Abstract
Purified recombinant proteins for use in biomedical research are invaluable to investigate protein function. However, purity varies in protein batches made in mammalian expression systems, such as CHO-cells or HEK293-cells. This study points to caution while investigating effects of proteins related to the transforming growth factor (TGF)-β superfamily. TGF-β itself is a very potent cytokine and has effects on cells in the femtomolar range. Thus, even very small amounts of contaminating TGF-β in purified protein batches may influence the experimental results given that receptors for TGF-β are present. When we attempted to characterize possible receptors for the TGF-β superfamily ligand GDF15, striking similarities between GDF15-induced activities and known TGF-β activities were found. However, differences between batches of GDF15 were a concern and finally led us to the conclusion that the measured effects were caused by TGF-β and not by GDF15. Our results emphasize that purified recombinant proteins must be used with caution and warrant proper controls. Notably, some conclusions made about GDF15 in already published papers may not be supported by the results shown. Awareness about this issue in the scientific community may prevent spreading of false positive results.
Highlights
Growth differentiation factor (GDF)-15, known as macrophage inhibitory cytokine (MIC)-1, is a distant member of the transforming growth factor (TGF)-β superfamily
We found that recombinant human GDF15 activated SMAD2, but not SMAD1/5 in the multiple myeloma cell line IH-1 (Fig 1A)
SB431542, is an inhibitor of ALK4, ALK5 and ALK7, the TGF-β family type 1 receptors that preferentially activate SMAD2 and/or SMAD3.[20]. We have previously shown that SB431542 inhibited activin A- and TGF-β-induced SMAD2 phosphorylation in myeloma cells,[17] and here we found that GDF15-induced activation of SMAD2 in the INA-6 myeloma cell line was inhibited by SB431542 (Fig 1B)
Summary
Growth differentiation factor (GDF)-15, known as macrophage inhibitory cytokine (MIC)-1, is a distant member of the transforming growth factor (TGF)-β superfamily. Members of the TGF-β superfamily are involved in regulating diverse biological processes, including apoptosis, proliferation, organ development and bone formation. The different ligands are divided into subgroups, including TGF-βs, bone morphogenetic proteins (BMPs), growth differentiation factors (GDFs), activins and inhibins, and nodal.[1] The different ligands signal through type 1 and type 2 receptors that are conserved single transmembrane serine/threonine kinase receptors. The formation of a ligand-receptor complex enables phosphorylation of intracellular SMAD transcription factors. The type of SMAD protein that is activated is determined by the type 1 receptors that are present in the ligand-bound signaling complex.
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