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C/EBPalpha inactivation in FAK-overexpressed HL-60 cells impairs cell differentiation
Ken-ichiro Hashimoto1, Yoshiko Sonoda, Masakazu Yamakado
1Department of Biochemistry, Kyoritsu University of Pharmacy, Shibakoen 1-5-30, Minato-ku, Tokyo, 105-8512, Japan.
Abstract:
We previously demonstrated that focal adhesion kinase (FAK)-overexpressed (HL-60/FAK) cells have marked resistance against various apoptotic stimuli such as oxidative stress, ionizing radiation and TNF-receptor-induced ligand (TRAIL) compared with vector-transfected (HL-60/Vect) cells. Here, we show that HL-60/FAK cells are highly resistant to all-trans retinoic acid (ATRA)-induced differentiation, whereas original HL-60 or HL-60/Vect cells are sensitive. Treatment with ATRA at 1 muM for 5 days markedly inhibited the proliferation and increased the expression of differentiation markers (CD38, CD11b) in HL-60/Vect cells, but showed no such effect in HL-60/FAK cells. Electrophoretic mobility shift assay (EMSA) using an oligonucleotide for the c/EBP consensus binding sequence showed that c/EBPalpha was activated in ATRA-treated HL-60/Vect cells but not in HL-60/FAK cells, indicating that c/EBPalpha activation by ATRA was impaired in HL-60/FAK cells. In addition, the association of retinoblastoma protein (pRb) and c/EBPalpha after treatment with ATRA was seen in HL-60/Vect cells but not in HL-60/FAK cells. Further, hyperphosphorylation of pRb was observed in HL-60/FAK cells. Finally, the introduction of FAK siRNA into HL-60/FAK cells resulted in the recovery of sensitivity to ATRA-induced differentiation, confirming that the inhibition of HL-60/FAK differentiation resulted from both the induction of pRb hyperphosphorylation and the inhibition of association of pRb and c/EBPalpha.
Insights
Focal adhesion kinase (FAK) overexpression in HL-60 cells confers resistance to all-trans retinoic acid (ATRA)-induced differentiation. This resistance is linked to impaired c/EBPalpha activation and altered retinoblastoma protein (pRb) interactions.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Focal adhesion kinase (FAK) overexpression confers resistance to various apoptotic stimuli.
- HL-60/FAK cells exhibit marked resistance to apoptotic stimuli compared to HL-60/Vect cells.
Purpose of the Study:
- To investigate the role of FAK in all-trans retinoic acid (ATRA)-induced differentiation of HL-60 cells.
- To elucidate the molecular mechanisms underlying FAK-mediated resistance to ATRA-induced differentiation.
Main Methods:
- Cell proliferation assays and differentiation marker analysis (CD38, CD11b) in HL-60/FAK and HL-60/Vect cells treated with ATRA.
- Electrophoretic mobility shift assay (EMSA) to assess c/EBPalpha activation.
- Western blot analysis to examine the association of retinoblastoma protein (pRb) and c/EBPalpha, and pRb phosphorylation status.
- FAK siRNA transfection to confirm the role of FAK in ATRA resistance.
Main Results:
- HL-60/FAK cells showed high resistance to ATRA-induced differentiation, unlike HL-60/Vect cells.
- ATRA treatment failed to inhibit proliferation or induce differentiation markers in HL-60/FAK cells.
- FAK overexpression impaired ATRA-induced activation of c/EBPalpha and the association of pRb with c/EBPalpha.
- HL-60/FAK cells exhibited hyperphosphorylation of pRb, which was reversed by FAK siRNA.
- FAK siRNA restored ATRA sensitivity in HL-60/FAK cells.
Conclusions:
- FAK overexpression confers resistance to ATRA-induced differentiation in HL-60 cells.
- This resistance mechanism involves FAK-induced pRb hyperphosphorylation and impaired pRb-c/EBPalpha complex formation.
- FAK plays a critical role in regulating cellular differentiation responses to ATRA.
