Steroidogenic pathway

Androgen-sensitive (LNCaP) and -independent PCA cells (C4-2B and 22RV1) express both mRNA and protein for LH and LH receptor (LHR). Exposure of these cells to LH for 4 hr increased the expression of several steroidogenic genes. Exposure for 10 days resulted in the increase of additional genes. At both time points, the upregulation of these genes was dose-dependent. This was mirrored by an increase in the expression of several key steroidogenic enzymes, including StAR, CYB5B, CYP11A, and 3βHSD. LH stimulated the production of progesterone and testosterone in LNCaP cells as measured by RIA. We have also demonstrated that treatment of LNCaP cells with LH enhanced their viability.

Granulosa cells were suspended in  mM Tris-HCl, pH , 6 M urea, 10% (v/v) glycerol, 2% (w/v) SDS, % (w/v) bromophenol blue, and freshly added 5% (v/v) β -mercaptoethanol and subjected to sonication on ice. Total protein content was quantified using Bradford assay (Bio-Rad Bradford Solution, USA). 20  μ g protein was loaded on 10% SDS-polyacrylamide gel electrophoresis under reducing conditions, along with prestained molecular weight markers. The separated proteins were electrophoretically transferred onto a nitrocellulose membrane (GE Healthcare) by a wet method (Bio-Rad, USA). The transfer was performed at a constant voltage (100 V) for 90 min in a buffer consisting of 25 mM Tris, 192 mM glycine, and 20% methanol. The membrane was then incubated for 1 h at room temperature in blocking buffer (TBS-containing 5% skimmed milk and % Tween-20). Then, the membranes were incubated overnight at 4°C with appropriate antibody at a dilution of 1 : 1000 in TBS-containing 5% skimmed milk and % Tween-20. They were washed in PBS-% Tween-20 and incubated for 1 h at room temperature with a horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG (final dilution 1 : 5000; Bangalore Genei) in TBS-containing 5% skimmed milk and % Tween-20. The signal was detected by ECL (enhanced chemiluminescence, Millipore Inc., USA).

The StAR protein was first identified, characterized and named by Dr. Douglas Stocco at Texas Tech University Health Sciences Center in 1994. [18] The role of this protein in lipoid CAH was confirmed the following year in collaboration with Dr. Walter Miller at the University of California, San Francisco . [19] All of this work follows the initial observations of the appearance of this protein and its phosphorylated form coincident with factors that caused steroid production by Dr. Nanette Orme-Johnson while at Tufts University . [20]

Steroidogenic pathway

steroidogenic pathway

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