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S. von Palffy et al.
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Figure 3. MSTNpp acts through mechanisms independent of myostatin in chronic myeloid leukemia (CML) and is produced by CML mesenchymal stromal cells (MSC). (A) Schematic illustration showing the MSTN precursor protein before and after enzymatic cleavage, generating MSTNpp and myostatin. (B) Bar graphs show- ing total cell numbers of CD34+ chronic phase CML cells after 7-day culture with 100 ng/mL of myostatin, 100 ng/mL of MSTNpp or no cytokine control. Two indi- vidual patient samples were used in triplicates. (C) Histogram showing binding of MSTNpp to the surface of KU812 cells, as compared to no cytokine control. (D) Bar graph showing relative MSTN gene expression of CD34+ cells, mononuclear cells (MNC) and cultured MSC from three chronic phase CML patients, in relation to expression in human skeletal muscle. (E) MSTNpp concentration in plasma of peripheral blood (PB) from 12 chronic phase CML patients, PB from four healthy donors and bone marrow (BM) from five healthy donors. The middle line in each bar indicates the mean plasma concentration. *P≤0.05; **P≤0.01.
cleavage of the precursor protein generates active and cir- culating MSTNpp and myostatin. Active myostatin is antagonized by MSTNpp by forming a latent complex which prevents binding of myostatin to its receptor (Figure 3A).22 To explore the mechanism by which MSTNpp exerts its growth promoting effects on CML cells, we first investigated if MSTNpp acts by blocking the activity of myostatin. CML CD34+ cells were cultured for seven days with or without myostatin and MSTNpp (Figure 3B). No difference in cell number was seen between myostatin cultured cells as compared to a no cytokine control, whereas MSTNpp, serving as a positive control, increased the total cell number. This finding indi- cates that myostatin does not exert a negative effect on CML-progenitor cell growth, suggesting that MSTNpp does not promote CML cells by inhibiting myostatin, but through another mechanism.
To investigate the possibility that MSTNpp acts direct-
ly on CML cells by binding to a receptor on the cell sur- face, KU812 cells were incubated with an anti-MSTNpp antibody together with MSTNpp, or with anti-MSTNpp antibody only, and analyzed by flow cytometry. MSTNpp was found to bind to the cell surface, as a shift in fluorescence intensity was observed when both MSTNpp and the anti-MSTNpp antibody were added to the cells (Figure 3C).
MSTNpp is produced by chronic myeloid leukemia mesenchymal stromal cells and is present in the plasma of chronic myeloid leukemia patients
It is known that MSTNpp is produced by muscle cells26 and is present in the blood of healthy individuals.27,28 To investigate whether the leukemic cells or the MSC within the marrow are another source of MSTNpp, we per- formed a MSTN RT-qPCR on CD34+ cells and MNC from primary CML patients, as well as on cultured MSC from
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haematologica | 2020; 105(8)