Fusion partners of NTRK3 affect subcellular localization of the fusion kinase and cytomorphology of melanocytes.

A subset of Spitz tumors harbor fusions of NTRK3 with ETV6, MYO5A, and MYH9. We evaluated a series of 22 melanocytic tumors in which an NTRK3 fusion was identified as part of the diagnostic workup. Tumors in which NTRK3 was fused to ETV6 occurred in younger patients were predominantly composed of epithelioid melanocytes and were classified by their histopathologic features as Spitz tumors.

Combined activation of MAP kinase pathway and β-catenin signaling cause deep penetrating nevi.

Deep penetrating nevus (DPN) is characterized by enlarged, pigmented melanocytes that extend through the dermis. DPN can be difficult to distinguish from melanoma but rarely displays aggressive biological behavior. Here, we identify a combination of mutations of the β-catenin and mitogen-activated protein kinase pathways as characteristic of DPN.

NTRK3 kinase fusions in Spitz tumours.

Oncogenic fusions in TRK family receptor tyrosine kinases have been identified in several cancers and can serve as therapeutic targets. We identified ETV6-NTRK3, MYO5A-NTRK3 and MYH9-NTRK3 fusions in Spitz tumours, and demonstrated that NTRK3 fusions constitutively activate the mitogen-activated protein kinase, phosphoinositide 3-kinase and phospholipase Cγ1 pathways in melanocytes. This signalling was inhibited by DS-6051a, a small-molecule inhibitor of NTRK1/2/3 and ROS1.

Alternative splicing of DENND1A, a PCOS candidate gene, generates variant 2.

Polycystic ovary syndrome (PCOS) is a common endocrinopathy characterized by hyperandrogenism and metabolic disorders. The excess androgens may be of both ovarian and adrenal origin. PCOS has a strong genetic component, and genome-wide association studies have identified several candidate genes, notably DENND1A, which encodes connecdenn 1, involved in trafficking of endosomes.

Potential role of increased oxygenation in altering perinatal adrenal steroidogenesis.

Background: At birth, the large fetal adrenal involutes rapidly, and the patterns of steroidogenesis change dramatically; the event(s) triggering these changes remain largely unexplored. Fetal abdominal viscera receive hypoxic blood having a partial pressure of oxygen of only ~2 kPa (20-23 mm Hg); perinatal circulatory changes change this to adult values (~20 kPa). We hypothesized that transition from fetal hypoxia to postnatal normoxia participates in altering perinatal steroidogenesis.

Quantitation of CYP24A1 enzymatic activity with a simple two-hybrid system.

Context: Mutations of the CYP24A1 gene encoding the 24-hydroxylase (24OHase) that inactivates metabolites of vitamin D can cause hypercalcemia in infants and adults; in vitro assays of 24OHase activity have been difficult.

Objective: We sought an alternative assay to characterize a CYP24A1 mutation in a young adult with bilateral nephrolithiasis and hypercalcemia associated with ingestion of excess vitamin D supplements and robust dairy intake for 5 years.

Methods: CYP24A1 exons were sequenced from leukocyte DNA.

The post-translational regulation of 17,20 lyase activity.

A single enzyme, microsomal P450c17, catalyzes the 17α-hydroxylase activity needed to make cortisol and the subsequent 17,20 lyase activity needed to produce the 19-carbon precursors of sex steroids. The biochemical decision concerning whether P450c17 stops after 17α-hydroxylation or proceeds to 17,20 lyase activity is largely dependent on three post-translational factors. First, 17,20 lyase activity is especially sensitive to the molar abundance of the electron-transfer protein P450 oxidoreductase (POR).

Phosphorylation of human cytochrome P450c17 by p38α selectively increases 17,20 lyase activity and androgen biosynthesis.

Cytochrome P450c17, a steroidogenic enzyme encoded by the CYP17A1 gene, catalyzes the steroid 17α-hydroxylation needed for glucocorticoid synthesis, which may or may not be followed by 17,20 lyase activity needed for sex steroid synthesis. Whether or not P450c17 catalyzes 17,20 lyase activity is determined by three post-translational mechanisms influencing availability of reducing equivalents donated by P450 oxidoreductase (POR). These are increased amounts of POR, the allosteric action of cytochrome b5 to promote POR-P450c17 interaction, and Ser/Thr phosphorylation of P450c17, which also appears to promote POR-P450c17 interaction.

Varied clinical presentations of seven patients with mutations in CYP11A1 encoding the cholesterol side-chain cleavage enzyme, P450scc.

Context: The cholesterol side-chain cleavage enzyme P450scc, encoded by CYP11A1, converts cholesterol to pregnenolone to initiate steroidogenesis. P450scc deficiency can disrupt adrenal and gonadal steroidogenesis, resembling congenital lipoid adrenal hyperplasia clinically and hormonally; only 12 such patients have been reported previously.

Objective: We sought to expand clinical and genetic experience with P450scc deficiency.

Distinguishing deficiencies in the steroidogenic acute regulatory protein and the cholesterol side chain cleavage enzyme causing neonatal adrenal failure.

Objectives: To determine the genetic basis of disordered steroidogenesis in Kuwaiti siblings.

Study Design: Two siblings (46,XX and 46,XY) had normal female external genitalia and severe glucocorticoid and mineralocorticoid deficiency presenting in the first month of life. Abdominal ultrasonography showed normal size adrenal glands, suggesting cholesterol side chain cleavage enzyme (P450scc) deficiency.

Expression of P450c17 in the human fetal nervous system.

P450c17 catalyzes steroid 17α-hydroxylase and 17,20 lyase activities. P450c17 is expressed in human fetal and postnatal adrenals and gonads and in the developing mouse nervous system, but little is known about its expression in the human nervous system. We obtained portions of 9-, 10-, and 11-wk gestation human fetuses and delineated the pattern of expression of P450c17 in their peripheral nervous systems by immunocytochemistry using the P450c17 antiserum previously used to characterize P450c17 in the mouse brain.

Transcriptional regulation of the human P450 oxidoreductase gene: hormonal regulation and influence of promoter polymorphisms.

P450 oxidoreductase (POR) is the flavoprotein that acts as the obligatory electron donor to all microsomal P450 enzymes, including those involved in hepatic drug metabolism as well as three steroidogenic P450 enzymes. The untranslated first exon of human POR was located recently, permitting analysis of human POR transcription. Expression of deletional mutants containing up to 3193 bp of the human POR promoter in human adrenal NCI-H295A and liver Hep-G2 cells located the proximal promoter at -325/-1 bp from the untranslated exon.

Partial defect in the cholesterol side-chain cleavage enzyme P450scc (CYP11A1) resembling nonclassic congenital lipoid adrenal hyperplasia.

Context: The cholesterol side-chain cleavage enzyme (P450scc), encoded by the CYP11A1 gene, converts cholesterol to pregnenolone to initiate steroidogenesis. Genetic defects in P450scc cause a rare autosomal recessive disorder that is clinically indistinguishable from congenital lipoid adrenal hyperplasia (lipoid CAH). Nonclassic lipoid CAH is a recently recognized disorder caused by mutations in the steroidogenic acute regulatory protein (StAR) that retain partial function.

Molecular regulation of human placental growth factor (PlGF) gene expression in placental villi and trophoblast cells is mediated via the protein kinase a pathway.

Cyclic 3',5'-adenosine monophosphate (cAMP) is a critical second messenger for human trophoblasts and regulates the expression of numerous genes. It is known to stimulate in vitro the fusion and differentiation of BeWo choriocarcinoma cells, which acquire characteristics of syncytiotrophoblasts. A DNA microarray analysis of BeWo cells undergoing forskolin-induced syncytialization revealed that among the induced genes, placental growth factor (PlGF) was 10-fold upregulated.

Consequences of POR mutations and polymorphisms.

P450 oxidoreductase (POR) transports electrons from NADPH to all microsomal cytochrome P450 enzymes, including steroidogenic P450c17, P450c21 and P450aro. Severe POR mutations A287P (in Europeans) and R457H (in Japanese) cause the Antley-Bixler skeletal malformation syndrome (ABS) plus impaired steroidogenesis (causing genital anomalies), but the basis of ABS is unclear. We have characterized the activities of ∼40 POR variants, showing that assays based on P450c17 activities, but not cytochrome c assays, correlate with the clinical phenotype.

Functional characterization of vasopressin receptor 2 mutations causing partial and complete congenital nephrogenic diabetes insipidus in Thai families.

Background: AVPR2 mutations cause most cases of nephrogenic diabetes insipidus (NDI); 211 AVPR2 mutations have been described, but only 7 are described causing partial NDI.

Methods: Two unrelated Thai boys had polyuria and polydipsia in infancy but had normal electrolytes and serum osmolality at 2 years of age. Patient 1 could not concentrate his urine in response to water deprivation or 1-desamino-8-D-arginine vasopressin (DDAVP); patient 2 could concentrate to approximately 600 mosm/l.

Human cytochrome p450c17: single step purification and phosphorylation of serine 258 by protein kinase a.

Cytochrome P450c17 (P450c17) is the single microsomal enzyme that catalyzes steroid 17alpha-hydroxylase and 17,20 lyase activities. The ratio of lyase to hydroxylase activity of human P450c17 determines whether steroidogenesis leads to the synthesis of cortisol or sex steroids. This ratio is regulated posttranslationally by factors that influence the efficiency of electron transfer from P450 oxidoreductase to P450c17.

Clinical, genetic, and enzymatic characterization of P450 oxidoreductase deficiency in four patients.

Context: P450 oxidoreductase (POR) deficiency causes disordered steroidogenesis; severe mutations cause genital ambiguity in both sexes plus the Antley-Bixler skeletal malformation syndrome, whereas mild mutations can cause adult infertility.

Objective: We describe four patients with POR deficiency and identify and characterize the activities of their mutations. A 46,XY male with micropenis and two 46,XX female infants with genital ambiguity presented with skeletal malformations, and a 46,XX adolescent presented with primary amenorrhea, elevated 17alpha-hydroxyprogesterone, and low sex steroids.

Novel P450c17 mutation H373D causing combined 17alpha-hydroxylase/17,20-lyase deficiency.

Context: Combined 17alpha-hydroxylase/17,20-lyase deficiency is a rare autosomal recessive form of congenital adrenal hyperplasia presenting with hypertension and sexual infantilism. This disorder is caused by defects in P450c17, encoded by the CYP17A1 gene.

Objective: We describe a 14-yr-old female with clinical and hormonal features of 17alpha-hydroxylase/17,20-lyase deficiency and identify and characterize the activities of her CYP17A1 mutations.

Natural and recombinant human glycodelin activate a proapoptotic gene cascade in monocyte cells.

Glycodelin-A (GdA) is a member of the superfamily of lipocalins and the predominant glycoprotein secreted by human and primate endometrium in the secretory and early pregnancy phases. GdA can inhibit NK cell activity, T cell proliferation, and chemotaxis of monocytes. Its physiological function is thought to mediate immunotolerance at the fetomaternal interface.

Pathways leading to phosphorylation of p450c17 and to the posttranslational regulation of androgen biosynthesis.

Cytochrome P450c17 (P450c17) is the single enzyme that catalyzes steroid 17alpha-hydroxylase and 17,20 lyase activities and hence is the crucial decision-making step that determines the class of steroid made in a steroidogenic cell. Although both activities are catalyzed on a single active site, the ratio of these activities is regulated by posttranslational events. Serine phosphorylation of P450c17 increases 17,20 lyase activity by increasing the enzyme's affinity for its redox partner, P450 oxidoreductase.

Selective estrogen receptor-beta agonists repress transcription of proinflammatory genes.

In addition to their role in the development and function of the reproductive system, estrogens have significant anti-inflammatory properties. Although both estrogen receptors (ERs) can mediate anti-inflammatory actions, ERbeta is a more desirable therapeutic target because ERalpha mediates the proliferative effects of estrogens on the mammary gland and uterus. In fact, selective ERbeta agonists have beneficial effects in preclinical models involving inflammation without causing growth-promoting effects on the uterus or mammary gland.

PPAR gamma represses VEGF expression in human endometrial cells: implications for uterine angiogenesis.

Unlabelled: Endometrial vasculature supports physiological uterine growth, embryonic implantation and endometrial pathology. Vascular endothelial growth factor (VEGF) is regulated by diverse developmental and hormonal signals, including eicosanoid ligands of PPARgamma. The action of natural and synthetic PPARgamma ligands on VEGF expression in primary and transformed human endometrial cell cultures was established by quantifying endogenous gene expression and transfected VEGF gene reporters.

All-trans retinoic acid inhibits vascular endothelial growth factor expression in a cell model of neutrophil activation.

Infiltrating neutrophil granulocytes are a particularly rich source of vascular endothelial growth factor (VEGF) in the endometrium and may contribute to the angiogenesis of endometriosis lesions. The objective of this study is to evaluate the expression and regulation of VEGF in endometrial neutrophils and in a model of neutrophil differentiation relevant to endometriosis. Immunohistochemistry was performed on endometriosis patient biopsies and cultured neutrophil-like HL-60 cells were assessed.

2,3,7,8-Tetrachlorodibenzo-p-dioxin increases glycodelin gene and protein expression in human endometrium.

Context: Glycodelin (GdA) is an immunosuppressive endometrial glycoprotein critical for embryonic implantation and pregnancy establishment.

Objective: The aim of the present study was to examine the effect of dioxin [2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)] on GdA production in human endometrial cells.

Design: Controlled endometrial explant (EE) and cell cultures were used in this study.