A size filter at the Golgi regulates apical membrane protein sorting.

Despite decades of research, apical sorting of epithelial membrane proteins remains incompletely understood. We noted that apical cytoplasmic domains are smaller than those of basolateral proteins; however, the reason for this discrepancy is unknown. Here we used a synthetic biology approach to investigate whether a size barrier at the Golgi apparatus might hinder apical sorting of proteins with large cytoplasmic tails. We focused on Crb3, Ace2 and Muc1 as representative apical proteins with short cytoplasmic tails. By incorporating a streptavidin-binding peptide, these proteins can be trapped in the endoplasmic reticulum until addition of biotin, which triggers synchronous release to the Golgi and subsequent transport to the apical cortex. Strikingly, increasing the size of their cytoplasmic domains caused partial mislocalization to the basolateral cortex and significantly delayed Golgi departure. Moreover, N-glycosylation of 'large' Crb3 was delayed, and 'small' Crb3 segregated into spatially distinct Golgi regions. Biologically, Crb3 forms a complex through its cytoplasmic tail with the Pals1 protein, which could also delay departure, but although associated at the endoplasmic reticulum and Golgi, Pals1 disassociated before Crb3 departure. Notably, a non-dissociable mutant Pals1 hampered the exit of Crb3. We conclude that, unexpectedly, a size filter at the Golgi facilitates apical sorting of proteins with small cytoplasmic domains and that timely release of Pals1, to reduce cytoplasmic domain size, is essential for normal Crb3 sorting.

A Size Filter Regulates Apical Protein Sorting.

Despite decades of research, apical sorting of epithelial membrane proteins remains incompletely understood. We noted that apical cytoplasmic domains are smaller than those of basolateral proteins; however, the reason for this discrepancy is unknown. We investigated whether a size barrier at the trans-Golgi network (TGN) might hinder apical sorting of proteins with large cytoplasmic tails. We focused on Crb3 and Ace2 as representative apical proteins with short cytoplasmic tails. By incorporating a streptavidin-binding peptide, these proteins can be trapped in the endoplasmic reticulum (ER) until addition of biotin, which triggers synchronous release to the Golgi and subsequent transport to the apical cortex. Strikingly, departure from the Golgi could be significantly delayed simply by increasing cytoplasmic bulk. Moreover, large and small Crb3 segregated into spatially distinct Golgi regions as detected by super resolution imaging. Biologically, Crb3 forms a complex through its cytoplasmic tail with the Pals1 protein, which could also delay departure, but although associated at the ER and Golgi, we found that Pals1 disassociates prior to Crb3 departure. Notably, a non-dissociable mutant Pals1 hampers the exit of Crb3. We conclude that an unexpected mechanism involving a size filter at the TGN facilitates apical sorting of proteins with small cytoplasmic domains and that timely release of Pals1, to reduce cytoplasmic domain size, is essential for the normal kinetics of Crb3 sorting.

Mutations in the exocyst component EXOC2 cause severe defects in human brain development.

The exocyst, an octameric protein complex, is an essential component of the membrane transport machinery required for tethering and fusion of vesicles at the plasma membrane. We report pathogenic variants in an exocyst subunit, EXOC2 (Sec5). Affected individuals have severe developmental delay, dysmorphism, and brain abnormalities; variability associated with epilepsy; and poor motor skills. Family 1 had two offspring with a homozygous truncating variant in EXOC2 that leads to nonsense-mediated decay of EXOC2 transcript, a severe reduction in exocytosis and vesicle fusion, and undetectable levels of EXOC2 protein. The patient from Family 2 had a milder clinical phenotype and reduced exocytosis. Cells from both patients showed defective Arl13b localization to the primary cilium. The discovery of mutations that partially disable exocyst function provides valuable insight into this essential protein complex in neural development. Since EXOC2 and other exocyst complex subunits are critical to neuronal function, our findings suggest that EXOC2 variants are the cause of the patients' neurological disorders.

Pyridoxamine reduces postinjury fibrosis and improves functional recovery after acute kidney injury.

Acute kidney injury (AKI) is a common and independent risk factor for death and chronic kidney disease (CKD). Despite promising preclinical data, there is no evidence that antioxidants reduce the severity of injury, increase recovery, or prevent CKD in patients with AKI. Pyridoxamine (PM) is a structural analog of vitamin B6 that interferes with oxidative macromolecular damage via a number of different mechanisms and is in a phase 3 clinical efficacy trial to delay CKD progression in patients with diabetic kidney disease. Because oxidative stress is implicated as one of the main drivers of renal injury after AKI, the ability of PM to interfere with multiple aspects of oxidative damage may be favorable for AKI treatment. In these studies we therefore evaluated PM treatment in a mouse model of AKI. Pretreatment with PM caused a dose-dependent reduction in acute tubular injury, long-term postinjury fibrosis, as well as improved functional recovery after ischemia-reperfusion AKI (IR-AKI). This was associated with a dose-dependent reduction in the oxidative stress marker isofuran-to-F2-isoprostane ratio, indicating that PM reduces renal oxidative damage post-AKI. PM also reduced postinjury fibrosis when administered 24 h after the initiating injury, but this was not associated with improvement in functional recovery after IR-AKI. This is the first report showing that treatment with PM reduces short- and long-term injury, fibrosis, and renal functional recovery after IR-AKI. These preclinical findings suggest that PM, which has a favorable clinical safety profile, holds therapeutic promise for AKI and, most importantly, for prevention of adverse long-term outcomes after AKI.

SIX2 Effects on Wilms Tumor Biology.

Wilms tumor (WT) blastema retains gene expression profiles characteristic of the multipotent nephron progenitor pool, or cap mesenchyme (CM), in the developing kidney. As a result, WT blastema and the CM are believed to represent contextual analogues of one another. Sine oculis homeobox 2 (SIX2) is a transcription factor expressed specifically in the CM, provides a critical mechanism for CM self-renewal, and remains persistently active in WT blastema, although its purpose in this childhood malignancy remains unclear. We hypothesized that SIX2, analogous to its function in development, confers a survival pathway to blastema, the putative WT stem cell. To test its functional significance in WT biology, wild-type SIX2 was overexpressed in the human WT cell line, WiT49. After validating this model, SIX2 effects on anchorage-independent growth, proliferation, invasiveness, canonical WNT pathway signaling, and gene expression of specific WNT pathway participants were evaluated. Relative to controls, WiT49 cells overexpressing SIX2 showed significantly enhanced anchorage-independent growth and early-passage proliferation representing surrogates of cell survival. Interestingly, overexpression of SIX2 generally repressed TCF/LEF-dependent canonical WNT signaling, which activates and coordinates both differentiation and stem pathways, but significantly heightened canonical WNT signaling through the survivin promoter, a mechanism that exclusively maintains the stem state. In summary, when overexpressed in a human WT cell line, SIX2 enhances cell survival and appears to shift the balance in WNT/ß-catenin signaling away from a differentiation path and toward a stem cell survival path.

CITED1 confers stemness to Wilms tumor and enhances tumorigenic responses when enriched in the nucleus.

Wilms tumor (WT) is the most common childhood kidney cancer and retains gene expression profiles reminiscent of the embryonic kidney. We have shown previously that CITED1, a transcriptional regulator that labels the self-renewing, multipotent nephron progenitor population of the developing kidney, is robustly expressed across all major WT disease and patient characteristics. In this malignant context, CITED1 becomes enriched in the nucleus, which deviates from its cytosolic predominance in embryonic nephron progenitors. We designed the current studies to test the functional and mechanistic effects of differential CITED1 subcellular localization on WT behavior. To mimic its subcellular distribution observed in clinical WT specimens, CITED1 was misexpressed ectopically in the human WT cell line, WiT49, as either a wild-type (predominantly cytosolic) or a mutant, but transcriptionally active, protein (two point mutations in its nuclear export signal, CITED1ΔNES; nuclear-enriched). In vitro analyses showed that CITED1ΔNES enhanced WiT49 proliferation and colony formation in soft agar relative to wild-type CITED1 and empty vector controls. The nuclear-enriched CITED1ΔNES cell line showed the greatest tumor volumes after xenotransplantation into immunodeficient mice (n=15 animals per cell line). To elucidate CITED1 gene targets in this model, microarray profiling showed that wild-type CITED1 foremost upregulated LGR5 (stem cell marker), repressed CDH6 (early marker of epithelial commitment of nephron progenitors), and altered expression of specific WNT pathway participants. In summary, forced nuclear enrichment of CITED1 in a human WT cell line appears to enhance tumorigenicity, whereas ectopic cytosolic expression confers stem-like properties and an embryonic phenotype, analogous to the developmental context.

Aberrant activation, nuclear localization, and phosphorylation of Yes-associated protein-1 in the embryonic kidney and Wilms tumor.

BACKGROUND: The Yes-associated-protein-1 (YAP1) is a novel, direct regulator of stem cell genes both in development and cancer. FAT4 is an upstream regulator that induces YAP1 cytosolic sequestering by phosphorylation (p-Ser 127) and therefore inhibits YAP1-dependent cellular proliferation. We hypothesized that loss of FAT4 signaling would result in expansion of the nephron progenitor population in kidney development and that YAP1 subcellular localization would be dysregulated in Wilms tumor (WT), an embryonal malignancy that retains gene expression profiles and histologic features reminiscent of the embryonic kidney. METHODS: Fetal kidneys from Fat4(-/-) mice were harvested at e18.5 and markers of nephron progenitors were investigated using immunohistochemical analysis. To examine YAP1 subcellular localization in WT, a primary WT cell line (VUWT30) was analyzed by immunofluorescence. Forty WT specimens evenly distributed between favorable and unfavorable histology (n = 20 each), and treatment failure or success (n = 20 each) was analyzed for total and phosphorylated YAP1 using immunohistochemistry and Western blot. RESULTS: Fat4(-/-) mouse fetal kidneys exhibit nuclear YAP1 with increased proliferation and expansion of nephron progenitor cells. In contrast to kidney development, subcellular localization of YAP1 is dysregulated in WT, with a preponderance of nuclear p-YAP1. By Western blot, median p-YAP1 quantity was 5.2-fold greater in unfavorable histology WT (P = 0.05). CONCLUSIONS: Fetal kidneys in Fat4(-/-) mice exhibit a phenotype reminiscent of nephrogenic rests, a WT precursor lesion. In WT, YAP1 subcellular localization is dysregulated and p-YAP1 accumulation is a novel biomarker of unfavorable histology.

SIX2 and CITED1, markers of nephronic progenitor self-renewal, remain active in primitive elements of Wilms' tumor.

PURPOSE: SIX2 and CITED1 are transcriptional regulators that specify self-renewing nephronic progenitor cells of the embryonic kidney. We hypothesized that SIX2, which promotes and maintains this stem cell population, and CITED1 remain active in Wilms' tumor (WT). METHODS: To evaluate expression domains and the pathogenic significance of SIX2 and CITED1 across WT, the Children's Oncology Group provided 40 WT specimens of stages I to IV (n = 10 per stage), which were enriched for unfavorable histology (n = 20) and treatment failure (relapse or death, n = 20). SIX2 and CITED1 protein expression was evaluated qualitatively (immunohistochemistry) and quantitatively (Western blot, or WB). Gene transcription was estimated using quantitative real-time polymerase chain reaction (qRT-PCR). RESULTS: SIX2 was visualized by immunohistochemistry in 36 (94.7%) of 38 specimens. Protein and messenger RNA expression of SIX2 were quantitatively similar across all stages of disease (P = .48 WB; P = 0.38 qPCR), in favorable or unfavorable histology (P = 0.51 WB; P = 0.58 qPCR), and in treatment failure or success (P = 0.86 WB; P = 0.49 qPCR). Although CITED1 expression paralleled SIX2 qualitatively, no quantitative correlation between SIX2 and CITED1 expression was observed (Spearman correlation coefficient, 0.28; P = 0.08). As in the fetal kidney, overlapping, but also distinct, WT cellular expression domains were observed between SIX2 and CITED1. CONCLUSION: SIX2 and CITED1 remain active across all disease characteristics of WT. Activity of these genes in WT potentially identifies a population of self-renewing cancer cells that exhibit an embryonic, stemlike phenotype. Taken together, these transcriptional regulators may be fundamental to WT cellular self-renewal and may represent targets for novel therapies that promote terminal differentiation.

Molecular characterization of Wilms' tumor from a resource-constrained region of sub-Saharan Africa.

Sub-Saharan African children have an increased incidence of Wilms' tumor (WT) and experience alarmingly poor outcomes. Although these outcomes are largely due to inadequate therapy, we hypothesized that WT from this region exhibits features of biological aggressiveness that may warrant broader implementation of high-risk therapeutic protocols. We evaluated 15 Kenyan WT (KWT) for features of aggressive disease (blastemal predominance and Ki67/cellular proliferation) and treatment resistance (anaplasia and p53 immunopositivity). To explore the additional biological features of KWT, we determined the mutational status of the CTNNB1/ß-catenin and WT1 genes and performed immunostaining for markers of Wnt pathway activation (ß-catenin) and nephronic progenitor cell self-renewal (WT1, CITED1 and SIX2). We characterized the proteome of KWT using imaging mass spectrometry (IMS). The results were compared to histology- and age-matched North American WT (NAWT) controls. For patients with KWT, blastemal predominance was noted in 53.3% and anaplasia in 13%. We detected increased loss to follow-up (p = 0.028), disease relapse (p = 0.044), mortality (p = 0.001) and nuclear unrest (p = 0.001) in patients with KWT compared to controls. KWT and NAWT showed similar Ki67/cellular proliferation. We detected an increased proportion of epithelial nuclear ß-catenin in KWT (p = 0.013). All 15 KWT specimens were found to harbor wild-type CTNNB1/ß-catenin, and one contained a WT1 nonsense mutation. WT1 was detected by immunostaining in 100% of KWT, CITED1 in 80% and SIX2 in 80%. IMS revealed a molecular signature unique to KWT that was distinct from NAWT. The African WT specimens appear to express markers of adverse clinical behavior and treatment resistance and may require alternative therapies or implementation of high-risk treatment protocols.

CITED1 expression in liver development and hepatoblastoma.

Hepatoblastoma, the most common pediatric liver cancer, consists of epithelial mixed embryonal/fetal (EMEF) and pure fetal histologic subtypes, with the latter exhibiting a more favorable prognosis. Few embryonal histology markers that yield insight into the biologic basis for this prognostic discrepancy exist. CBP/P-300 interacting transactivator 1 (CITED1), a transcriptional co-activator, is expressed in the self-renewing nephron progenitor population of the developing kidney and broadly in its malignant analog, Wilms tumor (WT). In this current study, CITED1 expression is detected in mouse embryonic liver initially on post-coitum day 10.5 (e10.5), begins to taper by e14.5, and is undetectable in e18.5 and adult livers. CITED1 expression is detected in regenerating murine hepatocytes following liver injury by partial hepatectomy and 3,5-diethoxycarbonyl-1,4-dihydrocollidine. Importantly, while CITED1 is undetectable in normal human adult livers, 36 of 41 (87.8%) hepatoblastoma specimens express CITED1, where it is enriched in EMEF specimens compared to specimens of pure fetal histology. CITED1 overexpression in Hep293TT human hepatoblastoma cells induces cellular proliferation and upregulates the Wnt inhibitors Kringle containing transmembrane protein 1 (KREMEN1) and CXXC finger protein 4 (CXXC4). CITED1 mRNA expression correlates with expression of CXXC4 and KREMEN1 in clinical hepatoblastoma specimens. These data show that CITED1 is expressed during a defined time course of liver development and is no longer expressed in the adult liver but is upregulated in regenerating hepatocytes following liver injury. Moreover, as in WT, this embryonic marker is reexpressed in hepatoblastoma and correlates with embryonal histology. These findings identify CITED1 as a novel marker of hepatic progenitor cells that is re-expressed following liver injury and in embryonic liver tumors.