Overexpression of EGR-1 modulates the activity of NF-κB and AP-1 in prostate carcinoma PC-3 and LNCaP cell lines

Abstract

Abstract. To address elements that might uniquely characterize EGR-1 mediated signaling, the expression of two transcription factors, namely, nuclear factor kappa B (NF-κB) and activator protein-1 (AP-1) were studied. PC-3 and LNCaP prostate carcinoma cell lines were transiently transfected with wildtype Egr-1 expression plasmid (pCMV-Egr-1) and treated with cisplatin and TPA. Overexpression of EGR-1 was found to induce nuclear expression of both, NF-κB and AP1. However, the intensity of the induced AP-1 and NF-κB was diminished after cisplatin treatment, but not after TPA. Our findings confirm that the overexpression of wild-type Egr-1 caused a marked increase in cell proliferation in PC-3 and LNCaP proliferation in a 14-day soft agar colony forming assay. In addition, luciferase reporter gene assay showed that the transcriptional activity of AP-1 and NF-κB in PC-3 and LNCaP prostate carcinoma cell lines was also modulated by the overexpression of EGR-1 in these cells using tandem repeated Luc-AP-1 and Luc-NF-κB. The activation of both NF-κB and AP-1 are key steps in the cascade of events following the activation of the EGR-1 gene. It was revealed that overexpression of EGR-1 selectively increased AP-1 and NF-κB activation, and that the activation of these nuclear factors appears to be essential for the induction of proliferation and anchorage independence in activated PC-3 and LNCaP cells. However, the mechanism underlying the modulation of AP-1 and NF-κB by the overexpression of EGR-1 is still unknown.

Inhibition of Egr-1 by siRNA in prostate carcinoma cell lines is associated with decreased expression of AP-1 and NF-κB

“…Our results show that indeed, without an external source of Egr-1, constitutive activation of NF-κB and AP-1 is associated with the expression of Egr-1, as knockdown of Egr-1 expression caused a significant inhibition of NF-κB and AP-1 activity and reduction in p21 and cIAP2 mRNA expression. This corroborates our previous studies that have suggested that forced expression of Egr-1 in PC-3 and LNCaP cells causes constitutive activation of NF-κB and AP-1 (38). The external stimulation of Egr-1 may be advantageous to proliferation, survival, motility and invasion, and resistance to cytotoxic drugs, when surviving in an ectopic environment, such as during seeding and growth in distant organs, such as the bone and lungs.…”

“…The phenotype of this inhibition is seen as the cell cycle arrest at the G1 to S-phase transition. This study complements and extends our previous study (38) in which a 5-10-fold increase in Egr-1 mRNA, NF-κB and AP-1 and protein levels in cell culture studies were observed. This compares to our finding that siRNA mediated silencing resulted in >98% reduction in Egr-1 mRNA and 91% reduction in Egr-1 protein in vitro, which led to dramatic changes in the cellular phenotype.…”

“…Our previous study demonstrated overexpression of Egr-1 in PC-3 and LNCaP prostate carcinoma cell lines caused pronounced increase in protein levels of NF-κB and AP-1 (38). In this study, we observed that depletion of the endogenous expression of Egr-1 (over 90%) by siRNA decreased PC-3 and LNCaP cell proliferation, cell growth and induced transcriptional arrest of the members of the related NF-κB and AP-1 nuclear factors.…”

“…Whether constitutive activation of NF-κB and AP-1 is a cause of Egr-1 production or whether constitutive production of Egr-1 elevates NF-κB and AP-1 activity is not clear. however, at least in PC-3 and LNCaP cells, it was recently elucidated that overexpression of Egr-1 results in increased NF-κB and AP-1 activity during normal and stressed conditions (38). Our results show that indeed, without an external source of Egr-1, constitutive activation of NF-κB and AP-1 is associated with the expression of Egr-1, as knockdown of Egr-1 expression caused a significant inhibition of NF-κB and AP-1 activity and reduction in p21 and cIAP2 mRNA expression.…”

Activation of MAP kinase family members triggered by TPA or ionomycin occurs via the protein phosphatase�4 pathway in Jurkat leukemia T cells

“…Membranes were incubated with SAP/JNK, p38 or ERK-1/2 antibody (dilution 1:1,000) and PP4 antibody (dilution 1:1,000) (Santa Cruz Biotechnology). Primary incubation was followed by incubations with HRP-conjugated secondary antibody (1:2,000; NEB), and detection was made with super signal chemiluminescent substrate (31)(32)(33)(34).…”