Versican is a large extracellular chondroitin sulfate proteoglycan that belongs to the family of lecticans. Alternativesplicing of versican generates at least four isoforms named V0, V1, V2, and V3. We show here that ectopic expression ofversican V1 isoform induced mesenchymal-epithelial transition (MET) in NIH3T3 fibroblasts, and inhibition of endogenousversican expression abolished the MET in metanephric mesenchyme. MET in NIH3T3 cells was demonstrated bymorphological changes and dramatic alterations in both membrane and cytoskeleton architecture. Molecular analysisshowed that V1 promoted a "switch" in cadherin expression from N-to E-cadherin, resulting in epithelial specificadhesion junctions. V1 expression reduced vimentin levels and induced expression of occludin, an epithelial-specificmarker, resulting in polarization of V1-transfected cells. Furthermore, an MSP (methylation-specific PCR) assay showedthat N-cadherin expression was suppressed through methylation of its DNA promoter. Exogenous expression of Ncadherinin V1-transfected cells reversed V1’s effect on cell aggregation. Reduction of E-cadherin expression by Snailtransfection and siRNA targeting E-cadherin abolished V1-induced morphological alteration. Transfection of an siRNAconstruct targeting versican also reversed the changed morphology induced by V1 expression. Silencing of endogenousversican prevented MET of metanephric mesenchyme. Taken together, our results demonstrate the involvement ofversican in MET: expression of versican is sufficient to induce MET in NIH3T3 fibroblasts and reduction of versicanexpression decreased MET in metanephric mesenchyme.

Members of the large aggregating chondroitin sulfate proteoglycans are characterized by anN-terminal fragment known as G1 domain, which is composed of an immunoglobulin (IgG)-like motifand two tandem repeats (TR). Previous studies have indicated that the expressed product of aggrecan G1domain was not secreted. Here we demonstrated that the inability of G1 secretion was associated with thetandem repeats but not the IgG-like motif, and specifically with TR1 of aggrecan. We also demonstratedthat the G2 domain, a domain unique to aggrecan, had a similar effect on product secretion. The sequenceof TR1 of G1 is highly conserved across species, which suggested similar functions played by thesemotifs. In a yeast two-hybrid assay, TR1 interacted with the calcium homeostasis endoplasmic reticulumprotein. Deletion/mutation experiments indicated that the N-terminal fragment of TR1, in particular, theamino acids H2R4 of this motif were key to its effect on product secretion. However, the N-terminal 55amino acids were required to exert this function. Taken together, our study suggests a possible molecularmechanism for the function of the tandem repeats in product processing.

Spontaneous loss of the Epstein-Barr virus (EBV) genome in the BL cell line Akata led to loss of tumorigenicityin SCID mice, suggesting an important oncogenic activity of EBV in B cells. We previously showed thatintroduction of the BARF1 gene into the human B-cell line Louckes induced a malignant transformation innewborn rats (M. X. Wei, J. C. Moulin, G. Decaussin, F. Berger, and T. Ooka, Cancer Res. 54:1843-1848, 1994).Since 1 to 2% of Akata cells expressed lytic antigens and expressed the BARF1 gene, we investigated whetherintroduction of the BARF1 gene into EBV-negative Akata cells can induce malignant transformation. Here weshow that BARF1-transfected, EBV-negative Akata cells activated Bcl2 expression and induced tumor formationwhen they were injected into SCID mice. In addition, when EBV-positive Akata cells expressing a low levelof BARF1 protein were injected into SCID mice, the expression of BARF1, as well as several lytic proteins, suchas EA-D, ZEBRA, and a 135-kDa DNA binding protein, increased in tumor cells while no latent LMP1 and lategp220-320 expression was observed in tumor cells. These observations suggest that the BARF1 gene may beinvolved in the conferral of tumorigenicity by EBV.

Versican is a large chondroitin sulfate proteoglycan belonging to the lectican family. Alternative splicing of versicangenerates at least four isoforms named V0, V1, V2, and V3. We have shown that the versican V1 isoform not only enhancedcell proliferation, but also modulated cell cycle progression and protected the cells from apoptosis. Futhermore, the V1isoform 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 theexpression of the proapoptotic protein Bad. By contrast, the V2 isoform exhibited opposite biological activities byinhibiting cell proliferation and down-regulated the expression of EGFR and cyclin A. Furthermore, V2 did not contributeapoptotic resistance to the cells. In light of these results, we are reporting opposite functions for the two versican isoformswhose expression is differentially regulated. Our studies suggest that the roles of these two isoforms are associated withthe subdomains CS and CS, respectively. These results were confirmed by silencing the expression of versican V1 withsmall interfering RNA (siRNA), which abolished V1-enhanced cell proliferation and V1-induced reduction of apoptosis.

The chondroitin sulfate proteoglycan versican is one of the major extracellular components in the developing and adultbrain. Here, we show that isoforms of versican play different roles in neuronal differentiation and neurite outgrowth.Expression of versican V1 isoform in PC12 cells induced complete differentiation, whereas expression of V2 induced anaborted differentiation accompanied by apoptosis. V1 promoted neurite outgrowth of hippocampal neurons, but V2 failedto do so. V1 transfection enhanced expression of epidermal growth factor receptor and integrins, and facilitated sustainedextracellular signal-regulated kinase/MAPK phosphorylation. Blockade of the epidermal growth factor receptor, _1integrin, or Src significantly inhibited neuronal differentiation. Finally, we demonstrated that versican V1 isoform alsopromoted differentiation of neural stem cells into neurons. Our results have implications for understanding how versicanregulates neuronal development, function, and repair.