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Context: In the human fetal adrenal gland (HFA), the inner fetal zone (FZ) secretes dehydroepiandrosterone sulfate. The function of the outer definitive zone (DZ) is less clear; however, the DZ phenotype is that of a reservoir of progenitor cells, many of which are mitotically active. Midkine (MK) is a heparin-binding growth factor with various bioactivities. Objective: The objective of this study was to investigate expression, proliferative effects, and ACTH regulation of MK in the HFA. Design and Setting: RNA, cryosections, and primary cell cultures from HFAs (14–24 wk) and adult adrenal RNA were used. Main Outcome Measures: The main outcome measures were MK mRNA levels (measured by quantitative real-time RT-PCR); MK localization (measured by immunostaining); MK proliferative effects and mechanism (measured by proliferation assays, flow cytometry, pharmacological interventions); and ACTH regulation (measured by quantitative real-time RT-PCR). Results: HFA MK mRNA levels were 4-fold higher than in adult adrenals (P &amp;lt; 0.05) and were comparable to levels in fetal and adult brains (positive controls). MK immunoreactivity was abundant throughout the HFA. Exogenous MK caused proliferation of isolated DZ cells but not FZ cells (72 h, P &amp;lt; 0.05). In contrast, basic fibroblast growth factor induced proliferation of cells from both zones. Pharmacological interventions indicated that MK-induced DZ cell proliferation may be mediated by phosphatidylinositol 3-kinase, MAPK kinase, and Src family kinases. ACTH (1 nm) increased MK mRNA by 3.5-fold (48 h, P &amp;lt; 0.01) in isolated FZ cells. Conclusions: MK likely plays a key role in HFA development. MK’s selective in vitro mitotic effects on DZ cells may provide insights into the mechanism underlying the distinct in vivo differences in mitotic activity between the DZ and FZ.

Midkine, a Heparin-Binding Growth Factor, Selectively Stimulates Proliferation of Definitive Zone Cells of the Human Fetal Adrenal Gland