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  • Original Paper
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Krüppel-like factor 5 mediates the transforming activity of oncogenic H-Ras

Abstract

Previous studies indicate that Krüppel-like factor 5 (KLF5), also known as intestinal-enriched Krüppel-like factor (IKLF), is a positive regulator of cell proliferation and gives rise to a transformed phenotype when overexpressed. Here we demonstrate that levels of KLF5 transcript and protein are significantly elevated in oncogenic H-Ras-transformed NIH3T3 cells. These cells display an accelerated rate of proliferation in both serum-containing and serum-deprived media and form anchorage-independent colonies in soft agar assays. H-Ras-transformed cells also contain elevated mitogen-activated protein kinase (MAPK) activity. When treated with inhibitors of MEK (MAPK kinase), H-Ras-transformed cells lose their growth advantage and no longer form colonies. Significantly, levels of KLF5 transcript and protein are substantially reduced in H-Ras-transformed cells treated with MEK inhibitors. Moreover, inhibition of KLF5 expression in H-Ras-transformed cells with KLF5-specific small interfering RNA (siRNA) leads to a decreased rate of proliferation and a significant reduction in colony formation. H-Ras-transformed cells also contain elevated levels of Egr1 that are diminished by MEK inhibitors. Inhibition of Egr1 by siRNA results in a reduced level of KLF5, indicating that Egr1 mediates the inductive action of MAPK on KLF5. Lastly, KLF5 activates expression of cyclin D1. These findings indicate that the increased expression of KLF5 in H-Ras-transformed cells is secondary to increased MAPK activity from H-Ras overexpression and that the elevated level of KLF5 is in part responsible for the proproliferative and transforming activities of oncogenic H-Ras.

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Accession codes

Accessions

GenBank/EMBL/DDBJ

Abbreviations

DMEM:

Dulbecco's modified Eagle's medium

ERK:

extracellular signal-regulated kinase

FBS:

fetal bovine serum

GAPDH:

glyceraldehydes-3-phosphate dehydrogenase

GKLF:

gut-enriched Krüppel-like factor

IKLF:

intestinal Krüppel-like factor

KLF:

Krüppel-like factor

MAPK:

mitogen-activated protein kinase

MEK:

MAPK/ERK kinase

PBS:

phosphate-buffered saline

PI:

propidium iodide

PMA:

phorbol-12-myristate-13-acetate

RT–PCR:

reverse transcription–polymerase chain reaction

siRNA:

small interfering RNA

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Acknowledgements

We thank Drs Raul Urrutia, Jerry Lingrel and Richard Pestell for kindly providing the various plasmid constructs in the study. This work was in part supported by grants from the National Institutes of Health (DK52230, DK64399 and CA84197). VWY is a recipient of a Georgia Cancer Coalition Distinguished Cancer Clinician Scientist Award.

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Correspondence to Vincent W Yang.

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Nandan, M., Yoon, H., Zhao, W. et al. Krüppel-like factor 5 mediates the transforming activity of oncogenic H-Ras. Oncogene 23, 3404–3413 (2004). https://doi.org/10.1038/sj.onc.1207397

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