A Rac1-Independent Role for P-Rex1 in Melanoblasts

guanine-nucleotide exchange factor 4-hydroxytamoxifen PIP3-dependent Rac-exchanger 1 TO THE EDITOR Given the recent discovery of RAC1-activating mutations in melanoma, and our finding that PIP3-dependent Rac-exchanger 1 (PREX1) is overexpressed and drives metastasis in this cancer, an important question is to establish whether the functions of P-Rex1 are mediated specifically by Rac alone (Lindsay et al., 2011Lindsay C.R. Lawn S. Campbell A.D. et al.P-Rex1 is required for efficient melanoblast migration and melanoma metastasis.Nat Commun. 2011; 2: 555Crossref PubMed Scopus (129) Google Scholar; Berger et al., 2012Berger M.F. Hodis E. Heffernan T.P. et al.Melanoma genome sequencing reveals frequent PREX2 mutations.Nature. 2012; 485: 502-506Crossref PubMed Scopus (561) Google Scholar). Here we describe a Rac1-independent in vivo role for P-Rex1 through identification and characterization of a mouse coat color phenotype. P-Rex1 is a guanine-nucleotide exchange factor (GEF) for Rac, whose primary cell function is induction of actin-mediated membrane ruffling and lamellipodia formation at the leading edge of cell migration (Welch et al., 2002Welch H.C. Coadwell W.J. Ellson C.D. et al.P-Rex1, a PtdIns(3,4,5)P3- and Gbetagamma-regulated guanine-nucleotide exchange factor for Rac.Cell. 2002; 108: 809-821Abstract Full Text Full Text PDF PubMed Scopus (433) Google Scholar; Hill et al., 2005Hill K. Krugmann S. Andrews D.R. et al.Regulation of P-Rex1 by phosphatidylinositol (3,4,5)-trisphosphate and Gbetagamma subunits.J Biol Chem. 2005; 280: 4166-4173Crossref PubMed Scopus (88) Google Scholar; Barber et al., 2007Barber M. Donald S. Thelen S. et al.Membrane translocation of P-Rex1 is mediated by G protein betagamma subunits and phosphoinositide 3-kinase.J Biol Chem. 2007; 282: 29967-29976Crossref PubMed Scopus (68) Google Scholar). To investigate this question we decided to examine the role of Rac1 and P-Rex1 in melanoblast development. Previously, we reported a “white belly” phenotype of mice with Prex1 deletion (Lindsay et al., 2011Lindsay C.R. Lawn S. Campbell A.D. et al.P-Rex1 is required for efficient melanoblast migration and melanoma metastasis.Nat Commun. 2011; 2: 555Crossref PubMed Scopus (129) Google Scholar). Impaired melanoblast migration was mostly responsible for this phenotype, with melanoblasts lacking at the most distal points of migration (belly and paws). Constitutive deletion of Rac1 is embryonically lethal, but a coat color defect of mice with melanocyte-specific RAC1 abrogation (Tyr::Cre Rac1fl/fl) has also been described; these mice have a larger belly spot on their ventral side, suggesting that alternative Rho-GTPases can be activated to enable melanoblast migration to the perimeter of the Tyr::Cre Racfl/fl white belly (Sugihara et al., 1998Sugihara K. Nakatsuji N. Nakamura K. et al.Rac1 is required for the formation of three germ layers during gastrulation.Oncogene. 1998; 17: 3427-3433Crossref PubMed Scopus (276) Google Scholar; Li et al., 2011Li A. Ma Y. Yu X. et al.Rac1 drives melanoblast organization during mouse development by orchestrating pseudopod-driven motility and cell-cycle progression.Dev Cell. 2011; 21: 722-734Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). A role for Rac1 in proliferation was also observed, as there was a marked reduction of melanoblast numbers in this phenotype. In line with these previous studies, and because mice with melanocyte-specific RAC1 abrogation require euthanization shortly after birth because of neurological problems, we used the same embryonic melanoblast reporter models to assess the downstream effects of P-Rex1 in vivo (Mackenzie et al., 1997Mackenzie M.A. Jordan S.A. Budd P.S. et al.Activation of the receptor tyrosine kinase Kit is required for the proliferation of melanoblasts in the mouse embryo.Dev Biol. 1997; 192: 99-107Crossref PubMed Scopus (269) Google Scholar; Mort et al., 2010Mort R.L. Hay L. Jackson I.J. Ex vivo live imaging of melanoblast migration in embryonic mouse skin.Pigment Cell Melanoma Res. 2010; 23: 299-301Crossref PubMed Scopus (28) Google Scholar; Li et al., 2011Li A. Ma Y. Yu X. et al.Rac1 drives melanoblast organization during mouse development by orchestrating pseudopod-driven motility and cell-cycle progression.Dev Cell. 2011; 21: 722-734Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). Melanocyte-specific reporter mouse strains employed were Tyr::Cre Z/EG, which drives green fluorescent protein expression in the melanoblast lineage, and DCT::β-galactosidase (otherwise referred to as DCT-lacZ). First, we hypothesized that, if the effects of P-Rex1 were mediated exclusively via Rac1, double mutant Tyr::Cre Rac1fl/fl; P-Rex1−/− mice would exhibit the same coat color phenotype as Tyr::Cre Racfl/fl mice alone. However, Tyr::Cre Racfl/fl; P-Rex1−/− mice display a dramatic alteration in coat color phenotype from Tyr::Cre Racfl/fl mice (n=7; Figure 1a). The ventral and dorsal coats of these mice are almost entirely white, with hypo-pigmented limbs and tail. Graying pigmented areas were only observed in the head coat. We concluded from this experiment that P-Rex1 and Rac1 together constitute fundamental signaling components of the mouse coat color phenotype, with minimal rescue of melanoblast development conferred by other GEFs or Rho-GTPases. It was also clear that P-Rex1 must be able to exert phenotypic effects other than via Rac1. To explore the Rac1-independent effects of P-Rex1 further, we crossed Tyr::Cre Racfl/fl; P-Rex1−/− mice with mice carrying the melanoblast reporter DCT-lacZ transgene (methods detailed in Lindsay et al., 2011Lindsay C.R. Lawn S. Campbell A.D. et al.P-Rex1 is required for efficient melanoblast migration and melanoma metastasis.Nat Commun. 2011; 2: 555Crossref PubMed Scopus (129) Google Scholar). Relative to Tyr::Cre Racfl/flmice or P-Rex1−/− embryos alone, Tyr::Cre Racfl/fl; P-Rex1−/− embryos at E15.5 displayed a substantial reduction in melanoblast numbers across their entire body (Figure 1b and c). To assess whether the cause of this reduction in melanoblast numbers could be accounted for by decreased proliferation ± increased cell death, we next treated our previously described primary immortalized Tyr::CrER2 INK4a−/− Racfl/fl melanocyte cell line with short interfering RNA to P-Rex1 (methods detailed in Li et al., 2012Li A. Ma Y. Jin M. et al.Activated mutant NRas(Q61K) drives aberrant melanocyte signaling, survival, and invasiveness via a Rac1-dependent mechanism.J Invest Dermatol. 2012; 132: 2610-2621Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). The use of this model system also allowed us to delete Rac1 function when these cells were treated with 4-hydroxytamoxifen (OHT). We first used western blotting to confirm that efficient P-Rex1 knockdown and OHT-induced Rac deletion were achieved (Figure 1d). There was no increase in cleaved caspase-3 evident in the absence of P-Rex1 and/or Rac, suggesting that the reduced cell numbers observed in Tyr::Cre Racfl/fl; P-Rex1−/− embryos were not accounted for by increased cell death (Figure 1d). Growth curves and anti-BrdU immunofluorescence of the same cell lines confirmed that there was a reduced proliferation in P-Rex1-depleted cells, both in the presence and absence of OHT (Figure 1e; Supplementary Figure S1a and b online). Taken together, these results suggest, in addition to our previously reported effects of Rac1 deletion on mouse coat color, that the loss of a Rac1-independent proliferative effect of P-Rex1 also contributes to the coat color phenotype observed in Tyr::Cre Racfl/fl; P-Rex1−/− mice (Li et al., 2011Li A. Ma Y. Yu X. et al.Rac1 drives melanoblast organization during mouse development by orchestrating pseudopod-driven motility and cell-cycle progression.Dev Cell. 2011; 21: 722-734Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). Download .pdf (.51 MB) Help with pdf files Supplementary Information In line with our previous characterization of the P-Rex1 knockout phenotype alone, we next decided to delineate whether the coat color phenotype of Tyr::Cre Racfl/fl; P-Rex1−/− mice could also be a consequence of reduced melanoblast migration (Lindsay et al., 2011Lindsay C.R. Lawn S. Campbell A.D. et al.P-Rex1 is required for efficient melanoblast migration and melanoma metastasis.Nat Commun. 2011; 2: 555Crossref PubMed Scopus (129) Google Scholar). To assess this, mice with the Z/EG double reporter transgene were crossed with Tyr::Cre Racfl/fl; P-Rex1−/−mice, driving green fluorescent protein expression in the melanoblast lineage (methods detailed in Lindsay et al., 2011Lindsay C.R. Lawn S. Campbell A.D. et al.P-Rex1 is required for efficient melanoblast migration and melanoma metastasis.Nat Commun. 2011; 2: 555Crossref PubMed Scopus (129) Google Scholar; Figure 2a). Live imaging of melanoblasts was performed using E15.5 embryo skin from each genotype (Figure 2b; Supplementary Movie 1, Supplementary Movie 2, Supplementary Movie 3). Consistent with our previous work, significant reductions in migration speed were observed between wild-type, P-Rex1−/− and Tyr::Cre Racfl/fl melanoblasts (Figure 2c and d). However, there was no significant difference in speed between Tyr::Cre Racfl/fl; P-Rex1−/− and Tyr::Cre Racfl/fl melanoblasts alone, suggesting that there was no change in migratory characteristics to account for the Tyr::Cre Racfl/fl; P-Rex1−/− phenotype (Figure 2c and d). These results were matched by similar differences between the same genotypes when Euclidean distance was measured (Figure 2e), as well as no observable change in cell morphology evident in Tyr::Cre Racfl/fl; P-Rex1−/− compared with Tyr::Cre Racfl/fl melanoblasts alone (Supplementary Figure S1c online; methods detailed in Helmy and Azim, 2012Helmy I.M. Azim AMA. Efficacy of ImageJ in the assessment of apoptosis.Diagn Pathol. 2012; 7: 15Crossref PubMed Scopus (42) Google Scholar). Finally, no cell death was seen in our melanoblast time-lapse movies of any genotype, again suggesting that P-Rex1 contributes to coat color phenotype by promoting cell proliferation using a Rac1-independent mechanism (Li et al., 2011Li A. Ma Y. Yu X. et al.Rac1 drives melanoblast organization during mouse development by orchestrating pseudopod-driven motility and cell-cycle progression.Dev Cell. 2011; 21: 722-734Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar; Supplementary Movies S1–3 online; methods detailed in Lindsay et al., 2011Lindsay C.R. Lawn S. Campbell A.D. et al.P-Rex1 is required for efficient melanoblast migration and melanoma metastasis.Nat Commun. 2011; 2: 555Crossref PubMed Scopus (129) Google Scholar).https://www.jidonline.org/cms/asset/1f6f6e64-2ed8-4736-8aae-103e186dfb34/mmc2.mp4Loading ... Download .mp4 (1.17 MB) Help with .mp4 files Supplementary Movie 1https://www.jidonline.org/cms/asset/cbb7682c-2cc8-48db-9a59-08272bab1a08/mmc3.mp4Loading ... Download .mp4 (0.93 MB) Help with .mp4 files Supplementary Movie 2eyJraWQiOiI4ZjUxYWNhY2IzYjhiNjNlNzFlYmIzYWFmYTU5NmZmYyIsImFsZyI6IlJTMjU2In0.eyJzdWIiOiJkZDAxNjQ1MjFhOWNmMGI4NGIxM2VkNmQxZTc3NzY4OSIsImtpZCI6IjhmNTFhY2FjYjNiOGI2M2U3MWViYjNhYWZhNTk2ZmZjIiwiZXhwIjoxNjc4NjUwNTQzfQ.HXpT6krwI_8GfvnIQn-1YBcIsGJ5_79WrNwLf21kaot02TKqg9Wzu9I6c3ag_v3ELWGp64i4s1CmdpbFPS81Tgwhd3b3ONyMBJC0DS0Wdt335etcQ5RzXttM-E_80H2u-9jNAjVGWsYPG0AEYjW6xl3MoAGakM29n_Cxj_I2H1K4TM_SkdogIeHF3nSS9SWl32JjPO4-alTffknIPgjzuk_ZUvojFQ0YdyfHmjjcwX5K4QxuGFODMIor-qPA-OdbpnBpuGZNOvnFJ4yW1zL-Ywv_Nysp1RYgJ-wOO1tVcYOH6hbF1LcWcRD43AwZ5llc809axHSUESGRqxlEyq38CA Download .mp4 (1.09 MB) Help with .mp4 files Supplementary Movie 3 To conclude, we have elucidated a proliferative role of P-Rex1 when Rac1 is deleted. As E15.5 migratory characteristics are not altered in the Tyr::Cre Racfl/fl; P-Rex1−/− double mutant embryos, this suggests that the role of P-Rex1 in migration is almost exclusively mediated via Rac1. Here we focused on E15.5 embryos, a useful time point to observe the late migratory effects observed with previously described Prex and Rac phenotypes (Li et al., 2011Li A. Ma Y. Yu X. et al.Rac1 drives melanoblast organization during mouse development by orchestrating pseudopod-driven motility and cell-cycle progression.Dev Cell. 2011; 21: 722-734Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar; Lindsay et al., 2011Lindsay C.R. Lawn S. Campbell A.D. et al.P-Rex1 is required for efficient melanoblast migration and melanoma metastasis.Nat Commun. 2011; 2: 555Crossref PubMed Scopus (129) Google Scholar). With a greater number of embryos, we would have performed further embryo time-point analyses at E13.5 to ensure there was no earlier melanoblast migratory deficit that could contribute to this phenotype, although even this experiment could not completely exclude such a possibility. One potential Rho-GTPase, RhoG, is a likely candidate for P-Rex1 interaction: it is the most structurally similar Rho-GTPase to Rac and has been shown to cooperate with Rac for induction of cell transformation (Roux et al., 1997Roux P. Gauthier-Rouvière C. Doucet-Brutin S. et al.The small GTPases Cdc42Hs, Rac1 and RhoG delineate Raf-independent pathways that cooperate to transform NIH3T3 cells.Curr Biol. 1997; 7: 629-637Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). Moreover, there are distinct regulatory and functional similarities between P-Rex1 and Vav proteins, which have been characterized as the predominant GEFs required for RhoG activation (Samson et al., 2010Samson T. Welch C. Monaghan-Benson E. et al.Endogenous RhoG is rapidly activated after epidermal growth factor stimulation through multiple guanine-nucleotide exchange factors.Mol Biol Cell. 2010; 21: 1629-1642Crossref PubMed Scopus (35) Google Scholar; Lawson et al., 2011Lawson C.D. Donald S. Anderson K.E. et al.P-Rex1 and Vav1 cooperate in the regulation of formyl-methionyl-leucyl-phenylalanine-dependent neutrophil responses.J Immunol. 2011; 186: 1467-1476Crossref PubMed Scopus (62) Google Scholar). Further studies are underway to investigate the phenotypes of these and other potential Rho-GTPases in melanoblast migration and melanomagenesis. Given that there is now considerable effort to generate Rac1 inhibitors, our data would suggest functions for proteins upstream of Rac1 that may become further therapeutic targets in melanoma. All experiments were conducted and approved in accordance with institutional and UK guidelines, and all animal studies were performed in accordance with local regulatory guidelines. This research was supported by a Medical Research Council clinical fellowship (CRL), CRUK, and a European Research Council consolidator grant (OJS). We thank Colin Nixon and Margaret O’Prey, as well as the biological services, histology, and imaging staff at the CRUK Beatson Institute for Cancer Research in general. We also thank Catherine Winchester for her assistance with checking the manuscript. Supplementary material is linked to the online version of the paper at http://www.nature.com/jid