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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online September 16, 2005 as doi:10.1096/fj.04-3327fje. |
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Department Dermatology, University of Cologne and Centre for Molecular Medicine, University of Cologne (CMMC), Cologne, Germany
2 Correspondence: Department Dermatology, University of Cologne and Centre for Molecular Medicine, University of Cologne (CMMC), Joseph-Stelzmann-Strasse 9, 50924 Cologne, Germany. E-mail: Ingo.Haase{at}uni-koeln.de
SPECIFIC AIM
Our study aims at identifying functions of the small GTPase Ras in cytoskeletal rearrangements and wound epithelialization by primary human epidermal keratinocytes.
PRINCIPAL FINDINGS
1. Ras induces keratinocyte shape changes
Activation of Ras signaling in primary keratinocytes by means of retroviral infection and transient transfection with activating, mutant H-ras constructs resulted in dramatic changes of keratinocyte shape. Activation of Ras by expression of V12Ras was associated with disappearance of ring-like actin cables at the cell periphery and increased spreading on collagen I as well as with enhanced ruffling of the cell membrane. Ras-induced spreading could be abrogated by incubation of cells with cytochalasin D, an inhibitor of actin polymerization (Fig. 1
).
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2. Ras-induced spreading is not caused by terminal keratinocyte differentiation
To test whether the shape changes observed were associated with terminal differentiation, we induced growth arrest in primary keratinocytes by treating them with 4 µg/mL mitomycin C for 2 h. These cells did not show gross morphological alterations 24 h after treatment but enlarged during subsequent days and started to express the terminal differentiation marker involucrin. When we treated these cells with cytochalasin D they remained large and spread out although actin filaments in these cells were fragmented suggesting that keratinocyte morphology after mitomycin-induced growth arrest is not maintained solely by reorganization of the actin cytoskeleton. In contrast, keratinocyte shape changes after V12Ras transfection could be completely inhibited by incubation with cytochalasin D (Fig. 1e
). To further investigate a possible correlation between keratinocyte shape changes upon Ras activation and terminal keratinocyte differentiation, numbers of transfected and untransfected keratinocytes that expressed the terminal differentiation marker involucrin were scored 30 h after transient transfection with V12Ras. At this time, V12Ras-induced shape changes were present (see Fig. 1
). The number of involucrin-positive keratinocytes was similar in the V12Ras transfected and in the untransfected population, indicating that until 30 h after transfection V12Ras did not stimulate initiation of the terminal differentiation program.
To analyze the influence of V12Ras on keratinocyte growth in our system, we infected primary human keratinocytes with retroviruses encoding mutants of H-Ras or empty vector and analyzed their growth over 14 days. Growth curves show that V12Ras-expressing keratinocytes did not proliferate less than empty vector controls, although spreading of keratinocytes on collagen-coated dishes was greatly enhanced. These results demonstrate that V12Ras-mediated keratinocyte shape changes are not the result of an induction of terminal differentiation.
3. Keratinocyte spreading is stimulated by Ras40C but not Ras35S or Ras37G
To test which Ras-related pathways were involved in the observed shape changes, we carried out spreading assays with keratinocytes transfected or infected with different mutant constructs that encode variants of activated Ras with different point mutations in their effector domains. These mutants stimulate different downstream signaling pathways selectively. Keratinocytes expressing V12Ras35S and V12Ras37G were not enlarged and did not show enhanced spreading when plated onto collagen-coated dishes. In contrast, V12Ras40C induced keratinocyte spreading, although not as extensively as V12Ras. As for V12Ras, V12Ras40C-induced spreading could be inhibited by incubation with cytochalasin D (data now shown).
4. Activated Ras stimulates wound closure in vitro
To investigate whether the observed keratinocyte shape changes were relevant in a more physiological setting, we carried out assays of wound epithelialization in vitro using keratinocytes retrovirally infected with different mutants of Ras that were growth arrested by mitomycin C treatment prior to the experiment. Transduction of keratinocytes with V12Ras stimulated wound epithelialization and resulted in a faster wound closure as compared with the empty vector control neo. A similar result was obtained with keratinocytes expressing V12Ras40C. In contrast, V12Ras35S and V12Ras37G had no significant effects on wound closure. We conclude that stimulation of Ras in keratinocytes leads to a proliferation-independent acceleration of wound closure in vitro that does not involve activation of the ERK cascade but is mediated by effectors that are also stimulated by V12Ras40C. The relevance of these findings is supported by our in situ analysis of a murine skin wound showing that keratinocytes in the epithelial tip adopt a shape similar to that observed in V12Ras-expressing keratinocytes in vitro (Fig. 2
).
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5. Rac, but not PI3-kinase activity is required for Ras-induced keratinocyte spreading
Phosphatidylinositol-3-kinase (PI3-K) is an effector of Ras in keratinocytes and thought to be stimulated by expression of V12Ras40C. We analyzed V12Ras-stimulated spreading of keratinocytes in the presence of the specific PI-3-kinase inhibitor LY294002. Surprisingly, most transfected keratinocytes still showed pronounced spreading in the presence of LY294002. This indicates that activation of PI3-K is not required for the cell shape changes observed after stimulation of Ras.
Another downstream effector of Ras known to be involved in the regulation of cytoskeletal actin dynamics is the small GTP binding protein Rac1. Incubation with toxin B dramatically inhibited spreading of V12Ras-expressing keratinocytes. To analyze the role of Rac more specifically, we inhibited Rac signaling by expression of a dominant negative mutant. Primary human keratinocytes expressing V12Ras from a retroviral promoter were transiently transfected with a plasmid encoding the dominant negative mutant of Rac1, N17Rac1, and spreading was determined as described. Cell spreading was reduced in V12Ras-infected keratinocytes expressing N17Rac. We also expressed a constitutively active mutant of Rac1, L61Rac1, in primary human keratinocytes using transient transfection. Cells transfected with this mutant showed enhanced spreading when plated onto collagen-coated dishes, similar to V12Ras transfected cells. This indicates that activity of Rac1 is sufficient to induce keratinocytes spreading and is required for V12Ras-stimulated spreading.
CONCLUSIONS AND SIGNIFICANCE
Activation of Ras leads to accelerated protrusion of the plasma membrane and formation of large lamellipodial structures. In previous studies, enlargement of keratinocytes after retroviral infection with an activated mutant of Ras was speculated to be associated with the induction of terminal differentiation. We provide evidence that Ras-induced keratinocyte shape changes are independent of terminal keratinocyte differentiation. This conclusion is based on the following. 1) Ras-induced shape changes can be inhibited by treatment with cytochalasin D, an inhibitor of actin polymerization, whereas shape changes accompanying terminal differentiation of keratinocytes after mitomycin C-induced growth arrest cannot be reversed by cytochalasin D treatment. 2) Transduction with an activating mutant of Ras does not result in an increased expression of the terminal differentiation marker involucrin. 3) Primary human keratinocytes do not show decreased proliferation after infection with a retroviral vector encoding an activating mutant of Ras.
Although V12Ras-expressing keratinocytes contained less polymerized actin than controls, shape changes were also dependent on actin polymerization. This indicates that Ras-induced spreading was accompanied by dynamic changes of the actin cytoskeleton. Using mutants of the Ras effector domain, we have found that protrusion of the cell membrane in keratinocytes expressing activated Ras is not a function of ERK activation or stimulation of Ral GDS. Our data suggest that keratinocyte spreading after H-Ras activation is a consequence of PI-3K-independent stimulation of the small GTPase Rac. A molecular mechanism mediating PI-3 K-independent Rac activation by Ras in cooperation with the GEF Tiam 1 has been proposed recently. An alternative pathway could be mediated by PREL1, a new protein that can bind to activated Ras and signals to the actin cytoskeleton via the Ena/VASP complex.
Using an in vitro wound healing assay we have shown that activation of Ras can lead to accelerated re-epithelialization of wounded keratinocyte monolayers in vitro. Consistent with our previous results showing that IGF-1 stimulates re-epithelialization of wounded keratinocyte monolayers more efficiently than EGF, V12Ras40C is more efficient in inducing wound closure than V12Ras35S. This may suggest that the capacity of epidermal keratinocytes to close a wound is determined more by their ability to spread than by random cell motility.
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-3327fje;
1 These authors contributed equally to this work. 
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