Quantification of active and total transforming growth factor-β levels in serum and solid organ tissues by bioassay

Transforming growth factor-β (TGF-β) is a multi-factorial peptide growth factor that has a vital role in the regulation of cell growth, differentiation, inflammation, and tissue repair. Quantification of biologically active TGF-β levels in tissues is crucial to illustrate mechanisms involved in various physiological and pathological processes, but direct measurement of bioactive TGF-β level in the tissue has been hampered by lack of reliable methods. Here, we introduced mink lung epithelial cell bioassay to quantify both active and total TGF-β levels in serum and protein lysates from solid organs in the mouse model.

Mink lung epithelial cells were stably transfected with plasminogen activator inhibitor-1 promoter/luciferase construct, in which bioactive TGF-β level was represented by luciferase activity. Serum total TGF-β levels were comparable between the bioassay and enzyme-linked immunosorbent assay (ELISA), but active TGF-β levels measured by ELISA were significantly lower than those obtained by the bioassay. Active and total TGF-β levels in the solid organs including heart, liver, and kidney were also measured. Total TGF-β levels were relatively comparable among these organs, but active TGF-β levels were slightly higher in hearts and kidneys than in livers. Positive luciferase activities in the bioassay were almost completely inhibited by adding pan-TGF-β neutralizing antibodies, suggesting its high specificity to bioactive TGF-β. We also measured myocardial TGF-β levels after myocardial infarction and sham control by the bioassay, and compared the values with those obtained by ELISA. The bioassay demonstrated that both active and total tissue TGF-β levels were significantly higher in post-myocardial infarction than in sham myocardium. ELISA was markedly less sensitive in detecting both active and total TGF-β levels than our bioassay and failed to show any statistically significant difference in TGF-β levels between myocardial infarction and sham myocardium.

Our data suggested that the bioassay was significantly more sensitive than ELISA in detecting active TGF-β in serum and both active and total TGF-β in solid organ tissues. The bioassay will be useful in investigating TGF-β profile in various solid organs in physiological and pathological conditions.