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DUSP5 and DUSP6 modulate corneal epithelial cell proliferation.

Wang Z, Reinach PS, Zhang F, Vellonen KS, Urtti A, Turner H, Wolosin JM - Mol. Vis. (2010)

Bottom Line: In DUSP6(+) Erk1/2 phosphorylation was prevented and proliferation rates decreased to less than 50%.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, SUNY State College of Optometry, New York, NY, USA.

ABSTRACT

Purpose: 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.

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Related in: MedlinePlus

Densitometric plots of the phosphorylated MAPK bands displayed in Figure 2. A and D: p.p38. B and E: pJNK1. C and E: pErk1. Top frames are for DUSP1 shRNA, bottom frames for DUSP5 shRNA. The values were normalized respective to loading controls, either total Erk or actin.
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f3: Densitometric plots of the phosphorylated MAPK bands displayed in Figure 2. A and D: p.p38. B and E: pJNK1. C and E: pErk1. Top frames are for DUSP1 shRNA, bottom frames for DUSP5 shRNA. The values were normalized respective to loading controls, either total Erk or actin.

Mentions: In the cells transduced with anti DUSP1 shRNAmir, the increases in DUSP1 were essentially abolished and all three TKs maintained high levels of activity throughout the first 60 min of post-EGF exposure. In the cells transduced with the DUSP5 shRNAmir, the DUSP5 rise was ified and Erk1/2 continuously increased over this period, but the phosphorylation changes in the JNK/SAPK and p38 kinases were not affected. The differences in phosphorylation in response to EGF between the cells transduced with shRNAs for DUSP1 and DUSP5 are shown in Figure 3 in densitometric plots of the results presented in Figure 2A,B.


DUSP5 and DUSP6 modulate corneal epithelial cell proliferation.

Wang Z, Reinach PS, Zhang F, Vellonen KS, Urtti A, Turner H, Wolosin JM - Mol. Vis. (2010)

Densitometric plots of the phosphorylated MAPK bands displayed in Figure 2. A and D: p.p38. B and E: pJNK1. C and E: pErk1. Top frames are for DUSP1 shRNA, bottom frames for DUSP5 shRNA. The values were normalized respective to loading controls, either total Erk or actin.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2927432&req=5

f3: Densitometric plots of the phosphorylated MAPK bands displayed in Figure 2. A and D: p.p38. B and E: pJNK1. C and E: pErk1. Top frames are for DUSP1 shRNA, bottom frames for DUSP5 shRNA. The values were normalized respective to loading controls, either total Erk or actin.
Mentions: In the cells transduced with anti DUSP1 shRNAmir, the increases in DUSP1 were essentially abolished and all three TKs maintained high levels of activity throughout the first 60 min of post-EGF exposure. In the cells transduced with the DUSP5 shRNAmir, the DUSP5 rise was ified and Erk1/2 continuously increased over this period, but the phosphorylation changes in the JNK/SAPK and p38 kinases were not affected. The differences in phosphorylation in response to EGF between the cells transduced with shRNAs for DUSP1 and DUSP5 are shown in Figure 3 in densitometric plots of the results presented in Figure 2A,B.

Bottom Line: In DUSP6(+) Erk1/2 phosphorylation was prevented and proliferation rates decreased to less than 50%.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, SUNY State College of Optometry, New York, NY, USA.

ABSTRACT

Purpose: 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.

Show MeSH
Related in: MedlinePlus