title: DUSP5 and DUSP6 modulate corneal epithelial cell proliferation.
abstract: OBJECTIVE
  Dual specificity phosphatases (DUSPs) modulate the duration and magnitude of phospho-activation of Erk1/2, p38 and JNK1/2, the terminal kinases (TKs) of the mitogen activated protein kinase (MAPK) cascades. Three DUSPs, DUSP1, DUSP5, and DUSP6, are overexpressed in ocular surface side population stem cells (SPSCs). Our objective was to identify the impact of these enzymes on TK phosphorylation and proliferation of corneal epithelial cells.
  
  
  METHODS
  SV40 immortalized (sv) and expanded fresh human corneal epithelial cells (efHCECs) were transduced with lentivectors to elicit expression of shRNAmir against DUSP1, DUSP5, and JNK1 to thereby create the DUSP1i, DUSP5i and JNKi cell sublines, or overexpress DUSP6 (henceforth DUSP6(+)), respectively. TK phosphorylation status and proliferation rates were determined by immunoblotting and (3)H thymidine uptake.
  
  
  RESULTS
  In both ef and svHCECs, EGF supplementation after a 24 h serum starvation caused a rapid 5-15 min spike in the phosphorylation of all three TK types. This was followed by gradual decreases to low phosphorylation levels within one h. These declines coincided with dramatic increases in DUSP1 and DUSP5 protein expression. In DUSP1i, the DUSP1 increase was abolished. All 3 TKs maintained high phosphorylation levels for at least 90 min and proliferation rates were unchanged from non-transduced cells. In DUSP5i, the DUSP5 protein increase was prevented, the post peak phosphorylation decrease occurred only on Erk1/2 and the proliferation rate increased by 50%-60%. In JNK1i, JNK1 was essentially knocked out and proliferation rates were also markedly elevated. At steady-state, DUSP1i maintained high levels of pJNK1/2 expression. In DUSP6(+) Erk1/2 phosphorylation was prevented and proliferation rates decreased to less than 50%.
  
  
  CONCLUSIONS
  DUSP5 and DUSP6 selectively control ERK pathway activity and proliferation. The lack of an effect of DUSP1 knockdown on proliferation can be attributed to its pan-MAPK effect. The expected augmented proliferative response due to enhanced and prolonged phosphorylation of Erk1/2 following DUSP1 knockdown does not occur because a pJNK1/2 antiproliferative effect is simultaneously unleashed.
authors.affiliation: Department of Biological Sciences, SUNY State College of Optometry, New York, NY, USA.
authors.forename: Zheng
authors.initials: Z
authors.lastname: Wang
authors.forename: Peter S
authors.initials: PS
authors.lastname: Reinach
authors.forename: Fan
authors.initials: F
authors.lastname: Zhang
authors.forename: Kati-Sisko
authors.initials: KS
authors.lastname: Vellonen
authors.forename: Arto
authors.initials: A
authors.lastname: Urtti
authors.forename: Helen
authors.initials: H
authors.lastname: Turner
authors.forename: J Mario
authors.initials: JM
authors.lastname: Wolosin
mesh_terms: D002461:Cell Line, Transformed; D049109:Cell Proliferation; D003720:Densitometry; D054638:Dual Specificity Phosphatase 1; D054642:Dual Specificity Phosphatase 6 / Q000235:genetics / Q000378:metabolism*; D054637:Dual-Specificity Phosphatases / Q000235:genetics / Q000378:metabolism*; D004847:Epithelial Cells / Q000166:cytology* / Q000201:enzymology*; D019573:Epithelium, Corneal / Q000166:cytology*; D015971:Gene Expression Regulation, Enzymologic; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D016086:Lentivirus / Q000235:genetics; D010766:Phosphorylation; D013936:Thymidine / Q000378:metabolism; D014161:Transduction, Genetic
chemical_list: C517019:DUSP1 protein, human; C442320:DUSP5 protein, human; C517035:DUSP6 protein, human; D054638:Dual Specificity Phosphatase 1; D054642:Dual Specificity Phosphatase 6; D054637:Dual-Specificity Phosphatases; D013936:Thymidine
references.citation: Cell Mol Life Sci. 1999 Aug 15;55(10):1230-54
references.pmid: 10487205
references.citation: Invest Ophthalmol Vis Sci. 2009 Sep;50(9):4162-72
references.pmid: 19324848
references.citation: Curr Opin Cell Biol. 1996 Apr;8(2):205-15
references.pmid: 8791420
references.citation: J Biol Chem. 2001 Mar 9;276(10):6905-8
references.pmid: 11238443
references.citation: Mol Cell Endocrinol. 2010 Jan 27;314(2):213-20
references.pmid: 19406201
references.citation: J Invest Dermatol. 1983 Jul;81(1 Suppl):33s-40s
references.pmid: 6345690
references.citation: Mol Endocrinol. 2007 Nov;21(11):2663-71
references.pmid: 17636038
references.citation: Nature. 2001 Mar 1;410(6824):37-40
references.pmid: 11242034
references.citation: Prog Retin Eye Res. 2000 Mar;19(2):223-55
references.pmid: 10674709
references.citation: Stem Cells. 2006 Jan;24(1):86-94
references.pmid: 16150918
references.citation: Invest Ophthalmol Vis Sci. 2009 Oct;50(10):4828-35
references.pmid: 19443725
references.citation: FASEB J. 1995 Jun;9(9):726-35
references.pmid: 7601337
references.citation: Biochem J. 2009 Mar 15;418(3):475-89
references.pmid: 19228121
references.citation: Cell. 1989 Apr 21;57(2):201-9
references.pmid: 2702690
references.citation: J Cell Sci. 2005 Apr 15;118(Pt 8):1715-24
references.pmid: 15811951
references.citation: Cancer Metastasis Rev. 2008 Jun;27(2):253-61
references.pmid: 18330678
references.citation: Apoptosis. 2010 Jul;15(7):864-76
references.pmid: 20411335
references.citation: Invest Ophthalmol Vis Sci. 2007 May;48(5):2050-61
references.pmid: 17460260
references.citation: Biochim Biophys Acta. 2007 Aug;1773(8):1299-310
references.pmid: 17188374
references.citation: Nat Med. 1997 Dec;3(12):1337-45
references.pmid: 9396603
references.citation: J Biol Chem. 2001 May 11;276(19):16491-500
references.pmid: 11278799
references.citation: Invest Ophthalmol Vis Sci. 2009 Dec;50(12):5630-8
references.pmid: 19608544
references.citation: Nat Rev Drug Discov. 2007 May;6(5):391-403
references.pmid: 17473844
references.citation: Invest Ophthalmol Vis Sci. 2009 Feb;50(2):604-13
references.pmid: 18824731
references.citation: Lab Invest. 1959 Jan-Feb;8(1):296-306; discussion 306-8
references.pmid: 13621628
references.citation: J Biol Chem. 2003 Jul 18;278(29):26715-21
references.pmid: 12738796
references.citation: EMBO J. 1999 Feb 1;18(3):664-74
references.pmid: 9927426
references.citation: J Pharm Sci. 2010 Feb;99(2):1087-98
references.pmid: 19623615
references.citation: Cell Signal. 2008 May;20(5):836-43
references.pmid: 18280112
references.citation: Invest Ophthalmol Vis Sci. 2008 Feb;49(2):539-49
references.pmid: 18234997
references.citation: Mol Cell Biol. 2005 Mar;25(5):1830-45
references.pmid: 15713638