These included alterations in cell structure and the disposition of cell adhesion/cytoskeletal factors, delay in denucleation, and reduced levels of alpha-catenin, pERK1/2, and MIP26. These fiber cell defects were recapitulated when Dlg-1 was disrupted only in fiber cells. These results suggest that Dlg-1 acts in a cell autonomous manner to regulate epithelial cell structure and fiber cell differentiation.

Examination of the pRb-dependent and pRb-independent functions of E7 in vivo.

High-risk human papillomaviruses encode two oncogenes, E6 and E7, expressed in nearly all cervical cancers. Although E7 protein is best known for its ability to inactivate the retinoblastoma tumor suppressor protein, pRb, many other activities for E7 have been proposed in in vitro studies. Herein, we describe studies that allowed us to define unambiguously the pRb-dependent and -independent activities of E7 for the first time in vivo. In these studies, we crossed mice transgenic for human papillomavirus 16 E7 to knock-in mice genetically engineered to express a mutant form of pRb (pRb(DeltaLXCXE)) that is selectively defective for binding E7. pRb inactivation was necessary for E7 to induce DNA synthesis and to overcome differentiation-dependent cell cycle withdrawal and DNA damage-induced cell cycle arrest. While most of E7's effects on epidermal differentiation were found to require pRb inactivation, a modest delay in terminal differentiation with resulting hyperplasia was observed in E7 mice on the Rb(DeltaLXCXE) mutant background. E7-induced p21 upregulation was also pRb dependent, and genetic Rb inactivation was sufficient to reproduce this effect. While E7-mediated p21 induction was partially p53 dependent, neither p53 nor p21 induction by E7 required p19(ARF). These data show that E7 upregulates the expression of p53 and p21 via pRb-dependent mechanisms distinct from the proposed p19-Mdm2 pathway. These results extend our appreciation of the importance of pRb as a relevant target for high-risk E7 oncoproteins.

Temporal regulation of VEID-7-amino-4-trifluoromethylcoumarin cleavage activity and caspase-6 correlates with organelle loss during lens development.

Lens fiber cell differentiation involves extensive reconstruction of the cell's architecture, including the degradation and elimination of all membrane-bound organelles via a process that has been likened to apoptosis. Using caspase reporter assays under conditions in which nonspecific cleavage of the reporter peptides by the proteasome has been inhibited, we investigated whether any specific caspase activities are temporally correlated with this process of organelle loss. Extracts from neonatal mouse lenses contained strong VEID-7-amino-4-trifluoromethylcoumarin (AFC) and minor IETD-AFC and LEVD-AFC cleavage activities, but no DEVD-AFC cleavage activity. Further testing suggested that the VEID-AFC and IETD-AFC cleavage activities were likely due to the same enzyme. In lens extracts from rat embryos, VEID-AFC cleavage activity increased during the period when organelles are eliminated, between embryonic days 15.5 and 18.5, whereas procaspase-6 protein levels decreased, suggesting that this enzyme is responsible for VEID-AFC cleavage. By contrast, in extracts from alpha AE7 transgenic mouse lenses in which apoptosis was induced, strong DEVD-AFC cleavage activity and activated caspase-3 protein were detected. Thus, within the same tissue, different caspase activities can predominate depending on the context, normal differentiation versus apoptosis. These results highlight the difference between normal fiber cell differentiation and apoptosis and the capacity of the lens to differentially regulate these two processes.

Requirement of PDZ-containing proteins for cell cycle regulation and differentiation in the mouse lens epithelium.

The roles of PDZ domain-containing proteins such as Dlg and Scrib have been well described for Drosophila; however, their requirement for mammalian development is poorly understood. Here we show that Dlg, Scrib, MAGI1, MAGI3, and MPDZ are expressed in the mouse ocular lens. We demonstrate that the increase in proliferation and defects in cellular adhesion and differentiation observed in epithelia of lenses that express E6, a viral oncoprotein that can bind to several PDZ proteins, including the human homologs of Dlg and Scrib, is dependent on E6's ability to bind these proteins via their PDZ domains. Analyses of lenses from mice carrying an insertional mutation in Dlg (dlg(gt)) show increased proliferation and proliferation in spatially inappropriate regions of the lens, a phenotype similar to that of lenses expressing E6. The results from this study indicate that multiple PDZ domain-containing proteins, including Dlg and Scrib, may be required for maintaining the normal pattern of growth and differentiation in the lens. Furthermore, the phenotypic similarities among the Drosophila dlg mutant, the lenses of dlg(gt) mice, and the lenses of E6 transgenic mice suggest that Dlg may have a conserved function in regulating epithelial cell growth and differentiation across species.

The PDZ ligand domain of the human papillomavirus type 16 E6 protein is required for E6's induction of epithelial hyperplasia in vivo.

Human papillomaviruses (HPVs) are the causative agent of warts. Infections with high-risk HPVs are associated with anogenital and head and neck cancers. One of the viral genes responsible for HPV's oncogenic activity is E6. Mice expressing the HPV-16 E6 protein in their epidermis (K14E6(WT)) develop epithelial hyperplasia and squamous carcinomas. Numerous cellular proteins interact with E6, some of which can be grouped based on common amino acid motifs in their E6-binding domains. One such group, the PDZ partners, including hDLG, hSCRIBBLE, MUPP1, and MAGI, bind to the carboxy-terminal four amino acids of E6 through their PDZ domains. E6's interaction with the PDZ partners leads to their degradation. Additionally, E6's binding to PDZ proteins has been correlated with its ability to transform baby rat kidney cells in tissue culture and to confer tumorigenicity onto cells in xenograft experiments. To address whether the ability of E6 to bind PDZ domain partners is necessary for E6 to confer epithelial hyperproliferation in vivo, we generated transgenic mice that express in stratified squamous epithelia a mutant of E6 lacking the last six amino acids at its carboxyl terminus, E6(Delta 146-151), from the human keratin 14 (K14) promoter. The K14E6(Delta 146-151) mice exhibit a radiation response similar to that of the K14E6(WT) mice, demonstrating that this protein, as predicted, retains an ability to inactivate p53. However, the K14E6(Delta 146-151) mice fail to display epithelial hyperplasia. These results indicate that an interaction of E6 with PDZ partners is necessary for its induction of epithelial hyperplasia.

p53 regulates apoptotic retinal ganglion cell death induced by N-methyl-D-aspartate.

PURPOSE: The tumor suppressor protein p53 plays a central role in regulating apoptosis in a variety of neuronal cell types. Previous studies have indicated that retinal ganglion cell (RGC) death induced by ischemia follows a p53-dependent pathway. Ischemia causes wide-spread damage to the retina, eliciting multiple different damaging pathways. We conducted experiments to specifically investigate the role of p53 in RGC death activated by overstimulation of the N-methyl-D-aspartate (NMDA) receptor, an ionotropic glutamate dependent calcium channel normally involved in glutamate neurotransduction. METHODS: RGC death was induced in both wild-type (CB6F1 or 129/Sv) and p53-deficient (129/Sv background) mice by a single intravitreal injection of either 40 or 160 nmol of NMDA into one eye leaving the other eye as an untreated control. Cell loss was quantified by comparing the number of surviving cells in the retinas from experimental eyes relative to the control eyes of the same animals. The accumulation of p53 mRNA in retinas was monitored by reverse-transcription PCR (RT-PCR) of retinal total RNA isolated from mice injected with 40 nmol of NMDA. The functional requirement for p53 was monitored in p53-deficient mice after intravitreal injection of 160 nmol of NMDA. Immunohistochemistry for cleaved poly(ADP-ribose) polymerase (PARP) was performed on p53-deficient mice after intravitreal injection of 160 nmol of NMDA. RESULTS: In wild-type CB6F1 mice, p53 mRNA levels are elevated within 3 h after NMDA injection. This accumulation correlates with the onset of changes in RGC nuclear morphology that precedes pyknosis, which occurs by 6 h. Mice (129/Sv) deficient for one or both alleles of p53 show no developmental change in RGC number, compared to wild-type animals (Mann-Whitney test, p=0.824), suggesting that p53 is not required for developmental programmed cell death of RGCs. In adult mice, however, p53-dependent changes in the rate of RGC death after exposure to 160 nmol of NMDA were observed. Four days after injection, p53+/+ and p53-/- mice exhibit statistically equivalent amounts of cell loss (p>0.1), while p53+/- mice have significantly attenuated cell loss (p<0.002), relative to the other groups. RGCs from NMDA-treated p53+/+ and p53-/- mice were analyzed further using immunohistochemistry to identify the cleavage products of poly(ADP-ribose) polymerase (PARP), a known substrate for caspases. Cleaved PARP was found in p53+/+ and p53+/- eyes, but not in p53-/- mice. CONCLUSIONS: Developmental RGC programmed cell death does not require p53. Selective overstimulation of the glutamate-dependent NMDA-receptor in adult mice activates a p53-dependent pathway of death in RGCs. The requirement for p53 is not absolute, however, because mice lacking this gene are able to execute an alternative pathway of cell death. Examination of the cleavage of PARP, which is a substrate for caspases, suggests that the p53-dependent pathway utilizes these proteases, but the p53-independent pathway does not.

Unique roles for E2F1 in the mouse lens in the absence of functional pRB proteins.

PURPOSE: Normal lens fiber cell differentiation requires functional retinoblastoma protein (pRB), because inactivation of this protein results in proliferation and apoptosis in normally postmitotic, differentiating fiber cells. Loss of either E2F1 or -3 can partially rescue the lens phenotype in Rb-deficient mice, implying that these E2Fs may have specific targets in this system. The purpose of this study was to determine what unique role E2F1 may play. METHODS: Expression of E2F family members and target genes was analyzed in the lenses of nontransgenic, E2F1-null, alphaAE7;E2F1-sufficient; and alphaAE7;E2F1-null mice by in situ hybridization, Northern blot analysis, and RT-PCR. RESULTS: In lenses of E2F1-null mice, there was no change in the expression of E2F-2 to -5 or their target genes, compared with E2F1-sufficient mice. However, in the lens of alphaAE7 mice where pRB proteins are inactivated, expression of E2F2 and -3a was increased. The E2F3a increase, but not that of E2F2, was dependent on E2F1. Expression of E2F target genes was increased with expression of E7 and expression of one of these, p19ARF, was E2F1 dependent. CONCLUSIONS: Although in the normal lens there do not appear to be unique roles for E2F1 that cannot be fulfilled by other E2F family members, in the absence of functional pRB proteins, E2F1 is specifically responsible for the increased expression of E2F3a and p19ARF. These findings suggest that E2F1 may be the preferred E2F regulating these target genes in the normal lens.

Decreased susceptibility to NMU-induced mammary carcinogenesis in transgenic rats carrying multiple copies of a rat ras gene driven by the rat Harvey ras promoter.

Ras protein over-expression has been observed in human breast cancers although the significance of Ras over-expression in the etiology of breast cancer is unknown and its contribution to breast cancer prognosis is still debated. In this study, the over-expression of both wild-type Harvey and Kirsten Ras proteins as contributors to rat mammary carcinogenesis were examined using a transgenic rat model. Three rat transgenic lines (designated HrHr transgenics) carrying three to six copies of wild-type rat Harvey ras driven by the wild-type rat Harvey ras promoter were produced. In addition, transgenic lines carrying either three or seven copies of the Kirsten ras gene under the same promoter (HrKr) were produced. No pathological changes in the mammary gland were observed in any of the HrHr or HrKr transgenic rat line heterozygotes. Two of the Ras transgenic lines, HrHr (R8) and HrKr (4334), had a significant reduction in NMU-induced rat mammary cancer when compared to their non-transgenic littermates. All five Ras transgenic lines developed fewer carcinomas than their non-transgenic littermates following NMU exposure. The percentage of NMU-induced G35 to A35 activating mutations in the endogenous Harvey ras gene in mammary carcinomas from the HrHr, HrKr transgenic rats and their non-transgenic littermates was similar ( approximately 50%). In contrast, less than 1% of the NMU-induced carcinomas in these Ras transgenic rats had an activating ras mutation in their transgenes. These findings highlight the potential of Ras to function as a modifier gene in repressing mammary carcinogenesis.

Deregulated cell cycle control in lens epithelial cells by expression of inhibitors of tumor suppressor function.

Previous studies have shown that cell cycle proteins such as retinoblastoma protein (pRB) are essential for cell cycle withdrawal in differentiating lens cells. However, little is known about which factors are critical for cell cycle control in the lens epithelial cells. Here we use the K14 promoter to direct expression of E6 and E7, oncogenes from human papillomavirus type 16, which are known to bind and inactivate p53 and pRB, as molecular tools to study cell cycle regulation in the lens epithelium of transgenic mice. Expression of either gene resulted in increased proliferation and apoptosis, and in the case of E6, a unique epithelial phenotype characterized by multilayering and intercellular vacuoles was observed. Lenses from mice expressing E7 mutants, which are defective in inactivating pRB proteins, were normal and the lens phenotype in the E6 mice was p53-independent. Thus, cell proliferation in the lens epithelium is controlled by multiple factors including, but not necessarily limited to, the pRB family.