Role Of PDZK1 Research Articles

MiR-101-3p-mediated role of PDZK1 in hepatocellular carcinoma progression and the underlying PI3K/Akt signaling mechanism

BackgroundThe molecular targets and associated mechanisms of hepatocellular carcinoma (HCC) have been widely studied, but the roles of PDZK1 in HCC are unclear. Therefore, the aim of this study is to explore the role and associated mechanisms of PDZK1 in HCC.ResultsIt was found that the expression of PDZK1 in HCC tissues was higher than that in paired paracancerous tissues. High expression of PDZK1 was associated with lymph node metastasis, degree of differentiation, and clinical stage. Upregulation of PDZK1 in HCC cells affected their proliferation, migration, invasion, apoptosis, and cell cycle, and also induced PI3K/AKT activation. PDZK1 is a downstream target gene of miR-101-3p. Accordingly, increase in the expression of miR-101-3p reversed the promotive effect of PDZK1 in HCC. Moreover, PDZK1 was found to accelerate cell proliferation and promote the malignant progression of HCC via the PI3K/AKT pathway.ConclusionOur study indicated that the miR-101-3p/PDZK1 axis plays a role in HCC progression and could be beneficial as a novel biomarker and new therapeutic target for HCC treatment.

9-LB: PDZK1 Inhibits Fatty Acid Uptake in Renal Tubular Epithelial Cell through FATP2 to Ameliorate Diabetic Kidney Disease

Background: Renal tubular injury is the driving force of diabetic kidney disease (DKD) and their targeting may be an effective means for prevention. PDZK1 is a scaffold protein that is highly expressed in proximal renal tubules and participates in renal tubular reabsorption. However, the role of PDZK1 in DKD with disorder of glucose and lipid metabolism is unclear. Methods: We generated mice lacking the Pdzk1 gene specifically in renal tubular epithelial cells by crossing Pdzk1 floxed mice with Pax8-Cre mice. We observed renal tubular injury and renal fibrosis in Pdzk1 Pax8-cre+ and Pdzk1 Pax8-cre- mice induced by high fat diet and streptozotin. In vitro, we elucidated that PDZK1 regulates fatty acid uptake and renal tubular injury. Results: In this study, proteomic analysis found that compared with the control, the expression of PDZK1 in kidney of DKD mice was significantly reduced. Compared with non-DKD patients, PDZK1 was also lower in renal tissue samples of patients with DKD. Pdzk1 Pax8-cre+ mice significantly increased lipid deposition of renal tubules, renal tubular injury and renal fibrosis compared with Pdzk1 Pax8-cre- mice induced by high fat diet and streptozotin. Knocked down PDZK1 increased intracellular lipid deposition in renal tubular epithelial cells stimulated with high glucose and palmitic acid. Knockdown PDZK1 could promote fatty acid uptake in renal tubular epithelial cells. PDZK1 interacted with fatty acid transporter 2 (FATP2) and inhibited the expression and localization of FATP2 on cell membrane. Conclusion: Diabetes stimulates the decreased expression of PDZK1 in renal tubular epithelial cells. The decrease of PDZK1 attenuates the inhibition of FATP2 localization on cell membrane, promoting ectopic lipid deposition and renal tubular injury, leading to DKD. This study will reveal the new regulatory mechanism of renal tubular injury and provide a new target for the treatment of DKD. Disclosure L. Yang: None. Y. Wang: None. Y. Xu: None. J. Ke: None. D. Zhao: None. Funding National Natural Science Foundation of China (82170454)

Retraction: PDZ-containing 1 acts as a suppressor of pancreatic cancer by regulating PTEN phosphorylation.

[This retracts the article DOI: 10.18632/oncotarget.20552.].

Abstract 50: High-Density Lipoprotein Protects Macrophages Against Apoptosis in a PDZK1-Dependent Manner

Macrophage apoptosis contributes to the formation of necrotic cores in atherosclerotic plaques, increasing the risk of plaque rupture, leading to clinical heart attack. Previous work from our group showed that HDL is able to protect macrophages against apoptosis, and that such protection requires HDL’s receptor protein scavenger receptor class B, type I (SR-BI). In this study, we hypothesize that this protection requires not only SR-BI, but also SR-BI’s adaptor protein PDZK1. We incubated mouse peritoneal macrophages with or without lipoproteins and triggered apoptosis with thapsigargin or tunicamycin. Cell apoptosis was assessed by annexinV, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and cleaved caspase-3 staining. Only low levels (~5% of cells) of apoptosis were observed in macrophages treated with thapsigargin or tunicamycin when they were cultured with lipoproteins. However thapsigargin or tunicamycin stimulated apoptosis increased ~4 fold when lipoproteins were removed from the culture medium. The addition of 50μg/ml of purified HDL (P<0.05), but not LDL, to lipoprotein-deficient medium suppressed apoptosis. However, HDL did not inhibit cell apoptosis when PDZK1 was deleted. Our time-course study showed that incubation with HDL increased phosphorylation of Akt1, Foxo3a and Erk1/2 and reduced Bim in wild type (WT), but not PDZK1-/- macrophages. In order to test the role of PDZK1 in vivo, LDLR-/- mice were transplanted with either PDZK1-/- (n=12) or WT (n=12) bone marrow and challenged with high fat diet to induce atherosclerosis. The cross-sections of aortic sinus were stained with H&E to visualize plaques. The relative necrotic core area was significantly larger (P<0.05) in PDZK1-/- group (98187.2±8076.1) than that in WT control (27279.6±16688.7). Also, compared to LDLR-/- mice transplanted with WT bone marrow, the ones with PDZK1-/- bone marrow have 2.2-fold increased TUNEL(+) (P<0.05) and 3.6-fold increased cleaved caspase-3(+) (P<0.01) macrophages in plaques. In conclusion, PDZK1 is required for the HDL-dependent protection against macrophage apoptosis. In atherosclerotic plaques, PDZK1 plays a role in protecting macrophages from apoptosis and suppressing the development of necrotic cores.

Correlation between PDZK1, Cdc37, Akt and breast cancer malignancy: the role of PDZK1 in cell growth through Akt stabilization by increasing and interacting with Cdc37.

PDZ domain containing 1 (PDZK1) is a scaffold protein that plays a role in the fate of several proteins. Estrogen can induce PDZK1 gene expression; however, our recent report showed that PDZK1 expression in the breast cancer cell line MCF-7 is indirect and involves insulin-like growth factor (IGF)-1 receptor function. Such a relationship was established in cell culture systems and human breast cancer tissues. Here we show that overexpression of PDZK1 promoted an increase in cyclin D1 and enhanced anchorage-independent growth of MCF-7 cells in the absence of 17β-estradiol, suggesting that PDZK1 harbors oncogenic activity. Indeed, PDKZ1 overexpression enhanced epidermal growth factor receptor (EGFR)-stimulated MEK/ERK1/2 signaling and IGF-induced Akt phosphorylation. PDZK1 appeared to play this role, in part, by stabilizing the integrity of the growth promoting factors Akt, human epidermal growth factor receptor 2 (Her2/Neu) and EGFR. Increased Akt levels occurred via a decrease in the ubiquitination of the kinase. PDZK1 overexpression was associated with resistance to paclitaxel/5-fluorouracil/etoposide only at low concentrations. Although the increased stability of Akt was sensitive to heat shock protein 90 (HSP90) inhibition, increased levels of the cochaperone cell division cycle 37 (Cdc37), as well as its ability to bind PDZK1, appear to play a larger role in kinase stability. Using human tissue microarrays, we show strong positive correlation between PDZK1, Akt and Cdc37 protein levels, and all correlated with human breast malignancy. There were no positive correlations between PDZK1 and Cdc37 at the mRNA levels, confirming our in vitro studies. These results demonstrate a relationship between PDZK1, Akt and Cdc37, and potentially Her2/Neu and EGFR, in breast cancer, representing a new axis that can be targeted therapeutically to reduce the burden of human breast cancer.

Role of PDZ Domain-Containing 1 (PDZK1) in MCF-7 Breast Cancer Cells.

Breast cancer is the most common cancer and second leading cause of cancer death in women in the U.S. Approximately 70% of the breast cancers are estrogen receptor (ER) positive at the time of presentation. Estrogen induced genes are either considered as markers for diagnosis or candidate oncogenes of breast cancer. PDZ domain-containing 1 (PDZK1) is expressed in ER positive primary breast cancers and PDZK1 expression is induced by estrogen in ER positive MCF-7 breast cancer cells. Chromosome 1 was reported to be involved in the anomalies of 50-60% of breast cancer. PDZK1 gene is localized in chromosome 1q21 and shows significant overexpression correlated to copy number increase in breast cancer cells. PDZK1 is involved in the maintenance of endothelial cell monolayer integrity, stabilization of scavenger receptor class B, type I (SR-BI) in the liver, and lipid metabolism. SR-BI is a physiological receptor for high density lipoprotein (HDL) and HDL was reported to increase MCF-7 breast cancer cell proliferation. However, the role of PDZK1, its signaling pathway after estrogen stimulation, and its interaction with SR-BI in breast cancer cells are not known. The objectives of this study were to determine whether 1) estrogen induced PDZK1 expression is mediated through ER, 2) PDZK1 binds to Src kinase, 3) PDZK1 expression affects cholesterol ester uptake from HDL by its interaction with SR-BI, and 4) PDZK1 binding proteins are induced by estrogen in the MCF-7 breast cancer cells. MCF-7 breast cancer cells were cultured with 1 nM estradiol-17β (E2). PDZK1 mRNA and protein expression was determined by real-time PCR and western blotting, respectively. Knockdown of ERα and ERβ was performed by specific siRNA. PDZK1 binding to Src kinase was determined by immunoprecipitation followed by western blotting. Uptake of cholesterol ester was determined by flowcytometer and by fluorescence microscope after incubating cells with fluorescent 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI)-labeled HDL. Expression of PDZK1 binding proteins of ATP-binding cassette, subfamily C, member 2 (ABCC2) and PDZK1 interacting protein 1 (PDZK1-IP1) mRNAs was determined by real-time PCR. PDZK1 mRNA and protein expression was detectable after 12h and 24h of 1 nM E2 treatment, respectively. Knockdown of ERα by siRNA reduced E2 stimulated PDZK1 expression to the basal level. Knockdown of ERβ by siRNA reduced PDZK1 expression less than that of ERα. Immunoprecipitation by Src antibody showed that Src binds to PDZK1 and ERα. Fluorescence detected after incubation with DiI-HDL in the presence of E2 stimulation was reduced by PDZK1 or SR-BI siRNA. Though E2 induced PDZK1 expression, but it did not stimulate the expression of ABCC2 or PDZK1-IP1 mRNA. In conclusion, estrogen induced PDZK1 expression is mediated by ER in ER positive MCF-7 breast cancer cells. PDZK1 may bind phosphorylated Src after estrogen stimulated ER binds to Src, since PDZK1 protein was induced 24h after estrogen stimulation. This reaction may occur on the cell membrane. Estrogen treatment may enhance cholesterol ester uptake from circulating HDL by SR-BI and it may stimulate cell proliferation. Since PDZK1 binding proteins of ABCC2 and PDZK1-IP1 were not induced by estrogen and therefore may not participate as scaffolding proteins in the signaling of PDZK1. This pathway may be targeted as a new therapeutic intervention blocking the effect of estrogen on cell proliferation. (poster)

Down regulation of small intestinal ion transport in PDZK1- (CAP70/NHERF3) deficient mice

The PDZ-binding protein PDZK1 (CAP70/PDZ-dc-1/NHERF3) in vitro binds to cystic fibrosis transmembrane conductance regulator (CFTR), the anion exchangers SLC26A3 and SLC26A6 and the Na(+)/H(+) exchanger NHE3, all of which are major transport proteins for intestinal anion secretion and salt absorption. This study was undertaken to search for a role of PDZK1 in regulating electrolyte transport in native murine small intestine. Short circuit current (I (SC)) and HCO-(3) secretory rate (J(HCO-)(3)) were measured to assess electrogenic anion secretion; (22)Na(+) fluxes to assess sodium absorption in isolated small intestine. NHE3, CFTR, as well as NHERF1, NHERF2, and PDZK1 messenger RNA (mRNA) expression levels, and NHE3 total enterocyte and brush border membrane (BBM) protein abundance were determined by quantitative polymerase chain reaction (PCR) and Western analysis. NHE3 localization was performed by immunohistochemistry. In pdzk1 -/- jejunal mucosa, basal net Na(+) absorption as well as the inhibition of Na(+) absorption by forskolin was significantly reduced. In pdzk1 -/- duodenal mucosa, identical basal I (SC) and (J(HCO-)(3)) but a significant, yet mild, reduction of forskolin-stimulated Delta(J(HCO-)(3)) and DeltaI (SC) was observed compared to +/+ tissue. Tissue conductance, morphological features, and the DeltaI (SC) and increase in (22)Na(+) absorption in response to luminal glucose was identical in pdzk1 +/+ and -/- small intestine, ruling out a general absorptive defect. While CFTR mRNA expression levels were unchanged, NHE3 mRNA expression levels were significantly increased in small intestinal mucosa of pdzk1 -/- mice. Total enterocyte and BBM abundance was not significantly different, suggesting an increased NHE3 turnover, possibly due to reduced NHE3 membrane retention time. Lack of the PDZ-adapter protein PDZK1 in murine small intestine causes a mild reduction in maximal CFTR activation, but a severe defect in electroneutral Na(+) absorption.

Role of PDZK1 in membrane expression of renal brush border ion exchangers.

Na-H exchanger NHE3 and Cl-anion exchanger CFEX (SLC26A6, PAT1) play principal roles in the reabsorption of Na and Cl in the proximal tubule of the mammalian kidney. The mechanisms by which NHE3 and CFEX are localized to and maintained in the brush border of the proximal tubule are largely unknown. To investigate the possible interaction of NHE3 and CFEX with the PDZ-domain-containing scaffolding protein PDZK1, we performed a series of in vitro interaction assays with GST-fusion proteins and native brush border membrane proteins. These studies demonstrated that, not only were NHE3 and CFEX capable of directly interacting with PDZK1, but that this interaction was mediated through their C-terminal PDZ-interaction sites. To determine whether PDZK1 interaction is essential for brush border localization of NHE3 and CFEX in vivo, we examined the expression of NHE3 and CFEX in kidneys of wild-type and PDZK1-null mutant mice by both Western analysis and immunocytochemistry. These studies indicated that, although brush border expression of NHE3 was unaffected by the loss of PDZK1, the expression of CFEX was markedly reduced. Finally, we assayed CFEX functional activity as Cl-oxalate exchange in brush border membrane vesicles and oxalate-stimulated volume absorption in microperfused proximal tubules. Consistent with the observed decrease in CFEX protein expression, both measures of CFEX functional activity were dramatically reduced in PDZK1-null animals. In conclusion, the scaffolding protein PDZK1 is essential for the normal expression and function of Cl-anion exchanger CFEX in the proximal tubule of the mammalian kidney.

Expression and regulation of the renal Na/phosphate cotransporter NaPi-IIa in a mouse model deficient for the PDZ protein PDZK1

Inorganic phosphate (P(i)) is reabsorbed in the renal proximal tubule mainly via the type-IIa sodium-phosphate cotransporter (NaPi-IIa). This protein is regulated tightly by different factors, among them dietary P(i) intake and parathyroid hormone (PTH). A number of PDZ-domain-containing proteins have been shown to interact with NaPi-IIa in vitro, such as Na(+)/H(+) exchanger-3 regulatory factor-1 (NHERF1) and PDZK1. PDZK1 is highly abundant in kidney and co-localizes with NaPi-IIa in the brush border membrane of proximal tubules. Recently, a knock-out mouse model for PDZK1 (Pdzk1(-/-)) has been generated, allowing the role of PDZK1 in the expression and regulation of the NaPi-IIa cotransporter to be examined in in vivo and in ex vivo preparations. The localization of NaPi-IIa and other proteins interacting with PDZK1 in vitro [Na(+)/H(+) exchanger (NHE3), chloride-formate exchanger (CFEX)/putative anion transporter-1 (PAT1), NHERF1] was not altered in Pdzk1(-/-) mice. The abundance of NaPi-IIa adapted to acute and chronic changes in dietary P(i) intake, but steady-state levels of NaPi-IIa were reduced in Pdzk1(-/-) under a P(i) rich diet. This was paralleled by a higher urinary fractional P(i) excretion. The abundance of the anion exchanger CFEX/PAT1 (SLC26A6) was also reduced. In contrast, NHERF1 abundance increased in the brush border membrane of Pdzk1(-/-) mice fed a high-P(i) diet. Acute regulation of NaPi-IIa by PTH in vivo and by PTH and activators of protein kinases A, C and G (PKA, PKC and PKG) in vitro (kidney slice preparation) was not altered in Pdzk1(-/-) mice. In conclusion, loss of PDZK1 did not result in major changes in proximal tubule function or NaPi-IIa regulation. However, under a P(i)-rich diet, loss of PDZK1 reduced NaPi-IIa abundance indicating that PDZK1 may play a role in the trafficking or stability of NaPi-IIa under these conditions.

The Multivalent PDZ Domain-containing Protein PDZK1 Regulates Transport Activity of Renal Urate-Anion Exchanger URAT1 via Its C Terminus

The urate-anion exchanger URAT1 is a member of the organic anion transporter (OAT) family that regulates blood urate level in humans and is targeted by uricosuric and antiuricosuric agents. URAT1 is expressed only in the kidney, where it is thought to participate in tubular urate reabsorption. We found that the multivalent PDZ (PSD-95, Drosophila discs-large protein, Zonula occludens protein 1) domain-containing protein, PDZK1 interacts with URAT1 in a yeast two-hybrid screen. Such an interaction requires the PDZ motif of URAT1 in its extreme intracellular C-terminal region and the first, second, and fourth PDZ domains of PDZK1 as identified by yeast two-hybrid assay, in vitro binding assay and surface plasmon resonance analysis (K(D) = 1.97-514 nM). Coimmunoprecipitation studies revealed that the wild-type URAT1, but not its mutant lacking the PDZ-motif, directly interacts with PDZK1. Colocalization of URAT1 and PDZK1 was observed at the apical membrane of renal proximal tubular cells. The association of URAT1 with PDZK1 enhanced urate transport activities in HEK293 cells (1.4-fold), and the deletion of the URAT1 C-terminal PDZ motif abolished this effect. The augmentation of the transport activity was accompanied by a significant increase in the V(max) of urate transport via URAT1 and was associated with the increased surface expression level of URAT1 protein from HEK293 cells stably expressing URAT1 transfected with PDZK1. Taken together, the present study indicates the novel role of PDZK1 in regulating the functional activity of URAT1-mediated urate transport in the apical membrane of renal proximal tubules.