Epidermal Ras blockade demonstrates spatially localized Ras promotion of proliferation and inhibition of differentiation

Abstract

While important in carcinogenesis, the role of Ras in normal self-renewing tissues such as epidermis is unclear. To address this, we altered Ras function in undifferentiated and differentiating epidermal layers. Ras blockade within undifferentiated basal epidermal cells leads to decreased integrin expression, diminished growth capacity and induction of differentiation. Ras blockade in post-mitotic suprabasal epidermis exerts no effect. In contrast, regulated Ras and Raf activation inhibits differentiation. These findings indicate that spatially restricted Ras/Raf signaling divides epidermis into an undifferentiated proliferative compartment and a differentiating post-mitotic compartment and suggest a new role for Ras in tissue homeostasis.

Introduction

Normal epidermis is a self-renewing tissue that maintains homeostasis via precise control of cellular proliferation. This stratified epithelial tissue contains a basal layer of mitotically active cells which cease proliferating, migrate outwards and activate expression of terminal differentiation genes. Previously identified positive growth influences, including those acting via receptor tyrosine kinases and integrins, may function in part through Ras GTPases (Shields et al., 2000).

Depending on the cell type and experimental conditions, Ras can either promote or inhibit differentiation via evolutionarily conserved effectors that include the Raf/MEK/MAP kinase cascade (Ewen, 2000; Shields et al., 2000). In muscle cells, it appears that Ras predominantly inhibits differentiation (Konieczny et al., 1989; Olson et al., 1987). In contrast, evidence that Ras promotes growth arrest and cellular differentiation has been obtained in cultured neuronal, adipocyte and myeloid cell lines (Crespo and Leon, 2000; Kozma et al., 1993; Qui and Green, 1992). In the case of myeloid cell lines, however, strong Ras signal has shown divergent results with respect to differentiation, possibly as a result of differences in cell culture conditions (Crespo and Leon, 2000). Contradictory data have also arisen regarding Ras effects in adipocytes indicating that Ras can induce adipocyte proliferation under some conditions instead of differentiation (Ruiz-Hidalgo et al., 1999). Therefore depending on the experimental setting in vitro, Ras can induce opposite effects, frustrating confident assignment of a physiologic role for Ras in a given tissue and underscoring the need for in vivo studies.

In skin, a role for Ras in epidermal carcinogenesis has been suggested. Activating mutations in Ras occur in neoplasms such as human squamous cell carcinoma (SCC) (Pierceall et al., 1991) and in experimental SCC induction in mice (Yuspa, 1994). Transgenic mice expressing active Ras mutants via promoter constructs that target suprabasal epidermal cells produce papillomas at sites of wounding over time that do not progress to carcinomas (Bailleul et al., 1990; Greenhalgh et al., 1993b). Basal layer promoter-driven active Ras expression from the K5 promoter, however, leads to growths that progress to undifferentiated neoplasias (Brown et al., 1998). These data point to Ras as a factor in epidermal cancer, however, they do not address a potential role for Ras in the physiologic control of growth and differentiation in epidermis.

In epidermal cells, conflicting data exists on the effect of Ras and its Raf/MEK/MAPK effectors on growth and differentiation. Several in vitro studies have reported that the pathway supports cellular proliferation and resists differentiation (Mainiero et al., 1997; Zhu et al., 1999). In contrast, other work demonstrated that constitutive Ras/Raf activation induces growth arrest and features of terminal differentiation in cultured murine keratinocytes (Lin and Lowe, 2001; Roper et al., 2001). In the latter case, two studies differ in reporting that growth arrest is either p19ARF-dependent (Lin and Lowe, 2001) or independent (Roper et al., 2001). In contrast to this, prior studies in murine keratinocytes had demonstrated that viral expression of Ras inhibits differentiation and stimulates growth (Dlugosz et al., 1994). Thus, work to date in cultured epidermal cells provides opposing and often directly contradictory findings regarding Ras impacts on epidermal growth and differentiation.

Published work to date is subject to several major challenges that may account for these disparities. First, most studies rely on inducing strong constitutive activation of the Ras/Raf/MEK/MAPK cascade components in a manner unlikely to occur in physiologic signaling. It is well known that this can produce discrepancies because Ras effects can differ dramatically depending on both signal strength and duration (Marshall, 1999). Second, these experiments were constrained to cultured cells which lack factors found in vivo that are necessary for normal signal integration such as matrix cues. Finally, similar to transgenic mice studies of Ras in epidermal neoplasia, these experiments focused only on strong gain-of-function unbalanced by loss-of-function studies. If Ras and its effectors play a physiologic role in epidermis, then approaches to block Ras may avoid potentially confounding effects of ectopic overexpression of constitutively active mutants.

Here we have used two general approaches to identify a spatially localized functional role for Ras in epidermis. First, we have blocked Ras function in vivo in both undifferentiated and differentiating epidermis using targeted expression of a dominant-negative Ras mutant. Second, we have both activated and blocked Ras/Raf activity at a range of signal strengths in primary epidermal cells. Our findings indicate that Ras/Raf signaling acts in a dose-dependent manner within undifferentiated cells to support proliferation while actively opposing entry into the post-mitotic differentiation pathway. These data support a model in which spatially restricted Ras activity divides epithelium into two distinct cellular compartments and suggest a major role for Ras in epidermal homeostasis.

Results

Altering epidermal Ras activity in transgenic mice

To study the effects of altering epidermal Ras function in vivo, we endeavored to inhibit Ras activity in this tissue compartment. Ras proteins demonstrate functional redundancy from yeast to mammals (Shields et al., 2000). Because of such redundancy, we used a dominant interference approach. First, we confirmed the ability of the dominant-negative Ras inhibitory mutant, RasN17, to inhibit Ras activation in epidermal cells. To do this, we directly measured the levels of active GTP-bound Ras in cells expressing either RasN17 or the constitutively GTP-bound active RasV12 mutant positive control. Both Ras mutants were expressed at similar levels (Figure 1a, lanes 3 and 4, middle panel) in primary human keratinocytes but exerted the expected opposite effects on Ras activation. RasN17 decreases levels of endogenous active Ras, confirming its ability to block Ras in epidermal cells (Figure 1a, lanes 1 and 3, top panel). In lacZ transduced controls, addition of epidermal growth factor (EGF) elevated GTP-bound Ras levels but to lower levels than those seen with RasV12 expression 24 h post-transduction (Figure 1a, lanes 2 and 4, top panel).

Figure 1
figure1

Altering epidermal Ras activity. (a) Active Ras assay. Primary human keratinocytes were transduced with retroviral expression vectors for dominant-negative RasN17, constitutively active RasV12 and lacZ control. 24 h later, active Ras was recovered by GST-Raf Binding Domain (RBD) affinity precipitation and detected by anti pan-Ras antibody. Note inhibition of GTP-bound Ras by RasN17 below the level of unstimulated control and induction by RasV12 (top, panel), although both show similar Ras overexpression (total Ras, second panel). lacZ transduced cells treated with EGF confirm the ability to detect induction of GTP-bound active Ras in these cells. (b) Altering epidermal Ras activity in transgenic mice. Mice transgenic for keratin 14 (K14) or keratin 1 (HK1)-targeted expression of dominant-negative RasN17 (N17) and active RasV12 (V12) were generated. Spatially localized expression of Ras proteins was confirmed immunochemically. Note expression of Ras protein predominantly in the basal layer of normal tissue, its suprabasal location in HK1-N17 tissue and enhanced basal layer expression in K14-N17 tissue. NL=normal littermate control; control=background obtained with no primary antibody; dotted line denotes the epidermal basement membrane zone (BMZ). Scale bars=30 μm. (c) Histology of K14-RasN17 and K14-RasV12 transgenic mice at birth. Note epidermal thinning and hyperkeratosis in K14-RasN17 skin in contrast to massive hyperplasia with absence of normally differentiated granular and cornified layers in K14-RasV12 tissue. Note the entirely normal appearance of HK1-N17 tissue. Scale bars=60 μm. (d) Proliferation marker expression in K14-RasN17 and wild-type littermate control (NL). In normal newborn epidermis (NL), note Ki-67 expression (brown nuclei, arrows) in almost all basal layer cells residing above the BMZ (dotted line) in contrast to spotty expression in K14-N17 skin. E=epidermis; HF=hair follicle. Scale bars=30 μm. (e) Lack of increased apoptotis in K14-N17 epidermis. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL) positive cells are noted with arrows. Note lack of increase in K14-N17 tissue compared to normal control. Scale bars=30 μm

Dominant-negative Ras was then targeted to the epidermis of transgenic mice. Two different promoters were used, the basal layer keratin 14 (K14) promoter that targets undifferentiated cells (Vassar et al., 1989) and the suprabasal layer keratin 1 (HK1) promoter targeting differentiating cells (Greenhalgh et al., 1993a). Genetically matched active K14-RasV12 transgenic mice controls were also generated (Table 1). As confirmed by immunostaining with anti-Ras antibodies recognizing both wild-type and mutant proteins, these promoters target Ras proteins to either undifferentiated basal layer cells or to those in the differentiating suprabasal layers as expected (Figure 1b). Severely affected K14-RasN17 mice displayed thin, shiny skin and limb defects (data not shown), dying within 1–3 days after birth while all HK1-RasN17 mice were entirely normal, suggesting that the primary site of epidermal Ras function resides within the basal layer. The differences in K14-RasN17 phenotypes within the same line correlated with gene copy number. In contrast to the massive epidermal hyperplasia with absence of differentiating layers that is seen in K14-RasV12 mice, which die at birth, newborn RasN17 epidermis was thin with an increase in thickness of the terminally differentiated stratum corneum (Figure 1c) and decreased numbers of actively cycling cells (Figure 1d). K14-RasN17 mice displayed normal transepidermal water loss (data not shown), confirming that barrier perturbation does not account for these findings.

Table 1 Summary of transgenic mice

Mildly affected K14-RasN17 mice expressing lower levels of epidermal RasN17 survived to adulthood, however, these mice spontaneously developed widespread superficial skin erosions by 6 weeks of age (data not shown). These erosions were not due to trauma or blistering and were characterized by non-healing loss of viable epidermis associated with infection and death. These findings were not associated with changes in the number of TUNEL(+) cells compared to control (Figure 1e), indicating that Ras blockade may affect proliferative epidermal self-renewal without affecting cell death.

Ras blockade leads to decreased epithelial cell proliferative capacity

The widespread epidermal erosions that develop in surviving transgenic mice with epidermal Ras blockade raise the possibility that Ras may help sustain proliferative capacity of epidermal progenitor cells. To test this, we used high efficiency (>99%) retroviral gene transfer without drug selection to primary human keratinocytes (Deng et al., 1998; Robbins et al., 2001). Ras function was altered by expressing wild-type or dominant-negative Ras and then the proliferative capacity of resulting clones was examined. High levels of active RasV12 have been observed to cause growth arrest of primary cells (Hahn et al., 1999; Serrano et al., 1997), including primary keratinocytes (Lin and Lowe, 2001). Therefore we used wild type H-Ras to control for induced protein expression and other potential non-specific effects as it does not produce strong levels of Ras signal unless activated by additional stimuli (data not shown). In keratinocytes seeded at low density (Zhu et al., 1999), Ras blockade greatly diminished the proportion of highly proliferative keratinocyte holoclones and increased the per cent of abortive colonies of <32 cells (Figure 2), consistent with a need for Ras in epithelial proliferative capacity.

Figure 2
figure2

Ras blockade leads to decreased epithelial cell proliferative capacity. Methylene blue stain of primary keratinocytes after high efficiency retroviral transduction with expression vectors for dominant-negative RasN17 (N17), wild type Ras (WT) and lacZ. The per cent of abortive colonies containing <32 cells obtained from triplicate independent transductions and cell platings±s.d. is noted below each representative plate

Ras promotes epidermal integrin expression

Both β4 and β1 integrin have been implicated as important in epidermal cell proliferative capacity and stem cell function (Li et al., 1998; Watt, 1998). To determine if Ras promotes expression of such progenitor markers, we studied expression of β4 and β1 integrin as a function of Ras activity. While Ras blockade is associated with decreased expression of β4 and β1 integrin, active Ras increases expression both in transgenic epidermis (Figure 3) and in primary cells in culture transduced at high efficiency with RasV12 retrovectors (data not shown). In the latter case, integrin upregulation occurs even during cell-cycle arrest induced by strong constitutive Ras signaling, indicating that integrin induction by Ras is not merely a byproduct of enhanced cellular proliferation.

Figure 3
figure3

Ras promotes epidermal integrin expression. β1 and β4 integrin expression was detected by immunostaining K14-RasN17 (N17), K14-RasV12 (V12) and normal littermate (NL) skin. Note decreased β1 and β4 integrin expression in K14-RasN17 skin and increased expression in K14-RasV12 skin extending up into suprabasal cells. Scale bars=30 μm

Ras suppresses epidermal differentiation

The histologic and cell proliferative findings above indicate that Ras inhibits differentiation in non-transformed epidermal cells and that Ras blockade allows unopposed differentiation to proceed along with a loss of proliferative capacity. Consistent with this hypothesis, Ras blockade leads to epidermis with high levels of differentiation markers such as K10, K1, loricrin, filaggrin and involucrin while epidermal Ras activation inhibits expression of these markers (Figure 4a and data not shown). To further examine this process, we studied Ras pathway impacts on differentiation in primary human keratinocytes in vitro. Normal cells were transduced with either RasV12, RasN17 or lacZ control. While active RasV12 inhibits both basal and calcium-induced levels of differentiation markers K1, K10 and involucrin, dominant-negative RasN17 dramatically increases their expression (Figure 4b and data not shown). In both cases, altering Ras function overrides the extracellular calcium differentiation signal, suggesting a potentially dominant role for Ras in cellular integration of differentiation stimuli and the need for a continual Ras signal to prevent cells from entering the terminal differentiation pathway.

Figure 4
figure4

Ras suppresses impacts differentiation and Ras blockade leads to induction of differentiation. (a) Expression of epidermal differentiation markers filaggrin and keratin 10 (K10) was detected by immunostaining. Note increased expression of differentiation markers in K14-RasN17 skin and decreased expression in K14-RasV12 skin. Scale bars=20 μm. (b) Cellular expression of epidermal differentiation marker keratin 1 (K1) protein in primary keratinocytes transduced with retrovectors for dominant-negative RasN17, active RasV12 and lacZ control and grown in basal media [−] or induced to differentiate with 1.5 mM calcium [+]. Antibodies to β-actin were used as a control for extract loading and quality. (c) Regulated dominant-negative Ras induces differentiation protein expression in a dose-dependent manner. Cells were transduced with ER:RasN17 and 4-hydroxytamoxifen (4OHT) added at 0, 0.5, 2, 5, 25 nm to lanes 3, 4, 5, 6 and 7, respectively. (d) Regulated active Ras inhibits differentiation protein expression in a dose-dependent manner. Cells were transduced with inducible ER:RasV12 and 4OHT added at 0, 0.5, 1, 5, 10 nm to lanes 3, 4, 5, 6 and 7, respectively

Control of differentiation via regulated activation and blockade of Ras

While demonstrated to inhibit differentiation in murine keratinocytes in earlier studies (Roop et al., 1986; Yuspa et al., 1983), Ras has also been observed to induce differentiation (Lin and Lowe, 2001; Roper et al., 2001). These latter studies where Ras promotes differentiation, however, may be confounded by the fact that cells underwent drug selection prior to analysis. This is important because Ras/Raf effects can vary with dose and drug selection can promote keratinocyte differentiation (Stockschlader et al., 1994). To avoid this pitfall, we expressed regulated active and dominant Ras mutants in primary human epidermal cells via a high efficiency gene transfer approach without drug selection (Deng et al., 1998).

Regulated Ras mutants were generated via N-terminal fusion of the estrogen receptor (ER) ligand binding domain, an approach used successfully to generate C-terminal Raf1 fusions conditionally responsive to 4-hydroxytamoxifen (4OHT) (Woods et al., 1997). Addition of 4OHT at a range of doses to cells expressing ER:RasV12 and ER:RasN17 was used to either activate or block Ras signal transduction, respectively. Regulated Ras function was confirmed by levels of phosphorylated downstream MAP kinases ERK1 and ERK2. Ras inhibition led to differentiation in a dose-dependent manner (Figure 4c). Induction of active Ras, on the other hand, inhibited differentiation marker expression, also in a dose-dependent manner (Figure 4d). In agreement with in vivo findings above, these regulated data suggest that Ras acts as a potent regulator of epidermal differentiation and that it inhibits entry into the differentiation program.

Downstream Ras effector pathways

Ras action can proceed via direct binding to initiators of its three major downstream effector cascades, Raf, RalGDS and PI3K (Gille and Downward, 1999; Shields et al., 2000). Which of these effector pathways contribute to Ras effects in epidermal cells? To begin to address this question, we utilized Ras mutants which predominantly induce either Raf, RalGDS or PI3K (White et al., 1995). When expressed in primary keratinocytes, the Raf-activating mutant was most potent at inhibiting differentiation gene expression (Figure 5a), suggesting that signaling through Raf may be important in Ras inhibition of differentiation. Because Ras does not activate the PI3K effector pathway in all cell types, we confirmed that Ras activity in primary epidermal cells leads to activation of the PI3K downstream effector, Akt, as measured by phosphorylation (Figure 5b). To further examine the role of Raf and PI3K effectors, we incubated primary cells with inhibitors of either the Ras/Raf downstream target MEK or PI3K. MEK inhibition with two distinct inhibitors strongly induced differentiation gene expression while PI3K inhibition by two different agents blocked differentiation marker expression (Figure 5c). This further supports a role for Ras action via Raf/MEK and indicates that known Ras effectors may promote divergent and even opposite effects on differentiation.

Figure 5
figure5

Ras effector impacts on differentiation. (a) Ras effector pathway mutants impact on differentiation marker expression. Extracts from primary keratinocytes expressing RasV12 also mutant at S35 (Raf pathway), G37 (RalGDS pathway) or C40 (PI3K pathway) were immunoblotted with antibodies to K1. (b) Active Ras induces activating phosphorylation of the PI3K effector Akt. Extracts from cells transduced with RasV12 and lacZ controls harvested at 24 and 48 h post-transduction were immunoblotted with antibody recognizing phosphorylated serine residue 473 on Akt. Note increased amounts of phosphorylated Akt as a function of Ras at each timepoint. (c) Impact of pharmacologic inhibition of MEK and PI3K on differentiation marker expression. Primary keratinocytes were incubated with MEK inhibitors PD098059 and U0126 or the PI3K inhibitors Wortmanin and LY294002 then K1 protein expression determined by immunoblotting. (d) Active MAPK in epidermis as a function of Ras activity. Immunofluorescence staining with polyclonal antibodies to phosphorylated ERK1 and ERK2 in transgenic mice; note basal layer expression in normal epidermis that is abolished in RasN17 and augmented in RasV12 tissue. Scale bars=10 μm

Consistent with these findings, phosphorylated active MAPKs, well characterized targets of Ras/Raf/MEK, are detected predominantly in basal layer cells within wild-type mouse epidermis (Figure 5d). While displaying comparable amounts of total MAPK, K14-RasN17 transgenic mouse epidermis shows minimal active MAPK, confirming inhibition of the Ras/Raf/MAPK cascade by RasN17 (Figure 5d and data not shown). In contrast, K14-RasV12 epidermis shows significant increases in active MAPK expression, both in the basal layer as well as in suprabasal layers where the K14 promoter is expressed within hyperproliferative epidermis (Figure 5d). In HK1-RasN17 epidermis, active MAPK expression is not observed in suprabasal layers as expected (data not shown). These data suggest that spatially restricted Raf/MEK/MAPK action within the basal layer of epidermis contributes to Ras epidermal effects and confirm transgenic epidermis with features of either blocked or enhanced function of this effector pathway.

Raf inducibly stimulates epithelial cell proliferation and inhibits differentiation

In contrast to our in vivo data indicating Ras promotes epidermal growth, we observed irreversible growth arrest after overexpression of constitutively active Ras and Raf1 in normal keratinocytes via retroviral transduction in vitro (data not shown), similar to that reported in other epithelial and non-epithelial cell types (Hahn et al., 1999; Serrano et al., 1997). These data suggest that cells possess a protective cell growth arrest mechanism against oncogenic Ras/Raf activation. The effects of Ras and its effectors, however, are known to vary depending on signal magnitude and duration (Halfar et al., 2001; Marshall, 1999; Roovers and Assoian, 2000). Retroviral gene transfer produces up to 100-fold more expression of delivered genes in our hands than does expression via keratin promoters in transgenic epidermis. This may account for the disparity observed between Ras growth effects in the two settings and suggested a dosage effect.

To see if signal strength could account for this disparity, we modulated signal magnitude through the Ras downstream effector Raf. To do this, we generated a retrovirus expressing a regulated Raf-1 fused to a mutant ligand binding domain of the murine estrogen receptor that can be regulated by exogenous tamoxifen (4OHT) (Woods et al., 1997). While 4OHT at low and high dose exerted no growth effects on control keratinocytes, in Raf:ER expressing keratinocytes low doses produced proliferation. In contrast, higher 4OHT doses inhibited cell growth (Figure 6a), indicating that Ras/Raf effects on growth of epidermal cells are dose-dependent. Raf also exerted signal strength-dependent suppression of calcium induced differentiation markers (Figure 6b,c), similar to inducible Ras.

Figure 6
figure6

Raf inducibly stimulates epithelial cell proliferation and inhibits differentiation. (a) Cell proliferation at low (10 nM) and higher (100 nM) concentrations of 4OHT after high efficiency transduction of primary keratinocytes with an inducible Raf-1:ER fusion. (b) Expression of differentiation marker K1 and active MAPKs ERK1 and ERK2 as a function of inducible Raf activity. (c) Raf activation inhibits K1 expression in a dose-dependent manner. (d) Blockade of Ras function through RasN17 is reversed by inducible activation of Raf:ER

The RasN17 mutant, while selective for Ras blockade, has also recently been shown capable of acting independently of Ras in some cell types by inhibiting the downstream transcription factor Elk-1 in NIH3T3 cells (Stewart and Guan, 2000). If RasN17 triggers differentiation through Ras blockade, then its effects should be reversed by activating pathway components downstream of Ras such as Raf. Inducible Raf thus offers the opportunity to determine whether RasN17 effects on epidermal differentiation occur via Ras inhibition or through unrelated actions on downstream effectors such as Elk-1. To test this, we co-expressed RasN17 with Raf:ER in primary human keratinocytes. Raf induction by 4OHT in this setting completely suppresses differentiation marker expression enhanced by RasN17 (Figure 6d). This confirms that RasN17 differentiation effects can be circumvented by activating downstream effectors of the Ras/Raf cascade. Furthermore, we confirmed that RasN17 exerts no effect on Elk-1 expression or Elk-1-driven reporter gene activity in keratinocytes (M Yamazaki et al., unpublished). These data suggest that the differentiation effects of the RasN17 mutant occur via blockade of the Ras/Raf pathway rather than through unrelated downstream mechanisms.

Discussion

Our findings suggest a model in which homeostatic control of epidermal growth and differentiation involves spatially localized Ras action that divides the epidermis into undifferentiated and differentiating cellular compartments (Figure 7). This model posits that Ras acts primarily within the basal layer cell compartment where proliferating cells reside. It predicts that its blockade there would lead to the findings of hypoplasia, induction of differentiation and proliferative exhaustion while its blockade in differentiating cells would exert no effect, consistent with our findings. In this setting, Ras signaling is necessary to maintain cells in the proliferative, undifferentiated state and a loss of Ras function leads to a decrease in proliferative capacity and subsequent entry into the terminal differentiation pathway. This model is based on our observation that basal layer Ras blockade leads to a thin, highly differentiated epidermis that appears to undergo spontaneous loss of proliferative self-renewal, resulting in widespread skin erosions. This model is also based on the observation that Ras activation produces the opposite picture, a highly proliferative undifferentiated epidermis expressing key integrins characteristic of the epidermal progenitor cell compartment.

Figure 7
figure7

Summary model of epidermal Ras action. Ras normally acts in progenitor cells [P] within the basal layer to support proliferative capacity and oppose differentiation (middle panel). Ras gain of function promotes expression of integrins and expands the compartment of progenitor cells of high proliferative capacity while opposing terminal differentiation (left panel). In the absence of Ras action, the progenitor cell population is depleted, integrin expression is decreased and cells enter the post-mitotic differentiated cell compartment (right panel). Unshaded cells are undifferentiated cells with active Ras signaling while shaded cells are post-mitotic. The intensity of shading reflects the degree of differentiation with the darkest cells the most differentiated

Ras blockade in stratified epithelium

We have attempted to block Ras pathway action in epidermis using a well characterized dominant interference approach. We have done this because of Ras redundancy in mammals, as evidenced by a lack of overt skin phenotypes in H-Ras−/− (Ise et al., 2000), N-Ras−/− (Umanoff et al., 1995), H-Ras−/− :N-Ras−/− (Esteban et al., 2001) mice and K-Ras−/− :N-Ras−/− chimeras (Johnson et al., 1997). Mice in which all three mammalian Ras isoforms are disrupted have not been successfully produced due to embryonic lethality which is even seen in K-Ras−/− : N-Ras−/− animals (Johnson et al., 1997). The fact that all Ras isoforms must be deleted in yeast in order to see a phenotype suggests that truly null Ras tissue in mammals may only be obtained by removing at least H, N and K-Ras simultaneously, and possibly additional Ras-related genes as well (Shields et al., 2000). Consistent with Ras redundancy, there is a compensatory increase in other Ras proteins in H-Ras−/− skin (Ise et al., 2000). The effectiveness and specificity of blockade by RasN17 has been confirmed in a number of ways. First, RasN17 decreases levels of endogenous GTP-bound Ras in epidermal cells. Second, this dominant-negative mutant also decreases levels of active MAPKs both in vitro and in vivo, indicating it inhibits the Ras/Raf/MEK/MAPK cascade. Third, pharmacologic blockade of the cascade using inhibitors to MEK recapitulates the effects of RasN17. Fourth, RasN17 effects on differentiation can be reversed by inducible Raf, suggesting that RasN17 does not directly activate differentiation in a Ras-independent manner. Finally, in epidermal cells RasN17 fails to alter the expression and transcriptional activity of Elk-1, a Ras-independent effect which has been observed in NIH3T3 cells. These findings indicate that RasN17 faithfully blocks the Ras/Raf/MEK/MAPK cascade in epidermal cells.

Spatial localization of Ras action

Although all Ras proteins are expressed in skin tissue extracts (Ise et al., 2000), the importance of the localization of Ras function within epidermis has not been characterized. Our data provides several lines of evidence to suggest that Ras regulation of growth and differentiation is spatially confined to basal epithelial cells attached to the basement membrane. First, we observe Ras protein is localized to basal layer cells within normal epidermis. Second, the use of promoters targeting differentiated versus undifferentiated cells demonstrated dramatically different impacts of altering Ras function depending on which layer of epidermis was targeted. Activating or blocking Ras function in basal layer cells produces dramatically opposing effects. On the other hand, altering Ras function in the suprabasal post-mitotic compartment via either activation (Bailleul et al., 1990; Greenhalgh et al., 1993b) or blockade as we have shown here, fails to directly impact the tissue, although in the case of the former, papillomas appear over time at sites of trauma. Finally, in addition to our promoter-targeted findings with transgenic mice, we have observed that phosphorylated MAPK is localized primarily within cells of the basal layer of normal epidermis. Such basal layer active MAPK is abolished in K14-RasN17 tissue, suggesting that a major site of Ras signaling may reside in this location and that Ras provides important regulatory stimuli for MAPK in epidermis.

Regulation of cellular proliferative capacity

Apparently contradictory data exists regarding the ability of the Ras/Raf/MAPK cascade to either inhibit growth of epidermal cells (Lin and Lowe, 2001; Roper et al., 2001) or to promote it (Roop et al., 1986; Zhu et al., 1999). We report both effects here. We have demonstrated that growth effects in epidermal cells are dependent on signal strength. Retroviral RasV12 expression is sufficient to cause growth arrest in epidermal cells. Short-term RasV12 expression leads to significantly elevated levels of active GTP-bound Ras. Such strong constitutive Ras/Raf signaling is known to trigger senescence via proteins such as ARF, p16INK4A and p53 in a wide variety of other cell types in what has been postulated to serve as a protection against inappropriate signaling by oncogenic Ras (Elenbaas et al., 2001; Ferbeyre et al., 2000; Hahn et al., 1999; Malumbres et al., 2000; Serrano et al., 1997). In this regard, we were unable to detect any differences in the expression of ARF or p53 proteins during growth arrest induced by high levels of Ras/Raf signaling (data not shown), indicating that this process may involve other cell cycle regulatory genes in human epidermal cells. We believe that prior studies in epidermal cells reporting growth inhibition by Ras/Raf are observing a protective response against high level signaling by oncogenic Ras/Raf and that this does not reflect the impact of physiologic activation in the tissue. Consistent with this possibility is the fact that these efforts used either retroviral overexpression of RasV12 (Lin and Lowe, 2001) or a strongly induced Raf:ER fusion with 200 nm 4OHT (Roper et al., 2001), twice the maximal level at which we observed growth inhibition.

Our data indicate that Ras function is required for epidermal self-renewal because Ras blockade in vivo leads to epidermal hypoplasia, widespread skin erosions and death. In support of this, Ras inhibition depletes the proliferative capacity of primary epidermal cells and low signal strengths of Raf activation stimulate proliferation. These findings are consistent with a recent study involving targeted disruption of the α-catenin gene which increases epidermal Ras and MAPK activity and epidermal proliferation in vivo (Vasioukhin et al., 2001). Similar findings were also recently reported in both human psoriasis and a transgenic mouse model with psoriatic features generated by suprabasal expression of integrin β1. In both cases induction of active MAPK was observed with hyperproliferation and inhibition of terminal differentiation; expression of active MEK in keratinocytes cultured in vitro on dermal equivalents induced similar features (Haase et al., 2001). A role for Ras/Raf/MEK/MAPK in epidermal proliferation is further supported by the finding that Ras supports keratinocyte proliferation in culture in a manner involving α6β4 integrin and Shc (Mainiero et al., 1997). In further agreement is work demonstrating that MAPK function supports both integrin expression and proliferation by epidermal progenitors (Zhu et al., 1999). Consistent with a role for Ras effectors in inducing integrins that support proliferative capacity, another recent study demonstrated that Raf and MEK increase expression of other integrins, namely α6 and β3, in NIH3T3 and endothelial cells (Woods et al., 2001). Acting synergistically with integrins, pro-proliferative growth factors such as EGF are the best characterized activators of Ras in epithelial cells (Mainiero et al., 1997), further supporting a role for Ras in epidermal growth promotion, however, the precise relationship of Ras and integrin signaling in epithelia remain to be determined. Finally, analysis of Raf targets in breast epithelial cells via gene expression profiling demonstrated that Raf's primary effects on growth regulatory genes were to induce genes promoting cellular proliferation (Schulze et al., 2001). Taken together, these data are consistent with a physiologic role for Ras in maintaining the proliferative capacity of epithelial progenitors in a process involving the Raf/MEK/MAPK effector cascade.

Regulation of differentiation

Ras/Raf signaling has been implicated in promoting differentiation of epidermal cells in some studies (Lin and Lowe, 2001; Roper et al., 2001) and opposing it in others (Dlugosz et al., 1994; Zhu et al., 1999). These prior effects have relied on in vitro analysis of Ras differentiation effects, which can generate conflicting results depending on the cell line and culture system used (Shields et al., 2000). In this regard, studies reporting differentiating effects of Ras/Raf occurred after the profound stress of cellular drug selection using agents such as G418 that promotes keratinocyte differentiation (Stockschlader et al., 1994).

Based on gain and loss of function experiments, both in vitro and in vivo, we believe that Ras signaling actively inhibits epidermal differentiation in cells early in the differentiation pathway. If this is true, then activation by a number of approaches should inhibit differentiation and blockade by several means should induce it, consistent with our observations. In vivo, expression of active Ras inhibits epidermal differentiation markers and leads to absence of normal differentiated granular and cornified layers. In vitro, unselected primary epidermal cells in which Ras and Raf signaling is activated at a range of signal strengths demonstrate inhibition of differentiation gene expression in a dose-dependent manner. Ras blockade in transgenic epidermis, on the other hand, induces high levels of differentiation markers and prominent stratum corneum formation suggesting that Ras inhibition leads to enhanced differentiation. Consistent with this, RasN17 expression in primary epidermal cells also induces differentiation markers and this can be triggered via an inducible ER:RasN17 fusion as well. Finally, pharmacologic blockade of MEK leads to differentiation gene induction. Taken together, these in vivo and in vitro data indicate that Ras signaling actively inhibits epidermal differentiation.

What is the role of downstream Ras effectors in inhibiting epidermal differentiation and are they merely a byproduct of those promoting proliferative capacity? Recent studies in transformed epithelial cells such as the aneuploid HaCaT cell line offer additional avenues whereby Ras effectors may oppose differentiation, such as by inhibiting intracellular calcium level elevation (Shi and Isseroff, 2000). Other studies with the HaCaT line indicate that MAPK is induced transiently during calcium-induced differentiation in a Ras-independent fashion (Schmidt et al., 2000). However, the majority of the latter data were generated using an aneuploid transformed cell line, a setting where Ras/Raf effects can differ significantly from non-transformed cells (Shields et al., 2000). Transient MAPK activation may be mechanistically distinct from that induced by Ras signaling via factors such as EGF. This is consistent with our findings as EGF activated Ras and inhibited the differentiation marker involucrin even in transformed HaCaT cells (Schmidt et al., 2000). Furthermore, divergent Ras effectors could exert differing effects on differentiation. In support of this, the epidermal differentiation marker cystatin A has recently been shown repressed by Ras/Raf/MEK/MAPK in human keratinocytes, consistent with our findings here, but induced by the alternate MEKK1/MKK7/JNK pathway (Takahashi et al., 2001). Our data supports such divergent effects by demonstrating that PI3K blockade inhibits differentiation marker expression. The definitive understanding of the specific roles of individual Ras effectors and how their specific effects are integrated in cellular decision-making regarding differentiation awaits further study.

In summary, our findings suggest that the Ras/Raf signaling cascade acts in a spatially restricted and signal strength-dependent fashion to promote epidermal proliferative capacity and to actively oppose the onset of differentiation. These data have implications for future efforts to understand the regulation of epithelial growth and differentiation and in developing new therapeutics for disorders of these processes.

Materials and methods

Cell growth and gene transfer

Amphotropic retrovectors were produced and human keratinocyte cell culture and gene transfer performed as described (Choate et al., 1996b; Deng et al., 1998; Kinsella and Nolan, 1996). Sequences for RasN17 (Feig and Cooper, 1988) and RasV12 (Serrano et al., 1997) were subcloned into the EcoRI site of LZRS (Kinsella and Nolan, 1996). The regulated Raf1 Raf:ER vector was generated by subcloning the XhoI-EcoRI fragment of constitutively active RafDD (Woods et al., 1997) and the EcoRI-NotI fragment of 4OHT responsive mutant estrogen receptor α, ERTM (Pelengaris et al., 1999) into XhoI-NotI sites of LZRS. The ER-Ras construct was made by subcloning the BamHI-EcoRI fragment of ERTM in frame upstream of RasV12 and RasN17 in the LZRS. High efficiency gene transfer was verified by immunofluorescence and Western blotting as previously described (Deng et al., 1998) using antibodies to Ras and ERα (Santa Cruz). MEK inhibitors were incubated with pre-confluent primary human keratinocytes for 24 h; PD098059 (20 μm; Calbiochem), U0126 (10 μm; Promega). For EGF studies, EGF was added at 50 ng/ml for 5 min.

Generation of transgenic animals

Sequences encoding RasN17 (Feig and Cooper, 1988) and RasV12 (Serrano et al., 1997) were subcloned downstream of a 2075-bp human keratin 14 (K14) promoter construct, which targets expression to keratinocytes within the basal epidermal layer (Vassar et al., 1989), and used to produce transgenic mice. Sequence encoding RasN17 and was also subcloned downstream of human keratin 1 (HK1) promoter, a construct that targets gene expression to keratinocytes in the suprabasal epidermal layers (Greenhalgh et al., 1993a), and used to generate additional transgenic mice. Transgene integration and copy number was confirmed by PCR followed by Southern blot analysis.

Protein expression

The following antibodies were used in immunoblotting and in immunostaining (Seitz et al., 1998); rabbit anti-Ras (Santa Cruz, 1 : 200), rabbit anti-MAPKs p44 ERK1 and p42 ERK2 (New England Biolabs, 1 : 50), rabbit anti-phosphoserine 473 Akt (Cell Signaling, 1 : 1000), rabbit anti-phosphorylated-MAPKs p44 ERK1 and p42 ERK2 (Promega, 1 : 1000), rat anti-α-6 integrin (Chemicon, 1 : 200), mouse anti-filaggrin (Dale et al., 1987) (Biomedical Technologies Inc, 1 : 50), mouse anti-K10 (Ivanyi et al., 1989) (DAKO, 1 : 50), anti-K1 (Babco, 1 : 2500), anti-loricrin (Babco, 1 : 200), FITC-conjugated goat-anti-mouse IgG (Sigma, 1 : 150), FITC-conjugated goat-anti-rabbit IgG (Sigma, 1 : 150), TRITC-conjugated rabbit-anti-rat IgG (Sigma, 1 : 200). For immunoblotting (Choate et al., 1996a), blots were stripped and re-probed with antibodies to β-actin (Santa Cruz, 1 : 4000) as an additional control for loading and extract quality. The active Ras pull-down assay was performed as described (de Rooij and Bos, 1997).

Flow cytometry

Forty-eight hours post transduction cells were resuspended in FACS buffer (PBS buffer containing 2% BSA, 0.1% sodium azide, 1 mM CaCl2 and 1 mM MgC2). Cells were incubated on ice with β1 and β4 integrins using polyclonal β1 (Santa Cruz 1 : 50) and β4 (Chemicon 1 : 500) antibodies for 30 min. Data was acquired using the FACSTAR program.

Skin barrier function analysis

Transgenic and control skin were analysed for transepidermal water loss levels using an evaporimeter (Servomed) (Choate et al., 1996b) in the peri-natal period.

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Acknowledgements

Supported by the USVA Office of Research and Development and AR43799 and AR45192, AR44012 from the NIAMS, NIH. M Dajee is a recipient of an NRSA Award from NIAMS. We thank M Lazarov, O Oro, J Ferrell, R Roth, Q Lin and D Kingsley for helpful discussions and pre-submission review. We thank E Fuchs for the K14 promoter, D Roop for the HK1 promoter, H Greulich for RasN17, M McMahon for Raf mutants, S Lowe for RasV12 and L Van Aelst for RasV12 S35, C40 and G37 mutants.

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Correspondence to Paul A Khavari.

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Keywords

  • epithelium
  • growth control
  • Ras
  • differentiation
  • skin

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