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.

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.

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 μg/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 α-smooth muscle actin and visualized by confocal microscopy, and TGF-β 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 (Ca²⁺) signaling in LX-2 cells treated with or without MSU crystals using caged inositol 1,4,5-triphosphate (IP₃). Finally, we confirmed an important role of the inflammasome in experimental liver fibrosis by the injection of carbon tetrachloride (CCl₄) 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-β and collagen1 mRNA and actin reorganization in HSCs from wild-type mice but not mice lacking inflammasome components. MSU crystals inhibited the release of Ca²⁺ via IP₃ 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 CCl₄ 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.

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.

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.