Investigation of KLF5 function in normal haemopoiesis.

Abstract

Krüppel-like factor 5 (KLF5) is a zinc-finger transcription factor known to have regulatory roles in the growth and differentiation of many adult tissues. In humans, KLF5 is located at 13q21-22, which is frequently lost in multiple tumour types, including tumours of the breast, endometrium, ovary and prostate where it is associated with loss of KLF5 expression. Little is known about the potential role of KLF5 in the haemopoietic system. Previous work by us and others has shown that KLF5 has a functional role in induction of differentiation of the myeloid compartment. In acute myeloid leukaemia (AML), our group has previously show that KLF5 expression is reduced relatively to normal CD34⁺ cells and that reduction of expression is associated with hypermethylation in intron 1. We also found that hypermethylation of KLF5 was associated with poor outcome, identifying KLF5 as an important target for further investigation in haemopoiesis and particularly the myeloid compartment. To extend the functional analysis of Klf5, an in vivo gene-ablation model was generated. As nonconditional Klf5 knockout mice (KO) die at embryonic day 8.5, pan-haemopoietic Klf5 conditional gene KO mice were generated by crossing Klf5fl/fl [fl/fl superscript] mice with Vav-cre transgenic mice. The Klf5fl/flVav-cre⁺ʹ⁻ [fl/fl superscript] and Klf5fl/fl [fl/fl superscript] mice were analysed at 3, 9 and 12 month of age for defects in steady state haemopoiesis. Peripheral blood (PB) analysis of 9 and 12 month old mice revealed that Klf5fl/flVav-cre⁺ʹ⁻ [fl/fl superscript] animals displayed significantly higher values for total white blood cell (WBC) count. To further characterise the changes in blood cell populations, flow cytometry was used with a range of different lineage antibody markers. The peripheral blood data indicated a decrease in the granulocytes of Klf5fl/flVav-cre⁺ʹ⁻ [fl/fl superscript] mice as well as an increase in the T-cell compartment. Interestingly, we also showed that the Klf5fl/flVav-cre⁺ʹ⁻ [fl/fl superscript] mice have increased numbers of blood and bone marrow eosinophils compared to Klf5fl/f [fl/fl superscript] mice. Consistently, we found significantly increased numbers of eosinophils in the lungs of Klf5fl/flVav-cre⁺ʹ⁻ [fl/fl superscript] mice compared to Klf5fl/fl [fl/fl superscript] mice. The spleen weight of Klf5fl/flVav-cre⁺ʹ⁻ [fl/fl superscript] mice was significantly higher compared to Klf5fl/fl [fl/fl superscript] mice. In addition, clonal assays conducted from the spleen of 9 and 12 month old mice showed a significant increase in colony number in the Klf5fl/flVav-cre⁺ʹ⁻ [fl/fl superscript]mice compared to Klf5fl/fl [fl/fl superscript] mice. This correlated with the flow cytometry data which showed a significant increase in haemopoietic stem cell (HSC) populations; short-term HSC (ST-HSC) and multipotent progenitor (MPP) in the spleen of Klf5fl/flVav-cre⁺ʹ⁻ [fl/fl superscript] mice. In summary, this study revealed multiple functional roles for Klf5 in haemopoiesis. Firstly, these studies demonstrated that as predicted losing Klf5 leads to alterations in the development of the myeloid compartment. Secondly, we showed that the stem cell compartments (ST-HSC and MPP) were significantly increased in the spleen of Klf5fl/flVav-cre⁺ʹ⁻ [fl/fl superscript] mice compared to Klf5fl/fl [fl/fl superscript] mice, which also correlated with extra-medullary splenic haemopoiesis and increased spleen size. Finally, the increase in T-cells for the Klf5fl/flVav-cre⁺ʹ⁻ [fl/fl superscript] mice suggests a possible previously unidentified functional role for Klf5 outside the myeloid compartment.