P27 Deficiency Results in Enhancing Hematopoiesis By Increasing IL22 Production from Bone Marrow Mesenchymal Stem Cells

Hematopoietic microenvironment plays an important role in hematopoiesis, in which the bone marrow mesenchymal stem cells (BM-MSCs) can directly or indirectly affect the hematopoietic cells and regulate hematopoietic function. It has been reported that bone marrow hematopoietic progenitor cells were increased in p27 deficient mice, however, it is unclear whether BM-MSCs are mediated enhancing hematopoiesis resulting from p27 deficiency.

To answer this question, the conditioned medium (CM) were harvested from BM-MSC cultures derived from WT and p27 knockout (KO) mice, respectively and added to the cultures of bone marrow cells derived from WT mice. After 4 days, the sca-1⁺ckit⁺Lin^- cells (hematopoietic stem cells, HSC) and sca-1⁺ckit⁺Lin⁺ cells (hematopoietic progenitor cells, HPC) in the resulting cells were analyzed by flow cytometry, and the assay for colony forming cells (CFCs) was performed and the colony numbers of the CFC were counted. Results showed that the HPC fraction and the number of CFCs were increased significantly, but the HSC fraction was not increased in the bone marrow cell cultures with the CM from p27 KO mice compared with those with the CM from WT mice.

To identify potential factors from the CM which enhance hematopoiesis caused by p27 deficiency, differences of protein expression profiles in the CM from the WT and p27 KO mice were examined by protein chip analysis. Five proteins were identified which were significantly increased in the CM from p27 KO mice, including interleukin-22 (IL-22), type I receptor of transforming growth factor-β, tumor necrosis factor- related apoptosis-inducing ligand, VE-cadherin and vascular endothelial growth factor B.

To determine whether IL22, as a paracrine factor, plays a role in stimulating hematopoiesis by activating JAK-STAT signal pathway mediated by IL22 receptor A1 (IL22RA1), we examined the effect of IL22 on HPCs and the expression of signal molecules of JAK-STAT pathway. Results showed that the expression levels of IL22 at both mRNA and protein levels were up-regulated significantly in BM-MSCs derived from p27 deficient mice compared with those from WT mice. The HPC fraction and the number of CFCs were increased significantly in bone marrow cultures in the present of IL22, and were decreased significantly in the cultures with the CM from p27 knockout mice containing IL22 antibody. The percentages of IL22RA1, STAT3 and p-STAT3-S727 positive HPCs were increased significantly in the bone marrow from p27 KO mice compared with that from WT mice.

Results in this study indicate that IL22, as a paracrine factor, might play an important role in stimulating hematopoiesis by activating JAK-STAT signal pathway mediated by IL22RA1.