Limits...
Raf-1 regulates Rho signaling and cell migration.

Ehrenreiter K, Piazzolla D, Velamoor V, Sobczak I, Small JV, Takeda J, Leung T, Baccarini M - J. Cell Biol. (2005)

Bottom Line: These defects are due to the hyperactivity and incorrect localization of the Rho-effector Rok-alpha to the plasma membrane.Raf-1 physically associates with Rok-alpha in wild-type (WT) cells, and reintroduction of either WT or kinase-dead Raf-1 in knockout fibroblasts rescues their defects in shape and migration.Thus, Raf-1 plays an essential, kinase-independent function as a spatial regulator of Rho downstream signaling during migration.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Genetics, Max F. Perutz Laboratories, University Departments at the Vienna Biocenter, 1030 Vienna, Austria.

ABSTRACT
Raf kinases relay signals inducing proliferation, differentiation, and survival. The Raf-1 isoform has been extensively studied as the upstream kinase linking Ras activation to the MEK/ERK module. Recently, however, genetic experiments have shown that Raf-1 plays an essential role in counteracting apoptosis, and that it does so independently of its ability to activate MEK. By conditional gene ablation, we now show that Raf-1 is required for normal wound healing in vivo and for the migration of keratinocytes and fibroblasts in vitro. Raf-1-deficient cells show a symmetric, contracted appearance, characterized by cortical actin bundles and by a disordered vimentin cytoskeleton. These defects are due to the hyperactivity and incorrect localization of the Rho-effector Rok-alpha to the plasma membrane. Raf-1 physically associates with Rok-alpha in wild-type (WT) cells, and reintroduction of either WT or kinase-dead Raf-1 in knockout fibroblasts rescues their defects in shape and migration. Thus, Raf-1 plays an essential, kinase-independent function as a spatial regulator of Rho downstream signaling during migration.

Show MeSH

Related in: MedlinePlus

Raf-1 ablation affects wound healing in vivo and keratinocytes motility, adhesion, and shape in vitro. (A) Hematoxylin/eosin-stained full thickness skin section, 12 d after wounding. The wound margins (arrows) have not yet met in the c-raf-1Δ/Δep animals. C, crust; E, newly formed epidermis; D, dermis. (B) Impaired migration in Raf-1 KO keratinocytes. Migration was assayed in a modified Boyden chamber assay using KGM (10 h), EGF (25 ng/ml, 15 h), or KGF (20 ng/ml, 15 h) as chemoattractants. (C) The percentage of cells adhering to different ECM components was determined in a 15-min adhesion assay (Fn, fibronectin; Ln, laminin; C1 and C4, collagens I and IV). Integrin-independent adhesion on BSA-coated surfaces was subtracted from the values plotted. Values are means (±SD, vertical bars) of three individual experiments. (D) Morphology of keratinocytes stained to visualize the actin cytoskeleton (rhodamine-conjugated phalloidin). Bar, 15 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2171799&req=5

fig2: Raf-1 ablation affects wound healing in vivo and keratinocytes motility, adhesion, and shape in vitro. (A) Hematoxylin/eosin-stained full thickness skin section, 12 d after wounding. The wound margins (arrows) have not yet met in the c-raf-1Δ/Δep animals. C, crust; E, newly formed epidermis; D, dermis. (B) Impaired migration in Raf-1 KO keratinocytes. Migration was assayed in a modified Boyden chamber assay using KGM (10 h), EGF (25 ng/ml, 15 h), or KGF (20 ng/ml, 15 h) as chemoattractants. (C) The percentage of cells adhering to different ECM components was determined in a 15-min adhesion assay (Fn, fibronectin; Ln, laminin; C1 and C4, collagens I and IV). Integrin-independent adhesion on BSA-coated surfaces was subtracted from the values plotted. Values are means (±SD, vertical bars) of three individual experiments. (D) Morphology of keratinocytes stained to visualize the actin cytoskeleton (rhodamine-conjugated phalloidin). Bar, 15 μm.

Mentions: We have used a Cre transgene expressed in the basal epidermal layer and follicular keratinocytes (Tarutani et al., 1997), to induce epidermis-specific ablation of the c-raf-1 gene. Primary keratinocytes derived from mice carrying an homozygous c-raf-1flox/flox (f/f) allele (Jesenberger et al., 2001) and the K5Cre transgene (c-raf-1Δ/Δep, deleted in epidermis) showed complete conversion of the c-raf-1flox to the c-raf-1Δ allele by PCR and lacked Raf-1 protein (Fig. 1, A and B). c-raf-1Δ/Δep mice were viable, fertile, and healthy. At 4 wk old, they displayed curled whiskers and a wavy fur (Fig. 1 C), a phenotype lost after the first hair cycle. The architecture of the epidermis (Fig. 1 D) as well as the expression of basal keratin and differentiation markers in the epidermis and hair follicles was indistinguishable in c-raf-1Δ/Δep mice and control littermates (not depicted). Thus, Raf-1 is not essential in epidermal development and homeostasis. Raf-1 ablation, however, markedly affected wound healing. Control mice closed and reepithelialized full thickness wounds (6-mm ∅) by day 9 after wounding, whereas >40% of the wound was still open in c-raf-1Δ/Δep mice at this time (not depicted). The wounds were not yet closed on day 12, when control mice had already completed both reepithelialization and clearance of cell debris underneath the wound crust (Fig. 2 A). c-raf-1Δ/Δep mice healed the wounds 4–5 d later, and the healed skin was indistinguishable from that of f/f mice (not depicted). The secondary responses of dermal components (granulation, inflammation, and neo-vascularization) were not affected by Raf-1 ablation in keratinocytes. The number of proliferating keratinocytes around the wound site was similarly unaffected (31 ± 10.8 Ki67+ cells in f/f, 32.5 ± 8.8 in Δ/Δep), and TUNEL staining of the migrating epithelial sheets did not show any apoptotic cells in c-raf-1Δ/Δep or control wounds (not depicted). Thus, lack of Raf-1 significantly delayed wound healing in the absence of proliferation or survival defects, suggesting that Raf-1 might be needed for keratinocyte migration.


Raf-1 regulates Rho signaling and cell migration.

Ehrenreiter K, Piazzolla D, Velamoor V, Sobczak I, Small JV, Takeda J, Leung T, Baccarini M - J. Cell Biol. (2005)

Raf-1 ablation affects wound healing in vivo and keratinocytes motility, adhesion, and shape in vitro. (A) Hematoxylin/eosin-stained full thickness skin section, 12 d after wounding. The wound margins (arrows) have not yet met in the c-raf-1Δ/Δep animals. C, crust; E, newly formed epidermis; D, dermis. (B) Impaired migration in Raf-1 KO keratinocytes. Migration was assayed in a modified Boyden chamber assay using KGM (10 h), EGF (25 ng/ml, 15 h), or KGF (20 ng/ml, 15 h) as chemoattractants. (C) The percentage of cells adhering to different ECM components was determined in a 15-min adhesion assay (Fn, fibronectin; Ln, laminin; C1 and C4, collagens I and IV). Integrin-independent adhesion on BSA-coated surfaces was subtracted from the values plotted. Values are means (±SD, vertical bars) of three individual experiments. (D) Morphology of keratinocytes stained to visualize the actin cytoskeleton (rhodamine-conjugated phalloidin). Bar, 15 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171799&req=5

fig2: Raf-1 ablation affects wound healing in vivo and keratinocytes motility, adhesion, and shape in vitro. (A) Hematoxylin/eosin-stained full thickness skin section, 12 d after wounding. The wound margins (arrows) have not yet met in the c-raf-1Δ/Δep animals. C, crust; E, newly formed epidermis; D, dermis. (B) Impaired migration in Raf-1 KO keratinocytes. Migration was assayed in a modified Boyden chamber assay using KGM (10 h), EGF (25 ng/ml, 15 h), or KGF (20 ng/ml, 15 h) as chemoattractants. (C) The percentage of cells adhering to different ECM components was determined in a 15-min adhesion assay (Fn, fibronectin; Ln, laminin; C1 and C4, collagens I and IV). Integrin-independent adhesion on BSA-coated surfaces was subtracted from the values plotted. Values are means (±SD, vertical bars) of three individual experiments. (D) Morphology of keratinocytes stained to visualize the actin cytoskeleton (rhodamine-conjugated phalloidin). Bar, 15 μm.
Mentions: We have used a Cre transgene expressed in the basal epidermal layer and follicular keratinocytes (Tarutani et al., 1997), to induce epidermis-specific ablation of the c-raf-1 gene. Primary keratinocytes derived from mice carrying an homozygous c-raf-1flox/flox (f/f) allele (Jesenberger et al., 2001) and the K5Cre transgene (c-raf-1Δ/Δep, deleted in epidermis) showed complete conversion of the c-raf-1flox to the c-raf-1Δ allele by PCR and lacked Raf-1 protein (Fig. 1, A and B). c-raf-1Δ/Δep mice were viable, fertile, and healthy. At 4 wk old, they displayed curled whiskers and a wavy fur (Fig. 1 C), a phenotype lost after the first hair cycle. The architecture of the epidermis (Fig. 1 D) as well as the expression of basal keratin and differentiation markers in the epidermis and hair follicles was indistinguishable in c-raf-1Δ/Δep mice and control littermates (not depicted). Thus, Raf-1 is not essential in epidermal development and homeostasis. Raf-1 ablation, however, markedly affected wound healing. Control mice closed and reepithelialized full thickness wounds (6-mm ∅) by day 9 after wounding, whereas >40% of the wound was still open in c-raf-1Δ/Δep mice at this time (not depicted). The wounds were not yet closed on day 12, when control mice had already completed both reepithelialization and clearance of cell debris underneath the wound crust (Fig. 2 A). c-raf-1Δ/Δep mice healed the wounds 4–5 d later, and the healed skin was indistinguishable from that of f/f mice (not depicted). The secondary responses of dermal components (granulation, inflammation, and neo-vascularization) were not affected by Raf-1 ablation in keratinocytes. The number of proliferating keratinocytes around the wound site was similarly unaffected (31 ± 10.8 Ki67+ cells in f/f, 32.5 ± 8.8 in Δ/Δep), and TUNEL staining of the migrating epithelial sheets did not show any apoptotic cells in c-raf-1Δ/Δep or control wounds (not depicted). Thus, lack of Raf-1 significantly delayed wound healing in the absence of proliferation or survival defects, suggesting that Raf-1 might be needed for keratinocyte migration.

Bottom Line: These defects are due to the hyperactivity and incorrect localization of the Rho-effector Rok-alpha to the plasma membrane.Raf-1 physically associates with Rok-alpha in wild-type (WT) cells, and reintroduction of either WT or kinase-dead Raf-1 in knockout fibroblasts rescues their defects in shape and migration.Thus, Raf-1 plays an essential, kinase-independent function as a spatial regulator of Rho downstream signaling during migration.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Genetics, Max F. Perutz Laboratories, University Departments at the Vienna Biocenter, 1030 Vienna, Austria.

ABSTRACT
Raf kinases relay signals inducing proliferation, differentiation, and survival. The Raf-1 isoform has been extensively studied as the upstream kinase linking Ras activation to the MEK/ERK module. Recently, however, genetic experiments have shown that Raf-1 plays an essential role in counteracting apoptosis, and that it does so independently of its ability to activate MEK. By conditional gene ablation, we now show that Raf-1 is required for normal wound healing in vivo and for the migration of keratinocytes and fibroblasts in vitro. Raf-1-deficient cells show a symmetric, contracted appearance, characterized by cortical actin bundles and by a disordered vimentin cytoskeleton. These defects are due to the hyperactivity and incorrect localization of the Rho-effector Rok-alpha to the plasma membrane. Raf-1 physically associates with Rok-alpha in wild-type (WT) cells, and reintroduction of either WT or kinase-dead Raf-1 in knockout fibroblasts rescues their defects in shape and migration. Thus, Raf-1 plays an essential, kinase-independent function as a spatial regulator of Rho downstream signaling during migration.

Show MeSH
Related in: MedlinePlus