Hyperosmotic Conditions Research Articles

Calcium-TonEBP1 signaling regulates the expression of matrix metalloproteinases in 29-kDA FN-f-treated chondrocytes

Purpose: Under hyperosmotic condition, tonicity-responsive enhancer-binding protein 1 (TonEBP1/nuclear factor of activated T-cells 5 (NFAT5)) modulates the expression of osmoprotective genes through Ca2+/calcineurin (CaN) signaling pathway. Fibronectin fragments (FN-fs), which are highly found in the synovial fluid of patients with osteoarthritis, induce matrix metalloproteinases (MMPs) expression via toll-like receptor-2 signaling pathway. In this study we examined whether 29-kDa FN-f has an influence on activation of Ca2+/TonEBP1 signaling pathway. Methods: Human articular chondrocytes were enzymatically isolated from articular cartilage and cultured in monolayer. The relative levels of mRNA and protein were analyzed by real-time quantitative reverse transcription-polymerase chain reaction and immuoblotting analysis, respectively. TonEBP1, CaN, and calmodulin (CaM) genes were knocked-down by transfection with small interfering RNAs (siRNAs). Results: The expressions of TonEBP1, CaM and CaN were significantly higher in human OA cartilage than normal cartilage. 29-kDa FN-f induced the expressions of TonEBP1, CaM and CaN at both mRNA and protein levels and then increased dephosphorylation of TonEBP1 and its translocation into the nucleus. Silencing of CaM, CaN, or TonEBP1 suppressed the expressions of MMP-1, -3, and -13 induced by 29-kDa FN-f, showing that CaM/CaN/TonEBP1 pathway may be implicated in 29-kDa FN-f-induced MMPs expression. In addition, inhibition of Ca2+ influx with BAPTA and thapsigargin revealed that Ca2+ is an important regulator in 29-kDa FN-f-induced MMPs expression. Furthermore, 29-kDa FN-f triggered the expression of MMPs via the phospholipase C/ phosphokinase C signaling axis. Conclusions: These results demonstrated that 29-kDa FN-f modulated the expression of MMPs through activation of Ca2+/CaM/CaN/TonEBP1 signaling pathway together with alteration of Ca2+ influx.

Deformation Microscopy for Dynamic Intracellular and Intranuclear Mapping of Mechanics with High Spatiotemporal Resolution

SUMMARYStructural heterogeneity is a hallmark of living cells that drives local mechanical properties and dynamic cellular responses. However, the robust quantification of intracellular mechanics is lacking from conventional methods. Here, we describe the development of deformation microscopy, which leverages conventional imaging and an automated hyperelastic warping algorithm to investigate strain history, deformation dynamics, and changes in structural heterogeneity within the interior of cells and cell nuclei. Using deformation microscopy, we found that partial or complete disruption of LINC complexes in cardiomyocytes in vitro and lamin A/C deficiency in myocytes in vivo abrogate dominant tensile loading in the nuclear interior. We also found that cells cultured on stiff substrates or in hyperosmotic conditions displayed abnormal strain burden and asymmetries at interchromatin regions, which are associated with active transcription. Deformation microscopy represents a foundational approach toward intracellular elastography, with the potential utility to provide mechanistic and quantitative insights in diverse mechanobiological applications.

The putative histone-like transcription factor FgHltf1 is required for vegetative growth, sexual reproduction, and virulence in Fusarium graminearum.

The target of rapamycin (TOR) signaling pathway plays critical roles in regulating vegetative development and virulence in Fusarium graminearum. Previously, we have demonstrated that the putative type 2A phosphatase FgPpg1, a downstream component of the pathway, is important for hyphal growth, sporulation, DON biosynthesis and virulence. Here, we report the identification of FgHLTF1 putatively encoding a histone-like transcription factor by the transcriptome analysis of an ΔFgppg1 mutant. The FgHLTF1 expression was significantly down-regulated by the deletion of FgPPG1 or treatment with rapamycin. Analysis of an F. graminearum strain expressing green fluorescent protein (GFP) revealed that FgHltf1-GFP fusion protein mainly localized to the nucleus. Targeted gene deletion mutants of FgHLTF1 exhibited a significant reduction in vegetative growth, sexual reproduction and virulence. Moreover, the growth of the ΔFghltf1 mutants was restricted by hyperosmotic stresses. Unlike the wild-type strain, the mutants showed anomalous subcellular translocation of FgHog1-GFP under hyperosmotic conditions, suggesting that FgHLTF1 is associated with the high osmolarity glycerol response pathway. Taken together, we conclude that FgHLTF1 is transcriptionally regulated by the TOR signaling pathway and plays important roles in regulating vegetative growth, sexual reproduction, virulence and hyperosmotic stresses in F. graminearum.

Function of L-Pipecolic Acid as Compatible Solute in Corynebacterium glutamicum as Basis for Its Production Under Hyperosmolar Conditions.

Pipecolic acid or L-PA is a cyclic amino acid derived from L-lysine which has gained interest in the recent years within the pharmaceutical and chemical industries. L-PA can be produced efficiently using recombinant Corynebacterium glutamicum strains by expanding the natural L-lysine biosynthetic pathway. L-PA is a six-membered ring homolog of the five-membered ring amino acid L-proline, which serves as compatible solute in C. glutamicum.Here, we show that de novo synthesized or externally added L-PA partially is beneficial for growth under hyper-osmotic stress conditions. C. glutamicum cells accumulated L-PA under elevated osmotic pressure and released it after an osmotic down shock. In the absence of the mechanosensitive channel YggB intracellular L-PA concentrations increased and its release after osmotic down shock was slower. The proline permease ProP was identified as a candidate L-PA uptake system since RNAseq analysis revealed increased proP RNA levels upon L-PA production. Under hyper-osmotic conditions, a ΔproP strain showed similar growth behavior than the parent strain when L-proline was added externally. By contrast, the growth impairment of the ΔproP strain under hyper-osmotic conditions could not be alleviated by addition of L-PA unless proP was expressed from a plasmid. This is commensurate with the view that L-proline can be imported into the C. glutamicum cell by ProP and other transporters such as EctP and PutP, while ProP appears of major importance for L-PA uptake under hyper-osmotic stress conditions.

Hyper-Osmotic Stress Elicits Membrane Depolarization and Decreased Permeability in Halotolerant Marine Debaryomyces hansenii Strains and in Saccharomyces cerevisiae.

The use of seawater and marine microorganisms can represent a sustainable alternative to avoid large consumption of freshwater performing industrial bioprocesses. Debaryomyces hansenii, which is a known halotolerant yeast, possess metabolic traits appealing for developing such processes. For this purpose, we studied salt stress exposure of two D. hansenii strains isolated from marine fauna. We found that the presence of sea salts during the cultivation results in a slight decrease of biomass yields. Nevertheless, higher concentration of NaCl (2 M) negatively affects other growth parameters, like growth rate and glucose consumption rate. To maintain an isosmotic condition, the cells accumulate glycerol as compatible solute. Flow cytometry analysis revealed that the osmotic adaptation causes a reduced cellular permeability to cell-permeant dye SYBR Green I. We demonstrate that this fast and reversible phenomenon is correlated to the induction of membrane depolarization, and occurred even in presence of high concentration of sorbitol. The decrease of membrane permeability induced by osmotic stress confers to D. hansenii resistance to cationic drugs like Hygromycin B. In addition, we describe that also in Saccharomyces cerevisiae the exposure to hyper-osmotic conditions induced membrane depolarization and reduced the membrane permeability. These aspects are very relevant for the optimization of industrial bioprocesses, as in the case of fermentations and bioconversions carried out by using media/buffers containing high nutrients/salts concentrations. Indeed, an efficient transport of molecules (nutrients, substrates, and products) is the prerequisite for an efficient cellular performance, and ultimately for the efficiency of the industrial process.

Mechanical Shielding in Plant Nuclei

In animal single cells, nuclear stiffness scales to the cell’s environment, questioning the physiological relevance of this finding in a multicellular context and the associated molecular mechanisms. Using the Arabidopsis root tip as a model system, and combining morphometry and micro-rheometry, we found that hyperosmotic stress decreases nuclear circularity and size and increases nuclear stiffness. These changes were accompanied by enhanced expression of mechanosensitive genes previously characterized in response to touch. These responses were reversible in the wild type, but were constitutively observed and more pronounced in gip1gip2, a mutant affected in the nuclear envelope GIPs proteins, homologs of MZT1. Such mutant even became resistant to lethal hyperosmotic conditions. Altogether we unravel an integrated nuclear response to hyperosmotic stress and propose a GIP-dependent mechanism for the mechanical protection of nuclei.

Living without the cell wall.

This study found that when filamentous actinomycetes are exposed to osmotic stress they extrude previously undetected cell wall-deficient cells, termed S-cells, that enable actinomycetes to thrive under hyperosmotic stress conditions.

Stress-driven increase in proline levels, and not proline levels themselves, correlates with the ability to withstand excess salt in a group of 17 Italian rice genotypes.

In most plant species, a rapid increase in free proline content occurs following exposure to hyperosmotic stress conditions. However, inconsistent results were reported concerning the role of such an increase on the plant response to water shortage or excess salt. Therefore, the possibility that proline accumulation may help the cell to withstand stress conditions, or that it simply represents a stress marker, is still a matter of debate. A possible relationship between proline accumulation and salt tolerance was investigated in a set of 17 Italian rice varieties. Rice seedlings were exposed to increasing salt concentrations during germination and early growth. The resulting levels of free proline were measured separately in shoots and roots and compared to those in untreated controls. Results were related to the corresponding ability of a given genotype to tolerate stress conditions. Neither absolute proline levels in untreated or in salt-stressed seedlings showed a straightforward relationship to the relative tolerance to salt, estimated as conductivity values able to reduce growth by 10 or 50%. Conversely, a highly significant correlation was found between the increase in proline levels in shoots and the ability to withstand stress. The results strengthen a recent hypothesis suggesting than an increase in proline metabolic rates, more than the resulting proline content, may help the cell to counteract the effects of abiotic stress conditions.

Induction of Osmolyte Pathways in Skeletal Muscle Inflammation: Novel Biomarkers for Myositis.

We recently identified osmolyte accumulators as novel biomarkers for chronic skeletal muscle inflammation and weakness, but their precise involvement in inflammatory myopathies remains elusive. In the current study, we demonstrate in vitro that, in myoblasts and myotubes exposed to pro-inflammatory cytokines or increased salt concentration, mRNA levels of the osmolyte carriers SLC5A3, SLC6A6, SLC6A12, and AKR1B1 enzyme can be upregulated. Induction of SLC6A12 and AKR1B1 was confirmed at the protein level using immunofluorescence and Western blotting. Gene silencing by specific siRNAs revealed that these factors were vital for muscle cells under hyperosmotic conditions. Pro-inflammatory cytokines activated mitogen-activated protein kinases, nuclear factor κB as well as nuclear factor of activated T-cells 5 mRNA expression. In muscle biopsies from patients with polymyositis or sporadic inclusion body myositis, osmolyte pathway activation was observed in regenerating muscle fibers. In addition, the osmolyte carriers SLC5A3 and SLC6A12 localized to subsets of immune cells, most notably to the endomysial macrophages and T-cells. Collectively, this study unveiled that muscle cells respond to osmotic and inflammatory stress by osmolyte pathway activation, likely orchestrating general protection of the tissue. Moreover, pro-inflammatory properties are attributed to SLC5A3 and SLC6A12 in auto-aggressive macrophages and T-cells in inflamed skeletal muscle.

A molecular dynamics approach towards evaluating osmotic and thermal stress in the extracellular environment

Objective: A molecular dynamics approach to understanding fundamental mechanisms of combined thermal and osmotic stress induced by thermochemical ablation (TCA) is presented.Methods: Structural models of fibronectin and fibronectin bound to its integrin receptor provide idealized models for the effects of thermal and osmotic stress in the extracellular matrix. Fibronectin binding to integrin is known to facilitate cell survival. The extracellular environment produced by TCA at the lesion boundary was modelled at 37 °C and 43 °C with added sodium chloride (NaCl) concentrations (0, 40, 80, 160, and 320 mM). Atomistic simulations of solvated proteins were performed using the GROMOS96 force field and TIP3P water model. Computational results were compared with the results of viability studies of human hepatocellular carcinoma (HCC) cell lines HepG2 and Hep3B under matching thermal and osmotic experimental conditions.Results: Cell viability was inversely correlated with hyperthermal and hyperosmotic stresses. Added NaCl concentrations were correlated with a root mean square fluctuation increase of the fibronectin arginylglycylaspartic acid (RGD) binding domain. Computed interaction coefficients demonstrate preferential hydration of the protein model and are correlated with salt-induced strengthening of hydrophobic interactions. Under the combined hyperthermal and hyperosmotic stress conditions (43 °C and 320 mM added NaCl), the free energy change required for fibronectin binding to integrin was less favorable than that for binding under control conditions (37 °C and 0 mM added NaCl).Conclusion: Results quantify multiple measures of structural changes as a function of temperature increase and addition of NaCl to the solution. Correlations between cell viability and stability measures suggest that protein aggregates, non-functional proteins, and less favorable cell attachment conditions have a role in TCA-induced cell stress.

Capacity of tissue water regulation is impaired in an osmoconformer living in impacted estuaries?

Estuarine osmoconformes rely on their ability to perform tissue and cell water regulation to cope with daily osmotic challenges that occur in the estuary. In addition, these animals currently must deal with pollutants present in the estuarine environment, which can disturb their capacity of water regulation. We collected the mangrove oyster Crassostrea rhizophorae in two tropical estuaries in the Northeast region of Brazil with different degrees of human interference: the Paraíba Estuary (impacted) and the Mamanguape Estuary (preserved). Tissue water content was analyzed after exposure to salinities 12, 24 and 36 for 24 h. Gill cell volume regulation was analyzed in vitro upon hypo- and hyper-osmotic conditions. We also analyzed gill MXR (multi-xenobiotic resistance) mechanism, as reference of environmental pollution. Gill and muscle of oysters from two sites of Paraíba Estuary, and from one site of Mamanguape Estuary were not able to maintain tissue water content upon hypo- and hyper-osmotic conditions. Gill cells of oyster from the same sites exhibited swelling followed by regulatory volume decrease upon hypo-osmotic condition. Gill MXR activity was increased in oysters from these sites. The best tissue and cell water regulation, and the lowest MXR activity, was found in oyster from downstream of Mamanguape Estuary, our reference site and the one most preserved. Tissue and cell water regulation proved to be a sensitive parameter to environmental pollution and could be considered as biomarker of aquatic contamination.

Hyperosmotic stress stimulates autophagy viatheNFAT5/mTOR pathway in cardiomyocytes.

Hyperosmotic stress may be initiated during a diverse range pathological circumstances, which in turn results in tissue damage. In this process, the activation of survival signaling, which has the capacity to restore cell homeostasis, determines cell fate. Autophagy is responsible for cell survival and is activated by various pathological stimuli. However, its interplay with hyperosmotic stress and its effect on terminally differentiated cardiac myocytes is unknown. Nuclear factor of activated T‑cells 5 (NFAT5), an osmo‑sensitive transcription factor, mediates the expression of cell survival associated‑genes under hyperosmotic conditions. The present study investigated whether NFAT5 signaling is required in hyperosmotic stress‑induced autophagy. It was demonstrated that the presence of a hyperosmotic stress induced an increase in NFAT5 expression, which in turn triggered autophagy through autophagy‑related protein 5 (Atg5) activation. By contrast, NFAT5 silencing inhibited DNA damage response 1 protein expression, which then initiated the activation of mammalian target of rapamycin signaling. Therefore, the balance between NFAT5‑induced apoptosis and autophagy may serve a critical role in the determination of the fate of cardiomyocytes under hyperosmotic stress. These data suggest that autophagy activation is a beneficial adaptive response to attenuate hyperosmotic stress‑induced cell death. Therefore, increasing autophagy through activation of NFAT5 may provide a novel cardioprotective strategy against hyperosmotic stress‑induced damage.

Coherent Feedforward Regulation of Gene Expression by Caulobacter σT and GsrN during Hyperosmotic Stress.

GsrN is a conserved small RNA that is under transcriptional control of the general stress sigma factor, σT, and that functions as a posttranscriptional regulator of Caulobacter crescentus survival under multiple stress conditions. We have defined features of GsrN structure that determine survival under hyperosmotic stress, and we have applied transcriptomic and proteomic methods to identify regulatory targets of GsrN under hyperosmotic conditions. The 5' end of GsrN, which includes a conserved cytosine-rich stem-loop structure, is necessary for cell survival after osmotic upshock. GsrN both activates and represses gene expression in this stress condition. Expression of an uncharacterized open reading frame predicted to encode a glycine zipper protein, osrP, is strongly activated by GsrN. Our data support a model in which GsrN physically interacts with osrP mRNA through its 5' C-rich stem-loop to enhance OsrP protein expression. We conclude that sigT, gsrN, and osrP form a coherent feedforward loop in which σT activates gsrN and osrP transcription during stress and GsrN activates OsrP protein expression at the posttranscriptional level. This study delineates transcriptional and posttranscriptional layers of Caulobacter gene expression control during hyperosmotic stress, uncovers a new regulatory target of GsrN, and defines a coherent feedforward motif in the Caulobacter general stress response (GSR) regulatory network.IMPORTANCE Bacteria inhabit diverse niches and must adapt their physiology to constant environmental fluctuations. A major response to environmental perturbation is to change gene expression. Caulobacter and other alphaproteobacteria initiate a complex gene expression program known as the general stress response (GSR) under conditions including oxidative stress, osmotic stress, and nutrient limitation. The GSR enables cell survival in these environments. Understanding how bacteria survive stress requires that we dissect gene expression responses, such as the GSR, at the molecular level. This study is significant, as it defines transcriptional and posttranscriptional layers of gene expression regulation in response to hyperosmotic stress. We further provide evidence that a coherent feedforward motif influences the system properties of the Caulobacter GSR pathway.

Multimarker response to salinity stress in two estuarine bivalves of different genetic diversity: Mya arenaria and Limecola balthica from the Gulf of Gdańsk (southern Baltic Sea)

AbstractThe estuarine bivalves Limecola balthica and Mya arenaria are common inhabitants of marine soft bottom habitats in the Northern Hemisphere. Both species are able to live under a wide range of environmental conditions including variable salinity. However, in L. balthica there is high genetic variability, and populations are often genetically adapted to local conditions. By contrast, genetic diversity in M. arenaria is low across the species’ geographic range, which attests to acclimatization to different conditions. We hypothesized that individuals of M. arenaria should perform better under osmotic stress. We tested this hypothesis by performing a 5‐week experiment that exposed individuals of both clam species to hypo‐ and hyperosmotic conditions. A multiple biomarker approach that included physiological, biochemical, and histological markers was used to assess bivalve performance. Exposure to the different salinities induced biological responses that particularly affected respiratory activity in both species tested, but these responses were much more pronounced in individuals of L. balthica. The results confirmed the hypothesis that the phenotypic plasticity of M. arenaria was more pronounced and reflected a different strategy of adapting to heterogeneous habitats.

Crystallizable W/O/W double emulsions made with milk fat: Formulation, stability and release properties

Food grade Water-in-oil-in-water W/O/W double emulsions with high encapsulation efficiency, controlled texture and triggered leakage are usually sought after in many food applications (e.g. functional foods) and still the subject of active research interest. For that purpose, we have used an edible crystallizable oil which is Anhydrous Milkfat (AMF) with high encapsulation properties, as recently demonstrated. The emulsions encapsulating a nutrient (MgCl2) were formulated using two surface-active species (polyglycerol polyricinoleate and sodium caseinate being also a chelating agent able to bind magnesium ions). The internal droplet and oil globule diameters were identical for all the systems. The stability of the double emulsions and the rate of magnesium release from the internal to the external aqueous phase were monitored during 24 days of storage at 4 °C, as a function of the formulation and globule volume fraction. It was found that in all cases, magnesium leakage occurred without film rupturing (no coalescence) and the double structure was preserved with time. On the other hand and at iso-osmotic conditions, the rate of Mg release was low (does not exceed 14% after 24 days of storage at 4 °C), independent on the concentration of sodium caseinate in the external aqueous phase, remained time independent and its level of release was lowered by increasing the globule volume fraction. Under moderate hypo osmotic conditions (∆P = +1.159 atm until +3.343 atm), the Mg release increased with the osmotic pressure gradient but remained also time independent. However, at large osmotic gradient (∆P = +5.503 atm), the Mg2+ release was triggered fastly and increased with time, in agreement with a no more protective structure, being beneficial for several food applications. The experimental data were interpreted and the permeation coefficient of Mg was determined within the frame of a mean-field model based on diffusion/permeation, integrating the transport in opposite direction than Mg2+, of the osmotic regulator, even occurring at different rates through the oil phase. Under hyper osmotic conditions (even large, ∆P = −7.502 atm), the Mg release was low, time independent and the double structure was usually preserved over time. So, this study allows to improve magnesium retention and its controlled release, in particular for nutritional and food applications.

The Influence of Hyperosmolarity in the Intervertebral Disc on the Proliferation and Chondrogenic Differentiation of Nucleus Pulposus-Derived Mesenchymal Stem Cells

Nucleus pulposus-derived mesenchymal stem cells (NP-MSCs) are suitable cell candidates for intervertebral disc (IVD) regeneration. However, little work has been done to determine the proliferation and chondrogenic differentiation of NP-MSCs in the hyperosmotic microenvironment of IVD. This study aimed to investigate the influence of the hyperosmolarity of IVD on the proliferation and chondrogenic differ­entiation of NP-MSCs. NP-MSCs were cultured in media of 300, 400, 430, and 500 mOsm/L, mimicking the osmotic pressures of serious degenerative, moderately degenerative, and healthy IVD. Cell proliferation was measured by CCK-8 assay. The expression of aggrecan, collagen I, and collagen II were measured by gene and protein expression analysis. Alcian blue and dimethylmethylene blue assay were used to investigate the accumulation of sulfate glycosaminoglycan. The regulation role of extracellular signal-regulated kinase (ERK) pathway was also analyzed. The results showed that, compared to 300 mOsm/L, hyperosmolarity of healthy IVD (430 and 500 mOsm/L) inhibited the proliferation and chondrogenic differentiation of NP-MSCs. The relative hypoosmotic condition of moderately degenerative IVD (400 mOsm/L) led to great proliferation and chondrogenic differentiation capacity. The ERK pathway was activated by the hyperosmolarity; inhibition of the ERK pathway abolished the difference in cell proliferation between the 300 mOsm/L and the hyperosmotic conditions, and enhanced chondrogenic differentiation. In conclusion, hyperosmolarity of IVD had a significant impact on the proliferation and chondrogenic differentiation of NP-MSCs. The ERK pathway was involved in the inhibition of proliferation and chondrogenic differentiation of NP-MSCs by the hyperosmolarity of IVD. The relative hypo-osmotic condition prevailing in degenerative discs offers a more permissive microenvironment for NP-MSCs.

Overexpression of Outer Membrane Protein X (OmpX) Compensates for the Effect of TolC Inactivation on Biofilm Formation and Curli Production in Extraintestinal Pathogenic Escherichia coli (ExPEC).

Our previous study showed that the inactivation of the efflux pump TolC could abolish biofilm formation and curli production of extraintestinal pathogenic Escherichia coli (ExPEC) strain PPECC42 under hyper-osmotic conditions. In this study we investigated the role of OmpX in biofilm formation and curli production of ExPEC PPECC42. Our data showed that OmpX disruption or overexpression didn't significantly affect the biofilm formation and curli production of the wild-type strain. However, in the tolC-deleted mutant, overexpressing OmpX suppressed the effect of TolC inactivation on ExPEC biofilm formation and curli production under hyper-osmotic growth conditions. Real-time qRT-PCR confirmed that OmpX overexpression affected curli production by regulating the transcription of the curli biosynthesis-related genes in the ΔtolC strain. Our findings suggest that OmpX is involved in biofilm formation and curli production.

TAG synthesis and storage under osmotic stress. A requirement for preserving membrane homeostasis in renal cells

Hyperosmolarity is a controversial signal for renal cells. It can induce cell stress or differentiation and both require an active lipid metabolism. We showed that hyperosmolarity upregulates phospholipid (PL) de novo synthesis in renal cells. PL synthesis requires fatty acids (FA), usually stored as triglycerides (TAG). PL and TAG de novo synthesis utilize the same initial biosynthetic route: sn-glycerol 3P (G3P) → phosphatidic acid (PA) → diacylglycerol (DAG). In the present work, we evaluate how such pathway contributes to PL and TAG synthesis in renal cells subjected to hyperosmolarity. Our results show an increase in PA and DAG formation under hyperosmotic conditions; augmented DAG production, due to lipin enzyme activity, lead to the increase of both TAG and PL. However, at early stages (24 and 48 h), most of the de novo synthesized DAG was directed to PL synthesis; longer treatments downregulated PL synthesis and the DAG formed was mainly driven to TAG synthesis. Hyperosmolarity induced ACC and FASN transcription which mediated FA de novo synthesis. New FA molecules were stored in TAG. Silencing experiments revealed that hyperosmotic-induction of lipin-1 and -2 was mediated by SREBP1. Interestingly, SREBP1 knockdown also dropped SREBP2, indicating a modulatory action between both isoforms. Impairing SREBP activity leads to a decline in TAG levels but not PL. Membrane homeostasis is maintained through the adequate PL synthesis and renewal and constitute a protective mechanism against hyperosmolarity. The present data reveal the relevance of TAG synthesis and storage for PL synthesis in renal cells.

COX-2 expression mediated by calcium-TonEBP signaling axis under hyperosmotic conditions serves osmoprotective function in nucleus pulposus cells

The nucleus pulposus (NP) of intervertebral discs experiences dynamic changes in tissue osmolarity because of diurnal loading of the spine. TonEBP/NFAT5 is a transcription factor that is critical in osmoregulation as well as survival of NP cells in the hyperosmotic milieu. The goal of this study was to investigate whether cyclooxygenase-2 (COX-2) expression is osmoresponsive and dependent on TonEBP, and whether it serves an osmoprotective role. NP cells up-regulated COX-2 expression in hyperosmotic media. The induction of COX-2 depended on elevation of intracellular calcium levels and p38 MAPK pathway, but independent of calcineurin signaling as well as MEK/ERK and JNK pathways. Under hyperosmotic conditions, both COX-2 mRNA stability and its proximal promoter activity were increased. The proximal COX-2 promoter (-1840/+123 bp) contained predicted binding sites for TonEBP, AP-1, NF-κB, and C/EBP-β. While COX-2 promoter activity was positively regulated by both AP-1 and NF-κB, AP-1 had no effect and NF-κB negatively regulated COX-2 protein levels under hyperosmotic conditions. On the other hand, TonEBP was necessary for both COX-2 promoter activity and protein up-regulation in response to hyperosmotic stimuli. Ex vivo disc organ culture studies using hypomorphic TonEBP+/- mice confirmed that TonEBP is required for hyperosmotic induction of COX-2. Importantly, the inhibition of COX-2 activity under hyperosmotic conditions resulted in decreased cell viability, suggesting that COX-2 plays a cytoprotective and homeostatic role in NP cells for their adaptation to dynamically loaded hyperosmotic niches.

Major traits of the senescent phenotype of nucleus pulposus intervertebral disc cells persist under the specific microenvironmental conditions of the tissue

Intervertebral discs (IVDs) are the joints of the spine, mainly consisting of extracellular matrix (ECM) with a low number of cells embedded therein. Low cellularity stems from nutrient deprivation due to the lack of blood supply, as well as from the hypoxic and hyperosmotic conditions prevailing in the tissue. Intervertebral disc degeneration (IDD) has been firmly connected with low back pain, a major age-related disease, whereas degenerated discs have been characterized by increased proteolytic activity and accumulation of senescent cells. While the catabolic phenotype of senescent IVD cells has been documented, whether this phenotype is preserved under the harsh conditions met in the IVD milieu has never been investigated. Here we showed that a combination of low glucose, hypoxia, high osmolality and absence of serum is anti-proliferative for young disc cells. In addition, we demonstrated for the first time that classical senescence markers, namely p16INK4a, p21WAF1 and ICAM-1, remain up-regulated in senescent cells under these conditions. Finally, up-regulation of the main senescence-associated ECM degrading enzymes, i.e. MMP-1, -2 and -3 was maintained in this strict environment. Conservation of IVD cells’ senescent phenotype under the actual conditions these cells are confronted with in vivo further supports their possible implication in IDD.