Dual inhibition of cMET and EGFR by microRNA-338-5p suppresses metastasis of esophageal squamous cell carcinoma.

MicroRNAs, as a group of post-transcriptional regulators, regulate multiple pathological processes including metastasis during tumor development. Here, we demonstrated the metastasis-suppressive function of microRNA (miR)-338-5p in esophageal squamous cell carcinoma (ESCC). Overexpression of miR-338-5p had inhibitory effect on invasive ability of ESCC cells and extracellular matrix degradation, whereas silencing miR-338-5p had opposite effects. Mechanistically, miR-338-5p directly targeted the 3' untranslated regions of hepatocellular growth factor receptor cMet (cMET) and epidermal growth factor receptor (EGFR). As a result, miR-338-5p inhibited the downstream signaling cascades of cMET and EGFR and repressed cMET- and EGFR-mediated ESCC cell invasion. Re-expression of cMET or EGFR in miR-338-5p overexpressing ESCC cells was sufficient to derepress ESCC cell invasion both in vitro and in vivo. We further showed that such manipulation downregulated the expression and secretion of matrix metalloproteinases 2 and 9, which resulted in impaired extracellular matrix degradation and cell invasion. Most importantly, systemic delivery of miR-338-5p mimic significantly inhibited metastasis of ESCC cells in nude mice. Taken together, our results uncovered a previously unknown mechanism through which miR-338-5p suppresses ESCC invasion and metastasis by regulating cMET/EGFR-matrix metalloproteinase 2/9 axis and highlighted the potential significance of miR-338-5p-based therapy in treating patients with metastatic ESCC.

MicroRNA-338-5p reverses chemoresistance and inhibits invasion of esophageal squamous cell carcinoma cells by targeting Id-1.

5-Fluorouracil (5-FU) is a chemotherapeutic agent commonly used to treat esophageal squamous cell carcinoma (ESCC), but acquisition of chemoresistance frequently occurs and the underlying mechanisms are not fully understood. We found that microRNA (miR)-338-5p was underexpressed in ESCC cells with acquired 5-FU chemoresistance. Forced expression of miR-338-5p in these cells resulted in downregulation of Id-1, and restoration of both in vitro and in vivo sensitivity to 5-FU treatment. The effects were abolished by reexpression of Id-1. In contrast, miR-338-5p knockdown induced 5-FU resistance in chemosensitive esophageal cell lines, and knockdown of both miR-338-5p and Id-1 resensitized the cells to 5-FU. In addition, miR-338-5p had suppressive effects on migration and invasion of ESCC cells. Luciferase reporter assay confirmed a direct interaction between miR-338-5p and the 3'-UTR of Id-1. We also found that miR-338-5p was significantly downregulated in tumor tissue and serum samples of patients with ESCC. Notably, low serum miR-338-5p expression level was associated with poorer survival and poor response to 5-FU/cisplatin-based neoadjuvant chemoradiotherapy. In summary, we found that miR-338-5p can modulate 5-FU chemoresistance and inhibit invasion-related functions in ESCC by negatively regulating Id-1, and that serum miR-338-5p could be a novel noninvasive prognostic and predictive biomarker in ESCC.

M6PR- and EphB4-Rich Exosomes Secreted by Serglycin-Overexpressing Esophageal Cancer Cells Promote Cancer Progression.

Accumulating evidence shows that exosomes participate in cancer progression. However, the functions of cancer cell exosome-transmitted proteins are rarely studied. Previously, we reported that serglycin (SRGN) overexpression promotes invasion and metastasis of esophageal squamous cell carcinoma (ESCC) cells. Here, we investigated the paracrine effects of exosomes from SRGN-overexpressing ESCC cells (SRGN Exo) on ESCC cell invasion and tumor angiogenesis, and used mass spectrometry to identify exosomal proteins involved. Cation-dependent mannose-6-phosphate receptor (M6PR) and ephrin type-B receptor 4 (EphB4) were pronouncedly upregulated in SRGN Exo. Upregulated exosomal M6PR mediated the pro-angiogenic effects of SRGN Exo both in vitro and in vivo, while augmented exosomal EphB4 mediated the pro-invasive effect of SRGN Exo on ESCC cells in vitro. In addition, in vitro studies showed that manipulation of M6PR expression affected the viability and migration of ESCC cells. Both M6PR and EphB4 expression levels were positively correlated with that of SRGN in the serum of patients with ESCC. High level of serum M6PR was associated with poor overall survival rates. Taken together, this study presents the first proof that exosomal M6PR and EphB4 play essential roles in tumor angiogenesis and malignancy, and that serum M6PR is a novel prognostic marker for ESCC patients.

microRNA-122 as a regulator of mitochondrial metabolic gene network in hepatocellular carcinoma.

Tumorigenesis involves multistep genetic alterations. To elucidate the microRNA (miRNA)-gene interaction network in carcinogenesis, we examined their genome-wide expression profiles in 96 pairs of tumor/non-tumor tissues from hepatocellular carcinoma (HCC). Comprehensive analysis of the coordinate expression of miRNAs and mRNAs reveals that miR-122 is under-expressed in HCC and that increased expression of miR-122 seed-matched genes leads to a loss of mitochondrial metabolic function. Furthermore, the miR-122 secondary targets, which decrease in expression, are good prognostic markers for HCC. Transcriptome profiling data from additional 180 HCC and 40 liver cirrhotic patients in the same cohort were used to confirm the anti-correlation of miR-122 primary and secondary target gene sets. The HCC findings can be recapitulated in mouse liver by silencing miR-122 with antagomir treatment followed by gene-expression microarray analysis. In vitro miR-122 data further provided a direct link between induction of miR-122-controlled genes and impairment of mitochondrial metabolism. In conclusion, miR-122 regulates mitochondrial metabolism and its loss may be detrimental to sustaining critical liver function and contribute to morbidity and mortality of liver cancer patients.

Significance of serglycin and its binding partners in autocrine promotion of metastasis in esophageal cancer.

Rationale: Little is known about the roles of proteoglycans in esophageal cancer. This study aims to investigate the roles and mechanisms of serglycin (SRGN) proteoglycan in promoting metastasis of esophageal squamous cell carcinoma (ESCC). Methods: Reverse phase protein array analysis was used to identify activated signaling pathways in SRGN-overexpressing cells. Chemokine array was used to identify differentially secreted factors from SRGN-overexpressing cells. Binding between SRGN and potential interacting partners was evaluated using proximity ligation assay and co-immunoprecipitation. The glycosaminoglycan (GAG) chains of SRGN were characterized using fluorophore-assisted carbohydrate electrophoresis. Tissue microarray and serum samples were used to determine the correlation of SRGN expression with clinicopathological parameters and patient survival. Results: In vitro and in vivo experiments showed that SRGN promoted invasion and metastasis in ESCC via activating ERK pathway, stabilizing c-Myc and upregulating the secretion of matrix metalloproteinases. SRGN-knockdown suppressed tumorigenic hallmarks. These SRGN-elicited functions were carried out in an autocrine manner by inducing the secretion of midkine (MDK), which was further identified as a novel binding partner of SRGN for the formation of a SRGN/MDK/CD44 complex. In addition, SRGN interacted with MDK and matrix metalloproteinase 2 in ESCC via its GAG chains, which were mainly decorated with chondroitin sulfate comprising of ∆di-4S and ∆di-6S CS. Clinically, high expression of serum SRGN in serum of patients with ESCC was an independent prognostic marker for poor survival. Conclusions: This study provides the first evidence that elevated serum SRGN has prognostic significance in patients with ESCC, and sheds light on the molecular mechanism by which elevated circulating SRGN in cancer patients might promote cancer progression.

HNF1α and CDX2 transcriptional factors bind to cadherin-17 (CDH17) gene promoter and modulate its expression in hepatocellular carcinoma.

Cadherin-17 (CDH17) belongs to the cell adhesion cadherin family with a prominent role in tumorigenesis. It is highly expressed in human hepatocellular carcinoma (HCC) and is proposed to be a biomarker and therapeutic molecule for liver malignancy. The present study aims to identify the transcription factors which interact and regulate CDH17 promoter activity that might contribute to the up-regulation of CDH17 gene in human HCC. A 1-kb upstream sequence of CDH17 gene was cloned and the promoter activity was studied by luciferase reporter assay. By bioinformatics analysis, deletion and mutation assays, and chromatin immunoprecipitation studies, we identified hepatic nuclear factor 1α (HNF1α) and caudal-related homeobox 2 (CDX2) binding sites at the proximal promoter region which modulate the CDH17 promoter activities in two HCC cell lines (Hep3B and MHCC97L). A consistent down-regulation of CDH17 and the two transcriptional activators (HNF1α and CDX2) expression was found in the liver of mouse during development, as well as in human liver cancer cells with less metastatic potential. Suppression of HNF1α and CDX2 expression by small interfering RNA (siRNA) significantly down-regulated expressions of CDH17 and its downstream target cyclin D1 and the viability of HCC cells in vitro. In summary, we identified the minimal promoter region of CDH17 that is regulated by HNF1α and CDX2 transcriptional factors. The present findings enhance our understanding on the regulatory mechanisms of CDH17 oncogene in HCC, and may shed new insights into targeting CDH17 expression as potential therapeutic intervention for cancer treatment.

Enhanced detection of early hepatocellular carcinoma by serum SELDI-TOF proteomic signature combined with alpha-fetoprotein marker.

BACKGROUND: Biomarkers for accurate diagnosis of early hepatocellular carcinoma (HCC) are limited in number and clinical validation. We applied SELDI-TOF-MS ProteinChip technology to identify serum profile for distinguishing HCC and liver cirrhosis (LC) and to compare the accuracy of SELDI-TOF-MS profile and alpha-fetoprotein (AFP) level in HCC diagnosis. PATIENTS AND METHODS: Serum samples were obtained from 120 HCC and 120 LC patients for biomarker discovery and validation studies. ProteinChip technology was employed for generating SELDI-TOF proteomic features and analyzing serum proteins/peptides. RESULTS: A diagnostic model was established by CART algorithm, which is based on 5 proteomic peaks with m/z values at 3324, 3994, 4665, 4795, and 5152. In the training set, the CART algorithm could differentiate HCC from LC subjects with a sensitivity and specificity of 98% and 95%, respectively. The results were assessed in blind validation using separate cohorts of 60 HCC and 60 LC patients, with an accuracy of 83% for HCC and 92% for LC patients. The diagnostic odd ratio (DOR) indicated that SELDI-TOF proteomic signature could achieve better diagnostic performance than serum AFP level at a cutoff of 20 ng/mL (AFP(20)) (92.72 vs 9.11), particularly superior for early-stage HCC (87% vs 54%). Importantly, a combined use of both tests could enhance the detection of HCC (sensitivity, 95%; specificity, 98%; DOR, 931). CONCLUSION: Serum SELDI-TOF proteomic signature, alone or in combination with AFP marker, promises to be a good tool for early diagnosis and/screening of HCC in at-risk population with liver cirrhosis.

Testicular cell junction: a novel target for male contraception.

Even though various contraceptive methods are widely available, the number of unwanted pregnancies is still on the rise in developing countries, pressurizing the already resource limited nations. One of the major underlying reasons is the lack of effective, low cost, and safe contraceptives for couples. During the past decade, some studies were performed using animal models to decipher if the Sertoli-germ cell junction in the testis is a target for male fertility regulation. Some of these study models were based on the use of hormones and/or chemicals to disrupt the hypothalamic-pituitary-testicular axis (e.g., androgen-based implants or pills) and others utilized a panel of chemical entities or synthetic peptides to perturb spermatogenesis either reversibly or non-reversibly. Among them, adjudin, a potential male contraceptive, is one of the compounds exerting its action on the unique adherens junctions, known as ectoplasmic specializations, in the testis. Since the testis is equipped with inter-connected cell junctions, an initial targeting of one junction type may affect the others and these accumulative effects could lead to spermatogenic arrest. This review attempts to cover an innovative theme on how male infertility can be achieved by inducing junction instability and defects in the testis, opening a new window of research for male contraceptive development. While it will still take much time and effort of intensive investigation before a product can reach the consumable market, these findings have provided hope for better family planning involving men.

Predicting prognosis in hepatocellular carcinoma after curative surgery with common clinicopathologic parameters.

BACKGROUND: Surgical resection is one important curative treatment for hepatocellular carcinoma (HCC), but the prognosis following surgery differs substantially and such large variation is mainly unexplained. A review of the literature yields a number of clinicopathologic parameters associated with HCC prognosis. However, the results are not consistent due to lack of systemic approach to establish a prediction model incorporating all these parameters. METHODS: We conducted a retrospective analysis on the common clinicopathologic parameters from a cohort of 572 ethnic Chinese HCC patients who received curative surgery. The cases were randomly divided into training (n = 272) and validation (n = 300) sets. Each parameter was individually tested and the significant parameters were entered into a linear classifier for model building, and the prediction accuracy was assessed in the validation set RESULTS: Our findings based on the training set data reveal 6 common clinicopathologic parameters (tumor size, number of tumor nodules, tumor stage, venous infiltration status, and serum alpha-fetoprotein and total albumin levels) that were significantly associated with the overall HCC survival and disease-free survival (time to recurrence). We next built a linear classifier model by multivariate Cox regression to predict prognostic outcomes of HCC patients after curative surgery This analysis detected a considerable fraction of variance in HCC prognosis and the area under the ROC curve was about 70%. We further evaluated the model using two other protocols; leave-one-out procedure (n = 264) and independent validation (n = 300). Both were found to have excellent prediction power. The predicted score could separate patients into distinct groups with respect to survival (p-value = 1.8e-12) and disease free survival (p-value = 3.2e-7). CONCLUSION: This described model will provide valuable guidance on prognosis after curative surgery for HCC in clinical practice. The adaptive nature allows easy accommodation for future new biomarker inputs, and it may serve as the foundation for future modeling and prediction for HCC prognosis after surgical treatment.

Comparative proteomic analysis of mouse livers from embryo to adult reveals an association with progression of hepatocellular carcinoma.

To identify potential oncofetal biomarkers that distinguish hepatocellular carcinoma (HCC) from healthy liver tissues, we compared and analyzed the proteomic profiles of mouse livers at different developmental stages. Fetal (E13.5, E16.5), newborn (NB), postnatal (3-week) and adult (3-month) livers were isolated and profiled by 2-D PAGE. Statistical analysis using linear regression and false discovery rate (FDR) revealed that 361 protein spots showed significant changes. Unsupervised hierarchical tree analysis segregated the proteins into fetal, NB, and postnatal-adult clusters. Distinctive protein markers were identified by MALDI-TOF/MS and the corresponding mRNA profiles were further determined by Q-PCR. Fetal markers (hPCNA, hHSP7C, hHEM6) and postnatal-adult markers (hARGI1, hASSY, hBHMT, hFABPL) were selected for testing against a panel of seven human hepatocyte/HCC cell lines and 59 clinical specimens. The fetal proteins were found to be overexpressed in the metastatic HCC cell lines and the tumor tissues, whereas the postnatal-adult proteins were expressed in non-tumor tissues and normal hepatocytes. This "Ying-Yang" pattern, as orchestrated by distinct fetal and adult markers, is hypothesized to indicate the progressive change of the liver from a growing, less-differentiated organ into a functional metabolic center. Thus, embryogenesis and tumorigenesis share certain oncofetal markers and adult "hepatic" phenotypes are lost in HCC.

Characterization of an acrosome protein VAD1.2/AEP2 which is differentially expressed in spermatogenesis.

The release of enzymes from the acrosome of the sperm head (acrosome reaction) starts the fertilization process and enables the spermatozoa to penetrate the zona pellucida of the oocytes. Defective acrosome reaction is one of the important causes of infertility in men. To investigate the molecular regulation of spermatogenesis in vivo, we used differential display reverse transcription-polymerase chain reaction to identify stage-specific genes in a retinol-supplemented vitamin-A deficiency (VAD) rat model and identified the VAD1.2 (acrosome-expressed protein 2, AEP2) gene, which was expressed strongly in the rat testis from post-natal day 32 to adult stage. The mouse VAD1.2 mRNA shared 85% and 67% sequence homology, and 74% and 38% amino acid homology, respectively, with the rat and human counterparts. VAD1.2 transcript was abundantly expressed in the rat seminiferous tubules at stage VIII-XII, and the protein was detected in the acrosome region of the round and elongated spermatids of mouse, human, monkey and pig. VAD1.2 co-localized with lectin-PNA to the acrosome region of spermatids. Interestingly, the expression of VAD1.2 protein in human testis diminished in patients with hypospermatogenesis, maturation arrest, undescended testis and Sertoli cell-only syndrome. Co-immunoprecipitation experiments followed by western blotting and mass spectrometry (MS-MS) identified syntaxin 1, beta-actin and myosin heavy chain (MHC) proteins as putative interacting partners. Taken together, the stage-specific expression of VAD1.2 in the acrosome of spermatids and the binding of VAD1.2 protein with vesicle forming (syntaxin 1) and structural (beta-actin and MHC) proteins suggest that VAD1.2 maybe involved in acrosome formation during spermiogenesis.

Nitric oxide and cyclic nucleotides: their roles in junction dynamics and spermatogenesis.

Spermatogenesis is a highly complicated process in which functional spermatozoa (haploid, 1n) are generated from primitive mitotic spermatogonia (diploid, 2n). This process involves the differentiation and transformation of several types of germ cells as spermatocytes and spermatids undergo meiosis and differentiation. Due to its sophistication and complexity, testis possesses intrinsic mechanisms to modulate and regulate different stages of germ cell development under the intimate and indirect cooperation with Sertoli and Leydig cells, respectively. Furthermore, developing germ cells must translocate from the basal to the apical (adluminal) compartment of the seminiferous epithelium. Thus, extensive junction restructuring must occur to assist germ cell movement. Within the seminiferous tubules, three principal types of junctions are found namely anchoring junctions, tight junctions, and gap junctions. Other less studied junctions are desmosome-like junctions and hemidesmosome junctions. With these varieties of junction types, testes are using different regulators to monitor junction turnover. Among the uncountable junction modulators, nitric oxide (NO) is a prominent candidate due to its versatility and extensive downstream network. NO is synthesized by nitric oxide synthase (NOS). Three traditional NOS, specified as endothelial NOS (eNOS), inducible NOS (iNOS), and neuronal NOS (nNOS), and one testis-specific nNOS (TnNOS) are found in the testis. For these, eNOS and iNOS were recently shown to have putative junction regulation properties. More important, these two NOSs likely rely on the downstream soluble guanylyl cyclase/cGMP/protein kinase G signaling pathway to regulate the structural components at the tight junctions and adherens junctions in the testes. Apart from the involvement in junction regulation, NOS/NO also participates in controlling the levels of cytokines and hormones in the testes. On the other hand, NO is playing a unique role in modulating germ cell viability and development, and indirectly acting on some aspects of male infertility and testicular pathological conditions. Thus, NOS/NO bears an irreplaceable role in maintaining the homeostasis of the microenvironment in the seminiferous epithelium via its different downstream signaling pathways.

Junction interaction in the seminiferous epithelium: regulatory roles of connexin-based gap junction.

Anchoring junction, tight junction (TJ), and gap junction (GJ) constitute three major junction types in mammalian testes. Connexin is the well-studied GJ protein. It forms the building block of connexon, which is composed of six connexin units. Connexon forms the functional GJ when pairing with counter-connexon from neighboring cells. In the testis, at least eleven connexins are associated with the Sertoli and germ cells of the seminiferous epithelium and the Leydig cells of the interstitium, modulating spermatogenesis and steroidogenesis, respectively. Significantly, connexins are recently speculated to act as regulators of other junctions in the testes using pan-connexin peptide model. This demonstrates that the loss of connexin function leads to a preferential degradation of occludin-based TJ, but not N-cadherin-based adherens junction (AJ), in the testis, despite the intermingled relationship of these three junctions at the site of blood-testis barrier. In the clinical aspects, connexins are shown to relate to male infertility and testicular dysfunctions. A panel of molecules and proteins and their associated protein kinases are actively participating in the regulation of connexin-mediated GJ and fine-tuning connexin-associated functions in the testis. Herein, we summarize the latest findings of connexins in the testis in the aspects of fertility, and testicular diseases, with emphasis on the unexplored roles of connexins in regulating other junction types. This can shed light on future studies in implicating the putative roles of connexins in the physiological functions of reproduction and the clinical aspects of male infertility. In addition, understanding the roles of connexins can advance the diagnosis and treatment of testicular dysfunction and infertility.

Cellular localization of sphingomyelin synthase 2 in the seminiferous epithelium of adult rat testes.

Sphingomyelin synthase 2 (SMS2) is an enzyme that catalyzes the conversion of phosphatidylcholine and ceramide to sphingomyelin and diacylglycerol, and it is crucial to cellular lipid metabolism. Using the technique of subtraction hybridization, we have isolated a full-length cDNA encoding SMS2 from rat testes, which shared 93 and 87% identity at the nucleotide level with SMS2 in mice and humans respectively. A specific polyclonal antibody was prepared against a 20 amino acid peptide of NH(2)-FSWPLSWPPGCFKSSCKKYS-COOH near the C-terminus of SMS2. Studies by RT-PCR and immunoblotting have shown that the expression of SMS2 was limited to late round spermatids and elongating spermatids, but it was not detected in late elongate spermatids and Sertoli cells. Furthermore, SMS2 was shown to associate with the developing acrosome beginning in late round spermatid through elongating spermatids (but not late elongate spermatids) and the cell membrane in studies using fluorescent microscopy and immunohistochemistry. These data were further confirmed by studies using immunogold electron microscopy. The expression of SMS2 in the seminiferous epithelium is stage-specific with its highest expression detected in the acrosome region in late round spermatids from stages VIII-IX, and also in the acrosome in elongating spermatids with diminished intensity in stages X-V; however, it was not found in the acrosome in elongate spermatids in stages VI-VIII. Collectively, these results suggest that SMS2 may play a crucial role in the lipid metabolism in acrosome formation and the plasma membrane restructuring from late round spermatids to early elongating spermatids.

Cancer cell-secreted IGF2 instigates fibroblasts and bone marrow-derived vascular progenitor cells to promote cancer progression.

Local interactions between cancer cells and stroma can produce systemic effects on distant organs to govern cancer progression. Here we show that IGF2 secreted by inhibitor of differentiation (Id1)-overexpressing oesophageal cancer cells instigates VEGFR1-positive bone marrow cells in the tumour macroenvironment to form pre-metastatic niches at distant sites by increasing VEGF secretion from cancer-associated fibroblasts. Cancer cells are then attracted to the metastatic site via the CXCL5/CXCR2 axis. Bone marrow cells transplanted from nude mice bearing Id1-overexpressing oesophageal tumours enhance tumour growth and metastasis in recipient mice, whereas systemic administration of VEGFR1 antibody abrogates these effects. Mechanistically, IGF2 regulates VEGF in fibroblasts via miR-29c in a p53-dependent manner. Analysis of patient serum samples showed that concurrent elevation of IGF2 and VEGF levels may serve as a prognostic biomarker for oesophageal cancer. These findings suggest that the Id1/IGF2/VEGF/VEGFR1 cascade plays a critical role in tumour-driven pathophysiological processes underlying cancer progression.

Endoplasmic reticulum chaperone prolyl 4-hydroxylase, beta polypeptide (P4HB) promotes malignant phenotypes in glioma via MAPK signaling.

Endoplasmic reticulum (ER) chaperone Prolyl 4-hydroxylase, beta polypeptide (P4HB) has previously been identified as a novel target for chemoresistance in glioblastoma multiforme (GBM). Yet its functional roles in glioma carcinogenesis remain elusive. In clinical analysis using human glioma specimens and Gene Expression Omnibus (GEO) profiles, we found that aberrant expression of P4HB was correlated with high-grade malignancy and an angiogenic phenotype in glioma. Furthermore, P4HB upregulation conferred malignant characteristics including proliferation, invasion, migration and angiogenesis in vitro, and increased tumor growth in vivo via the mitogen-activated protein kinase (MAPK) signaling pathway. Pathway analysis suggested genetic and pharmacologic inhibition of P4HB suppressed MAPK expression and its downstream targets were involved in angiogenesis and invasion. This is the first study that demonstrates the oncogenic roles of P4HB and its underlying mechanism in glioma. Since tumor invasion and Vascularisation are typical hallmarks in malignant glioma, our findings uncover a promising anti-glioma mechanism through P4HB-mediated retardation of MAPK signal transduction.

Kidney claudin-19: localization in distal tubules and collecting ducts and dysregulation in polycystic renal disease.

Tight junction (TJ) constitutes the barrier by controlling the passage of ions and molecules via paracellular pathway and the movement of proteins and lipids between apical and basolateral domains of the plasma membrane. Claudins, occludin, and junctional adhesion molecules are the major three transmembrane proteins at TJ. This study focuses a newly identified mammalian TJ gene, claudin-19, in kidneys. Mouse claudin-19 composes of 224 amino acids and shares 98.2% and 95% amino acid homology with rat and human, respectively; the most evolutionary-related claudins are claudin-1 and -7, which share approximately 75% DNA sequence homology with claudin-19. Claudin-19 is abundantly expressed in the mouse and rat kidneys among the organs examined by Northern blots, and to a much less extent, also found in brain by RT-PCR. Claudin-19 and zonula occludens-1 (ZO-1) are localized at junctional regions of Madin-Darby canine kidney (MDCK) cells by immunofluorescent microscopy. In addition, ZO-1 is found in the claudin-19-associated protein complexes in MDCK cells by co-immunoprecipitation. Using aquaporin-1 and aquaporin-2 antibodies as markers for different renal segment, strong expression of claudin-19 was observed in distal tubules of the cortex as well as in the collecting ducts of the medulla. To less extent, claudin-19 is also present in the proximal tubules (cortex) and in the loop of Henle (medulla). Furthermore, intense claudin-19 immunoreactivity is found co-localized with the ZO-1 in kidneys from postnatal day 15, day 45, and adult rats and mice. Similar localizations of claudin-19 and ZO-1 are also observed in human kidneys. Since these renal segments are mainly for controlling the paracellular cation transport, it is suggested that claudin-19 may participate in these processes. In human polycystic kidneys, decreased expression and dyslocalization of claudin-19 are noticed, suggesting a possible correlation between claudin-19 and renal disorders. Taken together, claudin-19 is a claudin isoform that is highly and specifically expressed in renal tubules with a putative role in TJ homeostasis in renal physiology.

Increased solubility of integrin betaA domain using maltose-binding protein as a fusion tag.

In proteomics research, generation of recombinant proteins in their native, soluble form with large quantity is often a challenging task. To tackle the expression difficulties, different expression vectors with distinct affinity fusion tags, i.e. pET-43.1a (N-utilization substance A tag), pMAL-cRI (maltose binding protein tag) (MBP tag), pGEX-4T-2 (glutathione S-transferase tag), and pET-15b (hexahistidine tag) were compared for their effects on the productivity and solubility, which were assessed by SDS-PAGE and immunoblotting, of the integrin betaA domain. The incubation temperatures were tested for its effects on these parameters. Our data suggested that MBP tag enhanced the yield and solubility of the betaA domain protein, which can also be recognized using an anti-CD18 antibody, at room temperature incubation. Thus, the nature of fusion partner chosen for expression in bacteria and its incubation temperature would significantly affect the yield and solubility of the recombinant target protein.

Regulation of Sertoli-germ cell adherens junction dynamics via changes in protein-protein interactions of the N-cadherin-beta-catenin protein complex which are possibly mediated by c-Src and myotubularin-related protein 2: an in vivo study using an androgen suppression model.

Using a well characterized model of cell-cell actin-based adherens junction (AJ) disruption by suppressing the intratesticular testosterone level in adult rats with testosterone-estradiol implants, we have confirmed earlier findings that Sertoli-germ cell AJ dynamics are regulated by the activation of kinases via putative signaling pathways but with some unexpected findings as follows. First, the loss of germ cells from the seminiferous epithelium during androgen suppression was associated with a surge in myotubularin-related protein 2 (MTMR2, a lipid phosphatase, in which adult MTMR2-/- mice were recently shown to be azoospermic because of the loss of cell adhesion function between germ and Sertoli cells); kinases: phosphatidylinositol 3-kinase, c-Src, and C-terminal Src kinase; adaptors: alpha-actinin, vinculin, afadin, and p130 Crk-associated protein; and AJ-integral membrane proteins at the ectoplasmic specialization (ES, a testis-specific cell-cell actin-based AJ type) site: N-cadherin, beta-catenin, integrin beta1, and nectin 3. Second, MTMR2, instead of structurally interacting with phosphatidylinositol 3-kinase, a protein and lipid kinase, was shown to associate only with c-Src, a nonreceptor protein tyrosine kinase, as demonstrated by both coimmunoprecipitation and fluorescent microscopy at the site of apical ES, but none of the kinases, adaptors, and AJ-integral proteins that were examined. Collectively, these results suggest that the MTMR2/c-Src is an important phosphatase/kinase protein pair in AJ dynamics in the testis. Because c-Src is known to associate with the cadherin/catenin protein complex at the ES in the testis, we next sought to investigate any changes in the protein-protein interactions of this protein complex during androgen suppression-induced germ cell loss. Indeed, there was a loss of N-cadherin and beta-catenin association, accompanied by a surge in Tyr phosphorylation of beta-catenin, during germ cell loss from the epithelium. Third, and perhaps the most important of all, during natural recovery of the epithelium after removal of testosterone-estradiol implants when spermatids were reattaching to Sertoli cells, an increase in N-cadherin and beta-catenin association was detected with a concomitant loss in the increased Tyr phosphorylation in beta-catenin. In summary, these results illustrate that the cadherin/catenin is a crucial cell adhesion complex that regulates AJ dynamics in the testis, and its functionality is likely modulated by the MTMR2/c-Src protein complex.

Regulation of ectoplasmic specialization dynamics in the seminiferous epithelium by focal adhesion-associated proteins in testosterone-suppressed rat testes.

Apical ectoplasmic specialization (ES) is a unique testis-specific cell-cell actin-based adherens junction type restricted to the Sertoli-round/elongating/elongate spermatid interface in the seminiferous epithelium. An endogenous testosterone (T) suppression model was used to study the regulation of apical ES dynamics in the testis. By providing sustained releases of T and estradiol using subdermal implants in rats, this treatment reduced endogenous testicular T level. This in turn led to sloughing of spermatids (step 8 and beyond) from the seminiferous epithelium, which can be reversed by removing the implants, or replacing them with a higher dose of T implants. This model thus allows us to study the restructuring events at the apical ES. It was shown that apical ES restructuring involved proteins that were usually restricted to the cell-matrix focal adhesion site in other epithelia. For instance, the protein levels of beta1-integrin, Tyr-phosphorylated focal adhesion kinase (p-FAK), and c-Src were induced during the T suppression-induced germ cell loss and recovery, implicating that these proteins are putative regulators of ES dynamics. Indeed, the formation of p-FAK/c-Src protein complex, but not their association with beta1-integrin, was stimulated during T suppression-induced germ cell loss. ERK, a MAPK known to regulate focal adhesion turnover, was also activated during the androgen suppression-induced spermatid loss and the early phase of the recovery when germ cells began to repopulate the epithelium. Collectively, these data suggest that the apical ES is a cell-cell adherens junction type with the characteristics of cell-matrix focal contacts. In addition to its role in conferring cell adhesion formation, the p-FAK/c-Src protein complex apparently also regulates apical ES disruption via the ERK signaling pathway.