M. Fatima Leite

Regulation of calcium signals in the nucleus by a nucleoplasmic reticulum

Calcium is a second messenger in virtually all cells and tissues. Calcium signals in the nucleus have effects on gene transcription and cell growth that are distinct from those of cytosolic calcium signals; however, it is unknown how nuclear calcium signals are regulated. Here we identify a reticular network of nuclear calcium stores that is continuous with the endoplasmic reticulum and the nuclear envelope. This network expresses inositol 1,4,5-trisphosphate (InsP3) receptors, and the nuclear component of InsP3-mediated calcium signals begins in its locality. Stimulation of these receptors with a little InsP3 results in small calcium signals that are initiated in this region of the nucleus. Localized release of calcium in the nucleus causes nuclear protein kinase C (PKC) to translocate to the region of the nuclear envelope, whereas release of calcium in the cytosol induces translocation of cytosolic PKC to the plasma membrane. Our findings show that the nucleus contains a nucleoplasmic reticulum with the capacity to regulate calcium signals in localized subnuclear regions. The presence of such machinery provides a potential mechanism by which calcium can simultaneously regulate many independent processes in the nucleus.

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.

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.

Hepatic DNA deposition drives drug‐induced liver injury and inflammation in mice

Drug‐induced liver injury (DILI) is an important cause of acute liver failure, with limited therapeutic options. During DILI, oncotic necrosis with concomitant release and recognition of intracellular content amplifies liver inflammation and injury. Among these molecules, self‐DNA has been widely shown to trigger inflammatory and autoimmune diseases; however, whether DNA released from damaged hepatocytes accumulates into necrotic liver and the impact of its recognition by the immune system remains elusive. Here we show that treatment with two different hepatotoxic compounds (acetaminophen and thioacetamide) caused DNA release into the hepatocyte cytoplasm, which occurred in parallel with cell death in vitro. Administration of these compounds in vivo caused massive DNA deposition within liver necrotic areas, together with an intravascular DNA coating. Using confocal intravital microscopy, we revealed that liver injury due to acetaminophen overdose led to a directional migration of neutrophils to DNA‐rich areas, where they exhibit an active patrolling behavior. DNA removal by intravenous DNASE1 injection or ablation of Toll‐like receptor 9 (TLR9)‐mediated sensing significantly reduced systemic inflammation, liver neutrophil recruitment, and hepatotoxicity. Analysis of liver leukocytes by flow cytometry revealed that emigrated neutrophils up‐regulated TLR9 expression during acetaminophen‐mediated necrosis, and these cells sensed and reacted to extracellular DNA by activating the TLR9/NF‐κB pathway. Likewise, adoptive transfer of wild‐type neutrophils to TLR9−/− mice reversed the hepatoprotective phenotype otherwise observed in TLR9 absence. Conclusion: Hepatic DNA accumulation is a novel feature of DILI pathogenesis. Blockage of DNA recognition by the innate immune system may constitute a promising therapeutic venue. (Hepatology 2015;61:348–360)

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.)

Effects of extracellular calcium concentration on the glutamate release by bioactive glass (BG60S) preincubated osteoblasts

Glutamate released by osteoblasts sharing similarities with its role in neuronal transmission is a very new scientific concept which actually changed the understanding of bone physiology. Since glutamate release is a calcium (Ca(2+))-dependent process and considering that we have previously demonstrated that the dissolution of bioactive glass with 60% of silicon (BG60S) can alter osteoblast Ca(2+)-signaling machinery, we investigated whether BG60S induces glutamate secretion in osteoblasts and whether it requires an increase in intracellular Ca(2+). Here we showed that the extracellular Ca(2+) increase due to BG60S dissolution leads to an intracellular Ca(2+) increase in the osteoblast, through the activation of an inositol 1,4,5-triphosphate receptor (InsP(3)R) and a ryanodine receptor (RyR). Additionally, we also demonstrated that glutamate released by osteoblasts can be profoundly altered by BG60S. The modulation of osteoblast glutamate released by the extracellular Ca(2+) concentration opens a new window in the field of tissue engineering, since many biomaterials used for bone repair are able to increase the extracellular Ca(2+) concentration due to their dissolution products.

The Spatial Distribution of Inositol 1,4,5-Trisphosphate Receptor Isoforms Shapes Ca2+ Waves

Cytosolic Ca2+ is a versatile second messenger that can regulate multiple cellular processes simultaneously. This is accomplished in part through Ca2+ waves and other spatial patterns of Ca2+ signals. To investigate the mechanism responsible for the formation of Ca2+ waves, we examined the role of inositol 1,4,5-trisphosphate receptor (InsP3R) isoforms in Ca2+ wave formation. Ca2+ signals were examined in hepatocytes, which express the type I and II InsP3R in a polarized fashion, and in AR4-2J cells, a nonpolarized cell line that expresses type I and II InsP3R in a ratio similar to what is found in hepatocytes but homogeneously throughout the cell. Expression of type I or II InsP3R was selectively suppressed by isoform-specific DNA antisense in an adenoviral delivery system, which was delivered to AR4-2J cells in culture and to hepatocytes in vivo. Loss of either isoform inhibited Ca2+ signals to a similar extent in AR4-2J cells. In contrast, loss of the basolateral type I InsP3R decreased the sensitivity of hepatocytes to vasopressin but had little effect on the initiation or spread of Ca2+ waves across hepatocytes. Loss of the apical type II isoform caused an even greater decrease in the sensitivity of hepatocytes to vasopressin and resulted in Ca2+ waves that were much slower and delayed in onset. These findings provide evidence that the apical concentration of type II InsP3Rs is essential for the formation of Ca2+ waves in hepatocytes. The subcellular distribution of InsP3R isoforms may critically determine the repertoire of spatial patterns of Ca2+ signals.

Nucleoplasmic calcium signaling and cell proliferation: calcium signaling in the nucleus.

Calcium (Ca2+) is an essential signal transduction element involved in the regulation of several cellular activities and it is required at various key stages of the cell cycle. Intracellular Ca2+ is crucial for the orderly cell cycle progression and plays a vital role in the regulation of cell proliferation. Recently, it was demonstrated by in vitro and in vivo studies that nucleoplasmic Ca2+ regulates cell growth. Even though the mechanism by which nuclear Ca2+ regulates cell proliferation is not completely understood, there are reports demonstrating that activation of tyrosine kinase receptors (RTKs) leads to translocation of RTKs to the nucleus to generate localized nuclear Ca2+ signaling which are believed to modulate cell proliferation. Moreover, nuclear Ca2+ regulates the expression of genes involved in cell growth. This review will describe the nuclear Ca2+ signaling machinery and its role in cell proliferation. Additionally, the potential role of nuclear Ca2+ as a target in cancer therapy will be discussed.

The insulin receptor translocates to the nucleus to regulate cell proliferation in liver

Insulins metabolic effects in the liver are widely appreciated, but insulins ability to act as a hepatic mitogen is less well understood. Because the insulin receptor (IR) can traffic to the nucleus, and Ca2+ signals within the nucleus regulate cell proliferation, we investigated whether insulins mitogenic effects result from activation of Ca2+‐signaling pathways by IRs within the nucleus. Insulin‐induced increases in Ca2+ and cell proliferation depended upon clathrin‐ and caveolin‐dependent translocation of the IR to the nucleus, as well as upon formation of inositol 1,4,5,‐trisphosphate (InsP3) in the nucleus, whereas insulins metabolic effects did not depend on either of these events. Moreover, liver regeneration after partial hepatectomy also depended upon the formation of InsP3 in the nucleus, but not the cytosol, whereas hepatic glucose metabolism was not affected by buffering InsP3 in the nucleus. Conclusion: These findings provide evidence that insulins mitogenic effects are mediated by a subpopulation of IRs that traffic to the nucleus to locally activate InsP3‐dependent Ca2+‐signaling pathways. The steps along this signaling pathway reveal a number of potential targets for therapeutic modulation of liver growth in health and disease. (Hepatology 2014;58:274–283)