Ilse Hofmann

p120 catenin is required for normal renal tubulogenesis and glomerulogenesis

Defects in the development or maintenance of tubule diameter correlate with polycystic kidney disease. Here, we report that absence of the cadherin regulator p120 catenin (p120ctn) from the renal mesenchyme prior to tubule formation leads to decreased cadherin levels with abnormal morphologies of early tubule structures and developing glomeruli. In addition, mutant mice develop cystic kidney disease, with markedly increased tubule diameter and cellular proliferation, and detached luminal cells only in proximal tubules. The p120ctn homolog Arvcf is specifically absent from embryonic proximal tubules, consistent with the specificity of the proximal tubular phenotype. p120ctn knockdown in renal epithelial cells in 3D culture results in a similar cystic phenotype with reduced levels of E-cadherin and active RhoA. We find that E-cadherin knockdown, but not RhoA inhibition, phenocopies p120ctn knockdown. Taken together, our data show that p120ctn is required for early tubule and glomerular morphogenesis, as well as control of luminal diameter, probably through regulation of cadherins.

p120-catenin is essential for terminal end bud function and mammary morphogenesis

Although p120-catenin (p120) is crucial for E-cadherin function, ablation experiments in epithelial tissues from different organ systems reveal markedly different effects. Here, we examine for the first time the consequences of p120 knockout during mouse mammary gland development. An MMTV-Cre driver was used to target knockout to the epithelium at the onset of puberty. p120 ablation was detected in approximately one-quarter of the nascent epithelium at the forth week post-partum. However, p120 null cells were essentially nonadherent, excluded from the process of terminal end bud (TEB) morphogenesis and lost altogether by week six. This elimination process caused a delay in TEB outgrowth, after which the gland developed normally from cells that had retained p120. Mechanistic studies in vitro indicate that TEB dysfunction is likely to stem from striking E-cadherin loss, failure of cell-cell adhesion and near total exclusion from the collective migration process. Our findings reveal an essential role for p120 in mammary morphogenesis.

Protein p0071 - an armadillo plaque protein that characterizes a specific subtype of adherens junctions.

In vertebrates, cell-cell-adhering proteins of both the punctum adhaerens and the desmosome type are characterized by transmembrane glycoproteins of the cadherin superfamily, and by cytoplasmic plaques, which comprise the common plaque protein plakoglobin ( [Cowin et al., 1986][1]; [Franke et al.,

Plakophilin-associated RNA-binding proteins in prostate cancer and their implications in tumor progression and metastasis

Both plakophilins (PKP) 1 and 3 play a role in the progression of prostate cancer. The RNA-binding proteins (RBPs) GAP-SH3-binding protein (G3BP), fragile-X-related protein 1 (FXR1), poly(A)-binding protein, cytoplasmic 1 (PABPC1), and up-frameshift factor 1 (UPF1) are associated with PKP3. All these RBPs have an impact on RNA metabolism. Until recently, the PKP-associated RBPs have not been analyzed in prostate cancer. In the current study, we showed by affinity purification that the PKP3-associated RBPs were also binding partners of PKP1. We examined the expression of PKP1/3-associated RBPs and PKP1/3 in prostate cell lines, tumor-free prostate, and 136 prostatic adenocarcinomas by immunofluorescence and immunoblot. All four RBPs G3BP, FXR1, UPF1, and PABPC1 were expressed in the glandular epithelium of the normal prostate. PKP1 and FXR1 were strongly reduced in tumor tissues with Gleason score >7 and diminished expression of PKP1 and FXR1 also appeared to be associated with a metastatic phenotype. Additionally, the predominant nuclear localization of UPF1 in normal glandular cells and low grade tumors was switched to a more cytoplasmic pattern in carcinomas with Gleason score >7. Our findings suggest that PKP1 and FXR1 may have a tumor-suppressive function and are downregulated in more aggressive tumors. Collectively, PKP1/3-associated RBPs FXR1 and UPF1 may have a functional role in prostate cancer progression and metastasis and highlight the potential importance of posttranscriptional regulation of gene expression and nonsense-mediated decay in cancer.

Plakophilin-3 is required for late embryonic amphibian development, exhibiting roles in ectodermal and neural tissues.

The p120-catenin family has undergone a significant expansion during the evolution of vertebrates, resulting in varied functions that have yet to be discerned or fully characterized. Likewise, members of the plakophilins, a related catenin subfamily, are found throughout the cell with little known about their functions outside the desmosomal plaque. While the plakophilin-3 (Pkp3) knockout mouse resulted in skin defects, we find larger, including lethal effects following its depletion in Xenopus. Pkp3, unlike some other characterized catenins in amphibians, does not have significant maternal deposits of mRNA. However, during embryogenesis, two Pkp3 protein products whose temporal expression is partially complimentary become expressed. Only the smaller of these products is found in adult Xenopus tissues, with an expression pattern exhibiting distinctions as well as overlaps with those observed in mammalian studies. We determined that Xenopus Pkp3 depletion causes a skin fragility phenotype in keeping with the mouse knockout, but more novel, Xenopus tailbud embryos are hyposensitive to touch even in embryos lacking outward discernable phenotypes, and we additionally resolved disruptions in certain peripheral neural structures, altered establishment and migration of neural crest, and defects in ectodermal multiciliated cells. The use of two distinct morpholinos, as well as rescue approaches, indicated the specificity of these effects. Our results point to the requirement of Pkp3 in amphibian embryogenesis, with functional roles in a number of tissue types.

Plakophilins 1 and 3 Bind to FXR1 and thereby influence the mRNA stability of desmosomal proteins

ABSTRACT Plakophilins 1 and 3 (PKP1/3) are members of the arm repeat family of catenin proteins and serve as structural components of desmosomes, which are important for cell-cell-adhesion. In addition, PKP1/3 occur as soluble proteins outside desmosomes, yet their role in the cytoplasm is not known. We found that cytoplasmic PKP1/3 coprecipitated with the RNA-binding proteins FXR1, G3BP, PABPC1, and UPF1, and these PKP1/3 complexes also comprised desmoplakin and PKP2 mRNAs. Moreover, we showed that the interaction of PKP1/3 with G3BP, PABPC1, and UPF1 but not with FXR1 was RNase sensitive. To address the cytoplasmic function of PKP1/3, we performed gain-and-loss-of-function studies. Both PKP1 and PKP3 knockdown cell lines showed reduced protein and mRNA levels for desmoplakin and PKP2. Whereas global rates of translation were unaffected, desmoplakin and PKP2 mRNA were destabilized. Furthermore, binding of PKP1/3 to FXR1 was RNA independent, and both PKP3 and FXR1 stabilized PKP2 mRNA. Our results demonstrate that cytoplasmic PKP1/3 are components of mRNA ribonucleoprotein particles and act as posttranscriptional regulators of gene expression.

Loss of Mpdz impairs ependymal cell integrity leading to perinatal‐onset hydrocephalus in mice

Hydrocephalus is a common congenital anomaly. LCAM1 and MPDZ (MUPP1) are the only known human gene loci associated with non‐syndromic hydrocephalus. To investigate functions of the tight junction‐associated protein Mpdz, we generated mouse models. Global Mpdz gene deletion or conditional inactivation in Nestin‐positive cells led to formation of supratentorial hydrocephalus in the early postnatal period. Blood vessels, epithelial cells of the choroid plexus, and cilia on ependymal cells, which line the ventricular system, remained morphologically intact in Mpdz‐deficient brains. However, flow of cerebrospinal fluid through the cerebral aqueduct was blocked from postnatal day 3 onward. Silencing of Mpdz expression in cultured epithelial cells impaired barrier integrity, and loss of Mpdz in astrocytes increased RhoA activity. In Mpdz‐deficient mice, ependymal cells had morphologically normal tight junctions, but expression of the interacting planar cell polarity protein Pals1 was diminished and barrier integrity got progressively lost. Ependymal denudation was accompanied by reactive astrogliosis leading to aqueductal stenosis. This work provides a relevant hydrocephalus mouse model and demonstrates that Mpdz is essential to maintain integrity of the ependyma.

Plakophilin 1-deficient cells upregulate SPOCK1: implications for prostate cancer progression

Plakophilin (PKP) 1 is frequently downregulated in prostate cancer and therefore may play a tumor-suppressive role. In the present study, we stably knocked down PKP1 in the non-neoplastic, prostatic BPH-1 cell line. In the PKP1-deficient cells, the expression of keratin 14 was lost, and the apoptosis rate was significantly reduced indicating that the cells acquired new biological capabilities. Moreover, we analyzed the gene expression profile of the PKP1-deficient BPH-1 cells. Among the genes that were significantly altered upon PKP1 knockdown, we noticed several extracellular matrix (ECM)-related genes and identified sparc/osteonectin, cwcv, and kazal-like domains proteoglycan 1 (SPOCK1/testican-1) as a gene of interest. SPOCK1 is a component of the ECM and belongs to a matricellular protein family named secreted protein, acidic, cysteine-rich (SPARC). The role of SPOCK1 in prostate cancer has not been clearly elucidated. We analyzed SPOCK1 mRNA expression levels in different cancer databases and characterized its expression in 136 prostatic adenocarcinomas by immunohistochemistry and western blot. SPOCK1 revealed a cytoplasmic localization in the glandular epithelium of the prostate and showed a significant upregulation of mRNA and protein in prostate tumor samples. Our findings support the hypothesis that PKP1 may have a tumor-suppressive function and suggest an important role of SPOCK1 in prostate tumor progression. Collectively, altered expression of PKP1 and SPOCK1 appears to be a frequent and critical event in prostate cancer.

Nuclear ARVCF Protein Binds Splicing Factors and Contributes to the Regulation of Alternative Splicing

Background: ARVCF occurs both at adherens junctions and in the nucleus, but little is known about its nuclear function. Results: ARVCF interacts with RNA-binding proteins involved in splicing and contributes to alternative splicing of specific transcripts. Conclusion: ARVCF influences splicing of specific pre-mRNAs. Significance: We propose a new role of nuclear ARVCF in post-transcriptional regulation of gene expression. The armadillo repeat protein ARVCF is a component of adherens junctions. Similar to related proteins, such as p120-catenin and β-catenin, with known signaling functions, localization studies indicate a cytoplasmic and a nuclear pool of ARVCF. We find that ARVCF interacts with different proteins involved in mRNA-processing: the splicing factor SRSF1 (SF2/ASF), the RNA helicase p68 (DDX5), and the heterogeneous nuclear ribonucleoprotein hnRNP H2. All three proteins bind to ARVCF in an RNA-independent manner. Furthermore, ARVCF occurs in large RNA-containing complexes that contain both spliced and unspliced mRNAs of housekeeping genes. By domain analysis, we show that interactions occur via the ARVCF C terminus. Overexpression of ARVCF, p68, SRSF1, and hnRNP H2 induces a significant increase in splicing activity of a reporter mRNA. Upon depletion of ARVCF followed by RNA sequence analysis, several alternatively spliced transcripts are significantly changed. Therefore, we conclude that nuclear ARVCF influences splicing of pre-mRNAs. We hypothesize that ARVCF is involved in alternative splicing, generating proteomic diversity, and its deregulation may contribute to diseased states, such as cancer and neurological disorders.

The proteins ARVCF and p0071 in renal cell carcinomas and their potential use in the diagnosis of renal tumours

graphs (Figure 1). We also use this system for postmortem coronary angiography. During autopsy the heart is isolated and inspected. First, a plain X-ray overview is recorded to evaluate the severity of calcified atherosclerosis. For contrast-based coronary angiography, a barium sulphate suspension (1 g ⁄ ml; Liquid polibar) is gently injected by hand pressure into the ostia of the right coronary artery and subsequently into the main left coronary artery. The use of barium sulphate ⁄ gelatin suspension is recommended to prevent settlement of the suspension in the vessel lumen. After each barium infusion, digital X-ray images are obtained in anterior-posterior and lateral directions (35 keV for 18 s). In this way, the use of the Faxitron specimen radiography system is fast and provides direct digital images during an autopsy. Because the images of the angiogram are directly available, the heart can be placed in the optimal position for good examination of all parts of the coronary tree. Digitization of the radiology is a big advance, with easier production of images for embedding in the post-mortem report, presentation at Mortality Audit, education and research. Digital imaging of post-mortem coronary angiography may have some limitations, such as the fact that the detection area of our Faxitron system is only 120 · 120 mm. Therefore only part of the coronary system of severely dilated hearts can be visualized in one photograph. In addition, performing coronary angiography in every autopsy could be too timeconsuming, particularly in the UK, where many general pathologists perform multiple sudden death autopsies per day in public mortuaries. This technique should therefore be reserved for selected cases. Lastly, the costs related to the acquisition of a digital Faxitron system are a possible limitation. To illustrate the use of a Faxitron specimen radiography system for post-mortem angiography, we present a case of sudden death of a 54-year-old man. Autopsy revealed a heart of 480 g with left ventricular hypertrophy. Post-mortem angiography using the Faxitron X-ray system showed an obstruction in the distal part of the dominant right coronary artery just proximal of the posterior descending branch (Figure 2A). Coronary dissection revealed a recent thrombus superimposed on a ruptured atherosclerotic plaque in the same part of the right coronary artery (Figure 2B,C). Histological evaluation of the heart showed infarction of the posterior wall with coagulative necrosis and influx of neutrophilic granulocytes. This case illustrates that digitalization of post-mortem coronary angiography is a welcome development in modern autopsy practice with direct availability of digital images.