Characterization of calcium signaling mechanisms in osteoblasts

Bone remodeling is controlled by the osteoclast, which resorbs bone, and the osteoblast, which synthesizes and secretes proteins that are eventually mineralized into bone. Ca$\sp{2+}$ homeostasis and signaling contribute to the function of nearly all cell types, and understanding both in the osteoblast is of importance given its secretory properties and interaction with osteoclasts. This study was undertaken to identify and investigate the physiology of the Ca$\sp{2+}$ signaling mechanisms present in osteoblasts. The Ca$\sp{2+}$ pumps, stores and channels present in osteoblasts were studied. RT-PCR cloning revealed that osteoblast-like cells express PMCA1b, an alternatively spliced transcript of the plasma membrane Ca$\sp{2+}$-ATPase. The PMCA1b isoform contains a consensus phosphorylation site for cAMP-dependent protein kinase A and a modified calmodulin binding domain. The regulation of osteoblast function by agents that act via cAMP-mediated pathways may involve alterations in the activity of the plasma membrane Ca$\sp{2+}$-ATPase.^ Calcium release from intracellular stores is a signaling mechanism used universally by cells responding to hormones and growth factors, and the compartmentalization and regulated release of calcium is cell-type specific. Fura-2 was employed to monitor intracellular Ca$\sp{2+}$. Thapsigargin and 2,5,-di-(tert-butyl)-1,4-benzohydroquinone (tBuHQ), two inhibitors of endoplasmic reticulum Ca$\sp{2+}$-ATPase activity, both emptied a single 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 Ca$\sp{2+}$ storage system present in osteoblasts is typical of a non-excitable cell type, despite these cells sharing characteristics of excitable cells such as voltage-sensitive Ca$\sp{2+}$ channels (VSCCs).^ VSCCs are important cell surface regulators of membrane permeability to Ca$\sp{2+}$. In non-excitable cells VSCCs act as cellular transducers of stimulus-secretion coupling, activators of intracellular proteins, and in control of cell growth and differentiation. Functional VSCCs have been shown to exist in osteoblasts, however, no molecular cloning has been reported. To obtain information concerning the molecular identity of the osteoblastic VSCC, we used an RT-PCR regional amplification approach. Sequencing of the products indicated that osteoblasts express at least two isoforms of the L-type VSCC, $\alpha 1\sb{\rm C-a}$ and the $\alpha 1\sb{\rm C-d}$, which share regions of identity to the $\alpha \sb{\rm 1C}$ isoform first identified in cardiac myocytes. The ability of $1,25(\rm OH)\sb2D\sb3$ and structural analogs to modulate expression of Ca$\sp{2+}$ channel mRNA was then investigated. Cells were cultured for 48 hr in the presence of $1,25(\rm OH)\sb2D\sb3$ or vitamin D analogs, and the levels of mRNA encoding VSCC $\alpha \sb{\rm 1C}$ were quantitated using a competitive RT-PCR assay. It was found that $1,25(\rm OH)\sb2D\sb3$ and analog BT reduced steady state levels of $\alpha \sb{\rm 1C}$ mRNA. Conversely, analog AT did not alter steady state levels of Ca$\sp{2+}$ channel mRNA. Since it has been shown previously that analog BT, but not AT, binds and activates the nuclear vitamin D receptor, these findings suggest that the down regulation of channel mRNA involves the nuclear receptor for $1,25(\rm OH)\sb2D\sb3$. ^

Structural studies of cytochrome P4501A1: Identification of substrate and cumene hydroperoxide binding regions in the active site of P4501A1 and characterization of the P4501A1 interaction with cytochrome P450 reductase

Cytochromes P450 are a superfamily of heme-thiolate proteins that function in a concert with another protein, cytochrome P450 reductase, as terminal oxidases of an enzymatic system catalyzing the metabolism of a variety of foreign compounds and endogenous substrates. In order to better understand P450s catalytic mechanism and substrate specificity, information about the structure of the active site is necessary. Given the lack of a crystal structure of mammalian P450, other methods have been used to elucidate the substrate recognition and binding site structure in the active center. In this project I utilized the photoaffinity labeling technique and site-directed mutagenesis approach to gain further structural insight into the active site of mammalian cytochrome P4501AI and examine the role of surface residues in the interaction of P4501A1 with the reductase. ^ Four crosslinked peptides were identified by photoaffinity labeling using diazido benzphetamine as a substrate analog. Alignment of the primary structure of cytochrome P4501A1 with that of bacterial cytochrome P450102 (the crystal structure of which is known) revealed that two of the isolated crosslinked peptides can be placed in the vicinity of heme (in the L helix region and β10-β11 sheet region of cytochrome P450102) and could be involved in substrate binding. The other two peptides were located on the surface of the protein with the label bound specifically to Lys residues that were proposed to be involved in reductase-P450 interaction. ^ Alternatively, it has been shown that some of the organic hydroperoxides can support P450 catalyzed reactions in the absence of NADPH, O2 and reductase. By means of photoaffinity labeling the cumene hydroperoxide binding region was identified. Using azidocumene as the photoaffinity label, the tripeptide T501-L502-K503 was shown to be the site where azidocumene covalently binds to P4501A1. The sequence alignment of cytochrome P4501A1 with cytochrome P450102 predicts that this region might correspond to β-sheet structure localized on the distal side of the heme ring near the I helix and the oxygen binding pocket. The role of Thr501 in the cumene hydroperoxide binding was confirmed by mutations of this residue and kinetic analysis of the effects of the mutations. ^ In addition, the role of two lysine residues, Lys271 and Lys279, in the interaction with reductase was examined by means of site-directed mutagenesis. The lysine residues were substituted with isoleucine and enzymatic activity of the wild type and the mutants were compared in reductase- and cumene hydroperoxide-supported systems. The lysine 279 residue has been shown to play a critical role in the P4501A1-reductase interaction. ^

Rapid effects of 1alpha,25-dihydroxyvitamin D3 on osteoblasts: Studies of membrane transducers that affect calcium signals

1,25-dihydroxyvitamin D3 [1,25(OH)2D 3] exerts pleiotropic effects on osteoblasts via both long-term nuclear receptor-mediated and rapid membrane-initiated pathways during bone remodeling and mineral homeostasis. This study explored the membrane transducers that mediate rapid effects of 1,25(OH)2D3 on osteoblasts, including sphingomyelinase (SMase) and L-type voltage sensitive calcium channels (VSCCs). ^ It was previously demonstrated that 1,25(OH)2D3 stimulates transmembrane influx of Ca2+ through VSCCs in ROS 17/2.8 osteoblasts, however the molecular identity of 1,25(OH)2D 3-regulated VSCC has not been known. In this study, on the basis of in vitro tests of three unique ribozymes specifically cleaving a1C mRNA, I transfected ROS 17/2.8 cells with vectors coding recombinant ribozyme modified with U1 snRNA structure, and successfully selected stable clonal cells in which the expression of a1C was strikingly reduced. Ca2+ influx studies in these cells compared to control transfectants showed selective attenuation of depolarization- and 1,25(OH)2D3-regulated Ca2+ responses. These results allow us to conclude that the cardiac ( a1C ) subtype of the L-type VSCC is the major membrane transducer of Ca 2+ influx in osteoblasts. ^ I also demonstrated that 1,25(OH)2D3 induces a rapid hydrolysis of membrane sphingomyelin (SM) in ROS 17/2.8 cells, with the concomitant generation of ceramide, detectable at 15 minute, and maximal at 1 hour after addition. Sphingosine, sphingosine-1-phosphate (SPP) and sphingosylphosphorylcholine (SPC), downstream products of SM hydrolysis, but not ceramide, elicit Ca 2+ release from intracellular stores. Considering ceramide, sphingosine, and SPP as second messengers modulating intracellular kinases or phosphatases, these findings implicate sphingolipid-signaling pathways in transducing rapid effects of 1,25(OH)2D3 on osteoblasts. In structure/function analyses of sphingolipid signaling, it was observed that psychosine elicits Ca2+ release from intracellular stores. This challenges the dogma that sphingosine phosphorylation permits mobilization of Ca2+ , because psychosine is a sphingosine analog galactosylated at 1-carbon, preventing phosphorylation at that site. Psychosine is the pathological metabolite found in patients with Krabbe's disease, suggesting that psychosine disrupts the physiological sphingolipid signaling by chronic release of Ca2+ from intracellular stores. ^ Slower SM turnover than Ca2+ influx through VSCCs in response to 1,25(OH)2D3 demonstrates ceramide does not mediate the 1,25(OH)2D3-induced Ca2+ signaling, a conclusion endorsed further by the failure of ceramide to induce Ca 2+ signaling. ^

Characterization of osteopontin charge forms produced by osteoblasts

Osteopontin (OPN) is a highly-phosphorylated extracellular matrix protein localized in bone, kidney, placenta, T-lymphocytes, macrophages, smooth muscle of the vascular system, milk, urine, and plasma. In ROS 17/2.8 osteoblast-like osteosarcoma cells, 1,25-dihydroxyvitamin D3 [1,25(OH)2D 3] regulates OPN at the transcriptional level resulting in increased steady state mRNA levels and increased production of OPN protein, maximal at 48 hours. Using ROS 17/2.8 cells as an osteoblast model, OPN was purified from culture medium after three hour treatments of either vehicle (ethanol) or 1,25(OH)2D3 via barium citrate precipitation followed by immunoaffinity chromatography. ^ Here, further evidence of regulation of OPN by 1,25(OH)2D 3 at the posttranslational level is presented. Prior to the up-regulation of OPN at the transcriptional level, 1,25(OH)2D3 induces a shift in OPN isoelectric point (pI) detected on two-dimensional gels from pI 4.6 to pI 5.1. Loading equal amounts of [32P]-labeled OPN recovered from ROS 17/2.8 cells exposed to 1,25(OH)2D3 or vehicle alone for three hours reveals that the shift from pI 4.6 to 5.1 is the result of reduced phosphorylation. Using structural analogs to 1,25(OH) 2D3, analog AT [25-(OH)-16-ene-23-yne-D3], which triggers Ca2+ influx through voltage sensitive Ca2+ channels but does not bind to the vitamin D receptor, mimicked the OPN pI shift while analog BT [1,25(OH)2-22-ene-24-cyclopropyl-D 3], which binds to the vitamin D receptor but does not allow Ca 2+ influx, did not. Inclusion of the Ca2+ channel blocker nifedipine also blocks the charge shift conversion of OPN. Further analysis of the signaling pathway initiated by 1,25(OH)2D3 reveals that inhibition of the cyclic 3′,5′ -adenosine monophosphate-dependent kinase, protein kinase A, or inhibition of the cyclic 3′,5′-guanine monophosphate-dependent kinase, protein kinase G, also prevents the charge shift conversion. ^ Isolation of OPN from rat femurs and tibiae provides evidence for the existence of these two OPN charge forms in vivo, evidenced by differential migration on isoelectric focusing gels and sodium dodecyl sulfate-polyacrylamide gels. Peptide sequencing of rat long bone fractions revealed the presence of a presumed dentin specific protein, dentin matrix protein-1 (DMP-1). Western blot analysis confirmed the existence of DMP-1 in these fractions. ^ Using the OPN charge forms in functional assays, it was determined that the charge forms have differential roles in both cell surface and mineralization functions. In cell attachment assays and Ca2+ influx assays using PC-3 prostate cancer cells, the pI 5.1 charge form of OPN was found to permit binding and increase intracellular Ca2+ concentrations of PC-3 cells. The increase in intracellular Ca2+ concentration was found to be integrin αvβ3-dependent. In mineralization assays, the pI 4.6 charge form of OPN promoted hydroxyapatite formation, while the pI 5.1 charge form had improved Ca2+ binding ability. ^ In conclusion, these findings suggest that 1,25(OH) 2D3 regulates OPN not only at the transcriptional level, but also plays a role in determination of the OPN phosphorylation state. The latter involves a short term (less than three hours) treatment and is associated with membrane-initiated Ca2+ influx. Functional assays utilizing the two OPN charge forms reveal the dependence of OPN post-translational state on its function. ^

Mechanisms of apoptosis in chronic lymphocytic leukemia (CLL)

Chronic lymphocytic leukemia (CLL) is an incurable disease characterized by the accumulation of terminally differentiated, mature B cells that do not progress beyond the G1 stage of cell cycle, suggesting that these cells possess intrinsic defects in apoptosis. Treatment relies heavily on chemotherapy (primarily nucleoside analogs and glucocorticoids) that may initially be effective in patients, but ultimately give rise to refractory, untreatable disease. The purpose of this study was to determine whether key components of the apoptotic machinery were intact in CLL lymphocytes, especially in patients refractory to therapy. ^ Activation of proteases has been shown to be at the core of the apoptotic pathway and this work demonstrates that protease activation is required for glucocorticoid and nucleoside analog-induced apoptosis in CLL cells. Inhibitors of serine proteases as well as caspase inhibitors blocked induced DNA fragmentation, and a peptide inhibitor of the nuclear scaffold (NS) protease completely suppressed both induced and spontaneous apoptosis. However, the NS protease inhibitor actually promoted several pro-apoptotic events, such as caspase activation, exposure of surface phosphatidylserine, and loss of mitochondrial membrane potential. These results suggested that the NS protease may interact with the apoptotic program in CLL cells at two separate points. ^ In order to further investigate the role of the NS protease in CLL, patient isolates were treated with proteasome inhibitors because of previous results suggesting that the ISIS protease might be a β subunit of the proteasome. Proteasome inhibitors induced massive DNA fragmentation in every patient tested, even in those resistant to the effects of glucocorticoid and nucleoside analogs in vitro. Several other features of apoptosis were also promoted by the proteasome inhibitor, including mitochondrial alterations such as release of cytochrome c and drops in mitochondrial membrane potential. Proteasome inhibitor-induced apoptosis was associated with inhibition of NFκB, a proteasome-regulated transcription factor that has been implicated in the suppression of apoptosis in a number of systems. The NS protease inhibitor also caused a decrease in active NFκB, suggesting that the proapoptotic effects of this agent might be due to depletion of NFκB. ^ Given these findings, the role of NFκB, in conferring survival in CLL was investigated. Glucocorticoid hormone treatment was shown to cause decreases in the activity of the transcription factor, while phorbol dibutyrate, which blocks glucocorticoid-induced DNA fragmentation, was capable of upregulating NFκB. Compellingly, introduction of an undegradable form of the constitutive NFκB inhibitor, IκB, caused DNA fragmentation in several patient isolates, some of which were resistant to glucocorticoid in vitro. Transcription of anti-apoptotic proteins by NFκB was postulated to be responsible for its effects on survival, but Bcl-2 levels did not fluctuate with glucocorticoid or proteasome inhibitor treatment. ^ The in vitro values generated from these studies were organized into a database containing numbers for over 250 patients. Correlation of relevant clinical parameters revealed that levels of spontaneous apoptosis in vitro differ significantly between Rai stages. Importantly, in vitro resistance to nucleoside analogs or glucocorticoids predicted resistance to chemotherapy in vivo, and inability to achieve remission. ^

SYNTHESIS AND BIOLOGICAL EVALUATION OF ANTITUMOR ALDOPHOSPHAMIDE ACETALS

Although the major metabolic pathways of cyclophosphamide are well established, the mechanism of antitumor drug selectivity is highly controversial. However, it is widely accepted that aldophosphamide, one of the primary metabolites, plays a crucial role in drug selectivity. In an attempt to gain a better understanding of the mechanism of selectivity of cyclophosphamide, a series of aldophosphamide analogs have been synthesized.^ The new analogs, unlike aldophosphamide, are relatively stable in neutral solution; however, they are converted rapidly to aldehydo intermediates in the presence of carboxylate esterase. Due to structural differences, these analogs may be classified into three different groups, arbitrarily designated as A, B, C, depending upon the facility with which the intermediate aldehydes form 4-hydroxy cyclic tautomers. The half-life of the aldehydo/4-hydroxy cyclic tautomeric mixture is longer for bis(acetoxy)aldophosphamide acetal I (a representative of group A), shorter for the n-ethyl analog III (B), and shortest for the N,N-dimethyl analog IV (C). The ratio of aldophosphamide: 4-hydroxycyclophosphamide at pseudoequilibrium is 1: 4 for compound I, 1: 2 for compound III and 0: 1 for compound IV. The therapeutic efficacy of these compounds are group A $>$ group B $>$ group C. It is apparent that the equilibrium position between the aldehydo and 4-hydroxy cyclic tautomers, which determines their stability, is a crucial determinant of both the cytotoxicity and antitumor selectivity. These findings, taken in conjunction with the aldehyde dehydrogenase selectivity hypothesis, may provide an explanation for the unique antitumor activity of cyclophosphamide. ^

A PHARMACOLOGICAL EVALUATION OF THE MECHANISM OF CYTOTOXICITY OF 9-BETA-D- XYLOFURANOSYLADENINE IN CHINESE HAMSTER OVARY CELLS

The biochemical determinants of cytotoxicity of the purine nucleoside analog, 9-(beta)-D-xylofuranosyladenine (xyl-A) were studied in wild-type Chinese hamster ovary cells and in nucleoside kinase deficient mutants. It was found that {('3)H}xyl-A was readily phosphorylated to the triphosphate level in both the wild-type and deoxycytidine kinase deficient mutant, but not by the adenosine kinase deficient cells. Values for the apparent Km and Vmax of this uptake process were 43.9 (mu)M and 118.7 nmol/min/10('9) cells, respectively. Cloning procedures indicated that the viability of CHO cells was decreased 90 per cent by a 5-hr incubation with 10 (mu)M xyl-A. However, the toxicity of xyl-A was increased 100-fold by the addition of a nontoxic concentration (10 (mu)M) of the adenosine deaminase inhibitor erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) to the medium. High-pressure liquid chromatographic analysis indicated that after 5 hr, the concentration of 9-(beta)-D-xylofuranosyladenine 5'-triphosphate (xyl-ATP) in cells incubated with xyl-A plus EHNA was 2.0 mM, four times greater than in those cells incubated with xyl-A alone. Incubation with xyl-A plus EHNA had no significant effect on the cellular concentrations of 5-phosphoribosyl-1-pyrophosphate after 1 hr whereas, treatment with 3'-dexoyadenosine (cordycepin) decreased the concentration of this metabolite. Determinations of the cellular nucleoside triphosphates indicated that under conditions that resulted in an intracellular accumulation of 500 (mu)M xyl-ATP, the endogenous concentrations of neither the ribonucleoside triphosphates nor deoxyribonucleoside triphosphates were significantly different from those of control cells. The ID(,50) for {('3)H}thymidine incorporation into DNA, 105 (mu)M xyl-ATP, was four-fold less than the ID(,50) for {('3)H}uridine incorporation into RNA suggesting that the process of DNA synthesis is more sensitive to the presence of xyl-ATP. When removed from exogenous xyl-A, CHO cells failed to recover their ability to synthesize RNA and DNA, although the intracellular xyl-ATP concentration decreased to less than 35 (mu)M. The selective inhibition of RNA synthesis by 6-azauridine did not prevent the expression of toxicity by xyl-ATP. However, the selective inhibition of DNA synthesis by ara-C significantly spared toxicity in cells that had accumulated an otherwise lethal concentration of xyl-ATP. It is shown that in cells which had accumulated 1.27 mM {('3)H}xyl-ATP, {('3)H}xyl-A was found to terminate cellular RNA chains at a frequency of 1.42 (mu)mol of {('3)H}xyl-A 3' termini per mol of mononucleotide. These results indicate that a general mechanism for the toxicity of xyl-A to CHO cells includes the cellular accumulation of xyl-ATP, which serves as a substrate for RNA synthesizing enzymes and subsequently is incorporated into nascent RNA transcripts as a chain terminator. A specific mechanism involving the premature termination of RNA primers required for the initiation of DNA synthesis is proposed to account for the inhibitory action of xyl-ATP on DNA synthesis. ^

BIOCHEMICAL DETERMINANTS IN THE CYTOTOXIC ACTION OF 9-BETA- D- ARABINOFURANOSYLADENINE

Studies with 9-(beta)-D-arabinfuranosyladenine (ara-A) have illustrated that there are numerous cell functions which are affected by the nucleoside analog and its metabolites. Although the precise mechanism responsible for the cytotoxicity of this drug is not known, it is presently thought tha

Asynchronous ECG time sampling: Saving bits with Golomb-Rice encoding

We present a technique for online compression of ECG signals using the Golomb-Rice encoding algorithm. This is facilitated by a novel time encoding asynchronous analog-to-digital converter targeted for low-power, implantable, long-term bio-medical sensing applications. In contrast to capturing the actual signal (voltage) values the asynchronous time encoder captures and encodes the time information at which predefined changes occur in the signal thereby minimizing the sensor's energy use and the number of bits we store to represent the information by not capturing unnecessary samples. The time encoder transforms the ECG signal data to pure time information that has a geometric distribution such that the Golomb-Rice encoding algorithm can be used to further compress the data. An overall online compression rate of about 6 times is achievable without the usual computations associated with most compression methods.

Expression and function of psoriasin (S100A7) and koebnerisin (S100A15) in the brain

The expression and function of psoriasin in the brain have been insufficiently characterized. Here, we show the induction of psoriasin expression in the central nervous system (CNS) after bacterial and viral stimulation. We used a pneumococcal meningitis in vivo model that revealed S100A15 expression in astrocytes and meningeal cells. These results were confirmed by a cell-based in vivo assay using primary rat glial and meningeal cell cultures. We investigated psoriasin expression in glial and meningeal cells using polyinosinic-polycytidylic acid, a synthetic analog of double-stranded RNA that mimics viral infection. Furthermore, previous results showed that antimicrobial peptides have not only bactericidal but also immunomodulatory functions. To test this statement, we used recombinant psoriasin as a stimulus. Glial and meningeal cells were treated with recombinant psoriasin at concentrations from 25 to 500 ng/ml. Treated microglia and meningeal cells showed phosphorylation of the extracellular signal-regulated kinase 1 (ERK1)/ERK2 (ERK1/2) signal transduction pathway. We demonstrated that this activation of ERK depends on RAGE, the receptor for advanced glycation end products. Furthermore, microglia cells treated with recombinant psoriasin change their phenotype to an enlarged shape. In conclusion, our results indicate an occurrence of psoriasin in the brain. An involvement of psoriasin as an antimicrobial protein that modulates the innate immune system after bacterial or viral stimulation is possible.

Molecular beacons with a homo-DNA stem: improving target selectivity

Molecular beacons (MBs) are stem-loop DNA probes used for identifying and reporting the presence and localization of nucleic acid targets in vitro and in vivo via target-dependent dequenching of fluorescence. A drawback of conventional MB design is present in the stem sequence that is necessary to keep the MBs in a closed conformation in the absence of a target, but that can participate in target binding in the open (target-on) conformation, giving rise to the possibility of false-positive results. In order to circumvent these problems, we designed MBs in which the stem was replaced by an orthogonal DNA analog that does not cross-pair with natural nucleic acids. Homo-DNA seemed to be specially suited, as it forms stable adenine-adenine base pairs of the reversed Hoogsteen type, potentially reducing the number of necessary building blocks for stem design to one. We found that MBs in which the stem part was replaced by homo-adenylate residues can easily be synthesized using conventional automated DNA synthesis. As conventional MBs, such hybrid MBs show cooperative hairpin to coil transitions in the absence of a DNA target, indicating stable homo-DNA base pair formation in the closed conformation. Furthermore, our results show that the homo-adenylate stem is excluded from DNA target binding, which leads to a significant increase in target binding selectivity