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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

Schematic description of proposed nuclear events associated with the transductions performed in this study. Erk1/2 phosphorylation causes nuclear translocation and activation of transcription factors that facilitate the G1 to S transition. JNK phosphorylation causes nuclear translocation and activation of antiproliferative events at some stage of the cell cycle (indicated by gray lines). The proliferation-enhancing event is blocked by pErk/2 dephosphorylation, by DUSP6 in the cytosol and DUSP5 and DUSP1 in the nuclei. The latter DUSP though, blocks JNK1/2 phosphorylation with high efficiency ifying its pro-proliferative action.
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f5: Schematic description of proposed nuclear events associated with the transductions performed in this study. Erk1/2 phosphorylation causes nuclear translocation and activation of transcription factors that facilitate the G1 to S transition. JNK phosphorylation causes nuclear translocation and activation of antiproliferative events at some stage of the cell cycle (indicated by gray lines). The proliferation-enhancing event is blocked by pErk/2 dephosphorylation, by DUSP6 in the cytosol and DUSP5 and DUSP1 in the nuclei. The latter DUSP though, blocks JNK1/2 phosphorylation with high efficiency ifying its pro-proliferative action.

Mentions: Gene microarray studies of SP stem cells from pig limbus [18] and human conjunctiva [17] and limbus (unpublished) revealed several features that have the potential to underpin this slow cycling phenotype. One of them is the high expression level of several DUSPs, relative to those of their nonSP counterparts. DUSP5 is the most prominently overexpressed DUSP in the two published studies. It is 10.6 times higher in the human conjunctival SP cell [17] than in the nonSP counterparts, 2.8 times better expressed in the pig limbus [18] and 21 times more in the human limbus (unpublished), respectively. DUSP1 is also overexpressed in these systems, but to a lesser extent. DUSP6 is overexpressed in the human system, but not in the porcine cells. Additionally, DUSP1 gene expression levels are intrinsically very high, similar to those of housekeeping enzymes such as β-actin (ACTB) or GAPDH [17]. These observations make it unlikely that the other more weakly expressed DUSPs are significant players in the regulation of MAPK dependent cell activities. In this context, the current results demonstrate that, a) DUSP5 affects the phosphorylation state of Erk1/2 in the presence of unmodified DUSP1 activity; b), sustained phosphorylation of Erk1/2 induced by inhibition of DUSP5 activity is associated with increased epithelial cell proliferation; and c) conversely, prevention of Erk1/2 phosphorylation by DUSP6, causes a major decrease in proliferation rates. In contrast to these seemingly straightforward relationships between Erk1/2 phosphorylation and proliferation rates, no measurable change in proliferation rates were seen when the Erk1/2 levels were augmented via DUSP1 shRNAmir vector, in either the sv or efHCEC systems. This latter result may be related to an intrinsic anti-proliferative feature of activated JNK1/2 in these cells, as suggested by the enhanced thymidine uptake rate observed in the JNK1 deficient sub line (Figure 4D). Thus, even though DUSP1 knockdown prolonged and enhanced Erk1/2 phosphorylation, its concomitant elevation of pJNK1/2 levels may ify the pro-proliferative effect of pErk1/2, suggesting the existence of set points for protein concentrations, affinities and activity rates intrinsically calibrated to buffer the pro-proliferative effect of Erk1/2 activation (Figure 5), as suggested by earlier studies [27]. We cannot discount involvement of p38 in addition or instead of JNK in such a putative regulatory MAPK cross stalk [28]. While we have not studied here the effect of the DUSP1 overexpression on responses observed in the ocular surface SPSCs [17,18], concurrent declines in pJNK1/2 formation may similarly provide a compensation whereby any negative effect on proliferation caused by reduced pErk1/2 levels is ified by the enhancement caused by decreased pJNK1/2 (Figure 2B). Additionally, based on the role of activated JNK1/2 on cellular responses to a variety of stress stimuli or pro-inflammatory cytokines, one would expect cells with high constitutive levels of DUSP1 gene to be better protected than untransduced cells from activation of intracellular pathways leading to apoptosis [29,30] or abnormal differentiation [31].


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)

Schematic description of proposed nuclear events associated with the transductions performed in this study. Erk1/2 phosphorylation causes nuclear translocation and activation of transcription factors that facilitate the G1 to S transition. JNK phosphorylation causes nuclear translocation and activation of antiproliferative events at some stage of the cell cycle (indicated by gray lines). The proliferation-enhancing event is blocked by pErk/2 dephosphorylation, by DUSP6 in the cytosol and DUSP5 and DUSP1 in the nuclei. The latter DUSP though, blocks JNK1/2 phosphorylation with high efficiency ifying its pro-proliferative action.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f5: Schematic description of proposed nuclear events associated with the transductions performed in this study. Erk1/2 phosphorylation causes nuclear translocation and activation of transcription factors that facilitate the G1 to S transition. JNK phosphorylation causes nuclear translocation and activation of antiproliferative events at some stage of the cell cycle (indicated by gray lines). The proliferation-enhancing event is blocked by pErk/2 dephosphorylation, by DUSP6 in the cytosol and DUSP5 and DUSP1 in the nuclei. The latter DUSP though, blocks JNK1/2 phosphorylation with high efficiency ifying its pro-proliferative action.
Mentions: Gene microarray studies of SP stem cells from pig limbus [18] and human conjunctiva [17] and limbus (unpublished) revealed several features that have the potential to underpin this slow cycling phenotype. One of them is the high expression level of several DUSPs, relative to those of their nonSP counterparts. DUSP5 is the most prominently overexpressed DUSP in the two published studies. It is 10.6 times higher in the human conjunctival SP cell [17] than in the nonSP counterparts, 2.8 times better expressed in the pig limbus [18] and 21 times more in the human limbus (unpublished), respectively. DUSP1 is also overexpressed in these systems, but to a lesser extent. DUSP6 is overexpressed in the human system, but not in the porcine cells. Additionally, DUSP1 gene expression levels are intrinsically very high, similar to those of housekeeping enzymes such as β-actin (ACTB) or GAPDH [17]. These observations make it unlikely that the other more weakly expressed DUSPs are significant players in the regulation of MAPK dependent cell activities. In this context, the current results demonstrate that, a) DUSP5 affects the phosphorylation state of Erk1/2 in the presence of unmodified DUSP1 activity; b), sustained phosphorylation of Erk1/2 induced by inhibition of DUSP5 activity is associated with increased epithelial cell proliferation; and c) conversely, prevention of Erk1/2 phosphorylation by DUSP6, causes a major decrease in proliferation rates. In contrast to these seemingly straightforward relationships between Erk1/2 phosphorylation and proliferation rates, no measurable change in proliferation rates were seen when the Erk1/2 levels were augmented via DUSP1 shRNAmir vector, in either the sv or efHCEC systems. This latter result may be related to an intrinsic anti-proliferative feature of activated JNK1/2 in these cells, as suggested by the enhanced thymidine uptake rate observed in the JNK1 deficient sub line (Figure 4D). Thus, even though DUSP1 knockdown prolonged and enhanced Erk1/2 phosphorylation, its concomitant elevation of pJNK1/2 levels may ify the pro-proliferative effect of pErk1/2, suggesting the existence of set points for protein concentrations, affinities and activity rates intrinsically calibrated to buffer the pro-proliferative effect of Erk1/2 activation (Figure 5), as suggested by earlier studies [27]. We cannot discount involvement of p38 in addition or instead of JNK in such a putative regulatory MAPK cross stalk [28]. While we have not studied here the effect of the DUSP1 overexpression on responses observed in the ocular surface SPSCs [17,18], concurrent declines in pJNK1/2 formation may similarly provide a compensation whereby any negative effect on proliferation caused by reduced pErk1/2 levels is ified by the enhancement caused by decreased pJNK1/2 (Figure 2B). Additionally, based on the role of activated JNK1/2 on cellular responses to a variety of stress stimuli or pro-inflammatory cytokines, one would expect cells with high constitutive levels of DUSP1 gene to be better protected than untransduced cells from activation of intracellular pathways leading to apoptosis [29,30] or abnormal differentiation [31].

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