RECEPTOR-BINDING OF TRANSFORMING GROWTH-FACTOR-BETA BY HUMAN FETAL ADRENAL-CELLS

We have shown previously that transforming growth factor beta 1 (TGF-b eta 1) is antimitotic for human fetal adrenal (HFA) cells in vitro and that this effect can be partially blocked by adrenocorticotropic horm one (ACTH). In the present study, we sought to determine whether ACTH might interfere with TGF-beta 1 action by means of reducing TGF-beta 1 binding to adrenal cells. We incubated adrenal cells with 50 pM I-125 -labeled TGF-beta 1 for 15 min to 3 h at 4 degrees C and found that th e binding of I-125-labeled TGF-beta 1 increased with time and could be inhibited in a dose-dependent manner by non-labeled TGF-beta 1 (0.05- 10 nM), but not with other relevant cytokines: IL6, TNF alpha, IGF-I, IGF-II, TGF-alpha, and EGF. Pretreatment of HFA cells with ACTH (0.009 -900 nM) for 4-24 h significantly increased specific I-125-labeled TGF -beta 1 binding compared to that in untreated cells; maximal increases in binding were achieved with 0.9 nM ACTH. This effect of ACTH could be mimicked by treatment of adrenal cells with dibutyryl cAMP (1 mM) o r forskolin (10 mu M). Scatchard analysis of data from ACTH-treated ce lls suggest the presence of two populations of TGF-beta 1 binding site s with different affinity and capacity of binding for the ligand. The high affinity sites had a K-d value Of 9.5 x 10(-10) M and a capacity of 5.4 x 10(4) sites/cell, while the low affinity binding structures h ad a K-d value of 4.7 x 10(-8) M and were present in a concentration o f 7.6 x 10(5) sites/cell. Up-regulation of TGF-beta 1 binding by ACTH or its agonists was not observed with cultured human fetal liver or ki dney cells. Incubation of HFA cells with cytochalasin D, which causes such cells to round as also occurs with ACTH, had no effect on binding of TGF-beta 1. Upon cross-linking of the I-125-labeled TGF-beta 1 bou nd to HFA cells by sulfosuccinimidyl suberate followed by solubilizati on and SDS-PAGE electrophoresis, we found TGF-beta 1 to be associated with three binding sites; the cross-linked complexes had apparent mole cular weights of >205, 65 and 45 kDa. Pre-incubation of HFA cells with ACTH increased the binding of labeled TGF-beta 1 to all three molecul ar weight substances. In control and ACTH-treated HFA cells (fetal zon e and neocortex) the predominant binder was the high molecular weight form. In contrast, the predominant form of TGF-beta 1 binding in kidne y and liver cells had a cross-linked weight of approximately 66 kDa; t here was little binding of TGF-beta 1 to the >205 kDa form. These resu lts indicate that HFA cells have specific binding sites for TGF-beta 1 that are heterogenous in size. Since ACTH and other agonists of prote in kinase A increased adrenal binding of TGF-beta 1, we suggest that A CTH reversal of TGF-beta 1 induced inhibition of adrenal growth is app arently not due to decreased binding of TGF-beta 1.