PAX-FKHR function as pangenes by simultaneously inducing and inhibiting myogenesis.

Alveolar rhabdomyosarcomas (ARMS) escape terminal differentiation despite exhibiting a skeletal muscle phenotype. To understand the role of the ARMS-specific PAX-FKHR proteins in myogenesis, we characterized their regulation of MyoD expression and function. Reporter assays show that PAX-FKHR transactivate MyoD expression through its 258 bp core enhancer. Gel-shift assays confirm that PAX-FKHR bind to core enhancer sequences showing similarity to consensus PAX3/PAX-FKHR-binding sites. We show that while PAX3-FKHR activates the expression of endogenous MyoD and myogenin proteins in transduced NIH3T3 fibroblasts, it inhibits them from terminally differentiating as shown by low myogenin and myosin heavy chain expression, and lack of myotube formation. Attenuation of MyoD transcriptional activity via phosphorylation coupled to the lack of cell cycle arrest is the underlying mechanism for the differentiation block. Lastly, we show that fibroblast growth factor receptor signaling likely mediates the inhibition of differentiation by PAX3-FKHR. In a single experimental system we demonstrate that PAX3-FKHR can simultaneously induce myogenesis while preventing its completion. We propose a model whereby PAX-FKHR commit a yet undefined precursor cell to the myogenic lineage while at the same time inhibit terminal differentiation, thereby contributing to ARMS formation.

Biological characterization of uncleavable plasma membrane-anchored human macrophage colony-stimulating factor.

The cell-surface form of human macrophage colony-stimulating factor (CSF-1(256), M-CSFalpha) is a plasma membrane-anchored transmembrane protein from which the soluble CSF-1 is released by ectodomain proteolytic cleavage. We have previously generated two forms of cell surface CSF-1 which failed to undergo the cleavage by deleting residues 161-165 or residues 159-165 in the extracellular juxtamembrane region (1). To determine the biologic significance of the ectodomain cleavage, we compared the biosynthesis and biologic activities of uncleavable mutant CSF-1 forms with those of the cleavable wild-type (WT) CSF-1. We found that the uncleavable CSF-1 forms were able to accumulate on cell surface at about threefold higher level than the cleavable WT CSF-1 did. We further demonstrated that the uncleavable plasma membrane-anchored forms of CSF-1 were biologically active in mediating the proliferation of CSF-1-dependent cells as well as the intercellular adhesion between CSF-1 receptor-bearing cells and CSF-1 expressing cells. Furthermore, the adhesive activity of uncleavable CSF-1 forms was about twofold stronger than that of WT CSF-1, which indicated that the ectodomain cleavage system plays an important role in regulating the biologic activities of membrane-anchored CSF-1.

Decreased in vitro production of 6-keto-prostaglandin F1 alpha by uterine arteries from postmenopausal women.

Cessation of ovarian function is associated with a marked increased in morbidity and mortality secondary to ischemic heart disease. Estrogen replacement has been shown to impart protection against ischemic heart disease. We hypothesized that estrogen may influence vascular production of vasodilators such as prostacyclin. To investigate this relationship we have measured the production of 6-keto-prostaglandin F1 alpha, and thromboxane B2 by superfused uterine arteries from pre- and postmenopausal women. Arterial specimens from healthy normotensive premenopausal (n = 5) and postmenopausal women (n = 5) were superfused for 5 hours. Production of 6-keto-prostaglandin F1 alpha reached steady state levels by 120 minutes and remained linear for the length of the experiment. Indomethacin (4 x 10(-5) mol/L) added at 120 minutes significantly decreased prostanoid production. In subsequent experiments, 17 beta-estradiol in concentrations of 10, 100, 1000 ng/ml was added to the superfusion media at 120 minutes. Total production of 6-keto-prostaglandin F1 alpha by premenopausal arteries superfused with neat media during the steady state interval (3 hours) was significantly greater than that of postmenopausal specimens (1.25 versus 0.27 ng/mg dry tissue, p less than 0.05). Thromboxane B2 levels were undetectable in spent media. However, the addition of 17 beta-estradiol did not alter production of 6-keto-prostaglandin F1 alpha. These data suggest that arterial production of prostacyclin is significantly decreased in uterine arteries from postmenopausal women, but in this in vitro model system estrogens did not affect vascular prostanoid production.