Dependent Regulatory Mechanism Research Articles

A simulation study on the role of mitochondria-sarcoplasmic reticulum Ca2+ interaction in cardiomyocyte energetics during exercise.

Previous studies demonstrated that the mitochondrial Ca2+ uniporter MCU and the Na+-Ca2+ exchanger NCLX exist in proximity to the sarcoplasmic reticulum (SR) ryanodine receptor RyR and the Ca2+ pump SERCA, respectively, creating a mitochondria-SR Ca2+ interaction. However, the physiological relevance of the mitochondria-SR Ca2+ interaction has remained unsolved. Furthermore, although mitochondrial Ca2+ has been proposed to be an important factor regulating mitochondrial energy metabolism, by activating NADH-producing dehydrogenases, the contribution of the Ca2+-dependent regulatory mechanisms to cellular functions under physiological conditions has been controversial. In this study, we constructed a new integrated model of human ventricular myocyte with excitation-contraction-energetics coupling and investigated systematically the contribution of mitochondria-SR Ca2+ interaction, especially focusing on cardiac energetics during dynamic workload transitions in exercise. Simulation analyses revealed that the spatial coupling of mitochondria and SR, particularly via mitochondrial Ca2+ uniport activity-RyR, was the primary determinant of mitochondrial Ca2+ concentration, and that the Ca2+-dependent regulatory mechanism facilitated mitochondrial NADH recovery during exercise and contributed to the stability of NADH in the workload transition by about 40%, while oxygen consumption rate and cytoplasmic ATP level were not influenced. We concluded that the mitochondria-SR Ca2+ interaction, created via the uneven distribution of Ca2+ handling proteins, optimizes the contribution of the mitochondrial Ca2+-dependent regulatory mechanism to stabilizing NADH during exercise. KEY POINTS: The mitochondrial Ca2+ uniporter protein MCU and the Na+-Ca2+ exchanger protein NCLX are reported to exist in proximity to the sarcoplasmic reticulum (SR) ryanodine receptor RyR and the Ca2+ pump SERCA, respectively, creating a mitochondria-SR Ca2+ interaction in cardiomyocytes. Mitochondrial Ca2+ (Ca2+ mit) has been proposed to be an important factor regulating mitochondrial energy metabolism, by activating NADH-producing dehydrogenases. Here we constructed an integrated model of a human ventricular myocyte with excitation-contraction-energetics coupling and investigated the role of the mitochondria-SR Ca2+ interaction in cardiac energetics during exercise. Simulation analyses revealed that the spatial coupling particularly via mitochondrial Ca2+ uniport activity-RyR is the primary determinant of Ca2+ mit concentration, and that the activation of NADH-producing dehydrogenases by Ca2+ mit contributes to NADH stability during exercise. The mitochondria-SR Ca2+ interaction optimizes the contribution of Ca2+ mit to the activation of NADH-producing dehydrogenases.

MiRNA-132-5p mediates a negative feedback regulation of IL-8 secretion through S100A8/A9 downregulation in neutrophil-like HL-60 cells.

Neutrophils are an important source of pro-inflammatory and immunomodulatory cytokines. This makes neutrophils efficient drivers of interactions with immune and non-immune cells to maintain homeostasis and modulate the inflammatory process by notably regulating the release of cytokines. Ca2+-dependent regulatory mechanism encompassing cytokine secretion by neutrophils are not still identified. In this context, we propose to define new insights on the role of Ca2+-binding proteins S100A8/A9 and on the regulatory role of miRNA-132-5p, which was identified as a regulator of S100A8/A9 expression, on IL-8 secretion. Differentiated HL-60 cells, a human promyelocytic leukemia cell line that can be induced to differentiate into neutrophil-like cells, were used as a model of human neutrophils and treated with N- formyl-methionyl-leucyl-phenylalanine (fMLF), a bacterial peptide that activates neutrophils. shRNA knockdown was used to define the role of selected targets (S100A8/A9 and miRNA-132-5p) on IL-8 secretion. Different types of cytokines engage different signaling pathways in the secretion process. IL-8 release is tightly regulated by Ca2+ binding proteins S100A8/A9. miRNA-132-5p is up-regulated over time upon fMLF stimulation and decreases S100A8/A9 expression and IL-8 secretion. These findings reveal a novel regulatory loop involving S100A8/A9 and miRNA-132-5p that modulates IL-8 secretion by neutrophils in inflammatory conditions. This loop could be a potential target for therapeutic intervention in inflammatory diseases.

The Regulation of Common Chinese Medicine Targeting Ferroptosis on a Variety of Clinical Diseases

Ferroptosis is an iron dependent regulatory mechanism of cell death, driven by Lipid peroxidation and iron dependent in cells. It is different from apoptosis, necrosis and autophagy in morphology, biochemistry and genetics. Ferroptosis is closely related to physiological and pathological processes such as cell growth and development, aging, and immunity and plays an important role in many diseases. In clinical practice, traditional Chinese medicine plays an increasingly important role in treating cancer. In recent years, although there have been more and more studies on the pathogenesis of Ferroptosis in traditional Chinese medicine, the role of Ferroptosis in the clinical treatment of traditional Chinese medicine remains to be further explored. Based on the research on the mechanism of Ferroptosis, this article mainly introduces the application of common traditional Chinese medicine in the research on the pathogenesis of Ferroptosis so as to help clinicians understand more about the status quo of traditional Chinese medicine in treating Ferroptosis related diseases.

Improved cell-type identification and comprehensive mapping of regulatory features with spatial epigenomics 96-channel microfluidic platform.

Gene expression is subject to epigenetic regulation and is dependent upon cellular context. Spatial omics tools can provide insight into cellular context; however, development has centered on spatial transcriptomics and proteomics. Deterministic barcoding in tissue for spatial omics sequencing (DBiT-seq) was the first spatial epigenomics platform at the cellular level. Here we present a comparison of spatial epigenomic profiling on both 50-channel and 96-channel platforms. The new 96-channel microfluidics chip design greatly improved precision in cell typing and identification of regulatory elements by spatial-ATAC-seq. Spatial mapping reveals complexity of glial cell and neuronal localization within brain structures as well as cis-regulatory elements controlling cellular function. This technology streamlines spatial analysis of the epigenome and contributes a new layer of spatial omics to uncover the context dependent regulatory mechanisms underpinning development, disease, and normal cellular function.

Breed dependent regulatory mechanisms of beneficial and non-beneficial fatty acid profiles in subcutaneous adipose tissue in cattle with divergent feed efficiency

The current study aimed to determine whether breed and feed efficiency affect the molecular mechanisms regulating beneficial and non-beneficial fatty acid profiles in subcutaneous adipose tissue of beef steers. Fatty acid profiling and RNA-Seq based transcriptome analysis were performed on subcutaneous adipose tissues collected from beef steers with three divergent breeds (Angus, ANG, n = 47; Charolais, CHAR, n = 48; Kinsella Composite, KC, n = 48) and different residual feed intake (RFI, a measure of feed efficiency). The comparison of fatty acid profiles showed that KC had higher beneficial FAs compared to the other two breeds. Distinct FA profiles between H-RFIfat and L-RFIfat steers was more obvious for KC steers, where H-RFIfat steers tended to have higher proportion of healthy FAs and lower proportion of the unhealthy FAs. A higher number of differentially expressed (DE) genes were observed for KC steers, whereas ANG and CHAR steers had a lower number of DE genes between H- and L-RFIfat steers. The association analyses of the gene expressions and FA profiles showed that 10 FA metabolism-associated genes together with the one upstream regulator (SREBF1) were associated with the proportion of C18:2n-6, total n-6, PUFA and PUFA/SFA for KC steers but not the other two breeds. Subcutaneous adipose tissue FA profiles and healthy FA index differed in cattle with divergent feed efficiency and such variation was unique for the three examined cattle breeds. Key FA metabolism-associated genes together with SREBF1 which is the upstream regulator of a set of genes involved in lipid metabolism may be of importance for genetic selection of meat with higher healthy FA index in beef cattle.

The core and accessory Hfq interactomes across Pseudomonas aeruginosa lineages

The major RNA-binding protein Hfq interacts with mRNAs, either alone or together with regulatory small noncoding RNAs (sRNAs), affecting mRNA translation and degradation in bacteria. However, studies tend to focus on single reference strains and assume that the findings may apply to the entire species, despite the important intra-species genetic diversity known to exist. Here, we use RIP-seq to identify Hfq-interacting RNAs in three strains representing the major phylogenetic lineages of Pseudomonas aeruginosa. We find that most interactions are in fact not conserved among the different strains. We identify growth phase-specific and strain-specific Hfq targets, including previously undescribed sRNAs. Strain-specific interactions are due to different accessory gene sets, RNA abundances, or potential context- or sequence- dependent regulatory mechanisms. The accessory Hfq interactome includes most mRNAs encoding Type III Secretion System (T3SS) components and secreted toxins in two strains, as well as a cluster of CRISPR guide RNAs in one strain. Conserved Hfq targets include the global virulence regulator Vfr and metabolic pathways involved in the transition from fast to slow growth. Furthermore, we use rGRIL-seq to show that RhlS, a quorum sensing sRNA, activates Vfr translation, thus revealing a link between quorum sensing and virulence regulation. Overall, our work highlights the important intra-species diversity in post-transcriptional regulatory networks in Pseudomonas aeruginosa.

METTL3-mediated RNA m6A Hypermethylation Promotes Tumorigenesis and GH Secretion of Pituitary Somatotroph Adenomas.

Pituitary growth hormone-secreting (GH) pituitary adenomas (PAs) cause mass effects and dysregulated hypersecretion of GH. However, somatic mutation burden is low in PAs. While progress has been made in identifying the epigenetic changes involved in GH-PA initiation, the precise details of its tumorigenesis in GH-PA patients remains to be elucidated. As N6-methyladenosine (m6A) has been shown to often play a critical role in various tumors, it represents a possible initiation point for the tumorigenesis of pituitary adenomas. However, the role of RNA methylation in GH adenomas remains unclear. Protein expression of m6A regulators was measured by immunohistochemistry. Global levels and distribution of m6A methylation were separately analyzed by m6A enzyme-linked immunosorbent assay and m6A sequencing (m6A-seq). RNA interference and lentivirus knockdown system were used to investigate the role of methyltransferase-like 3 (METTL3) and its m6A- dependent regulatory mechanism in tumor progression and GH secretion. We show that both METTL3 messenger RNA and protein expression are elevated in GH-PA samples when compared with both normal pituitary tissue specimens and nonsecreting pituitary adenomas. Levels of m6A modification increased in GH-PAs, and hypermethylated RNAs are involved in hormone secretion and cell development. Knockdown of METTL3 in GH3 cell line resulted in decreased cell growth and GH secretion. Importantly, we found that GNAS and GADD45γ act as the downstream targets in this process. Our findings strongly suggest that m6A methyltransferase METTL3 promotes tumor growth and hormone secretion by increasing expression of GNAS and GADD45γ in a m6A-dependent manner. Thus, METTL3 and the methylated RNAs constitute suitable targets for clinical treatment of GH-PAs.

Retracted: DNA methylation-mediated downregulation of PTPN3 attenuates to boost TGF-β signaling in osteosarcoma cells.

The misregulation of transforming growth factor-β (TGF-β) signaling can cause tumorigenesis, but the activation and suppression of TGF-β signaling are complicated biological processes. We discovered a protein tyrosine phosphatase nonreceptor 3 (PTPN3)-dependent regulatory mechanism in uncancerous osteoblast cells that involved a stabilizing PTPN3 interaction with the TGF-β type I receptor (TGFBR1), which impaired TGFBR1 ubiquitination by the Smurf2 (Smad ubiquitination regulatory factor 2) E3 ligase. TGFBR1 facilitated the phosphorylation of Smad2/3 (SMAD family member 2 and 3), and the phosphorylated Smad2/3 recruited Smad4 to assemble a complex that then was translocated into the nucleus to initiate gene transcription. By contrast, PTPN3 was significantly downregulated in osteosarcoma cells because the increased DNMT1 (DNA methyltransferase 1) caused a higher methylation level in the promoter of PTPN3. The decrease of PTPN3 failed to stabilize TGFBR1, causing the ubiquitination and degradation of TGFBR1 by Smurf2. The degradation of TGFBR1 impaired the phosphorylation of Smad2/3 and prevented the nuclear translocation of the Smad2/3/4 complex, thereby causing the dysregulation of TGF-β target genes and triggering tumorigenesis. Collectively, our results reveal that the DNA methylation-mediated downregulation of PTPN3 disables the stabilization of TGFBR1 and that the degradation of TGFBR1 by Smurf2 E3 ligase blocks Smad2/3/4-mediated gene expression that promotes the tumorigenesis of osteosarcoma.

Antiplatelet properties of Pim kinase inhibition are mediated through disruption of thromboxane A2 receptor signaling.

Pim kinases are upregulated in several forms of cancer, contributing to cell survival and tumor development, but their role in platelet function and thrombotic disease has not been explored. We report for the first time that Pim-1 kinase is expressed in human and mouse platelets. Genetic deletion or pharmacological inhibition of Pim kinase results in reduced thrombus formation but is not associated with impaired hemostasis. Attenuation of thrombus formation was found to be due to inhibition of the thromboxane A2 receptor as effects on platelet function were non-additive to inhibition caused by the cyclo-oxygenase inhibitor indomethacin or the thromboxane A2 receptor antagonist GR32191. Treatment with Pim kinase inhibitors caused reduced surface expression of the thromboxane A2 receptor and resulted in reduced responses to thromboxane A2 receptor agonists, indicating a role for Pim kinase in the regulation of thromboxane A2 receptor function. Our research identifies a novel, Pim kinase-dependent regulatory mechanism for the thromboxane A2 receptor and represents a new targeting strategy that is independent of cyclo-oxygenase-1 inhibition or direct antagonism of the thromboxane A2 receptor that, while attenuating thrombosis, does not increase bleeding.

Abstract 3518: SPARC related signaling genes in non-small cell lung cancer by RNA-sequencing transcriptome analysis

Abstract Non-small cell lung cancer (NSCLC) is the most common type of lung cancer, accounting for about 80 % cases of lung cancer and is the leading cause of cancer-related death in the United States and worldwide. SPARC (secreted protein acidic and rich in cysteine), a multicellular non-structural glycoprotein is known to be involved in the regulation of cell adhesion, cell proliferation as well as in tumorigenesis and metastasis in various cancers including NSCLC. SPARC contributes to matrix remodeling and turnover and is involved in modulating the tumor microenvironment. Previous studies have suggested that SPARC can be used as a prognostic factor for NSCLC and represents a highly promising therapeutic modality. In this study we used high-throughput RNA-sequencing transcriptome profiling analysis to delineate pathways involved in SPARC signaling. We assessed differentially expressed genes, novel genes, biological processes and biological signaling pathways in SPARC expressed NSCLC cell line. We identified a total of 3,280 upregulated and 1,608 downregulated differentially expressed genes with known functions in SPARC expressed NSCLC cells. KEGG enrichment analysis indicated that the differentially expressed genes were involved in cellular metabolic processes, regulation of biological processes or biological signaling pathways like cell cycle, spliceosome, apoptosis and WNT signaling pathway. The RNA-seq analysis further revealed that genes involved in apoptosis were significantly altered after SPARC modulation to NSCLC cells. Co-transfection experiments using SPARC shRNA indicated that SPARC modulation induced apoptosis via regulating PI3K/AKT signaling pathway. In summary, this study uncovers SPARC dependent regulatory mechanisms in NSCLC. Note: This abstract was not presented at the meeting. Citation Format: Sajani S. Lakka, Bhavesh K. Ahir. SPARC related signaling genes in non-small cell lung cancer by RNA-sequencing transcriptome analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3518.

Non aromatic hydrocarbon receptor dependent regulatory mechanism of cytochrome P4501A1 and its role in infection and inflammation

Infectious and inflammatory diseases are important diseases threatening human health. Without timely control, a series of complications will occur in patients, such as sepsis, inflammatory factor storm, and even lead to death. It has been found that cytochrome P4501A1 (CYP1A1) plays a key role in the development of infectious and inflammatory diseases through aromatic hydrocarbon receptor (AhR) dependent and non-dependent pathways in different cells and organs induced by different substances. The non AhR dependent regulatory mechanism of CYP1A1 and the different roles of CYP1A1 in infection and inflammation is reviewed in order to provide reference for further research on the relationship between CYP1A1 and infection and inflammation.

Genomic architecture of Shh-dependent cochlear morphogenesis.

The mammalian cochlea develops from a ventral outgrowth of the otic vesicle in response to Shh signaling. Mouse embryos lacking Shh or its essential signal transduction components display cochlear agenesis; however, a detailed understanding of the transcriptional network mediating this process is unclear. Here, we describe an integrated genomic approach to identify Shh-dependent genes and associated regulatory sequences that promote cochlear duct morphogenesis. A comparative transcriptome analysis of otic vesicles from mouse mutants exhibiting loss (Smoecko ) and gain (Shh-P1) of Shh signaling reveal a set of Shh-responsive genes partitioned into four expression categories in the ventral half of the otic vesicle. This target gene classification scheme provides novel insight into several unanticipated roles for Shh, including priming the cochlear epithelium for subsequent sensory development. We also mapped regions of open chromatin in the inner ear by ATAC-seq that, in combination with Gli2 ChIP-seq, identified inner ear enhancers in the vicinity of Shh-responsive genes. These datasets are useful entry points for deciphering Shh-dependent regulatory mechanisms involved in cochlear duct morphogenesis and establishment of its constituent cell types.

Menstrual blood-derived mesenchymal stem cells as a potential tool for cartilage regeneration

Purpose: Human mesenchymal stem cells (MSCs) are multipotent cells found in almost all human tissues. These cells are of great interest in regenerative medicine due to their high potential to differentiate into several directions including osteocytes, adipocytes and chondrocytes. Menstrual blood has been proposed as one of the perspective MSCs sources (MenSCs), due to their ease of access, as compared to the classical bone marrow MSCs (BMMSCs). As MSCs exhibit high chondrogenic differentiation potential, they ideally suit for therapeutic use in cartilage regeneration. Transforming growth factor β (TGF-β) has been considered as one of the major factors for induction of chondrogenic response in BMMSCs, however a member of the TGF-β superfamily Activin A also seems to contribute in the early stages of MSC chondrogenesis. Activin A seems necessary for the maintenance of self-renewal and pluripotency of MSCs. Furthermore there is an increasing body of evidence that Ca2+-dependent regulatory mechanisms can influence differentiation potential of chondrogenic progenitor cells, whereas Activin A was shown to increase free calcium concentrations in cells. Since Activin A also regulates menstrual cycle, we hypothesized that it may influence chondrogenic differentiation of MenSCs. The aim of this study was to characterize MenSCs and their chondrogenic differentiation capacity using two different growth factors - TGF-β3 and Activin A. The data was compared to the classical BMMSCs. Methods: BMMSCs and MenSCs were isolated from different healthy donors. Different cell surface markers (CD90, CD73, CD105, CD44, CD45, CD14, CD36, CD55, CD54, CD63, CD271, CD106, CD34, CD10, Stro1, Notch1) were analysed by flow cytometry. Cell proliferation capacity was determined by CCK-8 proliferation kit. Intracellular calcium was measured using fluorescent dye Cal-520. Adipogenic differentiation capacity was determined by Oil-Red staining and osteogenic differentiation by Alizarin Red staining. Chondrogenic differentiation (Activin A and TGF-b3 stimulation) was investigated by immuno- and histochemical staining with collagen II antibodies and safranin O, respectively. Results: In contrast to BMMSCs, MenSCs exhibited higher expression of CD10 and Notch1, and higher proliferative capacity. Furthermore, MenSCs exhibited higher level of intracellular calcium concentration as compared to BMMSCs. Adipogenic differentiation of MenSCs was weaker, whereas osteogenic differentiation capacity was higher compared to BMMSCs. Chondrogenic differentiation in BMMSCs was stronger with TGF-β3 than with Activin A, while MenSCs chondrogenic differentiation was remarkably more pronounced with Activin A than with TGF-β3. Conclusions: Taken together, our results demonstrate that MenSCs exhibit similar stem cell properties to BMMSCs, better proliferation potential and different regulatory mechanisms in chondrogenesis in vitro. Menstrual blood seems a convenient stem cells source for isolation of MSCs with chondrogenic differentiation potential, as does not require any invasive surgical interventions, and thus provides an option of repeated sample collection in the same donor, which make MenSCs a perspective tool in cartilage regenerative medicine.

Abstract 207: Electrically-induced Calcium Handling in Human Cardiac Progenitor Cells

The discovery of a resident population of cardiac stem cells known as cardiac progenitor cells (CPCs) has raised the question of the mechanisms involved in regenerative properties of this niche of cells. The physiology of human CPCs is not well understood, and a thorough understanding of the Ca 2+ handling ability of these cells is crucial in order to fully realize their regenerative potential. Therefore, to characterize the mechanism of electrically-induced Ca 2+ handling in human CPCs, we have used cells loaded with fluo-4 calcium dye and imaged using confocal microscopy to determine the spatial and temporal profile of cytosolic Ca 2+ upon electrical stimulation. Upon activation of electrical stimulation, a large increase in cytosolic Ca 2+ was observed followed by oscillations of the cytosolic Ca 2+ . The electrically-induced Ca 2+ oscillations were found to be dependent on the activity of L-type Ca 2+ channels (LTCC) and the presence of extracellular Ca 2+ . Furthermore, these oscillations could be abolished by pre-treatment of the cells with the inositol 1,4,5-trisphosphate receptor (IP 3 R) antagonist 2-APB. High-speed confocal imaging of the electrically-induced Ca 2+ oscillations in hCPCs revealed that these cells oscillate in a Ca 2+ wave-like fashion with activity originating in isolated regions of the cell and propagating throughout the cytosol and nucleus. In conclusion, we propose that Ca 2+ -dependent regulatory mechanisms can be exploited to influence their differentiation potential leading to improved function in cardiac regenerative medicine.

Cryopreserved Human Precision-Cut Lung Slices as a Bioassay for Live Tissue Banking. A Viability Study of Bronchodilation with Bitter-Taste Receptor Agonists.

Human precision-cut lung slices (hPCLSs) provide a unique ex vivo model for translational research. However, the limited and unpredictable availability of human lung tissue greatly impedes their use. Here, we demonstrate that cryopreservation of hPCLSs facilitates banking of live human lung tissue for routine use. Our results show that cryopreservation had little effect on overall cell viability and vital functions of immune cells, including phagocytes and T lymphocytes. In addition, airway contraction and relaxation in response to specific agonists and antagonists, respectively, were unchanged after cryopreservation. At the subcellular level, cryopreserved hPCLSs maintained Ca(2+)-dependent regulatory mechanisms for the control of airway smooth muscle cell contractility. To exemplify the use of cryopreserved hPCLSs in smooth muscle research, we provide evidence that bitter-taste receptor (TAS2R) agonists relax airways by blocking Ca(2+) oscillations in airway smooth muscle cells. In conclusion, the banking of cryopreserved hPCLSs provides a robust bioassay for translational research of lung physiology and disease.

Polarized Trafficking of AQP2 Revealed in Three Dimensional Epithelial Culture.

In renal collecting duct (CD) principal cells (PCs), vasopressin (VP) acts through its receptor, V2R, to increase intracellular cAMP leading to phosphorylation and apical membrane accumulation of the water channel aquaporin 2 (AQP2). The trafficking and function of basolaterally located AQP2 is, however, poorly understood. Here we report the successful application of a 3-dimensional Madin-Darby canine kidney (MDCK) epithelial model to study polarized AQP2 trafficking. This model recapitulates the luminal architecture of the CD and bi-polarized distribution of AQP2 as seen in kidney. Without stimulation, AQP2 is located in the subapical and basolateral regions. Treatment with VP, forskolin (FK), or 8-(4-Chlorophenylthio)-2′-O-methyladenosine 3′,5′-cyclic monophosphate monosodium hydrate (CPT-cAMP) leads to translocation of cytosolic AQP2 to the apical membrane, but not to the basolateral membrane. Treating cells with methyl-β-cyclodextrin (mβCD) to acutely block endocytosis causes accumulation of AQP2 on the basolateral membrane, but not on the apical membrane. Our data suggest that AQP2 may traffic differently at the apical and basolateral domains in this 3D epithelial model. In addition, application of a panel of phosphorylation specific AQP2 antibodies reveals the polarized, subcellular localization of differentially phosphorylated AQP2 at S256, S261, S264 and S269 in the 3D culture model, which is consistent with observations made in the CDs of VP treated animals, suggesting the preservation of phosphorylation dependent regulatory mechanism of AQP2 trafficking in this model. Therefore we have established a 3D culture model for the study of trafficking and regulation of both the apical and basolaterally targeted AQP2. The new model will enable further characterization of the complex mechanism regulating bi-polarized trafficking of AQP2 in vitro.

Cognitive Function and Calcium. Ca2+-dependent regulatory mechanism of circadian clock oscillation and its relevance to neuronal function

Circadian clock generates a variety of biological rhythms such as sleep/wake cycles and blood hormone rhythms. The circadian clock also bolsters daily mental activities. In fact, abnormalities of the circadian rhythms are found in several neurological disorders. The circadian clock has two important functions: (i) a cell-autonomous oscillatory function and (ii) a phase-adjusting function that synchronizes the clock oscillation with environmental cycling conditions such as light/dark cycle. Behavioral rhythms are controlled by the central clock in hypothalamic suprachiasmatic nucleus (SCN). The central clock orchestrates peripheral clocks in the other tissues via neuronal connection and/or actions of humoral factors. The molecular mechanism of the cell-autonomous clock is based on transcriptional feedback regulation of clock genes by their encoded products. Ca2+ is essential for not only the light response of the clock but also the cell autonomous oscillation mechanism. This article provides an overview of recent progress in studies of Ca2+-dependent regulatory mechanism of the molecular clockwork.

Purinergic signalling is required for calcium oscillations in migratory chondrogenic progenitor cells.

Osteoarthritis (OA) is the most common form of chronic musculoskeletal disorders. A migratory stem cell population termed chondrogenic progenitor cells (CPC) with in vitro chondrogenic potential was previously isolated from OA cartilage. Since intracellular Ca(2+) signalling is an important regulator of chondrogenesis, we aimed to provide a detailed understanding of the Ca(2+) homeostasis of CPCs. In this work, CPCs immortalised by lentiviral administration of the human telomerase reverse transcriptase (hTERT) and grown in monolayer cultures were studied. Expressions of all three IP3Rs were confirmed, but no RyR subtypes were detected. Ca(2+) oscillations observed in CPCs were predominantly dependent on Ca(2+) release and store replenishment via store-operated Ca(2+) entry; CPCs express both STIM1 and Orai1 proteins. Expressions of adenosine receptor mRNAs were verified, and adenosine elicited Ca(2+) transients. Various P2 receptor subtypes were identified; P2Y1 can bind ADP; P2Y4 is targeted by UTP; and ATP may evoke Ca(2+) transients via detected P2X subtypes, as well as P2Y1 and P2Y2. Enzymatic breakdown of extracellular nucleotides by apyrase completely abrogated Ca(2+) oscillations, suggesting that an autocrine/paracrine purinergic mechanism may drive Ca(2+) oscillations in these cells. As CPCs possess a broad spectrum of functional molecular elements of Ca(2+) signalling, Ca(2+)-dependent regulatory mechanisms can be supposed to influence their differentiation potential.

Soluble Beta-Amyloid Peptides, but Not Insoluble Fibrils, Have Specific Effect on Neuronal MicroRNA Expression

Recent studies indicate that soluble β-amyloid (sAβ) oligomers, rather than their fibrillar aggregates, contribute to the pathogenesis of Alzheimer's disease (AD), though the mechanisms of their neurotoxicity are still elusive. Here, we demonstrate that sAβ derived from 7PA2 cells exert a much stronger effect on the regulation of a set of functionally validated microRNAs (miRNAs) in primary cultured neurons than the synthetic insoluble Aβ fibrils (fAβ). Synthetic sAβ peptides at a higher concentration present comparable effect on these miRNAs in our neuronal model. Further, the sAβ-induced miR-134, miR-145 and miR-210 expressions are fully reversed by two selective N-methyl-d-aspartate (NMDA) receptor inhibitors, but are neither reversed by insulin nor by forskolin, suggesting an NMDA receptor-dependent, rather than PI3K/AKT or PKA/CREB signaling dependent regulatory mechanism. In addition, the repression of miR-107 expression by the sAβ containing 7PA2 CM is likely involved multiple mechanisms and multiple players including NMDA receptor, N-terminally truncated Aβ and reactive oxygen species (ROS).

Ca2+/S100 proteins inhibit the interaction of FKBP38 with Bcl-2 and Hsp90

FKBP38 (FK506-binding protein 38), a membrane-anchored TPR (tetratricopeptide repeat)-containing immunophilin, regulates signalling pathways such as cell survival, apoptosis, proliferation and metastasis. However, the mechanisms that regulate the activity of FKBP38 are, at present, poorly understood. We previously reported that Ca2+/S100 proteins directly associate with the TPR proteins, such as Hop [Hsp70 (heat-shock protein of 70 kDa)/Hsp90-organizing protein], kinesin-light chain, Tom70 (translocase of outer mitochondrial membrane 70), FKBP52, CyP40 (cyclophilin 40), CHIP (C-terminus of Hsc70-interacting protein) and PP5 (protein phosphatase 5), leading to the dissociation of the interactions of the TPR proteins with their target proteins. Therefore we have hypothesized that Ca2+/S100 proteins can interact with FKBP38 and regulate its function. In vitro binding studies demonstrated that S100A1, S100A2, S100A6, S100B and S100P specifically interact with FKBP38 and inhibit the interaction of FKBP38 with Bcl-2 and Hsp90. Overexpression of permanently active S100P in Huh-7 cells inhibited the interaction of FKBP38 with Bcl-2, resulting in the suppression of Bcl-2 stability. The association of the S100 proteins with FKBP38 provides a Ca2+-dependent regulatory mechanism of the FKBP38-mediated signalling pathways.