Dawidson Assis Gomes

Apoptotic hepatocyte DNA inhibits hepatic stellate cell chemotaxis via toll-like receptor 9†

Apoptosis of hepatocytes results in the development of liver fibrosis, but the molecular signals mediating this are poorly understood. Degradation and modification of nuclear DNA is a central feature of apoptosis, and DNA from apoptotic mammalian cells is known to activate immune cells via Toll‐like receptor 9 (TLR9). We tested if DNA from apoptotic hepatocytes can induce hepatic stellate cell (HSC) differentiation. Our data show that apoptotic hepatocyte DNA and cytidine‐phosphate‐guanosine oligonucleotides induced up‐regulation of transforming growth factor β1 and collagen 1 messenger RNA both in the human HSC line LX‐2 and in primary mouse HSCs. These effects were opposed by TLR9 antagonists. We have recently shown that adenosine inhibits HSC chemotaxis, and we now show that apoptotic hepatocyte DNA also inhibits platelet‐derived growth factor (PDGF)‐mediated HSC chemotaxis. Inhibition of HSC chemotaxis by PDGF was blocked by TLR9 antagonists, and was absent in primary HSCs from mice deficient in TLR9 or the TLR adaptor molecule MyD88. Stimulation of TLR9 on HSCs blocked signaling by the PDGF signaling molecule inositol 1,4,5‐triphosphate and reduced PDGF‐mediated increase in cytosolic Ca2+. Conclusion: DNA from apoptotic hepatocytes acts as an important mediator of HSC differentiation by (1) providing a stop signal to mobile HSCs when they have reached an area of apoptosing hepatocytes and (2) inducing a stationary phenotype‐associated up‐regulation of collagen production. (HEPATOLOGY 2007.)

The type III inositol 1,4,5-trisphosphate receptor preferentially transmits apoptotic Ca2+ signals into mitochondria.

There are three isoforms of the inositol 1,4,5- trisphosphate receptor (InsP3R), each of which has a distinct effect on Ca2+ signaling. However, it is not known whether each isoform similarly plays a distinct role in the activation of Ca2+-mediated events. To investigate this question, we examined the effects of each InsP3R isoform on transmission of Ca2+ signals to mitochondria and induction of apoptosis. Each isoform was selectively silenced using isoform-specific small interfering RNA in Chinese hamster ovary cells, which express all three InsP3R isoforms. ATP-induced cytosolic Ca2+ signaling patterns were altered, regardless of which isoform was silenced, but in a different fashion depending on the isoform. ATP also induced Ca2+ signals in mitochondria, which were inhibited more effectively by silencing the type III InsP3R than by silencing either the type I or type II isoform. The type III isoform also co-localized most strongly with mitochondria. When apoptosis was induced by activation of either the extrinsic or intrinsic apoptotic pathway, induction was reduced most effectively by silencing the type III InsP3R. These findings provide evidence that the type III isoform of the InsP3R plays a special role in induction of apoptosis by preferentially transmitting Ca2+ signals into mitochondria.

c-Met Must Translocate to the Nucleus to Initiate Calcium Signals

Hepatocyte growth factor (HGF) is important for cell proliferation, differentiation, and related activities. HGF acts through its receptor c-Met, which activates downstream signaling pathways. HGF binds to c-Met at the plasma membrane, where it is generally believed that c-Met signaling is initiated. Here we report that c-Met rapidly translocates to the nucleus upon stimulation with HGF. Ca2+ signals that are induced by HGF result from phosphatidylinositol 4,5-bisphosphate hydrolysis and inositol 1,4,5-trisphosphate formation within the nucleus rather than within the cytoplasm. Translocation of c-Met to the nucleus depends upon the adaptor protein Gab1 and importin β1, and formation of Ca2+ signals in turn depends upon this translocation. HGF may exert its particular effects on cells because it bypasses signaling pathways in the cytoplasm to directly activate signaling pathways in the nucleus.

Inflammasome-mediated regulation of hepatic stellate cells

The inflammasome is a cytoplasmic multiprotein complex that has recently been identified in immune cells as an important sensor of signals released by cellular injury and death. Analogous to immune cells, hepatic stellate cells (HSC) also respond to cellular injury and death. Our aim was to establish whether inflammasome components were present in HSC and could regulate HSC functionality. Monosodium urate (MSU) crystals (100 microg/ml) were used to experimentally induce inflammasome activation in LX-2 and primary mouse HSC. Twenty-four hours later primary mouse HSC were stained with alpha-smooth muscle actin and visualized by confocal microscopy, and TGF-beta and collagen1 mRNA expression was quantified. LX-2 cells were further cultured with or without MSU crystals for 24 h in a transwell chemotaxis assay with PDGF as the chemoattractant. We also examined inhibition of calcium (Ca(2+)) signaling in LX-2 cells treated with or without MSU crystals using caged inositol 1,4,5-triphosphate (IP(3)). Finally, we confirmed an important role of the inflammasome in experimental liver fibrosis by the injection of carbon tetrachloride (CCl(4)) or thioacetamide (TAA) in wild-type mice and mice lacking components of the inflammasome. Components of the inflammasome are expressed in LX-2 cells and primary HSC. MSU crystals induced upregulation of TGF-beta and collagen1 mRNA and actin reorganization in HSCs from wild-type mice but not mice lacking inflammasome components. MSU crystals inhibited the release of Ca(2+) via IP(3) in LX-2 cells and also inhibited PDGF-induced chemotaxis. Mice lacking the inflammasome-sensing and adaptor molecules, NLRP3 and apoptosis-associated speck-like protein containing CARD, had reduced CCl(4) and TAA-induced liver fibrosis. We concluded that inflammasome components are present in HSC, can regulate a variety of HSC functions, and are required for the development of liver fibrosis.

Nucleoplasmic Calcium Is Required for Cell Proliferation

Ca2+ signals regulate cell proliferation, but the spatial and temporal specificity of these signals is unknown. Here we use selective buffers of nucleoplasmic or cytoplasmic Ca2+ to determine that cell proliferation depends upon Ca2+ signals within the nucleus rather than in the cytoplasm. Nuclear Ca2+ signals stimulate cell growth rather than inhibit apoptosis and specifically permit cells to advance through early prophase. Selective buffering of nuclear but not cytoplasmic Ca2+ signals also impairs growth of tumors in vivo. These findings reveal a major physiological and potential pathophysiological role for nucleoplasmic Ca2+ signals and suggest that this information can be used to design novel therapeutic strategies to regulate conditions of abnormal cell growth.

Inheritance of DNA transferred from American trypanosomes to human hosts.

Interspecies DNA transfer is a major biological process leading to the accumulation of mutations inherited by sexual reproduction among eukaryotes. Lateral DNA transfer events and their inheritance has been challenging to document. In this study we modified a thermal asymmetric interlaced PCR by using additional targeted primers, along with Southern blots, fluorescence techniques, and bioinformatics, to identify lateral DNA transfer events from parasite to host. Instances of naturally occurring human infections by Trypanosoma cruzi are documented, where mitochondrial minicircles integrated mainly into retrotransposable LINE-1 of various chromosomes. The founders of five families show minicircle integrations that were transferred vertically to their progeny. Microhomology end-joining of 6 to 22 AC-rich nucleotide repeats in the minicircles and host DNA mediates foreign DNA integration. Heterogeneous minicircle sequences were distributed randomly among families, with diversity increasing due to subsequent rearrangement of inserted fragments. Mosaic recombination and hitchhiking on retrotransposition events to different loci were more prevalent in germ line as compared to somatic cells. Potential new genes, pseudogenes, and knockouts were identified. A pathway of minicircle integration and maintenance in the host genome is suggested. Thus, infection by T. cruzi has the unexpected consequence of increasing human genetic diversity, and Chagas disease may be a fortuitous share of negative selection. This demonstration of contemporary transfer of eukaryotic DNA to the human genome and its subsequent inheritance by descendants introduces a significant change in the scientific concept of evolutionary biology and medicine.

Nuclear Ca2+ regulates cardiomyocyte function

In the heart, cytosolic Ca(2+) signals are well-characterized events that participate in the activation of cell contraction. In contrast, nuclear Ca(2+) contribution to cardiomyocyte function remains elusive. Here, we examined functional consequences of buffering nuclear Ca(2+) in neonatal cardiomyocytes. We report that cardiomyocytes contain a nucleoplasmic reticulum, which expresses both ryanodine receptor (RyR) and inositol 1,4,5-trisphosphate receptor (InsP(3)R), providing a possible way for active regulation of nuclear Ca(2+). Adenovirus constructs encoding the Ca(2+) buffer protein parvalbumin were targeted to the nucleus with a nuclear localization signal (Ad-PV-NLS) or to the cytoplasm with a nuclear exclusion signal (Ad-PV-NES). A decrease in the amplitude of global Ca(2+) transients and RyR-II expression, as well as an increase in cell beating rate were observed in Ad-PV-NES and Ad-PV-NLS cells. When nuclear Ca(2+) buffering was imposed nuclear enlargement, increased calcineurin expression, NFAT translocation to the nucleus and subcellular redistribution of atrial natriuretic peptide were observed. Furthermore, prolongation of action potential duration occurred in adult ventricular myocytes. These results suggest that nuclear Ca(2+) levels underlie the regulation of specific protein targets and thereby modulate cardiomyocyte function. The local nuclear Ca(2+) signaling and the structures that control it constitute a novel regulatory motif in the heart.

Stem cells and calcium signaling.

The increasing interest in stem cell research is linked to the promise of developing treatments for many lifethreatening, debilitating diseases, and for cell replacement therapies. However, performing these therapeutic innovations with safety will only be possible when an accurate knowledge about the molecular signals that promote the desired cell fate is reached. Among these signals are transient changes in intracellular Ca(2+) concentration [Ca(2+)](i). Acting as an intracellular messenger, Ca(2+) has a key role in cell signaling pathways in various differentiation stages of stem cells. The aim of this chapter is to present a broad overview of various moments in which Ca(2+)-mediated signaling is essential for the maintenance of stem cells and for promoting their development and differentiation, also focusing on their therapeutic potential.

Insulin induces calcium signals in the nucleus of rat hepatocytes.

Insulin is an hepatic mitogen that promotes liver regeneration. Actions of insulin are mediated by the insulin receptor, which is a receptor tyrosine kinase. It is currently thought that signaling via the insulin receptor occurs at the plasma membrane, where it binds to insulin. Here we report that insulin induces calcium oscillations in isolated rat hepatocytes, and that these calcium signals depend upon activation of phospholipase C and the inositol 1,4,5‐trisphosphate receptor, but not upon extracellular calcium. Furthermore, insulin‐induced calcium signals occur in the nucleus, and are temporally associated with selective depletion of nuclear phosphatidylinositol bisphosphate and translocation of the insulin receptor to the nucleus. These findings suggest that the insulin receptor translocates to the nucleus to initiate nuclear, inositol 1,4,5‐trisphosphate‐mediated calcium signals in rat hepatocytes. This novel signaling mechanism may be responsible for insulins effects on liver growth and regeneration. (HEPATOLOGY 2008.)

Endothelial differentiation of human stem cells seeded onto electrospun polyhydroxybutyrate/polyhydroxybutyrate-co-hydroxyvalerate fiber mesh.

Tissue engineering is based on the association of cultured cells with structural matrices and the incorporation of signaling molecules for inducing tissue regeneration. Despite its enormous potential, tissue engineering faces a major challenge concerning the maintenance of cell viability after the implantation of the constructs. The lack of a functional vasculature within the implant compromises the delivery of nutrients to and removal of metabolites from the cells, which can lead to implant failure. In this sense, our investigation aims to develop a new strategy for enhancing vascularization in tissue engineering constructs. This studys aim was to establish a culture of human adipose tissue-derived stem cells (hASCs) to evaluate the biocompatibility of electrospun fiber mesh made of polyhydroxybutyrate (PHB) and its copolymer poly-3-hydroxybutyrate-co-3-hydroxyvalerate (PHB-HV) and to promote the differentiation of hASCs into the endothelial lineage. Fiber mesh was produced by blending 30% PHB with 70% PHB-HV and its physical characterization was conducted using scanning electron microscopy analysis (SEM). Using electrospinning, fiber mesh was obtained with diameters ranging 300 nm to 1.3 µm. To assess the biological performance, hASCs were extracted, cultured, characterized by flow cytometry, expanded and seeded onto electrospun PHB/PHB-HV fiber mesh. Various aspects of the cells were analyzed in vitro using SEM, MTT assay and Calcein-AM staining. The in vitro evaluation demonstrated good adhesion and a normal morphology of the hASCs. After 7, 14 and 21 days of seeding hASCs onto electrospun PHB/PHB-HV fiber mesh, the cells remained viable and proliferative. Moreover, when cultured with endothelial differentiation medium (i.e., medium containing VEGF and bFGF), the hASCs expressed endothelial markers such as VE-Cadherin and the vWF factor. Therefore, the electrospun PHB/PHB-HV fiber mesh appears to be a suitable material that can be used in combination with endothelial-differentiated cells to improve vascularization in engineered bone tissues.