DUSP5 and DUSP6 modulate corneal epithelial cell proliferation.
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.
Wang
Zheng
Z
Department of Biological Sciences, SUNY State College of Optometry, New York, NY, USA.
Reinach
Peter S
PS
Zhang
Fan
F
Vellonen
Kati-Sisko
KS
Urtti
Arto
A
Turner
Helen
H
Wolosin
J Mario
JM
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
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
Cell Mol Life Sci. 1999 Aug 15;55(10):1230-54
10487205
Invest Ophthalmol Vis Sci. 2009 Sep;50(9):4162-72
19324848
Curr Opin Cell Biol. 1996 Apr;8(2):205-15
8791420
J Biol Chem. 2001 Mar 9;276(10):6905-8
11238443
Mol Cell Endocrinol. 2010 Jan 27;314(2):213-20
19406201
J Invest Dermatol. 1983 Jul;81(1 Suppl):33s-40s
6345690
Mol Endocrinol. 2007 Nov;21(11):2663-71
17636038
Nature. 2001 Mar 1;410(6824):37-40
11242034
Prog Retin Eye Res. 2000 Mar;19(2):223-55
10674709
Stem Cells. 2006 Jan;24(1):86-94
16150918
Invest Ophthalmol Vis Sci. 2009 Oct;50(10):4828-35
19443725
FASEB J. 1995 Jun;9(9):726-35
7601337
Biochem J. 2009 Mar 15;418(3):475-89
19228121
Cell. 1989 Apr 21;57(2):201-9
2702690
J Cell Sci. 2005 Apr 15;118(Pt 8):1715-24
15811951
Cancer Metastasis Rev. 2008 Jun;27(2):253-61
18330678
Apoptosis. 2010 Jul;15(7):864-76
20411335
Invest Ophthalmol Vis Sci. 2007 May;48(5):2050-61
17460260
Biochim Biophys Acta. 2007 Aug;1773(8):1299-310
17188374
Nat Med. 1997 Dec;3(12):1337-45
9396603
J Biol Chem. 2001 May 11;276(19):16491-500
11278799
Invest Ophthalmol Vis Sci. 2009 Dec;50(12):5630-8
19608544
Nat Rev Drug Discov. 2007 May;6(5):391-403
17473844
Invest Ophthalmol Vis Sci. 2009 Feb;50(2):604-13
18824731
Lab Invest. 1959 Jan-Feb;8(1):296-306; discussion 306-8
13621628
J Biol Chem. 2003 Jul 18;278(29):26715-21
12738796
EMBO J. 1999 Feb 1;18(3):664-74
9927426
J Pharm Sci. 2010 Feb;99(2):1087-98
19623615
Cell Signal. 2008 May;20(5):836-43
18280112
Invest Ophthalmol Vis Sci. 2008 Feb;49(2):539-49
18234997
Mol Cell Biol. 2005 Mar;25(5):1830-45
15713638