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• W55243669 abstract "Models of epidermal carcinogenesis have demonstrated that Ras is a critical molecule involved in tumor initiation and progression. Previously, we have shown that RasGRP1 increases the susceptibility of mice to skin tumorigenesis when overexpressed in the epidermis by a transgenic approach, related to its ability to activate Ras. Moreover, RasGRP1 transgenic mice develop spontaneous papillomas and cutaneous squamous cell carcinomas, some of which appear to originate in sites of injury, suggesting that RasGRP1 may be responding to signals generated during the wound-healing process. In this study, we examined the response of the RasGRP1 transgenic animals to full-thickness incision wounding of the skin, and demonstrated that they respond by developing tumors along the wounded site. The tumors did not present mutations in the H-ras gene, but Rasgrp1 transgene dosage correlated with tumor susceptibility and size. Analysis of serum cytokines showed increased levels of granulocyte colony-stimulating factor in transgenic animals after wounding. Furthermore, in vitro experiments with primary keratinocytes showed that granulocyte colony-stimulating factor stimulated Ras activation, although RasGRP1 was dispensable for this effect. Since granulocyte colony-stimulating factor has been recently associated with proliferation of skin cancer cells, our results may help in the elucidation of pathways that activate Ras in the epidermis during tumorigenesis in the absence of oncogenic ras mutations. Models of epidermal carcinogenesis have demonstrated that Ras is a critical molecule involved in tumor initiation and progression. Previously, we have shown that RasGRP1 increases the susceptibility of mice to skin tumorigenesis when overexpressed in the epidermis by a transgenic approach, related to its ability to activate Ras. Moreover, RasGRP1 transgenic mice develop spontaneous papillomas and cutaneous squamous cell carcinomas, some of which appear to originate in sites of injury, suggesting that RasGRP1 may be responding to signals generated during the wound-healing process. In this study, we examined the response of the RasGRP1 transgenic animals to full-thickness incision wounding of the skin, and demonstrated that they respond by developing tumors along the wounded site. The tumors did not present mutations in the H-ras gene, but Rasgrp1 transgene dosage correlated with tumor susceptibility and size. Analysis of serum cytokines showed increased levels of granulocyte colony-stimulating factor in transgenic animals after wounding. Furthermore, in vitro experiments with primary keratinocytes showed that granulocyte colony-stimulating factor stimulated Ras activation, although RasGRP1 was dispensable for this effect. Since granulocyte colony-stimulating factor has been recently associated with proliferation of skin cancer cells, our results may help in the elucidation of pathways that activate Ras in the epidermis during tumorigenesis in the absence of oncogenic ras mutations. The role of Ras activation in non-melanoma skin cancer is well-documented, both from analysis of human squamous cell carcinomas (SCC)1Pierceall WE Goldberg LH Tainsky MA Mukhopadhyay T Ananthaswamy HN Ras gene mutation and amplification in human nonmelanoma skin cancers.Mol Carcinog. 1991; 4: 196-202Crossref PubMed Scopus (253) Google Scholar as well as from studies using mouse models of skin carcinogenesis.2Yuspa SH The pathogenesis of squamous cell cancer: lessons learned from studies of skin carcinogenesis–thirty-third G. H A Clowes Memorial Award lecture.Cancer Res. 1994; 54: 1178-1189PubMed Google Scholar, 3Yuspa SH The pathogenesis of squamous cell cancer: lessons learned from studies of skin carcinogenesis.J Dermatol Sci. 1998; 17: 1-7Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar In particular, the multistage carcinogenesis protocol on mouse skin has identified activating mutations in the ras proto-oncogene as the initiation event in skin neoplasms.4Balmain A Ramsden M Bowden GT Smith J Activation of the mouse cellular Harvey-ras gene in chemically induced benign skin papillomas.Nature. 1984; 307: 658-660Crossref PubMed Scopus (428) Google Scholar Interestingly, whereas Ras mutations are prevalent in mouse models of skin carcinogenesis, they have only been identified in 12% to 46% of sporadic human SCC samples, despite the fact that Ras is activated in the majority of the human SCC.1Pierceall WE Goldberg LH Tainsky MA Mukhopadhyay T Ananthaswamy HN Ras gene mutation and amplification in human nonmelanoma skin cancers.Mol Carcinog. 1991; 4: 196-202Crossref PubMed Scopus (253) Google Scholar, 5Spencer JM Kahn SM Jiang W DeLeo VA Weinstein IB Activated ras genes occur in human actinic keratoses, premalignant precursors to squamous cell carcinomas.Arch Dermatol. 1995; 131: 796-800Crossref PubMed Scopus (121) Google Scholar This suggests that other mechanisms of Ras activation play a role in the human disease. For example, epidermal growth factor receptor overexpression by amplification is known to occur in human SCC,6Derynck R The physiology of transforming growth factor-alpha.Adv Cancer Res. 1992; 58: 27-52Crossref PubMed Scopus (250) Google Scholar, 7Shimizu T Izumi H Oga A Furumoto H Murakami T Ofuji R Muto M Sasaki K Epidermal growth factor receptor overexpression and genetic aberrations in metastatic squamous-cell carcinoma of the skin.Dermatology. 2001; 202: 203-206Crossref PubMed Scopus (92) Google Scholar and epidermal growth factor receptor could lead to the biochemical stimulation of Ras. In animal models, overexpression of epidermal growth factor receptor ligands like transforming growth factor-α,8Vassar R Hutton ME Fuchs E Transgenic overexpression of transforming growth factor alpha bypasses the need for c-Ha-ras mutations in mouse skin tumorigenesis.Mol Cell Biol. 1992; 12: 4643-4653Crossref PubMed Scopus (101) Google Scholar or a dominant form of the Ras exchange factor Sos of seven less (SOS),9Sibilia M Fleischmann A Behrens A Stingl L Carroll J Watt FM Schlessinger J Wagner EF The EGF receptor provides an essential survival signal for SOS-dependent skin tumor development.Cell. 2000; 102: 211-220Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar act as an initiation event in the epidermis, further demonstrating that alterations in Ras upstream signals could lead to tumorigenesis in the skin via wild-type Ras activation. Biochemical activation of Ras in keratinocytes can be triggered by various extracellular stimuli, but in all cases it requires the participation of exchange factors that catalyze the GDP–GTP exchange. The best studied GDP–GTP exchange factor is SOS1, which is activated in response to receptor tyrosine kinase activation.10Li N Batzer A Daly R Yajnik V Skolnik E Chardin P Bar-Sagi D Margolis B Schlessinger J Guanine-nucleotide-releasing factor hSos1 binds to Grb2 and links receptor tyrosine kinases to Ras signalling.Nature. 1993; 363: 85-88Crossref PubMed Scopus (803) Google Scholar, 11Chardin P Camonis JH Gale NW van Aelst L Schlessinger J Wigler MH Bar-Sagi D Human Sos1: a guanine nucleotide exchange factor for Ras that binds to GRB2.Science. 1993; 260: 1338-1343Crossref PubMed Scopus (659) Google Scholar In recent years, we have identified a new GDP–GTP exchange factor in epidermal keratinocytes, RasGRP1, and demonstrated that it can transduce the activation of Ras by the diacylglycerol analog and potent skin tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA).12Rambaratsingh RA Stone JC Blumberg PM Lorenzo PS RasGRP1 represents a novel non-protein kinase C phorbol ester signaling pathway in mouse epidermal keratinocytes.J Biol Chem. 2003; 278: 52792-52801Crossref PubMed Scopus (36) Google Scholar The initial studies prompted us to investigate the role of RasGRP1 in the tumor promotion effects of phorbol esters in the multistage carcinogenesis protocol using a transgenic mouse model for overexpression of RasGRP1 under the keratin 5 promoter (K5.RasGRP1). Surprisingly, RasGRP1 overexpression did not increase the susceptibility to TPA in animals initiated by the carcinogen 7, 12-Dimethylbenz[a]anthracene (DMBA), but caused more malignant tumors than those observed in the wild-type mice, suggesting a role of RasGRP1 in tumor progression.13Luke CT Oki-Idouchi CE Cline JM Lorenzo PS RasGRP1 overexpression in the epidermis of transgenic mice contributes to tumor progression during multistage skin carcinogenesis.Cancer Res. 2007; 67: 10190-10197Crossref PubMed Scopus (19) Google Scholar This effect appeared strongly related to increases in active Ras, as RasGRP1 overexpression clearly leads to elevated RasGTP levels in mouse epidermal keratinocytes, both under basal as well as TPA stimulated conditions.13Luke CT Oki-Idouchi CE Cline JM Lorenzo PS RasGRP1 overexpression in the epidermis of transgenic mice contributes to tumor progression during multistage skin carcinogenesis.Cancer Res. 2007; 67: 10190-10197Crossref PubMed Scopus (19) Google Scholar Interestingly, the K5.RasGRP1 mice also produced tumors in response to TPA alone, implying that RasGRP1 overexpression can act as an initiation event. In addition to the response to chemical carcinogens and tumor promoters, we observed that the RasGRP1 transgenic colony developed spontaneous skin tumors over time, more frequently seen in animals housed in groups or with previous episodes of skin abrasion or injury.14Oki-Idouchi CE Lorenzo PS Transgenic overexpression of RasGRP1 in mouse epidermis results in spontaneous tumors of the skin.Cancer Res. 2007; 67: 276-280Crossref PubMed Scopus (34) Google Scholar This response resembled that of the Tg.Ac mice, which express v-H-ras under the ζ-globin promoter and generate tumors in response to tumor promoting stimuli like wounding and phorbol esters in absence of carcinogenic/initiation events.15Leder A Kuo A Cardiff RD Sinn E Leder P v-Ha-ras transgene abrogates the initiation step in mouse skin tumorigenesis: effects of phorbol esters and retinoic acid.Proc Nat Acad Sci. 1990; 87: 9178-9182Crossref PubMed Scopus (279) Google Scholar, 16Humble MC, Trempus CS, Spalding JW, Cannon RE, Tennant RW: Biological, cellular, and molecular characteristics of an inducible transgenic skin tumor model: a review. Oncogene 24:8217–8228Google Scholar The in vivo studies with the K5.RasGRP1 mice have implicated RasGRP1 as a novel link for Ras activation in epidermal keratinocytes and skin cancer. However, the signaling mechanisms contributing to RasGRP1 activation and spontaneous tumor formation, as well as the direct evidence for a role of skin wounding as a tumor promoter stimulus in the context of RasGRP1 overexpression, remained to be established. To address these questions, we have now characterized the tumorigenic response of the K5.RasGRP1 to full-thickness incision wounding of the skin. Our findings show that RasGRP1 overexpression in the epidermis conferred sensitivity to wounding-induced promotional stimuli. Furthermore, upon wounding, there was a significant increase in the levels of granulocyte colony-stimulating factor (G-CSF) in the circulation of transgenic mice compared with the wild-type animals. In vitro, G-CSF was able to stimulate Ras activation in keratinocytes in a very rapid fashion, although RasGRP1 appeared dispensable for this effect. Since G-CSF has been associated to the growth and progression of skin carcinoma cells, the data presented here may have implications for the understanding of RasGRP1-Ras signaling in skin tumor biology. The K5.RasGRP1 transgenic mice were previously generated and maintained in the FVB/N background.14Oki-Idouchi CE Lorenzo PS Transgenic overexpression of RasGRP1 in mouse epidermis results in spontaneous tumors of the skin.Cancer Res. 2007; 67: 276-280Crossref PubMed Scopus (34) Google Scholar RasGRP1 null mice (KO-RasGRP1), produced by inserting the Escherichia coli β-galactosidase gene and a neomycin cassette in exon 2 of Rasgrp1,17Dower NA Stang SL Bottorff DA Ebinu JO Dickie P Ostergaard HL Stone JC RasGRP is essential for mouse thymocyte differentiation and TCR signaling.Nat Immunol. 2000; 1: 317-321Crossref PubMed Scopus (12) Google Scholar were originally obtained in 129/J background and backcrossed for >10 generation to the FVB/N background. Wild-type mice were bred in house. All animal studies were done according to Institutional Animal Care and Use Committee guidelines at the University of Hawaii Animal Facility. Two cohorts based on genotype (wild-type and K5.RasGRP1 mice) were used for the studies. Each cohort had between 12 and 16 mice of mixed gender (equal number of males and females) between 6 to 8 weeks of age. Two days before wounding, the dorsal skin of the mice was shaved with electric clippers. Then, a full-thickness, 3-cm long incision was performed across the dorsal skin under isoflurane anesthesia. The wound was closed with five to seven surgical clips that were removed 7 days later. Mice were followed twice per week for a total of 10 weeks, to observe wound closure and tumor formation. Tumor size was measured with a caliper at least once a week. At the end of the protocol, the animals were euthanized by CO2 narcosis. Tumor samples were collected and processed for histology. Skin tumors were fixed in 4% paraformaldehyde for 24 hours, dehydrated, and maintained in 70% ethanol at 4°C until paraffin-embedded. H&E-stained slides were used for descriptive histopathology. Two 10-μm paraffin-embedded tumor sections were used for DNA extraction and ras mutation analysis. Briefly, after deparaffination, samples were digested with proteinase K followed by DNA extraction using the QIAamp DNA FFPE Tissue Kit (Qiagen, Valencia, CA). One hundred ng of DNA were used for a PCR reaction to amplify the H-ras sequence including codons 12, 13, and 61, using primers described by Vassar et al.8Vassar R Hutton ME Fuchs E Transgenic overexpression of transforming growth factor alpha bypasses the need for c-Ha-ras mutations in mouse skin tumorigenesis.Mol Cell Biol. 1992; 12: 4643-4653Crossref PubMed Scopus (101) Google Scholar Genomic DNA from SP-1 cells was used as a positive control for H-ras mutations. These cells (papilloma-derived keratinocytes generated by chemical-induced carcinogenesis in SENCAR mice) were obtained from Dr. Stuart Yuspa (National Cancer Institute, Bethesda, MD) and cultured as described for the primary keratinocytes (below). The PCR products were sequenced by the Advanced Studies in Genomics, Proteomics and Bioinformatics facility at University of Hawaii at Manoa. Primary cultures of mouse skin keratinocytes were prepared as described previously with modifications.14Oki-Idouchi CE Lorenzo PS Transgenic overexpression of RasGRP1 in mouse epidermis results in spontaneous tumors of the skin.Cancer Res. 2007; 67: 276-280Crossref PubMed Scopus (34) Google Scholar Briefly, keratinocytes were isolated from newborn epidermis by the trypsin flotation method18Hennings H Michael D Cheng C Steinert P Holbrook K Yuspa SH Calcium regulation of growth and differentiation of mouse epidermal cells in culture.Cell. 1980; 19: 245-254Abstract Full Text PDF PubMed Scopus (1507) Google Scholar and then plated at a density of 1.5 × 106 cells/60-mm cell culture dish coated with collagen I (Coating Matrix, Cascade Biologics-Invitrogen, Portland, OR) in Minimum Essential Medium (S-MEM) (Invitrogen, Carlsbad, CA) supplemented with 8% fetal bovine serum, non-essential amino acids, antibiotics/antimycotics, and CaCl2 to a final 0.3 mmol/L concentration. After a 24-hour incubation period, cultures were switched to a low calcium medium consisting of medium 154CF (Cascade Biologics-Invitrogen, Portland, OR) containing antibiotics/antimycotics and CaCl2 to a final 50 μmol/L concentration. This medium was supplemented with 2% Chelex-treated fetal bovine serum and a keratinocyte growth factor mix consisting of 0.2% bovine pituitary hormone, 5 μg/ml bovine insulin, 0.18 μg/ml hydrocortisone, 5 μg/ml bovine transferrin, and 0.2 ng/ml human epidermal growth factor (Cascade Biologics-Invitrogen, Portland, OR). Keratinocytes were used within 5 to 6 days after plating. Ten μg of genomic DNA from transgenic K5.RasGRP1 mice were digested with NheI/BamHI overnight and then separated on a 0.7% agarose gel, transferred to a nylon membrane, and hybridized with a probe for detection of the Rasgrp1 transgene. The probe was prepared by PCR of the transgene rat Rasgrp1 plasmid using primers previously described.14Oki-Idouchi CE Lorenzo PS Transgenic overexpression of RasGRP1 in mouse epidermis results in spontaneous tumors of the skin.Cancer Res. 2007; 67: 276-280Crossref PubMed Scopus (34) Google Scholar The PCR fragment (∼700 bp) was labeled with [α-32P]dCTP using the DecaLabel kit (Fermentas, Glen Burnie, MD) according to the manufacturer’s instructions, and used for hybridization following standard molecular biology procedures. Levels of GTP-loaded Ras (RasGTP) were measured by using the GST-RBD domain of Raf-1 as a probe in a pull-down assay. Briefly, primary keratinocytes were serum starved overnight (0.1% fetal bovine serum), treated with vehicle (0.1% bovine serum albumin in PBS) or 10 ng/ml mouse recombinant G-CSF (Leinco Technologies, Inc., St. Louis, MI) for 3 minutes, and immediately harvested on ice in lysis buffer containing 25 mmol/L Tris-HCl (pH 7.5), 150 mmol/L of NaCl, 5 mmol/L of MgCl2, 1 mmol/L of NaF, 1 mmol/L of sodium orthovanadate, 1% IGEPAL, 5% glycerol, and Mini Complete Roche-protease inhibitors (Roche Applied Science, Indianapolis, IN). Lysates were vortexed, incubated on ice for 5 minutes and then clarified by centrifugation at 13,000 rpm for 15 minutes at 4°C. Five hundred μg of lysate protein were incubated with GST-RBD-Raf-1 conjugated to glutathione beads for 1 hour with rotation in the cold. The affinity complexes were washed thrice with lysis buffer and then resuspended in 2× Laemmli buffer, boiled, and resolved on 15% acrylamide gels. Twenty-five μg of the total lysate protein were run in parallel as measurement of input of total Ras in the assay. Proteins were blotted onto nitrocellulose membranes and immunostaining was done using the pan anti-Ras clone RAS10 antibody (Calbiochem, San Diego, CA). RasGRP1 levels were evaluated by immunostaining using a monoclonal anti-RasGRP1 antibody (Santa Cruz Biotechnology, Santa Cruz, CA). For the in vitro wound assay, primary keratinocyte monolayers (80% to 90% confluence) were scratched with a pipette tip forming an eight-square grid pattern. Cells were harvested 5 minutes after wounding and used for a Ras pull down assay as described above. Fifty μg of total lysate protein were run in parallel as measurement of input of total Ras for the wound assay. Transgenic K5.RasGRP1 and wild-type animals were subjected to a full-thickness incision wound as described above. Blood was extracted from the mice at time 0 and 24 hours post-incision by cardiac puncture under isoflurane anesthesia. Serum was obtained by incubating the blood samples at room temperature for at least 2 hours followed by centrifugation. For the cytokine array experiment, serum samples were diluted 1:4 in dilution media provided in the array kit (TranSignal Mouse Cytokine Antibody Array 1.0, Panomics, Freemont, CA), and incubated for 2 hours at room temperature with gentle rotation. The remaining steps were done according to the manufacturer’s instructions. Our previous studies suggested a causal association between wounding and spontaneous skin tumors in the K5.RasGRP1 transgenic mice.14Oki-Idouchi CE Lorenzo PS Transgenic overexpression of RasGRP1 in mouse epidermis results in spontaneous tumors of the skin.Cancer Res. 2007; 67: 276-280Crossref PubMed Scopus (34) Google Scholar To investigate this association and gain further insight into the mechanisms of action of RasGRP1 in skin tumorigenesis, we used a full-thickness incision wounding protocol on wild-type and RasGRP1 transgenic mice. Two weeks after the dorsal incisions were performed, the wounds had healed in both groups, but raised lesions began to appear along the margins of the incision in a percentage of the K5.RasGRP1 mice. By the third week after wounding, tumors were clearly evident in the transgenic mice (Figure 1A). The tumor incidence at week #3 reached 50% in the transgenic group and did not vary significantly throughout the remaining 7 weeks of the protocol (Figure 1B). No tumors developed in the wild-type mice. Histopathological analysis revealed that the majority of the tumors generated in the K5.RasGRP1 mice were well-differentiated SCC, with only one papilloma and one trichoepithelioma, the latter observed in conjunction with an SCC (Figure 1C). Two transgenic mice in the protocol displayed a significantly elevated tumorigenic response, as compared with the rest of the tumor-bearing mice (Figure 2A). Whereas the transgenic animals used in this protocol were all deemed heterozygous, the differences in response among the animals prompted us to examine transgene copy numbers by Southern blot analysis. The results confirmed the transgenic status of all of the animals in the K5.RasGRP1 cohort (Figure 2B), but also indicated that the two mice with the elevated response had a higher transgene copy number than the rest of the transgenic mice in the group. Whether the nature of the copy number difference is due to homozygosis, remains to be determined; nevertheless, this finding suggests that Rasgrp1 gene dosage influences tumor susceptibility to wounding. Since many skin tumors generated in mouse models carry Ras activating mutations that participate as an initiation event, we examined the ras gene status in the tumors developed in K5.RasGRP1 mice in response to wounding. We focused on H-ras, as this is the most commonly mutated form of ras occurring in mouse skin papillomas and squamous cell carcinomas.19Balmain A Pragnell IB Mouse skin carcinomas induced in vivo by chemical carcinogens have a transforming Harvey-ras oncogene.Nature. 1983; 303: 72-74Crossref PubMed Scopus (334) Google Scholar Using a PCR approach followed by sequencing, we analyzed genomic DNA extracted from six SCC from the K5.RasGRP1 group for H-ras mutation at codons 12, 13, and 61. As control, we used genomic DNA extracted from a papilloma-derived keratinocyte cell line (SP-1), which carries a mutation in codon 61 of the H-ras gene.20Strickland JE Greenhalgh DA Koceva-Chyla A Hennings H Restrepo C Balaschak M Yuspa SH Development of murine epidermal cell lines which contain an activated rasHa oncogene and form papillomas in skin grafts on athymic nude mouse hosts.Cancer Res. 1988; 48: 165-169PubMed Google Scholar While the SP-1 cells showed the expected heterozygous T to A transversion in codon 61 of H-ras, no mutations in the H-ras proto-oncogene were present in the tumors (Figure 2C). We have previously seen that primary mouse keratinocytes derived from K5.RasGRP1 mice secreted G-CSF on in vitro wounding assays, while levels of this cytokine were negligible in conditioned medium derived from wounded wild-type keratinocyte monolayers.14Oki-Idouchi CE Lorenzo PS Transgenic overexpression of RasGRP1 in mouse epidermis results in spontaneous tumors of the skin.Cancer Res. 2007; 67: 276-280Crossref PubMed Scopus (34) Google Scholar This was an interesting observation, since G-CSF is a cytokine associated to skin neoplasia.21Obermueller E Vosseler S Fusenig NE Mueller MM Cooperative autocrine and paracrine functions of granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor in the progression of skin carcinoma cells.Cancer Res. 2004; 64: 7801-7812Crossref PubMed Scopus (98) Google Scholar Therefore, we sought to test if K5.RasGRP1 animals could secrete elevated levels of this cytokine on in vivo wounding of the skin. Using a mouse antibody array for cytokine detection, we found that serum levels of G-CSF under non wounded conditions were very low or below the limit of detection in both wild-type and K5.RasGRP1 mice (Figure 3). However, we detected a significant increase in G-CSF levels in the serum of K5.RasGRP1 mice 24-hour post wounding (Figure 3), providing evidence that skin wounding in transgenic RasGRP1 mice leads to increases in G-CSF secretion. ras oncogenes have been shown to cause induction of G-CSF in different cell types.22Demetri GD Ernst TJ Pratt 2nd, ES Zenzie BW Rheinwald JG Griffin JD Expression of ras oncogenes in cultured human cells alters the transcriptional and posttranscriptional regulation of cytokine genes.J Clin Invest. 1990; 86: 1261-1269Crossref PubMed Scopus (60) Google Scholar While RasGRP1 transgenic keratinocytes possess elevated levels of active Ras compared with wild-type cells,14Oki-Idouchi CE Lorenzo PS Transgenic overexpression of RasGRP1 in mouse epidermis results in spontaneous tumors of the skin.Cancer Res. 2007; 67: 276-280Crossref PubMed Scopus (34) Google Scholar those levels do not appear to be sufficient to trigger G-CSF secretion under control conditions. We then speculated that the increase in G-CSF levels from K5.RasGRP1 keratinocytes observed on wounding could be related to a spike of Ras activation induced by the wound-healing process. In fact, stimulation of Ras by wounding has been shown to occur in primary human keratinocytes.23Turchi L Chassot AA Rezzonico R Yeow K Loubat A Ferrua B Lenegrate G Ortonne JP Ponzio G Dynamic characterization of the molecular events during in vitro epidermal wound healing.J Invest Dermatol. 2002; 119: 56-63Crossref PubMed Scopus (55) Google Scholar To test if that was also the case under our experimental conditions, we subjected primary mouse keratinocytes to in vitro wounding and measured Ras active levels by pull down assay. As shown in Figure 4, levels of RasGTP, as well as active ERK1/2 (phospho ERK1/2), were rapidly increased after wounding. Because RasGTP is elevated in the K5.RasGRP1 cells, the further Ras activation caused by wounding could explain the effect on G-CSF secretion from the transgenic mice. G-CSF is known to stimulate Ras in hematopoietic cells,24Rausch O Marshall CJ Tyrosine 763 of the murine granulocyte colony-stimulating factor receptor mediates Ras-dependent activation of the JNK/SAPK mitogen-activated protein kinase pathway.Mol Cell Biol. 1997; 17: 1170-1179Crossref PubMed Scopus (72) Google Scholar thus G-CSF could also induce further Ras activation in keratinocytes, providing a positive feedback loop between Ras and G-CSF secretion, with RasGRP1 mediating the effect. To test this hypothesis, we compared RasGTP levels in primary keratinocytes derived from wild-type and K5.RasGRP1 animals upon G-CSF treatment. As shown in Figure 5A, G-CSF produced a significant activation of Ras in both wild-type and RasGRP1 transgenic keratinocytes and, as expected from the elevated RasGRP1 levels in the transgenic cells, activation of Ras was overall higher in the K5.RasGRP1 keratinocytes compared with the wild-type counterparts. However, two-way no matching analysis of variance test indicated that there was no effect of the genotype on the response to G-CSF (wild-type versus transgenic, P = 0.92, no significant), suggesting that RasGRP1 was dispensable in the effect of G-CSF on Ras. We have previously noticed that under high basal levels of RasGTP, further stimulation of RasGRP1—even with potent diacylglycerol mimetics—frequently produced only a minimal stimulation of Ras,25Tuthill MC Oki CE Lorenzo PS Differential effects of bryostatin 1 and 12-O-tetradecanoylphorbol-13-acetate on the regulation and activation of RasGRP1 in mouse epidermal keratinocytes.Mol Cancer Ther. 2006; 5: 602-610Crossref PubMed Scopus (17) Google Scholar suggesting that Ras activation could reach a plateau under our experimental conditions. Therefore, we wanted to confirm that the lack of effect of genotype on G-CSF-mediated Ras activation was not a result of an experimental artifact due to saturation of a response. To this end, we used primary keratinocytes derived from RasGRP1 null mice (KO-RasGRP1) in Ras pull down assays to evaluate the requirements for RasGRP1 in response to G-CSF. As shown in Figure 5B, the level of Ras stimulation induced by G-CSF in KO-RasGRP1 cells was significant and comparable with the stimulation observed in wild-type keratinocytes. Taken together, the results imply that RasGRP1 was not involved in the stimulation of Ras induced by G-CSF in mouse keratinocytes. In the present study we have demonstrated that transgenic mice for overexpression of RasGRP1 in epidermis are susceptible to wounding as a tumor promotional stimuli, developing cutaneous SCC with a short latency. The findings, together with our previous observations about the role of RasGRP1 in the multistage model of skin carcinogenesis, strongly suggest a role of RasGRP1 in the genesis of skin tumors derived from keratinocytes. RasGRP1 is one of the members of the RasGRP family, and functions as a guanine nucleotide exchange factor for Ras small GTPases, catalyzing their GDP–GTP exchange and leading to Ras activation.26Stone JC Regulation of Ras in lymphocytes: get a GRP.Biochem Soc Trans. 2006; 34: 858-861Crossref PubMed Scopus (63) Google Scholar In keratinocytes, RasGRP1 overexpression by transient transfection or by transgenic approach, leads to increases in the levels of basal RasGTP.12Rambaratsingh RA Stone JC Blumberg PM Lorenzo PS RasGRP1 represents a novel non-protein kinase C phorbol ester signaling pathway in mouse epidermal keratinocytes.J Biol Chem. 2003; 278: 52792-52801Crossref PubMed Scopus (36) Google Scholar, 14Oki-Idouchi CE Lorenzo PS Transgenic overexpression of RasGRP1 in mouse epidermis results in spontaneous tumors of the skin.Cancer Res. 2007; 67: 276-280Crossref PubMed Scopus (34) Google Scholar Therefore, one mechanism for the tumorigenic susceptibility of the K5.RasGRP1 transgenic mice could be the elevated levels of active Ras. Our data revealed no H-ras mutations in tumors originated on wounding of transgenic mice, and although mutations in other ras genes cannot be excluded, the more likely scenario is that the increase in RasGTP levels is a result of biochemical stimulation of wild-type Ras by RasGRP1. Additionally, we observed that Rasgrp1 gene dosage influences the tumor susceptibility and size in response to wounding, further supporting the idea that Ras activation depends on RasGRP1 in our transgenic mouse model. It has been previously documented that overexpression of active ras mutant genes in the epidermis leads to skin tumors27Brown K Quintanilla M Ramsden M Kerr IB Young S Balmain A v-ras genes from harvey and BALB murine sarcoma viruses can act as initiators of two-stage mouse skin carcinogenesis.Cell. 1986; 46: 447-456Abstract Full Text PDF PubMed Scopus (212) Google Scholar; however, the localization of the mutant within the epidermis influences the dependency on a tumor promoter stimulus for tumor formation and the malignancy of the tumors.28Bailleul B Surani MA White S Barton SC Brown K Blessing M Jorcano J Balmain A Skin hyperkeratosis and papilloma formation in transgenic mice expressing a ras oncogene from a suprabasal keratin promoter.Cell. 1990; 62: 697-708Abstract Full Text PDF PubMed Scopus (218) Google Scholar, 29Brown K Strathdee D Bryson S Lambie W Balmain A The malignant capacity of skin tumours induced by expression of a mutant H-ras transgene depends on the cell type targeted.Curr Biol. 1998; 8: 516-524Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar In this regard, while expression of a mutant ras in suprabasal keratinocytes depends on tumor promotion for papilloma formation, expression in basal keratinocytes using the keratin 5 promoter results in spontaneous tumors.29Brown K Strathdee D Bryson S Lambie W Balmain A The malignant capacity of skin tumours induced by expression of a mutant H-ras transgene depends on the cell type targeted.Curr Biol. 1998; 8: 516-524Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar One could predict that in our model, in which RasGRP1 is overexpressed under the keratin 5 promoter, tumor promotion could be dispensable. Still, tumor promotion is required. The possibility exists that overexpression of RasGRP1 alone does not lead to full Ras activation—at levels comparable with activation of mutant forms of Ras—and thus, a further stimulus is required to activate RasGRP1 and, in turn, Ras. The finding that G-CSF was elevated in the circulation of the RasGRP1 transgenic mice after wounding, together with our previous observation with primary keratinocytes subjected to the in vitro wounding assay,14Oki-Idouchi CE Lorenzo PS Transgenic overexpression of RasGRP1 in mouse epidermis results in spontaneous tumors of the skin.Cancer Res. 2007; 67: 276-280Crossref PubMed Scopus (34) Google Scholar supports the concept that G-CSF is at least in part produced by the transgenic epidermal keratinocytes in response to wounding. Previous studies have demonstrated that keratinocytes secretion of G-CSF can contribute to tumor growth and progression in SCC.21Obermueller E Vosseler S Fusenig NE Mueller MM Cooperative autocrine and paracrine functions of granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor in the progression of skin carcinoma cells.Cancer Res. 2004; 64: 7801-7812Crossref PubMed Scopus (98) Google Scholar Thus, the increase in G-CSF seen in the K5.RasGRP1 mice raises the possibility that this cytokine may be a critical contributing factor in the wounding-induced tumor formation in our transgenic mouse model. The fact that K5.RasGRP1-derived keratinocytes secret this cytokine also suggests that a stimulated RasGRP1-Ras pathway participates in the expression of G-CSF. In fact, ras oncogene expression can induced cytokines like G-CSF in human fibroblast and mesothelioma cells, and while the exact mechanism is still not understood, it involves in part the stabilization of cytokine transcripts.22Demetri GD Ernst TJ Pratt 2nd, ES Zenzie BW Rheinwald JG Griffin JD Expression of ras oncogenes in cultured human cells alters the transcriptional and posttranscriptional regulation of cytokine genes.J Clin Invest. 1990; 86: 1261-1269Crossref PubMed Scopus (60) Google Scholar Further studies are warranted to examine the role of G-CSF in keratinocytes in our model and the precise sequence of events that triggers RasGRP1-Ras activation in these cells." @default.
• W55243669 created "2016-06-24" @default.
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• W55243669 creator A5016810637 @default.
• W55243669 creator A5032630396 @default.
• W55243669 creator A5035059784 @default.
• W55243669 creator A5037054955 @default.
• W55243669 creator A5067087415 @default.
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• W55243669 date "2009-07-01" @default.
• W55243669 modified "2023-09-30" @default.
• W55243669 title "RasGRP1 Transgenic Mice Develop Cutaneous Squamous Cell Carcinomas in Response to Skin Wounding" @default.
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