calcimycin and Testicular-Neoplasms

The steroidogenic acute regulatory (StAR) protein, a 30-kDa mitochondrial factor, is a key regulator of steroid hormone biosynthesis, facilitating the transfer of cholesterol from the outer to the inner mitochondrial membrane. StAR protein expression is restricted to steroidogenic tissues, and it responds to hormonal stimulation through different second messenger pathways. The present study was designed to explore the mechanisms of extracellular calcium (Ca2+) involved in the hCG-stimulated expression of StAR protein and steroidogenesis in a mouse Leydig tumor cell line (mLTC-1). Extracellular Ca2+ (1.5 mmol/liter) enhanced the hCG (50 microg/liter)-induced increases in StAR messenger RNA (mRNA) and protein levels (1.7 +/- 0.3-fold; 4 h), as monitored by quantitative RT-PCR and immunoblotting. The potentiating effect of Ca2+ on the hCG-stimulated StAR response correlated with the acute progesterone (P) response. In accordance, omission of Ca2+ from the extracellular medium by specific Ca2+ chelators, EDTA or EGTA (4 mmol/liter each), markedly diminished the hCG-stimulated P production. The Ca2+ effect on hCG-induced StAR mRNA expression was dramatically suppressed by 10 micromol/liter verapamil, a Ca2+ channel blocker. The Ca2+-mobilizing agonist, potassium (K+; 4 mmol/liter), greatly increased the hCG responses of StAR expression and P production, which conversely were attenuated by Ca2+ antagonists, further supporting the involvement of intracellular free Ca2+ ([Ca2+]i) in these responses. The interaction of Ca2+ or K+ with hCG accounted for a clear increase in the StAR protein level (1.4-1.8-fold; 4 h) compared with that after hCG stimulation. Inhibition of protein synthesis by cycloheximide (CHX) drastically diminished the hCG-induced StAR protein content, indicating the requirement for on-going protein synthesis for hCG action. The transmembrane uptake of 45Ca2+ was increased by 26% with hCG and was strongly inhibited by verapamil. [Ca2+]i moderately augmented the response to hCG in fura-2/AM-loaded mLTC-1 cells within 30-40 sec, reaching a plateau within 1-3 min. Interestingly, the calcium ionophore (A23187) clearly increased (P < 0.01) StAR mRNA expression, in additive fashion with hCG. Northern hybridization analysis revealed four StAR transcripts at 3.4, 2.7, 1.6, and 1.4 kb, with the 1.6-kb band corresponding to the functional StAR protein; all of them were up-regulated 3- to 5-fold upon hCG stimulation, with a further increase in the presence o

Topics: Animals; Calcimycin; Calcium; Calcium Channel Blockers; Chorionic Gonadotropin; DAX-1 Orphan Nuclear Receptor; DNA-Binding Proteins; Fushi Tarazu Transcription Factors; Gene Expression; Homeodomain Proteins; Ionophores; Leydig Cell Tumor; Male; Mice; Phosphoproteins; Potassium; Progesterone; Receptors, Cytoplasmic and Nuclear; Receptors, Retinoic Acid; Repressor Proteins; RNA, Messenger; Second Messenger Systems; Steroidogenic Factor 1; Testicular Neoplasms; Transcription Factors; Transfection; Tumor Cells, Cultured; Verapamil

As part of an investigation into the role of leukotrienes in steroidogenesis, the formation of leukotriene B4 was investigated in purified Leydig cells from rat testes and from a tumour by using a sensitive radioimmunoassay. Detectable levels were found in both Leydig cell types (70 pg/10(6) cells) and these remain unchanged during incubation for 60 min at 32 degrees C. Addition of the Ca2+ ionophore A23187 increased LTB4 production more than 6-fold within 10 min whereas steroidogenesis was not increased until after 20 min. In the presence of luteinizing hormone or luteinizing hormone releasing hormone agonist no increase in LTB4 was detected in the testis Leydig cells whereas luteinizing hormone stimulated testosterone production from 3.2 +/- 0.1 to 148.9 +/- 7.5 ng/10(6) cells during the same time period. Similar results were obtained with the tumour Leydig cells. The LTB4 was found to be rapidly secreted by the cells in all experiments. The basal and A23187-stimulated levels were inhibited by nordihydroguaiaretic acid, a lipoxygenase inhibitor. It is concluded that LTB4 is produced in Leydig cells and can be stimulated by high calcium levels, but that it is probably not required for the control of steroidogenesis.

Topics: Animals; Calcimycin; Catechols; Leukotriene B4; Leydig Cell Tumor; Leydig Cells; Lipoxygenase Inhibitors; Luteinizing Hormone; Male; Masoprocol; Rats; Testicular Neoplasms; Testosterone