Abstract
Purpose: :
Membrane receptor activation induces phosphorylation of terminal kinases (tKs) of the Erk1/2, p38 and JNK cascades. The tKs transpose into the nucleus to activate their targets. Dual specificity phosphatases (DUSP), a 26 plus member family, modulate the duration and magnitude of tKs activation. This study identified the DUSPs in human corneal epithelial cells that modulate mitogenic responses to EGF through changes in ERK pathway activation.
Methods: :
DUSPs gene expression was profiled in both human corneal epithelium (HCE) and SV40-immortalized HCE cells (imHCEC) using microarrays. An expanded population of primary cells (prHCEC) was obtained by sequential passage in EpiLifetm. HCEC sublines expressing shRNAs for inhibition of DUSP1, DUSP5 and JNK1 gene expression (DUSP1i, DUSP5i and JNKi), were used pGIPz lentivectors (Open Biosystems). Transduced cells were selected based on strong puromycin resistance and GFP fluorescence conferred to the cells by the pGIPz expression. Western blots assessed the effects of shRNA expression on, phosphatase expression and target phosphorylation. Relative proliferation responses to EGF after serum starvation were determined by measuring [3H] thymidine uptake.
Results: :
DUSP gene expression levels in both HCE and imHCEC follow the order: DUSP1>>DUSP5>> DUSP4~DUSP6 >>DUSP14>>>all other DUSPs. In control, im and pr HCECs, EGF caused a rapid spike in the phosphorylation of all tKs, followed by decreases to near baseline levels, pre-EGF levels, which temporally coincided with dramatic increases in the levels of DUSP1 and 5 protein expression. In contrast, in DUSP1i, the DUSP1 increase was abolished and the tKs maintained a peak phosphorylation level for at least 2 hr; in DUSP5i the same temporal difference occurred exclusively in ERK tKs. In JNK1i, the target kinase was essentially knocked out. Proliferation measurements showed that in DUSP5i cells the EGF response was 3-fold larger than in control cells, whereas in DUSP1i it barely changed. Surprisingly, JNK1i cells displayed a dramatic larger than 5-fold increase in thymidine uptake.
Conclusions: :
DUSP5 selectively controls ERK pathway activity and proliferation. The lack of an effect of DUSP1 inhibition on proliferation can be attributed to its pan-MAPK effect. The expected pro-proliferative effect due to enhanced phosphorylation of ERK does not occur because a JNK antiproliferative effect is simultaneously unleashed.
Keywords: signal transduction: pharmacology/physiology • gene transfer/gene therapy • proliferation