Flowchart: Preparation: Jak2
 


Text Box: ACTH/cAMP                 

       

                                            

                                      

Text Box: JAK2 Cholesterol                                                                                                        

                                                                                      

 

 


                                

Text Box: EEC                                                  

                                                               

                                                             

Text Box: Bcl-xLText Box: Stat5                               

            

 

Blood. 2006 May 9; [Epub ahead of print]

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Constitutive activation of STAT5 and Bcl-xL overexpression can induce endogenous erythroid colony formation in human primary cells.

Garcon L, Rivat C, James C, Lacout C, Camara-Clayette V, Ugo V, Lecluse Y, Bennaceur-Griscelli A, Vainchenker W.

U 790 INSERM, Institut Gustave Roussy, 39 rue Camille Desmoulins, 94805 Villejuif, France.

The biological hallmark of PV is the formation of endogenous erythroid colonies (EEC) with an erythropoietin-independent erythroid differentiation. Recently it has been shown that an activating mutation of JAK2 (V617F) was at the origin of PV. In this work we studied whether the STAT5/Bcl-xL pathway could be responsible for EEC formation. A constitutively active form of STAT5 was transduced into human erythroid progenitors and induced an erythropoietin-independent terminal differentiation and EEC formation. Furthermore, Bcl-xL overexpression in erythroid progenitors was also able to induce erythroid colonies despite the absence of erythropoietin. Conversely, siRNA mediated STAT5 and Bcl- xL knock-down in human erythroid progenitors inhibited CFU-E formation in the presence of Epo. Altogether these results demonstrate that a sustained level of the sole Bcl-xL is capable to give rise to Epo independent erythroid colony formation and suggest that in PV patients, JAK2(V617F) may induce EEC via the STAT5/Bcl-xL pathway.

PMID: 16684963 [PubMed - as supplied by publisher]

Blood. 2006 May 2; [Epub ahead of print]

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JAK2V617F expression in murine hematopoietic cells leads to MPD mimicking human PV with secondary myelofibrosis.

Lacout C, Pisani DF, Tulliez M, Moreau Gachelin F, Vainchenker W, Villeval JL.

Inserm, U790, Universite Paris XI, Institut Gustave Roussy (IGR), Villejuif, France.

A JAK2(V617F) mutation is frequently found in several BCR/ABL-negative myeloproliferative disorders. To address the contribution of this mutant to the pathogenesis of these different myeloproliferative disorders, we used an adoptive transfer of marrow cells transduced with a retrovirus expressing JAK2(V617F) in recipient irradiated mice. Hosts were analyzed during 6 months after transplantation. For a period of 3 months, mice developed polycythemia, macrocytosis and usually peripheral blood granulocytosis. Transient thrombocytosis was only observed in a low expresser group. All mice displayed trilineage hyperplasia in marrow and spleen along with an amplification of myeloid and erythroid progenitor cells and a formation of endogenous erythroid colonies. After 3-4 months, polycythemia regressed, abnormally shaped red blood cells and platelets were seen in circulation and a deposition of reticulin fibers was observed in marrow and spleen. Development of fibrosis was associated with anemia, thrombocytopenia, high neutrophilia and massive splenomegaly. These features mimic human Polycythemia Vera and its evolution towards myelofibrosis. This work demonstrates that JAK2(V617F) is sufficient for polycythemia and fibrosis development and offers an in vivo model to assess novel therapeutic approaches for JAK2(V617F)-positive pathologies. Questions remain regarding the exact contribution of JAK2(V617F) in other myeloproliferative disorders.

PMID: 16670266 [PubMed - as supplied by publisher]

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

J Clin Endocrinol Metab. 2006 May 9; [Epub ahead of print]

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Leptin Interferes with ACTH/cAMP Signaling Possibly through a Janus kinase 2-Phosphatidylinositol 3-kinase/Akt-Phosphodiesterase 3-Cyclic AMP Pathway to Down-Regulate Cholesterol Side-Chain Cleavage Cytochrome P450 Enzyme in Human Adrenocortical NCI-H295 Cell Line.

Hsu HT, Chang YC, Chiu YN, Liu CL, Chang KJ, Guo IC.

Department of Veterinary Medicine, College of Bio-Resources and Agriculture, National Taiwan University, Taipei, Taiwan; Department of Surgery, Mackay Memorial Hospital, Taipei, Taiwan; Mackay Medicine, Nursing and Management College, Taipei, Taiwan; Department of Surgery, National Taiwan University Hospital, National Taiwan University, Taipei, Taiwan.

Context: Obesity has adverse effects on adrenocortical functions. Adipocyte-derived leptin, a biomarker molecule of obesity, may directly control adrenal steroidogenesis via an unclear mechanism. Objective: We studied the mechanism underlying leptin action on adrenal steroidogenesis in human adrenocortical NCI-H295 tumor cell line. Methods: Levels of progesterone, cortisol and cAMP were determined by ELISA. Western blotting was used to detect protein amounts of P450scc, JAK2, Akt and their phosphorylated forms. The mRNA expressions of P450scc and leptin receptors were measured by RT-PCR and real-time PCR. P450scc promoter activity was analyzed with a luciferase reporter system. Results: Cholera toxin mimicked ACTH action by increasing adrenal cAMP levels and steroid secretion. Leptin did not affect basal release but significantly inhibited ACTH/cholera toxin-induced steroid secretion. The concomitant inhibitions by leptin on cholera toxin-induced protein and ACTH/cholera toxin-induced mRNA expression of P450scc were confirmed. Leptin inhibited ACTH/cholera toxin-induced CYP11A1 promoter activity via a known cAMP-responsive region located between -1.7 kb and -1.5 kb. Leptin activated phosphorylations of JAK2 and Akt. Inhibitory effects of leptin on ACTH/cholera toxin-induced cAMP levels, CYP11A1 promoter activity and steroid secretion were blunted by either inhibitor of JAK2 (AG490) or PI3K/Akt (Wortmannin), as well as inhibitors of cAMP-degrading phosphodiesterases (PDEs), including non-specific IBMX and PDE3-specific SKF94836. Leptin failed to affect the inductions of CYP11A1 promoter activity and steroid secretion by PDE-non-hydrolyzable N(6)-MB-cAMP. Conclusions: Leptin interferes with ACTH/cAMP signaling possibly through a cAMP-degrading mechanism involving activation of JAK2, PI3K and PDE3 to down-regulate P450scc expression and consequent adrenal steroidogenesis.

PMID: 16684834 [PubMed - as supplied by publisher]

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

: J Mol Neurosci. 2005;25(1):105-17.

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Dystonia-associated forms of torsinA are deficient in ATPase activity.

Konakova M, Pulst SM.

Rose Moss Laboratory for Parkinson's Disease and Neurodegenerative Disorders, Burns and Allen Research Institute, Cedars Sinai Medical Center, Los Angeles, CA 90048, USA. konakova@protomech-inc.com

Early-onset dystonia is caused by mutations in the torsinA protein, a putative member of the AAA+ class of ATPases. In this study we have evaluated the ATPase activity of bacterially expressed wild-type torsinA and its disease-associated mutant forms. Upon overexpression in Escherichia coli, recombinant torsinA proteins were accumulated as insoluble inclusion bodies and required refolding to become soluble and catalytically active. The refolded wild-type and mutant torsinA proteins were capable of hydrolyzing ATP, but their specific ATPase activities differed significantly. Deletions of the amino acid residues E302/303 and F323-Y328 resulted in a decrease of ATPase activity to approximately 35% and approximately 75% of the wild-type level, respectively. ATPase activity of wild-type and mutant torsinA proteins was influenced by factors that varied with cell stress, such as temperature, pH, and ionic strength, and was inhibited by sodium vanadate. Our results provide the first direct evidence for a role of torsinA as an active ATPase and suggest that the mutations in torsinA might affect normal functions of the protein by reducing its enzymatic activity.

PMID: 15781971 [PubMed - indexed for MEDLINE]

 

 

J Neurosci. 2005 Nov 9;25(45):10502-9.

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Silencing primary dystonia: lentiviral-mediated RNA interference therapy for DYT1 dystonia.

Gonzalez-Alegre P, Bode N, Davidson BL, Paulson HL.

Department of Neurology, Carver College of Medicine, University of Iowa, Iowa City, Iowa 52242, USA. pedro-gonzalez-alegre@uiowa.edu

DYT1 is the most common inherited dystonia. Currently, there are no preventive or curative therapies for this dominantly inherited disease. DYT1 dystonia is caused by a common three-nucleotide deletion in the TOR1A gene that eliminates a glutamic acid residue from the protein torsinA. Recent studies suggest that torsinA carrying the disease-linked mutation, torsinA(DeltaE) acts through a dominant-negative effect by recruiting wild-type torsinA [torsinA(wt)] into oligomeric structures in the nuclear envelope. Therefore, suppressing torsinA(DeltaE) expression through RNA interference (RNAi) could restore the normal function of torsinA(wt), representing a potentially effective therapy regardless of the biological role of torsinA. Here, we have generated short hairpin RNAs (shRNAs) that mediate allele-specific suppression of torsinA(DeltaE) and rescue cells from its dominant-negative effect, restoring the normal distribution of torsinA(wt). In addition, delivery of this shRNA by a recombinant feline immunodeficiency virus effectively silenced torsinA(DeltaE) in a neural model of the disease. We further establish the feasibility of this viral-mediated RNAi approach by demonstrating significant suppression of endogenous torsinA in mammalian neurons. Finally, this silencing of torsinA is achieved without triggering an interferon response. These results support the potential use of viral-mediated RNAi as a therapy for DYT1 dystonia and establish the basis for preclinical testing in animal models of the disease.

PMID: 16280588 [PubMed - indexed for MEDLINE]

 

Exp Neurol. 2005 Dec;196(2):452-63. Epub 2005 Oct 20.

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Generation and characterization of Dyt1 DeltaGAG knock-in mouse as a model for early-onset dystonia.

Dang MT, Yokoi F, McNaught KS, Jengelley TA, Jackson T, Li J, Li Y.

Department of Molecular and Integrative Physiology, NeuroTech Group, Beckman Institute for Advanced Science and Technology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.

A trinucleotide deletion of GAG in the DYT1 gene that encodes torsinA protein is implicated in the neurological movement disorder of Oppenheim's early-onset dystonia. The mutation removes a glutamic acid in the carboxy region of torsinA, a member of the Clp protease/heat shock protein family. The function of torsinA and the role of the mutation in causing dystonia are largely unknown. To gain insight into these unknowns, we made a gene-targeted mouse model of Dyt1 DeltaGAG to mimic the mutation found in DYT1 dystonic patients. The mutated heterozygous mice had deficient performance on the beam-walking test, a measure of fine motor coordination and balance. In addition, they exhibited hyperactivity in the open-field test. Mutant mice also showed a gait abnormality of increased overlap. Mice at 3 months of age did not display deficits in beam-walking and gait, while 6-month mutant mice did, indicating an age factor in phenotypic expression as well. While striatal dopamine and 4-dihydroxyphenylacetic acid (DOPAC) levels in Dyt1 DeltaGAG mice were similar to that of wild-type mice, a 27% decrease in 4-hydroxy, 3-methoxyphenacetic acid (homovanillic acid) was detected in mutant mice. Dyt1 DeltaGAG tissues also have ubiquitin- and torsinA-containing aggregates in neurons of the pontine nuclei. A sex difference was noticed in the mutant mice with female mutant mice exhibiting fewer alterations in behavioral, neurochemical, and cellular changes. Our results show that knocking in a Dyt1 DeltaGAG allele in mouse alters their motor behavior and recapitulates the production of protein aggregates that are seen in dystonic patients. Our data further support alterations in the dopaminergic system as a part of dystonia's neuropathology.

PMID: 16242683 [PubMed - indexed for MEDLINE]

 

 

 

 

 

 

 

 

 

 

 

 

 

Parkinsonism Relat Disord. 2006 Jan;12(1):15-9. Epub 2005 Sep 29.

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DYT1 mutation in a cohort of Taiwanese primary dystonias.

Lin YW, Chang HC, Chou YH, Chen RS, Hsu WC, Wu WS, Weng YH, Lu CS.

Movement Disorders Unit, Department of Neurology, Chang Gung Memorial Hospital, Kweishan, Taoyuan, Taiwan.

To investigate the DYT1 gene mutation in Chinese ethnic, we examined a series of 200 patients with primary dystonias (11 familial and 189 sporadic), 53 of their asymptomatic relatives, 97 patients with familial or early-onset parkinsonism, and 200 healthy subjects. The GAG deletion at codon 946 was only found in three sporadic dystonia patients and seven of their asymptomatic familial members. The frequency of GAG deletion was 1.5% in dystonia patients, and was 6.7% in early-onset dystonias (< or = 26 years). We conclude that DYT1 mutation is a minor cause of primary dystonias in a cohort of Taiwanese population.

PMID: 16198613 [PubMed - indexed for MEDLINE