THE PHOSPHATASE INHIBITOR OKADAIC ACID STIMULATES THE TSH-INDUCED G(1)-S PHASE-TRANSITION IN THYROID-CELLS

Protein phosphorylation plays an essential role in regulating many cel lular processes in eukaryotes. Signal transduction mechanisms that are reversibly controlled by protein phosphorylation require also pro tei n phosphatases (PPs). Okadaic acid (OA), which is a potent inhibitor o f protein phosphatase 2A (PP2A) and protein phosphatase 1, elicits pho sphorylation of many proteins in unstimulated cells and induces differ ent cellular responses, including transcriptional activation, shape ch anges, and pseudomitotic state. In this study, the effects of OA on ra t thyroid cells (FRTL-5 strain) were analyzed to evaluate the role of serine/threonine phosphatases in hormone-induced thyroid cell prolifer ation. OA at a concentration range between 0.1 and 1 nM stimulated thy roid cell growth. Furthermore, 0.25 nM OA increased about 3.5-fold the thyrotropin (TSH)-induced DNA synthesis in quiescent cells. OA treatm ent also stimulated cell proliferation induced by drugs that mimic TSH effect, such as 8Br-cAMP and cholera toxin, suggesting that PP2A acti vity was relevant in the cAMP pathway activated by the hormone. Flow c ytometry experiments showed that OA significantly increased the fracti on of TSH-stimulated quiescent cells entering the S phase. In order to define the mechanisms underlying the observed stimulatory effect of O A on thyroid cell growth, expression of genes relevant in the G(1)-S p hase transition was evaluated. A a-fold increase in the level of cycli n D1 mRNA expression was found by Northern blot analysis in OA-treated cells, Although cdk2 gene expression was not modulated by the same OA treatment, an increase in Cdk2 protein was revealed by immunoprecipit ation experiments. Moreover, OA modifies the phosphorylation pattern o f the tumor suppressor retinoblastoma protein, a key event in the G(1) -S phase transition. Therefore, these experiments reveal that PP2A pho sphatases play an important role in thyroid cell growth and can act at multiple sites in the TSH pathways driving cells to S phase. (C) 1997 Academic Press.