Primary Renal Epithelial Cells Research Articles

121 LOSS OF TRANSGLUTAMINASE 2 (TG2) ACTIVITY PROMOTES RENAL CELL CARCINOMA (RCC)

You have accessJournal of UrologyKidney Cancer: Basic Research1 Apr 2011121 LOSS OF TRANSGLUTAMINASE 2 (TG2) ACTIVITY PROMOTES RENAL CELL CARCINOMA (RCC) Merve Erdem, Selcuk Erdem, Oner Sanli, Fikrettin Sahin, Isin Kilicarslan, and Dilek Telci Merve ErdemMerve Erdem Istanbul, Turkey More articles by this author , Selcuk ErdemSelcuk Erdem Istanbul, Turkey More articles by this author , Oner SanliOner Sanli Istanbul, Turkey More articles by this author , Fikrettin SahinFikrettin Sahin Istanbul, Turkey More articles by this author , Isin KilicarslanIsin Kilicarslan Istanbul, Turkey More articles by this author , and Dilek TelciDilek Telci Istanbul, Turkey More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2011.02.187AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES TG2 is a multifunctional enzyme that can decrease the migratory ability of cancer cells when secreted into the extracellular (EC) space through cross-linking the EC matrix proteins which then become resistant against the proteolytic degradation. Primary tumors of the liver, breast, and skin show reduced TG2 expression and activity in the tumor body resulting in progression of the primary tumor. However, increased TG2 expression in parallel with the integrin β1 (ITGB1) was detected in tumors isolated from metastatic sites. We previously showed that interaction of TG2 with syndecan-4 (SDC-4) and ITGB1 results in the loss of enzyme activity and promotes cell survival. Hence, the objective of this study was to investigate the importance of TG2 along with SDC-4 and ITGB1 in RCC progression. METHODS The expression level of TG2, ITGB1, and SDC-4 in the primary RCC cell line A-498 and control RPTEC (primary renal proximal tubule epithelial) cell line along with healthy and tumor tissue samples from 62 patients with RCC was evaluated by real time-PCR. TG2 activity in randomly selected patient samples and cell lines was measured by transamidation activity assay. RESULTS TG2 expression ratio showed a significant 2.3 fold decrease in A-498 cells when compared with the control RPTEC. A 3 fold decrease in TG2 expression levels were recorded in the 65% of primary RCC (P < 0.0001) regardless of the grade of the tumor. Rest of the tumors showed a 2.1 fold increase in the TG2 expression ratio, which was found to be in parallel with the increase in ITGB1 and SDC-4 expression levels in 87% of the cases. A-498 cells contained 2 fold less TG2 activity compared to RPTECs, while a remarkable decrease in TG2 activity was seen both in tumors with reduced TG2 expression and in tumors with a high TG2 expression in parallel with high ITGB1 and SDC4. CONCLUSIONS There was a statistically significant decrease in the expression and activity of TG2 in RCC cell line and tumors suggesting that the loss of the TG2 activity promotes RCC possibly through rendering the ECM more susceptible to proteolytic degradation. A subpopulation of RCC tumors showed increased TG2 expression but not activity in conjunction with increased ITGB1 and SDC4 expression where TG2 may be acting as a cell adhesion protein affecting the cell migration and survival. Whether increased TG2 expression in association with the ITGB1 and SDC4 expression predisposed this subpopulation of patients to metastasis will be determined with the patient follow-up in the future studies. © 2011 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 185Issue 4SApril 2011Page: e51 Advertisement Copyright & Permissions© 2011 by American Urological Association Education and Research, Inc.MetricsAuthor Information Merve Erdem Istanbul, Turkey More articles by this author Selcuk Erdem Istanbul, Turkey More articles by this author Oner Sanli Istanbul, Turkey More articles by this author Fikrettin Sahin Istanbul, Turkey More articles by this author Isin Kilicarslan Istanbul, Turkey More articles by this author Dilek Telci Istanbul, Turkey More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Functional Reorganization of Promyelocytic Leukemia Nuclear Bodies during BK Virus Infection

BK virus (BKV) is the causative agent for polyomavirus-associated nephropathy, a severe disease found in renal transplant patients due to reactivation of a persistent BKV infection. BKV replication relies on the interactions of BKV with many nuclear components, and subnuclear structures such as promyelocytic leukemia nuclear bodies (PML-NBs) are known to play regulatory roles during a number of DNA virus infections. In this study, we investigated the relationship between PML-NBs and BKV during infection of primary human renal proximal tubule epithelial (RPTE) cells. While the levels of the major PML-NB protein components remained unchanged, BKV infection of RPTE cells resulted in dramatic alterations in both the number and the size of PML-NBs. Furthermore, two normally constitutive components of PML-NBs, Sp100 and hDaxx, became dispersed from PML-NBs. To define the viral factors responsible for this reorganization, we examined the cellular localization of the BKV large tumor antigen (TAg) and viral DNA. TAg colocalized with PML-NBs during early infection, while a number of BKV chromosomes were adjacent to PML-NBs during late infection. We demonstrated that TAg alone was not sufficient to reorganize PML-NBs and that active viral DNA replication is required. Knockdown of PML protein did not dramatically affect BKV growth in culture. BKV infection, however, was able to rescue the growth of an ICP0-null herpes simplex virus 1 mutant whose growth defect was partially due to its inability to disrupt PML-NBs. We hypothesize that the antiviral functions of PML-NBs are inactivated through reorganization during normal BKV infection.

Shiga Toxin Subtypes Display Dramatic Differences in Potency

Purified Shiga toxin (Stx) alone is capable of producing systemic complications, including hemolytic-uremic syndrome (HUS), in animal models of disease. Stx includes two major antigenic forms (Stx1 and Stx2), with minor variants of Stx2 (Stx2a to -h). Stx2a is more potent than Stx1. Epidemiologic studies suggest that Stx2 subtypes also differ in potency, but these differences have not been well documented for purified toxin. The relative potencies of five purified Stx2 subtypes, Stx2a, Stx2b, Stx2c, Stx2d, and activated (elastase-cleaved) Stx2d, were studied in vitro by examining protein synthesis inhibition using Vero monkey kidney cells and inhibition of metabolic activity (reduction of resazurin to fluorescent resorufin) using primary human renal proximal tubule epithelial cells (RPTECs). In both RPTECs and Vero cells, Stx2a, Stx2d, and elastase-cleaved Stx2d were at least 25 times more potent than Stx2b and Stx2c. In vivo potency in mice was also assessed. Stx2b and Stx2c had potencies similar to that of Stx1, while Stx2a, Stx2d, and elastase-cleaved Stx2d were 40 to 400 times more potent than Stx1.

A system for the analysis of BKV non-coding control regions: Application to clinical isolates from an HIV/AIDS patient

The human polyomavirus BK virus (BKV) is an important opportunistic pathogen whose disease prevalence continues to increase with the growing immunocompromised population. To date, the major determinant of replication in cell culture has not been formally proven. BKV exists as archetype virus and rearranged variants, which are classified based on the DNA sequence of their non-coding control regions (NCCRs). The archetype BKV NCCR is divided into five blocks of sequence and rearranged variants contain deletions and duplications of these blocks. In this study, a genetic system was developed and used to identify the major determinant of replication ability in primary renal proximal tubule epithelial cells, the natural host cell of BKV. This system was also used to analyze NCCR variants isolated from an immunocompromised patient which contain assorted rearrangement patterns and functional differences. This study solidifies the NCCR as the major genetic determinant of BKV replication ability in vitro.

Bardet‐Biedl Sydrome (BBS) proteins play a role in Centrosome Amplification

Many cancers result from mitotic or apoptotic abnormalities. We and others have previously shown that BBS proteins are involved in centrosome organization and regulation. We observed centrosomal clusters in renal epithelial primary cells obtained from BBS mutant mice. Along with the extra centrosomes, we observed a marked increase in the amount of gamma‐tubulin in BBS renal epithelial cells compared to wild type controls. It was unclear if these phenomena were related to the cell culture or a hallmark of BBS mutations. Therefore, we compared BBS mutant and wild type primary renal epithelial cells in culture with kidney sections of the same mice. Although the culture of cells exacerbates the phenotype, we demonstrate a definitive increase in centrosome number within cells in fixed kidneys obtained from BBS mutant mice as compared to wild type controls. We hypothesize that BBS proteins are involved in the regulation or transport of proteins destined for proteosome degradation. Specifically targeting cancer cells with multiple centrosomes presents a viable and novel therapy. Confirming BBS proteins role in centrosome regulation may help to better understand tumorigenesis.This work was supported by the NIH and HHMI.

Decursin Prevents Cisplatin-Induced Apoptosis via the Enhancement of Antioxidant Enzymes in Human Renal Epithelial Cells

Adverse effects, nephrotoxicity and hepatotoxicity, of anticancer drugs such as cisplatin have limited the usage for cancer therapy. Therefore, development or identification of supplement agents in anticancer drugs is attractive to reduce side effects and enhance antitumor activity. Here, we found that decursin isolated from Angelica gigas showed protective effects of cisplatin-induced damage in normal human primary renal epithelial cells (HRCs). We found that decursin significantly blocked cisplatin-induced cytotoxicity by 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) assay in HRCs. Further, we found that decursin inhibited sub-G1 and cell death by suppression of cleavage of caspase-3, -9 and poly(ADP-ribose) polymerase (PARP) induced by cisplatin treatment in HRCs. Importantly, decursin effectively restored the activities of Cu/Zn superoxide dismutase (SOD), catalase and glutathione peroxidase in cisplatin-treated HRCs. Taken together, our findings demonstrate that decurcin prevents cisplatin-induced cytotoxicity and apoptosis through the activation of antioxidant enzymes in HRCs and suggest further that combination of decursin might suppressed adverse effects of anticancer drugs in cancer patients.

Xanthine Oxidoreductase Depletion Induces Renal Interstitial Fibrosis Through Aberrant Lipid and Purine Accumulation in Renal Tubules

Xanthine oxidoreductase (XOR) is an enzyme responsible for purine degradation, reactive oxygen species production, and adipogenesis. XOR gene-disrupted (XOR(-/-)) mice demonstrate renal failure and early death within several months. The aim of this study was to elucidate the mechanism of renal damage in XOR(-/-) mice and to determine the physiological role of XOR in the kidney. Histological analysis revealed that renal tubular damage in XOR(-/-) mice was accompanied by deposition of crystals and lipid-rich substances. Triglyceride content in renal homogenates was significantly increased in XOR(-/-) mice. The level of lipogenesis-related gene expression was comparable in XOR(+/+) and XOR(-/-) mice, whereas the expression of adipogenesis-related gene expression was significantly elevated in XOR(-/-) mice. Urinary excretions of xanthine and hypoxanthine were markedly elevated in XOR(-/-) mice. Immunohistochemical analysis, Western blotting, and real time RT-PCR revealed that various markers of fibrosis, inflammation, ischemia, and oxidative stress were increased in XOR(-/-) mice. Finally, we demonstrate that primary renal epithelial cells from XOR(-/-) mice are more readily transformed to myofibroblasts, which is a marker of increased epithelial mesenchymal transition. These results suggest that XOR gene disruption induced the depletion of uric acid and the accumulation of triglyceride-rich substances, xanthine, and hypoxanthine in the renal tubules. We believe that these changes contribute to a complex cellular milieu characterized by inflammation, tissue hypoxia, and reactive oxygen species production, ultimately resulting in renal failure through increased renal interstitial fibrosis.

Downregulation of α-galactosidase A upregulates CD77: functional impact for Fabry nephropathy

Anderson-Fabry disease, an inherited deficiency in the lysosomal enzyme alpha-galactosidase A, is characterized by the progressive accumulation of globotriaosylceramide (Gb3), also known as CD77. We sought to clarify the pathogenesis of Fabry disease by establishing a cell model of this disorder. The expression of alpha-galactosidase A was transiently silenced by RNA interference in HK2 and primary human renal epithelial cells and stably silenced in HK2 cells by retroviral transfection with small hairpin RNA. All of the silenced cells had histological similarities to cells of patients with Fabry disease. The cells had reduced viability, significant accumulation of intracellular Gb3, and a modest but significant increase in membranous Gb3 expression compared to nonsilenced cells. When silenced HK2 cells were reconstituted with agalsidase-alpha, a protein used for enzyme replacement therapy, they decreased their membranous CD77 expression to levels indistinguishable from those of nonsilenced cells. Because plasma and urinary Gb3 levels are not reliable biomarkers for Fabry disease, our study suggests that membranous CD77 levels mirror Gb3 tissue load and that CD77 expression levels may be used to monitor the efficacy of enzyme replacement therapy.

Early Events during BK Virus Entry and Disassembly

BK virus (BKV) is a nonenveloped, ubiquitous human polyomavirus that establishes a persistent infection in healthy individuals. It can be reactivated, however, in immunosuppressed patients and cause severe diseases, including polyomavirus nephropathy. The entry and disassembly mechanisms of BKV are not well defined. In this report, we characterized several early events during BKV infection in primary human renal proximal tubule epithelial (RPTE) cells, which are natural host cells for BKV. Our results demonstrate that BKV infection in RPTE cells involves an acidic environment relatively early during entry, followed by transport along the microtubule network to reach the endoplasmic reticulum (ER). A distinct disulfide bond isomerization and cleavage pattern of the major capsid protein VP1 was observed, which was also influenced by alterations in pH and disruption of trafficking to the ER. A dominant negative form of Derlin-1, an ER protein required for retro-translocation of certain misfolded proteins, inhibited BKV infection. Consistent with this, we detected an interaction between Derlin-1 and VP1. Finally, we show that proteasome function is also linked to BKV infection and capsid rearrangement. These results indicate that BKV early entry and disassembly are highly regulated processes involving multiple cellular components.

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.

Zag Expression during Aging Suppresses Proliferation after Kidney Injury

Recovery after acute kidney injury is impaired in the elderly, but mechanistic information regarding why this occurs is limited. In this study, aged mouse kidneys displayed a reduced epithelial proliferative reserve in vivo and in vitro. Microarray analysis identified increased expression of zinc-alpha (2)-glycoprotein (Zag) in aged proximal tubular cells. The addition of recombinant Zag to primary renal epithelial cell cultures decreased proliferation, whereas knockdown of Zag increased proliferation. In vivo, systemic small interference RNA suppressed expression of Zag in the mouse proximal tubule; this increased the rate of epithelial cell proliferation after renal ischemia/reperfusion in aged mice but also increased parenchymal fibrosis. These results demonstrate that increased Zag expression in the aged kidney acts to suppress the proliferative response to injury and introduce Zag as a modifier of the aging phenotype.

Cidofovir Inhibits Polyomavirus BK Replication in Human Renal Tubular Cells Downstream of Viral Early Gene Expression

The human polyomavirus BK (BKV) causes nephropathy and hemorrhagic cystitis in kidney and bone marrow transplant patients, respectively. The anti-viral cidofovir (CDV) has been used in small case series but the effects on BKV replication are unclear, since polyomaviruses do not encode viral DNA polymerases. We investigated the effects of CDV on BKV(Dunlop) replication in primary human renal proximal tubule epithelial cells (RPTECs). CDV inhibited the generation of viral progeny in a dose-dependent manner yielding a 90% reduction at 40 microg/mL. Early steps such as receptor binding and entry seemed unaffected. Initial large T-antigen transcription and expression were also unaffected, but subsequent intra-cellular BKV DNA replication was reduced by >90%. Late viral mRNA and corresponding protein levels were also 90% reduced. In uninfected RPTECs, CDV 40 microg/mL reduced cellular DNA replication and metabolic activity by 7% and 11% in BrdU and WST-1 assays, respectively. BKV infection increased DNA replication to 142% and metabolic activity to 116%, respectively, which were reduced by CDV 40 microg/mL to levels of uninfected untreated RPTECs. Our results show that CDV inhibits BKV DNA replication downstream of large T-antigen expression and involves significant host cell toxicity. This should be considered in current treatment and drug development.

Reduction of Foxp3-expressing regulatory T cell infiltrates during the progression of renal allograft rejection in a mouse model

Background Regulatory T (Treg) cells are the immune suppressors in the maintenance of immune homeostasis and tolerance to self and non-self antigens, and may have therapeutic potential in the treatment of transplant rejection in patients. However, Treg cell development and action are poorly understood in transplantation. In this study, the association of CD4 +Foxp3 + infiltrates within renal allograft tissue with graft survival was investigated in a mouse model. Methods Kidney donors from C57BL/6J mice (H-2 b) were transplanted to bilaterally nephrectomized Balb/c recipient mice (H-2 d). Treg cells were examined with FACS and immunohistochemical staining. Results Here we showed that without any immunosuppressive regimen, kidney allografts were mostly rejected from 20 to 60 days after transplantation. During the progression of allograft rejection Foxp3 + Treg phenotype infiltrates were significantly diminished, while intragraft expression of TGF-β1, IL-6, IL-17 and IL-23 was up-regulated. The regulatory function of CD4 +CD25 + infiltrates was confirmed by their suppressive activity in mixed lymphocyte reaction. Further in vitro studies indicated that primary renal tubular epithelial cell (TEC) cultures produced high levels of IL-6 in response to allogeneic lymphocyte or IL-17 stimulation, and neutralization of IL-6 increased CD4 +CD25 +Foxp3 + cells in co-cultures with TEC. Conclusion Diminution of Foxp3 + Treg infiltrates associates with renal allograft rejection, and neutralization of IL-6 activity enhances Foxp3 + Treg cell differentiation. Our findings suggest that increase in intragraft IL-6 may down-regulate infiltrating Foxp3 + Treg cells.

Involvement of ezrin in cold storage preservation injury in renal proximal tubule epithelial cells: Possible structural and signaling roles

Renal transplantation is the treatment of choice for patients with end‐stage renal disease. Preservation injury is caused by ischemia/reperfusion and hypothermia during cold storage, which leads to early graft loss and delayed graft function. Advancements in organ preservation are limited by our understanding of basic root mechanisms that cause this injury. We hypothesize that sub‐lamellar cytoskeletal protein failure is involved and studied this in isolated rabbit renal proximal tubules and epithelial cells. Tubules cold stored in UW solution released ezrin from cytoskeletal actin attachments. Ezrin phosphorylation at T567 and Y353 decreased with progressive cold storage ischemia, which controls both ezrin binding to the cytoskeleton and survival signaling through Akt, respectively. Tubule epithelial cell death at reperfusion after cold storage ischemia was almost exclusively by apoptosis (annexin‐V FACS analysis). Transfection of primary renal epithelial cells with wild‐type mammalian ezrin cDNA increased survival and viability after cold storage ischemia and reperfusion and increased Akt phosphorylation (IHC). Conversely, inhibition of ezrin expression with specific siRNA duplexes decreased cell survival and viability. In conclusion, inactivation of ezrin during preservation may cause renal preservation injury by altering membrane structure and by reducing survival signaling.

Cytoskeletal Involvement in Hypothermic Renal Preservation Injury

Cytoskeletal degradation occurs in warm renal ischemia and reperfusion and during hypothermia. The purpose of this study was to determine cytoskeletal changes during cold storage preservation injury in renal tubules and to determine whether these changes contribute to the injury. Isolated canine renal proximal tubules or their primary epithelial cultures were cold-stored in University of Wisconsin solution and reperfused in vitro to simulate renal cold storage preservation injury. Assays for cytoskeletal protein degradation and viability together with biologically active cytoskeletal agents and molecular interventions were used to test this hypothesis. Progressively increasing the cold storage time of isolated renal tubules in University of Wisconsin solution caused proportional disassembly of both ezrin and Na/K adenosine triphosphatase proteins from their cytoskeletal attachments. The sublamellar structural protein, fodrin, was metabolized to products consistent with calpain hydrolysis during preservation. Time-dependent deterioration in tubule membrane function (organic cation transporter-1 transport activity) was observed during cold preservation, and this was mimicked in fresh tubules by chemically induced cytoskeletal disruption with cytochalasin-D treatment. Cold preservation decreased total tubulin content. Taxol slowed this loss by preventing tubulin depolymerization, which improved membrane function and tubule viability. The viability of primary renal epithelial cells was enhanced by overexpression of ezrin (transfection) and diminished with specific ezrin small interfering RNA knockdown during cold preservation injury. Hypothermic storage preservation causes disruption of key cytoskeletal elements in kidney tubules, which contributes causally to the injury at rewarming.

Hypoxia promotes fibrogenesis in vivo via HIF-1 stimulation of epithelial-to-mesenchymal transition

Hypoxia has been proposed as an important microenvironmental factor in the development of tissue fibrosis; however, the underlying mechanisms are not well defined. To examine the role of hypoxia-inducible factor-1 (HIF-1), a key mediator of cellular adaptation to hypoxia, in the development of fibrosis in mice, we inactivated Hif-1alpha in primary renal epithelial cells and in proximal tubules of kidneys subjected to unilateral ureteral obstruction (UUO) using Cre-loxP-mediated gene targeting. We found that Hif-1alpha enhanced epithelial-to-mesenchymal transition (EMT) in vitro and induced epithelial cell migration through upregulation of lysyl oxidase genes. Genetic ablation of epithelial Hif-1alpha inhibited the development of tubulointerstitial fibrosis in UUO kidneys, which was associated with decreased interstitial collagen deposition, decreased inflammatory cell infiltration, and a reduction in the number of fibroblast-specific protein-1-expressing (FSP-1-expressing) interstitial cells. Furthermore, we demonstrate that increased renal HIF-1alpha expression is associated with tubulointerstitial injury in patients with chronic kidney disease. Thus, we provide clinical and genetic evidence that activation of HIF-1 signaling in renal epithelial cells is associated with the development of chronic renal disease and may promote fibrogenesis by increasing expression of extracellular matrix-modifying factors and lysyl oxidase genes and by facilitating EMT.

The Cysteine-Rich Domain Protein KCP Is a Suppressor of Transforming Growth Factor β/Activin Signaling in Renal Epithelia

The transforming growth factor beta (TGF-beta) superfamily, including the bone morphogenetic protein (BMP) and TGF-beta/activin A subfamilies, is regulated by secreted proteins able to sequester or present ligands to receptors. KCP is a secreted, cysteine-rich (CR) protein with similarity to mouse Chordin and Xenopus laevis Kielin. KCP is an enhancer of BMP signaling in vertebrates and interacts with BMPs and the BMP type I receptor to promote receptor-ligand interactions. Mice homozygous for a KCP null allele are hypersensitive to developing renal interstitial fibrosis, a disease stimulated by TGF-beta but inhibited by BMP7. In this report, the effects of KCP on TGF-beta/activin A signaling are examined. In contrast to the enhancing effect on BMPs, KCP inhibits both activin A- and TGF-beta1-mediated signaling through the Smad2/3 pathway. These inhibitory effects of KCP are mediated in a paracrine manner, suggesting that direct binding of KCP to TGF-beta1 or activin A can block the interactions with prospective receptors. Consistent with this inhibitory effect, primary renal epithelial cells from KCP mutant cells are hypersensitive to TGF-beta and exhibit increased apoptosis, dissociation of cadherin-based cell junctions, and expression of smooth muscle actin. Furthermore, KCP null animals show elevated levels of phosphorylated Smad2 after renal injury. The ability to enhance BMP signaling while suppressing TGF-beta activation indicates a critical role for KCP in modulating the responses between these anti- and profibrotic cytokines in the initiation and progression of renal interstitial fibrosis.

Gene Expression Programs in Response to Hypoxia: Cell Type Specificity and Prognostic Significance in Human Cancers

BackgroundInadequate oxygen (hypoxia) triggers a multifaceted cellular response that has important roles in normal physiology and in many human diseases. A transcription factor, hypoxia-inducible factor (HIF), plays a central role in the hypoxia response; its activity is regulated by the oxygen-dependent degradation of the HIF-1α protein. Despite the ubiquity and importance of hypoxia responses, little is known about the variation in the global transcriptional response to hypoxia among different cell types or how this variation might relate to tissue- and cell-specific diseases.Methods and FindingsWe analyzed the temporal changes in global transcript levels in response to hypoxia in primary renal proximal tubule epithelial cells, breast epithelial cells, smooth muscle cells, and endothelial cells with DNA microarrays. The extent of the transcriptional response to hypoxia was greatest in the renal tubule cells. This heightened response was associated with a uniquely high level of HIF-1α RNA in renal cells, and it could be diminished by reducing HIF-1α expression via RNA interference. A gene-expression signature of the hypoxia response, derived from our studies of cultured mammary and renal tubular epithelial cells, showed coordinated variation in several human cancers, and was a strong predictor of clinical outcomes in breast and ovarian cancers. In an analysis of a large, published gene-expression dataset from breast cancers, we found that the prognostic information in the hypoxia signature was virtually independent of that provided by the previously reported wound signature and more predictive of outcomes than any of the clinical parameters in current use.ConclusionsThe transcriptional response to hypoxia varies among human cells. Some of this variation is traceable to variation in expression of the HIF1A gene. A gene-expression signature of the cellular response to hypoxia is associated with a significantly poorer prognosis in breast and ovarian cancer.

A noninvasive transfer system for polarized renal tubule epithelial cell sheets using temperature-responsive culture dishes

We used temperature-responsive culture dishes onto which the temperature-responsive polymer, poly(Nisopropylacrylamide), was covalently grafted for tissue engineering. Confluent cells harvested as intact sheets from these surfaces by simple temperature reduction can be transferred to various surfaces including additional culture dishes, other cell sheets, and tissues. In order to examine the maintenance of cell polarity, Madin-Darby canine kidney cells and human primary renal proximal tubule epithelial cells which had developed apical-basal cell polarity in culture, were subjected to cell sheet transfer. This functional and structural cell polarity, which is susceptible to treatment with trypsin, was examined by immunohistochemistry and transmission electron microscopy. Using our cell-sheet method, the noninvasive transfer of these cell sheets retaining typical distributions of Na+/K+-ATPase, GLUT-1, SGLT-1, aquaporin-1, neutral endopeptidase and dipeptidylendopeptidase IV, could be achieved. The transferred cell sheets also developed numerous microvilli and tight junctions at the apical and lateral membranes, respectively. For biochemical analysis, immunoblotting of occludin, a transmembrane protein that composes tight junctions, was conducted and results confirmed that occludin remained intact after cell sheet transfer. This two-dimensional cell sheet manipulation method promises to be useful for tissue engineering as well as in the investigation of epithelial cell polarity.

Amino Acid-stimulated Ca2+ Oscillations Produced by the Ca2+-sensing Receptor Are Mediated by a Phospholipase C/Inositol 1,4,5-Trisphosphate-independent Pathway That Requires G12, Rho, Filamin-A, and the Actin Cytoskeleton

The G protein-coupled Ca(2+)-sensing receptor (CaR) is an allosteric protein that responds to two different agonists, Ca(2+) and aromatic amino acids, with the production of sinusoidal or transient oscillations in intracellular Ca(2+) concentration ([Ca(2+)](i)). Here, we examined whether these differing patterns of [Ca(2+)](i) oscillations produced by the CaR are mediated by separate signal transduction pathways. Using real time imaging of changes in phosphatidylinositol 4,5-biphosphate hydrolysis and generation of inositol 1,4,5-trisphosphate in single cells, we found that stimulation of CaR by an increase in the extracellular Ca(2+) concentration ([Ca(2+)](o)) leads to periodic synthesis of inositol 1,4,5-trisphosphate, whereas l-phenylalanine stimulation of the CaR does not induce any detectable change in the level this second messenger. Furthermore, we identified a novel pathway that mediates transient [Ca(2+)](i) oscillations produced by the CaR in response to l-phenylalanine, which requires the organization of the actin cytoskeleton and involves the small GTPase Rho, heterotrimeric proteins of the G(12) subfamily, the C-terminal region of the CaR, and the scaffolding protein filamin-A. Our model envisages that Ca(2+) or amino acids stabilize unique CaR conformations that favor coupling to different G proteins and subsequent activation of distinct downstream signaling pathways.