The nuclear factor-kappa B pathway regulates cytochrome P450 3A4 protein stability.

We have previously observed that CYP3A4 protein levels are suppressed by inhibition of the proteasome in primary cultured hepatocytes. Because this result is opposite of what would be expected if CYP3A4 were degraded by the proteasome, it seemed likely that there might be another protein susceptible to proteasomal degradation that regulated CYP3A4 expression. In this study, we evaluated whether the nuclear factor-kappaB (NF-kappaB) pathway was involved in that process. Our model system used an adenovirus system to express CYP3A4 protein in HepG2 cells, which are derived from human cancer cells. Similar to results in primary hepatocytes, the inhibition of the proteasome with N-benzoyloxycarbonyl (Z)-Leu-Leu-leucinal (MG132) suppresses CYP3A4 protein levels. We also found that MG132 treatment had a broad affect on the NF-kappaB pathway, including down-regulation of NF-kappaB DNA binding activity and IkappaB kinase (IKK)alpha levels and up-regulation of IKKbeta and inhibitory kappaB levels. Treatment of the HepG2 cells with several structurally distinct NF-kappaB inhibitors also suppressed CYP3A4 protein levels. When the HepG2 cells were treated with cycloheximide, a general inhibitor of protein synthesis, the loss of CYP3A4 protein was accelerated by cotreatment with either proteasome or NF-kappaB inhibitors. These results indicate that NF-kappaB activity regulated CYP3A4 protein stability, and they suggest that the NF-kappaB pathway was responsible for the decrease in CYP3A4 protein levels that resulted from the proteasomal inhibition.

Expression of avian Ca2+-ATPase in cultured mouse myogenic cells.

cDNA encoding Ca2+-ATPase was cloned from a chicken skeletal muscle library. The cDNA (termed FCa) comprised 3,239 base pairs, including an open reading frame encoding 994 amino acids which showed the highest degree of homology with the adult rabbit fast-twitch Ca2+-ATPase isoform (C. J. Brandl, S. de Leon, D. R. Martin, and D. H. MacLennan, J. Biol. Chem. 262:3768-3774, 1987). Radiolabeled FCa hybridized to a 3.2-kilobase transcript in chicken skeletal muscle RNA but not to cardiac muscle RNA, which confirmed its identity as encoding the fast Ca2+-ATPase isoenzyme. FCa was transfected into the mouse myogenic line C2C12, from which a protein of 100 kilodaltons was immunopurified by using a monoclonal antibody specific for the avian fast Ca2+-ATPase. Immunofluorescence microscopy of a line (designated C2FCa2) stably expressing the avian Ca2+-ATPase localized the protein to the nuclear envelope and a population of cytoplasmic vesicles. A similar pattern was observed when C2FCa2 cells were stained with DiOC6(3), a cyanine dye that labels endoplasmic reticulum and mitochondria (M. Terasaki, J. Song, J. R. Wong, M. J. Weiss, and L. B. Chen, Cell 38:101-108, 1984). We conclude that the avian Ca2+-ATPase fast isoform is expressed and correctly targeted to the endoplasmic reticulum in mouse C2C12 cells.

Inhibitors of ER Ca(2+)-ATPase activity deplete the ATP- and thrombin-sensitive Ca2+ pool in UMR 106-01 osteosarcoma cells.

While calcium release from intracellular stores is a signaling mechanism used universally by cells responding to hormones and growth factors, the compartmentalization and regulated release of calcium is cell type-specific. We employed thapsigargin and 2,5,-di-(tert-butyl)-1,4-benzohydroquinone (tBuHQ), two inhibitors of endoplasmic reticulum (ER) Ca(2+)-ATPase activity which block the transport of Ca2+ into intracellular stores, to characterize free Ca2+ compartmentalization in UMR 106-01 osteoblastic osteosarcoma cells. Each drug elicited transient increases in cytosolic free Ca2+ ([Ca2+]i), followed by a stable plateau phase which was elevated above the control [Ca2+]i. The release of Ca2+ from intracellular stores was coupled to an increased plasma membrane Ca2+ permeability which was not due to L-type Ca2+ channels. Thapsigargin and tBuHQ emptied the intracellular calcium pool which was released in response to either ATP or thrombin, identifying it as the inositol 1,4,5-trisphosphate-sensitive calcium store. The results of sequential and simultaneous additions of thapsigargin and tBuHQ indicate that both drugs depleted the same Ca2+ store and inhibited the same Ca(2+)-ATPase activity.

Osteoblasts express the PMCA1b isoform of the plasma membrane Ca(2+)-ATPase.

We report here that osteoblasts and osteoblast-like osteosarcoma cells express PMCA1b, an alternatively spliced transcript of plasma membrane Ca(2+)-ATPase. Synthetic oligonucleotide pairs were designed based upon unique regions of the cDNA encoding known PMCA isoforms (PMCA1-3) and used as primers in PCR-mediated amplification of cDNA synthesized from ROS 17/2.8 osteosarcoma cell RNA. A product was observed only when PMCA1-specific primers were present; no products were seen with PMCA2 or PMCA3 primers unless cDNA synthesized from rat brain RNA was present. Examination of the cDNA encoding the C terminus of PMCA1 from ROS 17/2.8 cells revealed that the mRNA is spliced to yield the PMCA1b isoform, a Ca(2+)-ATPase containing a consensus phosphorylation site for cAMP-dependent protein kinase A and a modified calmodulin binding domain. PMCA1b was also detected in UMR-106-01 osteosarcoma cells and unpassaged primary rat calvarial osteoblasts. These results suggest that the regulation of osteoblast function by agents that act via cAMP-mediated pathways may involve alterations in the activity of the plasma membrane Ca(2+)-ATPase.

A role for G protein-coupled lysophospholipid receptors in sphingolipid-induced Ca2+ signaling in MC3T3-E1 osteoblastic cells.

Sphingolipids have been proposed to modulate cell function by acting as intracellular second messengers and through binding to plasma membrane receptors. Exposure of MC3T3-E1 osteoblastic cells to sphingosine (SPH), sphingosine-1-phosphate (SPP), or sphingosylphosphorylcholine (SPC) led to the release of Ca2+ from the endoplasmic reticulum (ER) and acute elevations in cytosolic-free Ca2+ ([Ca2+]i). Desensitization studies suggest that SPP and SPC bind plasma membrane endothelial differentiation gene (Edg) receptors for lysophosphatidic acid (LPA). Consistent with the coupling of Edg receptors to G proteins, SPP- and SPC-induced Ca2+ signaling was inhibited by pretreatment of the cells with pertussis toxin (PTx). Of the Edg receptors known to bind SPH derivatives in other cell types, MC3T3-E1 cells were found to express transcripts encoding Edg-1 and Edg-5 but not Edg-3, Edg-6, or Edg-8. In contrast to SPP and SPC, the ability of SPH to elicit [Ca2+]i elevations was affected neither by prior exposure of cells to LPA nor by PTx treatment. However, LPA-induced Ca2+ signaling was blocked in MC3T3-E1 cells previously exposed to SPH. Elevations in [Ca2+]i were not evoked by SPP or SPC in cells treated with 2-aminoethoxydiphenylborate (2-APB), an inhibitor of inositol 1,4,5-trisphosphate (IP3)-gated Ca2+ channels in the ER. No effect of 2-APB was observed on SPH-or LPA-induced [Ca2+]i elevations. The data support a model in which SPP and SPC bind Edg-1 and/or Edg-5 receptors in osteoblasts leading to the release of Ca2+ from the ER through IP3-gated channels.

Phylogenetic examination of vertebrate central nervous system myelin proteins by electro-immunoblotting.

Central nervous system (CNS) myelin proteins from vertebrate classes were examined by immunoblotting with antisera against mammalian CNS myelin proteins. Higher vertebrates possessed proteolipid (PLP), DM-20 and Wolfgram (WP) proteins, except that DM-20 was missing in amphibia. Fish CNS myelins contained neither PLP nor WP; instead they bound antisera to mammalian peripheral nervous system P0 protein. All classes carried myelin basic protein, but only mammals exhibited a component equivalent to rat 21.5K (21,500 dalton). These phylogenetic data are consistent with major changes in CNS myelin protein composition at the transition from fishes to higher vertebrates.

Mutation of highly conserved arginine residues disrupts the structure and function of annexin V.

BACKGROUND: Annexins are a family of structurally related proteins that bind to phospholipid membranes in a Ca(2+)-dependent manner. Annexins are characterized by highly conserved canonical domains of approximately 70 amino acids. Annexin V contains four such domains. Each of these domains has a highly conserved arginine (R). METHODS: To evaluate the role of the conserved arginines in the molecular structure of annexin V, negatively charged amino acids were substituted for arginines at positions R43, R115, R199, and R274 using site-directed mutagenesis. RESULTS: Mutants R199D and R274E were rapidly degraded when expressed in bacteria, and were not further characterized. R43E exhibited an electrophoretic mobility similar to the wild-type protein, while R115E migrated significantly in a slower fashion, suggesting a less compact conformation. R43E and R115E exhibited much greater susceptibility to proteolytic digestion than the wild type. While Ca(2+)-dependence for phospholipid binding was similar in both mutants (half-maximal 50-80 microM Ca2+), R43E and R115E exhibited a 6- and 2-fold decrease in phospholipid affinity, respectively. Consistent with the different phospholipid affinities of the annexins, a phospholipid-dependent clotting reaction, the activated partial thromboplastin time (aPTT), was significantly prolonged by the wild-type protein and mutants R115E and R115A. The aPTT was unaffected by R43E. CONCLUSIONS: Our data suggest that mutation of these highly conserved arginine residues in each of the four canonical domains of annexin have differential effects on the phospholipid binding, tertiary structure, and proteolytic susceptibility of annexin V. The site I mutation, R43E, produced a large decrease in phospholipid affinity associated with an increase in proteolytic susceptibility. The site II mutation, R115E, produced a small change in phospholipid binding but a significant modification of electrophoretic mobility. Our data suggest that highly conserved arginine residues are required to stabilize the tertiary structure of annexin V by establishing hydrogen bonds and ionic bridges.

Turnover of the catalytic subunit of Na,K-ATPase in HTC cells.

A polyclonal antibody to the catalytic subunit of rat kidney Na,K-ATPase has been raised in rabbits and used to analyze the turnover of the subunit in the rat hepatoma cell line HTC. It had been shown previously (Baumann, H., and Doyle, D. (1978) J. Biol. Chem. 253, 4408-4418) that the membrane proteins of these cells displayed multicomponent turnover kinetics, the minority of the surface proteins turning over with a half-time of about 20 h and the remainder with a half-time of about 100 h. That the antibody precipitated both the alpha (catalytic) and beta (glycosylated) subunits of the Na,K-ATPase from Triton extracts of HTC cells could be demonstrated following metabolic labeling of the cells with either [3H]leucine or a mixture of [3H] mannose and [3H]fucose, but following labeling with [35S]methionine radioactivity was found only in the alpha subunit of the precipitates. Incorporation of [35S]methionine into the alpha subunit could be detected 2 min after addition of the isotope to the cell suspension. Then and at all times thereafter the label was recoverable only from the particulate fraction of a 150,000 X g 60-min centrifugation; no labeled alpha subunit was ever detected in the supernatant fraction. By quantitative densitometry of radioautographs of sodium dodecyl sulfate-polyacrylamide gels of labeled antibody precipitates, it could be shown in pulse-chase experiments that the specific activity of the alpha subunit remained unchanged for 3-4 h (transit time) after the pulse was initiated and that the activity subsequently decayed exponentially with a half-time of 18 h. In a population growing with a generation time (tG) of 33 h, this decay corresponds to a turnover rate constant of 0.49/tG. The catalytic subunit is among those membrane proteins with a rapid turnover rate.

The sarcoplasmic reticulum Ca(2+)-ATPase, SERCA1a, contains endoplasmic reticulum targeting information.

The fast-twitch skeletal muscle Ca(2+)-ATPase isoenzyme, SERCA1a, is localized in chick skeletal myotubes to both the sarcoplasmic reticulum (SR) and to the nuclear envelope, an extension of the endoplasmic reticulum (ER). The ER labeling remained after cycloheximide treatment, indicating that it did not represent newly synthesized SERCA1a in transit to the SR. Expression of the cDNA encoding SERCA1a in cultured non-muscle cells led to the localization of the enzyme in the ER, as indicated by organelle morphology and the co-localization of SERCA1a with the endogenous ER luminal protein, BiP. Immunopurification analysis showed that SERCA1a was not bound to BiP, nor was any degradation apparent. Thus, the SR Ca(2+)-ATPase appears to contain ER targeting information.

Biosynthesis and insertion of Wolfgram protein into optic nerve membranes.

Antibodies against pig brain Wolfgram protein (WP) were prepared and utilized in the analysis of WP biosynthesis in membranes from optic nerves of 20 day-old rats. Newly synthesized WP appeared rapidly (less than 5 min) in myelin and in a non-myelin microsome fraction and accumulated in both thereafter. Monensin did not affect the insertion of WP in either membrane fraction. These results are consistent with biosynthesis of WP on free ribosomes.

Effects of sphingosine derivatives on MC3T3-E1 pre-osteoblasts: psychosine elicits release of calcium from intracellualr stores.

Sphingosine, sphingosine-1-phosphate (SPP) and sphingosylphosphorylcholine (SPC) each elicited transient elevations in intracellular free Ca2+ ([Ca2+]i) as a result of Ca2+ release from an intracellular store in MC3T3-E1 pre-osteoblasts. No elevation in [Ca2+]i was detected in response to the application of N-acetyl sphingosine. Psychosine (1-galactosyl sphingosine) also caused Ca2+ release from intracellular stores, which suggests that phosphorylation of the 1-carbon of sphingosine is not required for its action as a Ca2+ release agonist in MC3T3-E1 cells. Sequential additions of combinations of sphingosine, SPP, SPC and psychosine revealed Ca2+ transients only after the application of the first sphingolipid, despite the fact that the intracellular Ca2+ stores refilled. This indicates that each agent desensitized the MC3T3-E1 cells to the action of the others and suggests that all of the sphingolipids tested employ the same receptor or a common intracellular messenger.

Na,K-ATPase expression in C2C12 cells during myogenesis: minimal contribution of alpha 2 isoform to Na,K transport.

Cells of the murine skeletal muscle line, C2C12, undergo differentiation from mononuclear myoblasts to multinuclear myotubes that express a number of proteins associated with striated muscle. We examined the relationship between the abundance of the mRNAs encoding the fast-twitch Ca-ATPase and the alpha isoforms of Na,K-ATPase and the subsequent expression of their respective polypeptides. Both the mRNA and protein levels of the alpha 1 isoform remained constant throughout differentiation. In contrast, the content of mRNAs encoding the alpha 2 isoform and fast-twitch Ca-ATPase increased coordinately with the abundance of their corresponding polypeptides during myotube development. Despite the dramatic increase in alpha 2 expression, estimates of in vitro Na,K-ATPase activity and assessments of in vivo transport activity suggest that alpha 2 contributes little to ionic homeostasis in C2C12 myotubes.

Down-regulation of L-type Ca2+ channel transcript levels by 1,25-dihyroxyvitamin D3. Osteoblastic cells express L-type alpha1C Ca2+ channel isoforms.

Osteoblast Ca2+ channels play a fundamental role in controlling intracellular and systemic Ca2+ homeostasis. A reverse transcription-polymerase chain reaction strategy was used to determine the molecular identity of voltage-sensitive calcium channels present in ROS 17/2.8 osteosarcoma cells. The amino acid sequences encoded by the two resultant PCR products matched the alpha1C-a and the alpha1C-d isoforms. The ability of 1, 25-dihydroxyvitamin D3 (1,25(OH)2D3) and structural analogs to modulate expression of voltage-sensitive calcium channel mRNA transcripts was then investigated. ROS 17/2.8 cells were cultured for 48 h in the presence of either 1,25(OH)2D3,1,24-dihydroxy-22-ene-24-cyclopropyl D3 (analog BT) or 25-hydroxy-16-ene-23-yne-D3 (analog AT), and the levels of mRNA encoding alpha1C were quantitated using a competitive reverse transcription-polymerase chain reaction assay. We found that 1, 25(OH)2D3 and analog BT reduced steady state levels of alpha1C mRNA. Conversely, the Ca2+-mobilizing analog AT did not alter steady state levels of voltage-sensitive calcium channel mRNA. Since analog BT, but not analog AT, binds and transcriptionally activates the nuclear receptor for 1,25(OH)2D3, these findings suggest that the down-regulation of voltage-sensitive calcium channel mRNA levels may involve the nuclear receptor.

1,25 dihydroxyvitamin D(3) activates sphingomyelin turnover in ROS17/2.8 osteosarcoma cells without sphingolipid-induced changes in cytosolic Ca(2+).

1,25-Dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] initiates the hydrolysis of sphingomyelin in ROS 17/2.8 osteosarcoma cells with the resultant generation of cell-associated ceramide. Increases in ceramide levels were detectable at 15 min and maximal one hour after exposure of cells to 1,25(OH)(2)D(3). Neither 1,25(OH)(2)D(3) nor exogenous ceramide elicited a change in cytosolic free Ca(2+) ([Ca(2+)](i)). Transient elevations in [Ca(2+)](i) were observed when cells were exposed to exogenous sphingosine, but there was no detectable conversion of ceramide to sphingosine in 1, 25(OH)(2)D(3)-treated cells. Ceramide also did not stimulate Ca(2+) uptake across ROS 17/2.8 cell plasma membranes. Collectively, these results suggest that 1,25(OH)(2)D(3) activates sphingomyelin turnover in ROS 17/2.8 osteosarcoma cells but that the sphingolipid metabolite ceramide is not responsible for 1,25(OH)(2)D(3)-induced activation of plasma membrane Ca(2+) channels.

Expression of dihydropyridine binding sites in renal epithelial cells.

It has recently been shown that rabbit kidney epithelial cells (proximal tubule) possess two dihydropyridine (DHP)-sensitive calcium entry channels (1, 2). To evaluate the properties of the DHP binding sites, the binding of the DHP, [3H]PN200-110, was studied in epithelial cell membrane fractions (proximal tubule) of rabbit kidney cortex. High-affinity binding sites for the DHP were observed in both basolateral and apical membranes and in a membrane microsomal preparation from rabbit primary cultures of proximal tubule cells (cultured PT). In an extended analysis of the basolateral membrane preparation, two high-affinity binding sites were evident with binding dissociation constants, Kd, of 0.005 and 0.75 nM. The Kd values are similar to that observed for L-type calcium channel alpha1-subunits. Using a homology-based cloning strategy, a 388-base fragment of an alpha1-subunit was cloned from RNA isolated from rabbit cultured PT cells and freshly isolated proximal tubules and found to encode a protein identical to the cardiac form of the L-type alpha1-subunit (alpha1C-subunit). It is concluded that renal epithelial cells express high-affinity dihydropyridine receptors and that the receptors may be components of plasma membrane calcium channels, including L-type calcium channels, that control calcium entry in these cells.

1,25 (OH)2D3 enhances PTH-induced Ca2+ transients in preosteoblasts by activating L-type Ca2+ channels.

We previously demonstrated electrophysiologically that 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] shifts the activation threshold of L-type Ca2+ channels in osteoblasts toward the resting potential and prolongs mean open time. Presently, we used single-cell Ca2+ imaging to study the combined effects of 1,25(OH)2D3 and parathyroid hormone (PTH) during generation of Ca2+ transients in fura 2-loaded MC3T3-E1 cells. Pretreatment with 1,25(OH)2D3 concentrations, which alone did not produce Ca2+ transients, consistently enhanced Ca2+ responses to PTH. Enhancement was dose dependent over the range of 1 to 10 nM and was blocked by pretreatment with 5 microM nitrendipine during pretreatment. A 1,25(OH)2D3 analog that activates L-type channels and shifts their activation threshold also enhanced PTH responses. In contrast, an analog devoid of membrane Ca2+ effects did not enhance PTH-induced Ca2+ transients. The PTH-induced Ca2+ transient involved activation of a dihydropyridine-insensitive cation channel that was inhibited by Gd3+. Together, these data suggest that 1,25(OH)2D3 increases osteoblast responsiveness to PTH through rapid modification of L-type Ca2+ channel gating properties, whose activation enhances Ca2+ entry through other channels such as the PTH-responsive, Gd(3+)-sensitive cation channel.

Observations on the origins of plasma membrane in rat deciduoma.

An NADH-ferricyanide reductase activity resistant to inactivation by cytochemical procedures was examined during decidualization of rat endometrium. Resistant activity was restricted to plasma membranes, distal elements of the Golgi apparatus, and discoid cisternae and cytoplasmic vesicles of decidual cells of endometrium of the pseudopregnant rat on days 3, 4, 5, 7, and 9, after mating. The procedure reduced or eliminated any evidence of NADH-ferricyanide reductase activity from other cellular components such as endoplasmic reticulum, nuclei, and mitochondria. The observations of the glutaraldehyde-resistant reductase in both plasma membranes and discoid cisternae may indicate a role for the latter in the biosynthesis of plasm membranes during decidualization when massive cell proliferation and membrane biosynthesis occur. The origin of the discoid cisternae is tentatively ascribed to the mature faces of the Golgi apparatus.

Voltage-sensitive calcium channels in osteoblasts: mediators of plasma membrane signalling events.

Voltage-Sensitive Calcium Channels (VSCCs) are important cell surface regulators of membrane permeability to Ca2+. In non-excitable cells such as osteoblasts, VSCCs act as cellular transducers of stimulus-secretion coupling, activators of intracellular proteins, and in control of cell growth and differentiation. To obtain information concerning the molecular identity of the osteoblastic VSCC, we used an RT-PCR regional amplification approach. Effort focused on the large membrane-spanning alpha 1 subunit that is responsible for ion translocation. We designed primers using conserved alpha 1 subunit sequences common to many channel isoforms from various tissues and species. The generation of several RT-PCR products from various osteoblastic cell lines and subsequent sequencing of these products indicated that osteoblasts express at least two variants of the L-type VSCC which share regions of identity to the CaCh2 isoform first identified in cardiac myocytes. Our work provides the first molecular evidence for the existence and identity of VSCCs present in osteoblasts.

Annexin VI is associated with calcium-sequestering organelles.

Annexin VI is a member of a Ca(2+)-dependent, phospholipid-binding protein family. Although functions for this annexin have been proposed from in vitro studies, most remain controversial. Díaz-Muñoz et al. (J Biol Chem 265:15894, 1990) demonstrated that annexin VI modified, in a Ca(2+)-dependent manner, the gating behavior of the sarcoplasmic reticulum Ca(2+)-release channel, reconstituted into artificial bilayers, by increasing both the open probability and the mean open time. This effect was specific to the trans chamber, which represents the luminal side of the sarcoplasmic reticulum. In agreement with those findings, we show herein that annexin VI produced no effect on Ca(2+)-uptake or -release by intact heavy sarcoplasmic reticulum vesicles (analogous to the cis chamber). We also used monospecific antibodies to evaluate the subcellular localization of annexin VI by immunofluorescent microscopy. Studies in rat skeletal muscle suggest that annexin VI is present surrounding individual myofibrils. Double immunolocalization studies with cultured muscle cells (chick myotubes) using anti-annexin VI and anti-SR Ca(2+)-ATPase antibodies demonstrated superimposable staining patterns. In non-muscle tissue (normal rat kidney (NRK) cells), a punctate, perinuclear anti-annexin VI staining pattern was observed. Collectively, these data suggest that annexin VI may play a regulatory role in the Ca(2+)-release/uptake cycle in the sarcoplasmic reticulum as well as in non-muscle organelles, a key process in stimulus-response systems.