CLONING OF A MEMBRANE-SPANNING PROTEIN WITH EPIDERMAL GROWTH FACTOR-LIKE REPEAT MOTIFS FROM ADRENAL GLOMERULOSA CELLS

The three zones of adrenal cortex are thought to arise from a single m ultipotential stem cell, but the mechanisms underlying the zonal diffe rentiation during embryonic development of adrenal cortex are poorly u nderstood. Employing subtraction cloning strategy, we isolated three d istinct clones that were specifically expressed in the rat glomerulosa zone. One clone, named zona glomerulosa specific clone, encoded a mem brane-spanning protein with a signal peptide at the N-terminus, six ep idermal growth factor-like repeat motifs, and a transmembrane domain n ear the C-terminus. It was identified as a rat homolog of preadipocyte factor-1 (Pref-1), a factor involved in maintaining the undifferentia ted status of preadipocyte. Immunohistochemical studies confirmed the presence of Pref-1 protein in the glomerulosa zone. Detailed examinati on revealed that the zone is divided into two layers; the first is a f ew-cells-thick layer present underneath the capsule (expressing both P ref-1 protein and aldosterone synthase cytochrome P450), and the secon d layer is beneath the first (containing Pref-1 protein but not aldost erone synthase). Moreover, another cell layer was found beneath the se cond layer and above the fasciculata zone, whose cells contained no Pr ef-1 protein, aldosterone synthase, or 11 beta-hydroxylase. These find ings suggest that a recently reported aldosterone synthase- and 11 bet a-hydroxylase-less cell layer between the two zones (see Ref. 16) is c omposed of two kinds of cell: Pref-1 protein-positive and -negative ce lls. The level of Pref-1 message in the adrenal glands of animals havi ng various pituitary-adrenal axis activities, as well as various plasm a salt concentrations, correlated with the total number of glomerulosa cells. However, the specific content of Pref-1 message in a cell was fairly constant. When the adrenal gland was surgically enucleated and the remaining capsule regenerated, the level of Pref-1 transcript was significantly suppressed at the early phase. At this phase, only a min or population of the cortical cells expressed Pref-1 protein, most of these cells already expressing a fasciculata/reticularis-specific mark er, inner zone antigen. These findings suggest that the capsular cells , mostly composed of the glomerulosa cells, may have potential for dif ferentiating into other zones' cells, and the down-regulation of Pref- 1 expression may be an important step in the adrenal zonal differentia tion.