A cAMP-specific phosphodiesterase (PDE8B) that is mutated in adrenal hyperplasia is expressed widely in human and mouse tissues: a novel PDE8B isoform in human adrenal cortex

Abstract

Bilateral adrenocortical hyperplasia (BAH) is the second most common cause of corticotropin-independent Cushing syndrome (CS). Genetic forms of BAH have been associated with complex syndromes such as Carney Complex and McCune-Albright syndrome or may present as isolated micronodular adrenocortical disease (iMAD) usually in children and young adults with CS. A genome-wide association study identified inactivating phosphodiesterase (PDE) 11A (PDE11A)-sequencing defects as low-penetrance predisposing factors for iMAD and related abnormalities; we also described a mutation (c.914A > C/H305P) in cyclic AMP (cAMP)-specific PDE8B, in a patient with iMAD. In this study we further characterize this mutation; we also found a novel PDE8B isoform that is highly expressed in the adrenal gland. This mutation is shown to significantly affect the ability of the protein to degrade cAMP in vitro. Tumor tissues from patients with iMAD and no mutations in the coding PDE8B sequence or any other related genes (PRKAR1A, PDE11A) showed downregulated PDE8B expression (compared to normal adrenal cortex). Pde8b is detectable in the adrenal gland of newborn mice and is widely expressed in other mouse tissues. We conclude that PDE8B is another PDE gene linked to iMAD; it is a candidate causative gene for other adrenocortical lesions linked to the cAMP signaling pathway and possibly for tumors in other tissues.

Figure 1

Computed tomography (CT) of the adrenal gland of our patient (CAR 559.03) and her father (CAR 559.01) who is also carrier of the H305P mutation in PDE8B.

Figure 2

A) Conservation of His at position 305 of PDE across different species; B) Gel electrophoresis on the products generated by 5′ RACE; the adrenal specific fragment that was further used to identify the 5′ end of the novel isoform is shown; C) Nucleotide and amino acid sequence of the novel exon; D) PDE8B isoforms generated by alternative splicing; the newly identified exon is shown in red; E) expression of the novel isoform across panel of 17 normal human tissues.

Figure 3

In vitro experiments with PDE8B ORF construct; asterisk indicates significance of the observed difference between the wild type and mutant constructs (P<0.05).

Figure 4

Western blot analysis of PDE8B in 3 normal (N) and 6 tumor adrenocortical (AT) tissue samples from patients with no mutations in the coding sequence of PDE8B. Lower protein levels in the tumor samples compared to the normal tissues were observd. The protein expression is controlled by beta-actin.

Figure 5

A) Brown PDE8B immunostaining (arrows) in the mouse embryo on day 15.5, seen in a whole body section (Br – brain; H – heart; Li – liver; Ad – adipose tissue, upper arrow indicates the hibernating gland); B) Brown PDE8B immunostaining (arrows) in the adipose tissue; upper arrow indicates the hibernating gland (B – bone; M – skeletal muscle; Sk – skin); C) Immunostaining in embryonic liver showing brown labeling forming the islets in between the liver cells (arrows); D) PDE8B in the heart ventricle (HV) forming small spots between muscle fibers (arrows). The atria (Au) do not show any labeling; E) Brown PDE8B immunostaining in the adipose tissue of the hibernating gland of a newborn mouse, arrow indicates the hibernating gland; F) PDE8B immunostaining (arrows) in the heart ventricle and liver of a newborn mouse; G) The wall of the heart ventricle; PDE8B labeling (arrow) forms a network around the muscle fibers; H) Liver; PDE8B labeling (arrow) seen as a heterogeneous distribution throughout the liver parenchyma; I) Kidney tubules, most likely distal tubules, but not glomeruli, showing PDE8B labeling (arrow); Gl – glomerulus; K – kidney; T – tubules; J) Intense PDE8B immunostaining (arrow) in the adipose tissue; R- rib; K) PDE8B immunostaining (arrow) in the liver perenchyma; blood vessels, including branches of the portal vein and hepatic artery, and bile duct remain unlabeled (BD – bile duct; H – hepatocyte; HA – hepatic artery; PV – portal vein branch); L) PDE8B immunostaining (arrow) in the brown adipose tissue surrounding the thymus; thymus and blood vessels remain unstained (BV – blood vessel; Th – thymus); M) Light PDE8B immunostaining (arrow) in the epithelial cells of the bronchioles (Br – bronchiole); N) PDE8B immunostaining (arrow) in the seminiferous tubules of the adult mouse testis showing spermatogonia to be slightly labeled, spermatozoa seem to be unstained (SfT – seminiferous tubule; Sg – spermatogonia; Sz – spermatozoa).