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RhoA is dispensable for skin development, but crucial for contraction and directed migration of keratinocytes.

Jackson B, Peyrollier K, Pedersen E, Basse A, Karlsson R, Wang Z, Lefever T, Ochsenbein AM, Schmidt G, Aktories K, Stanley A, Quondamatteo F, Ladwein M, Rottner K, van Hengel J, Brakebusch C - Mol. Biol. Cell (2011)

Bottom Line: Rho-inhibiting toxins further increased multinucleation of RhoA- 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- keratinocytes.

View Article: PubMed Central - PubMed

Affiliation: Biomedical Institute, BRIC, University of Copenhagen, 2200 Copenhagen, Denmark.

ABSTRACT
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- 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- 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- 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.

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Generation of mice with a keratinocyte-restricted deletion of the RhoA gene. (A) Targeting scheme showing the wild-type RhoA gene (wt), the targeting construct (vector), the recombined RhoA gene with floxed neo-TK cassette (floxed with neo; white box), the floxed RhoA gene (floxed), and the knockout allele (ko) after homozygous recombination of the targeting construct and removal of the floxed exon 3. The respective alleles were distinguished by HindIII digestion and Southern blot hybridization with an external 5′ probe, resulting in the detection of the indicated fragment sizes (examples for the Southern blot hybridization are shown at the right side of the scheme). (B) Mice with a keratinocyte-restricted deletion of the RhoA gene (ko) show no obvious phenotype. (C) Western blot analysis reveals efficient loss of RhoA but compensatory increase of RhoB protein in the epidermis of RhoA-deficient mice (ko), compared to controls (con; n = 3; *p < 0.05). (D) Hematoxylin and eosin–stained paraffin sections of back skin of 5-mo-old RhoA fl/fl K5 Cre (ko) and control (con) mice indicate normal skin structure.
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Figure 1: Generation of mice with a keratinocyte-restricted deletion of the RhoA gene. (A) Targeting scheme showing the wild-type RhoA gene (wt), the targeting construct (vector), the recombined RhoA gene with floxed neo-TK cassette (floxed with neo; white box), the floxed RhoA gene (floxed), and the knockout allele (ko) after homozygous recombination of the targeting construct and removal of the floxed exon 3. The respective alleles were distinguished by HindIII digestion and Southern blot hybridization with an external 5′ probe, resulting in the detection of the indicated fragment sizes (examples for the Southern blot hybridization are shown at the right side of the scheme). (B) Mice with a keratinocyte-restricted deletion of the RhoA gene (ko) show no obvious phenotype. (C) Western blot analysis reveals efficient loss of RhoA but compensatory increase of RhoB protein in the epidermis of RhoA-deficient mice (ko), compared to controls (con; n = 3; *p < 0.05). (D) Hematoxylin and eosin–stained paraffin sections of back skin of 5-mo-old RhoA fl/fl K5 Cre (ko) and control (con) mice indicate normal skin structure.

Mentions: To study the specific role of RhoA in skin, we generated mice with a conditional knockout of the RhoA gene by using the Cre-loxP system (Figure 1A). Homologously recombined clones were identified by Southern blot with an external probe (Figure 1A; floxed with neo). After transient transfection with Cre recombinase to remove the neo-TK cassette and selection for fialuridine (FIAU)-resistant cells that had lost the TK gene, floxed clones were detected by Southern blot with an external probe (Figure 1A; floxed). RhoA fl/fl mice were generated by blastocyst injection of floxed ES cells and subsequent crossing with K5 Cre mice (Ramirez et al., 2004) to obtain mice with a keratinocyte-restricted deletion of the RhoA gene. Offspring were routinely genotyped by genomic PCR (Figure 1B).


RhoA is dispensable for skin development, but crucial for contraction and directed migration of keratinocytes.

Jackson B, Peyrollier K, Pedersen E, Basse A, Karlsson R, Wang Z, Lefever T, Ochsenbein AM, Schmidt G, Aktories K, Stanley A, Quondamatteo F, Ladwein M, Rottner K, van Hengel J, Brakebusch C - Mol. Biol. Cell (2011)

Generation of mice with a keratinocyte-restricted deletion of the RhoA gene. (A) Targeting scheme showing the wild-type RhoA gene (wt), the targeting construct (vector), the recombined RhoA gene with floxed neo-TK cassette (floxed with neo; white box), the floxed RhoA gene (floxed), and the knockout allele (ko) after homozygous recombination of the targeting construct and removal of the floxed exon 3. The respective alleles were distinguished by HindIII digestion and Southern blot hybridization with an external 5′ probe, resulting in the detection of the indicated fragment sizes (examples for the Southern blot hybridization are shown at the right side of the scheme). (B) Mice with a keratinocyte-restricted deletion of the RhoA gene (ko) show no obvious phenotype. (C) Western blot analysis reveals efficient loss of RhoA but compensatory increase of RhoB protein in the epidermis of RhoA-deficient mice (ko), compared to controls (con; n = 3; *p < 0.05). (D) Hematoxylin and eosin–stained paraffin sections of back skin of 5-mo-old RhoA fl/fl K5 Cre (ko) and control (con) mice indicate normal skin structure.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3046057&req=5

Figure 1: Generation of mice with a keratinocyte-restricted deletion of the RhoA gene. (A) Targeting scheme showing the wild-type RhoA gene (wt), the targeting construct (vector), the recombined RhoA gene with floxed neo-TK cassette (floxed with neo; white box), the floxed RhoA gene (floxed), and the knockout allele (ko) after homozygous recombination of the targeting construct and removal of the floxed exon 3. The respective alleles were distinguished by HindIII digestion and Southern blot hybridization with an external 5′ probe, resulting in the detection of the indicated fragment sizes (examples for the Southern blot hybridization are shown at the right side of the scheme). (B) Mice with a keratinocyte-restricted deletion of the RhoA gene (ko) show no obvious phenotype. (C) Western blot analysis reveals efficient loss of RhoA but compensatory increase of RhoB protein in the epidermis of RhoA-deficient mice (ko), compared to controls (con; n = 3; *p < 0.05). (D) Hematoxylin and eosin–stained paraffin sections of back skin of 5-mo-old RhoA fl/fl K5 Cre (ko) and control (con) mice indicate normal skin structure.
Mentions: To study the specific role of RhoA in skin, we generated mice with a conditional knockout of the RhoA gene by using the Cre-loxP system (Figure 1A). Homologously recombined clones were identified by Southern blot with an external probe (Figure 1A; floxed with neo). After transient transfection with Cre recombinase to remove the neo-TK cassette and selection for fialuridine (FIAU)-resistant cells that had lost the TK gene, floxed clones were detected by Southern blot with an external probe (Figure 1A; floxed). RhoA fl/fl mice were generated by blastocyst injection of floxed ES cells and subsequent crossing with K5 Cre mice (Ramirez et al., 2004) to obtain mice with a keratinocyte-restricted deletion of the RhoA gene. Offspring were routinely genotyped by genomic PCR (Figure 1B).

Bottom Line: Rho-inhibiting toxins further increased multinucleation of RhoA- 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- keratinocytes.

View Article: PubMed Central - PubMed

Affiliation: Biomedical Institute, BRIC, University of Copenhagen, 2200 Copenhagen, Denmark.

ABSTRACT
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- 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- 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- 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.

Show MeSH
Related in: MedlinePlus