JNKs function as CDK4-activating kinases by phosphorylating CDK4 and p21.

Cyclin D-CDK4/6 are the first cyclin-dependent kinase (CDK) complexes to be activated by mitogenic/oncogenic pathways. They have a central role in the cell multiplication decision and in its deregulation in cancer cells. We identified T172 phosphorylation of CDK4 rather than cyclin D accumulation as the distinctly regulated step determining CDK4 activation. This finding challenges the view that the only identified metazoan CDK-activating kinase, cyclin H-CDK7-Mat1 (CAK), which is constitutively active, is responsible for the activating phosphorylation of all cell cycle CDKs. We previously showed that T172 phosphorylation of CDK4 is conditioned by an adjacent proline (P173), which is not present in CDK6 and CDK1/2. Although CDK7 activity was recently shown to be required for CDK4 activation, we proposed that proline-directed kinases might specifically initiate the activation of CDK4. Here, we report that JNKs, but not ERK1/2 or CAK, can be direct CDK4-activating kinases for cyclin D-CDK4 complexes that are inactivated by p21-mediated stabilization. JNKs and ERK1/2 also phosphorylated p21 at S130 and T57, which might facilitate CDK7-dependent activation of p21-bound CDK4, however, mutation of these sites did not impair the phosphorylation of CDK4 by JNKs. In two selected tumor cells, two different JNK inhibitors inhibited the phosphorylation and activation of cyclin D1-CDK4-p21 but not the activation of cyclin D3-CDK4 that is mainly associated to p27. Specific inhibition by chemical genetics in MEFs confirmed the involvement of JNK2 in cyclin D1-CDK4 activation. Therefore, JNKs could be activating kinases for cyclin D1-CDK4 bound to p21, by independently phosphorylating both CDK4 and p21.

The role of cyclic AMP and its effect on protein kinase A in the mitogenic action of thyrotropin on the thyroid cell.

Cyclic AMP has been shown to inhibit cell proliferation in many cell types and to activate it in some. The latter has been recognized only lately, thanks in large part to studies on the regulation of thyroid cell proliferation in dog thyroid cells. The steps that led to this conclusion are outlined. Thyrotropin activates cyclic accumulation in thyroid cells of all the studied species and also phospholipase C in human cells. It activates directly cell proliferation in rat cell lines, dog, and human thyroid cells but not in bovine or pig cells. The action of cyclic AMP is responsible for the proliferative effect of TSH. It accounts for several human diseases: congenital hyperthyroidism, autonomous adenomas, and Graves' disease; and, by default, for hypothyroidism by TSH receptor defect. Cyclic AMP proliferative action requires the activation of protein kinase A, but this effect is not sufficient to explain it. Cyclic AMP action also requires the permissive effect of IGF-1 or insulin through their receptors, mostly as a consequence of PI3 kinase activation. The mechanism of these effects at the level of cyclin and cyclin-dependent protein kinases involves an induction of cyclin D3 by IGF-1 and the cyclic AMP-elicited generation and activation of the cyclin D3-CDK4 complex.

Phosphatidylinositol 3-kinase, protein kinase B and ribosomal S6 kinases in the stimulation of thyroid epithelial cell proliferation by cAMP and growth factors in the presence of insulin.

The proliferation of most normal cells depends on the co-operation of several growth factors and hormones, each with a specific role, but the key events involved in the action of each necessary stimulant remain largely uncharacterized. In the present study, the pathways involved in the mechanism(s) of co-operation have been investigated in primary cultures of dog thyroid epithelial cells. In this physiologically relevant system, thyroid stimulating hormone (TSH) acting through cAMP, epidermal growth factor (EGF) and phorbol esters (such as PMA) induce DNA synthesis. Their effect requires stimulation of the insulin-like growth factor-1 (IGF-1) receptor by either IGF-1 or insulin, which are not themselves mitogenic agents. In contrast, hepatocyte growth factor (HGF) is itself fully mitogenic. The results of the study demonstrate that cAMP, EGF, HGF and PMA stimulate p70 ribosomal S6 kinase (p70 S6 kinase). However, insulin/IGF-1 also stimulate p70 S6 kinase. Thus stimulation of p70 S6 kinase might be necessary, but is certainly not sufficient, for the induction of DNA synthesis and is not specific for any stimulated pathway. In contrast, phosphatidylinositol 3-kinase (PI 3-kinase) and protein kinase B (PKB) activation by insulin and HGF is strong and sustained, whereas it is weak and transient with EGF and absent in the presence of TSH or PMA. These findings suggest that: (i) stimulation of PI 3-kinases and/or PKB is not involved in the cAMP-dependent pathways leading to thyrocyte proliferation, or in the action of PMA, (ii) the stimulation of the PI 3-kinase/PKB pathway may account for the permissive action of insulin/IGF-1 in the proliferation of these cells, and (iii) the stimulation of this pathway by HGF may explain why this agent does not require insulin or IGF-1 for its mitogenic action.

IGF-1 or insulin, and the TSH cyclic AMP cascade separately control dog and human thyroid cell growth and DNA synthesis, and complement each other in inducing mitogenesis.

The regular doubling of cell mass, and therefore of cell protein content, is required for repetitive cell divisions. Preliminary observations have shown that in dog thyrocytes insulin induces protein accumulation but not DNA synthesis, while TSH does not increase protein accumulation but triggers DNA synthesis in the presence of insulin. We show here that EGF and phorbol myristate ester complement insulin action in the same way. HGF is the only factor activating both protein accumulation and DNA synthesis. The effects of insulin on protein accumulation and in permitting the TSH effect are reproduced by IGF-1 and are mediated, at least in part by the IGF-1 receptor. The concentration effect curves are similar for both effects. Similar results are obtained in human thyrocytes. They reflect true cell growth, as shown by increases in RNA content and cell size. Carbachol and fetal calf serum also stimulate protein synthesis and accumulation without triggering DNA synthesis, but they are not permissive for the mitogenic effects of TSH or of the general adenylate cyclase activator, forskolin. Moreover the mitogenic effect of TSH greatly decreased in cells deprived of insulin for 2 days although these cells remain hypertrophic. Hypertrophy may therefore be necessary for cell division, but it is not sufficient to permit it. Three different mechanisms can therefore be distinguished in the mitogenic action of TSH: (1) the increase of cell mass (hypertrophy) induced by insulin or IGF-1; (2) the permissive effect of insulin or IGF-1 on the mitogenic effect of TSH which may involve both the increase of cell mass and the induction of specific proteins such as cyclin D3 and (3) the mitogenic effect of the TSH cyclic AMP cascade proper.

Phosphorylation of the three Rb protein family members is a common step of the cAMP-, the growth factor, and the phorbol ester-mitogenic cascades but is not necessary for the hypertrophy induced by insulin.

Thyrotropin (TSH) through the cAMP cascade and in the presence of insulin induces the proliferation of dog thyroid cells. In this work, it is shown that TSH via cAMP causes the phosphorylation of the three members of the pRb family, pRb, p107, and p130, with the same kinetics as those observed when these cells are stimulated by mitogens acting through a tyrosine kinase receptor or through activation of kinase C. It is the first described point of convergence of cAMP-dependent and -independent mitogenic pathways in dog thyrocytes and suggests that the phosphorylation of the three proteins may be involved in the initiation of DNA synthesis in these cells. We also show that insulin, which induces hypertrophy and is permissive for the TSH mitogenic action, does not provoke the phosphorylation of any pRb family member, suggesting that none of these phosphorylations is required for this effect.

Thyrotropin via cyclic AMP induces insulin receptor expression and insulin Co-stimulation of growth and amplifies insulin and insulin-like growth factor signaling pathways in dog thyroid epithelial cells.

Despite the similarity of their receptors and signal transduction pathways, insulin is regarded as a regulator of glucose, protein, and lipid metabolism, whereas insulin-like growth factors (IGF-I and IGF-II) mainly act as mitogenic hormones. In the dog thyroid primary culture model, the triggering of DNA synthesis by thyrotropin (TSH) through cAMP, or by cAMP-independent factors including epidermal growth factor, hepatocyte growth factor and phorbol esters, requires insulin or IGFs as comitogenic factors. In the present study, in TSH-treated cells, IGF-I receptors and insulin receptors were paradoxically equivalent in their capacity to elicit the comitogenic pathway, which, however, was mediated only by IGF-I receptors in dog thyroid cells stimulated by cAMP-independent mitogens. Moreover, prior cell exposure to TSH or forskolin increased their responsiveness to insulin, IGF-I, and IGF-II, as seen on DNA synthesis and activation of a common insulin/IGF signaling pathway. To understand these observations, binding characteristics and expression of insulin and IGF-I receptors were examined. To analyze IGF-I receptor characteristics, the unexpected interference of a huge presence of IGF-binding proteins at the cell membrane was avoided using labeled Long R3 IGF-I instead of IGF-I. Strikingly, TSH, through cAMP, time-dependently induced insulin binding and insulin receptor mRNA and protein accumulation without any effect on IGF-I receptors. These findings constitute a first example of an induction of insulin receptor gene expression by a cAMP-mediated hormone. In dog thyroid cells, this allows low physiological insulin concentrations to act as a comitogenic factor and might explain in part the enhanced responsiveness to IGFs in response to TSH. This raises the possibility that TSH-insulin interactions may play a role in the regulation of thyroid growth and function in vivo.

c-Myc expression is controlled by the mitogenic cAMP-cascade in thyrocytes.

In dog thyroid epithelial cells in primary culture, thyrotropin (TSH), acting through cAMP, induces proliferation and differentiation expression, whereas epidermal growth factor (EGF) and phorbol esters induce proliferation and dedifferentiation. In these cells, we have detailed the regulation by cAMP of the c-myc protooncogene mRNA and protein. The cAMP signaling pathway induces a biphasic increase of c-myc mRNA and protein. c-Myc protein accumulation follows the abundance and kinetics of its mRNA expression. Using in vitro elongation of nascent transcripts to measure transcription and actinomycin D (AcD) chase experiments to study mRNA stability, we have shown that in the first phase cAMP releases a transcriptional elongation block. No modification of transcriptional initiation was observed. After 30 min of treatment with TSH, c-myc mRNA was also stabilized. During the second phase, cAMP stabilization of the mRNA disappears and transcription is again shut off. Thus, in a tissue in which it stimulates proliferation and specific gene expression, cAMP regulates biphasically c-myc expression by mechanisms operating at the transcriptional and posttranscriptional levels.

Mitogenic, dedifferentiating, and scattering effects of hepatocyte growth factor on dog thyroid cells.

Hepatocyte growth factor (HGF)/scatter factor (SF) is a potent mitogenic factor or motility factor in different cells, acting through the tyrosine kinase receptor encoded by the met protooncogene. In the present work, we demonstrate the powerful mitogenic activity of this growth factor on dog thyroid cells in primary culture. This effect, maximal at 50 ng/ml, was superior to those of other thyroid mitogenic agents, such as TSH, forskolin, and epidermal growth factor (EGF). HGF inhibited both TSH- and forskolin-stimulated iodide uptake (a thyroid-specific differentiation marker) in the same way as EGF. However, as with basic fibroblast growth factor, this dedifferentiating action appeared only during the growing phase concomitantly with the enhanced proliferation. HGF treatment also markedly decreased TSH receptor and thyroglobulin messenger RNA levels, two other markers of differentiated thyrocytes. Besides its proliferative and dedifferentiating effects, HGF enhanced the motility of the cultured thyroid cells. Concerning the mechanism of its action, we showed that HGF had no effect on basal cAMP levels, but like EGF and 12-O-tetradecanoyl-phorbol 13-acetate, it induced the rapid tyrosine phosphorylation of mitogen-activated protein kinases p42 and p44. These data establish HGF as the strongest mitogenic agent for dog thyroid cells and may explain the important role of met oncogene expression in human thyroid tumors.