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Suppression of Egr-1 expression in human oral squamous carcinoma cells by okadaic acid
H Okamura1, H Morimoto, M Fujita
1Department of Histology and Oral Histology, School of Dentistry, The University of Tokushima, 3-18-15, Kuramoto, Tokushima 770-8504, Japan.
Abstract:
We examined the expression of early growth response-1 (Egr-1) gene in human oral squamous carcinoma cell lines SCCKN and SCC-25 cells and human osteoblastic cell lines Saos-2 and MG63 cells treated with okadaic acid, a potent inhibitor of protein phosphatases type 1 and type 2A. Western blot analysis revealed that Egr-1 was strongly expressed in SCCKN cells and that okadaic acid decreased the expression of Egr-1 protein in these cells. However, Egr-1 was expressed at lower levels in SCC-25, Saos-2, and MG63 cells and transiently increased with the okadaic acid treatment. Suppression of Egr-1 protein expression in okadaic acid-treated SCCKN cells stemmed from the suppression of the Egr-1 mRNA level, as determined by the RT-RCR method. Formaldehyde-fixed and alcohol-permeabilized cultured SCCKN cells were reacted with the anti-Egr-1 antibody using immunohistochemical methods. Intense fluorescence was observed in the nuclei of the control SCCKN cells interacted with anti-Egr-1 antibody. However, only a weak reaction was observed in the nuclei in SCCKN cells treated with okadaic acid. A gel mobility shift assay showed that treatment of SCCKN cells with okadaic acid suppressed Egr-1 binding to the DIG-labeled Egr-1 consensus oligonucleotide probe. The present results indicate that the alteration of phosphorylation states in SCCKN cells regulates Egr-1 binding to its consensus sequence and its expression at the transcriptional level.
Insights
Okadaic acid affects early growth response-1 (Egr-1) gene expression differently in oral cancer and osteoblast cells. In SCCKN oral cancer cells, okadaic acid suppresses Egr-1, impacting its DNA binding and transcription.
Area of Science:
- Molecular Biology
- Cancer Research
- Cell Signaling
Background:
- Early growth response-1 (Egr-1) is a transcription factor involved in cellular processes.
- Okadaic acid is a specific inhibitor of protein phosphatases type 1 and 2A.
- Differential regulation of Egr-1 may play a role in oral squamous cell carcinoma.
Purpose of the Study:
- To investigate the effect of okadaic acid on Egr-1 expression in human oral squamous carcinoma and osteoblastic cell lines.
- To elucidate the mechanisms underlying Egr-1 regulation by protein phosphatases.
Main Methods:
- Western blot analysis to assess Egr-1 protein levels.
- RT-PCR to determine Egr-1 mRNA expression.
- Immunohistochemistry to visualize Egr-1 localization.
- Gel mobility shift assay to evaluate Egr-1 DNA binding activity.
Main Results:
- Okadaic acid strongly suppressed Egr-1 expression in SCCKN cells, affecting both mRNA levels and protein expression.
- In contrast, okadaic acid transiently increased Egr-1 expression in SCC-25, Saos-2, and MG63 cells.
- Okadaic acid treatment reduced Egr-1 binding to its consensus DNA sequence in SCCKN cells.
Conclusions:
- Alterations in phosphorylation states, modulated by okadaic acid, regulate Egr-1 binding to DNA.
- These changes in phosphorylation affect Egr-1 expression at the transcriptional level in SCCKN cells.
- The findings suggest a role for protein phosphatase activity in controlling Egr-1-mediated gene expression in oral cancer cells.
