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A Rac1-Independent Role for P-Rex1 in Melanoblasts

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A Rac1-Independent Role for P-Rex1 in Melanoblasts CR Lindsay et al. Role of Rac1 and P-Rex1 in Melanoblast Development REFERENCES inflammatory gene profiles. Cytometry B Clin and is an endothelial adhesion receptor. Cytom 78:88–95 JImmunol179:6673–85 Bardin N, Anfosso F, Masse´ JM, et al. (2001) Identification of CD146 as a component Elshal MF, Khan SS, Raghavachari N, et al. (2007) Kagami S, Rizzo HL, Lee JJ et al. (2010) Circulating of the endothelial junction involved in the A unique population of effector memory Th17, Th22, and Th1 cells are increased in control of cell-cell cohesion. Blood 98: lymphocytes identified by CD146 having a psoriasis. J Invest Dermatol 130:1373–83 3677–84 distinct immunophenotypic and genomic pro- Larochelle C, Cayrol R, Kebir H et al. (2012) file. BMC Immunol 8:29 Dagur PK, Biancotto A, Wei L, et al. (2011) Melanoma cell adhesion molecule identifies MCAM-expressing CD4( þ ) T cells in Elshal MF, Khan SS, Takahashi Y et al. (2005) encephalitogenic T lymphocytes and pro- peripheral blood secrete IL-17 A and CD146 (Mel-CAM), an adhesion marker of motes their recruitment to the central nervous are significantly elevated in inflammatory endothelial cells, is a novel marker of lym- system. Brain 135(Part 10):2906–24 autoimmune diseases. J Autoimmun 37: phocyte subset activation in normal periph- Mehta NN, Yu Y, Saboury B et al. (2011) Systemic 319–27 eral blood. Blood 106:2923–4 and vascular inflammation in patients with Dagur PK, Tatlici G, Gourley M, et al. (2010) Guezguez B, Vigneron P, Lamerant N et al. (2007) moderate to severe psoriasis as measured CD146 þ T lymphocytes are increased in Dual role of melanoma cell adhesion mole- by [18 F]-fluorodeoxyglucose positron emis- both the peripheral circulation and in cule (MCAM)/CD146 in lymphocyte endothe- sion tomography-computed tomography the synovial effusions of patients with various lium interaction: MCAM/CD146 promotes (FDG-PET/CT): a pilot study. Arch Dermatol musculoskeletal diseases and display pro- rolling via microvilli induction in lymphocyte 147:1031–9 A Rac1-Independent Role for P-Rex1 in Melanoblasts Journal of Investigative Dermatology (2015) 135, 314–318; doi:10.1038/jid.2014.323; published online 11 September 2014 TO THE EDITOR melanoblasts lacking at the most distal melanoblast lineage, and DCT::b- Given the recent discovery of RAC1- points of migration (belly and paws). galactosidase (otherwise referred to as activating mutations in melanoma, and Constitutive deletion of Rac1 is DCT-lacZ). our finding that PIP3-dependent Rac- embryonically lethal, but a coat color First, we hypothesized that, if the exchanger 1 (PREX1) is overexpressed defect of mice with melanocyte-specific effects of P-Rex1 were mediated exclu- and drives metastasis in this cancer, an RAC1 abrogation (Tyr::Cre Rac1fl/fl)has sively via Rac1, double mutant Tyr::Cre important question is to establish whe- also been described; these mice have a Rac1fl/fl;P-Rex1À / À mice would exhibit ther the functions of P-Rex1 are medi- larger belly spot on their ventral side, the same coat color phenotype as ated specifically by Rac alone (Lindsay suggesting that alternative Rho-GTPases Tyr::Cre Racfl/fl mice alone. However, et al., 2011; Berger et al., 2012). Here can be activated to enable melanoblast Tyr::Cre Racfl/fl; P-Rex1 À / À mice dis- we describe a Rac1-independent in vivo migration to the perimeter of the play a dramatic alteration in coat color role for P-Rex1 through identification Tyr::Cre Racfl/fl white belly (Sugihara phenotype from Tyr::Cre Racfl/fl mice and characterization of a mouse coat et al., 1998; Li et al., 2011). A role for (n ¼ 7; Figure 1a). The ventral and dorsal color phenotype. P-Rex1 is a guanine- Rac1 in proliferation was also observed, coats of these mice are almost entirely nucleotide exchange factor (GEF) for as there was a marked reduction white, with hypo-pigmented limbs and Rac, whose primary cell function is of melanoblast numbers in this tail. Graying pigmented areas were only induction of actin-mediated membrane phenotype. In line with these previous observed in the head coat. We con- ruffling and lamellipodia formation at studies, and because mice with melano- cluded from this experiment that P-Rex1 the leading edge of cell migration cyte-specific RAC1 abrogation require and Rac1 together constitute fundamen- (Welch et al., 2002; Hill et al., 2005; euthanization shortly after birth because tal signaling components of the mouse Barber et al., 2007). of neurological problems, we used the coat color phenotype, with minimal To investigate this question we same embryonic melanoblast reporter rescue of melanoblast development decided to examine the role of Rac1 models to assess the downstream conferred by other GEFs or Rho- and P-Rex1 in melanoblast develop- effects of P-Rex1 in vivo (Mackenzie GTPases. It was also clear that P-Rex1 ment. Previously, we reported a ‘‘white et al., 1997; Mort et al., 2010; Li must be able to exert phenotypic effects belly’’ phenotype of mice with Prex1 et al., 2011). Melanocyte-specific other than via Rac1. deletion (Lindsay et al., 2011). Impaired reporter mouse strains employed were To explore the Rac1-independent melanoblast migration was mostly Tyr::Cre Z/EG, which drives green effects of P-Rex1 further, we crossed responsible for this phenotype, with fluorescent protein expression in the Tyr::Cre Racfl/fl;P-Rex1À / À mice with mice carrying the melanoblast reporter DCT-lacZ transgene (methods detailed in Abbreviations: GEF, guanine-nucleotide exchange factor; OHT, 4-hydroxytamoxifen; PREX1, PIP3- Lindsay et al., 2011). Relative to Tyr::Cre fl/fl À / À dependent Rac-exchanger 1 Rac mice or P-Rex1 embryos fl/fl À / À Accepted article preview online 30 July 2014; published online 11 September 2014 alone, Tyr::Cre Rac ;P-Rex1 314 Journal of Investigative Dermatology (2015), Volume 135 CR Lindsay et al. Role of Rac1 and P-Rex1 in Melanoblast Development P-Rex1–/– Tyr::Cre Rac1fl/fl P-Rex1–/–; Tyr::Cre Rac1fl/fl 15,000 Control P-Rex1–/– ** ** 10,000 * ** 5,000 ** Tyr::Cre Rac1fl/fl P-Rex1–/– Number of melanoblasts ** Tyr::Cre Rac1fl/fl 0 Ctr Rac1 f/f P-Rex1–/– Rac1 f/f P-Rex1–/– Ctr Ctr siPrex1–2 siPrex1–2 OHT siPrex1–4 DMSO siPrex1–4 80 Staurosporin Ctr siP-Rex1–2 siP-Rex1–4 Ctr siP-Rex1–2 siP-Rex1–4 8 ) ** 4 ** ) P-Rex1 4 60 6 Rac 1/2/3 40 4 Actin ** * DCT 20 2 ** Number of cells (10 CC3 Number of cells (10 0 0 DMSO OHT 0 246 0246 Days Days Figure 1. P-Rex1 and Rac1 are fundamental components of a mouse coat color phenotype. (a) Ventral coats of P-Rex1 À / À , Tyr::Cre Rac1fl/fl and P-Rex1 À / À ; Tyr::Cre Rac1fl/flmice. Final photomicrograph shows dorsal and head coat of Tyr::Cre Rac1fl/fl;P-Rex1 À / À mice. All mice were bred from a pure genetic background (C57Bl6). A total of 7 Tyr::Cre Rac1fl/fl;P-Rex1 À / À mice were bred; as Tyr::Cre Rac1fl/fl mice have a limited life span and no breeding potential, Tyr::Cre Rac1fl/ þ mice were crossed with P-Rex1 À / À mice to produce approximately one in four Tyr::Cre Rac1fl/fl;P-Rex1 À / À pups. (b) Representative photos of DCT-lacZ melanoblast distribution observed in wild-type (control), P-Rex1 À / À , Tyr::Cre Rac1fl/fl,andTyr::Cre Rac1fl/fl;P-Rex1 À / À embryos at E15.5. Black interrupted lines represent the area used for melanoblast quantification shown in c. White demarcated lines represent typical areas of melanoblast sparing visualized for each phenotype (n ¼ 3). (c) Number of melanoblasts in wild-type (Ctr), P-Rex1 À / À (‘P-Rex1 À / À ’), Tyr::Cre Rac1fl/fl (‘Rac1 f/f’), and Tyr::Cre Rac1fl/fl;P-Rex1 À / À (‘Rac1 f/f P-rex1 À / À ’) embryos at E15.5 (X5 embryos). Lower quartile, median, and upper quartile are shown. **Po0.01 by t-test; *Po0.05 by t-test. (d) Western blots of the primary melanocyte cell line, small interfering RNA (siRNA) treated as indicated, then treated with DMSO or 4-hydroxytamoxifen (OHT) for 5 days were probed with antibodies as indicated; Ctr, control siRNA–treated cells. (e) Growth curve of the primary melanocyte cell line, siRNA-treated as indicated, then treated with DMSO or OHT. Each point (mean±SEM) is from three replicates of three independent experiments. Asterisks represent difference between control siRNA–treated cells and P-Rex1 siRNA–treated cells at the same time point; Ctr, control siRNA-treated cells. www.jidonline.org 315 CR Lindsay et al. Role of Rac1 and P-Rex1 in Melanoblast Development Control P-Rex1–/– Tyr::Cre Rac1fl/fl P-Rex1–/– Tyr::Cre Rac1fl/fl 1 1 1 1 3 2 2 2 3 4 3 5 5 4 4 5 2 6 7 6 7 6 3 0′ 5′ 10′ 15′ 20′ 25′ Control P-Rex1–/– Tyr::Cre Rac1fl/fl P-Rex1–/– Tyr::Cre Rac1fl/fl 80 Control 80 P-Rex1–/– 60 60 40 40 ) m) 20 m) 20 –1 μ μ 0.8 80 0 0 m) μ -Axis ( –20 -Axis ( –20 y y –40 m min 0.6 60 ** –40 μ ** * –60 –60 ** NS –80 –80 0.4 ** NS 40 ** –50 50 –50 50 ** ** x-Axis (μm) x-Axis (μm) 0.2 ** ** 20 80 fl/fl 80 –/– Tyr::Cre Rac1 P-Rex1 0.0 Euclidean distance ( 0 60 60 Tyr::Cre Rac1fl/fl speed ( Migration 40 40 Ctr –/– –/– Ctr –/– –/– m) m) 20 20 μ μ Rac1 f/f Rac1 f/f 0 0 P-Rex1 P-Rex1 -Axis ( -Axis ( –20 –20 y y –40 –40 Rac1 f/f P-Rex1 Rac1 f/f P-Rex1 –60 –60 –80 –80 –50 50 –50 50 x-Axis (μm) x-Axis (μm) Figure 2.
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