Inhibition of thyrotropin-stimulated DNA synthesis by microinjection of inhibitors of cellular Ras and cyclic AMP-dependent protein kinase.

Abstract

Microinjection of a dominant interfering mutant of Ras (N17 Ras) caused a significant reduction in thyrotropin (thyroid-stimulating hormone [TSHJ)-stimulated DNA synthesis in rat thyroid cells. A similar reduction was observed following injection of the heat-stable protein kinase inhibitor of the cyclic AMPdependent protein kinase. Coinjection of both inhibitors almost completely abolished TSH-induced DNA synthesis. In contrast to TSH, overexpression of cellular Ras protein did not stimulate the expression of a cyclic AMP response element-regulated reporter gene. Similarly, injection of N17 Ras had no effect on TSHstimulated reporter gene expression. Moreover, overexpression of cellular Ras protein stimulated similar levels of DNA synthesis in the presence or absence of the heat-stable protein kinase inhibitor. Together, these results suggest that in Wistar rat thyroid cells, a full mitogenic response to TSH requires both Ras and cyclic APK-dependent protein kinase.The ras gene family is one member of a nucleotide-binding superfamily which participates in a diverse range of cellular activities, including growth, differentiation, and protein trafficking. Experimental evidence has shown that Ras is involved in growth factor-stimulated proliferation in many cell types. Sequencing and crystallographic data revealed that Ras shares structural homology with heterotrimeric G proteins. Ras and heterotrimeric G proteins are also functionally related; both contain intrinsic GTPase activity and alternate between active and inactive states, depending on whether GTP or GDP is bound. Oncogenic forms of the Ras protein are generated by mutations which reduce the protein's normal GTPase activity or eliminate the enhancement of its GTPase activity by GTPase-activating protein (GAP) (43). One result of these mutations is a constitutively active signaling molecule. Oncogenic Ras stimulates proliferation in fibroblasts and differentiation in PC12 cells (4,14,35). Conversely, disruption of Ras function with anti-Ras antibodies or a dominant interfering mutant of Ras (N17 Ras) blocks mitogenic responses to platelet-derived growth factor, epidermal growth factor, insulin, and serum in fibroblasts (6, 7, 31) and neuronal differentiation induced by nerve growth factor in PC12 cells (39).One common feature of Ras-regulated signaling has been its role in downstream events initiated by receptor tyrosine kinases. Although many growth factors stimulate proliferation through receptors containing (or associated with) tyrosine kinase activity, there are a large number of mitogenic peptides and hormones which stimulate proliferation through G protein-coupled receptors (10,17,25,28) (30). TSH treatment activates adenylyl cyclase activity, resulting in increased levels of intracellular cyclic AMP (cAMP) and activation of the cAMP-dependent protein kinase (cAPK). This pathway is thought to be the primary signaling pathway induced by TSH in most thyroid cells. Thyroid follicular cells also respond to alterations in Ras activity. Several investigator...

Ras signaling through PI3K confers hormone-independent proliferation that is compatible with differentiation

“…Although the mitogenic activity of TSH is Ras-dependent (Kupperman et al, 1993), oncogenic Ras impairs the ability of TSH to stimulate thyroid-speci®c gene expression. De-di erentiation occurs in Ras-transformed thyroid cells (Fusco et al, 1987;Avvedimento et al, 1989) and in cells acutely injected with Ras proteins (Kupperman et al, 1996), indicating that even transient expression of activated Ras induces de-di erentiation.…”

“…TSH-stimulated proliferation is Ras- (Kupperman et al, 1993) and PI3K (Cass et al, 1999) dependent. Based on this, we examined whether Ras-mediated activation of PI3K was su cient to stimulate TSHindependent proliferation.…”

“…DNA synthesis was monitored by BrdU incorporation (LaMorte et al, 1992;Kupperman et al, 1993). Inhibitors were added 2 h prior to BrdU addition.…”

“…TSH stimulates the expression of thyroid-speci®c genes and proliferation through cAMP-mediated signaling pathways. In Wistar rat thyroid (WRT) cells, the mitogenic activity of TSH is Ras-dependent, and the requirement for Ras maps downstream from cAMP (Kupperman et al, 1993). Expression of oncogenic Hor K-Ras in thyroid cells confers TSH-independent proliferation (Fusco et al, 1987;Lemoine et al, 1990;Meinkoth et al, 1991), and mutations in all three cellular Ras genes have been identi®ed in human thyroid tumors (Fagin, 1994).…”

The p85 regulatory subunit of PI3K mediates TSH–cAMP–PKA growth and survival signals

“…Stimulation of growth by cyclic AMP-protein kinase A (cAMP-PKA) in selected cell types, as in thyroid cells, is tightly dependent on Ras and Phosphatidylinositol 3-kinase (PI3K) (Kupperman et al, 1993;Al-Alawi et al, 1995;Miller et al, 1998). Also, cAMP-PKA are powerful survival signals in all cell types tested (Affaitati et al, 2003;Wang et al, 2005).…”