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.

p53 promotes VEGF expression and angiogenesis in the absence of an intact p21-Rb pathway.

There is growing evidence that the p53 tumour suppressor downregulates vascular endothelial growth factor (VEGF) expression, although the underlying mechanisms remain unclear and controversial. Here we provide insights from in vitro experiments and in vivo xenotransplantation assays that highlight a dual role for p53 in regulating VEGF during hypoxia. Unexpectedly, and for the first time, we demonstrate that p53 rapidly induces VEGF transcription upon hypoxia exposure by binding, in an HIF-1α-dependent manner, to a highly conserved and functional p53-binding site within the VEGF promoter. However, during sustained hypoxia, p53 indirectly downregulates VEGF expression via the retinoblastoma (Rb) pathway in a p21-dependent manner, which is distinct from its role in cell-cycle regulation. Our findings have important implications for cancer therapy, especially for tumours that harbour wild-type TP53 and a dysfunctional Rb pathway.

Cancer stem cells: a reality, a myth, a fuzzy concept or a misnomer? An analysis.

The concept of cancer stem cells (CSC) embodies two aspects: the stem cell as the initial target of the oncogenic process and the existence of two populations of cells in cancers: the CSC and derived cells. The second is discussed in this review. CSC are defined as cells having three properties: a selectively endowed tumorigenic capacity, an ability to recreate the full repertoire of cancer cells of the parent tumor and the expression of a distinctive repertoire of surface biomarkers. In operational terms, the CSC are among all cancer cells those able to initiate a xenotransplant. Other explicit or implicit assumptions exist, including the concept of CSC as a single unique infrequent population of cells. To avoid such assumptions, we propose to use the operational term tumor-propagating cells (TPC); indeed, the cells that initiate transplants did not initiate the cancer. The experimental evidence supporting the explicit definition is analyzed. Cancers indeed contain a fraction of cells mainly responsible for the tumor development. However, there is evidence that these cells do not represent one homogenous population. Moreover, there is no evidence that the derived cells result from an asymmetric, qualitative and irreversible process. A more general model is proposed of which the CSC model could be one extreme case. We propose that the TPC are multiple evolutionary selected cancer cells with the most competitive properties [maintained by (epi-)genetic mechanisms], at least partially reversible, quantitative rather than qualitative and resulting from a stochastic rather than deterministic process.

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.

Regulation of thyroid cell proliferation by TSH and other factors: a critical evaluation of in vitro models.

TSH via cAMP, and various growth factors, in cooperation with insulin or IGF-I stimulate cell cycle progression and proliferation in various thyrocyte culture systems, including rat thyroid cell lines (FRTL-5, WRT, PC Cl3) and primary cultures of rat, dog, sheep and human thyroid. The available data on cell signaling cascades, cell cycle kinetics, and cell cycle-regulatory proteins are thoroughly and critically reviewed in these experimental systems. In most FRTL-5 cells, TSH (cAMP) merely acts as a priming/competence factor amplifying PI3K and MAPK pathway activation and DNA synthesis elicited by insulin/IGF-I. In WRT cells, TSH and insulin/IGF-I can independently activate Ras and PI3K pathways and DNA synthesis. In dog thyroid primary cultures, TSH (cAMP) does not activate Ras and PI3K, and cAMP must be continuously elevated by TSH to directly control the progression through G(1) phase. This effect is exerted, at least in part, via the cAMP-dependent activation of the required cyclin D3, itself synthesized in response to insulin/IGF-I. This and other discrepancies show that the mechanistic logics of cell cycle stimulation by cAMP profoundly diverge in these different in vitro models of the same cell. Therefore, although these different thyrocyte systems constitute interesting models of the wide diversity of possible mechanisms of cAMP-dependent proliferation in various cell types, extrapolation of in vitro mechanistic data to TSH-dependent goitrogenesis in man can only be accepted in the cases where independent validation is provided.

Respective roles of carbamylcholine and cyclic adenosine monophosphate in their synergistic regulation of cell cycle in thyroid primary cultures.

The stimulation of thyroid cell proliferation by TSH through cAMP depends on permissive comitogenic factors, generally the insulin-like growth factors and insulin. In dog thyroid primary cultures, the use of the phosphodiesterase-resistant analog of cAMP (Bu)(2)cAMP instead of TSH allowed to unveil a potent comitogenic activity of carbamylcholine, which can substitute for insulin and was shown to mimic insulin action on cell cycle regulatory proteins. Like insulin, carbamylcholine induced the accumulation of cyclin D3 and overcame the repression by cAMP of this protein, which was shown 1) to be essential for cell cycle progression by means of microinjections of a neutralizing antibody; and 2) to be rate limiting for the cAMP-dependent assembly of cyclin D3-cdk4 complexes, their nuclear translocation and the phosphorylation of pRb. Relative to insulin, carbamylcholine offers the significant experimental advantage that its signaling cascades can be immediately deactivated by the muscarinic antagonist atropine. In the presence of carbamylcholine, the elimination of (Bu)(2)cAMP blocked within 2 h the entry of cells into DNA synthesis phase, but the addition of atropine still permitted the entry of cells in S phase. These data support our view that the progression in G1 phase stimulated by cAMP consists of at least two essential actions that are clearly dissociated: in a first stage, depending on the supportive activity of an agent that stimulates the required cyclin D3 accumulation, cAMP induces the assembly and nuclear translocation of cyclin D3-cdk4 complexes, and then cAMP can exert alone the last crucial control that determines the cell commitment toward DNA replication.

TSH induces insulin receptors that mediate insulin costimulation of growth in normal human thyroid cells.

The mitogenic/goitrogenic effects of thyrotropin (TSH) on human thyrocytes in vitro and in vivo depend on permissive comitogenic effects of insulin-like growth factors (IGFs), which are mimicked in vitro by the low-affinity binding of high supraphysiological concentrations of insulin to IGF-I receptors. Contrary to general assumption, we show here that very low concentrations of insulin, acting through insulin receptors but not IGF-I receptors, can also support the stimulation of DNA synthesis by TSH in primary cultures of normal human thyrocytes. Moreover, TSH through cAMP increases the content of insulin receptors demonstrated by Western blotting and the cells' responsiveness to low insulin concentrations. These observations provide the first in vitro evidence in normal human thyroid cells of a functional interaction between TSH and insulin acting through its own receptor.

TSH and cAMP do not signal mitogenesis through Ras activation.

Ras activation by receptor tyrosine kinases or serpentine receptors is generally considered to be essential for G1 phase progression and mitogenesis. In the physiologically relevant model of primary dog thyrocytes, the accumulation of the GTP-bound form of Ras constituted an early convergence point of various mitogenic or comitogenic stimuli including EGF, HGF, phorbol esters, insulin and carbachol. By contrast, the basal level of GTP-Ras was slightly reduced by TSH and forskolin and did not increase during the TSH/cAMP-dependent progression into G1 phase. This rules out a role for the activation of Ras as a signal in the mitogenesis elicited by TSH via cAMP in these cells.

Transforming growth factor beta(1) selectively inhibits the cyclic AMP-dependent proliferation of primary thyroid epithelial cells by preventing the association of cyclin D3-cdk4 with nuclear p27(kip1).

Dog thyroid epithelial cells in primary culture constitute a physiologically relevant model of positive control of DNA synthesis initiation and G0-S prereplicative phase progression by cAMP as a second messenger for thyrotropin (thyroid-stimulating hormone [TSH]). As previously shown in this system, the cAMP-dependent mitogenic pathway differs from growth factor cascades as it stimulates the accumulation of p27(kip1) but not cyclins D. Nevertheless, TSH induces the nuclear translocations and assembly of cyclin D3 and cdk4, which are essential in cAMP-dependent mitogenesis. Here we demonstrate that transforming growth factor beta(1) (TGFbeta(1)) selectively inhibits the cAMP-dependent cell cycle in mid-G1 and various cell cycle regulatory events, but it weakly affects the stimulation of DNA synthesis by epidermal growth factor (EGF), hepatocyte growth factor, serum, and phorbol esters. EGF+serum and TSH did not interfere importantly with TGFbeta receptor signaling, because they did not affect the TGFbeta-induced nuclear translocation of Smad 2 and 3. TGFbeta inhibited the phosphorylation of Rb, p107, and p130 induced by TSH, but it weakly affected the phosphorylation state of Rb-related proteins in EGF+serum-treated cells. TGFbeta did not inhibit c-myc expression. In TSH-stimulated cells, TGFbeta did not affect the expression of cyclin D3, cdk4, and p27(kip1), nor the induced formation of cyclin D3-cdk4 complexes, but it prevented the TSH-induced relocalization of p27(kip1) from cdk2 to cyclin D3-cdk4. It prevented the nuclear translocations of cdk4 and cyclin D3 without altering the assembly of cyclin D3-cdk4 complexes probably formed in the cytoplasm, where they were prevented from sequestering nuclear p27(kip1) away from cdk2. This study dissociates the assembly of cyclin D3-cdk4 complexes from their nuclear localization and association with p27(kip1). It provides a new mechanism of regulation of proliferation by TGFbeta, which points out the subcellular location of cyclin D-cdk4 complexes as a crucial factor integrating mitogenic and antimitogenic regulations in an epithelial cell in primary culture.

Cyclin D3 accumulation and activity integrate and rank the comitogenic pathways of thyrotropin and insulin in thyrocytes in primary culture.

The proliferation of most normal cells depends on the synergistic interaction of several growth factors and hormones, but the cell cycle basis for this combined requirement remains largely uncharacterized. We have addressed the question of the requirement for insulin/IGF-1 also observed in many cell culture systems in the physiologically relevant system of primary cultures of dog thyroid epithelial cells stimulated by TSH, which exerts its mitogenic activity only via cAMP. The induction of cyclin A and cdc2, the phosphorylation of cdk2, the nuclear translocation of cdk4 and the assembly of cyclin D3-cdk4 complexes required the synergy of TSH and insulin. Cyclin D3 (the most abundant cyclin D) was necessary for the proliferation stimulated by TSH in the presence of insulin as shown by microinjection of a neutralizing antibody. Cyclin D3 accumulation and activity were differentially regulated by insulin and TSH, which points out this cyclin as an integrator that ranks these comitogenic pathways as supportive and activatory, respectively. Paradoxically TSH alone strongly repressed cyclin D3 accumulation. This inhibition was overridden by insulin, which markedly stimulated cyclin D3 mRNA and protein accumulation, but failed to assemble cyclin D3-cdk4 complexes in the absence of TSH. TSH unmasked the DCS-22 epitope of cyclin D3 and assembled cyclin D3-cdk4 in the presence of insulin. These data demonstrate that cyclin D synthesis and cyclin D-cdk assembly can be dissociated and complementarily regulated by different agents and signalling pathways.

Nature of the critical labile event that controls RB phosphorylation in the cyclic AMP-dependent cell cycle of thyrocytes in primary culture.

This study addresses the nature of the critical labile event that couples at restriction point mitogenic cascades with the autonomous part of the cell cycle. In primary cultures of dog thyroid epithelial cells, kinetic experiments indicate that a labile cAMP-dependent event positively controls a late G1 commitment to DNA replication and RB phosphorylation. As previously shown in this system, the cAMP-dependent mitogenic pathway differs from the most generally envisaged growth factor cascades as it stimulates the accumulation of p27(kip1) but not of cyclins D. Nevertheless, cyclin D3 and CDK4 activity are essential in the cAMP-dependent mitogenesis, and cAMP unmasks the DCS-22 epitope of cyclin D3 and induces the nuclear translocations and assembly of cyclin D3 and CDK4 in a complex that also contains p27(kip1). Unexpectedly, the washing out of forskolin rapidly arrested S phase entry and the accumulation of hyperphosphorylated RB, but did not reverse any of the above events associated with cyclin D3-CDK4 activation. This implies that even after induction of stable nuclear cyclin D3-CDK4 complexes, dog thyrocytes still depend on cAMP for RB phosphorylation and commitment to DNA synthesis, which suggests that a key labile event responsible for a last control of restriction point passage remains to be uncovered, in the cAMP-dependent cell cycle of dog thyrocytes and possibly other systems.

Critical residues of the homeodomain involved in contacting DNA bases also specify the nuclear accumulation of thyroid transcription factor-1.

The N-terminal end of thyroid transcription factor-1 (TTF-1) homeodomain is composed of a stretch of five basic amino-acids that is conserved in both POU- and NK2-class homeodomains and constitutes a functional nuclear localization signal. By analyzing the cellular distribution of fusion proteins, composed of a jellyfish green fluorescent variant and different parts of TTF-1, we show here that the presence of this basic sequence is not sufficient by itself to confer complete nuclear accumulation. By mutagenesis, we identified a second region located in the center of the DNA recognition helix of the homeodomain that is also able to specify a predominantly nuclear localization of the chimeric proteins, independently of the presence of the basic NLS. The destruction, by mutagenesis, of both the basic stretch and the motif in the DNA recognition helix led to the total loss of nuclear accumulation, indicating that complete nuclear accumulation of TTF-1 results from the concerted action of these two proteic signals. Both of the regions of the homeodomain that are involved in nuclear targeting also encompass critical amino-acids responsible for DNA binding site recognition, as evidenced by the loss of DNA binding activity in vitro upon mutagenesis. Specifically, residues in the central part of the DNA recognition helix are involved in contacting bases in the major groove of DNA and are the most conserved in homeodomain proteins, suggesting that this part of the homeodomain could play a general role in the nuclear localization of members of this family of proteins.

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.

A requirement for cyclin D3-cyclin-dependent kinase (cdk)-4 assembly in the cyclic adenosine monophosphate-dependent proliferation of thyrocytes.

In different systems, cyclic adenosine monophosphate (cAMP) either blocks or promotes cell cycle progression in mid to late G1 phase. Dog thyroid epithelial cells in primary culture constitute a model of positive control of DNA synthesis initiation and G0-S prereplicative phase progression by cAMP as a second messenger for thyrotropin (TSH). The cAMP-dependent mitogenic pathway is unique as it is independent of mitogen-activated protein kinase activation and differs from growth factor-dependent pathways at the level of the expression of several protooncogenes/transcription factors. This study examined the involvement of D-type G1 cyclins and their associated cyclin-dependent kinase (cdk4) in the cAMP-dependent G1 phase progression of dog thyroid cells. Unlike epidermal growth factor (EGF)+serum and other cAMP-independent mitogens, TSH did not induce the accumulation of cyclins D1 and D2 and partially inhibited the basal expression of the most abundant cyclin D3. However, TSH stimulation enhanced the nuclear detection of cyclin D3. This effect correlated with G1 and S phase progression. It was found to reflect both the unmasking of an epitope of cyclin D3 close to its domain of interaction with cdk4, and the nuclear translocation of cyclin D3. TSH and EGF+serum also induced a previously undescribed nuclear translocation of cdk4, the assembly of precipitable cyclin D3-cdk4 complexes, and the Rb kinase activity of these complexes. Previously, cdk4 activity was found to be required in the cAMP-dependent mitogenic pathway of dog thyrocytes, as in growth factor pathways. Here, microinjections of a cyclin D3 antibody showed that cyclin D3 is essential in the TSH/ cAMP-dependent mitogenesis, but not in the pathway of growth factors that induce cyclins D1 and D2. The present study (a) provides the first example in a normal cell of a stimulation of G1 phase progression occurring independently of an enhanced accumulation of cyclins D, (b) identifies the activation of cyclin D3 and cdk4 through their enhanced assembly and/or nuclear translocation, as first convergence steps of the parallel cAMP-dependent and growth factor mitogenic pathways, and (c) strongly suggests that this new mechanism is essential in the cAMP-dependent mitogenesis, which provides the first direct demonstration of the requirement for cyclin D3 in a G1 phase progression.

Activation of cyclic AMP-dependent kinase is required but may not be sufficient to mimic cyclic AMP-dependent DNA synthesis and thyroglobulin expression in dog thyroid cells.

Thyrotropin (TSH), via a cyclic AMP (cAMP)-dependent pathway, induces cytoplasmic retractions, proliferation, and differentiation expression in dog thyroid cells. The role of cAMP-dependent protein kinase (PKA) in the induction of these events was assessed by microinjection into living cells. Microinjection of the heat-stable inhibitor of PKA (PKI) inhibited the effects of TSH, demonstrating that activation of PKA was required in this process. Overexpression of the catalytic (C) subunit of PKA brought about by microinjection of the expression plasmid pC alpha ev or of purified C subunit itself was sufficient to mimic the cAMP-dependent cytoplasmic changes and thyroperoxidase mRNA expression but not to induce DNA synthesis and thyroglobulin (Tg) expression. The cAMP-dependent morphological effect was not observed when C subunit was coinjected with the regulatory subunit (RI or RII subunit) of PKA. To mimic the cAMP-induced PKA dissociation into free C and R subunits, the C subunit was coinjected with the regulation-deficient truncated RI subunit (RIdelta1-95) or with wild-type RI or native RII subunits, followed by incubation with TSH at a concentration too low to stimulate the cAMP-dependent events by itself. Although the cAMP-dependent morphology changes were still observed, neither DNA synthesis nor Tg expression was stimulated in these cells. Taken together, these data suggest that in addition to PKA activation, another cAMP-dependent mechanism could exist and play an important role in the transduction of the cAMP signal in thyroid cells.

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.

Paradoxical accumulation of the cyclin-dependent kinase inhibitor p27kip1 during the cAMP-dependent mitogenic stimulation of thyroid epithelial cells.

In different systems, cAMP either blocks or promotes cell cycle progression in mid to late G1 phase. Dog thyroid epithelial cells in primary culture constitute a model of positive control of DNA synthesis initiation and G0-S pre-replicative phase progression by cyclic AMP (cAMP) as a second messenger for thyrotropin (TSH). We report here that TSH markedly increases the expression of p27kip1, the inhibitor of the cell cycle and cyclin-dependent kinases. This effect was prevented by the concomitant administration of the cAMP-independent mitogens, epidermal growth factor (EGF)+serum. EGF+serum also slightly inhibited the weak basal accumulation of p27kip1. Nevertheless, in the case of stimulation by TSH alone, the cAMP-dependent cell cycle progression was fully compatible with the enhanced expression of p27kip1. This observation is paradoxical since a decrease of p27kip1 is generally associated with growth stimulation in other systems, and since a similar cAMP-dependent increase of p27kip1 in macrophages has been found responsible for mid-G1 cell cycle arrest. The opposite regulation of p27kip1 in response to TSH or EGF+serum in dog thyroid epithelial cells suggests a major difference at mid to late G1 stages between cAMP-dependent and cAMP-independent mitogenic pathways.