Versican modulates gap junction intercellular communication

Versican is a large chondroitin sulfate proteoglycan and belongs to the family of lecticans. Versican possesses two globular domains, G1 and G3 domain, separated by a CS-attachment region. The CS-attachment region present in the middle region is divided into two spliced domains named CSalpha and beta. Alternative splicing of versican generates at least four versican isoforms named V0, V1, V2, and V3. We have successfully cloned the full-length cDNA of chick versican isoforms V1 and V2 and found that versican isoform V1 induced mesenchymal-epithelial transition in NIH3T3 cells. Mesenchymal-epithelial transition induced by V1 in NIH3T3 cells is characterized by expression of E-cadherin and occludin, two epithelial markers, and reduced expression of fibroblastic marker vimentin (Sheng et al., 2006, Mol Biol Cell. 17, 2009-2020). In the present studies, we found that versican V1 isoform not only induced cell transition, but also increased intercellular communication via gap junction channels composed of connexin proteins. Our results showed that V1 induces plasma membrane localization of connexin 43, resulting in increased cell communication. This was further confirmed by blocking assays. Gap junctions mediated the transfer of small cytoplasmic molecules and the diffusion of second messenger molecules between adjacent cells. The ability of versican in regulating gap junction implied a potential role of versican in coordinating functions.     

Bone morphogenic protein-7 induces mesenchymal to epithelial transition in adult renal fibroblasts and facilitates regeneration of injured kidney

In the kidney, a unique plasticity exists between epithelial and mesenchymal cells. During kidney development, the metanephric mesenchyme contributes to emerging epithelium of the nephron via mesenchymal to epithelial transition (MET). In the injured adult kidney, renal epithelia contribute to the generation of fibroblasts via epithelial-mesenchymal transition, facilitating renal fibrosis. Recombinant human bone morphogenic protein (BMP)-7, a morphogen that is essential for the conversion of epithelia from condensing mesenchyme during kidney development, enhances the repair of tubular structures in the kidney. In this setting, BMP-7 inhibits epithelial-mesenchymal transition involving adult renal epithelial tubular cells and decreases secretion of type I collagen by adult renal fibroblasts. In search of a mechanism behind the ability of BMP-7 to repair damaged renal tubules, we hypothesized that systemic treatment with BMP-7 might induce MET involving adult renal fibroblasts in the injured kidney, generating functional epithelial cells. Here we report that BMP-7 induces formation of epithelial cell aggregates in adult renal fibroblasts associated with reacquisition of E-cadherin expression and decreased motility, mimicking the effect of BMP-7 on embryonic metanephric mesenchyme to generate epithelium. In addition, we provide evidence that BMP-7-mediated repair of renal injury is associated with MET involving adult renal interstitial fibroblasts in mouse models for renal fibrosis. Collectively, these findings suggest that adult renal fibroblasts might retain parts of their original embryonic imprint and plasticity, which can be re-engaged by systemic administration of BMP-7 to mediate repair of tubular injury in a fibrotic kidney.     

Versican 3′-untranslated region (3′UTR) promotes dermal wound repair and fibroblast migration by regulating miRNA activity

Versican is an extracellular chondroitin sulfate proteoglycan which functions as a structural molecule but can also regulate a variety of cellular activities. This study was designed to explore the roles of versican in the process of dermal wound repair. To elevate levels of versican, we ectopically expressed the versican 3′-untranslated region (3′UTR) as a competitive endogenous RNA to modulate expression of versican. We demonstrated that wounds closed faster in transgenic mice expressing the versican 3′UTR, as compared to those in wildtype mice. We stably expressed versican 3′UTR in NIH3T3 fibroblasts and found that the 3′UTR-transfected cells showed increased migratory capacity relative to vector-transfected cells. Interestingly, we found that the 3′UTRs of versican and β-catenin shared common microRNAs (miRNAs) including miR-185, miR-203*, miR-690, miR-680, and miR-434-3p. Luciferase assays showed that all of these miRNAs could target the 3′UTRs of both versican and β-catenin, when the luciferase constructs contained fragments harboring the miRNA binding sites. As a consequence, expression of both versican and β-catenin was up-regulated, which was confirmed in vitro and in vivo. Transfection with small interfering RNAs (siRNAs) targeting the versican 3′UTR abolished the 3′UTR's effects on cell migration and invasion. Taken together, these results demonstrate that versican plays important roles in wound repair and that versican messenger RNAs (mRNAs) could compete with endogenous RNAs for regulating miRNA functions.     

Interleukin-11 promotes the progress of gastric carcinoma via abnormally expressed versican

Versican, a ubiquitous component of the extracellular matrix (ECM), accumulates both in tumor stroma and cancer cells and is highly regulated by various cytokines. The aberrant expression of versican and its isoforms is known to modulate cell proliferation, differentiation, and migration, all of which are features of the invasion and metastasis of cancer; versican is also known to favour the homeostasis of the ECM. Interleukin-11 (IL-11) is an important cytokine that exhibits a wide variety of biological effects in gastric cancer development. Here, we analysed the expression of versican isoforms and found that the major isoforms expressed by both gastric carcinoma tissue and gastric cell lines were V0 and V1, and V1 was significantly higher in gastric carcinoma tissue. The treatment of the gastric cell lines AGS and MKN45 with rhIL-11 resulted in a significant increase in the expression of V0 and V1. Exogenous IL-11 increased migration in AGS and MKN45 cells, whereas these effects were reversed when the expression of V0 and V1 were abolished by siRNA targeting versican V0/V1. Collectively, these findings suggest that the abnormally expressed versican and its isoforms participate, at least in part, in the progress of gastric carcinoma triggered by IL-11.     

IQ-domain GTPase-activating protein 1 regulates beta-catenin at membrane ruffles and its role in macropinocytosis of N-cadherin and adenomatous polyposis coli

Beta-catenin is an integral component of E-cadherin dependent cell-cell junctions. Here we show that beta-catenin co-localizes with IQ-domain GTPase-activating protein 1 (IQGAP1), adenomatous polyposis coli (APC), and N-cadherin at actin-positive membrane ruffles in NIH 3T3 fibroblasts. We used deletion mapping to identify the membrane ruffle-targeting region of beta-catenin, localizing it to amino acids 47-217, which overlap the IQGAP1 binding site. Knockdown by small interference RNA (siRNA) revealed IQGAP1-dependent membrane targeting of beta-catenin, APC, and N-cadherin. Transient overexpression of IQGAP1 or N-cadherin increased beta-catenin at membrane ruffles. IQGAP1/APC regulates cell migration, and using a wound healing assay we demonstrate that siRNA-mediated loss of beta-catenin also caused a modest reduction in the rate of cell migration. More significantly, we discovered that beta-catenin is internalized by Arf6-dependent macropinocytosis near sites of membrane ruffling. The beta-catenin macropinosomes co-stained for APC, N-cadherin, and to a lesser extent IQGAP1, and internalization of each binding partner was abrogated by siRNA-dependent knockdown of beta-catenin. In addition, beta-catenin macropinosomes co-localized with the lysosomal marker, lysosome associated membrane protein 1, consistent with their recycling by the late endosomal machinery. Our findings expand on current knowledge of beta-catenin function. We propose that in motile cells beta-catenin is recruited by IQGAP1 and N-cadherin to active membrane ruffles, wherein beta-catenin mediates the internalization and possible recycling of the membrane-associated proteins N-cadherin and APC.     

In vitro mesenchymal-epithelial transition in NIH3T3 fibroblasts results in onset of low-dose radiation hypersensitivity coupled with attenuated connexin-43 response

Background

Mesenchymal-to-epithelial transition (MET) is associated with altered cell adhesion patterns. Independent studies showed that cellular adhesion regulates low-dose hyper-radiosensitivity (HRS), a phenomenon reported widely in tumour cells. Therefore, present study aimed to investigate whether MET and associated cellular adhesion alterations affect cellular radiosensitivity.

MicroRNA-506-3p initiates mesenchymal-to-epithelial transition and suppresses autophagy in osteosarcoma cells by directly targeting SPHK1

Osteosarcoma (OS) is the most common malignant bone tumor. In cancer cells, autophagy is related to epithelial-to-mesenchymal transition (EMT). Although microRNA (miR)-506-3p has been demonstrated to act as a tumor suppressor in OS, its role in regulating the EMT process and autophagy remains unknown. The results showed that miR-506-3p directly inhibited the expression of sphingosine kinase 1 (SPHK1) in 143B and SaOS-2 cells. The invasive capability of OS cells was reduced following miR-506-3p mimics transfection, and restored when SPHK1 was overexpressed simultaneously. Further, miR-506-3p mimics initiated mesenchymal-to-epithelial transition (MET) – E-cadherin expression was upregulated, whilst vimentin and fibronectin were downregulated. The basal autophagy flux (LC3II/I) was suppressed by miR-506-3p mimics. The alterations induced by miR-506-3p mimics were partly reversed by SPHK1 overexpression or treatment of rapamycin. Meanwhile, treatment of SPHK1-transfected cells with 3-methyladenine inhibited EMT. The data suggest that miR-506-3p initiates MET and suppresses autophagy in OS cells by targeting SPHK1.     

STAT1 activation regulates proliferation and differentiation of renal progenitors

We have shown previously that activation of STAT1 contributes to the pathogenesis of Wilms tumor. This neoplasm caricatures metanephric development and is believed to originate from embryonic renal mesenchymal progenitors that lose their ability to undergo mesenchymal–epithelial transition (MET). Therefore, we hypothesized that STAT1 is also activated and functional during metanephric development. Here we have demonstrated that both STAT1 and STAT3 are activated during normal development of the embryonic kidney. Furthermore, activation of STAT1 stimulated the proliferation of metanephric mesenchymal cells, but it prevented MET and tubulogenesis induced by leukemia inhibitory factor, which preferentially activates STAT3. Consistent with its negative regulation of metanephric mesenchymal differentiation, inhibition of STAT1 activation with protein kinase CK2 inhibitor TBB or RNAi-mediated knockdown of STAT1 promoted differentiation of metanephric progenitors and abolished the effect of cytokine-induced STAT1 activation in these cells. Additionally, a cell-permeable peptide that inhibits STAT1-mediated transactivation by targeting the STAT1 N-domain also blocked cytokine-induced STAT1-dependent proliferation in metanephric progenitors and promoted LIF-induced MET and tubulogenesis. Finally, the STAT1 peptide inhibitor caused the down regulation of survival/anti-apoptotic factors, Mcl-1 and Hsp-27, and induced apoptosis in renal tumor cells with constitutively active STAT1, indicating that STAT1 is required for these cells to survive. These findings show that both metanephric progenitors and renal tumor cells utilize a STAT1-dependent mechanism for growth or survival.     

沉默PPP3CA对后肾间充质细胞MET转化、凋亡、增殖及迁移的影响

肾脏是人体重要器官,肾脏发育对肾脏的形成和功能至关重要,其中后肾间充质细胞 (Metanephric mesenchyme,MM) 间质-上皮转化 (Mesenchymal-epithelial transition,MET) 是肾单位形成的关键环节。qRT-PCR和Western blotting实验检测蛋白质磷酸酶3催化亚基α (Protein phosphatase 3 catalytic subunit alpha,PPP3CA) 在不同状态MM细胞株mK3、mK4中的表达谱及对MET标志蛋白调控作用;采用慢病毒包装方式构建稳定敲低PPP3CA的mK4细胞株;采用CCK-8、EdU实验、细胞划痕实验、流式细胞技术分别检测PPP3CA对上皮样后肾间充质细胞株mK4细胞生长、迁移、凋亡的调控作用。PPP3CA在mK4细胞中表达量较间质样后肾间充质细胞mK3更高,敲低PPP3CA后,检测MET标志物及细胞生物学行为,结果显示敲低PPP3CA显著上调上皮细胞标志物E-cadherin表达,促进MET过程,且促进细胞凋亡,抑制细胞增殖和迁移。此外,敲低PPP3CA促进ERK1/2磷酸化,提示PPP3CA生物学功能的调控机制可能与其去磷酸化ERK1/2蛋白相关。以上结果提示PPP3CA在MM细胞MET转化和生物学行为调节中发挥重要功能,为发现和解析肾发育过程中潜在的关键调节因子提供了新的理论基础。     

The Novel Catenin p120Binds Classical Cadherins and Induces an Unusual Morphological Phenotype in NIH3T3 Fibroblasts

p120^cas(CAS) is a tyrosine kinase substrate whose phosphorylation has been implicated in cell transformation by Src and in ligand-induced signaling through the EGF, PDGF, and CSF-1 receptors. More recently, CAS has been shown to associate with E-cadherin and its cofactors (catenins), molecules that are involved in cell adhesion. Although both CAS and β-catenin contain armadillo repeat domains (Arm domains), the amino acid identity between these proteins in this region is only 22%, and it is not yet clear whether CAS will emulate other catenins by associating with other members of the cadherin family. Here we report that in addition to binding E-cadherin, wild-type CAS associated with N-cadherin and P-cadherin. Transient transfection of cloned CAS isoforms into MDCK epithelial cells indicated that CAS1 and CAS2 isoforms are equally capable of binding to E-cadherin even though these cells preferentially express CAS2 isoforms. In addition, CAS colocalized with N-cadherin in NIH3T3 cells and analysis of CAS mutantsin vivoindicated that the CAS–N-cadherin interaction requires an intact CAS Arm domain. The data suggest that CAS–cadherin interactions in general are dictated by the conserved armadillo repeats and are not heavily influenced by sequences added outside the Arm domain by alternative splicing. Interestingly, overexpression of CAS in NIH3T3 cells induced a striking morphological phenotype characterized by the presence of long dendrite-like processes. This branching phenotype was specific for CAS, since (i) overexpression of the stucturally similar β-catenin had little effect on cell morphology, and (ii) the branching was abolished by deletions in the CAS Arm domain. Our data indicate that, like other catenins, CAS is a cofactor for multiple members of the cadherin family. However, the dramatically distinct phenotype exhibited by fibroblasts overexpressing CAS, versus β-catenin, support recent data suggesting that these catenins have fundamentally different and possibly opposing roles in cadherin complexes.     

The roles of versican V1 and V2 isoforms in cell proliferation and apoptosis

Versican is a large chondroitin sulfate proteoglycan belonging to the lectican family. Alternative splicing of versican generates at least four isoforms named V0, V1, V2, and V3. We have shown that the versican V1 isoform not only enhanced cell proliferation, but also modulated cell cycle progression and protected the cells from apoptosis. Futhermore, the V1 isoform was able to not only activate proto-oncogene EGFR expression and modulate its downstream signaling pathway, but also induce p27 degradation and enhance CDK2 kinase activity. As well, the V1 isoform down-regulated the expression of the proapoptotic protein Bad. By contrast, the V2 isoform exhibited opposite biological activities by inhibiting cell proliferation and down-regulated the expression of EGFR and cyclin A. Furthermore, V2 did not contribute apoptotic resistance to the cells. In light of these results, we are reporting opposite functions for the two versican isoforms whose expression is differentially regulated. Our studies suggest that the roles of these two isoforms are associated with the subdomains CSbeta and CSalpha, respectively. These results were confirmed by silencing the expression of versican V1 with small interfering RNA (siRNA), which abolished V1-enhanced cell proliferation and V1-induced reduction of apoptosis.     

BMP-2 Induces Versican and Hyaluronan That Contribute to Post-EMT AV Cushion Cell Migration

Distal outgrowth and maturation of mesenchymalized endocardial cushions are critical morphogenetic events during post-EMT atrioventricular (AV) valvuloseptal morphogenesis. We explored the role of BMP-2 in the regulation of valvulogenic extracellular matrix (ECM) components, versican and hyaluronan (HA), and cell migration during post-EMT AV cushion distal outgrowth/expansion. We observed intense staining of versican and HA in AV cushion mesenchyme from the early cushion expansion stage, Hamburger and Hamilton (HH) stage-17 to the cushion maturation stage, HH stage-29 in the chick. Based on this expression pattern we examined the role of BMP-2 in regulating versican and HA using 3D AV cushion mesenchymal cell (CMC) aggregate cultures on hydrated collagen gels. BMP-2 induced versican expression and HA deposition as well as mRNA expression of versican and Has2 by CMCs in a dose dependent manner. Noggin, an antagonist of BMP, abolished BMP-2-induced versican and HA as well as mRNA expression of versican and Has2. We further examined whether BMP-2-promoted cell migration was associated with expression of versican and HA. BMP-2- promoted cell migration was significantly impaired by treatments with versican siRNA and HA oligomer. In conclusion, we provide evidence that BMP-2 induces expression of versican and HA by AV CMCs and that these ECM components contribute to BMP-2-induced CMC migration, indicating critical roles for BMP-2 in distal outgrowth/expansion of mesenchymalized AV cushions.     

The effects of modifying RhoA and Rac1 activities on heterotypic contact inhibition of locomotion

Contact inhibition of locomotion (CIL) occurs when a cell ceases moving in the same direction following contact with another cell. Homotypic and heterotypic CIL occur between cells of the same and different types, respectively. Using Abercrombie's confronted explants assay we studied the effect of changing Rac1 or RhoA activities on heterotypic CIL between NIH3T3 and chicken heart fibroblasts. Both dominant active (L61) and dominant negative (N17) Rac1 expressed in NIH3T3 cells resulted in loss of heterotypic CIL. N17Rac1 expression caused RhoA activation. Increasing RhoA activity directly (V14RhoA) or indirectly (downregulation of N-cadherin or p120-catenin) also resulted in loss of CIL. High RhoA activity has been associated with tumour invasion and our results are consistent with loss of heterotypic CIL playing a role.     

SALL4 as an Epithelial-Mesenchymal Transition and Drug Resistance Inducer through the Regulation of c-Myc in Endometrial Cancer

SALL4 plays important roles in the development and progression of many cancers. However, the role and molecular mechanism of SALL4 in endometrial cancer remain elusive. In the present research, we have demonstrated that the expression of SALL4 was upregulated in endometrial cancer and correlated positively with tumor stage, metastases and poor survival of patients. The overexpression of SALL4 promoted the invasiveness in endometrial cancer cells, as indicated by the upregulation of mesenchymal cell marker N-cadherin and downregulation of the epithelial marker E-cadherin, and invasion assays in vitro. Additionally, there was also an increase in drug resistance in these cell models due to the upregulation of ATP-binding cassette multidrug transporter ABCB1 expression. Moreover, we also found that ABCB1 was critical for SALL4-induced drug resistance. In contrast, SALL4 knockdown restored drug sensitivity, reversed EMT, diminished cell metastasis and suppressed the downregulation of E-cadherin and the upregulation of N-cadherin and ABCB1. Furthermore, we showed that SALL4 upregulated c-Myc expression and c-Myc was a direct target for SALL4 by ChIP assay, depletion of c-Myc with siRNA abolished the SALL4-induced downregulation of E-cadherin, upregulation of N-cadherin and ABCB1, suggesting that c-Myc was a downstream target for SALL4 and required for SALL4-induced EMT, invasion and drugs resistance in endometrial cancer cells. These results indicated that SALL4 could induce EMT and resistance to antineoplastic drugs through the regulation of c-Myc. SALL4 and c-Myc may be novel therapeutic targets for endometrial cancer.     

An epithelial precursor is regulated by the ureteric bud and by the renal stroma

Kidney epithelia develop from the metanephric mesenchyme after receiving inductive signals from the ureteric bud and from the renal stroma. However, it is not clear how these signals induce the different types of epithelia that make up the nephron. To investigate inductive signaling, we have isolated clusters of epithelial progenitors from the metanephric mesenchyme, thereby separating them from the renal stroma. When the isolated progenitors were treated with the ureteric bud factor LIF, they expressed epithelial proteins (ZO-1, E-cadherin, laminin alpha(5)) and produced nephrons (36 glomeruli with 58 tubules), indicating that they are the target of inductive signaling from the ureteric bud, and that renal stroma is not absolutely required for epithelial development in vitro. In fact, stroma-depleted epithelial progenitors produced sevenfold more glomeruli than did intact metanephric mesenchyme (5 glomeruli, 127 tubules). Conversely, when epithelial progenitors were treated with both LIF and proteins secreted from a renal stromal cell line, glomerulogenesis was abolished but tubular epithelia were expanded (0 glomeruli, 47 tubules). Hence, by isolating epithelial progenitors from the metanephric mesenchyme, we show that they are targeted by factors from the ureteric bud and from the renal stroma, and that epithelial diversification is stimulated by the ureteric bud and limited by renal stroma.     

采用RNAi技术抑制PeroxiredoxinⅠ的表达

Peroxiredoxin(Prx)属于抗氧化蛋白超家族,广泛存在于原核生物和真核生物中[1,2].哺乳动物的Prx蛋白家族包括6个成员,PrxⅠ—Ⅵ.PrxⅠ定位于细胞质中,在多种组织中表达.它还是一个可诱导的蛋白,在氧化应急条件下其表达显著增高,并高表达于一些恶性肿瘤细胞中[3~5].该蛋白的生化