Abstract
cAMP-mediated cell proliferation is a complex process that involves multiple pathways. Using a cAMP-dependent cell system, FRTL-5 thyroid cells, we have previously demonstrated the existence of a precise autocrine loop in the control of cell proliferation that involves the positive effector thyrotropin (TSH) and the general inhibitor somatostatin. In search of the regulatory mechanisms responsible for the TSH and somatostatin control of cell proliferation, we analyzed the cell cycle regulatory proteins and the cellular pathways involved in the action of both signals. The results show that specific inhibition of cAMP-dependent protein kinase (PKA) and phosphatidylinositol (PI) 3-kinase blocks independently TSH-induced FRTL-5 cell proliferation and that somatostatin interferes with both signals. Each pathway activates different proteins required for G(1)/S progression. Thus, PKA is responsible for the TSH-induction of 3-hydroxy-3-methylglutaryl-CoA reductase mRNA levels, RhoA activation, and down-regulation of p27(kip1). These correlated events are necessary for FRTL-5 cell proliferation after TSH stimulation. Moreover, TSH through PKA pathway increases cyclin-dependent kinase 2 levels, whereas PI 3-kinase signaling increases cyclin E levels. Together, both pathways finally converge, increasing the formation and activation of cyclin E x cyclin-dependent kinase 2 complexes and the phosphorylation of the retinoblastoma protein, two important steps in the transition from G(1) to S phase in growth-stimulated cells. Somatostatin exerts its antiproliferative effect inhibiting more upstream the TSH stimulation of PKA and PI 3-kinase, interfering with the TSH-mediated increases of intracellular cAMP levels by inactivation of adenylyl cyclase activity. Together, these results suggest the existence of a PKA-dependent pathway and a new PKA-independent PI 3-kinase pathway in the TSH/cAMP-mediated proliferation of FRTL-5 thyroid cells.
Highlights
CAMP-mediated cell proliferation is a complex process that involves multiple pathways
protein kinase (PKA) and PI 3-kinase Are Involved in TSH-induced FRTL-5 Thyroid Cell Proliferation—We previously described the existence of an autocrine loop in the control of FRTL-5 thyroid cell proliferation that involves TSH, the main regulator of thyroid cell proliferation, and somatostatin, a cytostatic agent [8]
It is widely accepted that cAMP is the main mediator of TSH stimulation of thyroid cell growth [28, 43], increasing evidence suggests that the mechanism of TSH action involves more than the PKA classical signal transduction pathway [44]
Summary
Cyclin-dependent kinase; Rb, retinoblastoma; TSH, thyrotropin; SSTR2, specific somatostatin receptor type 2; HMG-CoA, 3-hydroxy-3-methylglutaryl-coenzyme A; PI, phosphatidylinositol; GFP, green fluorescent protein; PKA, cAMP-dependent protein kinase. TGF-1 interference with TSH action has been studied in FRTL-5 cells [13]; the mechanism of interference between somatostatin and TSH is unknown It has recently been demonstrated, in another system, that somatostatin interferes with the insulin-mediated induction of cell cycle proteins by activating p27kip1 [18]. P27kip has been implicated in G1 arrest induced by inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase [19, 20] These inhibitors interfere with cell cycle progression by suppressing the isoprenylation of proteins [21,22,23]. Somatostatin prevents TSH modulation of p27kip11⁄7Cdk association, cyclin E1⁄7Cdk kinase activity, and the phosphorylation of Rb. somatostatin blocks the TSH-mediated induction of HMG-CoA-reductase mRNA levels as well as RhoA activation, two of the decisive events in FRTL-5 cells growth, which we show are mediated by PKA but not by PI 3-kinase. The mechanism by which somatostatin interferes with TSH effects involves inhibition of the adenylyl cyclase activity and the consecutive decrease in TSHinduced intracellular cAMP levels
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