Thymosin β4, a negative regulator of hematopoietic progenitors produced by bone marrow endothelial cells

Abstract

For further study the hematopoietic inhibitors which elaborated by bone marrow endothelial cells (a cell line, published previously), the serum-free bone marrow endothelial cell conditioned medium (BMEC-cm) was collected. The components of different molecular weight (MW) > 10 KD, < 10 KD, < 0.5 KD were separated from BMEC-cm by means of serial ultrafiltration. In the component of MW > 10 KD, the solutes of MW < 10 KD were already removed by repeated filtrations. The effects of BMEC-cm as a whole and its ultrafiltration-prepared components on hematopoietic progenitors were investigated in vitro by culturing the cells in a liquid culture system contained growth factors. The results showed that BMEC-cm significantly increased the number of HPP-CFC but exerted much less influence on CFU-GM. Meanwhile the component of MW > 10 KD significantly increased the number of CFU-GM but decreased the number of HPP-CFC. Both the components of MW < 10 KD (or Tβ4 as control) and the component of MW < 0.5 KD (or AcSDKP as control) significantly decreased the number of HPP-CFC and CFU-GM. The results of HPLC analysis showed that the thymosin β4-like material and AcSDKP-like material were present in < 10 KD and < 0.5 KD component respectively. The detection of expression of mRNA demonstrated that thymosin β4 mRNA was strongly expressed in BMEC. ELISA assays of BMEC-cm showed that BMEC elaborated high levels of Tβ4 and AcSDKP. The above results suggested that Tβ4 (MW = 4982) may be one of the inhibitors existed in < 10 KD component and AcSDKP (MW = 487) which can be generated from Tβ4 by a one-step enzymatic cleavage may be one of the inhibitors existed in < 0.5 KD component. Since both Tβ4 (or < 10 KD BMEC-cm) and AcSDKP (or < 0.5 KD BMEC-cm) inhibite hematopoietic progenitors entering into cell cycle. Hence, they may be useful in protecting hematopoietic progenitors from the damaging effect of cytotoxic agents and may be useful also in enhancing the expansion of early hematopoietic progenitors meanwhile decreasing their differentiation when they were employed together with growth factors in liquid cultures.