Cunxi Li

Fibrocystin/Polyductin Modulates Renal Tubular Formation by Regulating Polycystin-2 Expression and Function

Autosomal recessive polycystic kidney disease is caused by mutations in PKHD1, which encodes the membrane-associated receptor-like protein fibrocystin/polyductin (FPC). FPC associates with the primary cilia of epithelial cells and co-localizes with the Pkd2 gene product polycystin-2 (PC2), suggesting that these two proteins may function in a common molecular pathway. For investigation of this, a mouse model with a gene-targeted mutation in Pkhd1 that recapitulates phenotypic characteristics of human autosomal recessive polycystic kidney disease was produced. The absence of FPC is associated with aberrant ciliogenesis in the kidneys of Pkhd1-deficient mice. It was found that the COOH-terminus of FPC and the NH2-terminus of PC2 interact and that lack of FPC reduced PC2 expression but not vice versa, suggesting that PC2 may function immediately downstream of FPC in vivo. PC2-channel activities were dysregulated in cultured renal epithelial cells derived from Pkhd1 mutant mice, further supporting that both cystoproteins function in a common pathway. In addition, mice with mutations in both Pkhd1 and Pkd2 had a more severe renal cystic phenotype than mice with single mutations, suggesting that FPC acts as a genetic modifier for disease severity in autosomal dominant polycystic kidney disease that results from Pkd2 mutations. It is concluded that a functional and molecular interaction exists between FPC and PC2 in vivo.

Conditional Mutation of Pkd2 Causes Cystogenesis and Upregulates β-Catenin

Loss of polycystin-2 (PC2) in mice (Pkd2(-/-)) results in total body edema, focal hemorrhage, structural cardiac defects, abnormal left-right axis, hepatorenal and pancreatic cysts, and embryonic lethality. The molecular mechanisms by which loss of PC2 leads to these phenotypes remain unknown. We generated a model to allow targeted Pkd2 inactivation using the Cre-loxP system. Global inactivation of Pkd2 produced a phenotype identical to Pkd2(-/-) mice with undetectable PC2 protein and perinatal lethality. Using various Cre mouse lines, we found that kidney, pancreas, or time-specific deletion of Pkd2 led to cyst formation. In addition, we developed an immortalized renal collecting duct cell line with inactive Pkd2; these cells had aberrant cell-cell contact, ciliogenesis, and tubulomorphogenesis. They also significantly upregulated beta-catenin, axin2, and cMyc. Our results suggest that loss of PC2 disrupts normal behavior of renal epithelial cells through dysregulation of beta-catenin-dependent signaling, revealing a potential role for this signaling pathway in PC2-associated ADPKD.

Polycystin-2 expression is regulated by a PC2-binding domain in the intracellular portion of fibrocystin.

Autosomal dominant (ADPKD) and autosomal recessive (ARPKD) polycystic kidney disease are caused by mutations in Pkd1/Pkd2 and Pkhd1, which encode polycystins (PCs) and fibrocystin/polyductin (FPC). Our recent study reported that a deficiency in FPC increases the severity of cystic disease in Pkd2 mutants and down-regulates PC2 in vivo, but the precise molecular mechanism of these effects is unknown (Kim, I., Fu, Y., Hui, K., Moeckel, G., Mai, W., Li, C., Liang, D., Zhao, P., Ma, J., Chen, X.-Z., George, A. L., Jr., Coffey, R. J., Feng, Z. P., and Wu, G. (2008) J. Am. Soc. Nephrol. 19, 455–468). In this study, through the use of deletion and mutagenesis strategies, we identified a PC2-binding domain in the intracellular C terminus of FPC and an FPC-binding domain in the intracellular N terminus of PC2. These binding domains provide a molecular basis for the physical interaction between PC2 and FPC. In addition, we also found that physical interaction between the binding domains of PC2 and FPC is able to prevent down-regulation of PC2 induced by loss of FPC. In vivo, we generated a mouse model of ADPKD with hypomorphic Pkd2 alleles (Pkd2nf3/nf3) and show that PC2 down-regulation is accompanied by a phenotype similar to that of Pkhd1–/– mice. These findings demonstrate a common mechanism underlying cystogenesis in ADPKD and ARPKD and provide insight into the molecular relationship between PC2 and FPC.

Excess PLAC8 promotes an unconventional ERK2-dependent EMT in colon cancer.

The epithelial-to-mesenchymal transition (EMT) transcriptional program is characterized by repression of E-cadherin (CDH1) and induction of N-cadherin (CDH2), and mesenchymal genes like vimentin (VIM). Placenta-specific 8 (PLAC8) has been implicated in colon cancer; however, how PLAC8 contributes to disease is unknown, and endogenous PLAC8 protein has not been studied. We analyzed zebrafish and human tissues and found that endogenous PLAC8 localizes to the apical domain of differentiated intestinal epithelium. Colon cancer cells with elevated PLAC8 levels exhibited EMT features, including increased expression of VIM and zinc finger E-box binding homeobox 1 (ZEB1), aberrant cell motility, and increased invasiveness. In contrast to classical EMT, PLAC8 overexpression reduced cell surface CDH1 and upregulated P-cadherin (CDH3) without affecting CDH2 expression. PLAC8-induced EMT was linked to increased phosphorylated ERK2 (p-ERK2), and ERK2 knockdown restored cell surface CDH1 and suppressed CDH3, VIM, and ZEB1 upregulation. In vitro, PLAC8 directly bound and inactivated the ERK2 phosphatase DUSP6, thereby increasing p-ERK2. In a murine xenograft model, knockdown of endogenous PLAC8 in colon cancer cells resulted in smaller tumors, reduced local invasion, and decreased p-ERK2. Using MultiOmyx, a multiplex immunofluorescence-based methodology, we observed coexpression of cytosolic PLAC8, CDH3, and VIM at the leading edge of a human colorectal tumor, supporting a role for PLAC8 in cancer invasion in vivo.

Myristoylated Naked2 Antagonizes Wnt-β-Catenin Activity by Degrading Dishevelled-1 at the Plasma Membrane

In Drosophila, naked cuticle is an inducible antagonist of the Wnt-β-catenin pathway, likely acting at the level of Dishevelled (Dsh/Dvl), an essential component of this pathway. The mechanism by which naked cuticle and its two vertebrate orthologs, Naked1 (NKD1) and Naked2 (NKD2), inhibit Dvl function is unknown. NKD2 is myristoylated, a co-translational modification that leads to its plasma membrane localization. In contrast, myristoylation-deficient G2A NKD2 is cytoplasmic. Herein we show that the ability of Nkd2/NKD2 to antagonize Wnt-β-catenin activity during zebrafish embryonic development and in mammalian HEK293 cells is myristoylation-dependent. NKD2 and Dvl-1 interact and co-localize at the lateral membrane of polarized epithelial cells. In reciprocal overexpression and siRNA knockdown experiments, NKD2 and Dvl-1 destabilize each other via enhanced polyubiquitylation; this effect is also dependent upon Naked2 myristoylation. Cell fractionation and ubiquitylation assays indicate that endogenous NKD2 interacts with a slower migrating, ubiquitylated form of Dvl-1 in plasma membrane fractions. These results provide a mechanism by which NKD2 antagonizes Wnt signaling: myristoylated NKD2 interacts with Dvl-1 at the plasma membrane, and this interaction leads to their mutual ubiquitin-mediated proteasomal degradation.

Use of Fluorescence-activated Vesicle Sorting for Isolation of Naked2-associated, Basolaterally Targeted Exocytic Vesicles for Proteomics Analysis

By interacting with the cytoplasmic tail of a Golgi-processed form of transforming growth factor-α (TGFα), Naked2 coats TGFα-containing exocytic vesicles and directs them to the basolateral corner of polarized epithelial cells where the vesicles dock and fuse in a Naked2 myristoylation-dependent manner. These TGFα-containing Naked2-associated vesicles are not directed to the subapical Sec6/8 exocyst complex as has been reported for other basolateral cargo, and thus they appear to represent a distinct set of basolaterally targeted vesicles. To identify constituents of these vesicles, we exploited our finding that myristoylation-deficient Naked2 G2A vesicles are unable to fuse at the plasma membrane. Isolation of a population of myristoylation-deficient, green fluorescent protein-tagged G2A Naked2-associated vesicles was achieved by biochemical enrichment followed by flow cytometric fluorescence-activated vesicle sorting. The protein content of these plasma membrane de-enriched, flow-sorted fluorescent G2A Naked2 vesicles was determined by LC/LC-MS/MS analysis. Three independent isolations were performed, and 389 proteins were found in all three sets of G2A Naked2 vesicles. Rab10 and myosin IIA were identified as core machinery, and Na+/K+-ATPase α1 was identified as an additional cargo within these vesicles. As an initial validation step, we confirmed their presence and that of three additional proteins tested (annexin A1, annexin A2, and IQGAP1) in wild-type Naked2 vesicles. To our knowledge, this is the first large scale protein characterization of a population of basolaterally targeted exocytic vesicles and supports the use of fluorescence-activated vesicle sorting as a useful tool for isolation of cellular organelles for comprehensive proteomics analysis.

Naked1 Antagonizes Wnt Signaling by Preventing Nuclear Accumulation of β-Catenin

Cyto-nuclear shuttling of β-catenin is at the epicenter of the canonical Wnt pathway and mutations in genes that result in excessive nuclear accumulation of β-catenin are the driving force behind the initiation of many cancers. Recently, Naked Cuticle homolog 1 (Nkd1) has been identified as a Wnt-induced intracellular negative regulator of canonical Wnt signaling. The current model suggests that Nkd1 acts between Disheveled (Dvl) and β-catenin. Here, we employ the zebrafish embryo to characterize the cellular and biochemical role of Nkd1 in vivo. We demonstrate that Nkd1 binds to β-catenin and prevents its nuclear accumulation. We also show that this interaction is conserved in mammalian cultured cells. Further, we demonstrate that Nkd1 function is dependent on its interaction with the cell membrane. Given the conserved nature of Nkd1, our results shed light on the negative feedback regulation of Wnt signaling through the Nkd1-mediated negative control of nuclear accumulation of β-catenin.

lncRNA MIR100HG-derived miR-100 and miR-125b mediate cetuximab resistance via Wnt/β-catenin signaling

De novo and acquired resistance, which are largely attributed to genetic alterations, are barriers to effective anti-epidermal-growth-factor-receptor (EGFR) therapy. To generate cetuximab-resistant cells, we exposed cetuximab-sensitive colorectal cancer cells to cetuximab in three-dimensional culture. Using whole-exome sequencing and transcriptional profiling, we found that the long non-coding RNA MIR100HG and two embedded microRNAs, miR-100 and miR-125b, were overexpressed in the absence of known genetic events linked to cetuximab resistance. MIR100HG, miR-100 and miR-125b overexpression was also observed in cetuximab-resistant colorectal cancer and head and neck squamous cell cancer cell lines and in tumors from colorectal cancer patients that progressed on cetuximab. miR-100 and miR-125b coordinately repressed five Wnt/β-catenin negative regulators, resulting in increased Wnt signaling, and Wnt inhibition in cetuximab-resistant cells restored cetuximab responsiveness. Our results describe a double-negative feedback loop between MIR100HG and the transcription factor GATA6, whereby GATA6 represses MIR100HG, but this repression is relieved by miR-125b targeting of GATA6. These findings identify a clinically actionable, epigenetic cause of cetuximab resistance.

Loss of Bicc1 impairs tubulomorphogenesis of cultured IMCD cells by disrupting E-cadherin-based cell-cell adhesion

The Bicaudal-C (Bic-C) gene was originally discovered in Drosophila melanogaster. The gene product Bic-C is thought to serve as an RNA-binding molecule targeting diverse proteins at the post-transcriptional level. Recent research has shown this gene to be conserved in many species, from Caenorhabditis elegans to humans. Disruption of this protein can disturb the normal migration direction of the anterior follicle cell of Drosophila oocytes, while mutation of a mouse Bicc1 (a mouse homologue of Bic-C) results in phenotypes mimicking human hereditary polycystic kidney disease (PKD). However, the cellular function of Bicc1 gene products in mammalian systems remains largely unknown. In this study, we established stable IMCD (mouse inner medullary collecting duct) cell lines, in which Bicc1 was silenced by short hairpin RNA inhibition (shRNA). We show that inhibition of Bicc1 disrupted normal tubulomorphogenesis and induced cystogenesis of IMCD cells grown in three dimensional cultures. To determine what factors contributed to the defect, we systematically examined biological changes of Bicc1-silenced IMCD cells. We found that the cells had significant defects in E-cadherin-based cell-cell adhesion, along with abnormalities in actin cytoskeleton organization, cell-extracellular matrix interactions, cell proliferation, and apoptosis. These findings suggest that lack of Bicc1 leads to disruption of normal cell-cell junctions, which in turn impedes establishment of epithelial polarity. These cellular defects may initiate abnormal tubulomorphogenesis and cystogenesis of IMCD cells grown in vitro. The observation of aberrant cellular behaviors in Bicc1-silenced IMCD cells reveal functions for Bicc1 in renal epithelial cells and provides insight into a potential pathogenic mechanism of polycystic kidney disease.

EGF receptor-independent action of TGF-alpha protects Naked2 from AO7-mediated ubiquitylation and proteasomal degradation

Naked family members (Drosophila Naked Cuticle and mammalian Naked1 and Naked2) have been identified as inducible antagonists of canonical Wnt signaling. We recently reported that Naked2, but not Naked1, interacts with the cytoplasmic tail of TGF-α, thereby coating TGF-α-containing exocytic vesicles and directing these vesicles to the basolateral corner of polarized epithelial cells. Here, we show that Naked2 is a short-lived protein with a half-life of 60 min caused by its rapid ubiquitin-mediated proteasomal degradation. Overexpression of TGF-α stabilizes Naked2 protein in an EGF receptor (EGFR)-independent manner; a physical interaction between the cytoplasmic tail of TGF-α and Naked2 is necessary and sufficient for this protection. We have identified a RING finger protein, AO7/RNF25, as a ubiquitin ligase for Naked2, and we have shown that overexpression of TGF-α reduces binding of AO7 to Naked2. These results identify an EGFR-independent action of TGF-α, in which it protects Naked2 from proteasomal degradation, thus ensuring its delivery to the basolateral surface of polarized epithelial cells.