Summary Lineage conversion of differentiated cells in response to hormonal feedback has yet to be described. To investigate this we studied the adrenal cortex, which is composed of functionally distinct concentric layers that develop postnatally, the outer zona Glomerulosa (zG) and the inner zona Fasiculata (zF). These layers have separate functions, are continuously renewed in response to physiological demands and are regulated by discrete hormonal feedback loops. Their cellular origin, lineage relationship and renewal mechanism, however, remain poorly understood. Cell fate mapping and gene deletion studies using zG-specific Cre expression demonstrate that differentiated zG cells undergo lineage conversion into zF cells. In addition, zG maintenance is dependent on the master transcriptional regulator Steroidogenic Factor 1 (SF-1) and zG-specific Sf-1 deletion prevents lineage conversion. These findings demonstrate that adrenocortical zonation and regeneration result from lineage conversion and may provide a paradigm for homeostatic cellular renewal in other tissues.

alpha-Tocopherol modulates two major signal transduction pathways centered on protein kinase C and phosphatidylinositol 3-kinase. Changes in the activity of these key kinases are associated with changes in cell proliferation, platelet aggregation, and NADPH-oxidase activation. Several genes are also regulated by tocopherols partly because of the effects of tocopherol on these two kinases, but also independently of them. These genes can be divided in five groups: Group 1. Genes that are involved in the uptake and degradation of tocopherols: alpha-tocopherol transfer protein, cytochrome P450 (CYP3A), gamma-glutamyl-cysteine synthetase heavy subunit, and glutathione-S-transferase. Group 2. Genes that are implicated with lipid uptake and atherosclerosis: CD36, SR-BI, and SR-AI/II. Group 3. Genes that are involved in the modulation of extracellular proteins: tropomyosin, collagen-alpha-1, MMP-1, MMP-19, and connective tissue growth factor. Group 4. Genes that are connected to adhesion and inflammation: E-selectin, ICAM-1 integrins, glycoprotein IIb, IL-2, IL-4, IL-1b, and transforming growth factor-beta (TGF-beta). Group 5. Genes implicated in cell signaling and cell cycle regulation: PPAR-gamma, cyclin D1, cyclin E, Bcl2-L1, p27, CD95 (APO-1/Fas ligand), and 5a-steroid reductase type 1. The transcription of p27, Bcl2, alpha-tocopherol transfer protein, cytochrome P450 (CYP3A), gamma-glutamyl-cysteine sythetase heavy subunit, tropomyosin, IL-2, and CTGF appears to be upregulated by one or more tocopherols. All the other listed genes are downregulated. Gene regulation by tocopherols has been associated with protein kinase C because of its deactivation by alpha-tocopherol and its contribution in the regulation of a number of transcription factors (NF-kappaB, AP1). A direct participation of the pregnane X receptor (PXR) / retinoid X receptor (RXR) has been also shown. The antioxidant-responsive element (ARE) and the TGF-beta-responsive element (TGF-beta-RE) appear in some cases to be implicated as well.

Mutations in NADPH P450 oxidoreductase (POR) cause a broad spectrum of human disease with abnormalities in steroidogenesis. We have studied the impact of P450 reductase mutations on the activity of CYP19A1. POR supported CYP19A1 activity with a calculated Km of 126 nm for androstenedione and a Vmax of 1.7 pmol/min. Mutations R457H and V492E located in the FAD domain of POR that disrupt electron transfer caused a complete loss of CYP19A1 activity. The A287P mutation of POR decreased the activities of CYP17A1 by 60-80% but had normal CYP19A1 activity. Molecular modeling and protein docking studies suggested that A287P is involved in the interaction of POR:CYP17A1 but not in the POR:CYP19A1 interaction. Mutations C569Y and V608F in the NADPH binding domain of POR had 49 and 28% of activity of CYP19A1 compared with normal reductase and were more sensitive to the amount of NADPH available for supporting CYP19A1 activity. Substitution of NADH for NADPH had a higher impact on C569Y and V608F mutants of POR. Similar effects were obtained at low/high (5.5/8.5) pH, but using octanol to limit the flux of electrons from POR to CYP19A1 inhibited activity supported by all variants. High molar ratios of KCl also reduced the CYP19A1 supporting activities of C569Y and V608F mutants of POR to a greater extent compared to normal POR and A287P mutant. Because POR supports many P450s involved in steroidogenesis, bone formation, and drug metabolism, variations in the effects of POR mutations on specific enzyme activities may explain the broad clinical spectrum of POR deficiency.

Human sexual determination is initiated by a cascade of genes that lead to the development of the fetal gonad. Whereas development of the female external genitalia does not require fetal ovarian hormones, male genital development requires the action of testicular testosterone and its more potent derivative dihydrotestosterone (DHT). The "classic" biosynthetic pathway from cholesterol to testosterone in the testis and the subsequent conversion of testosterone to DHT in genital skin is well established. Recently, an alternative pathway leading to DHT has been described in marsupials, but its potential importance to human development is unclear. AKR1C2 is an enzyme that participates in the alternative but not the classic pathway. Using a candidate gene approach, we identified AKR1C2 mutations with sex-limited recessive inheritance in four 46,XY individuals with disordered sexual development (DSD). Analysis of the inheritance of microsatellite markers excluded other candidate loci. Affected individuals had moderate to severe undervirilization at birth; when recreated by site-directed mutagenesis and expressed in bacteria, the mutant AKR1C2 had diminished but not absent catalytic activities. The 46,XY DSD individuals also carry a mutation causing aberrant splicing in AKR1C4, which encodes an enzyme with similar activity. This suggests a mode of inheritance where the severity of the developmental defect depends on the number of mutations in the two genes. An unrelated 46,XY DSD patient carried AKR1C2 mutations on both alleles, confirming the essential role of AKR1C2 and corroborating the hypothesis that both the classic and alternative pathways of testicular androgen biosynthesis are needed for normal human male sexual differentiation.