RNA interference reveals a requirement for both p18INK4c and p27Kip1 in B lymphopoiesis

Abstract

Cyclin-dependent kinase inhibitors (CKIs) p18(INK4c) (p18) and p27(Kip1) (p27) were reported to be able to modulate self-renewal and differentiation of hematopoietic stem cells (HSCs) and hematopoietic progenitor cells, and regulate the lineage cell proliferation and maturation into the terminal elements; however, whether p18 and p27 in HSCs affect the development of lineage cells, especially B lymphocytes, in the reconstituted blood is unknown. Here we employed small-interference RNA (siRNA) technique, which provides a powerful tool for tissue-targeted knockdown of genes, to evaluate the biological functions of the p18 and p27 in the hematopoiesis process. We knocked down the expression of p18, p27 alone or both in the isolated sca-1(+) bone marrow cells by lentiviral vector-based siRNA system, and transplanted these cells into lethally irradiated C57BL/6J mice to evaluate the effect of these two genes on reconstituted lymphocyte development. The knockdown of p18 or p27 alone or both was proved to be effective as verified by western blotting. FACS analysis results showed that compared with the control group, the B lymphocytes were both significantly lower in p18, p27 alone and especially in both p18 and p27 knockdown group in reconstituted peripheral blood; and the B lymphocytes showed similar trend in bone marrow. Interestingly, the differentiation to T cells was not greatly changed, only with the dramatic decrease of the CD4/CD8 ratio. Overexpression of the antiapoptotic protein Bcl2 could not rescue the B lymphopoiesis. All these results demonstrate that p18 and p27 are collaboratively involved in B lymphopoiesis, and simultaneous knockdown of p18 and p27 probably blocks the differentiation from HSCs to B lymphocytes, but not triggers apoptosis of B cell precursors.