Abstract
The early growth response gene-1 (Egr-1) is a transcription factor that plays an important role in cell growth and differentiation. It has been known that Egr-1 expression is down-regulated in many types of tumor tissues, including human fibrosarcoma HT1080 cells, and introduction of the Egr-1 gene into HT1080 cells inhibits cell growth and tumorigenic potential. Trifluoperazine (TFP), a phenothiazine class calmodulin antagonist, is known to inhibit DNA synthesis and cell proliferation and potentially important in antitumor activities. To understand the regulatory mechanism of Egr-1, we investigated the effect of TFP on expression of Egr-1 in HT1080 cells. Herein, we report that Egr-1 expression was increased by TFP in synergy with serum at the transcriptional level. Both the Ca(2+)/calmodulin-dependent protein kinase II inhibitor KN62 and the calcineurin inhibitor cyclosporin A enhanced TFP-dependent increase of Egr-1, suggesting that the Ca(2+)/calmodulindependent pathway plays a role in regulation of Egr-1 expression in HT1080 cells. The TFP-stimulated increase of the Egr-1 protein was preferentially inhibited by the MEK-specific inhibitor PD98059. In addition, activation of human Egr-1 promoter and the transcriptional activation of the ternary complex factor Elk-1 induced by TFP were inhibited both by pretreatment of PD98059 and by expression of the dominant-negative RasN17. These results indicate that the Ras/MEK/Erk/Elk-1 pathway is necessary for TFP-induced Egr-1 expression. We propose that the calmodulin antagonist TFP stimulates Egr-1 gene expression by modulating Ras/MEK/Erk and activation of the Elk-1 pathway in human fibrosarcoma HT1080 cells.
Highlights
Ca2ϩ is an important intracellular messenger in many biological processes
We investigated whether inhibition of DNA synthesis by calmodulin antagonists is related to a gain in early growth response gene-1 (Egr-1) expression
These results indicate that costimulation with serum and calmodulin antagonist has a synergistic effect on expression of Egr-1 in serum-starved HT1080 cells
Summary
CaMK, calmodulin-dependent protein kinase; TFP, trifluoperazine; TCF, ternary complex factor; DMEM, Dulbecco’s modified Eagle’s medium; MOPS, 4-morpholinepropanesulfonic acid; kb, kilobase(s); GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PCR, polymerase chain reaction; CMV, cytomegalovirus; PMA, phorbol 12-myristate 13-acetate; CPZ, chlorpromazine; SRE, serum response element; PKC, protein kinase C; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; MEK, MAPK/ERK kinase; JNK, c-Jun N-terminal kinase; KSR, kinase suppressor of Ras. Stimulation of Egr-1 Expression by TFP reduced in a number of tumor cells [21, 22], and loss of expression is closely associated with tumor formation in mammalian cells and tissues [22]. Stable expression of Egr-1 inhibited cell proliferation and soft agar growth in NIH3T3 cells transformed with v-sis, indicating that Egr-1 functions as a tumor suppressor [23]. We examined the effect of TFP on expression of the tumor suppressor Egr-1 in human fibrosarcoma HT1080 cells. Our results revealed that treatment of cells with serum and TFP increased levels of both mRNA and the protein of Egr-1. The increment was associated with the increase of Egr-1 promoter activity through activation of the ternary complex factor (TCF) Elk-1
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