Evidence For Involvement Of Type Research Articles

Metabolic adaptation to the chronic loss of Ca2+ signaling induced by KO of IP3 receptors or the mitochondrial Ca2+ uniporter

Calcium signaling is essential for regulating many biological processes. Endoplasmic reticulum inositol trisphosphate receptors (IP3Rs) and the mitochondrial Ca2+ uniporter (MCU) are key proteins that regulate intracellular Ca2+ concentration. Mitochondrial Ca2+ accumulation activates Ca2+-sensitive dehydrogenases of the tricarboxylic acid (TCA) cycle that maintain the biosynthetic and bioenergetic needs of both normal and cancer cells. However, the interplay between calcium signaling and metabolism is not well understood. In this study, we used human cancer cell lines (HEK293 and HeLa) with stable KOs of all three IP3R isoforms (triple KO [TKO]) or MCU to examine metabolic and bioenergetic responses to the chronic loss of cytosolic and/or mitochondrial Ca2+ signaling. Our results show that TKO cells (exhibiting total loss of Ca2+ signaling) are viable, displaying a lower proliferation and oxygen consumption rate, with no significant changes in ATP levels, even when made to rely solely on the TCA cycle for energy production. MCU KO cells also maintained normal ATP levels but showed increased proliferation, oxygen consumption, and metabolism of both glucose and glutamine. However, MCU KO cells were unable to maintain ATP levels and died when relying solely on the TCA cycle for energy. We conclude that constitutive Ca2+ signaling is dispensable for the bioenergetic needs of both IP3R TKO and MCU KO human cancer cells, likely because of adequate basal glycolytic and TCA cycle flux. However, in MCU KO cells, the higher energy expenditure associated with increased proliferation and oxygen consumption makes these cells more prone to bioenergetic failure under conditions of metabolic stress.

Functional Inositol 1,4,5-Trisphosphate Receptors Assembled from Concatenated Homo- and Heteromeric Subunits

Vertebrate genomes code for three subtypes of inositol 1,4,5-trisphosphate (IP3) receptors (IP3R1, -2, and -3). Individual IP3R monomers are assembled to form homo- and heterotetrameric channels that mediate Ca(2+) release from intracellular stores. IP3R subtypes are regulated differentially by IP3, Ca(2+), ATP, and various other cellular factors and events. IP3R subtypes are seldom expressed in isolation in individual cell types, and cells often express different complements of IP3R subtypes. When multiple subtypes of IP3R are co-expressed, the subunit composition of channels cannot be specifically defined. Thus, how the subunit composition of heterotetrameric IP3R channels contributes to shaping the spatio-temporal properties of IP3-mediated Ca(2+) signals has been difficult to evaluate. To address this question, we created concatenated IP3R linked by short flexible linkers. Dimeric constructs were expressed in DT40-3KO cells, an IP3R null cell line. The dimeric proteins were localized to membranes, ran as intact dimeric proteins on SDS-PAGE, and migrated as an ∼1100-kDa band on blue native gels exactly as wild type IP3R. Importantly, IP3R channels formed from concatenated dimers were fully functional as indicated by agonist-induced Ca(2+) release. Using single channel "on-nucleus" patch clamp, the channels assembled from homodimers were essentially indistinguishable from those formed by the wild type receptor. However, the activity of channels formed from concatenated IP3R1 and IP3R2 heterodimers was dominated by IP3R2 in terms of the characteristics of regulation by ATP. These studies provide the first insight into the regulation of heterotetrameric IP3R of defined composition. Importantly, the results indicate that the properties of these channels are not simply a blend of those of the constituent IP3R monomers.

Genetic evidence for involvement of type 1, type 2 and type 3 inositol 1,4,5-trisphosphate receptors in signal transduction through the B-cell antigen receptor.

Stimulation of B-cell antigen receptor (BCR) induces a rapid increase in cytoplasmic free calcium due to its release from intracellular stores and influx from the extracellular environment. Inositol 1,4,5-trisphosphate receptors (IP3Rs) are ligand-gated channels that release intracellular calcium stores in response to the second messenger, inositol 1,4,5-trisphosphate. Most hematopoietic cells, including B cells, express at least two of the three different types of IP3R. We demonstrate here that B cells in which a single type of IP3R has been deleted still mobilize calcium in response to BCR stimulation, whereas this calcium mobilization is abrogated in B cells lacking all three types of IP3R. Calcium mobilization by a transfected G protein-coupled receptor (muscarinic M1 receptor) was also abolished in only triple-deficient cells. Capacitative Ca2+ entry, stimulated by thapsigargin, remains unaffected by loss of all three types of IP3R. These data establish that IP3Rs are essential and functionally redundant mediators for both BCR- and muscarinic receptor-induced calcium mobilization, but not for thapsigargin-induced Ca2+ influx. We further show that the BCR-induced apoptosis is significantly inhibited by loss of all three types of IP3R, suggesting an important role for Ca2+ in the process of apoptosis.

No evidence for involvement of type 1 collagen structural genes in ‘genetic predisposition’ to alcoholic cirrhosis

Type 1 collagen is the predominant collagen in cirrhotic livers. Each type 1 collagen molecule contains three subunits, two are identical (the α1 chains) and the sequence of the third (α2) is very similar. They are encoded at the non-synthenic loci, COL1A1 and COL1A2 and restriction site dimorphisms have been described at each locus. Genetic factors have been invoked as a basis for increased susceptibility to alcoholic cirrhosis. One hypothesis is that genetically determined differences in type 1 collagen may be involved in this predisposition. We have examined this by analysing restriction fragment length polymorphisms at each type 1 collagen locus in leucocyte DNA from 56 unrelated patients with alcoholic cirrhosis and 74 local unrelated healthy controls. Based on the presence or absence of these restriction site dimorphisms four possible haplotypes were generated at COL1A1 and COL1A2. We found no significant difference in allele frequencies between alcoholic cirrhotics and controls and, unlike a previous small study, we found no particular haplotype of either gene was associated with alcoholic cirrhosis. Our study provides no evidence for involvement of type 1 collagen structural genes in a genetic predisposition to cirrhosis in alcoholics.

No evidence for involvement of type I collagen structural genes in “Genetic predisposition” to alcoholic cirrhosis

No evidence for involvement of type I collagen structural genes in “Genetic predisposition” to alcoholic cirrhosis

The evidence for involvement of type C RNA tumor viruses in human acute leukemia.

RNA- and reverse transcriptase (R.T.)-containing viruses have been etiologically related to leukemia in several species of animals. There is no convincing report of isolation of a similar virus from man. In the past few years, however, biochemical evidence for three components thought to be characteristic of type C viruses has been detected in human acute leukemic cells. This includes the following: 1) An enzyme with physical and biochemical properties so far indistinguishable from viral R.T. has been purified from these cells. It was also recently shown that this enzyme is immunologically closely related to R.T. purified from type C primate RNA tumor viruses, much less to murine virus R.T., and not at all to avian or feline. 2) RNA has been detected in these cells with some nucleotide sequence relatedness to the genomic RNA of some murine and primate type-C viruses. 3) The DNA product of the endogenous R.T. of human leukemic cells has nucleotide sequences related to murine and especially primate type C sarcoma viruses. Thus, certain virus-like components exist in human leukemic cells in a functional form within ribonucleoprotein particles which resemble animal type C viruses. However, if they represent virus, they are defective—not released under ordinary conditions—and a causal relationship to human neoplasia, if any, has not been shown and may be impossible to prove.