IMMUNOLOCALIZATION OF TRANSFORMING GROWTH FACTOR-BETA-1 IN THE BOVINE ADRENAL-CORTEX USING ANTIPEPTIDE ANTIBODIES

Eight- to 12-amino acid long peptides, representing fragments of transforming growth factor-beta-1 (TGF-beta-1) and TGF-beta-2, were selected on the basis of their potential immunogenicity and were used to generate polyclonal antibodies. Anti-TGF-beta-1-(91-103) antibodies recognized specifically TGF-beta-1, prevented TGF-beta-1 binding to NRK-49F cells, and neutralized the biological activity of TGF-beta-1 in adrenocortical cells (consisting in the inhibition of angiotensin-II-induced cortisol production). Antibodies raised against TGF-beta-2-(65-73) appeared to recognize TGF-beta-2 with a better affinity than TGF-beta-1, but were unable to block the binding of either TGF-beta-1 or TGF-beta-2 to their receptors or to inhibit their biological activity. These observations are in line with a prominant role of the C-terminal domain of TGF-beta-1 in its interaction with its receptor(s) and, hence, in its biological activity. Using anti-TGF-beta-1-(91-103) antibodies, we could localize immunoreactive TGF-beta-1-like material in the cortex of adult bovine adrenal glands. No reactivity was detected in the capsule or adrenal medulla. The reactivity was maximal in the zona fasciculata/reticularis and weaker in the zona glomerulosa. TGF-beta-like material was present in a latent form in the conditioned medium from primary cultures of bovine adrenocortical cells. These cells secreted about 5 ng heat-activatable TGF-beta-like material/24 h of culture.10(6) cells. Taken together with our previous reports that bovine adrenocortical cells possess high affinity TGF-beta-1 receptors and secrete a TGF-beta-1-like molecule under a latent form, the present observations further support the hypothesis that TGF-beta-1 or a closely immunologically related protein acts as an autocrine regulator of adrenocortical steroidogenic functions.