Limits...
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.

Show MeSH

Related in: MedlinePlus

Effects of DUSP1, DUSP5, and DUSP6 expression changes on the steady-state and post activation time course of terminal kinase phosphorylation in svHCEC and efHCECs cells. A: Effect of DUSP1 shRNAmir. The antisense agent prevented the post EGF activation increases in DUSP1 protein and concomitantly abolished or diminished the decline in the initial EGF induced phosphorylations of Erk1/2, JNK1/2, and p38 normally occurring in the control. B. Effect of DUSP5 shRNAmir. The antisense agent completely blocked the post-EGF increase in DUSP5 protein levels and abolished the post peak decline in pErk1/2 phosphorylation. This agent did not visibly modify the time course of the phosphorylation responses of JNK1/2 or p38. C. Effects of overexpression of DUSP6. Phosphorylation of Erk1/2 in response to EGF was virtually abolished. The effects on the other two terminal kinases were nil or minimal. D. Comparison of the effects of shRNAmirs against DUSP1 and DUSP5 expression on the phosphorylation of the terminal kinases in cells growing in log phase (50%–80% confluent) in whole culture medium. Both antisense interventions increased pErk1/2 steady-state levels. DUSP1 selectively increased pJNK1/2 phosphorylation. Neither had an effect on p-p38 steady-state expression. E. Effect of expression of JNK1 shRNAmir on the expression of JNK1 protein in svHCEC cells. F. Experiment showing that the permanent transduction with DUSP5 shRNA does not interfere with the rise in DUSP1 protein that occurs after cell reactivation with EGF. G. Effect of DUSP5 shRNAmir on pErk1/2 phosphorylation in the efHCECs cells. Blots A-E are representative of two or more repeats.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2927432&req=5

f2: Effects of DUSP1, DUSP5, and DUSP6 expression changes on the steady-state and post activation time course of terminal kinase phosphorylation in svHCEC and efHCECs cells. A: Effect of DUSP1 shRNAmir. The antisense agent prevented the post EGF activation increases in DUSP1 protein and concomitantly abolished or diminished the decline in the initial EGF induced phosphorylations of Erk1/2, JNK1/2, and p38 normally occurring in the control. B. Effect of DUSP5 shRNAmir. The antisense agent completely blocked the post-EGF increase in DUSP5 protein levels and abolished the post peak decline in pErk1/2 phosphorylation. This agent did not visibly modify the time course of the phosphorylation responses of JNK1/2 or p38. C. Effects of overexpression of DUSP6. Phosphorylation of Erk1/2 in response to EGF was virtually abolished. The effects on the other two terminal kinases were nil or minimal. D. Comparison of the effects of shRNAmirs against DUSP1 and DUSP5 expression on the phosphorylation of the terminal kinases in cells growing in log phase (50%–80% confluent) in whole culture medium. Both antisense interventions increased pErk1/2 steady-state levels. DUSP1 selectively increased pJNK1/2 phosphorylation. Neither had an effect on p-p38 steady-state expression. E. Effect of expression of JNK1 shRNAmir on the expression of JNK1 protein in svHCEC cells. F. Experiment showing that the permanent transduction with DUSP5 shRNA does not interfere with the rise in DUSP1 protein that occurs after cell reactivation with EGF. G. Effect of DUSP5 shRNAmir on pErk1/2 phosphorylation in the efHCECs cells. Blots A-E are representative of two or more repeats.

Mentions: Figure 2 summarizes the effects of the lentivector-induced modification of DUSP activity on the phosphorylation levels of all three TKs in the svHCEC. Unless indicated otherwise, cells were starved of growth factors by incubating them for 24 h in base media (D/F12 for svHCECs, complement-free EpiLife for the efHCECs using multiple replicate cultures. Next, cells were reactivated by the addition of either 10 ng/ml EGF in the case of the svHCEC or EpiLife complement (contains bovine pituitary extract and insulin) in the case of the efHCECs. Cultures were collected just before reactivation or 5, 15, 30, and 60 min thereafter. For the DUSP1 shRNA experiments, the five control and five transduced cell time points were run side by side, each with MW markers. For the DUSP5 shRNA and the DUSP6 ORF, to allow the side by side time course comparison to be present in a single 10-well gel, we ran separately one gel for the 0–5 min time points and another for the 5–60 min comparisons.


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)

Effects of DUSP1, DUSP5, and DUSP6 expression changes on the steady-state and post activation time course of terminal kinase phosphorylation in svHCEC and efHCECs cells. A: Effect of DUSP1 shRNAmir. The antisense agent prevented the post EGF activation increases in DUSP1 protein and concomitantly abolished or diminished the decline in the initial EGF induced phosphorylations of Erk1/2, JNK1/2, and p38 normally occurring in the control. B. Effect of DUSP5 shRNAmir. The antisense agent completely blocked the post-EGF increase in DUSP5 protein levels and abolished the post peak decline in pErk1/2 phosphorylation. This agent did not visibly modify the time course of the phosphorylation responses of JNK1/2 or p38. C. Effects of overexpression of DUSP6. Phosphorylation of Erk1/2 in response to EGF was virtually abolished. The effects on the other two terminal kinases were nil or minimal. D. Comparison of the effects of shRNAmirs against DUSP1 and DUSP5 expression on the phosphorylation of the terminal kinases in cells growing in log phase (50%–80% confluent) in whole culture medium. Both antisense interventions increased pErk1/2 steady-state levels. DUSP1 selectively increased pJNK1/2 phosphorylation. Neither had an effect on p-p38 steady-state expression. E. Effect of expression of JNK1 shRNAmir on the expression of JNK1 protein in svHCEC cells. F. Experiment showing that the permanent transduction with DUSP5 shRNA does not interfere with the rise in DUSP1 protein that occurs after cell reactivation with EGF. G. Effect of DUSP5 shRNAmir on pErk1/2 phosphorylation in the efHCECs cells. Blots A-E are representative of two or more repeats.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: Effects of DUSP1, DUSP5, and DUSP6 expression changes on the steady-state and post activation time course of terminal kinase phosphorylation in svHCEC and efHCECs cells. A: Effect of DUSP1 shRNAmir. The antisense agent prevented the post EGF activation increases in DUSP1 protein and concomitantly abolished or diminished the decline in the initial EGF induced phosphorylations of Erk1/2, JNK1/2, and p38 normally occurring in the control. B. Effect of DUSP5 shRNAmir. The antisense agent completely blocked the post-EGF increase in DUSP5 protein levels and abolished the post peak decline in pErk1/2 phosphorylation. This agent did not visibly modify the time course of the phosphorylation responses of JNK1/2 or p38. C. Effects of overexpression of DUSP6. Phosphorylation of Erk1/2 in response to EGF was virtually abolished. The effects on the other two terminal kinases were nil or minimal. D. Comparison of the effects of shRNAmirs against DUSP1 and DUSP5 expression on the phosphorylation of the terminal kinases in cells growing in log phase (50%–80% confluent) in whole culture medium. Both antisense interventions increased pErk1/2 steady-state levels. DUSP1 selectively increased pJNK1/2 phosphorylation. Neither had an effect on p-p38 steady-state expression. E. Effect of expression of JNK1 shRNAmir on the expression of JNK1 protein in svHCEC cells. F. Experiment showing that the permanent transduction with DUSP5 shRNA does not interfere with the rise in DUSP1 protein that occurs after cell reactivation with EGF. G. Effect of DUSP5 shRNAmir on pErk1/2 phosphorylation in the efHCECs cells. Blots A-E are representative of two or more repeats.
Mentions: Figure 2 summarizes the effects of the lentivector-induced modification of DUSP activity on the phosphorylation levels of all three TKs in the svHCEC. Unless indicated otherwise, cells were starved of growth factors by incubating them for 24 h in base media (D/F12 for svHCECs, complement-free EpiLife for the efHCECs using multiple replicate cultures. Next, cells were reactivated by the addition of either 10 ng/ml EGF in the case of the svHCEC or EpiLife complement (contains bovine pituitary extract and insulin) in the case of the efHCECs. Cultures were collected just before reactivation or 5, 15, 30, and 60 min thereafter. For the DUSP1 shRNA experiments, the five control and five transduced cell time points were run side by side, each with MW markers. For the DUSP5 shRNA and the DUSP6 ORF, to allow the side by side time course comparison to be present in a single 10-well gel, we ran separately one gel for the 0–5 min time points and another for the 5–60 min comparisons.

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