Chondrocyte channel transcriptomics

Expression patterns of Notch receptors and their ligands in human osteoarthritic and healthy articular cartilage

Maxi-K also known as BK channels, Slo1 or KCa1.1 channels, are one type of calcium-activated potassium channels that have large single channel conductance of 100–300 pS. Their most important physiological property is dual regulation through membrane voltage and intracellular Ca2+. 1 The complexity of this channel function mirrors the complexity of its protein structure. The amino acid sequence includes the integral membrane pore shared by all K+ channels, the integral membrane voltage sensor domains present in voltage-dependent channels, and a cytoplasmic domain (CTD) consisting of approximately 800 amino acids per subunit, which accounts for the C-terminal two thirds of the entire channel. The CTD structure confers upon the BK channel its ability to respond to changes in intracellular Ca2+. 2-5 It is also the source of functional heterogeneity through alternate splicing, polymorphisms, phosphorylation, and protein interactions, which modulate BK channel activity. 5-8 These channels modulate several physiological events, like blood pressure, smooth muscle relaxation or electrical tuning of hair cells in the cochlea and have a leading role in many pathophysiological conditions such as epilepsy, ischemic stroke, cognitive disorders, and the behavioral response to alcohol, to give only a few examples.9, 10 Studies involving activation and inactivation with pharmacological and genetic tools, including global, and tissue-specific knockouts, have implicated Maxi-K channels in cardiac function, neuroprotection, and cardio-protection from ischemia-reperfusion (IR) injury, in addition to IR-induced inflammation and mucosal barrier disruption in the small intestine. 11 It is also known that Maxi-K channels function as neuronal calcium sensors and contribute to the control of cellular excitability and the regulation of neurotransmitter release.9 Numerous Maxi-K channel blockers and activators are used to identify these channels and study their functions. Some of the most common Maxi-K channel modulators include tetraethylamonium (TEA), paxilline, penitrem A, charybdotoxin, iberiotoxin, indoles, benzimidazolones, biarylthioureas, anthraquinone analogs, tetrahydroquinolines, terpenes, benzofuroindoles, anilinoanthraquinones and quinoline. 9, 12-15 Both, the structural variety presented by the main modulators of the Maxi-K channel and the large number of pathophysiological conditions in which they are involved open a powerful research niche for the treatment of multiple pathologies. References Cui, J.; Yang, H.; Lee, U. S. Molecular mechanisms of BK channel activation. Cellular and Molecular Life Sciences. 2009, 66, 852-875. Ge, L.; Hoa, N. T.; Wilson, Z.; Arismendi-Morillo, G.; Kong, X.; Tajhya, R. B.; Beeton, C.; Jadus, M. R. Big Potassium (BK) ion channels in biology, disease and possible targets for cancer immunotherapy. Int. Immunopharmacol. 2014, 22, 427-443. Wallner, M.; Meera, P.; Toro, L. Determinant for β-subunit regulation in high-conductance voltage-activated and Ca2 -sensitive K channels: an additional transmembrane region at the N terminus. Proceedings of the National Academy of Sciences. 1996, 93, 14922-14927. Atkinson, N. S.; Robertson, G. A.; Ganetzky, B. A component of calcium-activated potassium channels encoded by the Drosophila slo locus. Science. 1991, 253, 551-555. Zang, K.; Zhang, Y.; Hu, J.; Wang, Y. The large conductance calcium-and voltage-activated potassium channel (BK) and epilepsy. CNS & Neurological Disorders-Drug Targets (Formerly Current Drug Targets-CNS & Neurological Disorders). 2018, 17, 248-254. Miller, C. An overview of the potassium channel family. Genome Biol. 2000, 1, 1-5. Lee, U. S.; Cui, J. BK channel activation: structural and functional insights. Trends Neurosci. 2010, 33, 415-423. Currò, D. The modulation of potassium channels in the smooth muscle as a therapeutic strategy for disorders of the gastrointestinal tract. Advances in protein chemistry and structural biology. 2016, 104, 263-305. Gribkoff, V. K.; Starrett Jr, J. E.; Dworetzky, S. I. Maxi-K potassium channels: form, function, and modulation of a class of endogenous regulators of intracellular calcium. Neuroscientist. 2001, 7, 166-177. Hermann, A.; Sitdikova, G. F.; Weiger, T. M. Oxidative stress and maxi calcium-activated potassium (BK) channels. Biomolecules. 2015, 5, 1870-1911. Goswami, S. K.; Ponnalagu, D.; Hussain, A. T.; Shah, K.; Karekar, P.; Gururaja Rao, S.; Meredith, A. L.; Khan, M.; Singh, H. Expression and activation of BKCa channels in mice protects against ischemia-reperfusion injury of isolated hearts by modulating mitochondrial function. Frontiers in cardiovascular medicine. 2019, 5, 194. Hannigan, K. I.; Large, R. J.; Bradley, E.; Hollywood, M. A.; Sergeant, G. P.; McHale, N. G.; Thornbury, K. D. Effect of a novel BKCa opener on BKCa currents and contractility of the rabbit corpus cavernosum. American Journal of Physiology-Cell Physiology. 2016, 310, C284-C292. Ibrahim, Z. G.; Elrewey, H. A. S. Rubidium Efflux Assay for the Determination of Calcium Activated Potassium Channel Activity. American International Journal of Biology and Life Sciences. 2020, 2, 18-27. Maqoud, F.; Cetrone, M.; Mele, A.; Tricarico, D. Molecular structure and function of big calcium-activated potassium channels in skeletal muscle: pharmacological perspectives. Physiological genomics. 2017, 49, 306-317. N'gouemo, P. Targeting BK (big potassium) channels in epilepsy. Expert opinion on therapeutic targets. 2011, 15, 1283-1295.

†Role of DNA methylation on 15-Lipoxygenase-1 gene expression in Osteoarthritis

IntroductionWe saw a lack of data on the biomechanical behavior of degenerated articular cartilage (OA) compared with that of healthy cartilage, even though the susceptibility to wear and tear of articular cartilage plays a key role in the progression of osteoarthritis (OA). Therefore, we performed a comparison between naturally occurring OA and healthy cartilage from pigs, before and after tribological stress.AimThe aim of the study was to compare OA-cartilage with healthy cartilage and to analyze the resilience to tribological shear stress, which will be measured as height loss (HL), and to friction forces of the cartilage layers. The findings will be substantiated in macro- and microscopical evaluations before and after tribological exposure.MethodsWe assessed stifle joints of fifteen old and sixteen young pigs from the local abattoir radiologically, macroscopically and histologically to determine possible OA alterations. We put pins from the femoral part of the joints and plates from the corresponding tibial plateaus in a pin-on-plate tribometer under stress for about two hours with about 1108 reciprocating cycles under a pressure of approximately 1 MPa. As a surrogate criterion of wear and tear, the HL was recorded in the tribometer. The heights of the cartilage layers measured before and after the tribological exposure were compared histologically. The condition of the cartilage before and after the tribological exposure was analyzed both macroscopically with an adapted ICRS score and microscopically according to Little et al. (2010). We assessed the friction forces acting between the surfaces of the cartilage pair–specimens.ResultsArticular cartilage taken from old pigs showed significant degenerative changes compared to that taken from the young animals. The macroscopic and microscopic scores showed strong alterations of the cartilage after the tribological exposure. There was a noticeable HL of the cartilage specimens after the first 100 to 300 cycles. The HL after tribological exposure was lower in the group of the old animals with 0.52 mm ± 0.23 mm than in the group of the young animals with 0.86 mm ± 0.26 mm (p < 0.0001). The data for the HL was validated by the histological height measurements with 0.50 mm ± 0.82 mm for the old and 0.79 mm ±0.53 mm for the young animals (p = 0.133). The friction forces measured at the cartilage of the old animals were 2.25 N ± 1.15 N and 1.89 N ± 1.45 N of the young animals (p = 0.3225).ConclusionUnlike articular cartilage from young pigs, articular cartilage from old pigs showed OA alterations. Tribological shear stress exposure revealed that OA cartilage showed less HL than healthy articular cartilage. Tribological stress exposure in a pin–on–plate tribometer seemed to be an appropriate way to analyze the mechanical stability of articular cartilage, and the applied protocol could reveal weaknesses of the assessed cartilage tissue. Friction and HL seemed to be independent parameters when degenerated and healthy articular cartilage were assessed under tribological exposure in a pin–on- plate tribometer.

The diverse microarray datasets that have become available over the past several years represent a rich opportunity and challenge for biological data mining. Many supervised and unsupervised methods have been developed for the analysis of individual microarray datasets. However, integrated analysis of multiple datasets can provide a broader insight into genetic regulation of specific biological pathways under a variety of conditions. To aid in the analysis of such large compendia of microarray experiments, we present Microarray Experiment Functional Integration Technology (MEFIT), a scalable Bayesian framework for predicting functional relationships from integrated microarray datasets. Furthermore, MEFIT predicts these functional relationships within the context of specific biological processes. All results are provided in the context of one or more specific biological functions, which can be provided by a biologist or drawn automatically from catalogs such as the Gene Ontology (GO). Using MEFIT, we integrated 40 Saccharomyces cerevisiae microarray datasets spanning 712 unique conditions. In tests based on 110 biological functions drawn from the GO biological process ontology, MEFIT provided a 5% or greater performance increase for 54 functions, with a 5% or more decrease in performance in only two functions.

The real-time system of the JT-60 has a cascade structure with the major and the minor control loops. The multi-variable feedback control is adopted in the major control loop in order to adjust non-linear effects which will appear in the high-/beta/region. By applying the concept of 'phase shift control' to the control program of the major control loop, a flexible tokamak discharge control is expected to be performed with taking account of machine protection.

A simplified measurement of degraded collagen in tissues: application in healthy, fibrillated and osteoarthritic cartilage.

Spiral ganglion neurons possess a rich repertoire of ion channels that underlies differential firing properties. Previous studies have shown that voltage-gated K+ channels (Kv1.1, Kv3.1, Kv4.2, and BK) are arranged in a tonotopic manner along the length of cochlea. The present study will expand the analysis to three additional classes of ion channels: two-pore-domain K+ channels (K2Ps), voltage-gated Ca2+ channels (VGCCs), and Ca2+-activated K+ channels; in order to determine their contribution to the tonotopic organization in these primary afferent neurons. Transcript profiling was first used to survey candidate subunits. A subset of K2P subunit transcripts (TASK-1, TASK-3, THIK-2, TRAAK, TREK-1, and TWIK-1) were identified in the spiral ganglion; of which TWIK-1 was the most abundantly expressed and was confirmed to localize to neurons. Of the ten VGCC subunits, eight transcripts (Cav1.2, Cav1.3, Cav2.1, Cav2.2, Cav2.3, Cav3.1, Cav3.2, and Cav3.3) were expressed. Immunocytochemistry showed that three subunits (Cav1.3, Cav2.2, and Cav3.3) were specifically localized to neurons and tonotopically distributed, which provided insights to the electrophysiological results that demonstrated the involvement of both HVA and LVA VGCCs in differential regulation of action potential duration and latency, respectively. Lastly, of the Ca2+-activated K+ channel subunits, BKalpha, BKbeta1, BKbeta2, BKbeta4, SK1, SK2, and SK3 transcripts were identified. Examination of the two BKalpha splice sites showed only a single prominent ZERO variant, which suggested that BK beta-subunit regulation may possibly be the main mechanisms of BK channel contribution to the firing properties. Antibody labeling of BKbeta4 subunit showed uniform distribution. Future work will further investigate the remaining beta-subunits. Taken together, K2Ps, VGCCs, and Ca2+-activated K+ channels could all potentially, although not necessary directly, influence neuronal excitability and action potential waveforms, thus demonstrating the complexity in underlying diversity of spiral ganglion neuron firing patterns.

One of the promising therapeutic uses of the potassium channel openers is in the management of cerebral vasospasm, a prolonged vasoconstriction of major cerebral arteries which follows aneurysmal subarachnoid haemorrhage. In this review, we first summarize the properties of potassium channels in cerebral vascular smooth muscle. Calcium-activated and ATP-dependent potassium channels are the major potassium channels identified in the cerebrovascular smooth muscle and both are believed to play a role in the regulation of cerebrovascular smooth muscle tone. The calcium-activated potassium channels can be activated by depolarization, by elevation of internal calcium and by some vasodilators. Some neuropeptides and potassium channel openers open the ATP-dependent potassium channels and produce vasodilation. We then review the effects of both synthetic and endogenous potassium channel openers in the cerebrovascular system, discuss their efficacy in the management of models of cerebrovascular spasm, and outline the clinical promise of these agents.

Activation of annexin II and V expression, terminal differentiation, mineralization and apoptosis in human osteoarthritic cartilage

In cartilage, collagen type II is of particular importance as its breakdown results in the irreversible loss of structural integrity of the tissue. Within the protease, the collagenases have a major involvement in this collagen network degradation. Evidence demonstrates that collagenase-3 is the major enzyme accounting for collagen degradation in osteoarthritic (OA) cartilage. Collagenase-3 has a greater effect (five to 10 times) on type II collagen than collagenase-1, and, in OA, is localized predominantly in the lower intermediate and deep layers of the cartilage, where type II collagen fibers are of the largest size and chondrocytes possess the most efficient capacity to reconstitute the extracellular matrix. Collagenase is upregulated in OA cartilage and is suggested to be implicated in cartilage remodeling in pathological conditions. Various factors induce its transcription, including proinflammatory cytokines and growth factors such as IL-1β, IL-17, tumor necrosis factor alpha, transforming growth factor beta and hepatocyte growth factor. Interestingly, we recently reported that transforming growth factor beta, but not IL-1β, treatment of normal cartilage mimicked the in situ collagenase-3 distribution in OA cartilage. The proximal promoter sequence contains a TATA box, as well as AP-1, Ets/PEA-3, and OSE-2 binding sites. The AP-1 site is essential for both basal and proinflammatory cytokine inducible transcription, and the PEA-3 site exerts a cooperative effect. We also demonstrated that some AP-1 proteins play a different role in terms of collagenase-3 production according to the stimulator. Indeed, IL-17-induced collagenase-3 resulted in FosB activation, whereas IL-1β stimulated c-Fos, which may explain the different capacities of these cytokines in producing this enzyme. Our data also suggest that JunB protein plays a rate-limiting step in cytokine-induced collagenase-3 production in OA chondrocytes. Moreover, we recently identified a novel protein-binding site on the collagenase-3 promoter that appears to be directly implicated in the repression of its basal transcription. This site was designated AGRE for AG-rich element. This site was not found in other human metalloprotease genes or in the mouse collagenase. Contrary to the other human collagenase genes that are transcribed into one mature mRNA, human cells expressed collagenase-3 transcripts of 3.0, 2.5 and 2.2/2.0 kb as demonstrated by northern blot. We recently identified five different collagenase-3 RNA species in humans; each could be translated in a cellular environment, indicating that they could be synthesized in response to specific cellular events. For two of the RNA species, the enzyme synthesized would differ from the original collagenase-3 and will have potentially different function/activity. Moreover, one of the transcripts appears to be an alternative transcription start site. Start sites are known to regulate gene expression by affecting the level of transcription initiation, the translation efficiency of the mRNA produced, and the generation of protein isoforms differing at their amino termini, or may respond differently to the cellular environment. At present, a therapeutic intervention based on the inhibition of metalloproteases is under intensive investigation, and collagenase-3 appears to be an attractive target for the development of disease-modifying OA drugs. However, the human collagenase-3 is subjected to different levels of regulation and constitutes a more complex system than originally thought. It would be interesting to verify whether compounds that block only one pathway, such as the synthesis/activity of the original collagenase-3, are sufficient to block all the effects of this enzyme, or whether all the enzyme transcripts should rather be targeted in order to achieve maximum therapeutic efficacy.

Hepatocyte growth factor/scatter factor is not a potent regulator of anabolic and catabolic gene expression in adult human articular chondrocytes

A flavonoid-rich diet has been associated with a lower incidence of cardiovascular diseases, probably because of the antioxidant and vasoactive properties of flavonoids. Indeed, many flavonoids show vasorelaxing properties, due to different and often not yet completely clarified mechanisms of action. Among them, the activation of vascular potassium channels has been indicated as a possible pathway, accounting, at least in part, for the vasodilatory action of some flavonoid derivatives, such as apigenin and dioclein. Therefore, this work aims at evaluating, on in vitro isolated rat aortic rings, the endothelium-independent vasorelaxing effects of a number of flavonoid derivatives, to identify a possible activation of calcium-activated and/or ATP-sensitive potassium channels and to indicate some possible structure-activity relationships. Among the several flavonoids submitted to the pharmacological assay, only baicalein and quercetagetin were almost completely ineffective, while quercetin, hesperidin, quercitrin and rhoifolin exhibited only a partial vasorelaxing effect. On the contrary, acacetin, apigenin, chrysin, hesperetin, luteolin, pinocembrin, 4'-hydroxyflavanone, 5-hydroxyflavone, 5-methoxyflavone, 6-hydroxyflavanone and 7-hydroxyflavone, belonging to the chemical classes of flavones and flavanones, showed full vasorelaxing effects. The vasodilatory activity of hesperetin, luteolin, 5-hydroxyflavone and 7-hydroxyflavone were antagonised by tetraethylammonium chloride, indicating the possible involvement of calcium-activated potassium channels. Moreover, iberiotoxin clearly antagonised the effects of 5-hydroxyflavone, indicating the probable importance of a structural requirement (the hydroxy group in position 5) for a possible interaction with large-conductance, calcium-activated potassium channels. Finally, glibenclamide inhibited the vasorelaxing action of luteolin and 5-hydroxyflavone, suggesting that ATP-sensitive potassium channels may also be involved in their mechanism of action.

Dynamic modulation of ion channel expression, localization, and/or function drives plasticity in intrinsic neuronal excitability. Voltage-gated Kv2.1 potassium channels are constitutively maintained in a highly phosphorylated state in neurons. Increased neuronal activity triggers rapid calcineurin-dependent dephosphorylation, loss of channel clustering, and hyperpolarizing shifts in voltage-dependent activation that homeostatically suppress neuronal excitability. These changes are reversible, such that rephosphorylation occurs after removal of excitatory stimuli. Here, we show that cyclin-dependent kinase 5 (CDK5), a Pro-directed Ser/Thr protein kinase, directly phosphorylates Kv2.1, and determines the constitutive level of Kv2.1 phosphorylation, the rapid increase in Kv2.1 phosphorylation upon acute blockade of neuronal activity, and the recovery of Kv2.1 phosphorylation after stimulus-induced dephosphorylation. We also demonstrate that although the phosphorylation state of Kv2.1 is also shaped by the activity of the PP1 protein phosphatase, the regulation of Kv2.1 phosphorylation by CDK5 is not mediated through the previously described regulation of PP1 activity by CDK5. Together, these studies support a novel role for CDK5 in regulating Kv2.1 channels through direct phosphorylation.

Voltage gated potassium ion (Kv) channels regulate action potentials of the nervous system by responding to changes in transmembrane voltage, enabling K+ transport across the membrane to restore cells to their resting potential. Comprised of four identical subunits, Kv channels contain four voltage sensing domains arranged on the periphery of a central pore domain. Each voltage sensor is comprised of four transmembrane helices, numbered S1 through S4. The S4 helix, containing four to six highly-conserved, positively-charged arginine or lysine residues, is responsible for voltage sensitivity in Kv channels. The pore domain consists of two transmembrane helices, S5 and S6. The S5 helix constitutes the periphery of the pore domain and is believed to be relatively immobile. The S6 helices, lining the interior of the channel, gate the protein and regulate K+ permeation. Because each subunit of Kv channels contains six transmembrane helices, they are often referred to as 6TM Kv channels. The depolarization of an action potential is initiated as sodium ions enter the cell. At the cellular resting potential of -70 mV, potassium ion channels are closed, and the S4 helix is in its “down” state. As the electrochemical gradient changes, the S4 helices of Kv channels begin to reorient within the membrane. At the peak of the action potential (roughly +20 mV), the S4 helices exist in their “up” state. This conformational transition of the S4 helix is coupled to the pore domain via the S4-S5 linker, a short, amphipathic helix along the intracellular membrane-water interface. By bridging the C-terminus of the voltage sensor to the N-terminus of the pore domain, the S4-S5 linker couples the voltage sensitivity of the voltage sensor to K+ conduction in the pore domain. Because they begin opening at voltages less than 0 mV, all crystal structures of Kv channels contain an open pore domain. With no structure in the closed conformation, the mechanism of gating in Kv channels remains unclear. Nevertheless, significant biophysical studies have revealed insights into both the closed conformation and the gating transition itself. In this dissertation, I will explore questions relevant to the gating mechanism in voltage gated potassium ion channels through fully-atomistic molecular dynamics (MD) simulations. First, in Chapter 2, I will address the potential role of the 310 helical conformation found in the C-terminal end of S4 in the crystal structures of Kv channels. Spanning eight or more residues, these 310 helices are both uncharacteristically long and conserved in K+ channel crystal structures. By simulating the Kv1.2/2.1 chimera channel’s voltage sensor embedded in a lipid bilayer, I find that an alpha to 310 helical interconversion of the S4 helix reproduces many experimental measurements of the open and closed states of Kv channels. In Chapter 3, I perform molecular dynamics simulations of the entire Kv1.2/2.1 chimera channel. First, I examine the impact of an alpha to 310 helical interconversion of the S4 helix on the pore domain of the channel. Though the results are consistent with the results in Chapter 2 (and the corresponding experimental measurements), I find that this secondary structural modification is insufficient to influence the pore domain of the channel on the timescale of my simulations. In the second half of Chapter 3, I use molecular dynamics simulations to generate a closed state model of the Kv1.2/2.1 chimera from luminescence resonance energy transfer (LRET) measurements of the closed conformation of KvAP. The resulting structure is indeed closed, and also recapitulates a number of experimentally determined measurements of the closed channel. In Chapter 4, I focus on the pore domain. First, using targeted molecular dynamics simulations, I generate a transition between a closed model of the KvAP linker and pore domain to the open conformation. Then, using an umbrella sampling method, I quantify the energetics of the gating transition in KvAP and assess the physiological implications. In agreement with experimental studies of Kv channel energetics, I find that the open pore is roughly 2.7 kcal/mol lower in free energy than the closed conformation. The targeted molecular dynamics and umbrella sampling simulations reveal additional insights into the gating mechanism of KvAP. Lastly, in Chapter 5, I use MD simulations to gain insights into the binding mechanism of VSTx1, a Kv channel inhibitor. By using the experimentally determined neutron scattering density profile of the VSTx1 toxin bound to a lipid bilayer as a restraint for molecular dynamics simulations, I recreate the experimental scattering density profile, and also offer insight into the binding of VSTx1 to a lipid membrane.