ABSTRACT
Primary keratinocytes are an important tool to investigate the molecular mechanism underlying the skin phenotype of mice with null mutations in Rho GTPase genes. If the RhoA gene deletion is conditional, the knockout can be induced in vitro by transfection with cre-IRES-GFP and sorting for GFP positive cells by flow cytometry. Such in vitro knockout will allow determining the cell autonomous functions of the Rho GTPase, independent of any in vivo interactions. Using the same method, also other expression vectors or knockdown constructs can be introduced into primary mouse keratinocytes.
Subject(s)
Gene Knockout Techniques , Keratinocytes/enzymology , rho GTP-Binding Proteins/genetics , Animals , Cell Separation/methods , Flow Cytometry , Mice , Primary Cell Culture/methods , TransfectionABSTRACT
RhoA is a small guanosine-5'-triphosphatase (GTPase) suggested to be essential for cytokinesis, stress fiber formation, and epithelial cell-cell contacts. In skin, loss of RhoA was suggested to underlie pemphigus skin blistering. To analyze RhoA function in vivo, we generated mice with a keratinocyte-restricted deletion of the RhoA gene. Despite a severe reduction of cofilin and myosin light chain (MLC) phosphorylation, these mice showed normal skin development. Primary RhoA-null keratinocytes, however, displayed an increased percentage of multinucleated cells, defective maturation of cell-cell contacts. Furthermore we observed increased cell spreading due to impaired RhoA-ROCK (Rho-associated protein kinase)-MLC phosphatase-MLC-mediated cell contraction, independent of Rac1. Rho-inhibiting toxins further increased multinucleation of RhoA-null cells but had no significant effect on spreading, suggesting that RhoB and RhoC have partially overlapping functions with RhoA. Loss of RhoA decreased directed cell migration in vitro caused by reduced migration speed and directional persistence. These defects were not related to the decreased cell contraction and were independent of ROCK, as ROCK inhibition by Y27632 increased directed migration of both control and RhoA-null keratinocytes. Our data indicate a crucial role for RhoA and contraction in regulating cell spreading and a contraction-independent function of RhoA in keratinocyte migration. In addition, our data show that RhoA is dispensable for skin development.
Subject(s)
Cell Movement , Keratinocytes/enzymology , Keratinocytes/pathology , Skin/enzymology , Skin/growth & development , rhoA GTP-Binding Protein/metabolism , Actin Depolymerizing Factors/metabolism , Animals , Cell Count , Cell Differentiation , Cytokinesis , Epidermis/growth & development , Epidermis/metabolism , Epidermis/pathology , Epidermis/ultrastructure , Focal Adhesions/metabolism , Gene Deletion , Giant Cells/cytology , Intercellular Junctions/metabolism , Membrane Proteins/metabolism , Mice , Myosin Light Chains/metabolism , Myosin-Light-Chain Phosphatase/metabolism , Occludin , Organ Specificity , Phosphorylation , Skin/pathology , Skin/ultrastructure , Stress Fibers/metabolism , Wound Healing , rac1 GTP-Binding Protein/metabolism , rho-Associated Kinases/deficiency , rho-Associated Kinases/metabolismABSTRACT
N-WASP is a cytoplasmic molecule mediating Arp2/3 nucleated actin polymerization. Mice with a keratinocyte-specific deletion of the gene encoding N-WASP showed normal interfollicular epidermis, but delayed hair-follicle morphogenesis and abnormal hair-follicle cycling, associated with cyclic alopecia and prolonged catagen and telogen phases. The delayed anagen onset correlated with an increased expression of the cell-cycle inhibitor p21CIP, and increased activity of the TGFbeta pathway, a known inducer of p21CIP expression. Primary N-WASP-null keratinocytes showed reduced growth compared with control cells and enhanced expression of the gene encoding the cell-cycle inhibitor p15INK4B, a TGFbeta target gene. Inhibition of TGFbeta signaling blocked overexpression of p15INK4B and restored proliferation of N-WASP-deficient keratinocytes in vitro. However, induction of N-WASP gene deletion in vitro did not result in obvious changes in TGFbeta signaling or growth of keratinocytes, indicating that the in vivo environment is required for the phenotype development. These data identify the actin nucleation regulator N-WASP as a novel element of hair-cycle control that modulates the antiproliferative and pro-apoptotic TGFbeta pathway in keratinocytes in vivo and in vitro.
Subject(s)
Alopecia/genetics , Cyclin-Dependent Kinase Inhibitor p15/biosynthesis , Cyclin-Dependent Kinase Inhibitor p21/biosynthesis , Keratinocytes/metabolism , Wiskott-Aldrich Syndrome Protein, Neuronal/metabolism , Actin Cytoskeleton , Alopecia/pathology , Alopecia/physiopathology , Animals , Cell Cycle/genetics , Cell Proliferation , Cells, Cultured , Cyclin-Dependent Kinase Inhibitor p15/genetics , Cyclin-Dependent Kinase Inhibitor p21/genetics , Hair Follicle/growth & development , Hair Follicle/pathology , Keratinocytes/pathology , Mice , Morphogenesis/genetics , Signal Transduction , Transforming Growth Factor beta/metabolism , Wiskott-Aldrich Syndrome Protein, Neuronal/geneticsABSTRACT
Differentiation of skin stem cells into hair follicles (HFs) requires the inhibition of beta-catenin degradation, which is controlled by a complex containing axin and the protein kinase GSK3beta. Using conditional gene targeting in mice, we show now that the small GTPase Cdc42 is crucial for differentiation of skin progenitor cells into HF lineage and that it regulates the turnover of beta-catenin. In the absence of Cdc42, degradation of beta-catenin was increased corresponding to a decreased phosphorylation of GSK3beta at Ser 9 and an increased phosphorylation of axin, which is known to be required for binding of beta-catenin to the degradation machinery. Cdc42-mediated regulation of beta-catenin turnover was completely dependent on PKCzeta, which associated with Cdc42, Par6, and Par3. These data suggest that Cdc42 regulation of beta-catenin turnover is important for terminal differentiation of HF progenitor cells in vivo.
