Rgnef (p190RhoGEF) knockout inhibits RhoA activity, focal adhesion establishment, and cell motility downstream of integrins.
BACKGROUND
Cell migration is a highly regulated process that involves the formation and turnover of cell-matrix contact sites termed focal adhesions. Rho-family GTPases are molecular switches that regulate actin and focal adhesion dynamics in cells. Guanine nucleotide exchange factors (GEFs) activate Rho-family GTPases. Rgnef (p190RhoGEF) is a ubiquitous 190 kDa GEF implicated in the control of colon carcinoma and fibroblast cell motility.


RESULTS
Rgnef exon 24 floxed mice (Rgnef(flox)) were created and crossed with cytomegalovirus (CMV)-driven Cre recombinase transgenic mice to inactivate Rgnef expression in all tissues during early development. Heterozygous Rgnef(WT/flox) (Cre+) crosses yielded normal Mendelian ratios at embryonic day 13.5, but Rgnef(flox/flox) (Cre+) mice numbers at 3 weeks of age were significantly less than expected. Rgnef(flox/flox) (Cre+) (Rgnef-/-) embryos and primary mouse embryo fibroblasts (MEFs) were isolated and verified to lack Rgnef protein expression. When compared to wildtype (WT) littermate MEFs, loss of Rgnef significantly inhibited haptotaxis migration, wound closure motility, focal adhesion number, and RhoA GTPase activation after fibronectin-integrin stimulation. In WT MEFs, Rgnef activation occurs within 60 minutes upon fibronectin plating of cells associated with RhoA activation. Rgnef-/- MEF phenotypes were rescued by epitope-tagged Rgnef re-expression.


CONCLUSIONS
Rgnef-/- MEF phenotypes were due to Rgnef loss and support an essential role for Rgnef in RhoA regulation downstream of integrins in control of cell migration.
Moores Cancer Center, University of California San Diego, La Jolla, California, United States of America.
Nichol L G
NL
Miller
Christine
C
Lawson
Xiao Lei
XL
Chen
Ssang-Taek
ST
Lim
David D
DD
Schlaepfer
D000704:Analysis of Variance; D000818:Animals; D002465:Cell Movement / Q000502:physiology*; D017931:DNA Primers / Q000235:genetics; D004622:Embryo, Mammalian / Q000166:cytology; D005347:Fibroblasts / Q000378:metabolism; D005353:Fibronectins / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D022001:Focal Adhesions / Q000378:metabolism*; D005838:Genotype; D020662:Guanine Nucleotide Exchange Factors / Q000235:genetics* / Q000378:metabolism; D007150:Immunohistochemistry; D016023:Integrins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014945:Wound Healing / Q000502:physiology*; D020666:ras-GRF1 / Q000235:genetics* / Q000378:metabolism; D020742:rhoA GTP-Binding Protein / Q000378:metabolism*
D017931:DNA Primers; D005353:Fibronectins; D020662:Guanine Nucleotide Exchange Factors; D016023:Integrins; C513037:Rasgrf1 protein, mouse; D020666:ras-GRF1; D020742:rhoA GTP-Binding Protein