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. 2000 Feb 1;97(3):1281-6.
doi: 10.1073/pnas.97.3.1281.

Expression of reelin in adult mammalian blood, liver, pituitary pars intermedia, and adrenal chromaffin cells

N R Smalheiser  1 , E Costa, A Guidotti, F Impagnatiello, J Auta, P Lacor, V Kriho, G D Pappas
Affiliations

Expression of reelin in adult mammalian blood, liver, pituitary pars intermedia, and adrenal chromaffin cells

N R Smalheiser et al. Proc Natl Acad Sci U S A. .

Abstract

Reelin regulates telencephalic and cerebellar lamination during mammalian development and is expressed in several structures of the adult brain; however, only traces of reelin were believed to be in peripheral tissues. Because reelin structurally resembles extracellular matrix proteins, and because many of these proteins are expressed in blood, we hypothesized that reelin also might be detectable in the circulation. Reelin (420 kDa) and two reelin-like immunoreactive bands (310 and 160 kDa) are expressed in serum and platelet-poor plasma of rats, mice, and humans, but these three bands were not detectable in serum of homozygous reeler (rl/rl) mice. Reelin plasma levels in heterozygous (rl/+) mice were half of those in wild-type littermates. Western blotting and immunocytochemistry using antireelin mAbs indicated that reelin-like immunoreactivity was expressed in a subset of chromaffin cells within the rat adrenal medulla and in a subset of cells coexpressing alpha-melanocyte-stimulating hormone within the pituitary pars intermedia. However, surgical removal of adrenal or pituitary failed to decrease the amount of reelin (420-kDa band) expressed in serum. Adult liver expressed one-third of the reelin mRNA concentration expressed in adult mouse cerebral cortex. Full-length reelin protein was detectable in liver extracts in situ; acutely isolated liver cells also secreted full-length reelin in vitro. Liver appears to be a prime candidate to produce and maintain the circulating reelin pool. It now becomes relevant to ask whether circulating reelin has a physiologic role on one or more peripheral target tissues.

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Figures

Figure 1
Figure 1
Reelin-like immunoreactivity in serum, adrenal, and brain extracts of various species. (A) Serum from an adult rat (loaded at 1, 2, 3, and 4 μl per lane) was Western-blotted with G10 antibody. (B) Serum from an adult human (loaded at 1, 2, and 3 μl per lane) was blotted with antibody 142. (C–E) No reelin-like immunoreactivity is detectable in serum, adrenal glands, or brain extracts of rl/rl mice. (C) Adult mice (4–6 months old). Lanes: 1, wild-type +/+ mouse; 2, blank; 3 and 4, rl/rl mice; 5, blank; 6, wild-type mouse. (D) Three-week-old mice were sacrificed and perfused with saline, and adrenal extracts were prepared. Lanes: 1 and 2, rl/+ mice; 3, blank; 4–6, rl/rl mice; 7, blank; 8, rl/+ mouse. Protein was adjusted to ≈60 μg in lanes 1–6 and 140 μg in lane 8. (E) Brain extracts of the same mice shown in D. Protein was adjusted to ≈70 μg in lanes 1–2 and ≈140 μg in lanes 3–8. Marks indicate 420-, 310-, and 160-kDa bands.
Figure 2
Figure 2
Adult rat adrenal gland reacted with antireelin antibody G10. (A) Low-power photo. C, adrenal cortex; M, adrenal medulla. Scattered clusters of chromaffin cells are positive, whereas the cortex is negative. (Bar = 0.4 mm.) (B) Immunoelectron microscopy of chromaffin cells. Dense reelin-like immunoreactivity is observed in a subset of chromaffin granules. Arrowheads show reelin-positive granules; arrows show reelin-negative granules. Asterisks indicate reelin-like immunoreactivity within the extracellular space. (Bar = 0.5 μm.)
Figure 3
Figure 3
Reelin-like immunostaining in adult rat adrenal medulla and pituitary pars intermedia viewed by confocal microscopy. (A) Adrenal chromaffin cells, stained with antireelin antibody 142. (Bar = 20 μm.) (B) Pituitary pars intermedia, stained with antireelin antibody G10. (C) Double labeling of pituitary pars intermedia cells for reelin and α-MSH. (Left) Antibody G10. (Bar = 10 μm.) (Center) Anti-MSH. (Right) Overlay shows that a subset of the α-MSH-positive cells express reelin.
Figure 4
Figure 4
Immunoblotting of adult rat adrenal and pituitary glands. Rats were perfused extensively with saline, and then glands from several animals were pooled and extracted. An equal amount of protein (50 μg) was loaded on each lane. (A) Western blotted with antireelin antibody G10. (B) Same blot reblotted with anti-DAB1 antibody. Lanes: 1, adrenal gland; 2, intermediate-posterior pituitary dissected as a unit; 3, anterior pituitary. Marks indicate the position of molecular mass standards at 207, 121, and 81 kDa.
Figure 5
Figure 5
Low-power photo of pituitary (intermediate and posterior lobes). (A) Reacted with antireelin antibody G10. Scattered clusters of cells are observed throughout the intermediate lobe (I), whereas the posterior lobe (P) is negative. (B) Reacted with anti-DAB1 antibody. The intermediate lobe is negative, whereas strong immunoreactivity is observed throughout the posterior lobe. (Bar = 0.2 mm.)
Figure 6
Figure 6
Reelin-immunoreactive bands in liver in situ and in vitro. (A) Adult rat liver extracted and Western-blotted by using antireelin antibody G10. Lane 1: calibration standard (cerebellar granule cell-conditioned medium). Lane 2: liver extract. (B) Adult rat liver was dissociated into single cells by perfusion with collagenase, followed by sieving to remove tissue clumps. Cells were plated in collagen-coated dishes at ≈80% confluence, allowed to recover in full feeding medium containing serum for 4 hr, rinsed five times in minimal serum-free medium (Williams' E medium with BSA, glutamine, and antibiotics), and then incubated with this medium with changes immediately, after 5 hr, and overnight. Conditioned medium was harvested, spun to remove cellular debris, and assayed for reelin by Western blotting. Lanes: 1 and 2, conditioned medium collected overnight from duplicate culture wells shows that reelin was released primarily as the full-length, 420-kDa reelin gene product; 3 and 4, same as lanes 1 and 2 but incubated in the presence of dexamethasone (2 μM); 5, calibration standard (cerebellar granule cell-conditioned medium). As a control, no reelin-like immunoreactivity was observed in the conditioned medium incubated with liver cells momentarily, and more reelin was observed after overnight incubation than after 5 hr, indicating that the release of reelin was time-dependent (not shown). Extracts of the liver cells examined after overnight incubation also contained a predominant, 420-kDa reelin band that was decreased in the presence of dexamethasone (not shown).
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