Evidence that TNF-β (lymphotoxin α) can activate the inflammatory environment in human chondrocytes

Introduction

Inflammatory cytokines play a key role in the pathogenesis of joint diseases such as rheumatoid arthritis (RA). Current therapies target mainly tumor necrosis factor α (TNF-α) as this has proven benefits. However, a large number of patients do not respond to or become resistant to anti-TNF-α therapy. While the role of TNF-α in RA is quite evident, the role of TNF-β, also called lymphotoxin-α (LT-α), is unclear. In this study we investigated whether TNF-β and its receptor play a role in chondrocytes in the inflammatory environment.

Methods

An in vitro model of primary human chondrocytes was used to study TNF-β-mediated inflammatory signaling.

Results

Cytokine-induced inflammation enhances TNF-β and TNF-β-receptor expression in primary human chondrocytes accompanied by the up-regulation of inflammatory (cyclooxygenase-2), matrix degrading (matrix metalloproteinase-9 and -13) and apoptotic (p53, cleaved caspase-3) signaling pathways, all known to be regulated by NF-κB. In contrast, anti-TNF-β, similar to the natural NF-κB inhibitor (curcumin, diferuloylmethane) or the knockdown of NF-κB by using antisense oligonucleotides (ASO), suppressed IL-1β-induced NF-κB activation and its translocation to the nucleus, and abolished the pro-inflammatory and apoptotic effects of IL-1β. This highlights, at least in part, the crucial role of NF-κB in TNF-β-induced-inflammation in cartilage, similar to that expected for TNF-α. Finally, the adhesiveness between TNF-β-expressing T-lymphocytes and the responding chondrocytes was significantly enhanced through a TNF-β-induced inflammatory microenvironment.

Conclusions

These results suggest for the first time that TNF-β is involved in microenvironment inflammation in chondrocytes during RA parallel to TNF-α, resulting in the up-regulation of NF-κB signaling and activation of pro-inflammatory activity.     

MS4A15 drives ferroptosis resistance through calcium-restricted lipid remodeling

Ferroptosis is an iron-dependent form of cell death driven by biochemical processes that promote oxidation within the lipid compartment. Calcium (Ca²⁺) is a signaling molecule in diverse cellular processes such as migration, neurotransmission, and cell death. Here, we uncover a crucial link between ferroptosis and Ca²⁺ through the identification of the novel tetraspanin MS4A15. MS4A15 localizes to the endoplasmic reticulum, where it blocks ferroptosis by depleting luminal Ca²⁺ stores and reprogramming membrane phospholipids to ferroptosis-resistant species. Specifically, prolonged Ca²⁺ depletion inhibits lipid elongation and desaturation, driving lipid droplet dispersion and formation of shorter, more saturated ether lipids that protect phospholipids from ferroptotic reactive species. We further demonstrate that increasing luminal Ca²⁺ levels can preferentially sensitize refractory cancer cell lines. In summary, MS4A15 regulation of anti-ferroptotic lipid reservoirs provides a key resistance mechanism that is distinct from antioxidant and lipid detoxification pathways. Manipulating Ca²⁺ homeostasis offers a compelling strategy to balance cellular lipids and cell survival in ferroptosis-associated diseases.Subject terms: Lipidomics, Cancer genetics, Cell biology     

Ablation of cdk4 and cdk6 affects proliferation of basal progenitor cells in the developing dorsal and ventral forebrain

Little is known about the molecular players driving proliferation of neural progenitor cells (NPCs) during embryonic mouse development. Here, we demonstrate that proliferation of NPCs in the developing forebrain depends on a particular combination of cell cycle regulators. We have analyzed the requirements for members of the cyclin‐dependent kinase (cdk) family using cdk‐deficient mice. In the absence of either cdk4 or cdk6, which are both regulators of the G1 phase of the cell cycle, we found no significant effects on the proliferation rate of cortical progenitor cells. However, concomitant loss of cdk4 and cdk6 led to a drastic decrease in the proliferation rate of NPCs, specifically the basal progenitor cells of both the dorsal and ventral forebrain at embryonic day 13.5 (E13.5). Moreover, basal progenitors in the forebrain of Cdk4;Cdk6 double mutant mice exhibited altered cell cycle characteristics. Cdk4;cdk6 deficiency led to an increase in cell cycle length and cell cycle exit of mutant basal progenitor cells in comparison to controls. In contrast, concomitant ablation of cdk2 and cdk6 had no effect on the proliferation of NCPs. Together, our data demonstrate that the expansion of the basal progenitor pool in the developing telencephalon is dependent on the presence of distinct combinations of cdk molecules. Our results provide further evidence for differences in the regulation of proliferation between apical and basal progenitors during cortical development. © 2018 Wiley Periodicals, Inc. Develop Neurobiol 78: 660–670, 2018     

Knocking Down of Isoprene Emission Modifies the Lipid Matrix of Thylakoid Membranes and Influences the Chloroplast Ultrastructure in Poplar

Isoprene is a small lipophilic molecule with important functions in plant protection against abiotic stresses. Here, we studied the lipid composition of thylakoid membranes and chloroplast ultrastructure in isoprene-emitting (IE) and nonisoprene-emitting (NE) poplar (Populus × canescens). We demonstrated that the total amount of monogalactosyldiacylglycerols, digalactosyldiacylglycerols, phospholipids, and fatty acids is reduced in chloroplasts when isoprene biosynthesis is blocked. A significantly lower amount of unsaturated fatty acids, particularly linolenic acid in NE chloroplasts, was associated with the reduced fluidity of thylakoid membranes, which in turn negatively affects photosystem II photochemical efficiency. The low photosystem II photochemical efficiency in NE plants was negatively correlated with nonphotochemical quenching and the energy-dependent component of nonphotochemical quenching. Transmission electron microscopy revealed alterations in the chloroplast ultrastructure in NE compared with IE plants. NE chloroplasts were more rounded and contained fewer grana stacks and longer stroma thylakoids, more plastoglobules, and larger associative zones between chloroplasts and mitochondria. These results strongly support the idea that in IE species, the function of this molecule is closely associated with the structural organization and functioning of plastidic membranes.Isoprene is globally the most abundant biogenic hydrocarbon constitutively emitted from many plant species (Guenther et al., 2012). It has been proposed that leaf isoprene emission is an important adaptation for plants, conferring tolerance to different environmental constraints (Vickers et al., 2009; Loreto and Schnitzler, 2010; Loreto and Fineschi, 2014). However, biogenic isoprene emission represents a nontrivial carbon loss in plants, particularly under stress conditions (Fang et al., 1996; Brilli et al., 2007; Teuber et al., 2008; Ghirardo et al., 2014), and the reason(s) why plants emit isoprene are still ambiguous, and the true role of isoprene emission remains elusive. Different approaches and techniques have been used to determine whether and how the cost of this expensive carbon emission is matched by the accomplishment of the physiological function in planta. It has been shown that isoprene might quench and/or regulate reactive oxygen and nitrogen species formation (Behnke et al., 2010a; Velikova et al., 2012), thereby indirectly providing a general antioxidant action (for review, see Vickers et al., 2009; Loreto and Schnitzler, 2010) and stabilizing thylakoid membrane structures due to the lipophilic properties of this molecule (Sharkey et al., 2001; Velikova et al., 2011).Protein and pigment-protein complexes are assembled and embedded in a lipid matrix, which has a unique lipid composition. The thylakoid lipid bilayer of chloroplasts is characterized by a high proportion of galactolipids with one (monogalactosyldiacylglycerol [MGDG]) or two (digalactosyldiacylglycerol [DGDG]) Gal molecules (Joyard et al., 2010). MGDGs are the primary constituents (approximately 50%) of thylakoid membrane glycerolipids, followed by DGDGs (approximately 30%), sulfoquinovosyldiacylglycerol (approximately 5%–12%), and phosphatidylglycerol (approximately 5%–12%; Kirchhoff et al., 2002). Galactolipids contain a large proportion of polyunsaturated fatty acids, and consequently, the thylakoid membrane is a relatively fluid system (Gounaris and Barber, 1983) compared with other biological membranes. The fluidity of the thylakoid membrane is essential for photosynthetic processes.The thylakoid membranes are highly organized internal membrane chloroplast systems that conduct the light reactions of photosynthesis. These membranes comprise pigments and proteins organized in complexes. Thylakoid membranes are arranged into stacked and unstacked regions called grana and stroma thylakoids, respectively, differentially enriched in PSI and PSII complexes (Mustárdy et al., 2008). The spatial separation of the PSI and PSII complexes in the stacked and unstacked membrane regions and the macromolecular organization of PSII in stacked grana thylakoids are self-organizing processes and important features to maintain the functional integrity of the photosynthetic apparatus (Kirchhoff et al., 2007).It is not known how changes in the lipid matrix affect lipid-protein interactions and vice versa, and how membrane macroorganization ensures the efficient diffusion of protein complexes associated with plant adaptation to the changing environment remains elusive. The isoprene impact on thylakoid intactness and functionality has been assessed using different biophysical techniques (Velikova et al., 2011). Thermoluminescence data demonstrated that the position of the main peak (Qb peak; Sane, 2004) was upshifted approximately 10° in isoprene-emitting (IE) plants, suggesting modifications in the lipid environment due to the presence of isoprene in heterologous Arabidopsis (Arabidopsis thaliana) plants expressing the isoprene synthase gene from poplar (Populus spp.). It was also shown that isoprene improves the stability of PSII light-harvesting complex II (LHCII) through the modification of pigment-protein complex organization in thylakoid membranes (Velikova et al., 2011). Moreover, we recently showed that knocking down isoprene emission in poplar remodels the chloroplast proteome (Velikova et al., 2014). The lack of isoprene resulted in the down-regulation of proteins associated with the light reactions of photosynthesis, redox regulation, and oxidative stress defenses and several proteins responsible for lipid metabolism (Velikova et al., 2014).In this study, we focused on the lipid composition of thylakoid membranes in IE and nonisoprene-emitting (NE) poplar (Populus × canescens) leaves. Specifically, we determined whether the translational suppression of isoprene synthase in NE leaves influences the lipid matrix of thylakoids and how this phenomenon affects membrane structure and function. Here, we provide evidence that the suppression of isoprene biosynthesis in poplar (1) reduced total galactolipids, phospholipids (PLs), and linolenic fatty acid (18:3), (2) altered the chloroplast ultrastructure, and (3) stimulated photoprotective mechanisms.     

Influence of the Charge at D85 on the Initial Steps in the Photocycle of Bacteriorhodopsin

Studies have shown that trans-cis isomerization of retinal is the primary photoreaction in the photocycle of the light-driven proton pump bacteriorhodopsin (BR) from Halobacterium salinarum, as well as in the photocycle of the chloride pump halorhodopsin (HR). The transmembrane proteins HR and BR show extensive structural similarities, but differ in the electrostatic surroundings of the retinal chromophore near the protonated Schiff base. Point mutation of BR of the negatively charged aspartate D85 to a threonine T (D85T) in combination with variation of the pH value and anion concentration is used to study the ultrafast photoisomerization of BR and HR for well-defined electrostatic surroundings of the retinal chromophore. Variations of the pH value and salt concentration allow a switch in the isomerization dynamics of the BR mutant D85T between BR-like and HR-like behaviors. At low salt concentrations or a high pH value (pH 8), the mutant D85T shows a biexponential initial reaction similar to that of HR. The combination of high salt concentration and a low pH value (pH 6) leads to a subpopulation of 25% of the mutant D85T whose stationary and dynamic absorption properties are similar to those of native BR. In this sample, the combination of low pH and high salt concentration reestablishes the electrostatic surroundings originally present in native BR, but only a minor fraction of the D85T molecules have the charge located exactly at the position required for the BR-like fast isomerization reaction. The results suggest that the electrostatics in the native BR protein is optimized by evolution. The accurate location of the fixed charge at the aspartate D85 near the Schiff base in BR is essential for the high efficiency of the primary reaction.     

Angiotensin II type 1 receptor regulation and differential trophic effects on rat cardiac myofibroblasts after acute myocardial infarction

Fibroblast growth in the scar and surviving tissue is a key element of the remodeling post myocardial infarction. The regulation of fibroblast growth after acute myocardial infarction remains to be determined. Recently, Angiotensin II has been demonstrated to be a mitogen for neonatal cardiac fibroblasts. In this study adult rat cardiac fibroblasts were isolated from different regions of the infarcted rat heart and Angiotensin II effects examined. Adult Wistar-rats were sham operated or left coronary artery ligated. After 4 days, hearts were removed and fibroblasts from sham operated, infarct- and non-infarct regions of the left ventricle isolated. Radioligand binding studies were performed and cell number, cell area, total protein, and AT(1) receptor mRNA after stimulation determined. Radioligand binding studies demonstrated that myofibroblasts expressed a single class of high affinity Angiotensin II AT(1) receptors. Myofibroblasts from the infarct area revealed a lower maximal binding capacity, compared to sham operated myocardium. Conversely, myofibroblasts from the non-infarct area had a higher expression of Angiotensin II AT(1) receptor mRNA compared to sham operated myofibroblasts. Angiotensin II (1 microM, 48 h) increased cell-number in sham operated and non-infarct, but not in infarct myofibroblasts. Angiotensin II elevated total protein in sham operated, non-infarct, and infarct myofibroblasts. In addition, Angiotensin II increased cell area in sham operated and infarct myofibroblasts. These data demonstrate that Angiotensin II acted as a mitogen in sham operated and non-infarct myofibroblasts and stimulated hypertrophy in infarct myofibroblasts. These regional different effects of Angiotensin II might participate in the remodeling post myocardial infarction.     

Local adaptations to frost in marginal and central populations of the dominant forest tree Fagus sylvatica L. as affected by temperature and extreme drought in common garden experiments

Local adaptations to environmental conditions are of high ecological importance as they determine distribution ranges and likely affect species responses to climate change. Increased environmental stress (warming, extreme drought) due to climate change in combination with decreased genetic mixing due to isolation may lead to stronger local adaptations of geographically marginal than central populations. We experimentally observed local adaptations of three marginal and four central populations of Fagus sylvatica L., the dominant native forest tree, to frost over winter and in spring (late frost). We determined frost hardiness of buds and roots by the relative electrolyte leakage in two common garden experiments. The experiment at the cold site included a continuous warming treatment; the experiment at the warm site included a preceding summer drought manipulation. In both experiments, we found evidence for local adaptation to frost, with stronger signs of local adaptation in marginal populations. Winter frost killed many of the potted individuals at the cold site, with higher survival in the warming treatment and in those populations originating from colder environments. However, we found no difference in winter frost tolerance of buds among populations, implying that bud survival was not the main cue for mortality. Bud late frost tolerance in April differed between populations at the warm site, mainly because of phenological differences in bud break. Increased spring frost tolerance of plants which had experienced drought stress in the preceding summer could also be explained by shifts in phenology. Stronger local adaptations to climate in geographically marginal than central populations imply the potential for adaptation to climate at range edges. In times of climate change, however, it needs to be tested whether locally adapted populations at range margins can successfully adapt further to changing conditions.     

Complex bird clocks.

The circadian pacemaking system of birds comprises three major components: (i) the pineal gland, which rhythmically synthesizes and secretes melatonin; (ii) a hypothalamic region, possibly equivalent to the mammalian suprachiasmatic nuclei; and (iii) the retinae of the eyes. These components jointly interact, stabilize and amplify each other to produce a highly self-sustained circadian output. Their relative contribution to overt rhythmicity appears to differ between species and the system may change its properties even within an individual depending, for example, on its state in the annual cycle or its photic environment. Changes in pacemaker properties are partly mediated by changes in certain features of the pineal melatonin rhythm. It is proposed that this variability is functionally important, for instance, for enabling high-Arctic birds to retain synchronized circadian rhythms during the low-amplitude zeitgeber conditions in midsummer or for allowing birds to adjust quickly their circadian system to changing environmental conditions during migratory seasons. The pineal melatonin rhythm, apart from being involved in generating the avian pacemaking oscillation, is also capable of retaining day length information after isolation from the animal. Hence, it appears to participate in photoperiodic after-effects. Our results suggest that complex circadian clocks have evolved to help birds cope with complex environments.     

Size‐dependent insect flight energetics at different sugar supplies

Under most circumstances, large body size confers a higher fitness and is positively selected, whereas selection against large size is empirically poorly documented. Physiologically, according to the ¾ power law, larger animals have lower relative but higher absolute energy demands, such that large body size may become disadvantageous, particularly under fast locomotion in food‐limited environments. After a period of initial feeding on different sugar concentrations, we investigated size‐dependent energy content (reserves) at baseline and of females unflown (i.e. resting) or flown for 18 h in two (replicate) insect species: the yellow dung fly Scathophaga stercoraria and the yellow fever mosquito Aedes aegypti. Tethered adults of various sizes were tested in a flight mill. In both species, teneral glycogen, sugar, and lipid content increased with sugar availability, and isometrically or even hyper‐allometrically (slope > 1) with body size. Activity treatment also revealed the expected consumption effects. Both species increased their flight distance with sugar supply, although only larger mosquitoes flew longer. Crucially, larger females of both species disproportionately exhausted more glycogen and sugars (but not lipid) during flight. The mosquitoes appeared to adjust their flight more finely to their size‐dependent energy reserves at all sugar availabilities, whereas, in the dung flies, size‐dependent energy demands were detectable only with a low but not with an overly high sugar supply. Although we found a greater absolute and relative locomotory energy demand for the larger flies, which is in agreement with interspecific patterns in insects, this was (more than) compensated by their greater baseline energy reserves, resulting in the greater net flying capacity of larger individuals. Consequently, we found no evidence for energetic mechanisms limiting the performance of large flying insects under food limitation. The differences between the two species presumably relate to mosquitoes inherently being long distance flyers and dung flies being short distance flyers. © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, ●● , ●●–●●.