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Address correspondence to Paul A. Khavari, Program in Epithelial Biology, 269 Campus Dr., Rm. 2145, Stanford, CA 94305. Tel.: (650) 725-5266. Fax: (650) 723-8762. E-mail: khavari{at}CMGM.stanford.edu
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Abstract |
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Key Words: Gab1; SHP-2; Ras; epidermis; skin
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Introduction |
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Recent data suggests that these discrepancies could be due to differences in Ras signal strength in cultured cells (Dajee et al., 2002). In tissue, recent work suggests that Ras may act in a spatially localized fashion within basal layer cells to promote proliferative capacity and oppose differentiation (Dajee et al., 2002). Targeting active Ras mutants to epidermis of transgenic mice generates hyperplastic, undifferentiated epidermis (Bailleul et al., 1990; Greenhalgh et al., 1993; Brown et al., 1998; Dajee et al., 2002). Redundancy and embryonic lethality have hindered generation of tissue deficient in all three H, N, and K-Ras isoforms (Umanoff et al., 1995; Johnson et al., 1997; Ise et al., 2000; Esteban et al., 2001). However, expression of a dominant-negative Ras mutant that lowers levels of active Ras/MAPK in epidermis leads to premature differentiation and proliferative failure (Dajee et al., 2002). Together, these data suggest a tentative model in which Ras acts within epidermis to support proliferative capacity and oppose terminal differentiation. Compelling additional support for this model requires alterations in nonredundant components acting either upstream or downstream of Ras in epidermis.
The relative importance of signaling elements controlling Ras function and mediating its effects can vary depending on the cell type (Shields et al., 2000). Receptors for ligands that include growth factors and matrix proteins can activate Ras in many settings through membrane proximal proteins that include Shc, Grb2, and the guanine nucleotide exchange factor Sos (Schlessinger, 2000; Shields et al., 2000). In epidermis, examples of such receptors implicated in Ras induction include certain growth factor receptor tyrosine kinases and integrins (Mainiero et al., 1997; Zhu et al., 1999; Sibilia et al., 2000). Of interest, mice with targeted deletion of several receptors capable of activating Ras display the reduced epidermal proliferation seen with transgenic Ras blockade, suggesting their potential involvement in epidermal Ras signaling. Examples of these receptors include the EGF receptor (EGFR)* (Sibilia and Wagner, 1995; Threadgill et al., 1995) and ß1 integrin (Brakebusch et al., 2000). Major effectors that initiate signaling cascades downstream of Ras include Raf family members, phosphoinositide 3-kinases (PI3Ks), and RalGDS proteins (Shields et al., 2000). However, the involvement of specific regulatory and effector molecules important in Ras function, depends on cellular setting and tissue type, and the factors important in epidermal Ras signaling are not clearly defined.
Among proteins acting upstream of Ras are the multisubstrate docking protein Gab1 and the SHP-2 tyrosine phosphatase. Gab1 is a member of a docking protein family (Hibi and Hirano, 2000; Guy et al., 2002; Liu and Rohrschneider, 2002) that recruits multiple signaling proteins after binding to and phosphorylation by selected receptor tyrosine kinases (Holgado-Madruga et al., 1996; Weidner et al., 1996; Lock et al., 2000). Less well characterized as an upstream regulator of Ras function than the Shc/Grb2 proteins, Gab1 appears to play a role in activation of Ras effectors, including the MAPK and PI3K signaling cascades. Although growth factordriven MAPK activation can proceed normally in Shc-/- cells (Lai and Pawson, 2000), it is impaired in Gab1-/- cells (Itoh et al., 2000; Sachs et al., 2000). Gab1 induction of the MAPK pathway in response to growth factors such as EGF is dependent on binding to the SHP-2 protein tyrosine phosphatase (Cunnick et al., 2001). SHP-2 contains two src homology (SH)2 domains at its NH2 terminus and is encoded by the PTPN11 gene recently shown mutated in Noonan syndrome, a disease characterized by facial dysmorphology, growth retardation, and cardiac defects (Feng, 1999; Tartaglia et al., 2001). Gab1 activates SHP-2 by targeting it to the membrane in a process dependent on the NH2-terminal pleckstrin homology (PH) domain of Gab1 (Cunnick et al., 2002). Although SHP-2 can dephosphorylate Gab1, full characterization of the substrates important for SHP-2 function has not yet been accomplished (Yu et al., 2002). Gab1 and SHP-2 knock-out mice die during embryogenesis, hindering the study of adult tissues null for these proteins (Saxton et al., 1997; Itoh et al., 2000; Sachs et al., 2000). However, analysis of in utero and chimeric tissue suggests that Gab1 and SHP-2 play a role in the morphogenesis of epithelial tissues (Qu et al., 1999; Itoh et al., 2000). The degree to which these proteins influence epidermal growth and differentiation through proteins such as Ras is currently unknown.
Here, we provide evidence supporting a role for Gab1 and SHP-2 in promoting Ras/MAPK signaling to enhance epidermal cell proliferation and oppose differentiation. In epidermal cells, overexpression of wild-type Gab1 and SHP-2 extends the duration of MAPK activation in response to EGF. In contrast, dominant-negative Gab1 and SHP-2 mutants reduce endogenous basal levels of active Ras and MAPK and induce differentiation, a process that can be reversed by coexpression of active Ras. In vivo, disruption of Gab1 function in Gab1-/- postnatal epidermis obtained by embryo grafting and in tissue expressing dominant-negative Gab1 and SHP-2 leads to decreased proliferation and enhanced differentiation. Consistent with this, Gab1-/- epidermis displays diminished levels of active Ras and MAPK. These data indicate that Gab1 and SHP-2 function as nonredundant positive regulators of epidermal Ras function to promote cell proliferation and oppose terminal differentiation.
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Results |
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Altered Ras/MAPK activity and epidermal growth and differentiation in postnatal Gab1-/- tissue
In studying the effects of loss of function of Gab1 and SHP-2, genetic ablation studies are an important complement to expression of transdominant molecules. Shc/Grb2-mediated signal transmission from EGFR to Ras may operate in a partially distinct manner from that mediated by SHP-2 (Shi et al., 2000), and the relative degree to which these two mechanisms function in epidermis is unknown. If Gab1/SHP-2 function is important in maintaining physiologic levels of active Ras, then its disruption should lead to diminished levels of GTP-bound Ras. To test this, we analyzed active Ras levels in epidermis isolated from Gab1-/- embryos. Levels of active Ras and its downstream target MAPKs were decreased in Gab1-/- epidermis compared with Gab1+/+ control (Fig. 7 a). Thus, Gab1 plays a nonredundant role in positively modulating Ras/MAPK activity in epidermis.
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Discussion |
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Combined with induction of differentiation by Gab1/SHP-2 inhibition, these findings suggest that Gab1 and SHP-2 may inhibit differentiation by contributing to sustained Ras/MAPK signaling in epidermal cells. In this regard, the role of Gab1/SHP-2 may be to facilitate basal levels of Ras/MAPK activation in response to factors such as EGF, whereas other proteins, such as Sos/Grb2/Shc, mediate responses to strong growth factor stimulation. In agreement with this possibility is the recent observation that both dominant-negative Ras and pharmacologic inhibition of MEK/MAPK triggers keratinocyte differentiation in the absence of other stimuli (Dajee et al., 2002). These data thus suggest an important role for Gab1 and SHP-2 as necessary in maintaining the undifferentiated state within the epidermis through promotion of Ras/MAPK signaling.
Precise roles for specific receptor tyrosine kinases in controlling growth and differentiation in the epidermis have not been fully defined, and we believe it is unlikely that a single receptor such as EGFR is the sole control point for these processes. In epidermal cells, we observed that Gab1 tyrosine phosphorylation and binding to SHP-2 along with Ras activation is enhanced in response to EGF and can be blocked by a selective inhibitor of EGFR function. In addition to EGFR, integrins such as ß1 appear to promote epidermal proliferation and inhibit differentiation (Brakebusch et al., 2000; Haase et al., 2001), although characterization of integrin effects in isolation from their important roles in adhesion requires further study. Similar to EGFR, specific integrins may rely at least in part on Ras for intracellular signal transmission (Mainiero et al., 1997; Zhu et al., 1999); however, a potential role for Gab1SHP-2 in integrin signaling has not been systematically examined. SHP-2 does not appear to transmit signal from EGFR to Ras through the better characterized Shc, Grb2, and Sos pathways, but evidence suggests that Gab1 itself may (Shi et al., 2000). Additional studies are required to examine the relative contributions of each of these elements to epidermal growth and differentiation.
Our data suggest that a mechanistic process involving EGFR, Gab1, SHP-2, Ras, Raf, MEK, and MAPK may operate in a manner that is spatially confined to the basal layer of this stratified epithelium. Consistent with this possibility, EGFR, Ras, and active MAPK protein are localized in the undifferentiated basal layer of epidermis (Fukuyama and Shimizu, 1991; Dajee et al., 2002) (Fig. S1 available at http://www.jcb.org/cgi/content/full/jcb.200205017/DC1). Currently, available antibodies to Gab1 and SHP-2 do not function well in tissue immunostaining as judged by failure to give specific signals with skin tissue with either absence or overexpression of these proteins (unpublished data). However, we have shown here that both of these proteins are expressed in undifferentiated epidermal cells, and it has been demonstrated previously that the Gab1 promoter is active in the basal layer of epidermis (Itoh et al., 2000). Since the basal epidermal layer houses the proliferative pool of undifferentiated cells responsible for epidermal self-renewal, such a model of spatially localized action would predict that augmenting the function of components of this pathway in this location would enhance epidermal proliferation. The current work indicates that this prediction holds true for Gab1 and SHP-2. We and others have reported similar effects for Ras (Bailleul et al., 1990; Greenhalgh et al., 1993; Brown et al., 1998; Dajee et al., 2002).
Regarding other components of the Ras/MAPK pathway, epidermal expression of active MEK1 (Haase et al., 2001) also augments proliferation and inhibits differentiation. In further support of our model, interference with function of these proteins leads to diminished proliferation and epidermal hypoplasia. Examples of this include the observations reported here for Gab1 and SHP-2 and previously described EGFR-/- mice (Sibilia and Wagner, 1995; Threadgill et al., 1995) and transgenic mice overexpressing a dominant-negative Ras in epidermis (Dajee et al., 2002). In the case of the latter, hypoproliferation was only observed when dominant-negative Ras was targeted to the basal layer and not suprabasal layer cells, underscoring the spatial localization and basal layer cell intrinsic nature of this process. Together, these data support a model in which epidermal proliferation is influenced by the action of a signaling process involving EGFR, Gab1, SHP-2, Ras, Raf, MEK, and MAPK whose spatial localization helps divide epidermis into a proliferative, undifferentiated compartment and a postmitotic differentiating compartment.
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Materials and methods |
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Protein expression
Keratinocytes were lysed in lysis buffer (25 mM Hepes, pH 7.5, 150 mM NaCl, 1% NP-40, 10 mM MgCl2, 1mM EDTA, 10% glycerol) with protease inhibitors and denatured by boiling with 20 µg of extract loaded per lane. Antibodies were obtained from the following sources: Gab1 and pTyr (Upstate Biotechnology), SHP-2 and c-myc (Santa Cruz Biotechnology, Inc.), pan-Ras (Oncogene Research Products), involucrin and human keratin 1 (Babco), and phospho-MAPK and MAPK (Cell Signaling). Immunoblots were stripped and reprobed with antibodies to ß-actin (Santa Cruz Biotechnology, Inc.) as an additional control for loading and extract quality. Results were quantitated using a GS-710 Calibrated Imaging Densitometer (Bio-Rad Laboratories), and data were normalized to loading control for the same sample lane.
Immunoprecipitation and quantitation of active Ras
500 µg of cell lysate protein was precleared with equilibrated protein A beads (Sigma-Aldrich) and incubated with anti-Gab1 or antiSHP-2 antibody for 4 h. Immune complexes were precipitated with protein A/G beads (Sigma-Aldrich) and subjected to immunoblotting. The active Ras-GTP pull-down assay was performed under nonsaturated conditions as described (de Rooij and Bos, 1997). Briefly, 150 µl Escherichia coli GST-RBD lysate was incubated with 30 µl glutathione-sepharose beads (Amersham Biosciences) at room temperature for 30 min with shaking. After washing the sepharose beads, 500 µg of epidermal cell extract was added at 4°C for 1 h with shaking. After three washes, the samples were subjected to 12% SDS-PAGE. Levels of active Ras protein were detected by a pan-Ras antibody (Oncogene Research Products) and quantitated as noted above.
Histology and immunofluorescence
Genetically engineered human epidermis was regenerated on CB.17 scid/scid mice after gene transfer as described (Choate et al., 1996; Robbins et al., 2001). E17.5 Gab1-/- and wild-type embryonic mice skin was grafted following the same procedure. Four mice were grafted and analyzed per group. At 35 wk after grafting, skin tissue was excised and subjected to analysis. For histological examination, skin tissue was fixed in 4% paraformaldehyde overnight, embedded in paraffin, and 5-µm sections were stained with hematoxylin and eosin. For immunostaining, 5-µm skin cryosections were allowed to air dry for 30 min and permeabilized with cold acetone for 10 min. Sections were blocked with 10% horse serum for 1 h and treated with the primary antibody for 1 h at room temperature. Slides were then washed three times with PBS and incubated for 30 min with secondary antibodies. After three washes with PBS, slides were mounted in Vectashield (Vector Laboratories) and examined under a ZEISS 100M Axiovert microscope. The following panel of antibodies was used in immunostaining: antihuman K1, antimouse K10, and antimouse involucrin (Babco); antihuman Ki-67 (LabVision); antimouse Ki-67 (Dako); antiintegrin 6 (Chemicon); FITC-conjugated goat antimouse IgG, FITC-conjugated goat antirabbit IgG and FITC-conjugated rabbit antirat IgG (Sigma-Aldrich); and Cy3-conjugated goat antirat IgG (Jackson ImmunoResearch Laboratories).
Online supplemental material
Figs. S1 and S2 are available at http://www.jcb.org/cgi/content/full/jcb.200205017/DC1. Fig. S1 shows that Ras and EGFR are expressed in the undifferentiated epidermal basal layer. Immunostaining of normal human epidermal tissue with antibodies to Ras and EGFR (green) and the superbasal layer differentiation marker involucrin (red) are shown; the dotted line denotes the basement membrane zone. Fig. S2 shows the histology and immunostaining of proliferation marker Ki-67 and differentiation marker keratin 10 of Gab1 E17.5 embryo skin.
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Footnotes |
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* Abbreviations used in this paper: E, embryonic day; EGFR, EGF receptor; IGF, insulin-like growth factor-1; PDGF, platelet-derived growth factor; PH, pleckstrin homology; PI3K, phosphoinositide 3-kinase; SH, src homology.
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Acknowledgments |
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This research was supported by the United States Veterans Affairs Office of Research and Development and grant nos. AR43799, AR45192, AR44012 from the National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health.
Submitted: 6 May 2002
Revised: 11 July 2002
Accepted: 29 August 2002
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