Krüppel-like factor 5 promotes mitosis by activating the cyclin B1/Cdc2 complex during oncogenic Ras-mediated transformation

Abstract

We previously showed that the zinc finger-containing transcription factor Krüppel-like factor 5 (KLF5) is important in mediating transformation by oncogenic H-Ras through induction of cyclin D1 expression and acceleration of the G1/S transition of the cell cycle. Here we present evidence of a role for KLF5 in accelerating mitotic entry in H-Ras-transformed NIH3T3 fibroblasts. When compared with non-transformed parental NIH3T3 cells, H-Ras-transformed fibroblasts exhibit an increase in mitotic index, levels of cyclin B1 and Cdc2, and cyclin B1/Cdc2 kinase activity. Inhibition of KLF5 expression in H-Ras-transformed cells with KLF5-specific small interfering RNA (siRNA) results in a decrease in each of the aforementioned parameters, with a concomitant reduction in the transforming potential of the cells. Conversely, over-expression of KLF5 in NIH3T3 cells leads to an increase in the promoter activity of the genes encoding cyclin B1 and Cdc2. These results indicate that KLF5 accelerates mitotic entry in H-Ras-transformed cells by transcriptionally activating cyclin B1 and Cdc2, which leads to an increase in cyclin B1/Cdc2 kinase activity. Extending our previous observation that KLF5 activates cyclin D1 transcription to promote G1/S transition, our current results further support a crucial function for KLF5 in mediating cellular transformation caused by oncogenic H-Ras.

Fig. 1

KLF5 increases mitotic activity in oncogenic H-Ras-transformed NIH3T3 cells. (A) NIH3T3 cells, untransfected Ras7 cells, and Ras 7 cells transfected with non-specific (NS) control siRNA or KLF5-specific siRNA from day 1 to day 3 were stained with the Hoechst 33258 dye to label those cells in mitosis. The mitotic index is the percent of cells in mitosis among a total of 500 cells counted. N = 4; **P < 0.01 by two-tailed Student's t test. (B) Cells were immuno-stained with a phospho-histone H3 antibody to label mitotic cells. Hoechst dye was used to stain nuclei. Mitotic index is represented as percent of phospho-histone H3-positive cells among a total of 1000 cells counted. N = 3; *P < 0.05; **P < 0.01 by two-tailed Student's t test.

Fig. 2

KLF5 accelerates G₂/M transition of the cell cycle in H-Ras-transformed NIH3T3 cells. (A) Immuno-staining was performed with a phospho-Ser/Thr-Pro MPM2 antibody to identify cells in the M phase of cell cycle. Cells were then stained with PI to measure the DNA content and subjected to FACS analysis. The percentages of MPM2-positive cells among all cells analyzed were plotted at different time points. N = 3; *P < 0.05; **P < 0.01 by two-tailed Student's t test. (B) The ratio of M-phase population to G₂/M-phase population was calculated by dividing the number of MPM2-positive cells by those in the G₂/M phase (i.e., cells with a DNA content of 4N). N = 3; *P < 0.05; **P < 0.01 by two-tailed Student's t test.

Fig. 3

Effect of KLF5 inhibition by siRNA on cyclin B1, Cdc2, and cyclin B1/Cdc2 kinase activity in H-Ras transformed cells. (A) The levels of KLF5, cyclin B1, and Cdc2, in NIH3T3 cells, untransfected Ras7 cells, and Ras7 cells transfected with non-specific (NS) control siRNA or KLF5-specific siRNA, were determined by Western blot analysis. Actin serves as a loading control. (B) The cyclin B1/Cdc2 kinase activity was measured by immunoprecipitation with a Cdc2 antibody followed by in vitro kinase reaction using histone H1 as a substrate. P-histone H1 is the phosphorylated product.

Fig. 4

KLF5 increases cyclin B1 and Cdc2 promoter activity. NIH3T3 cells were co-transfected with a luciferase plasmid containing cyclin B1 promoter (cyclin B1 promoter-luc) [30] or Cdc2 promoter (Cdc2 promoter-luc) [32] and either a control vector (pMT3 vector) or with a KLF5-expression vector (pMT3-KLF5). Luciferase activity was determined 2 days following transfection and normalized to the internal control, Renilla luciferase. N = 4; **P < 0.001 when compared to PMT3 vector-transfected cells using two-tailed Student's t test.

Fig. 5

A model for the role of KLF5 in mediating H-Ras-induced transformation. The induction of KLF5 by H-Ras is mediated by MAPK and Egr-1 as previously demonstrated [13]. KLF5 then activates the transcription of the genes encoding cyclin D1 [13] and cyclin B1/Cdc2 (this study). The combined effect is an acceleration of the cell cycle, which eventually results in cellular transformation.