title: Arhgap28 is a RhoGAP that inactivates RhoA and downregulates stress fibers.
abstract: The small GTPase RhoA is a major regulator of actin reorganization during the formation of stress fibers; thus identifying molecules that regulate Rho activity is necessary for a complete understanding of the mechanisms that determine cell contractility. Here, we have identified Arhgap28 as a Rho GTPase activating protein (RhoGAP) that switches RhoA to its inactive form. We generated an Arhgap28-LacZ reporter mouse that revealed gene expression in soft tissues at E12.5, pre-bone structures of the limb at E15.5, and prominent expression restricted mostly to ribs and limb long bones at E18.5 days of development. Expression of recombinant Arhgap28-V5 in human osteosarcoma SaOS-2 cells caused a reduction in the basal level of RhoA activation and disruption of actin stress fibers. Extracellular matrix assembly studies using a 3-dimensional cell culture system showed that Arhgap28 was upregulated during Rho-dependent assembly of the ECM. Taken together, these observations led to the hypothesis that an Arhgap28 knockout mouse model would show a connective tissue phenotype, perhaps affecting bone. Arhgap28-null mice were viable and appeared normal, suggesting that there could be compensation from other RhoGAPs. Indeed, we showed that expression of Arhgap6 (a closely related RhoGAP) was upregulated in Arhgap28-null bone tissue. An upregulation in RhoA expression was also detected suggesting that Arhgap28 may be able to additionally regulate Rho signaling at a transcriptional level. Microarray analyses revealed that Col2a1, Col9a1, Matn3, and Comp that encode extracellular matrix proteins were downregulated in Arhgap28-null bone. Although mutations in these genes cause bone dysplasias no bone phenotype was detected in the Arhgap-28 null mice. Together, these data suggest that the regulation of Rho by RhoGAPs, including Arhgap28, during the assembly and development of mechanically strong tissues is complex and may involve multiple RhoGAPs.
authors.affiliation: Wellcome Trust Centre for Cell-Matrix Research, University of Manchester, Manchester, United Kingdom.
authors.forename: Ching-Yan Chloé
authors.initials: CY
authors.lastname: Yeung
authors.affiliation: Wellcome Trust Centre for Cell-Matrix Research, University of Manchester, Manchester, United Kingdom.
authors.forename: Susan H
authors.initials: SH
authors.lastname: Taylor
authors.affiliation: Wellcome Trust Centre for Cell-Matrix Research, University of Manchester, Manchester, United Kingdom.
authors.forename: Richa
authors.initials: R
authors.lastname: Garva
authors.affiliation: Wellcome Trust Centre for Cell-Matrix Research, University of Manchester, Manchester, United Kingdom.
authors.forename: David F
authors.initials: DF
authors.lastname: Holmes
authors.affiliation: Faculty of Life Sciences, University of Manchester, Manchester, United Kingdom.
authors.forename: Leo A
authors.initials: LA
authors.lastname: Zeef
authors.affiliation: Department of Dermatology, Oulu Center for Cell-Matrix Research, University of Oulu, Oulu, Finland.
authors.forename: Raija
authors.initials: R
authors.lastname: Soininen
authors.affiliation: Wellcome Trust Centre for Cell-Matrix Research, University of Manchester, Manchester, United Kingdom.
authors.forename: Raymond P
authors.initials: RP
authors.lastname: Boot-Handford
authors.affiliation: Wellcome Trust Centre for Cell-Matrix Research, University of Manchester, Manchester, United Kingdom.
authors.forename: Karl E
authors.initials: KE
authors.lastname: Kadler
mesh_terms: D000199:Actins; D000818:Animals; D003599:Cytoskeleton / Q000235:genetics; D005109:Extracellular Matrix / Q000235:genetics*; D005347:Fibroblasts; D018507:Gene Expression Regulation, Developmental; D006801:Humans; D051379:Mice; D018345:Mice, Knockout; D015398:Signal Transduction; D022502:Stress Fibers / Q000235:genetics*; D020741:rho GTP-Binding Proteins / Q000096:biosynthesis* / Q000235:genetics; D020742:rhoA GTP-Binding Protein / Q000235:genetics / Q000378:metabolism
chemical_list: D000199:Actins; C526380:RhoA protein, mouse; D020741:rho GTP-Binding Proteins; D020742:rhoA GTP-Binding Protein