Rac1 plays a role in CXCL12 but not CCL3-induced chemotaxis and Rac1 GEF inhibitor NSC23766 has off target effects on CXCR4

Shirley Mills, Lesley Howell, Andrew Beekman, Leanne Stokes, Anja Mueller

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Abstract

Cell mi­gra­tion to­wards a chemo­tac­tic stim­u­lus re­lies on the re-arrange­ment of the cy­toskele­ton, which is trig­gered by ac­ti­va­tion of small G pro­teins RhoA, Rac1 and Cd­c42, and leads to for­ma­tion of lamel­lopo­dia and actin poly­meri­sa­tion amongst other ef­fects. Here we show that Rac1 is im­por­tant for CX­CR4 in­duced chemo­taxis but not for CCR1/​CCR5 in­duced chemo­taxis. For CX­CL12-in­duced mi­gra­tion via CX­CR4, breast can­cer MCF-7 cells are re­liant on Rac1, sim­i­larly to THP-1 mono­cytes and Ju­rkat T-cells. For CCL3-in­duced mi­gra­tion via CCR1 and/​or CCR5, Rac1 sig­nalling does not reg­u­late cell mi­gra­tion in ei­ther sus­pen­sion or ad­her­ent cells. We have con­firmed the in­volve­ment of Rac1 with the use of a spe­cific Rac1 block­ing pep­tide. We also used a Rac1 in­hibitor EHT 1864 and a Rac1-GEF in­hibitor NSC23766 to probe the im­por­tance of Rac1 in chemo­taxis. Both in­hibitors did not block CCL3-in­duced chemo­taxis, but they were able to block CX­CL12-in­duced chemo­taxis. This con­firms that Rac1 ac­ti­va­tion is not es­sen­tial for CCL3-in­duced mi­gra­tion, how­ever NSC23766 might have sec­ondary ef­fects on CX­CR4. This small mol­e­cule ex­hibits ag­o­nis­tic fea­tures in in­ter­nal­i­sa­tion and cAMP as­says, whereas it acts as an an­tag­o­nist for CX­CR4 in mi­gra­tion and cal­cium re­lease as­says. Our find­ings strongly sug­gest that Rac1 ac­ti­va­tion is not nec­es­sary for CCL3 sig­nalling, and re­veal that NSC23766 could be a novel CX­CR4 re­cep­tor lig­and.
Original languageEnglish
Pages (from-to)88–96
Number of pages9
JournalCellular Signalling
Volume42
Early online date16 Oct 2017
DOIs
Publication statusPublished - Jan 2018

Keywords

  • Chemokine receptor
  • Chemotaxis
  • Antagonist
  • Rac1
  • Ligand bias

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