Protein kinase A-dependent and -independent signaling pathways contribute to cyclic AMP-stimulated proliferation.

The effects of cyclic AMP (cAMP) on cell proliferation are cell type specific. Although the growth-inhibitory effects of cAMP have been well studied, much less is known regarding how cAMP stimulates proliferation. We report that cAMP stimulates proliferation through both protein kinase A (PKA)-dependent and PKA-independent signaling pathways and that phosphatidylinositol 3-kinase (PI3K) is required for cAMP-stimulated mitogenesis. In cells where cAMP is a mitogen, cAMP-elevating agents stimulate membrane ruffling, Akt phosphorylation, and p70 ribosomal S6 protein kinase (p70s6k) activity. cAMP effects on ruffle formation and Akt were PKA independent but sensitive to wortmannin. In contrast, cAMP-stimulated p70s6k activity was repressed by PKA inhibitors but not by wortmannin or microinjection of the N-terminal SH2 domain of the p85 regulatory subunit of PI3K, indicating that p70s6k and Akt can be regulated independently. Microinjection of highly specific inhibitors of PI3K or Rac1, or treatment with the p70s6k inhibitor rapamycin, impaired cAMP-stimulated DNA synthesis, demonstrating that PKA-dependent and -independent pathways contribute to cAMP-mediated mitogenesis. Direct elevation of PI3K activity through microinjection of an antibody that stimulates PI3K activity or stable expression of membrane-localized p110 was sufficient to confer hormone-independent DNA synthesis when accompanied by elevations in p70s6k activity. These findings indicate that multiple pathways contribute to cAMP-stimulated mitogenesis, only some of which are PKA dependent. Furthermore, they demonstrate that the ability of cAMP to stimulate both p70s6k- and PI3K-dependent pathways is an important facet of cAMP-regulated cell cycle progression.

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

Hormones are specialized mitogens that stimulate proliferation in their differentiated target cells. Thyrotropin (TSH), the physiologic regulator of thyroid cells, stimulates cAMP-mediated proliferation and thyroid-specific gene expression. The mitogenic effects of TSH require Ras, therefore Ras activation should be compatible with the maintenance of thyroid differentiation. However, expression of activated Ras extinguishes the differentiated phenotype of thyroid cells. One explanation for this apparent paradox is the selective utilization of Ras effector pathways. We tested the hypothesis that Ras signaling through PI3K mediates the mitogenic effects of TSH in cells which retain their differentiated character. Expression of a Ras effector mutant (RasV12S35) that signals preferentially through Raf-1, although sufficient to confer TSH-independent proliferation, abolished hormone-regulated expression of thyroglobulin and the sodium/iodide symporter. In contrast, expression of a Ras mutant (RasV12C40) that binds selectively to PI3K conferred TSH-independent proliferation without marked effects on thyroid-specific gene expression. Unlike the inhibitory effects of TSH on the proliferation of RasV12S35-expressing cells, TSH enhanced RasV12C40-stimulated proliferation by further increasing the activity of p70s6k, an important mediator of the mitogenic effects of TSH and RasV12C40. These results demonstrate that channeling Ras-dependent signals to PI3K confers TSH with the ability to stimulate proliferation in differentiated cells. Oncogene (2000) 19, 924 - 932.

Differential effects of cyclic adenosine 3',5'-monophosphate on p70 ribosomal S6 kinase.

cAMP exerts differential effects on mitogenic signaling pathways. In many cells, cAMP inhibits growth factor-stimulated MAPK activity and proliferation. In others, cAMP promotes growth. TSH stimulates proliferation through elevations in cAMP in thyroid follicular cells. This mitogenic pathway is dependent upon both protein kinase A and Ras, but not upon Raf-1, mitogen-activated protein kinase kinase, or mitogen-activated protein kinase. We report that TSH, acting through cAMP, activates pp70s6k and that this activity is required for TSH-stimulated DNA synthesis. A similar role for pp70s6k in cAMP-mediated mitogenesis was observed in secondary rat Schwann cells and in Swiss3T3 fibroblasts, two additional cell types that respond to cAMP with growth. In contrast, cAMP elevation did not activate pp70s6k in NIH3T3 or REF52 fibroblasts, cells in which cAMP fails to stimulate proliferation. Together, these results suggest that pp70s6k plays an important and general role in cAMP-mediated proliferation.

Differential effects of acute and chronic exposure to interferon-gamma on cyclic adenosine 3',5'-monophosphate response element-regulated gene expression.

TSH stimulates proliferation and maintains differentiated function in thyroid follicular cells. The mitogenic activity and the stimulatory effects of TSH on thyroid-specific gene expression are impaired by interferon-gamma (IFNgamma); however, the mechanisms for these effects have not been elucidated in detail. We examined the effects of IFNgamma on acute responses to TSH in rat thyroid cells. IFNgamma did not impair TSH-stimulated p70/p85 ribosomal protein S6 kinase (p70/p85s6k) activity or cAMP response element (CRE)-regulated gene expression, although it inhibited DNA synthesis and thyroglobulin expression, effects measured over a more prolonged time course than those on kinase activity and reporter gene expression. Unexpectedly, when cells were chronically exposed to IFNgamma, CRE-lacZ promoter activity was decreased, whereas other cAMP-mediated signals, such as p70/p85s6k activity and CRE-binding protein phosphorylation, were unaffected. Activating protein-1-regulated promoters were also impaired by IFNgamma treatment, but with kinetics that differed from those of CRE-regulated promoters. Neither acute nor chronic treatment with interleukin-1beta impaired cAMP signaling, indicating that the effects of IFNgamma are specific. These studies identify CRE- and activating protein-1-regulated promoters as targets of IFNgamma in thyroid cells and fibroblasts. IFNgamma-mediated inhibition of these promoters, in addition to those containing thyroid-specific transcription factor-1-binding sites, may contribute to the profound effects of IFNgamma on thyroid cells.

The role of nitrate and ammonium ions and light on the induction of nitrate reductase in maize leaves.

Corn seedlings (Zea mays cv W64A x W182E) were grown hydroponically, in the presence or absence of NO(3) (-), with or without light and with NH(4)Cl as the only N source. In agreement with earlier results nitrate reductase (NR) activity was found only in plants treated with both light and NO(3) (-). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by transfer of the proteins to nitrocellulose paper and reaction with antibodies prepared against a pure NR showed that crude extracts prepared from light-grown plants had a polypeptide of approximately 116 kilodaltons (the subunit size for NR) when NO(3) (-) was present in the growth medium. Crude extracts from plants grown in the dark did not have the 116 kilodalton polypeptide, although smaller polypeptides, which reacted with NR-immunoglobulin G, were sometimes found at the gel front. When seedlings were grown on Kimpack paper or well washed sand, NR activity was again found only when the seedlings were exposed to light and NO(3) (-). Under these conditions, however, a protein of about 116 kilodaltons, which reacted with the NR antibody was present in light-grown plants whether NO(3) (-) was added to the system or not. The NR antibody cross-reacting protein was also seen in hydroponically grown plants when NH(4)Cl(-) was the only added form of nitrogen. These results indicate that the induction of an inactive NR-protein precursor in corn is mediated either by extremely low levels of NO(3) (-) or by some other unidentified factor, and that higher levels of NO(3) (-) are necessary for converting the inactive NR cross-reacting protein to a form of the enzyme capable of reducing NO(3) (-) to NO(2) (-).