Conservation and expression of a novel alternatively spliced Evi1 exon.

The Evi1 transcriptional repressor protein is expressed in a developmentally regulated manner, is essential for normal development, participates in regulating cell proliferation and differentiation of cells of haemopoietic and neuronal origin and contributes to the progression of leukaemia. In this report we describe a new murine Evi1 gene transcript (Delta105) that contains two alternatively spliced regions encoding a 9 amino acid insertion (Rp+9) within the repressor domain (Rp) and a 105 amino acid C-terminal truncation. Abundant levels of the 105 amino acid truncated protein are observed in murine leukaemia cells. The combined primary sequence alterations do not affect the DNA binding, transcriptional repressor or CtBP2 protein binding properties of Evi1 but they do reduce its transforming and cell proliferation stimulating activities. Reduced transforming activity is most likely due to the C-terminal truncation as the activity of Evi1 containing either Rp or Rp+9 is indistinguishable. Both isoforms exist in all murine tissues and cell lines examined. However, only the Rp+9 alternative splice variant is also found in humans and other vertebrates. Murine and human forms of Evi1 with Rp or Rp+9 exist. The additional 9 amino acids are encoded by a conserved 27 nucleotide exon, the overall structural organisation of the gene being preserved in the two species. The function of the Rp+9 and Delta105 splice variants is unknown although the conservation of Rp+9 throughout evolution in vertebrate species suggests it is essential to the broad spectrum of biological activities attributed to this developmentally essential protein.

The Evi1 proto-oncoprotein blocks endomitosis in megakaryocytes by inhibiting sustained cyclin-dependent kinase 2 catalytic activity.

The 3q21q26 syndrome leukaemias are characterised by dystrophic megakaryocytes, elevated platelet counts, ectopic EVI1 protein production and poor prognosis. To investigate the molecular basis of this disease, we developed a model system to examine the biological activity of EVI1 in a megakaryocyte progenitor cell line. For this purpose, Evi1 was conditionally expressed in human erythroleukaemia cells (HEL) that progress along the megakaryocyte lineage in the presence of 12-O-tetradecanoylphorbol 13-acetate (TPA). TPA-stimulated HEL cells normally undergo: (1) growth arrest; (2) altered morphology; (3) endomitosis and (4) characteristic changes in gene expression, including reduction of the erythroid-specific glycophoryn A and elevation of the specific glycoproteins GPIIIa and GPVI. Enforced Evi1 expression alone had no effect upon HEL cell proliferation or differentiation but a phenotype was manifest upon stimulation to differentiate. Evi1-expressing, TPA-treated HEL cells still showed growth arrest, had reduced and enhanced glycophoryn A and GPIIIa mRNA's, respectively, but failed to significantly elevate GPVI mRNA. This was accompanied by inhibition of endomitosis and altered cell morphology. Sustained CDK2 catalytic activity, typically associated with megakaryocyte endomitosis, was dramatically decreased in TPA-stimulated Evi1-expressing HEL cells because of significantly reduced levels of cyclin A. Therefore, enforced Evi1 expression could inhibit megakaryocyte differentiation although retention of some characteristic molecular changes, in combination with a block in endomitosis and altered morphology, suggest a defect in lineage progression. These results suggest that ectopic Evi1 expression contributes to a defective megakaryocyte differentiation programme and is likely to contribute to the phenotype observed in 3q21q26 syndrome leukaemias.

Calcineurin activates NF-kappaB in skeletal muscle C2C12 cells.

Calcineurin (CnA) is an important signalling molecule in skeletal muscle, in the promotion of differentiation, slow-fibre phenotype and possibly fibre hypertrophy. We found that stable expression of constitutively active CnA in muscle C2C12 cells strongly activated NF-kappaB, a key mediator of muscle wasting. NF-kappaB activation by CnA was associated with elevated phospho-IkappaBalpha, and could be repressed by specific genetic (porZAKI-4 and porDSCR1) and chemical (cyclosporin A) inhibitors of CnA, but tumour necrosis factor-alpha (TNF-alpha) appeared not to be a key component in the cross-talk. Functionally, CnA-induced NF-kappaB activation seemed to interfere with terminal muscle differentiation. We therefore showed a functional interaction between the CnA and NF-kappaB pathways in skeletal muscle cells, which involved opposing phenotypic effects of CnA.

Quantifying the temporospatial expression of postnatal porcine skeletal myosin heavy chain genes.

Postnatal skeletal muscle fiber type is commonly defined by one of four major myosin heavy chain (MyHC) gene isoforms (slow/I, 2a, 2x, and 2b) that are expressed. We report on the novel use of combined TaqMan quantitative real-time RT-PCR and image analysis of serial porcine muscle sections, subjected to in situ hybridization (ISH) and immunocytochemistry (IHC), to quantify the mRNA expression of each MyHC isoform within its corresponding fiber type, termed relative fiber type-restricted expression. This versatile approach will allow quantitative temporospatial comparisons of each MyHC isoform among muscles from the same or different individuals. Using this approach on porcine skeletal muscles, we found that the relative fiber type-restricted expression of each postnatal MyHC gene showed wide spatial and temporal variation within a given muscle and between muscles. Marked differences were also observed among pig breeds. Notably, of the four postnatal MyHC isoforms, the 2a MyHC gene showed the highest relative fiber type-restricted expression in each muscle examined, regardless of age, breed, or muscle type. This suggests that although 2a fibers are a minor fiber type, they may be disproportionately more important as a determinant of overall muscle function than was previously believed.