The effect of food vehicles on in vitro performance of pantoprazole sodium delayed release sprinkle formulation.

Certain patient populations, including children, the elderly or people with dysphagia, find swallowing whole medications such as tablets and capsules difficult. To facilitate oral administration of drugs in such patients, a common practice is to sprinkle the drug products (e.g., usually after crushing the tablet or opening the capsule) on food vehicles before consumption which improves swallowability. Thus, evaluation of the impact of food vehicles on the potency and stability of the administered drug product is important. The aim of the current study was to evaluate the physicochemical properties (viscosity, pH, and water content) of common food vehicles used for sprinkle administration (e.g., apple juice, applesauce, pudding, yogurt, and milk) and their impacts on the in vitro performance (i.e., dissolution) of pantoprazole sodium delayed release (DR) drug products. The food vehicles evaluated exhibited marked difference in viscosity, pH and water content. Notably, the pH of the food as well as the interaction between food vehicle pH and drug-food contact time were the most significant factors affecting the in vitro performance of pantoprazole sodium DR granules. For example, the dissolution of pantoprazole sodium DR granules sprinkled on food vehicles of low pH (e.g., apple juice or applesauce) for short durations remained unchanged compared with the control group (i.e., without mixing with food vehicles). However, use of high pH food vehicles (e.g., milk) with prolonged contact time (e.g., 2 h) resulted in accelerated pantoprazole release, drug degradation and loss of potency. Overall, a thorough assessment of physicochemical properties of food vehicles and formulation characteristics are a necessary part of the development of sprinkle formulations.

Selective Targeting of Bromodomains of the Bromodomain-PHD Fingers Family Impairs Osteoclast Differentiation.

Histone acetyltransferases of the MYST family are recruited to chromatin by BRPF scaffolding proteins. We explored functional consequences and the therapeutic potential of inhibitors targeting acetyl-lysine dependent protein interaction domains (bromodomains) present in BRPF1-3 in bone maintenance. We report three potent and selective inhibitors: one (PFI-4) with high selectivity for the BRPF1B isoform and two pan-BRPF bromodomain inhibitors (OF-1, NI-57). The developed inhibitors displaced BRPF bromodomains from chromatin and did not inhibit cell growth and proliferation. Intriguingly, the inhibitors impaired RANKL-induced differentiation of primary murine bone marrow cells and human primary monocytes into bone resorbing osteoclasts by specifically repressing transcriptional programs required for osteoclastogenesis. The data suggest a key role of BRPF in regulating gene expression during osteoclastogenesis, and the excellent druggability of these bromodomains may lead to new treatment strategies for patients suffering from bone loss or osteolytic malignant bone lesions.

Studies of OC-STAMP in Osteoclast Fusion: A New Knockout Mouse Model, Rescue of Cell Fusion, and Transmembrane Topology.

The fusion of monocyte/macrophage lineage cells into fully active, multinucleated, bone resorbing osteoclasts is a complex cell biological phenomenon that utilizes specialized proteins. OC-STAMP, a multi-pass transmembrane protein, has been shown to be required for pre-osteoclast fusion and for optimal bone resorption activity. A previously reported knockout mouse model had only mononuclear osteoclasts with markedly reduced resorption activity in vitro, but with paradoxically normal skeletal micro-CT parameters. To further explore this and related questions, we used mouse ES cells carrying a gene trap allele to generate a second OC-STAMP null mouse strain. Bone histology showed overall normal bone form with large numbers of TRAP-positive, mononuclear osteoclasts. Micro-CT parameters were not significantly different between knockout and wild type mice at 2 or 6 weeks old. At 6 weeks, metaphyseal TRAP-positive areas were lower and mean size of the areas were smaller in knockout femora, but bone turnover markers in serum were normal. Bone marrow mononuclear cells became TRAP-positive when cultured with CSF-1 and RANKL, but they did not fuse. Expression levels of other osteoclast markers, such as cathepsin K, carbonic anhydrase II, and NFATc1, were not significantly different compared to wild type. Actin rings were present, but small, and pit assays showed a 3.5-fold decrease in area resorbed. Restoring OC-STAMP in knockout cells by lentiviral transduction rescued fusion and resorption. N- and C-termini of OC-STAMP were intracellular, and a predicted glycosylation site was shown to be utilized and to lie on an extracellular loop. The site is conserved in all terrestrial vertebrates and appears to be required for protein stability, but not for fusion. Based on this and other results, we present a topological model of OC-STAMP as a 6-transmembrane domain protein. We also contrast the osteoclast-specific roles of OC- and DC-STAMP with more generalized cell fusion mechanisms.

Deletion of Orai1 alters expression of multiple genes during osteoclast and osteoblast maturation.

Store-operated Ca(2+) entry (SOCE) is a major Ca(2+) influx pathway in most non-excitable cell types and Orai1 was recently identified as an essential pore-subunit of SOCE channels. Here we investigate the physiological role of Orai1 in bone homeostasis using Orai1-deficient mice (Orai1(-/-)). Orai1(-/-) mice developed osteopenia with decreased bone mineral density and trabecular bone volume. To identify the nature and origin of the bone defect, bone-resorbing osteoclasts and bone-forming osteoblasts from Orai1(-/-) mice were examined. Orai1-mediated SOCE was completely abolished in Orai1(-/-) osteoclast precursor cells and osteoclastogenesis in vitro from Orai1(-/-) mice was impaired due to a defect in cell fusion of pre-osteoclasts. Also, resorption activity in vitro was comparable but the size of pits formed by Orai1(-/-) osteoclasts was smaller. We next assessed the role of Orai1 in osteoblast differentiation and function by using a pre-osteoblast cell line, as well as primary osteoblasts from wild-type and Orai1(-/-) mice. SOCE in MC3T3-E1 pre-osteoblastic cells was inactivated by lentiviral overexpression of a pore-dead Orai1 mutant. Lack of SOCE in MC3T3-E1 had no effect on alkaline phosphatase staining and expression but substantially inhibited mineralized nodule formation. Consistent with this finding, Orai1-mediated SOCE was markedly reduced in Orai1(-/-) osteoblast precursor cells and osteoblastogenesis in vitro from Orai1(-/-) stromal cells showed impaired mineral deposition but no change in differentiation. This indicates that Orai1 is involved in the function but not in the differentiation of osteoblasts. Together, these results suggest that Orai1 plays a critical role in bone homeostasis by regulating both osteoblasts and osteoclasts.

Orai1-mediated calcium entry plays a critical role in osteoclast differentiation and function by regulating activation of the transcription factor NFATc1.

Bone diseases such as postmenopausal osteoporosis are primarily caused by excessive formation and activity of osteoclasts (OCLs). Receptor activator of nuclear factor-κB ligand (RANKL) is a key initiating cytokine for OCL differentiation and function. RANKL induces calcium (Ca(2+)) oscillations, resulting in selective and robust induction of nuclear factor of activated T cells c1 (NFATc1), a Ca(2+)-responsive transcription factor that drives osteoclastogenesis. Store-operated Ca(2+) entry (SOCE) is a major Ca(2+) influx pathway in most nonexcitable cell types and is activated by any stimulus that depletes Ca(2+) stores in the endoplasmic reticulum. Although the role of Orai1, a SOCE channel in the plasma membrane, in maintaining Ca(2+) oscillations and transactivation of NFAT in other cell types is well known, its contribution to osteoclastogenesis remains unclear. We show here that silencing of the Orai1 gene with viral delivery of shRNA reduces SOCE and inhibits RANKL-induced osteoclastogenesis of RAW264.7 cells, a murine monocyte/macrophage cell line, by suppressing the induction of NFATc1. This was accompanied by defective induction of OCL-specific genes, such as tartrate-resistant acid phosphatase and immunoreceptor OCL-associated receptor, which are known to be direct transcriptional targets of NFATc1 during osteoclastogenesis. In addition, maturation of OCLs was abrogated by defective cell fusion of pre-OCLs depleted of Orai1, consistent with defective RANKL-mediated induction of d2 isoform of vacuolar ATPase V(o) domain that is involved in cell fusion of pre-OCLs. We found that the functional bone resorbing capacity was severely impaired in OCLs depleted of Orai1, potentially related to the observed decrease in the induction of cathepsin K, a major bone matrix degrading protease. Our results indicate that Orai1 plays a critical role in the differentiation and function of OCLs, suggesting that Orai1 might be a potential therapeutic target for the treatment or prevention of bone loss caused by OCLs.

Calcium signaling in osteoclasts.

It has long been known that many bone diseases, including osteoporosis, involve abnormalities in osteoclastic bone resorption. As a result, there has been intense study of the mechanisms that regulate both the differentiation and bone resorbing function of osteoclast cells. Calcium (Ca(2+)) signaling appears to play a critical role in the differentiation and functions of osteoclasts. Cytoplasmic Ca(2+) oscillations occur during RANKL-mediated osteoclastogenesis. Ca(2+) oscillations provide a digital Ca(2+) signal that induces osteoclasts to up-regulate and autoamplify nuclear factor of activated T cells c1 (NFATc1), a Ca(2+)/calcineurin-dependent master regulator of osteoclastogenesis. Here we review previous studies on Ca(2+) signaling in osteoclasts as well as recent breakthroughs in understanding the basis of RANKL-induced Ca(2+) oscillations, and we discuss possible molecular players in this specialized Ca(2+) response that appears pivotal for normal bone function. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.

Activation and regulation of store-operated calcium entry.

The process of store-operated Ca(2+) entry (SOCE), whereby Ca(2+) influx across the plasma membrane is activated in response to depletion of intracellular Ca(2+) stores in the endoplasmic reticulum (ER), has been under investigation for greater than 25 years; however, only in the past 5 years have we come to understand this mechanism at the molecular level. A surge of recent experimentation indicates that STIM molecules function as Ca(2+) sensors within the ER that, upon Ca(2+) store depletion, rearrange to sites very near to the plasma membrane. At these plasma membrane-ER junctions, STIM interacts with and activates SOCE channels of the Orai family. The molecular and biophysical data that have led to these findings are discussed in this review, as are several controversies within this rapidly expanding field.

STIM1 is a calcium sensor specialized for digital signaling.

When cells are activated by calcium-mobilizing agonists at low, physiological concentrations, the resulting calcium signals generally take the form of repetitive regenerative discharges of stored calcium, termed calcium oscillations [1]. These intracellular calcium oscillations have long fascinated biologists as a mode of digitized intracellular signaling. Recent work has highlighted the role of calcium influx as an essential component of calcium oscillations [2]. This influx occurs through a process known as store-operated calcium entry, which is initiated by calcium sensor proteins, STIM1 and STIM2, in the endoplasmic reticulum [3]. STIM2 is activated by changes in endoplasmic reticulum calcium near the resting level, whereas a threshold of calcium depletion is required for STIM1 activation [4]. Here we show that, surprisingly, it is STIM1 and not STIM2 that is exclusively involved in calcium entry during calcium oscillations. The implication is that each oscillation produces a transient drop in endoplasmic reticulum calcium and that this drop is sufficient to transiently activate STIM1. This transient activation of STIM1 can be observed in some cells by total internal reflection fluorescence microscopy. This arrangement nicely provides a clearly defined and unambiguous signaling system, translating a digital calcium release signal into calcium influx that can signal to downstream effectors.

Calcium influx mechanisms underlying calcium oscillations in rat hepatocytes.

UNLABELLED: The process of capacitative or store-operated Ca(2+) entry has been extensively investigated, and recently two major molecular players in this process have been described. Stromal interacting molecule (STIM) 1 acts as a sensor for the level of Ca(2+) stored in the endoplasmic reticulum, and Orai proteins constitute pore-forming subunits of the store-operated channels. Store-operated Ca(2+) entry is readily demonstrated with protocols that provide extensive Ca(2+) store depletion; however, the role of store-operated entry with modest and more physiological cell stimuli is less certain. Recent studies have addressed this question in cell lines; however, the role of store-operated entry during physiological activation of primary cells has not been extensively investigated, and there is little or no information on the roles of STIM and Orai proteins in primary cells. Also, the nature of the Ca(2+) influx mechanism with hormone activation of hepatocytes is controversial. Hepatocytes respond to physiological levels of glycogenolytic hormones with well-characterized intracellular Ca(2+) oscillations. In the current study, we have used both pharmacological tools and RNA interference (RNAi)-based techniques to investigate the role of store-operated channels in the maintenance of hormone-induced Ca(2+) oscillations in rat hepatocytes. Pharmacological inhibitors of store-operated channels blocked thapsigargin-induced Ca(2+) entry but only partially reduced the frequency of Ca(2+) oscillations. Similarly, RNAi knockdown of STIM1 or Orai1 substantially reduced thapsigargin-induced calcium entry, and more modestly diminished the frequency of vasopressin-induced oscillations. CONCLUSION: Our findings establish that store-operated Ca(2+) entry plays a role in the maintenance of agonist-induced oscillations in primary rat hepatocytes but indicate that other agonist-induced entry mechanisms must be involved to a significant extent.

The cytosolic N-terminus of presenilin-1 potentiates mouse ryanodine receptor single channel activity.

Ryanodine receptors (RyRs) amplify intracellular Ca(2+) signals by massively releasing Ca(2+) from intracellular stores. Exaggerated chronic Ca(2+) release can trigger cellular apoptosis underlying a variety of neurodegenerative diseases. Aberrant functioning of presenilin-1 (PS1) protein instigates Ca(2+)-dependent apoptosis, providing a basis for the "calcium hypothesis" of Alzheimer's disease (AD). To get insight into this problem, we hypothesized that the previously reported physical interaction between RyR and PS1 modulates functional properties of the RyR. We generated a soluble cytoplasmic N-terminal fragment of PS1 comprising the first 82 amino acid (PS1 NTF(1-82)), the candidate for interaction with putative cytoplasmic modulatory sites of the RyR, and studied its effect on single channel currents of mouse brain RyRs incorporated in lipid bilayers. PS1 NTF(1-82) strongly increased both mean currents (EC(50)=12nM, Hill coefficient (n(H)) approximately 1) and open probability for higher sublevels for single RyR channels (EC(50)=7nM, n(H) approximately 2). Bell-shaped Ca(2+)-activation curve remained unchanged, suggesting that PS1 NTF(1-82) allosterically potentiates RyRs, but that the channel still requires Ca(2+) for activation. Corroborating such an independent mechanism, the RyR potentiation by PS1 NTF(1-82) was overridden by receptor desensitization at high [Ca(2+)] (pCa>5). This potentiation of RyR by PS1 NTF(1-82) reveals a new mechanism of physiologically relevant PS1-regulated Ca(2+) release from intracellular stores, which could be alternative or additional to recently reported intracellular Ca(2+) leak channels formed by PS1 holoproteins.

Differential inositol 1,4,5-trisphosphate receptor signaling in a neuronal cell line.

Differential intracellular distribution of the three pharmacologically and biophysically distinct types of IP3Rs can lead to different subcellular Ca2+ transients each coupled to discrete intracellular functions. Here, we report the functional localization of differentially distributed IP3 receptor types in the commonly-used hippocampal cell line HT22. The distinct subcellular localization and Ca2+ signaling properties of these receptors determine the potential role of specific IP3 receptor types in cellular function. By utilizing immunochemistry, we conclude that HT22 cells express all three IP3 receptors with types 1 and 3 being expressed predominantly in the endoplasmic reticulum and perinuclear regions and type 2 being expressed predominantly in the nuclear envelope. Optical imaging studies using the Ca2+-sensitive indicator dye fluo-3 show that nuclear IP3 responses have greater amplitude and faster kinetics than cytosolic IP3 responses corresponding to the biophysical characteristics of the differentially distributed receptor types. These results support the hypothesis that differentially distributed IP3R isotypes mediate distinct cellular functions through differential, organelle-specific Ca2+ signaling.

Homer proteins control neuronal differentiation through IP(3) receptor signaling.

Neurons expand, sustain or prune their dendritic trees during ontogenesis [Cline, H.T. (2001). Dendritic arbor development and synaptogenesis. Curr. Opin. Neurobiol. 11, 118-126; Wong, W.T. and Wong, R.O.L. (2000) Rapid dendritic movements during synapse formation and rearrangement. Curr. Opin. Neurobiol. 10, 118-124] which critically depends on neuronal activity [Wong, W.T., Faulkner-Jones, B.E., Sanes, J.R. and Wong, R.O.L. (2000) Rapid dendritic remodeling in the developing retina: dependence on neurotransmission and reciprocal regulation by Rac and Rho. J. Neurosci. 20, 5024-5036; Li, Z., Van Aelst, L. and Cline, H.T. (2000) Rho GTPases regulate distinct aspects of dendritic arbor growth in Xenopus central neurons in vivo. Nat. Neurosci. 3, 217-225; Wong, W.T. and Wong, R.O.L. (2001) Changing specificity of neurotransmitter regulation of rapid dendritic remodeling during synaptogenesis. Nat. Neurosci. 4, 351-352.] and sub-cellular Ca(2+) signals [Lohmann, C., Myhr, K.L. and Wong, R.O. (2002) Transmitter-evoked local calcium release stabilizes developing dendrites, Nature 418, 177-181.]. The role of synaptic clustering proteins connecting both processes is unclear. Here, we show that expression levels of Vesl-1/Homer 1 isoforms critically control properties of Ca(2+) release from intracellular stores and dendritic morphology of CNS neurons. Vesl-1L/Homer 1c, an isoform with a functional WH1 and coiled-coil domain, but not isoforms missing these features were capable of potentiating intracellular calcium signaling activity indicating that such regulatory interactions function as a general paradigm in cellular differentiation and are subject to changes in expression levels of Vesl/Homer isoforms.

Polycystin-1 can interact with homer 1/Vesl-1 in postnatal hippocampal neurons.

Polycystin-1 (PC-1) has been identified as critical to development of the nervous system, but the significance of PC-1 expression in neurons remains undefined, and little is known of its roles outside the kidney, where mutation results in autosomal dominant polycystic kidney disease (ADPKD). In kidney, PC-1 interacts with cadherins, catenins, and its cognate calcium channel polycystin-2 (PC-2), which in turn interacts with a number of actin-regulatory proteins. Because some of the proteins that interact with PC-1 in kidney also participate in synaptic remodeling and plasticity in the hippocampus, we decided to test PC-1's potential to interact with a recently discovered type of plasticity-associated protein (homer 1a/Vesl-1S) in postnatal mouse hippocampus. Homer 1a/Vesl-1S is an activity-induced protein believed to participate in synaptic remodeling/plasticity responses to temporal lobe seizure and learning. Here we report the following. 1) PC-1 contains a homer-binding motif (PPxxF), which lies within its purported cytoplasmic domain. 2) Immunoreactivity for PC-1 (PC-1-ir) is highly colocalized with homer 1a immunoreactivity (H1a-ir) in primary cultured hippocampal neurons. 3) PC-1-ir and H1a-ir are present and appear to be colocalized in mouse hippocampus but not cortex on postnatal day 2 (P2), when higher frequencies of spontaneous activity are normal for hippocampus compared with cortex. 4) An endogenous PC-1-ir band with the correct size for the reported C-terminal G-protein-sensitive domain cleavage product of PC-1 (approximately 150 kDa) coimmunoprecipitates with endogenous homer 1/Vesl-1 proteins from mouse brain, suggesting that PC-1 can interact with homer 1/Vesl-1 proteins in postnatal hippocampal neurons.

Effects of Vesl/Homer proteins on intracellular signaling.

The clustering of signaling molecules at specialized cellular sites allows cells to effectively convert extracellular signals into intracellular signals and to produce a concerted functional output with specific temporal and spatial patterns. A prime example for these molecules and their effects on cellular signaling are the postsynaptic density proteins of the central nervous system. Recently, one group of these proteins, the Vesl/Homer protein family has received increased attention because of its unique molecular properties that allow both the clustering and functional modulation of a plethora of different binding proteins. Within multiprotein signaling complexes, Vesl/Homer proteins influence proteins as diverse as metabotropic glutamate receptors; transient receptor potential channels; intra-cellular calcium channels; the scaffolding protein, Shank; small GTPases; transcription factors; and cytoskeletal proteins. Furthermore, interaction with such functionally relevant proteins also links Vesl/Homer proteins indirectly to an even larger group of cellular effector proteins, putting the Vesl/Homer proteins at the crossroads of several critical intracellular signaling processes. In addition to the initial reports of Vesl/Homer protein expression in the central nervous system, members of this protein family have now been identified in other excitable cells in various muscle types and in a large number of nonexcitable cells. The widespread expression of Vesl/Homer proteins in different organs and their functional importance in cellular protein signaling complexes is further evidenced by their conservation in organisms from Drosophila to humans.

Vesl/Homer proteins regulate ryanodine receptor type 2 function and intracellular calcium signaling.

Cellular signaling proteins such as metabotropic glutamate receptors, Shank, and different types of ion channels are physically linked by Vesl (VASP/Ena-related gene up-regulated during seizure and LTP)/Homer proteins [Curr. Opin. Neurobiol. 10 (2000) 370; Trends Neurosci. 23 (2000) 80; J. Cell Sci. 113 (2000) 1851]. Vesl/Homer proteins have also been implicated in differentiation and physiological adaptation processes [Nat. Neurosci. 4 (2001) 499; Nature 411 (2001) 962; Biochem. Biophys. Res. Commun. 279 (2000) 348]. Here we provide evidence that a Vesl/Homer subtype, Vesl-1L/Homer-1c (V-1L), reduces the function of the intracellular calcium channel ryanodine receptor type 2 (RyR2). In contrast, Vesl-1S/Homer-1a (V-1S) had no effect on RyR2 function but reversed the effects of V-1L. In live cells, in calcium release studies and in single-channel electrophysiological recordings of RyR2, V-1L reduced RyR2 activity. Important physiological functions and pharmacological properties of RyR2 are preserved in the presence of V-1L. Our findings demonstrate that a protein-protein interaction between V-1L and RyR2 is not only necessary for organizing the structure of intracellular calcium signaling proteins [Curr. Opin. Neurobiol. 10 (2000) 370; Trends Neurosci. 23(2000)80; J. Cell Sci. 113 (2000) 1851; Nat Neurosci. 4 (2001) 499; Nature 411 (2001) 962; Biochem. Biophys. Res. Commun. 279 (2000) 348; Nature 386 (1997) 284], but that V-1L also directly regulates RyR2 channel activity by changing its biophysical properties. Thereby it may control cellular calcium homeostasis. These observations suggest a novel mechanism for the regulation of RyR2 and calcium-dependent cellular functions.

Differential functional interaction of two Vesl/Homer protein isoforms with ryanodine receptor type 1: a novel mechanism for control of intracellular calcium signaling.

Vesl/Homer proteins physically link proteins that mediate cellular signaling [Curr. Opin. Neurobiol. 10 (2000) 370; Trends Neurosci. 23 (2000) 80; J. Cell Sci. 113 (2000) 1851] and thereby influence cellular function [Nat. Neurosci. 4 (2001) 499; Nature 411 (2001) 962]. A previous study reported that Vesl-1L/Homer-1c (V-1L) controls the gain of the intracellular calcium activated calcium channel ryanodine receptor type 1 (RyR1) channel [J. Biol Chem. 277 (2002) 44722]. Here, we show that the function of RyR1 is differentially regulated by two isoforms of Vesl-1/Homer-1, V-1L and Vesl-1S/Homer-1a (V-1S). V-1L increases the activity of RyR1 while important regulatory functions and pharmacological characteristics are preserved. V-1S alone had no effect on RyR1, even though, like V-1L, it is directly bound to the channel. However, V-1S dose-dependently decreased the effects of V-1L on RyR1, providing a novel mechanism for the regulation of intracellular calcium channel activity and calcium homeostasis by changing expression levels of Vesl/Homer proteins.