Ramon Bartrons

TIGAR, a p53-Inducible Regulator of Glycolysis and Apoptosis

The p53 tumor-suppressor protein prevents cancer development through various mechanisms, including the induction of cell-cycle arrest, apoptosis, and the maintenance of genome stability. We have identified a p53-inducible gene named TIGAR (TP53-induced glycolysis and apoptosis regulator). TIGAR expression lowered fructose-2,6-bisphosphate levels in cells, resulting in an inhibition of glycolysis and an overall decrease in intracellular reactive oxygen species (ROS) levels. These functions of TIGAR correlated with an ability to protect cells from ROS-associated apoptosis, and consequently, knockdown of endogenous TIGAR expression sensitized cells to p53-induced death. Expression of TIGAR may therefore modulate the apoptotic response to p53, allowing survival in the face of mild or transient stress signals that may be reversed or repaired. The decrease of intracellular ROS levels in response to TIGAR may also play a role in the ability of p53 to protect from the accumulation of genomic damage.

PFK-2/FBPase-2: maker and breaker of the essential biofactor fructose-2,6-bisphosphate

Fructose-2,6-bisphosphate is responsible for mediating glucagon-stimulated gluconeogenesis in the liver. This discovery has led to the realization that this compound plays a significant role in directing carbohydrate fluxes in all eukaryotes. Biophysical studies of the enzyme that both synthesizes and degrades this biofactor have yielded insight into its molecular enzymology. Moreover, the metabolic role of fructose-2,6-bisphosphate has great potential in the treatment of diabetes.

6-Phosphofructo-2-kinase (pfkfb3) gene promoter contains hypoxia-inducible factor-1 binding sites necessary for transactivation in response to hypoxia

The up-regulation of glycolysis to enhance the production of energy under reduced pO2 is a hallmark of the hypoxic response. A key regulator of glycolytic flux is fructose-2,6-bisphosphate, and its steady state concentration is regulated by the action of different isozymes product of four genes (pfkfb1–4). pfkfb3 has been found in proliferating cells and tumors, being induced by hypoxia. To understand the organization of cis-acting sequences that are responsible for the oxygen-regulated pfkfb3 gene, we have studied its 5′-flanking region. Extensive analysis of the 5′ pfkfb3 promoter sequence revealed the presence of putative consensus binding sites for various transcription factors that could play an important role in pfkfb3 gene regulation. These DNA consensus sequences included estrogen receptor, hypoxia response element (HRE), early growth response, and specific protein 1 putative binding sites. Promoter deletion analysis as well as putative HREs sequences (wild type and mutated) fused to a c-fos minimal promoter unit constructs demonstrate that the sequence located from -1269 to -1297 relative to the start site is required for hypoxia-inducible factor 1 (HIF-1) induction. The effective binding of HIF-1 transcription factor to the HREs at -1279 and -1288 was corroborated by electrophoretic mobility shift assay and biotinylated oligonucleotide pull-down. In addition, HIF-1α null mouse embryo fibroblasts transfected with a full-length pfkfb3 promoter-luciferase reporter construct further demonstrated that HIF-1 protein was critically involved for hypoxia transactivation of this gene. Altogether, these results demonstrate that pfkfb3 is a hypoxia-inducible gene that is stimulated through HIF interaction with the consensus HRE site in its promoter region.

Amino Acids Activate Mammalian Target of Rapamycin Complex 2 (mTORC2) via PI3K/Akt Signaling

The activity of mammalian target of rapamycin (mTOR) complexes regulates essential cellular processes, such as growth, proliferation, or survival. Nutrients such as amino acids are important regulators of mTOR complex 1 (mTORC1) activation, thus affecting cell growth, protein synthesis, and autophagy. Here, we show that amino acids may also activate mTOR complex 2 (mTORC2). This activation is mediated by the activity of class I PI3K and of Akt. Amino acids induced a rapid phosphorylation of Akt at Thr-308 and Ser-473. Whereas both phosphorylations were dependent on the presence of mTOR, only Akt phosphorylation at Ser-473 was dependent on the presence of rictor, a specific component of mTORC2. Kinase assays confirmed mTORC2 activation by amino acids. This signaling was functional, as demonstrated by the phosphorylation of Akt substrate FOXO3a. Interestingly, using different starvation conditions, amino acids can selectively activate mTORC1 or mTORC2. These findings identify a new signaling pathway used by amino acids underscoring the crucial importance of these nutrients in cell metabolism and offering new mechanistic insights.

JunB is involved in the inhibition of myogenic differentiation by bone morphogenetic protein-2.

Bone morphogenetic proteins (BMPs) constitute a family of multifunctional growth and differentiation factors structurally related to transforming growth factor-β. BMPs were first identified by their osteoinductive effects, inducing ectopic bone formation when implanted in skeletal muscle, and have an important role as regulators of skeletal development in vivo. In vitro, BMP-2 is able to transdifferentiate myogenic C2C12 cells into the osteoblastic phenotype. In this report, we show that the osteoinductive effects of BMP-2 in C2C12 cells are mediated by bone morphogenetic protein receptor type-IA in combination with both activin receptor type II and bone morphogenetic protein receptor type II. We also analyzed the expression levels of nuclear protooncogenes to understand early transcriptional events induced by BMP-2. We show thatjunB is an immediate early gene induced by BMP-2 and transforming growth factor-β. BMP-2 induces transcriptional activation of JunB expression as early as 30 min after ligand addition, reaching maximal levels after 90 min. Increase of JunB mRNA correlates with a higher AP-1 binding activity. Furthermore, ectopic overexpression of JunB is sufficient to inhibit expression of myoblast differentiation markers in C2C12 cells. These data, taken together, show the involvement of JunB in the early steps of inhibition of myogenic differentiation induced by transforming growth factor-β family members.

A zinc-finger transcription factor induced by TGF-β promotes apoptotic cell death in epithelial Mv1Lu cells

Transforming growth factor‐β (TGF‐β) superfamily members constitute a group of multifunctional factors that are able to stimulate apoptotic cell death in a variety of cells. In this report, we show that a zinc‐finger transcription factor (TIEG) is an immediate early gene transcriptionally induced by TGF‐β in the epithelial Mv1Lu cell line. We also demonstrate that, mimicking TGF‐β effects, ectopic overexpression of TIEG is sufficient to trigger the apoptotic cell program in these cells, which is preceded by a decrease of Bcl‐2 protein levels. Finally, apoptotic events elicited by TIEG overexpression can be effectively prevented by ectopic co‐expression of Bcl‐2. On the basis of these results we suggest that induction of TIEG expression has a role in the pro‐apoptotic properties of TGF‐β.

PFKFB3 gene silencing decreases glycolysis, induces cell‐cycle delay and inhibits anchorage‐independent growth in HeLa cells

The high rate of glycolysis despite the presence of oxygen in tumor cells (Warburg effect) suggests an important role for this process in cell division. The glycolytic rate is dependent on the cellular concentration of fructose 2,6‐bisphosphate (Fru‐2,6‐P2), which, in turn, is controlled by the bifunctional enzyme 6‐phosphofructo‐2‐kinase/fructose‐2,6‐bisphosphatase (PFK‐2). The ubiquitous PFK‐2 isoenzyme (uPFK‐2, alternatively named UBI2K5 or ACG) coded by the pfkfb3 gene is induced by different stimuli (serum, progesterone, insulin, hypoxia, etc.) and has the highest kinase/phosphatase activity ratio amongst all PFK‐2 isoenzymes discovered to date, which is consistent with its role as a powerful activator of glycolysis. uPFK‐2 is expressed in brain, placenta, transformed cells and proliferating cells. In the present work, we analyze the impact of small interfering RNA (siRNA)‐induced silencing of uPFK‐2 on the inhibition of cell proliferation. HeLa cells treated with uPFK‐2 siRNA showed a decrease in uPFK‐2 RNA levels measured at 24 h. uPFK‐2 protein levels were severely depleted at 48–72 h when compared with cells treated with an unrelated siRNA, correlating with decreased glycolytic activity, Fru‐2,6‐P2, lactate and ATP concentrations. These metabolic changes led to reduced viability, cell‐cycle delay and an increase in the population of apoptotic cells. Moreover, uPFK‐2 suppression inhibited anchorage‐independent growth. The results obtained highlight the importance of uPFK‐2 on the regulation of glycolysis, on cell viability and proliferation and also on anchorage‐independent growth. These data underscore the potential for uPFK‐2 as an effective tumor therapeutic target.

BMP-2 decreases Mash1 stability by increasing Id1 expression

In neural development, bone morphogenetic proteins (BMPs) restrict neuronal differentiation, thereby promoting the maintenance of progenitor cells or even inducing astrocytogenesis. We report that exposure of neuroendocrine lung carcinoma cells to BMP‐2 leads to a rapid decline in steady‐state levels of Mash1 protein and some neuron‐specific markers. BMP‐2 induces a post‐transcriptional decrease in Mash1 levels through enhanced degradation. We demonstrate that Mash1 protein stability is tightly regulated by the E47/Id1 expression ratio. Transient induction of Id1 by BMP‐2 negatively correlates with Mash1 levels. Furthermore, an ectopic increase in Id1 levels is sufficient to induce degradation of either ectopic or endogenous Mash1, whereas expression of Mash1 in Id1‐deficient cells or overexpression of E47 makes Mash1 levels refractory to the addition of BMP‐2. Furthermore, we show that the E47/Id1 expression ratio also regulates CK2‐mediated phosphorylation of Mash1 on Ser152, which increases interaction of Mash1–E47 heterodimers. We propose a novel mechanism in which the balance between Id and E protein levels regulates not only the transcriptional function but also protein stability of the neurogenic bHLH transcription factor Mash1.

p38 Regulates Expression of Osteoblast-specific Genes by Phosphorylation of Osterix

Osterix, a zinc finger transcription factor, is specifically expressed in osteoblasts and osteocytes of all developing bones. Because no bone formation occurs in Osx-null mice, Osterix is thought to be an essential regulator of osteoblast differentiation. We report that, in several mesenchymal and osteoblastic cell types, BMP-2 induces an increase in expression of the two isoforms of Osterix arising from two alternative promoters. We identified a consensus Sp1 sequence (GGGCGG) as Osterix binding regions in the fibromodulin and the bone sialoprotein promoters in vitro and in vivo. Furthermore, we show that Osterix is a novel substrate for p38 MAPK in vitro and in vivo and that Ser-73 and Ser-77 are the regulatory sites phosphorylated by p38. Our data also demonstrate that Osterix is able to increase recruitment of p300 and Brg1 to the promoters of its target genes fibromodulin and bone sialoprotein in vivo and that it directly associates with these cofactors through protein-protein interactions. Phosphorylation of Osterix at Ser-73/77 increased its ability to recruit p300 and SWI/SNF to either fibromodulin or bone sialoprotein promoters. We therefore propose that Osterix binds to Sp1 sequences on target gene promoters and that its phosphorylation by p38 enhances recruitment of coactivators to form transcriptionally active complexes.

BMP2 induction of actin cytoskeleton reorganization and cell migration requires PI3-kinase and Cdc42 activity

Bone morphogenetic proteins (BMPs) are potent regulators of several cellular events. We report that exposure of C2C12 cells to BMP2 leads to an increase in cell migration and a rapid rearrangement of the actin filaments into cortical protrusions. These effects required independent and parallel activation of the Cdc42 small GTPase and the α-isoform of the phosphoinositide 3-kinase (PI3Kα), because ectopic expression of a dominant-negative form of Cdc42 or distinct pharmacological PI3K inhibitors abrogated these responses. Furthermore, we demonstrate that BMP2 activates different group I and group II PAK isoforms as well as LIMK1 with similar kinetics to Cdc42 or PI3K activation. BMP2 activation of PAK and LIMK1, measured by either kinase activity or with antibodies raised against phosphorylated residues at their activation loops, were abolished by blocking PI3K-signaling pathways. Together, these findings suggest that Cdc42 and PI3K signals emanating from BMP receptors are involved in specific regulation of actin assembly and cell migration.