Lipid raft interacting galectin 8 regulates primary ciliogenesis.

Primary cilium is a specialized sensory organelle that transmits environmental information into cells. Its length is tightly controlled by various mechanisms such as the frequency or the cargo size of the intraflagellar transport trains which deliver the building materials such as tubulin subunits essential for the growing cilia. Here, we show the sialoglycan interacting galectin 8 regulates the process of primary ciliogenesis. As the epithelia become polarized, there are more galectin 8 being apically secreted and these extracellular galectin 8 molecules apparently bind to a lipid raft enriched domain at the base of the primary cilia through interacting with lipid raft components, such as GD3 ganglioside and scaffold protein caveolin 1. Furthermore, the binding of galectin 8 at this critical region triggers rapid growth of primary cilia by perturbing the barrier function of the transition zone (TZ). Our study also demonstrates the functionality of this barrier depends on intact organization of lipid rafts at the cilia as genetically knockout of Cav1 and pharmacologically inhibition of lipid raft both phenocopy the effect of apical addition of recombinant galectin 8; that is, rapid elongation of primary cilia and redistribution of cilia proteins from TZ to the growing axoneme. Indeed, as cilia elongated, endogenous galectin 8, caveolin 1, and TZ component, TMEM231, also transited from the TZ to the growing axoneme. We also noted that the interaction between caveolin 1 and TMEM231 could be perturbed by exogenous galectin 8. Taken together, we proposed that galectin 8 promoted primary cilia elongation through impeding the barrier function of the TZ by interfering with the interaction between caveolin 1 and TMEM231.

Corneal Biomechanical Characteristics in Osteogenesis Imperfecta With Collagen Defect.

Purpose: To identify the characteristic corneal biomechanical properties of osteogenesis imperfecta (OI), and to compare the corneal biomechanical properties between OI and keratoconus. Methods: We included 46 eyes of 23 patients with OI, 188 eyes of 99 keratoconus patients, and 174 eyes of 92 normal controls to compare corneal biomechanical parameters between OI corneas, keratoconus, and normal controls by using Corneal Visualization Scheimpflug Technology (Corvis ST). Results: Patients with OI had significantly higher Corvis biomechanical index (CBI) (P < 0.001), higher tomographic and biomechanical index (TBI) (P = 0.040), lower Corvis Biomechanical Factor (CBiF) (P = 0.034), and lower stiffness parameter at first applanation (SP-A1) (P < 0.001) compared with normal controls. In contrast, OI group showed lower CBI (P < 0.001), lower TBI (P < 0.001), higher CBiF (P < 0.001), and higher SP-A1 (P = 0.020) than keratoconus group. Notably, the stress-strain index (SSI) was not significantly different between the OI and normal controls (P = 1.000), whereas keratoconus showed the lowest SSI compared with OI group (P = 0.025) and normal controls (P < 0.001). Conclusions: Although the corneal structures of OI patients are less stable and easier to deform as compared to those of the control group, there is no significant difference in material stiffness observed between the OI and normal controls. In contrast, the corneas of keratoconus showed not only lower structural stability and higher deformability but also lower material stiffness compared with those of OI cornea and normal controls. Translational Relevance: The biomechanical alterations are different between OI corneas and keratoconus.

Corvis Biomechanical Factor Facilitates the Detection of Primary Angle Closure Glaucoma.

Purpose: To characterize the corneal biomechanical properties of primary angle closure glaucoma (PACG) and to investigate the diagnostic performance of combining corneal biomechanical parameters and anterior segment parameters in detecting PACG. Methods: This retrospective cross-sectional study evaluated 79 and 81 eyes of normal controls and patients with PACG, respectively. Corvis Biomechanical Factor (CBiF) and anterior chamber volume (ACV) were measured using the Corvis ST and Pentacam, respectively. We performed multivariable logistic regression, adjusted for age, sex, central corneal thickness, intraocular pressure, and ACV to evaluate the effect of CBiF on PACG. The area under the receiver operating curve (AUC) was calculated to compare the diagnostic performance of ACV, CBiF, and ACV-CBiF combination for detecting PACG. Results: The median CBiF of the control and PACG groups was 6.61 (interquartile range [IQR], 6.39-6.88) and 6.20 (IQR, 5.93-6.48), respectively (P < 0.001). A lower CBiF, suggestive of decreased corneal biomechanical stability, increased the odds of PACG (odds ratio, 0.029; 95% confidence interval [CI], 0.003-0.266; P = 0.002) in the multivariable logistic regression model. The ACV-CBiF combination yielded the highest AUC (0.934; 95% CI, 0.882-0.968) compared with ACV alone (0.878; 95% CI, 0.823-0.928). The ACV-CBiF combination had significantly higher discriminatory ability than that of ACV alone (DeLong test, P = 0.004). Conclusions: Lower CBiF and ACV may act as independent predictors for PACG. Combining ACV and CBiF may enhance detection of PACG. Translational Relevance: The combination of corneal biomechanical parameters and anterior segment parameters enhances the detection of PACG.

Inhibition of Rho-associated protein kinase activity enhances oxidative phosphorylation to support corneal endothelial cell migration.

Corneal endothelial cell (CEC) dysfunction causes corneal edema and severe visual impairment that require transplantation to restore vision. To address the unmet need of organ shortage, descemetorhexis without endothelial keratoplasty has been specifically employed to treat early stage Fuchs endothelial corneal dystrophy, which is pathophysiologically related to oxidative stress and exhibits centrally located corneal guttae. After stripping off central Descemet's membrane, rho-associated protein kinase (ROCK) inhibitor has been found to facilitate CEC migration, an energy-demanding task, thereby achieving wound closure. However, the correlation between ROCK inhibition and the change in bioenergetic status of CECs remained to be elucidated. Through transcriptomic profiling, we found that the inhibition of ROCK activity by the selective inhibitor, ripasudil or Y27632, promoted enrichment of oxidative phosphorylation (OXPHOS) gene set in bovine CECs (BCECs). Functional analysis revealed that ripasudil, a clinically approved anti-glaucoma agent, enhanced mitochondrial respiration, increased spare respiratory capacity, and induced overexpression of electron transport chain components through upregulation of AMP-activated protein kinase (AMPK) pathway. Accelerated BCEC migration and in vitro wound healing by ripasudil were diminished by OXPHOS and AMPK inhibition, but not by glycolysis inhibition. Correspondingly, lamellipodial protrusion and actin assembly that were augmented by ripasudil became reduced with additional OXPHOS or AMPK inhibition. These results indicate that ROCK inhibition induces metabolic reprogramming toward OXPHOS to support migration of CECs.

Host Immune Response and Associated Clinical Features in a Primary Cytomegalovirus Eye Infection Model Using Anterior Chamber Inoculation.

Purpose: To investigate the pathogenesis of cytomegalovirus (CMV)-associated anterior segment infection in immunocompetent hosts and evaluate the effects of ganciclovir and glucocorticoid treatment in management of the disease. Methods: We used an inoculation model to reproduce CMV anterior segment infection in immunocompetent rats. Flow cytometry, cytokine analysis, histopathological sections, and quantitative polymerase chain reaction were performed to investigate the immune response after CMV infection. The effects of ganciclovir and glucocorticoid treatment were also assessed. Results: Anterior chamber inoculation of CMV in rats provoked characteristic pathological features of human CMV anterior segment infection. The innate and adaptive immunity sequentially developed in an anterior segment after inoculation, and the elevation of intraocular pressure (IOP) was highly associated with ocular infiltration and inflammation. Early ocular immune response reduced virus DNA in the anterior segment and alleviated viral lymphadenopathy. Early intervention with ganciclovir enhanced the release of cytokines associated with T response and facilitated recruitment of NKT and T cells in drainage lymph nodes. Glucocorticoid treatment, alone or combined with ganciclovir, decreased elevation of IOP but also impeded DNA clearance. Conclusions: The inoculation model reproduced characteristic pathological features of human CMV anterior segment infection. The use of glucocorticoid in current practice may hinder viral clearance, and ganciclovir therapy can assist cytokine expression to combat the virus.

Endocytosis of peroxiredoxin 1 links sterile inflammation to immunoparalysis in pediatric patients following cardiopulmonary bypass.

After cardiopulmonary bypass (CPB), the occurrence of systemic inflammatory response is often accompanied by a persistent compensatory anti-inflammatory response syndrome that can lead to a compromised immune competence termed immunoparalysis, rendering the patients susceptible to infections which is a leading complication following cardiac surgery. However, the underlying mechanisms of CPB-elicited immunoparalysis remain obscure. In this study we showed that peroxiredoxin 1 (Prdx1), a putative cytosolic antioxidant, was released immediately after CPB in a cohort of pediatric patients receiving congenital cardiac surgery. This increased Prdx1 was correlated to a reduced human leukocyte antigen-DR expression and an elevated interleukin-10 (IL-10) production, as well as a hypo-responsiveness of macrophages to endotoxin and a higher incidence of nosocomial infection. We demonstrated that substitution of Ser83 for Cys83 prevented Prdx1 from oligomerization and subsequent binding and internalization to macrophages. These effects mitigated Prdx1-induced IL-10 induction and endotoxin tolerance. Furthermore, after engagement with toll-like receptor (TLR) 4, clathrin-dependent endocytosis is crucial for Prdx1 to elicit IL-10 production in phagocytes. Congruently, inhibition of Prdx1/TLR4 endocytosis in phagocytes reversed the Prdx1/IL-10-mediated hypo-responsiveness to endotoxin. Our findings unveiled the possible mechanisms by which Prdx1 undertakes to cause immunoparalysis, and targeting endocytosis of Prdx1 could be a novel therapeutic approach for postoperative infections associated with CPB.

Effect of 3-Hydroxy-3-Methyl-Glutaryl-Coenzyme A Reductase Inhibitors on the Meibomian Gland Morphology in Patients with Dyslipidemia.

PURPOSE: Previous studies have suggested an association between dyslipidemia and meibomian gland dysfunction (MGD). The aim of this prospective, nonrandomized clinical study is to evaluate the possible association of dyslipidemia and its treatment with meibomian gland (MG) morphologic changes by standardized meibography. DESIGN: Prospective, nonrandomized clinical study. METHODS: Two groups of participants were enrolled: group 1, comprised of patients under regular 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase inhibitor (statin) treatment for dyslipidemia, and group 2, those with newly diagnosed dyslipidemia who were under lifestyle interventions. Meibography was performed at baseline and at both the 6- and 12-month visits and were graded by meiboscores. Participants underwent slit lamp examination for signs of changes in meibum quality and MG lid morphologic features. The Ocular Surface Disease Index questionnaire was given to measure subjective symptoms of ocular surface disease. Dry eye parameters including tear meniscus height, noninvasive first and average tear film break-up time, and Schirmer test results were also recorded. RESULTS: Ninety-eight participants completed this longitudinal study over 12 months. There were statistically significant changes in total meiboscores (P = .01) and upper eyelid meiboscores (P = .012), lid margin abnormality scores (P = .0059), and meibum quality (P = .0002) in the statin group during follow-up visits. Similar changes of upper eyelid meiboscores (P = .046) and meibum quality (P = .046) were noted in the nonstatin group. CONCLUSION: Meibomian gland atrophy and deterioration of meibum quality continued in the long term among participants with dyslipidemia even under statin usage.

Exploring a peptidomimetic approach of N-cadherin in modulating fibroblast growth factor receptor signaling for corneal endothelial regeneration.

Endothelial rejection and a critical shortage of corneal transplants present an unmet medical need in corneal regeneration research area. Although basic fibroblast growth factor (bFGF) is a potent mitogenic factor for corneal ex vivo expansion, it is also a morphogen eliciting unfavorable endothelial-mesenchymal transition (EnMT) of corneal endothelial cells. A pharmacological reagent that retains the beneficial proliferative effect while lacking the EnMT effect of bFGF would be of great potential in corneal regeneration. In present study, we demonstrated that bFGF not only activated the canonical fibroblast growth factor receptor 1 (FGFR1) tyrosine kinase pathway, but also further upregulated matrix metalloproteinase activity to cleave N-cadherin into N-terminus and C-terminus fragments, which activated the classical FGFR1 tyrosine kinase pathway and a cryptic ß-catenin pathway to affect corneal proliferation and EnMT, respectively. We generated the synthetic peptides resembling a critical motif in the ectodomain of N-cadherin and found these peptides enhanced downstream proliferative signaling of FGFR1 but without seemingly EnMT effect. The potential of these peptides can be demonstrated on both ex vivo cell culture and in vivo rat cryo-injury model. Our study indicated this peptidomimetic approach of N-cadherin can stimulate corneal regeneration and offer a promising therapeutic option to treat corneal endothelial dysfunction.

Targeting non-muscle myosin II promotes corneal endothelial migration through regulating lamellipodial dynamics.

Corneal endothelial cell (CEC) dysfunction causes corneal edema that may lead to blindness. In addition to corneal transplantation, simple descemetorhexis has been proposed to treat centrally located disease with adequate peripheral cell reserve, but promoting the centripetal migration of CECs is pivotal to this strategy. Here, we show that targeting non-muscle myosin II (NMII) activity by Y27632, a ROCK inhibitor, or blebbistatin, a selective NMII inhibitor, promotes directional migration of CECs and accelerates in vitro wound healing. The lamellipodial protrusion persistence is increased, and actin retrograde flow is decreased after NMII inhibition. Counteracting lamellipodial protrusion by actin-related protein 2/3 (ARP2/3) inhibitor abolishes this migration-promoting effect. Although both Y27632 and blebbistatin accelerate wound healing, cell junctional integrity and barrier function are better preserved after blebbistatin treatment, leading to more rapid corneal deturgescence in rabbit corneal endothelial wounding model. Our findings indicate that NMII is a promising therapeutic target in the treatment of CEC dysfunction. KEY MESSAGES: NMII inhibition promotes directional migration and wound healing of CECs in vitro. Lamellipodial protrusion persistence is increased after NMII inhibition. Selective NMII inhibitor preserves junctional integrity better than ROCK inhibitor. Selective NMII inhibitor accelerates corneal deturgescence after wounding in vivo.

Novel Interleukin-10 Gene Polymorphism Is Linked to Gestational Diabetes in Taiwanese Population.

Objective: The association of interleukin-10 (IL-10) polymorphism with diabetes and its complication was recently established, while there were few researches considering the potential role of IL-10 in gestational diabetes (GDM). This study aimed to investigate the association between IL-10 gene rs1800896 (-1082 A/G), rs1800871 (-819 T/C), rs1800872 (-592 A/C), and rs3021094 (3388 A/C) single nucleotide polymorphisms (SNPs) and GDM susceptibility. Methods: This study included 72 GDM patients and 100 healthy pregnant women. Direct sequencing of the products from polymerase chain reactions of the extracted genomic DNA from study subjects were conducted for analyzing IL-10 gene polymorphism and further genotype frequencies were compared. Plasma IL-10 concentration was measured by ELISA method. Results: The results revealed no significant difference in -592 A/C, -819 T/C, and -1082 A/G genotypes. Significantly increased prevalence of A allele (P = 0.028, OR = 1.69, 95% CI = 1.081-2.64) and A/A genotype (P = 0.031, OR = 2.881, 95% CI = 1.145-7.250) at a previously un-characterized rs3021094 SNP were discovered in the GDM group. Increased IL-10 levels and insulin resistance were also related to the genotype of rs3021094. The risk of GDM was increased when IL-10 level was over 6.5 pg/ml. Conclusion: Our study demonstrated that A allele and A/A genotype of rs3021094 SNP in IL-10 gene were linked to increased risk for GDM, IL-10 plasma level and insulin resistance, which could be potential targets for early screening and detection of GDM.

Galectin-8 regulates targeting of Gp135/podocalyxin and lumen formation at the apical surface of renal epithelial cells.

Establishment of apical-basal polarity, through correct targeting of polarity determinants to distinct domains of the plasma membrane, is a fundamental process for the development of functioning epithelial tubules. Here we report that galectin (Gal)-8 regulates apical-basal polarity of Madin-Darby canine kidney (MDCK) cells via apical targeting of 135-kDa glycoprotein (Gp135). Gal-8 interacts with newly synthesized Gp135 in a glycan-dependent manner. Gal-8 knockdown induces aberrant lumens at the lateral domain and mistargeting of Gp135 to this structure, thus disrupting the kidney epithelial polarity of MDCK cells, which organize lumens at the apical surface. The O-glycosylation deletion mutant of Gp135 phenocopies the effect of Gal-8 knockdown, which suggests that Gal-8 is the decoding machinery for the apical sorting signals of Gp135 residing at its O-glycosylation-rich region. Collectively, our results reveal a new role of Gal-8 in the development of luminal organs by regulating targeting of apical polarity protein Gp135.-Lim, H., Yu, C.-Y., Jou, T.-S. Galectin-8 regulates targeting of Gp135/podocalyxin and lumen formation at the apical surface of renal epithelial cells.

Early measurement of IL-10 predicts the outcomes of patients with acute respiratory distress syndrome receiving extracorporeal membrane oxygenation.

Patients diagnosed with acute respiratory distress syndrome are generally severely distressed and associated with high morbidity and mortality despite aggressive treatments such as extracorporeal membrane oxygenation (ECMO) support. To identify potential biomarker of predicting value for appropriate use of this intensive care resource, plasma interleukin-10 along with relevant inflammatory cytokines and immune cell populations were examined during the early and subsequent disease courses of 51 critically ill patients who received ECMO support. High interleukin-10 levels at the time of ECMO installation and during the first 6 hours after ECMO support of these patients stand as a promising biomarker associated with grave prognosis. The initial interleukin-10 level is correlated to other conventional risk evaluation scores as a predictive factor for survival, and furthermore, elevated interleukin-10 levels are also related to a delayed recovery of certain immune cell populations such as CD14+CD16+, CD14+TLR4+ monocytes, and T regulator cells. Genetically, high interleukin-10 is associated to two polymorphic nucleotides (-592 C and -819 C) at the interleukin-10 gene promoter area. Our finding provides prognostic and mechanistic information on the outcome of severely respiratory distressed patients, and potentially paves the strategy to develop new therapeutic modality based on the principles of precision medicine.

In Vitro and In Vivo Models to Study Corneal Endothelial-mesenchymal Transition.

Corneal endothelial cells (CECs) play a crucial role in maintaining corneal clarity through active pumping. A reduced CEC count may lead to corneal edema and diminished visual acuity. However, human CECs are prone to compromised proliferative potential. Furthermore, stimulation of cell growth is often complicated by gradual endothelial-mesenchymal transition (EnMT). Therefore, understanding the mechanism of EnMT is necessary for facilitating the regeneration of CECs with competent function. In this study, we prepared a primary culture of bovine CECs by peeling the CECs with Descemet's membrane from the corneal button and demonstrated that bovine CECs exhibited the EnMT process, including phenotypic change, nuclear translocation of ß-catenin, and EMT regulators snail and slug, in the in vitro culture. Furthermore, we used a rat corneal endothelium cryoinjury model to demonstrate the EnMT process in vivo. Collectively, the in vitro primary culture of bovine CECs and in vivo rat corneal endothelium cryoinjury models offers useful platforms for investigating the mechanism of EnMT.

Peroxiredoxin 1 induces inflammatory cytokine response and predicts outcome of cardiogenic shock patients necessitating extracorporeal membrane oxygenation: an observational cohort study and translational approach.

BACKGROUND: Extracellular peroxiredoxin 1 (Prdx1) has been implicated to play a pivotal role in regulating inflammation; however, its function in tissue hypoxia-induced inflammation, such as severe cardiogenic shock patients, has not yet been defined. Thus, the objective of this study was to test the hypothesis that Prdx1 possesses prognostic value and instigates systemic inflammatory response syndrome in cardiogenic shock patients undergoing extracorporeal membrane oxygenation (ECMO) support. METHODS: We documented the early time course evolution of circulatory Prdx1, hypoxic marker carbonic anhydrase IX, inflammatory cytokines including IL-6, IL-8, IL-10, MCP-1, TNF-α, IL-1ß, and danger signaling receptors (TLR4 and CD14) in a cohort of cardiogenic shock patients within 1 day after ECMO support. In vitro investigations employing cultured murine macrophage cell lines and human monocytes were applied to clarify the relationship between Prdx1 and inflammatory response. RESULTS: Prdx1 not only peaked earlier than all the other cytokines we studied during the initial course, but also predicted a worse outcome in patients who had higher initial Prdx1 plasma levels. The Prdx1 levels in patients positively correlated with hypoxic markers carbonic anhydrase IX and lactate, and inflammatory cytokines. In vitro study demonstrated that hypoxia/reoxygenation induced Prdx1 release from human monocytes and enhanced the responsiveness of the monocytes in Prdx1-induced cytokine secretions. Furthermore, functional inhibition by Prdx1 antibody implicated a crucial role of Prdx1 in hypoxia/reoxygenation-induced IL-6 secretion. CONCLUSIONS: Prdx1 release during the early phase of ECMO support in cardiogenic shock patients is associated with the development of systemic inflammatory response syndrome and poor clinical outcomes. Thus, circulating Prdx1 provides not only prognostic information but may be a promising target against ischemia/reperfusion injury.

Ras-activated RSK1 phosphorylates EBP50 to regulate its nuclear localization and promote cell proliferation.

Differential subcellular localization of EBP50 leads to its controversial role in cancer biology either as a tumor suppressor when it resides at the membrane periphery, or a tumor facilitator at the nucleus. However, the mechanism behind nuclear localization of EBP50 remains unclear. A RNA interference screening identified the downstream effector of the Ras-ERK cascade, RSK1, as the molecule unique for nuclear transport of EBP50. RSK1 binds to EBP50 and phosphorylates it at a conserved threonine residue at position 156 (T156) under the regulation of growth factor. Mutagenesis experiments confirmed the significance of T156 residue in nuclear localization of EBP50, cellular proliferation, and oncogenic transformation. Our study sheds light on a possible therapeutic strategy targeting at this aberrant nuclear expression of EBP50 without affecting the normal physiological function of EBP50 at other subcellular localization.

Inhibition of matrix metalloproteinase activity reverses corneal endothelial-mesenchymal transition.

Ex vivo culture or regeneration of corneal endothelial cells often is subjected to gradual endothelial-mesenchymal transition and loss of function. Here, we found that during ex vivo culture, bovine corneal endothelial cells underwent endothelial-mesenchymal transition and had an up-regulated expression and activity of matrix metalloproteinases. Inhibition of matrix metalloproteinase activity in confluent bovine corneal endothelial cells decreased the level of endothelial-mesenchymal transition regulators: snail and slug. The phosphorylation and degradation of the key Wnt signaling pathway modulator active ß-catenin also were accelerated with the broad-spectrum matrix metalloproteinase inhibitor Marimastat, which may result from decreased N-cadherin shedding and increased intact N-cadherin molecules on the cell membrane. Intracameral injection of Marimastat also suppressed basic fibroblast growth factor-induced endothelial-mesenchymal transition in a rat corneal endothelium cryo-injury model and significantly diminished the corneal edema. Our study indicated that inhibition of matrix metalloproteinase activity can reverse endothelial-mesenchymal transition and preserve the function of corneal endothelial cells both during ex vivo culture and in vivo. This may offer a potential therapeutic target in regenerative medicine for the treatment of corneal endothelial dysfunctions.

Inhibition of Rho-associated kinase relieves C5a-induced proteinuria in murine nephrotic syndrome.

Childhood nephrotic syndrome is mainly caused by minimal change disease which is named because only subtle ultrastructural alteration could be observed at electron microscopic level in the pathological kidney. Glomerular podocytes are presumed to be the target cells whose protein sieving capability is compromised by a yet unidentified permeability perturbing factor. In a cohort of children with non-hereditary idiopathic nephrotic syndrome, we found the complement fragment C5a was elevated in their sera during active disease. Administration of recombinant C5a induced profound proteinuria and minimal change nephrotic syndrome in mice. Purified glomerular endothelial cells, instead of podocytes, were demonstrated to be responsible for the proteinuric effect elicited by C5a. Further studies depicted a signaling pathway involving Rho/Rho-associated kinase/myosin activation leading to endothelial cell contraction and cell adhesion complex breakdown. Significantly, application of Rho-associated kinase inhibitor, Y27632, prevented the protein leaking effects observed in both C5a-treated purified endothelial cells and mice. Taken together, our study identifies a previously unknown mechanism underlying nephrotic syndrome and provides a new insight toward identifying Rho-associated kinase inhibition as an alternative therapeutic option for nephrotic syndrome.

Aberrant nuclear localization of EBP50 promotes colorectal carcinogenesis in xenotransplanted mice by modulating TCF-1 and ß-catenin interactions.

Dysregulation of canonical Wnt signaling is thought to play a role in colon carcinogenesis. ß-Catenin, a key mediator of the pathway, is stabilized upon Wnt activation and accumulates in the nucleus, where it can interact with the transcription factor T cell factor (TCF) to transactivate gene expression. Normal colonic epithelia express a truncated TCF-1 form, called dnTCF-1, that lacks the critical ß-catenin-binding domain and behaves as a transcriptional suppressor. How the cell maintains a balance between the two forms of TCF-1 is unclear. Here, we show that ERM-binding phosphoprotein 50 (EBP50) modulates the interaction between ß-catenin and TCF-1. We observed EBP50 localization to the nucleus of human colorectal carcinoma cell lines at low cell culture densities and human primary colorectal tumors that manifested a poor clinical outcome. In contrast, EBP50 was primarily membranous in confluent cell lines. Aberrantly located EBP50 stabilized conventional ß-catenin/TCF-1 complexes and connected ß-catenin to dnTCF-1 to form a ternary molecular complex that enhanced Wnt/ß-catenin signaling events, including the transcription of downstream oncogenes such as c-Myc and cyclin D1. Genome-wide analysis of the EBP50 occupancy pattern revealed consensus binding motifs bearing similarity to Wnt-responsive element. Conventional chromatin immunoprecipitation assays confirmed that EBP50 bound to genomic regions highly enriched with TCF/LEF binding motifs. Knockdown of EBP50 in human colorectal carcinoma cell lines compromised cell cycle progression, anchorage-independent growth, and tumorigenesis in nude mice. We therefore suggest that nuclear EBP50 facilitates colon tumorigenesis by modulating the interaction between ß-catenin and TCF-1.

Src-dependent phosphorylation of ROCK participates in regulation of focal adhesion dynamics.

When a cell migrates, the RhoA-ROCK-mediated contractile signal is suppressed in the leading edge to allow dynamic adhesions for protrusion. However, several studies have reported that RhoA is indeed active in the leading edge of a migrating cell during serum stimulation. Here, we present evidence that regulation of ROCKII phosphorylation at the Y722 site in peripheral focal contacts is crucial for controlling the turnover of the focal adhesion (FA) complex uncoupled from RhoA activation during serum-stimulated migration. However, this phosphorylation control is dispensable for migration when RhoA is downregulated in cells treated with platelet-derived growth factor (PDGF). We further present evidence that ROCKII is phosphorylated by Src in FAs and this phosphorylation event decreases RhoA binding activity of ROCKII. Lack of this regulatory control leads to sustained myosin-mediated contractility and FA elongation during lysophosphatidic acid (LPA) stimulation. Altogether, our data suggest that Src-dependent ROCKII phosphorylation provides a means of tuning contractility required for FAs dynamics when RhoA is active.

Podocalyxin EBP50 ezrin molecular complex enhances the metastatic potential of renal cell carcinoma through recruiting Rac1 guanine nucleotide exchange factor ARHGEF7.

Podocalyxin was initially identified in glomerular podocytes to critically maintain the structural and functional integrity of the glomerular ultrafiltrative apparatus. Lately, it has emerged as a malignant marker in tumors arising from a variety of tissue origins. By immunohistochemistry, we identified that 9.6% of renal cell carcinoma patients overexpress this protein. This subset of patients had significantly shorter disease-specific and overall survivals, and, importantly, we established podocalyxin overexpression as an independent prognostic factor for latent distant metastasis with multivariate analysis. Podocalyxin down-regulation by small interfering RNA led to defective migration in model renal tubular cells, which was corrected by re-expression of podocalyxin. The activity of the small GTPase Rac1, a well-characterized modulator of cell migration, was diminished by podocalyxin knock-down. Conversely, podocalyxin overexpression in human embryonic kidney cells up-regulated Rac1 activity, which depended on a complex formed by podocalyxin, ERM-binding phosphoprotein 50, ezrin, and ARHGEF7, a Rac1 activator. Therefore, podocalyxin can serve as a biomarker to identify renal cell carcinoma patients with higher metastatic potential for more aggressive intervention at earlier clinical stages.