Marcela Sosa-Garrocho

Determining and understanding the control of glycolysis in fast‐growth tumor cells

Control analysis of the glycolytic flux was carried out in two fast‐growth tumor cell types of human and rodent origin (HeLa and AS‐30D, respectively). Determination of the maximal velocity (Vmax) of the 10 glycolytic enzymes from hexokinase to lactate dehydrogenase revealed that hexokinase (153–306 times) and phosphfructokinase‐1 (PFK‐1) (22–56 times) had higher over‐expression in rat AS‐30D hepatoma cells than in normal freshly isolated rat hepatocytes. Moreover, the steady‐state concentrations of the glycolytic metabolites, particularly those of the products of hexokinase and PFK‐1, were increased compared with hepatocytes. In HeLa cells, Vmax values and metabolite concentrations for the 10 glycolytic enzyme were also significantly increased, but to a much lesser extent (6–9 times for both hexokinase and PFK‐1). Elasticity‐based analysis of the glycolytic flux in AS‐30D cells showed that the block of enzymes producing Fru(1,6)P2 (i.e. glucose transporter, hexokinase, hexosephosphate isomerase, PFK‐1, and the Glc6P branches) exerted most of the flux control (70–75%), whereas the consuming block (from aldolase to lactate dehydrogenase) exhibited the remaining control. The Glc6P‐producing block (glucose transporter and hexokinase) also showed high flux control (70%), which indicated low flux control by PFK‐1. Kinetic analysis of PFK‐1 showed low sensitivity towards its allosteric inhibitors citrate and ATP, at physiological concentrations of the activator Fru(2,6)P2. On the other hand, hexokinase activity was strongly inhibited by high, but physiological, concentrations of Glc6P. Therefore, the enhanced glycolytic flux in fast‐growth tumor cells was still controlled by an over‐produced, but Glc6P‐inhibited hexokinase.

Inhibitory Smad7: Emerging Roles in Health and Disease

Smad7 is an inhibitory Smad protein that blocks Transforming Growth Factor-beta (TGF-β) signaling through a negative feedback loop, also capable of mediating the crosstalk between TGF-β and other signaling pathways. Smad7 mRNA and protein levels are upregulated after TGF-β signaling; subsequently, Smad7 protein binds TGF-β type I receptor blocking R-Smad phosphorylation and eventually TGF-β signaling. Because of this inhibitory function, Smad7 can antagonize diverse cellular processes regulated by TGF-β such as cell proliferation, differentiation, apoptosis, adhesion and migration. Smad7 induction by different cytokines, besides TGF-β, is also critical for crosstalk/integration of a variety of signaling pathways, and relevant in the pathology of some diseases. Thus, Smad7 plays a key role in the control of various physiological events, and even in some pathological processes including fibrosis and cancer. This review highlights the main known functions of Smad7 with a particular focus on the relevance that alterations of Smad7 function may have in homeostasis, also describing some Smad7 emerging roles in the development of several human diseases that identify this protein as a potential therapeutic target.

Transforming growth factor-β/SMAD Target gene SKIL is negatively regulated by the transcriptional cofactor complex SNON-SMAD4.

Background: Human SKIL gene encodes for SNON, a negative regulator of the TGF-β/SMAD pathway. Results: We provide a molecular mechanism of transcriptional regulation of SKIL gene expression by TGF-β/SMADs. Conclusion: Transcriptional cofactor complex SNON-SMAD4 negatively controls the expression of SKIL gene. Significance: The formation and function of complex SNON-SMAD4 are impaired in cancer cells lacking SMAD4, which affects TGF β-target gene regulation. The human SKI-like (SKIL) gene encodes the SMAD transcriptional corepressor SNON that antagonizes TGF-β signaling. SNON protein levels are tightly regulated by the TGF-β pathway: whereas a short stimulation with TGF-β decreases SNON levels by its degradation via the proteasome, longer TGF-β treatment increases SNON levels by inducing SKIL gene expression. Here, we investigated the molecular mechanisms involved in the self-regulation of SKIL gene expression by SNON. Bioinformatics analysis showed that the human SKIL gene proximal promoter contains a TGF-β response element (TRE) bearing four groups of SMAD-binding elements that are also conserved in mouse. Two regions of 408 and 648 bp of the human SKIL gene (∼2.4 kb upstream of the ATG initiation codon) containing the core promoter, transcription start site, and the TRE were cloned for functional analysis. Binding of SMAD and SNON proteins to the TRE region of the SKIL gene promoter after TGF-β treatment was demonstrated by ChIP and sequential ChIP assays. Interestingly, the SNON-SMAD4 complex negatively regulated basal SKIL gene expression through binding the promoter and recruiting histone deacetylases. In response to TGF-β signal, SNON is removed from the SKIL gene promoter, and then the activated SMAD complexes bind the promoter to induce SKIL gene expression. Subsequently, the up-regulated SNON protein in complex with SMAD4 represses its own expression as part of the negative feedback loop regulating the TGF-β pathway. Accordingly, when the SNON-SMAD4 complex is absent as in some cancer cells lacking SMAD4 the regulation of some TGF-β target genes is modified.

Angiotensin II increases mRNA levels of all TGF-β isoforms in quiescent and activated rat hepatic stellate cells

AII (angiotensin II) is a vasoactive peptide that plays an important role in the development of liver fibrosis mainly by regulating profibrotic cytokine expression such as TGF‐β (transforming growth factor‐β). Activated HSCs (hepatic stellate cells) are the major cell type responsible for ECM (extracellular matrix) deposition during liver fibrosis and are also a target for AII and TGF‐β actions. Here, we studied the effect of AII on the mRNA levels of TGF‐β isoforms in primary cultures of rat HSCs. Both quiescent and activated HSCs were stimulated with AII for different time periods, and mRNA levels of TGF‐β1, TGF‐β2 and TGF‐β3 isoforms were evaluated using RNaseI protection assay. The mRNA levels of all TGF‐β isoforms, particularly TGF‐β2 and TGF‐β3, were increased after AII treatment in activated HSCs. In addition, activated HSCs were able to produce active TGF‐β protein after AII treatment. The mRNA expression of TGF‐β isoforms induced by AII required both ERK1/2 and Nox (NADPH oxidase) activation but not PKC (protein kinase C) participation. ERK1/2 activation induced by AII occurs via AT1 receptors, but independently of either PKC and Nox activation or EGFR (epidermal growth factor receptor) transactivation. Interestingly, AII has a similar effect on TGF‐β expression in quiescent HSCs, although it has a smaller but significant effect on ERK1/2 activation in these cells.

Hyperinsulinemia is Associated with Increased Soluble Insulin Receptors Release from Hepatocytes.

It has been generally assumed that insulin circulates freely in blood. However it can also interact with plasma proteins. Insulin receptors are located in the membrane of target cells and consist of an alpha and beta subunits with a tyrosine kinase cytoplasmic domain. The ectodomain, called soluble insulin receptor (SIR) has been found elevated in patients with diabetes mellitus. We explored if insulin binds to SIRs in circulation under physiological conditions and hypothesize that this SIR may be released by hepatocytes in response to high insulin concentrations. The presence of SIR in rat and human plasmas and the culture medium of hepatocytes was explored using Western blot analysis. A purification protocol was performed to isolated SIR using affinity, gel filtration, and ion exchange chromatographies. A modified reverse hemolytic plaque assay was used to measure SIR release from cultured hepatocytes. Incubation with 1 nmol l−1 insulin induces the release of the insulin receptor ectodomains from normal rat hepatocytes. This effect can be partially prevented by blocking protease activity. Furthermore, plasma levels of SIR were higher in a model of metabolic syndrome, where rats are hyperinsulinemic. We also found increased SIR levels in hyperinsulinemic humans. SIR may be an important regulator of the amount of free insulin in circulation. In hyperinsulinemia, the amount of this soluble receptor increases and this could lead to higher amounts of insulin bound to this receptor, rather than free insulin, which is the biologically active form of the hormone. This observation could enlighten the mechanisms of insulin resistance.

Plasma membrane calcium ATPase isoform 3 expression in single cells isolated from rat liver

The plasma membrane Ca2+-ATPase (PMCA) located in the hepatocyte is a controversial molecule in itself since it displays different features to those regarded as canonical for P-type Ca2+-ATPases, and from which transcript expression as well as catalytic activity continues to be under active investigation. Our aim in this study was to explore at a first glance, pmca isoform distribution using isolated parenchymal and non-parenchymal cells from rat liver tissue. Expression of pmca transcripts was analyzed in fresh or cell-enriched culture preparations, confirming pmca1 and pmca4 as the housekeeping isoforms in all cell types studied (hepatocytes, Kupffer cells, and stellate cells). However, for the first time we show expression of pmca3 transcripts edited at two different sites in both hepatocytes and non-parenchymal cells. Interestingly, employing non-parenchymal cells we demonstrate the specific expression of pmca3e transcripts previously considered nearly exclusive of excitable tissues. Real-time PCR quantification shows a significant decrease of pmca3 transcripts in cultured Kupffer and hepatic stellate cells in comparison with fresh cells. The presence of pmca2 along with pmca3 in all liver cell types studied suggests that high affinity isoforms are relevant to the adequate management of calcium in liver tissue, particularly when hepatic cells become activated by diverse stimuli.

Novel Regulation of Ski Protein Stability and Endosomal Sorting by Actin Cytoskeleton Dynamics in Hepatocytes

Background: The Ski oncoprotein and tumor suppressor is a negative regulator of the antimitotic TGF-β/Smad pathway. Results: The Ski protein is localized in the nucleus and cytoplasm of hepatocytes. Ski protein stability is controlled differentially by actin cytoskeleton dynamics. Conclusion: TGF-β/Smads and GPCR/actin cytoskeleton-dynamic signals regulate Ski protein stability via the proteasome in hepatocytes. Significance: Stabilization of Ski protein may favor the proliferation of regenerating hepatocyte. TGF-β-induced antimitotic signals are highly regulated during cell proliferation under normal and pathological conditions, such as liver regeneration and cancer. Up-regulation of the transcriptional cofactors Ski and SnoN during liver regeneration may favor hepatocyte proliferation by inhibiting TGF-β signals. In this study, we found a novel mechanism that regulates Ski protein stability through TGF-β and G protein-coupled receptor (GPCR) signaling. Ski protein is distributed between the nucleus and cytoplasm of normal hepatocytes, and the molecular mechanisms controlling Ski protein stability involve the participation of actin cytoskeleton dynamics. Cytoplasmic Ski is partially associated with actin and localized in cholesterol-rich vesicles. Ski protein stability is decreased by TGF-β/Smads, GPCR/Rho signals, and actin polymerization, whereas GPCR/cAMP signals and actin depolymerization promote Ski protein stability. In conclusion, TGF-β and GPCR signals differentially regulate Ski protein stability and sorting in hepatocytes, and this cross-talk may occur during liver regeneration.

Actin-cytoskeleton polymerization differentially controls the stability of Ski and SnoN co-repressors in normal but not in transformed hepatocytes.

BACKGROUND Ski and SnoN proteins function as transcriptional co-repressors in the TGF-β pathway. They regulate cell proliferation and differentiation, and their aberrant expression results in altered TGF-β signalling, malignant transformation, and alterations in cell proliferation. METHODS We carried out a comparative characterization of the endogenous Ski and SnoN protein regulation by TGF-β, cell adhesion disruption and actin-cytoskeleton rearrangements between normal and transformed hepatocytes; we also analyzed Ski and SnoN protein stability, subcellular localization, and how their protein levels impact the TGF-β/Smad-driven gene transcription. RESULTS Ski and SnoN protein levels are lower in normal hepatocytes than in hepatoma cells. They exhibit a very short half-life and a nuclear/cytoplasmic distribution in normal hepatocytes opposed to a high stability and restricted nuclear localization in hepatoma cells. Interestingly, while normal cells exhibit a transient TGF-β-induced gene expression, the hepatoma cells are characterized by a strong and sustained TGF-β-induced gene expression. A novel finding is that Ski and SnoN stability is differentially regulated by cell adhesion and cytoskeleton rearrangements in the normal hepatocytes. The inhibition of protein turnover down-regulated both Ski and SnoN co-repressors impacting the kinetic of expression of TGF-β-target genes. CONCLUSION Normal regulatory mechanisms controlling Ski and SnoN stability, subcellular localization and expression are altered in hepatocarcinoma cells. GENERAL SIGNIFICANCE This work provides evidence that Ski and SnoN protein regulation is far more complex in normal than in transformed cells, since many of the normal regulatory mechanisms are lost in transformed cells.

Calcium-sensing receptor inhibits TGF-β-signaling by decreasing Smad2 phosphorylation

Calcium‐sensing receptor (CaSR) contributes to maintain homeostatic levels of extracellular calcium. In addition, CaSR controls other cellular activities such as proliferation and migration, particularly in cells not related to extracellular calcium homeostasis, potentially by cross‐talking with parallel signaling pathways. Here we report that CaSR attenuates transforming growth factor‐β (TGF‐β)‐signaling in hepatic C9 cells and in transfected HEK293 cells. Wild type CaSR interferes with TGF‐β‐dependent Smad2 phosphorylation and induces its proteasomal degradation, resulting in a decrease of TGF‐β‐dependent transcriptional activity, whereas an inactivating CaSR mutant does not transduce an inhibitory effect of extracellular calcium on TGF‐β signaling. Attenuation of TGF‐β signaling in response to extracellular calcium is linked to Rab11‐dependent CaSR‐trafficking with the intervention of CaSR carboxyl‐terminal tail. Our data suggest that CaSR might regulate TGF‐β‐dependent cellular responses mediated by TGF‐β signaling inhibition.

Downregulation of SnoN oncoprotein induced by antibiotics anisomycin and puromycin positively regulates transforming growth factor-β signals.

BACKGROUND SnoN and Ski proteins function as Smad transcriptional corepressors and are implicated in the regulation of diverse cellular processes such as proliferation, differentiation and transformation. Transforming growth factor-β (TGF-β) signaling causes SnoN and Ski protein degradation via proteasome with the participation of phosphorylated R-Smad proteins. Intriguingly, the antibiotics anisomycin (ANS) and puromycin (PURO) are also able to downregulate Ski and SnoN proteins via proteasome. METHODS We explored the effects of ANS and PURO on SnoN protein downregulation when the activity of TGF-β signaling was inhibited by using different pharmacological and non-pharmacological approaches, either by using specific TβRI inhibitors, overexpressing the inhibitory Smad7 protein, or knocking-down TβRI receptor or Smad2 by specific shRNAs. The outcome of SnoN and Ski downregulation induced by ANS or PURO on TGF-β signaling was also studied. RESULTS SnoN protein downregulation induced by ANS and PURO did not involve the induction of R-Smad phosphorylation but it was abrogated after TGF-β signaling inhibition; this effect occurred in a cell type-specific manner and independently of protein synthesis inhibition or any other ribotoxic effect. Intriguingly, antibiotics seem to require components of the TGF-β/Smad pathway to downregulate SnoN. In addition, SnoN protein downregulation induced by antibiotics favored gene transcription induced by TGF-β signaling. CONCLUSIONS ANS and PURO require TGF-β/Smad pathway to induce SnoN and Ski protein downregulation independently of inducing R-Smad2 phosphorylation, which facilitates TGF-β signaling. GENERAL SIGNIFICANCE Antibiotic analogs lacking ribotoxic effects are useful as pharmacological tools to study TGF-β signaling by controlling Ski and SnoN protein levels.