Protein phosphatase inhibitors arrest cell cycle and reduce branching morphogenesis in fetal rat lung cultures

Protein phosphatase 2A (PPBA) is a key signal transduction intermediate in the regulation of cellular proliferation and differentiation in vitro. Howe ver the role of PP2A in the context of a developing organ is unknown. To ex plore the role of PP2A in the regulation of lung development, we studied th e effect of PP2A inhibition on new airway branching, induction of apoptosis , DNA synthesis, and expression of epithelial marker genes in whole organ e xplant cultures of embryonic (E14) rat lung. Microdissected lung primordia were cultured in medium containing one of either two PP2A inhibitors, okada ic acid (OA, 0-9 nM) or cantharidin (Can, 0-3,600 nM), or with the PP2B inh ibitor deltamethrin (Del, 0-10 mu M) as a control for a PP2A-specific effec t for 48 h. PP2A inhibition with OA and Can significantly inhibited airway branching and overall lung growth. PP2B inhibition with Del did not affect lung growth or new airway development. Histologically, both PP2A- and PP2B- inhibited explants were similar to controls. Increased apoptosis was not th e mechanism of decreased lung growth and new ah-way branching inasmuch as O A-treated explant sections subjected to the terminal deoxynucleotidyltransf erase dUTP nick end labeling reaction demonstrated a decrease in apoptosis. However, PP2A inhibition with OA increased DNA content and 5-bromo-2'-deox yuridine uptake that correlated with a G(2)/M cell cycle arrest. PP2A inhib ition also resulted in altered differentiation of the respiratory epitheliu m as evidenced by decreased mRNA levels of the early epithelial marker surf actant protein C. These findings suggest that inhibition of protein phospha tases with OA and Can halted mesenchymal cell cycle progression and reduced branching morphogenesis in fetal rat lung explant culture.