Impaired Cell Adhesion Research Articles

Intracellular targeting of annexin A2 inhibits tumor cell adhesion, migration, and in vivo grafting

Cytoskeletal-associated proteins play an active role in coordinating the adhesion and migration machinery in cancer progression. To identify functional protein networks and potential inhibitors, we screened an internalizing phage (iPhage) display library in tumor cells, and selected LGRFYAASG as a cytosol-targeting peptide. By affinity purification and mass spectrometry, intracellular annexin A2 was identified as the corresponding binding protein. Consistently, annexin A2 and a cell-internalizing, penetratin-fused version of the selected peptide (LGRFYAASG-pen) co-localized and specifically accumulated in the cytoplasm at the cell edges and cell-cell contacts. Functionally, tumor cells incubated with LGRFYAASG-pen showed disruption of filamentous actin, focal adhesions and caveolae-mediated membrane trafficking, resulting in impaired cell adhesion and migration in vitro. These effects were paralleled by a decrease in the phosphorylation of both focal adhesion kinase (Fak) and protein kinase B (Akt). Likewise, tumor cells pretreated with LGRFYAASG-pen exhibited an impaired capacity to colonize the lungs in vivo in several mouse models. Together, our findings demonstrate an unrecognized functional link between intracellular annexin A2 and tumor cell adhesion, migration and in vivo grafting. Moreover, this work uncovers a new peptide motif that binds to and inhibits intracellular annexin A2 as a candidate therapeutic lead for potential translation into clinical applications.

Characterization of the role of RILP in cell migration.

Rab-interacting lysosomal protein (RILP) is a regulator of late stages of endocytosis. Recent work proved that depletion of RILP promotes migration of breast cancer cells in wound healing assay, whereas its overexpression influences re-arrangements of actin cytoskeleton. Here, we further characterized the role of RILP in cell migration by analyzing several aspects of this process. We showed that RILP is fundamental also for migration of lung cancer cells regulating cell velocity. RILP silencing did not affect Golgi apparatus nor microtubules reorientation during migration. However, both RILP over-expression and expression of its mutated form, RILPC33, impair cell adhesion and spreading. In conclusion, our results demonstrate that RILP has important regulatory roles in cell motility affecting migration velocity but also in cell adhesion and cell spreading.

Amphiphilic Nucleating Agents to Enhance Calcium Phosphate Growth on Polymeric Surfaces.

Poly(ε-caprolactone) (PCL) is an aliphatic polyester widely explored in the preparation of guided bone regeneration (GBR) membranes because of its interesting mechanical properties and biodegradability. However, PCL high hydrophobicity often impairs cell adhesion and proliferation as well as calcium phosphate growth, all of which are crucial to achieving suitable bone-tissue integration. In this work, aimed at achieving less-hydrophobic surfaces, amphiphilic molecules were added at low concentrations to the polymeric dope solutions that generated the GBR membranes. During membrane formation, these molecules migrate to the solution/air interface in such a way that, upon liquid-solid phase transition, the negatively charged heads are exposed while the apolar tails are anchored to the polymer bulk. As a consequence, these molecules became nucleating agents for subsequent calcium phosphate growth using an alternating soaking process. Herein, PCL porous membranes containing different amphiphilic molecules, such as stearic acid and bis(2-ethylhexyl) phosphate, were investigated. This new, simple, and atoxic method to superficially treat polymeric membranes could be extended to a wide range of polymers and applications.

The Role of Titanium Surface Nanostructuring on Preosteoblast Morphology, Adhesion, and Migration.

Surface structuring of titanium-based implants is known to modulate the behavior of adherent cells, but the influence of different nanotopographies is poorly understood. The aim is to investigate preosteoblast proliferation, adhesion, morphology, and migration on surfaces with similar surface chemistry but distinct nanotopographical features. Sonochemical treatment and anodic oxidation are employed to fabricate disordered, mesoporous titania (TMS) and ordered titania nanotubular (TNT) topographies on titanium, respectively. Morphological evaluation reveals that cells are polygonal and well-spread on TMS, but display an elongated, fibroblast-like morphology on TNT surfaces, while they are much flatter on glass. Both nanostructured surfaces impair cell adhesion, but TMS is more favorable for cell growth due to its support of cell attachment and spreading in contrast to TNT. A quantitative wound healing assay in combination with live-cell imaging reveals that cell migration on TMS surfaces has a more collective character than on other surfaces, probably due to a closer proximity between neighboring migrating cells on TMS. The results indicate distinctly different cell adhesion and migration on ordered and disordered titania nanotopographies, providing important information that can be used in optimizing titanium-based scaffold design to foster bone tissue growth and repair while allowing for the encapsulation of drugs into porous titania layer.

Cinnamomum cassia extracts reverses TGF-β1-induced epithelial-mesenchymal transition in human lung adenocarcinoma cells and suppresses tumor growth in vivo

Metastasis is the most common cause of cancer-related mortality in patients, and epithelial-mesenchymal transition (EMT) is essential for cancer metastasis and antidrug resistance. Cinnamomum cassia has several antioxidative, anti-inflammatory, and anticancer biological effects. However, the anti-EMT effect of C. cassia in human lung carcinoma is rarely reported. In this study, we determined whether C. cassia extracts (CCE) reduces the EMT and tumor growth of human lung adenocarcinoma cells. CCE inhibited the transforming growth factor (TGF)-β1-induced cell motility and invasiveness of A549 and H1299 cells by repressing matrix metalloproteinase-2 and urokinase-type plasminogen activator as well as impaired cell adhesion to collagen. CCE also affected the TGF-β1-induced EMT by downregulating the expression of vimentin and fibronectin and upregulating E-cadherin. The nude mice xenograft model showed that CCE reduced A549 tumor growth. Thus, CCE possesses antimetastatic activity of A549 and H1299 cells by affecting EMT and suppressing A549 tumor growth in vivo. This result suggested that CCE could be used as an antimetastatic agent or as an adjuvant for anticancer therapy.

Orphan G protein-coupled receptor GPRC5A modulates integrin β1-mediated epithelial cell adhesion

ABSTRACTG-Protein Coupled Receptor (GPCR), Class C, Group 5, Member A (GPRC5A) has been implicated in several malignancies. The underlying mechanisms, however, remain poorly understood. Using a panel of human cell lines, we demonstrate that CRISPR/Cas9-mediated knockout and RNAi-mediated depletion of GPRC5A impairs cell adhesion to integrin substrates: collagens I and IV, fibronectin, as well as to extracellular matrix proteins derived from the Engelbreth-Holm-Swarm (EHS) mouse sarcoma (Matrigel). Consistent with the phenotype, knock-out of GPRC5A correlated with a reduced integrin β1 (ITGB1) protein expression, impaired phosphorylation of the focal adhesion kinase (FAK), and lower activity of small GTPases RhoA and Rac1. Furthermore, we provide the first evidence for a direct interaction between GPRC5A and a receptor tyrosine kinase EphA2, an upstream regulator of FAK, although its contribution to the observed adhesion phenotype is unclear. Our findings reveal an unprecedented role for GPRC5A in regulation of the ITGB1-mediated cell adhesion and it's downstream signaling, thus indicating a potential novel role for GPRC5A in human epithelial cancers.

Aqueous extract of Polygonum bistorta modulates proteostasis by ROS-induced ER stress in human hepatoma cells

Hepatocellular carcinoma (HCC) remains the leading cause of cancer mortality with limited therapeutic targets. The endoplasmic reticulum (ER) plays a pivotal role in maintaining proteostasis in normal cells. However, alterations in proteostasis are often found in cancer cells, making it a potential target for therapy. Polygonum bistorta is used in traditional Chinese medicine owing to its anticancer activities, but the molecular and pharmacological mechanisms remain unclear. Using hepatoma cells as a model system, this study demonstrated that P. bistorta aqueous extract (PB) stimulated ER stress by increasing autophagosomes but by blocking degradation, followed by the accumulation of ubiquitinated proteins and cell apoptosis. In addition, an autophagy inhibitor did not enhance ubiquitinated protein accumulation whereas a reactive oxygen species (ROS) scavenger diminished both ubiquitinated protein accumulation and ligand-stimulated epidermal growth factor receptor (EGFR) expression, suggesting that ROS generation by PB may be upstream of PB-triggered cell death. Nevertheless, PB-exerted proteostasis impairment resulted in cytoskeletal changes, impairment of cell adhesion and motility, and inhibition of cell cycle progression. Oral administration of PB delayed tumour growth in a xenograft model without significant body weight loss. These findings indicate that PB may be a potential new alternative or complementary medicine for HCC.

Glycogen storage disease type Ib neutrophils exhibit impaired cell adhesion and migration

Glycogen storage disease type Ib (GSD-Ib), characterized by impaired glucose homeostasis, neutropenia, and neutrophil dysfunction, is an inherited autosomal recessive disorder caused by a deficiency in the glucose-6-phosphate transporter (G6PT). Neutrophils play an essential role in the defense against invading pathogens. The recruitment of neutrophils towards the inflammation sites in response to inflammatory stimuli is a tightly regulated process involving rolling, adhesion, and transmigration. In this study, we investigated the role of G6PT in neutrophil adhesion and migration using in vivo and in vitro models. We showed that the GSD-Ib (G6pt−/−) mice manifested severe neutropenia in both blood and bone marrow, and treating G6pt−/− mice with granulocyte colony-stimulating factor (G-CSF) corrected neutropenia. However, upon thioglycolate challenge, neutrophils from both untreated and G-CSF-treated G6pt−/−mice exhibited decreased ability to migrate to the peritoneal cavity. In vitro migration and cell adhesion of G6PT-deficient neutrophils were also significantly impaired. Defects in cell migration were not due to enhanced apoptosis or altered fMLP receptor expression. Remarkably, the expression of the β2 integrins CD11a and CD11b, which are critical for cell adhesion, was greatly decreased in G6PT-deficient neutrophils. This study suggests that deficiencies in G6PT cause impairment in neutrophil adhesion and migration via aberrant expression of β2 integrins, and our finding should facilitate the development of novel therapies for GSD-Ib.

3-Hydroxyflavone inhibits human osteosarcoma U2OS and 143B cells metastasis by affecting EMT and repressing u-PA/MMP-2 via FAK-Src to MEK/ERK and RhoA/MLC2 pathways and reduces 143B tumor growth in vivo

Many natural flavonoids have cytostatic and apoptotic properties; however, we little know whether the effect of synthetic 3-hydroxyflavone on metastasis and tumor growth of human osteosarcoma. Here, we tested the hypothesis that 3-hydroxyflavone suppresses human osteosarcoma cells metastasis and tumor growth. 3-hydroxyflavone, up to 50 μM without cytotoxicity, inhibited U2OS and 143B cells motility, invasiveness and migration by reducing matrix metalloproteinase (MMP)-2 and urokinase-type plasminogen activator (u-PA) and also impaired cell adhesion to gelatin. 3-hydroxyflavone significantly reduced p-focal adhesion kinase (FAK) Tyr397, p-FAK Tyr925, p-steroid receptor coactivator (Src), p-mitogen/extracellular signal-regulated kinase (MEK)1/2, p-myosin light chain (MLC)2 Ser19, epithelial cell adhesion molecule, Ras homolog gene family (Rho)A and fibronectin expressions. 3-hydroxyflavone also affected the epithelial-mesenchymal transition (EMT) by down-regulating expressions of Vimentin and α-catenin with activation of the transcription factor Slug. In nude mice xenograft model and tail vein injection model showed that 3-hydroxyflavone reduced 143B tumor growth and lung metastasis. 3-hydroxyflavone possesses the anti-metastatic activity of U2OS and 143B cells by affecting EMT and repressing u-PA/MMP-2 via FAK-Src to MEK/ERK and RhoA/MLC2 pathways and suppresses 143B tumor growth in vivo. This may lead to clinical trials of osteosarcoma chemotherapy to confirm the promising result in the future.

Bile acids destabilise HIF-1α and promote anti-tumour phenotypes in cancer cell models

BackgroundThe role of the microbiome has become synonymous with human health and disease. Bile acids, as essential components of the microbiome, have gained sustained credibility as potential modulators of cancer progression in several disease models. At physiological concentrations, bile acids appear to influence cancer phenotypes, although conflicting data surrounds their precise physiological mechanism of action. Previously, we demonstrated bile acids destabilised the HIF-1α subunit of the Hypoxic-Inducible Factor-1 (HIF-1) transcription factor. HIF-1 overexpression is an early biomarker of tumour metastasis and is associated with tumour resistance to conventional therapies, and poor prognosis in a range of different cancers.MethodsHere we investigated the effects of bile acids on the cancer growth and migratory potential of cell lines where HIF-1α is known to be active under hypoxic conditions. HIF-1α status was investigated in A-549 lung, DU-145 prostate and MCF-7 breast cancer cell lines exposed to bile acids (CDCA and DCA). Cell adhesion, invasion, migration was assessed in DU-145 cells while clonogenic growth was assessed in all cell lines.ResultsIntracellular HIF-1α was destabilised in the presence of bile acids in all cell lines tested. Bile acids were not cytotoxic but exhibited greatly reduced clonogenic potential in two out of three cell lines. In the migratory prostate cancer cell line DU-145, bile acids impaired cell adhesion, migration and invasion. CDCA and DCA destabilised HIF-1α in all cells and significantly suppressed key cancer progression associated phenotypes; clonogenic growth, invasion and migration in DU-145 cells.ConclusionsThese findings suggest previously unobserved roles for bile acids as physiologically relevant molecules targeting hypoxic tumour progression.

Role of Myeloma-Derived MIF in Myeloma Cell Adhesion to Bone Marrow and Chemotherapy Response.

Multiple myeloma (MM) remains an incurable cancer characterized by accumulation of malignant plasma cells in the bone marrow (BM). The mechanism underlying MM homing to BM is poorly elucidated. The clinical significance of migration inhibitory factor (MIF) expression was examined by analyzing six independent gene expression profile databases of primary MM cells using the Student's t test and Kaplan-Meier test. Enzyme-linked immunosorbent assay was used to examine MIF expression. In vivo bioluminescent imaging was used to determine MM cell localization and treatment efficacy in human MM xenograft mouse models, with three to four mice per group. MM cell attachment to BM stromal cells (BMSCs) was monitored by cell adhesion assay. MIF regulation of the expression of adhesion molecules was determined by chromatin immunoprecipitation (ChIP) assay. Statistical tests were two-sided. High levels of MIF were detected in MM BM (MIF level in BM plasma: healthy = 10.72 ± 5.788 ng/mL, n = 5; MM = 1811 ± 248.7 ng/mL, n = 10; P < .001) and associated with poor survival of patients (Kaplan-Meier test for MM OS: 87 MIF(high) patients, 86 MIF(low) patients, P = .02). Knocking down MIF impaired MM cell adhesion to BMSCs in vitro and led to formation of extramedullary tumors in SCID mice. MIF acted through surface receptor CXCR4 and adaptor COPS5 to regulate the expression of adhesion molecules ALCAM, ITGAV, and ITGB5 on MM cells. More importantly, MIF-deficient MM cells were sensitive to chemotherapy in vitro when cocultured with BMSCs and in vivo. MIF inhibitor 4-IPP sensitized MM cells to chemotherapy. MIF is an important player and a novel therapeutic target in MM. Inhibiting MIF activity will sensitize MM cells to chemotherapy.

Cystinosin deficiency causes podocyte damage and loss associated with increased cell motility

The involvement of the glomerulus in the pathogenesis of cystinosis, caused by loss-of-function mutations in cystinosin (CTNS, 17p13), is a matter of controversy. Although patients with cystinosis demonstrate glomerular lesions and high-molecular-weight proteinuria starting from an early age, a mouse model of cystinosis develops only signs of proximal tubular dysfunction. Here we studied podocyte damage in patients with cystinosis by analyzing urinary podocyte excretion and by in vitro studies of podocytes deficient in cystinosin. Urine from patients with cystinosis presented a significantly higher amount of podocytes compared with controls. In culture, cystinotic podocytes accumulated cystine compatible with cystinosin deficiency. The expression of podocyte specific genes CD2AP, podocalyxin, and synaptopodin and of the WT1 protein was evident in all cell lines. Conditionally immortalized podocyte lines of 2 patients with different CTNS mutations had altered cytoskeleton, impaired cell adhesion sites, and increased individual cell motility. Moreover, these cells showed enhanced phosphorylation of both Akt1 and Akt2 (isoforms of protein kinase B). Inhibition of Akt by a specific inhibitor (Akti inhibitor 1/2) resulted in normalization of the hypermotile phenotype. Thus, our study extends the list of genetic disorders causing podocyte damage and provides the evidence of altered cell signaling cascades resulting in impaired cell adhesion and enhanced cell motility in cystinosis.

αTAT1 downregulation induces mitotic catastrophe in HeLa and A549 cells.

α-Tubulin acetyltransferase 1 (αTAT1) controls reversible acetylation on Lys40 of α-tubulin and modulates multiple cellular functions. αTAT1 depletion induced morphological defects of touch receptor neurons in Caenorhabditis elegans and impaired cell adhesion and contact inhibition in mouse embryonic fibroblasts, however, no morphological or proliferation defects in human RPE-hTERT cells were found after αTAT1-specific siRNA treatment. Here, we compared the effect of three αTAT1-specific shRNAs on proliferation and morphology in two human cell lines, HeLa and A549. The more efficient two shRNAs induced mitotic catastrophe in both cell lines and the most efficient one also decreased F-actin and focal adhesions. Further analysis revealed that αTAT1 downregulation increased γ-H2AX, but not other DNA damage markers p-CHK1 and p-CHK2, along with marginal change in microtubule outgrowth speed and inter-kinetochore distance. Overexpression of αTAT1 could not precisely mimic the distribution and concentration of endogenous acetylated α-tubulin (Ac-Tu), although no overt phenotype change was observed, meanwhile, this could not completely prevent αTAT1 downregulation-induced deficiencies. We therefore conclude that efficient αTAT1 downregulation could impair actin architecture and induce mitotic catastrophe in HeLa and A549 cells through mechanisms partly independent of Ac-Tu.

MiR-9-5p, miR-675-5p and miR-138-5p Damages the Strontium and LRP5-Mediated Skeletal Cell Proliferation, Differentiation, and Adhesion.

This study was designed to evaluate the effects of strontium on the expression levels of microRNAs (miRNAs) and to explore their effects on skeletal cell proliferation, differentiation, adhesion, and apoptosis. The targets of these miRNAs were also studied. Molecular cloning, cell proliferation assay, cell apoptosis assay, quantitative real-time PCR, and luciferase reporter assay were used. Strontium altered the expression levels of miRNAs in vitro and in vivo. miR-9-5p, miR-675-5p, and miR-138-5p impaired skeletal cell proliferation, cell differentiation and cell adhesion. miR-9-5p and miR-675-5p induced MC3T3-E1 cell apoptosis more specifically than miR-138-5p. miR-9-5p, miR-675-5p, and miR-138-5p targeted glycogen synthase kinase 3 β (GSK3β), ATPase Aminophospholipid Transporter Class I Type 8A Member 2 (ATP8A2), and Eukaryotic Translation Initiation Factor 4E Binding Protein 1 (EIF4EBP1), respectively. Low-density lipoprotein receptor-related protein 5 (LRP5) played a positive role in skeletal development. miR-9-5p, miR-675-5p, and miR-138-5p damage strontium and LRP5-mediated skeletal cell proliferation, differentiation, and adhesion, and induce cell apoptosis by targeting GSK3β, ATP8A2, and EIF4EBP1, respectively.

ΔNp63α Transcriptionally Regulates the Expression of CTEN That Is Associated with Prostate Cell Adhesion.

p63 is a member of the p53 transcription factor family and a linchpin of epithelial development and homeostasis. p63 drives the expression of many target genes involved in cell survival, adhesion, migration and cancer. In this study, we identify C-terminal tensin-like (CTEN) molecule as a downstream target of ΔNp63α, the predominant p63 isoform expressed in epithelium. CTEN belongs to the tensin family and is mainly localized to focal adhesions, which mediate many biological events such as cell adhesion, migration, proliferation and gene expression. Our study demonstrate that ΔNp63 and CTEN are both highly expressed in normal prostate epithelial cells and are down-regulated in prostate cancer. In addition, reduced expression of CTEN and ΔNp63 is correlated with prostate cancer progression from primary tumors to metastatic lesions. Silencing of ΔNp63 leads to decreased mRNA and protein levels of CTEN. ΔNp63α induces transcriptional activity of the CTEN promoter and a 140-bp fragment upstream of the transcription initiation site is the minimal promoter region required for activation. A putative binding site for p63 is located between -61 and -36 within the CTEN promoter and mutations of the critical nucleotides in this region abolish ΔNp63α-induced promoter activity. The direct interaction of ΔNp63α with the CTEN promoter was demonstrated using a chromatin immunoprecipitation (ChIP) assay. Moreover, impaired cell adhesion caused by ΔNp63α depletion is rescued by over-expression of CTEN, suggesting that CTEN is a downstream effector of ΔNp63α-mediated cell adhesion. In summary, our findings demonstrate that ΔNp63α functions as a trans-activation factor of CTEN promoter and regulates cell adhesion through modulating CTEN. Our study further contributes to the potential regulatory mechanisms of CTEN in prostate cancer progression.

Knockdown of EHF inhibited the proliferation, invasion and tumorigenesis of ovarian cancer cells.

Ovarian cancer is the most lethal gynecologic malignancy worldwide. ETS homologous factor (EHF), a member of E26 transformation specific (ETS) transcription factors, has been reported overexpressed in ovarian cancer. However, the molecular mechanism underlying the biological function of EHF in ovarian cancer is still unclear. Here, we found that EHF was elevated in ovarian cancer tissues compared with non-tumorous tissues. Moreover, high EHF expression level was correlated with short survival time of patients with ovarian cancer. Knockdown of EHF in ovarian cancer cells, SKOV3 and OVCAR3, significantly inhibited cell proliferation and increased cells population in G1 phase. The proteins promoting cell cycles (Cyclin B1, Cyclin D1, and PCNA) were down-regulated and the protein negatively regulating cell cycle progression (P21) was up-regulated after EHF knockdown. Moreover, inhibition of EHF in ovarian cancer cells dramatically induced cell apoptosis, but impaired cell adhesion and cell invasion. Furthermore, phosphorylation levels of ERK and AKT were notably reduced in EHF knockdown cells. Finally, in vivo data showed that knockdown of EHF inhibited tumor growth in nude mice. Our data indicates that EHF could be a potential prognosis marker for ovarian cancer and work as an oncogene by targeting ERK and AKT signaling, which can serve as a new target for ovarian cancer treatment. © 2015 Wiley Periodicals, Inc.

Melatonin protects ADSCs from ROS and enhances their therapeutic potency in a rat model of myocardial infarction.

Myocardial infarction (MI) is a major cause of death and disability worldwide. In the last decade, mesenchymal stem cells (MSCs) based cell therapy has emerged as a promising therapeutic strategy. Although great advance have been made using MSCs to treat MI, the low viability of transplanted MSCs severely limits the efficiency of MSCs therapy. Here, we show evidence that ex vivo pre-treatment with melatonin, an endogenous hormone with newly found anti-oxidative activity, could improve survival and function of adipose tissue derived MSCs (ADSCs) in vitro as well as in vivo. ADSCs with 5 μM melatonin pre-treatment for 24 hrs showed increased expression of the antioxidant enzyme catalase and Cu/Zn superoxide dismutase (SOD-1), as well as pro-angiogenic and mitogenic factors like insulin-like growth factor 1, basic fibroblast growth factor, hepatocyte growth factor (HGF), epidermal growth factor. Furthermore, melatonin pre-treatment protected MSCs from reactive oxygen species (ROS) induced apoptosis both directly by promoting anti-apoptosis kinases like p-Akt as well as blocking caspase cascade, and indirectly by restoring the ROS impaired cell adhesion. Using a rat model of MI, we found that melatonin pre-treatment enhanced the viability of engrafted ADSCs, and promoted their therapeutic potency. Hopefully, our results may shed light on the design of more effective therapeutic strategies treating MI by MSCs in clinic.

P53 enables metabolic fitness and self-renewal of nephron progenitor cells

Contrary to its classic role in restraining cell proliferation, we demonstrate here a divergent function of p53 in the maintenance of self-renewal of the nephron progenitor pool in the embryonic mouse kidney. Nephron endowment is regulated by progenitor availability and differentiation potential. Conditional deletion of p53 in nephron progenitor cells (Six2Cre+;p53fl/fl) induces progressive depletion of Cited1+/Six2+ self-renewing progenitors and loss of cap mesenchyme (CM) integrity. The Six2(p53-null) CM is disorganized, with interspersed stromal cells and an absence of a distinct CM-epithelia and CM-stroma interface. Impaired cell adhesion and epithelialization are indicated by decreased E-cadherin and NCAM expression and by ineffective differentiation in response to Wnt induction. The Six2Cre+;p53fl/fl cap has 30% fewer Six2(GFP+) cells. Apoptotic index is unchanged, whereas proliferation index is significantly reduced in accordance with cell cycle analysis showing disproportionately fewer Six2Cre+;p53fl/fl cells in the S and G2/M phases compared with Six2Cre+;p53+/+ cells. Mutant kidneys are hypoplastic with fewer generations of nascent nephrons. A significant increase in mean arterial pressure is observed in early adulthood in both germline and conditional Six2(p53-null) mice, linking p53-mediated defects in kidney development to hypertension. RNA-Seq analyses of FACS-isolated wild-type and Six2(GFP+) CM cells revealed that the top downregulated genes in Six2Cre+;p53fl/fl CM belong to glucose metabolism and adhesion and/or migration pathways. Mutant cells exhibit a ∼50% decrease in ATP levels and a 30% decrease in levels of reactive oxygen species, indicating energy metabolism dysfunction. In summary, our data indicate a novel role for p53 in enabling the metabolic fitness and self-renewal of nephron progenitors.

A new class of orthosteric uPAR·uPA small-molecule antagonists are allosteric inhibitors of the uPAR·vitronectin interaction.

The urokinase receptor (uPAR) is a GPI-anchored cell surface receptor that is at the center of an intricate network of protein-protein interactions. Its immediate binding partners are the serine proteinase urokinase (uPA), and vitronectin (VTN), a component of the extracellular matrix. uPA and VTN bind at distinct sites on uPAR to promote extracellular matrix degradation and integrin signaling, respectively. Here, we report the discovery of a new class of pyrrolone small-molecule inhibitors of the tight ∼1 nM uPAR·uPA protein-protein interaction. These compounds were designed to bind to the uPA pocket on uPAR. The highest affinity compound, namely 7, displaced a fluorescently labeled α-helical peptide (AE147-FAM) with an inhibition constant Ki of 0.7 μM and inhibited the tight uPAR·uPAATF interaction with an IC50 of 18 μM. Biophysical studies with surface plasmon resonance showed that VTN binding is highly dependent on uPA. This cooperative binding was confirmed as 7, which binds at the uPAR·uPA interface, also inhibited the distal VTN·uPAR interaction. In cell culture, 7 blocked the uPAR·uPA interaction in uPAR-expressing human embryonic kidney (HEK-293) cells and impaired cell adhesion to VTN, a process that is mediated by integrins. As a result, 7 inhibited integrin signaling in MDA-MB-231 cancer cells as evidenced by a decrease in focal adhesion kinase (FAK) phosphorylation and Rac1 GTPase activation. Consistent with these results, 7 blocked breast MDA-MB-231 cancer cell invasion with IC50 values similar to those observed in ELISA and surface plasmon resonance competition studies. Explicit-solvent molecular dynamics simulations show that the cooperativity between uPA and VTN is attributed to stabilization of uPAR motion by uPA. In addition, free energy calculations revealed that uPA stabilizes the VTNSMB·uPAR interaction through more favorable electrostatics and entropy. Disruption of the uPAR·VTNSMB interaction by 7 is consistent with the cooperative binding to uPAR by uPA and VTN. Interestingly, the VTNSMB·uPAR interaction was less favorable in the VTNSMB·uPAR·7 complex suggesting potential cooperativity between 7 and VTN. Compound 7 provides an excellent starting point for the development of more potent derivatives to explore uPAR biology.

P53 Enables metabolic fitness and self-renewal of nephron progenitor cells.

Contrary to its classic role in restraining cell proliferation, we demonstrate here a divergent function of p53 in the maintenance of self-renewal of the nephron progenitor pool in the embryonic mouse kidney. Nephron endowment is regulated by progenitor availability and differentiation potential. Conditional deletion of p53 in nephron progenitor cells (Six2Cre(+);p53(fl/fl)) induces progressive depletion of Cited1(+)/Six2(+) self-renewing progenitors and loss of cap mesenchyme (CM) integrity. The Six2(p53-null) CM is disorganized, with interspersed stromal cells and an absence of a distinct CM-epithelia and CM-stroma interface. Impaired cell adhesion and epithelialization are indicated by decreased E-cadherin and NCAM expression and by ineffective differentiation in response to Wnt induction. The Six2Cre(+);p53(fl/fl) cap has 30% fewer Six2(GFP(+)) cells. Apoptotic index is unchanged, whereas proliferation index is significantly reduced in accordance with cell cycle analysis showing disproportionately fewer Six2Cre(+);p53(fl/fl) cells in the S and G2/M phases compared with Six2Cre(+);p53(+/+) cells. Mutant kidneys are hypoplastic with fewer generations of nascent nephrons. A significant increase in mean arterial pressure is observed in early adulthood in both germline and conditional Six2(p53-null) mice, linking p53-mediated defects in kidney development to hypertension. RNA-Seq analyses of FACS-isolated wild-type and Six2(GFP(+)) CM cells revealed that the top downregulated genes in Six2Cre(+);p53(fl/fl) CM belong to glucose metabolism and adhesion and/or migration pathways. Mutant cells exhibit a ∼ 50% decrease in ATP levels and a 30% decrease in levels of reactive oxygen species, indicating energy metabolism dysfunction. In summary, our data indicate a novel role for p53 in enabling the metabolic fitness and self-renewal of nephron progenitors.