T618I CSF3R mutations in chronic neutrophilic leukemia induce oncogenic signals through aberrant trafficking and constitutive phosphorylation of the O-glycosylated receptor form.

Activating mutations in the membrane-proximal region of the colony-stimulating factor 3 receptor (CSF3R) are a hallmark of chronic neutrophilic leukemia (CNL) with the T618I mutation being most common. The mechanisms underlying constitutive activation of the T618I CSF3R and its signal propagation are poorly understood. Ligand-independent activation of the T618I CSF3R has previously been attributed to loss of receptor O-glycosylation and increased receptor dimerization. Here, we show that the T618I CSF3R is indeed glycosylated but undergoes enhanced spontaneous internalization and degradation that results in a marked decrease in its surface expression. Inhibition of the proteasome dramatically increases expression of the O-glycosylated T618I CSF3R. We also demonstrate that the O-glycosylated wild-type CSF3R is tyrosine phosphorylated in response to ligand but constitutively phosphorylated in cells expressing T618I CSF3R. Constitutive tyrosine phosphorylation of the O-glycosylated T618I receptor form correlated with activation of JAK2 and both the mutant receptor and JAK2 were found to be constitutively ubiquitinated. These observations provide novel insights into the mechanisms of oncogenic signaling by T618I CSF3R mutations in CNL.

Molecular evaluation of proliferative-phase endometrium may provide insight about the underlying causes of infertility in women with endometriosis.

PURPOSE: To determine if endometrial gene expression is different in women with endometriosis-related infertility and fertile women. METHODS: Prospective study of mid-follicular phase endometrium in 47 subjects in two phases: microarray study of 10 infertile women with endometriosis and five fertile controls, and a quantitative real-time PCR (qRT-PCR) study of 27 infertile women with endometriosis and 15 fertile controls. Gene expression was determined by DNA microarray, and qRT-PCR used for 12 "promising" genes based on the microarray analysis. RESULTS: Compared to fertile controls, women with stage I-II endometriosis had 23, and women with stage III-IV had 35 genes that were significantly up- or down-regulated by microarray. However, using qRT-PCR, only chemokine ligand (CXCL) 13 was significantly down-regulated and somatostatin was significantly up-regulated with early endometriosis, and only CXCL 14 was significantly down-regulated with advanced endometriosis compared to fertile controls. CONCLUSIONS: Our findings indicate that the pattern of gene expression in proliferative-phase endometrium is different when comparing tissue from patients with endometriosis versus fertile controls. Recognition of these endometrial alterations could be helpful to diagnose and stage endometriosis, and may provide insight to explain why conception rates are low in women with endometriosis.

Circadian rhythms in acute intermittent porphyria--a pilot study.

BACKGROUND: Acute intermittent porphyria (AIP) is an inherited disorder of haem synthesis wherein a partial deficiency of porphobilinogen (PBG) deaminase (PBGD) with other factors may give rise to biochemical and clinical manifestations of disease. The biochemical hallmarks of active AIP are relative hepatic haem deficiency and uncontrolled up-regulation of hepatic 5-aminolevulinic acid (ALA) synthase-1 (ALAS1) with over-production of ALA and PBG. The treatment of choice is intravenous haem, which restores the deficient regulatory haem pool of the liver and represses ALAS1. Recently, haem has been shown to influence circadian rhythms by controlling their negative feedback loops. We evaluated whether subjects with AIP exhibited an altered circadian profile. MATERIALS AND METHODS: Over a 21-h period, we measured levels of serum cortisol, melatonin, ALA, PBG and mRNA levels (in peripheral blood mononuclear cells) of selected clock-controlled genes and genes involved in haem synthesis in 10 Caucasian (European-American) women who were either postmenopausal or had been receiving female hormone therapy, six of whom have AIP and four do not and are considered controls. RESULTS: Four AIP subjects with biochemical activity exhibited higher levels of PBG and lower levels and dampened oscillation of serum cortisol, and a trend for lower levels of serum melatonin, than controls or AIP subjects without biochemical activity. Levels of clock-controlled gene mRNAs showed significant increases over baseline in all subjects at 5 a.m. and 11 p.m., whereas mRNA levels of ALAS1, ALAS2 and PBGD were increased only at 11 p.m. in subjects with active AIP. CONCLUSIONS: This pilot study provides evidence for disturbances of circadian markers in women with active AIP that may trigger or sustain some common clinical features of AIP.

Heme status affects human hepatic messenger RNA and microRNA expression.

AIM: To assess effects of heme on messenger RNA (mRNA) and microRNA (miRNA) profiles of liver cells derived from humans. METHODS: We exposed human hepatoma cell line Huh-7 cells to excess iron protoporphyrin (heme) (10 µmol/L) or induced heme deficiency by addition of 4, 6-dioxoheptanoic acid (500 µmol/L), a potent inhibitor of aminolevulinic acid dehydratase, for 6 h or 24 h. We harvested total RNA from the cells and performed both mRNA and miRNA array analyses, with use of Affymetrix chips, reagents, and instruments (human genome U133 plus 2.0 and miRNA 2.0 arrays). We assessed changes and their significance and interrelationships with Target Scan, Pathway Studios, and Ingenuity software. RESULTS: Changes in mRNA levels were most numerous and striking at 6 h after heme treatment but were similar and still numerous at 24 h. After 6 h of heme exposure, the increase in heme oxygenase 1 gene expression was 60-fold by mRNA and 88-fold by quantitative reverse transcription-polymerase chain reaction. We found striking changes, especially up-regulation by heme of nuclear erythroid-2 related factor-mediated oxidative stress responses, protein ubiquitination, glucocorticoid signaling, P53 signaling, and changes in RNAs that regulate intermediary metabolism. Fewer mRNAs were down-regulated by heme, and the fold decreases were less exuberant than were the increases. Notable decreases after 24 h of heme exposure were patatin-like phospholipase domain-containing protein 3 (-6.5-fold), neuronal PAS domain protein 2 (-1.93-fold), and protoporphyrinogen oxidase (-1.7-fold). CONCLUSION: Heme excess exhibits several toxic effects on liver and kidney, which deserve study in humans and in animal models of the human porphyrias or other disorders.