Proteasomal inhibition alters the trafficking of the neurotrophin receptor TrkA.

Neurotrophin receptors of the Trk family promote neuronal survival. The signal transduction of Trk receptors is regulated by endosomal trafficking. Monoubiquitination of receptor tyrosine kinases is an established signal for sorting of internalized receptors to late endosomes. The NGF receptor TrkA is sorted to late endosomes and undergoes ubiquitination, indicating a so far undefined regulatory role of proteasomal activity in the trafficking of TrkA. Surprisingly, we found that proteasomal inhibition alters the trafficking of TrkA from the late endosomal sorting pathway to the recycling pathway. Many neurodegenerative diseases are associated with impaired proteasomal activity. Thus, our study suggests that missorting of neurotrophic receptors might contribute to neuronal death in those neurodegenerative diseases that are known to be associated with impaired proteasomal function.

SOCS-mediated downregulation of mutant Jak2 (V617F, T875N and K539L) counteracts cytokine-independent signaling.

Recently, mutations in the gene of Janus kinase 2 (Jak2) were discovered in patients suffering from chronic myeloproliferative disorders (MPD) and leukemia. As suppressors of cytokine signaling (SOCS) proteins are potent feedback inhibitors of Jak-mediated signaling, we investigated their role in signal transduction through constitutively active Jak2 mutants. We selected two mutants, Jak2-V617F and Jak2-K539L, found in patients with MPDs and Jak2-T875N identified in acute megakaryoblastic leukemia. We found SOCS family members to be induced through Jak2-V617F in human leukemia cell lines expressing the mutant allele and in stable HEK transfectants inducibly expressing constitutively active Jak2 mutants. SOCS proteins were recruited to the membrane and bound to the constitutively active Jaks. In contrast to wild-type Jak2, the mutant proteins were constitutively ubiquitinated and degraded through the proteasome. Taken together, we show a SOCS-mediated downregulation of the constitutively active, disease-associated mutant Jak2 proteins. Furthermore, a threshold level of mutant Jak expression has to be overcome to allow full cytokine-independent constitutive activation of signaling proteins, which may explain progression to homozygocity in MPDs as well as gene amplification in severe phenotypes and leukemia.

A new C-terminal located mutation (V272ter) in the PIT-1 gene manifesting with severe congenital hypothyroidism. Possible functionality of the PIT-1 C-terminus.

OBJECTIVE: We describe a newborn with clinical signs of severe hypothyroidism and combined pituitary hormone deficiency due to a new mutation in the PIT-1 gene. PATIENT AND METHODS: Endocrine stimulation test revealed a deficiency for PRL, TSH and GH, suggesting a defect in the pituitary transcription factor PIT-1. Genetic analysis of the PIT-1 gene was performed by exon-specific PCR, followed by SSCP mutation screening and DNA sequencing of the abnormal migrating fragments. RESULTS: DNA sequencing revealed a new mutation (V272ter) in direct neighborhood to a known mutational hot spot (R271W) in the C-terminal part of the PIT-1 molecule. CONCLUSIONS: Whereas the R271W mutation has a dominant negative effect on the mutant protein, the newly described mutation is inherited in an autosomal-recessive way. The biological consequences of these two different mutations are discussed.

Idiopathic growth hormone deficiency: a vanishing diagnosis?

Some non-organic causes for growth hormone (GH) deficiency (GHD) can be attributed to genetic defects within the hypothalamo-pituitary axis. Using modern molecular biology techniques micromutations within the GH and GH-releasing hormone receptor genes have been detected as a rare cause of isolated GHD. Combined pituitary hormone deficiencies (CPHD), on the other hand, are associated with defects that manifest during the organogenesis of the anterior pituitary gland. In recent years an increasing number of patients with CPHD has been reported, showing mutations within pituitary transcription factors Pit-1, Prop-1 and HesX1. Such defects can be observed with different frequencies in patients. Some disorders, such as CPHD due to Pit-1 mutations, display a hormonal phenotype that seems more or less invariable. In most other forms of genetic CPHD both the combination and severity of anterior pituitary hormone deficiencies vary considerably. Ongoing research concentrates on factors involved in the differentiation and proliferation of cells that belong to the hypothalamo-pituitary growth axis. As not every possible candidate turns out to be a frequent cause of GHD or CPHD in humans, it will be many more years before the term 'idiopathic' becomes a vanishing attribute to the clinical diagnosis of pituitary insufficiency.

Combined pituitary hormone deficiency: role of Pit-1 and Prop-1.

During fetal development of the anterior pituitary gland, a number of sequential processes occur that affect cell differentiation and proliferation. Molecular analyses have revealed several steps that are required for pituitary cell line specification and have identified specific factors that control these steps. The gene encoding the pituitary transcription factor 1 (Pit-1) is expressed during differentiation steps that take place quite late in the development of the anterior pituitary gland. Clinically, patients with mutations of the PIT1 gene are characterized by severe deficiencies in growth hormone (GH) and prolactin (PRL), and often develop secondary hypothyroidism. A second pituitary transcription factor is known as Prophet of Pit-1 (Prop-1), and a mutation of the Prop1 gene has been detected in Ames dwarf mice. Several Prop1 mutations have been identified that structurally affect the 'paired-like' DNA-binding domain of the Prop-1 protein molecule. Patients with PROP1 mutations show combined pituitary hormone deficiency. These patients exhibit secondary hypogonadism in addition to the deficiencies of GH, PRL and thyroid-stimulating hormone (TSH) also seen in patients with PIT1 mutations. Although all are in the subnormal range, the levels of GH, PRL and TSH in patients with PROP1 mutations are, on average, slightly higher than in patients with PIT1 mutations. Some degree of hypocortisolism may necessitate cortisol substitution in patients with PROP1 mutations.

Abnormal postprandial duodenal chyme transport in patients with long standing insulin dependent diabetes mellitus.

BACKGROUND: Patients with long standing diabetes mellitus frequently have upper gut dysmotility. Gastroparesis has been well studied, whereas detailed data on duodenal motor function are limited. AIMS: To characterise postprandial duodenal chyme transport in such patients. METHODS: Intraluminal multiple impedance measurement, recently introduced as a novel technique for investigation of chyme transport, was used to study postprandial duodenal chyme flow in 10 patients with long standing insulin dependent diabetes mellitus with gastroparesis, and 10 healthy volunteers. RESULTS: Four distinct transport patterns of chyme, termed bolus transport events (BTEs), were found in both groups and could be characterised as: short distance propulsive; simple long distance propulsive; retrograde; and complex long distance propulsive. Diabetic patients had significantly lower numbers of propulsive BTEs (p < 0.01), and higher proportions of retrograde BTEs and complex long distance BTEs (p < 0.05) than control subjects, whereas the proportion of simple long distance BTEs was significantly lower (p < 0.05). The mean propagation velocities of the BTEs were similar in both groups. CONCLUSION: Abnormal postprandial duodenal chyme transport was found in patients with long standing insulin dependent diabetes mellitus. This is characterised by transport disorganisation and may result in disturbed chyme clearance.

Chyme transport patterns in human duodenum, determined by multiple intraluminal impedancometry.

To get information about the mechanisms involved in chyme transport during the fasting and postprandial states, the novel procedure of multiple intraluminal impedancometry was evaluated in 14 healthy subjects (6 during fasting, 8 after a test meal). All main features of the migrating motor complex (MMC) cycle were determined. During phase II of the MMC cycle and the postprandial period, different transport patterns of chyme, termed bolus transport events (BTEs), were determined. These were 1) simple long-distance propulsive transport (spreading distance > 16 cm), 2) short-distance propulsive transport, and 3) retrograde transport. A significantly lower number of BTEs was recorded during fasting than postprandially. Short-distance propulsive BTEs predominated during fasting (72%), and long-distance propulsive BTEs predominated after the test meal (76%). Retrograde BTEs were recorded during fasting (4%) and postprandially (8%). In the latter state, complex long-distance propulsive BTEs were also observed (5%), consisting of multiple components. A major proportion of gastric contents was found to be continuously transported to jejunum. In conclusion, impedancometry enables us to determine patterns and parameters of chyme transport during fasting and postprandial states.