A Transgenic Mouse Model of Merkel Cell Virus Small Tumor Antigen

Merkel cell carcinoma (MCC), a primary cutaneous neoplasm, originates in the mechanoreceptor Merkel cells in the basal layer of the epidermis. Risk factors include UV exposure, advanced age and immunosuppression, suggesting an infectious etiology. MCC incidence in the US is rising, with approximately 1500 cases per year. The non-­‐enveloped, double-­‐stranded DNA Merkel cell polyomavirus (MCV) is responsible for approximately 80% of MCC cases. The virus was discovered by subjecting MCC tissue samples to digital transcriptome subtraction, in which mRNA is isolated, the human transcripts subtracted in silico and the remaining transcripts compared to viral sequences. MCV expresses differentially spliced Large (LT), Small (sT) and 57 kT tumor antigens from the T antigen early locus, similar to other polyomaviruses such as SV40. Both LT and sT are critical for transformation. LT is a helicase responsible for replication of the viral genome, however in integrated viral genomes it is either truncated or mutated to eliminate its replicative functions. sT contributes to transformation via hyperphosphorylation and inhibition of the cap-­‐dependent translation inhibitor 4E-­‐BP1. The function of 57 kT remains unknown. Knockdown of LT induces necroptosis of MCV-­‐ positive MCC cells, whereas sT expression in rodent Rat-­‐1 cells is transformative. v Being that sT is the transformative agent in rodent cells, it would be of interest to develop a mouse model expressing sT in a tissue-­‐specific manner to determine whether tumor formation occurs. Indeed, several mouse models of SV40 T antigen have been developed over the past decades, each resulting in tissue-­‐specific tumor formation. We developed a MCV sT transgenic mouse model, in which a lox-­‐stop-­‐lox sT is expressed via an ER-­‐inducible Cre gene under the control of the ubiquitin promoter. Upon tamoxifen-­‐ induced MCV sT expression, ER-­‐Cre-­‐positive mice demonstrate severe weight loss, ruffled fur and a hunched posture, necessitating euthanasia. Western blotting reveals sT expression in several tissues, whereas TUNEL staining shows significant cell death. While we were unable to observe transformation, we believe this drastic phenotype demonstrates the validity of our MCV sT transgenic mouse model and warrants further investigation into the mechanism of death

Requirement for a Uroplakin 3a-like protein in the development of zebrafish pronephric tubule epithelial cell function, morphogenesis, and polarity

Uroplakin (UP)3a is critical for urinary tract development and function; however, its role in these processes is unknown. We examined the function of the UP3a-like protein Upk3l, which was expressed at the apical surfaces of the epithelial cells that line the pronephric tubules (PTs) of the zebrafish pronephros. Embryos treated with upk3l-targeted morpholinos showed decreased pronephros function, which was attributed to defects in PT epithelial cell morphogenesis and polarization including: loss of an apical brush border and associated phospho-ERM proteins, apical redistribution of the basolateral Na+/K+-ATPase, and altered or diminished expression of the apical polarity complex proteins Prkcz (atypical protein kinase C zeta) and Pard3 (Par3). Upk3l missing its C-terminal cytoplasmic domain or containing mutations in conserved tyrosine or proline residues did not rescue, or only partially rescued the effects of Upk3l depletion. Our studies indicate that Upk3l promotes epithelial polarization and morphogenesis, likely by forming or stimulating interactions with cytoplasmic signaling or polarity proteins, and that defects in this process may underlie the pathology observed in UP3a knockout mice or patients with renal abnormalities that result from altered UP3a expression. © 2012 Mitra et al

The CT of Upk3l is critical for its function.

<p>(A) Clustal W alignment of the TM and CT region of hUP3a, xUP3a and Upk3l. The amino acids that comprise the TM and CT domain are highlighted, and conserved functional motifs and residues are underlined or shaded, respectively. (B) Phenotypes of embryos injected with CNT-MO, <i>upk3l</i>-MO alone, or <i>upk3l</i>-MO co-injected with mCherry mRNA and mRNAs encoding MO-resistant versions of <i>upk3l</i>ΔCT, <i>upk3l</i>P₂₅₈L, <i>upk3l</i>Y₂₅₁F, or <i>upk3l</i>Y₂₅₁D. Microinjection of MO-resistant mRNA alone at the same doses did not produce detectable phenotypic abnormalities. (C) Morphological phenotypes associated with embryos injected with 5 ng CNT-MO (<i>n</i> = 100), 3 ng of <i>upk3l</i>-MO (<i>n</i> = 100), or 3 ng of <i>upk3l</i>-MO and 100 pg of <i>upk3l</i>, <i>upk3l</i>ΔCT, <i>upk3l</i>P₂₅₈L, <i>upk3l</i>Y₂₅₁F, or <i>upk3l</i>Y₂₅₁D mRNA (n≥50). (D) Localization of FLAG-tagged Upk3l, Upk3lΔCT, Upk3lP₂₅₈L, Upk3lY₂₅₁F, or Upk3lY₂₅₁D in MDCK cells co-stained for actin and nuclei. Upk3lY₂₅₁F and Upk3lY₂₅₁D showed a predominantly intracellular localization and their low levels of expression necessitated an 1.5-fold increase in photomultiplier voltage above that used for the other samples.</p

Pronephric clearance, heart rate, and pericardial area in control and morphant embryos.

<p>(A) Clearance of 70 kDa-TRITC dextran injected into the common cardinal vein of 54-hpf control (top panel) or morphant (bottom panel) embryos. The fluorescence intensity of the sampled area (circled) was measured in each embryo 0, 5, and 24 h following dextran injection. (B) The heart rate, pericardial area, and the dextran retention was recorded for each embryo and the values relative to t = 0 (post dextran injection) were calculated. Data are reported as mean ± SEM (n = 25). (C) Uptake of 500 kDa or 10 kDa FITC-dextran in control or morphant PT epithelial cells. Tubule lumens are marked by asterisks, while internalized dextran is indicated by arrows. Note the incomplete ring of actin in morphant PT cells.</p

p53 ablation is critical for MCV sT transformation of MEF cells.

<p>(A) MCV sT induces soft agar colony formation in the absence of p53 in MEF cells. MEF cells isolated form <i>Ubc</i>^{<i>CreERT2</i>}<i>; ROSA</i>^<i>sT</i>, <i>ROSA</i>^<i>sT</i>, <i>Ubc</i>^{<i>CreERT2</i>}<i>; p53</i>^{<i>flox/flox</i>} and two <i>Ubc</i>^{<i>CreERT2</i>}<i>; ROSA</i>^<i>sT</i><i>; p53</i>^{<i>flox/flox</i>} embryos from littermate were treated with 500nM 4OHTMX over 7 days, and cells were subjected to soft agar colony formation assay. Asterisks(*) indicate statistical significance <i>p<0</i>.<i>05</i>. (B) Soft agar colonies induced by p53 ablation. p53 ablation alone did not lead to colony formation. (C) Expression of MCV sT in MEFs from <i>Ubc</i>^{<i>CreERT2</i>}<i>; ROSA</i>^<i>sT</i><i>; p53</i>^{<i>flox/flox</i>}.</p

MCV sT transgenic mice develop tumors in a p53 null setting.

<p>(A) p53 ablation does not rescue mice from MCV sT-induced lethality. Kaplan-Meier curve of low dose (0.02 mg/g) TMX injected <i>Ubc</i>^{<i>CreERT2</i>}<i>; ROSA</i>^<i>sT</i><i>; p53</i>^{<i>flox/flox</i>} mice (Solid blue line, n = 26) and <i>Ubc</i>^{<i>CreERT2</i>}<i>; p53</i>^{<i>flox/flox</i>} mice (dotted blue line, n = 10). Pink lines indicate the survival of <i>Ubc</i>^{<i>CreERT2</i>}<i>; ROSA</i>^<i>sT</i><i>(solid line)</i> and <i>Ubc</i>^{<i>CreERT2</i>} (dotted line) mice with wild type p53 as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142329#pone.0142329.g001" target="_blank">Fig 1B</a> for comparison with p53 knockout background (blue lines). (B) MCV sT expression produces tumors <i>in vivo</i> in <i>p53</i> null setting. 80% (4/5) of mice that survived over 60 days after TMX injection develop grossly visible tumor in the spleen and liver. Representative spleen and liver tissues with tumor nodules from p53.7F are shown with corresponding normal tissues from a C57BL/6 control mouse. (C) Immunoblotting of MCV sT protein expression in liver and spleen tissues from mouse p53.7F) with macroscopic tumors. Spleen, muscle and ear tissues consistently maintained sT expression over 60 days after TMX injection. sT expression was detectable in liver tissues by immunoblotting only from liver with visible nodules (mouse p53.7F). MCV sT protein was detected using CM8E6 antibody, and Hsp/Hsc70 expression was used as a loading control. (D) The top panel shows anaplastic neoplasia in the spleen and liver. A range of proliferative changes was observed in the kidney, where distal tubular epithelia are most severely affected, but glomeruli (black arrowhead), proximal tubular epithelia (*), and interstitial tissues are relatively spared of proliferative changes. The middle panel shows a representative immunohistochemical staining of sT protein expression in liver tumor, spleen tumor and kidney tissues from <i>Ubc</i>^{<i>CreERT2</i>}<i>; ROSA</i>^<i>sT</i><i>; p53</i>^{<i>flox/flox</i>} mice. Tissue samples were immunostained with MCV sT (CM5E1) antibody. (E) The top panel shows a sT-induced liver tumor with immunoreactivity to α-smooth muscle actin (ASMA) and bottom panel shows K14 positivity in scattered tumor cells.</p