Opposing Roles of Syndecan-1 and Syndecan-2 in Polyethyleneimine-mediated Gene Delivery

Sébastien Paris,Alina Burlacu,Yves Durocher

doi:10.1074/jbc.m705424200

Abstract

Polyethyleneimines (PEIs) are efficient non-viral vectors for gene transfer. Heparan sulfate proteoglycans have been proposed to be the cell-surface receptors for PEI.DNA complexes (polyplexes). Here, we investigated if syndecan-1 (SDC1) and syndecan-2 (SDC2) are involved in PEI-mediated transfection. Following addition of polyplexes to HEK293 cells, green fluorescent protein-tagged SDCs rapidly formed clusters with PEI that were dependent of lipid raft integrity. However, although SDC1 overexpression slightly enhanced PEI-mediated gene expression, SDC2 dramatically inhibited it. Confocal microscopy analysis showed that SDC1.polyplex endocytosis occurred within minutes after addition of polyplexes, whereas SDC2.polyplex endocytosis took hours. Expression of SDC1 cytoplasmic deletion mutants revealed that the SDC1 cytoplasmic tail is required for gene expression, but not for clustering or endocytosis, whereas overexpression of SDC1/SDC2 chimeras showed that the SDC2 ectodomain is responsible for the inhibitory effect on gene transfer. This study provides evidence that SDCs may have opposing effects on PEI-mediated transfection.

Highlights

Cations, enabling the rapid production of recombinant proteins and viral vectors [5]
Using cytoplasmic domain deletion mutants of SDC1 and SDC1/SDC2 chimeras, we evaluated the role of their various domains in polyplex-mediated endocytosis and transgene expression
The green fluorescent protein (GFP)-tagged truncated form of CD4 (⌬CD4), which lacks the helix in the cytoplasmic domain, preventing its internalization by Nef [35], was used as a nonHSPG control transmembrane protein

Summary

EXPERIMENTAL PROCEDURES

Reagents and Antibodies—The following antibodies were used: anti-syndecan-1 (N-18), anti-syndecan-2 (L-18), and anti-syndecan-3 (D-19) (Santa Cruz Biotechnology, Inc.) and anti-syndecan-4 (F94-8G3) (Dr Guido David, University of Leuven, Leuven, Belgium). BPEI (10 mg/ml) was mixed with 2 mg/ml RITC in 0.5 M bicarbonate buffer (pH 9.0) and incubated at room temperature under constant agitation for 2 h. Two micrograms of BPEI (for fluorescence microscopy and flow cytometry analysis) or RITC-labeled BPEI (for confocal microscopy analysis) was added to 1 ␮g of plasmid DNA (pTT5 for fluorescence and confocal microscopy analysis or pTT5-DsRed for flow cytometry analysis); both had been previously diluted in 100 ␮l of hybridoma serum-free medium (Invitrogen). Red fluorescent protein (DsRed)-expressing cells were determined by flow cytometry using an EPICS Profile II (Beckman Coulter) equipped with a 15-milliwatt argon ion laser. Colocalization Experiments and Microscopy—Colocalization of fluorescent proteins and BPEI-RITC was performed using a confocal microscope. After washing cells with fresh medium, plated cells were transfected with BPEI-RITC for the indicated periods of time. Zeiss LSM 510 META confocal system version 3.2 software and IrfanView version 3.85 were used for image acquisition by confocal microscopy and conventional microscopy, respectively

RESULTS

Not Required for Polyplex Endocytosis but Is Necessary for Gene

DISCUSSION