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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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Normal differentiation and cell–cell junctions in RhoA- epidermis. Cryosections of back skin of 5-mo-old mice were analyzed for the presence of keratin 14 (a, a′), keratin 10 (b, b′), loricrin (c, c′), keratin 6 (d, d′), E-cadherin (e, e′), desmoplakin (f, f′), and ZO-1 (g, g′). Counterstaining for α6 integrin indicates the DEJs (a-d, a′-d′). Nuclei are visualized by DAPI. (Bar = 50 μm.)
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Figure 2: Normal differentiation and cell–cell junctions in RhoA- epidermis. Cryosections of back skin of 5-mo-old mice were analyzed for the presence of keratin 14 (a, a′), keratin 10 (b, b′), loricrin (c, c′), keratin 6 (d, d′), E-cadherin (e, e′), desmoplakin (f, f′), and ZO-1 (g, g′). Counterstaining for α6 integrin indicates the DEJs (a-d, a′-d′). Nuclei are visualized by DAPI. (Bar = 50 μm.)

Mentions: Histological investigation of the dorsal skin of adult mice by hematoxylin and eosin staining revealed no obvious morphological differences between epidermis and hair follicles of control and ko mice and no blistering (Figure 1D). Analysis of keratinocyte differentiation by immunofluorescence staining revealed a normal pattern of differentiation. Keratin 14, a marker of basal keratinocytes, was similarly distributed in ko and control skin (Figure 2, a and a′). α6 integrin, a subunit of the laminin receptor α6β4 integrin, was detected primarily at the basal side of the basal keratinocytes in control and ko epidermis, suggesting normal polarization and attachment to the basement membrane in the absence of RhoA (Figure 2). Also, keratin 10, a marker for suprabasal keratinocytes, and loricrin, a protein associated with cornified cell envelope, displayed normal distribution in RhoA ko mice (Figure 2, b, b′, c, and c′). Finally, staining for keratin 6, a marker of hyperproliferation and stress in the interfollicular epidermis, was not detectable in control and RhoA ko interfollicular epidermis (Figure 2, d and d’).


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)

Normal differentiation and cell–cell junctions in RhoA- epidermis. Cryosections of back skin of 5-mo-old mice were analyzed for the presence of keratin 14 (a, a′), keratin 10 (b, b′), loricrin (c, c′), keratin 6 (d, d′), E-cadherin (e, e′), desmoplakin (f, f′), and ZO-1 (g, g′). Counterstaining for α6 integrin indicates the DEJs (a-d, a′-d′). Nuclei are visualized by DAPI. (Bar = 50 μm.)
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 2: Normal differentiation and cell–cell junctions in RhoA- epidermis. Cryosections of back skin of 5-mo-old mice were analyzed for the presence of keratin 14 (a, a′), keratin 10 (b, b′), loricrin (c, c′), keratin 6 (d, d′), E-cadherin (e, e′), desmoplakin (f, f′), and ZO-1 (g, g′). Counterstaining for α6 integrin indicates the DEJs (a-d, a′-d′). Nuclei are visualized by DAPI. (Bar = 50 μm.)
Mentions: Histological investigation of the dorsal skin of adult mice by hematoxylin and eosin staining revealed no obvious morphological differences between epidermis and hair follicles of control and ko mice and no blistering (Figure 1D). Analysis of keratinocyte differentiation by immunofluorescence staining revealed a normal pattern of differentiation. Keratin 14, a marker of basal keratinocytes, was similarly distributed in ko and control skin (Figure 2, a and a′). α6 integrin, a subunit of the laminin receptor α6β4 integrin, was detected primarily at the basal side of the basal keratinocytes in control and ko epidermis, suggesting normal polarization and attachment to the basement membrane in the absence of RhoA (Figure 2). Also, keratin 10, a marker for suprabasal keratinocytes, and loricrin, a protein associated with cornified cell envelope, displayed normal distribution in RhoA ko mice (Figure 2, b, b′, c, and c′). Finally, staining for keratin 6, a marker of hyperproliferation and stress in the interfollicular epidermis, was not detectable in control and RhoA ko interfollicular epidermis (Figure 2, d and d’).

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