Syk Signaling Pathway studies indicated that PTTG facilitates G1/S transition

single ventricular chamber. Four of Syk Signaling Pathway 18 Tg:Pomc Pttg transgenic fish also exhibited coexisting overt pericardial effusion. Taken together, corticotroph targeted PTTG overexpression in Tg:pomc pttg zebrafish results in ACTH dependent hypercortisolism and metabolic disruptions mimicking features of mammalian Cushing disease. Corticotroph PTTG Overexpression Induces Cyclin E. Our previous studies indicated that PTTG facilitates G1/S transition by acting coordinately with Sp1 to up regulate cyclin D expression in human choriocarcinoma cells. To understand the mechanism for zebrafish corticotroph PTTG overexpression inducing altered G1/S transition, we analyzed expression of key G1/S cell cycle regulators by real time PCR in adult Tg:Pomc Pttg and WT pituitary glands.
Whereas expression of pituitary cyclin D, p21, and p27 were not different between WT and Tg:Pomc Pttg, cyclin E mRNA levels were more than doubled in the Tg:Pomc Pttg pituitary. Cyclin E up regulation has been associated with poor clinical outcomes inhuman malignancies. In the adult pituitary, cyclinE is undetectable in normal cells but preferentially up regulated in tumors of corticotroph, but not other, lineage. In murine pituitary POMC cells, cyclin E overexpression collaborates with p27kip1 null mutation to increase cell proliferation, centrosome instability, and tumor formation. Up regulated cyclin E is also associated with loss of Brg1 observed in approximately one third of human corticotroph adenomas.
Enhanced pituitary cyclin E mRNA levels observed in Tg:Pomc Pttg fish may not represent protein expression, but the minute adult zebrafish pituitary size technically hampered analysisof protein expressionbyWestern blot. We therefore determined whether PTTG regulates cyclin E expression in mammalian AtT20 corticotroph tumor cells that express abundant endogenous PTTG and cyclin E proteins. Suppression of endogenous PTTG expression with a PTTG specific siRNA resulted in decreased cyclin E expression and enhanced p27kip1 levels, whereas p21 expression was not changed. These observations suggest that PTTG up regulation ofcyclin E and down regulation of p27kip1 in pituitary corticotroph tumor cells occurs independently of p21. In Vivo Testing of CDK/Cyclin Inhibitors in Tg:Pomc Pttg Zebrafish. Zebrafish pituitary POMC cell differentiation starts at the anterior neural ridge by 20 hpf, and is completed within the mature pituitary by 48 hpf.
Within the first few days of embryonic development, our transgenic fish shown here recapitulate hallmark features of Cushing disease, i.e, lineage specific corticotroph expansion with partial Gc resistance. The observed G1/S alteration, cyclin E up regulation, and neoplastic corticotroph changes led us to screen small molecule CDK inhibitors with different spectra of inhibitory selectivity, including flavopiridol, R roscovitine, olomoucine, PD 0332991, and CAY10572. Tg:Pomc Pttg, POMC eGFP double transgenic embryos were exposed to each compound added to the embryo culture medium. Although flavopiridol retarded early embryonic development before corticotroph ontogeny occurred, in vivo treatment of zebrafish embryos with R roscovitine, olomoucine, PD 0332991, and CAY10572 starting at 18 hpf caused no apparent growth defect by 40 hpf. Strikingly, R ro