Progesterone action in a murine Leydig tumor cell line (mLTC-1), possibly through a nonclassical receptor type

In a recent report we demonstrated that a high (micromolar) concentration o f progesterone (P) specifically down-regulates LH receptor (R) expression a nd function in murine Leydig tumor cells. The aim of the present study was to characterize further the putative novel R, mediating these P effects in the murine Leydig tumor cell line, mLTC-1. The binding of [H-3]P to these c ells revealed a high (K-d, similar to 9.3 nmol/liter) and a low affinity (K -d, similar to 284 nmol/liter) component, and the binding displayed with sp ecificity (P > dehydroepiandrosterone > 17-OHP). The binding was apparently different from that of the classical nuclear PR in the following ways. 1) The P/glucocorticoid antagonist RU 486 did not compete with [3H]P binding t o the mLTC-1 cells. 2) No expression of the classical PR messenger RNA was detected, despite clear P binding to these cells, by Northern hybridization or RT-PCR. 3) An antibody against the C-terminal end of the classical PR ( alpha c-262) revealed in mLTC-1 cells several molecular size protein bands between 45-57 kDa on Western hybridization, whereas these immunoreactive pr oteins were faintly recognized by another antibody (alpha-PR) directed towa rd the NH2-terminal region of the classical PR. The sizes of the immunoreac tive molecules were relatively similar to those detected using the same ant ibodies in human sperm lysates, but were at variance with the classical PR (120, 94, and 60 kDa), detected with these antibodies in human uterus. The immunoreactive proteins bound peroxidase-labeled-P, which could be displace d in the presence of a 10-fold excess of free P. 4) An immediate increase i n the intracellular free calcium level was observed after P treatment in cu ltured mLTC-1 cells, whereas it also increased the Ca-45(2+) entry within 1 5 min in these cells. 5) Increasing doses of P (0.1-10 mu mol/liter) demons trated significant inhibition of LH receptor messenger RNA levels in a dose -dependent manner in mLTC-1 cells. In conclusion, a nonclassical PR is expr essed and functional in these cells, and it is clearly distinct from the cl assical nuclear PR. It is apparent that recently reported inhibitory effect s of P on LH receptor gene expression and function are mediated through thi s novel type PR in mouse Leydig cells.