Tumor necrosis factor-alpha (TNF) blocks LH-stimulated
androstenedione production by immature rat theca-interstitial cells (
TIC) in vitro. The mechanism for TNF inhibition of LH-induced
androstenedione is unknown and was investigated. LH stimulation of
androstenedione synthesis in
TIC is mediated via a cAMP-dependent signaling pathway. LH-stimulated cAMP in
TIC-
conditioned medium was reduced in a biphasic manner by TNF at 1 and 48 h, but not at 4 and 24 h. To determine whether inhibition of cAMP resulted from TNF interference of LH binding,
TIC were given TNF for 24 and 48 h, and LH binding was determined. TNF inhibited LH binding at 24 and 48 h. Scatchard analysis revealed a TNF-induced decrease in
LH receptor number without altered affinity.
TIC were given TNF and cAMP analogs [N6-benzoyl-cAMP,
8-thiomethyl-cAMP, 8-(6-aminohexyl)amino-cAMP, and N6-2'-O-(Bu)2cAMP], which selectively activate
cAMP-dependent protein kinase (PKA) type I and/or PKA type II, respectively. At 48 and 96 h, TNF blocked
androstenedione production stimulated by all combinations of cAMP analogs; however,
androstenedione synthesis recovered by 48 h after removal of TNF. Peak PKA activity in
TIC was observed at 30 min in the presence of LH or cAMP analogs. LH- or cAMP analog-directed PKA activity was inhibited after concomitant exposure to TNF; however, a 24-h pretreatment with TNF did not affect cAMP analog-stimulated PKA activity. The results indicate that in the modulation of steroidogenesis, TNF acts at multiple sites in the PKA pathway. First, TNF suppresses LH-stimulated cAMP production by
TIC. Secondly, inhibition of cAMP may result from TNF attenuation of LH binding, and thirdly, TNF inhibits PKA activity of
TIC and, thus, attenuates
androstenedione production.