The mineral dissolution function of osteoclasts is dispensable for hypertrophic cartilage degradation during long bone development and growth

During long bone development and post-natal growth, the cartilaginous model of the skeleton is progressively replaced by bone, a process known as endochondral ossification. In the primary spongiosa, osteoclasts degrade the mineralized cartilage produced by hypertrophic chondrocytes to generate cartilage trabeculae that osteoblasts embed in bone matrix. This leads to the formation of the trabecular bone network of the secondary spongiosa that will undergo continuous remodeling. Osteoclasts are specialized in mineralized tissue degradation, with the combined ability to solubilize hydroxyapatite and to degrade extracellular matrix proteins. We reported previously that osteoclasts lacking Dock5 could not degrade bone due to abnormal podosome organization and absence of sealing zone formation. Consequently, adult Dock5^−/− mice have increased trabecular bone mass. We used Dock5^−/− mice to further investigate the different functions of osteoclast during endochondral bone formation. We show that long bones are overall morphologically normal in developing and growing Dock5^−/− mice. We demonstrate that Dock5^−/− mice also have normal hypertrophic cartilage and cartilage trabecular network. Conversely, trabecular bone volume increased progressively in the secondary spongiosa of Dock5^−/− growing mice as compared to Dock5^+/+ animals, even though their osteoclast numbers were the same. In vitro, we show that Dock5^−/− osteoclasts do present acidic compartments at the ventral plasma membrane and produce normal amounts of active MMP9, TRAP and CtsK for matrix protein degradation but they are unable to solubilize minerals. These observations reveal that contrarily to bone resorption, the ability of osteoclasts to dissolve minerals is dispensable for the degradation of mineralized hypertrophic cartilage during endochondral bone formation.     

New Selective Peptidyl Di(chlorophenyl) Phosphonate Esters for Visualizing and Blocking Neutrophil Proteinase 3 in Human Diseases

The function of neutrophil protease 3 (PR3) is poorly understood despite of its role in autoimmune vasculitides and its possible involvement in cell apoptosis. This makes it different from its structural homologue neutrophil elastase (HNE). Endogenous inhibitors of human neutrophil serine proteases preferentially inhibit HNE and to a lesser extent, PR3. We constructed a single-residue mutant PR3 (I217R) to investigate the S4 subsite preferences of PR3 and HNE and used the best peptide substrate sequences to develop selective phosphonate inhibitors with the structure Ac-peptidyl^P(O-C₆H₄-4-Cl)₂. The combination of a prolyl residue at P4 and an aspartyl residue at P2 was totally selective for PR3. We then synthesized N-terminally biotinylated peptidyl phosphonates to identify the PR3 in complex biological samples. These inhibitors resisted proteolytic degradation and rapidly inactivated PR3 in biological fluids such as inflammatory lung secretions and the urine of patients with bladder cancer. One of these inhibitors revealed intracellular PR3 in permeabilized neutrophils and on the surface of activated cells. They hardly inhibited PR3 bound to the surface of stimulated neutrophils despite their low molecular mass, suggesting that the conformation and reactivity of membrane-bound PR3 is altered. This finding is relevant for autoantibody binding and the subsequent activation of neutrophils in granulomatosis with polyangiitis (formerly Wegener disease). These are the first inhibitors that can be used as probes to monitor, detect, and control PR3 activity in a variety of inflammatory diseases.     

Neurovascular protection by post‐ischemic intravenous injections of the lipoxin A4 receptor agonist,BML‐111, in a rat model of ischemic stroke

Resolution of inflammation is an emerging new strategy to reduce damage following ischemic stroke. Lipoxin A₄ (LXA₄) is an anti‐inflammatory, pro‐resolution lipid mediator with high affinity binding to ALX, the lipoxin A₄ receptor. Since LXA₄ is rapidly inactivated, potent analogs have been created, including the ALX agonist BML‐111. We hypothesized that post‐ischemic intravenous administration of BML‐111 would provide protection to the neurovascular unit and reduce neuroinflammation in a rat stroke model. Animals were subjected to 90 min of middle cerebral artery occlusion (MCAO) and BML‐111 was injected 100 min and 24 h after stroke onset and animals euthanized at 48 h. Post‐ischemic treatment with BML‐111 significantly reduced infarct size, decreased vasogenic edema, protected against blood–brain barrier disruption, and reduced hemorrhagic transformation. Matrix metalloproteinase‐9 and matrix metalloproteinase‐3 were significantly reduced following BML‐111 treatment. Administration of BML‐111 dramatically decreased microglial activation, as seen with CD68, and neutrophil infiltration and recruitment, as assessed by levels of myeloperoxidase and intracellular adhesion molecule‐1. The tight junction protein zona occludens‐1 was protected from degradation following treatment with BML‐111. These results indicate that post‐ischemic activation of ALX has pro‐resolution effects that limit the inflammatory damage in the cerebral cortex and helps maintain blood–brain barrier integrity after ischemic stroke.

     

Trigger Factor from the Psychrophilic Bacterium Psychrobacter frigidicola Is a Monomeric Chaperone

In eubacteria, trigger factor (TF) is the first chaperone to interact with newly synthesized polypeptides and assist their folding as they emerge from the ribosome. We report the first characterization of a TF from a psychrophilic organism. TF from Psychrobacter frigidicola (TF_Pf) was cloned, produced in Escherichia coli, and purified. Strikingly, cross-linking and fluorescence anisotropy analyses revealed it to exist in solution as a monomer, unlike the well-characterized, dimeric E. coli TF (TF_Ec). Moreover, TF_Pf did not exhibit the downturn in reactivation of unfolded GAPDH (glyceraldehyde-3-phosphate dehydrogenase) that is observed with its E. coli counterpart, even at high TF/GAPDH molar ratios and revealed dramatically reduced retardation of membrane translocation by a model recombinant protein compared to the E. coli chaperone. TF_Pf was also significantly more effective than TF_Ec at increasing the yield of soluble and functional recombinant protein in a cell-free protein synthesis system, indicating that it is not dependent on downstream systems for its chaperoning activity. We propose that TF_Pf differs from TF_Ec in its quaternary structure and chaperone activity, and we discuss the potential significance of these differences in its native environment.     

The erratic mitochondrial clock: variations of mutation rate, not population size, affect mtDNA diversity across birds and mammals

Background  

During the last ten years, major advances have been made in characterizing and understanding the evolution of mitochondrial DNA, the most popular marker of molecular biodiversity. Several important results were recently reported using mammals as model organisms, including (i) the absence of relationship between mitochondrial DNA diversity and life-history or ecological variables, (ii) the absence of prominent adaptive selection, contrary to what was found in invertebrates, and (iii) the unexpectedly large variation in neutral substitution rate among lineages, revealing a possible link with species maximal longevity. We propose to challenge these results thanks to the bird/mammal comparison. Direct estimates of population size are available in birds, and this group presents striking life-history trait differences with mammals (higher mass-specific metabolic rate and longevity). These properties make birds the ideal model to directly test for population size effects, and to discriminate between competing hypotheses about the causes of substitution rate variation.     

Improved Energy Supply Regulation in Chronic Hypoxic Mouse Counteracts Hypoxia-Induced Altered Cardiac Energetics

Background

Hypoxic states of the cardiovacular system are undoubtedly associated with the most frequent diseases of modern time. Therefore, understanding hypoxic resistance encountered after physiological adaptation such as chronic hypoxia, is crucial to better deal with hypoxic insult. In this study, we examine the role of energetic modifications induced by chronic hypoxia (CH) in the higher tolerance to oxygen deprivation.

Methodology/Principal Findings

Swiss mice were exposed to a simulated altitude of 5500 m in a barochamber for 21 days. Isolated perfused hearts were used to study the effects of a decreased oxygen concentration in the perfusate on contractile performance (RPP) and phosphocreatine (PCr) concentration (assessed by ³¹P-NMR), and to describe the integrated changes in cardiac energetics regulation by using Modular Control Analysis (MoCA). Oxygen reduction induced a concomitant decrease in RPP (−46%) and in [PCr] (−23%) in Control hearts while CH hearts energetics was unchanged. MoCA demonstrated that this adaptation to hypoxia is the direct consequence of the higher responsiveness (elasticity) of ATP production of CH hearts compared with Controls (−1.88±0.38 vs −0.89±0.41, p<0.01) measured under low oxygen perfusion. This higher elasticity induces an improved response of energy supply to cellular energy demand. The result is the conservation of a healthy control pattern of contraction in CH hearts, whereas Control hearts are severely controlled by energy supply.

Conclusions/Significance

As suggested by the present study, the mechanisms responsible for this increase in elasticity and the consequent improved ability of CH heart metabolism to respond to oxygen deprivation could participate to limit the damages induced by hypoxia.     

Cardiomyocytes hypertrophic status after myocardial infarction determines distinct types of arrhythmia: Role of the ryanodine receptor

The mechanisms responsible for sudden cardiac death in heart failure (HF) are unclear. We investigated early and delayed afterdepolarizations (EADs, DADs) in HF. Cardiomyocytes were enzymatically isolated from the right ventricle (RV) and the septum of rats 8 weeks after myocardial infarction (MI) and sham-operated animals. Membrane capacitance, action potentials (AP) and ionic currents were measured by whole-cell patch-clamp. The [Ca²⁺]_i transients and Ca²⁺ sparks were recorded with Fluo-4 during fluorescence measurements. Arrhythmia was triggered in 40% of MI cells (not in sham) using trains of 5 stimulations at 2.0 Hz. EADs and DADs occurred in distinct cell populations both in the RV and the septum. EADs occurred in normal-sized PMI cells (<230 pF), whereas DADs occurred in hypertrophic PMI cells (>230 pF). All cells exhibited prolonged APs due to reduced I_to current. However, additional modifications in Ca²⁺-dependent ionic currents occurred in hypertrophic cells: a decrease in the inward rectifier K⁺ current I_K1, and a slowing of L-type Ca²⁺ current inactivation which was responsible for the lack of adaptation of APs to abrupt changes in the pacing rate. The occurrence of spontaneous Ca²⁺ sparks, reflecting ryanodine receptor (RyR2) diastolic activity, increased with hypertrophy. The [Ca²⁺]_i transient amplitude, sarcoplasmic reticulum (SR) Ca²⁺ load and Ca²⁺ sparks amplitude were all inversely correlated with cell size. We conclude that the trophic status of cardiomyocytes determines the type of cellular arrhythmia in MI rats, based on differential electrophysiological remodeling which may reflect early-mild and late-severe or differential modifications in the RyR2 function.     

The Changing Fate of a Secretory Glycoprotein in Developing Maize Endosperm

Zeins are the major storage proteins in maize (Zea mays) endosperm, and their accumulation in zein bodies derived from the endoplasmic reticulum is well characterized. In contrast, relatively little is known about post-Golgi compartments or the trafficking of vacuolar proteins in maize endosperm, specifically the presence of globulins in structures resembling protein storage vacuoles that appear in early to mid-stage seed development. We investigated this pathway by expressing and analyzing a recombinant reporter glycoprotein during endosperm maturation, using a combination of microscopy and sensitive glycopeptide analysis. Specific N-glycan acceptor sites on the protein were followed through the stages of grain development, revealing a shift from predominantly paucimannosidic vacuolar glycoforms to predominantly trimmed glycan structures lacking fucose. This was accompanied by a change in the main subcellular localization of the protein from large protein storage vacuole-like post-Golgi organelles to the endoplasmic reticulum and zein bodies. The endogenous storage proteins corn α-globulin and corn legumin-1 showed a similar spatiotemporal profile both in transgenic plants expressing the reporter glycoprotein and in wild-type plants. This indicates that the shift of the intracellular trafficking route, as observed with our reporter glycoprotein, may be a common strategy in maize seed development.Storage proteins in cereal seeds accumulate in different compartments of the endosperm cell, and their abundance and distribution varies according to the species. While in most cereals prolamins are the more abundant class of storage proteins, small-grain species (e.g. wheat [Triticum aestivum], oat [Avena sativa], and barley [Hordeum vulgare]) may contain variable proportions of both prolamins and globulins, and these are delivered to the protein storage vacuole (PSV) via Golgi-dependent and Golgi-independent pathways (Wettstein, 1980; Levanony et al., 1992; Herman and Schmidt, 2004; Takahashi et al., 2005; Cameron-Mills and von Tosi et al., 2009). In rice (Oryza sativa), where globulins and prolamins accumulate in distinct storage compartments, most globulins (mainly glutelins) accumulate in PSVs whereas prolamins aggregate into dense protein bodies within the rough endoplasmic reticulum (ER) and remain in ER-derived organelles (Okita and Rogers, 1996). Maize (Zea mays) stores mainly prolamins (zeins) comprised in three zein subfamilies (α, γ, and δ) that form ER-derived zein bodies. Mature zein bodies consist of a central core of α and δ zeins, while γ zeins are mainly found in the periphery (Lending and Larkins, 1989). Small amounts of globulins also accumulate in maize endosperm, i.e. corn α-globulin (CAG) and corn legumin-1 (CL-1; Woo et al., 2001). Unlike legumin homologs in other plant species including cereals, CL-1 lacks the canonical asparaginyl endopeptidase cleavage sequence (Woo et al., 2001), so it is not cleaved into α and β chains (Yamagata et al., 2003). CAG has been observed in small, PSV-like compartments within the maize endosperm cell (Woo et al., 2001) and a similar fate has been predicted for CL-1 (Yamagata et al., 2003). The identification and localization of globulins in maize indicates the presence of storage vacuoles in maize endosperm, but it does not address the question whether the size and number of these organelles is significant in maize, whether they change morphologically during seed maturation, and how proteins reach this destination.Proteins may reach the PSV by different routes, and in some species storage protein trafficking appears to undergo changes during seed development. For example, in the context of 2S and 11S storage protein trafficking in pumpkin (Cucurbita pepo) and castor bean (Ricinus communis) it has been proposed that seed developmental stages may be important in determining the transport routes to the PSV (Vitale and Hinz, 2005). A seed-development-mediated change in the trafficking route of wheat prolamins has been suggested earlier as well (Shy et al., 2001; Tosi et al., 2009). One approach to study such change in trafficking routes along seed maturation is to scrutinize the glycosylation pattern of proteins destined to the PSV, taking advantage of the fact that the intracellular trafficking route of a glycoprotein determines its final N-glycan structures (Lerouge et al., 1998).The first stage of N-glycosylation (which takes place in the ER) involves the cotranslational addition of a precursor oligosaccharide (Glc₃Man₉GlcNAc₂) that is modified by various glycosidases and glycosyltransferases to form the final glycan structure as the protein migrates through the endomembrane system (Lis and Sharon, 1993; Lerouge et al., 1998). ER-resident glycoproteins contain high-Man-type N-glycans whereas proteins passing though the Golgi apparatus contain complex-type N-glycans that include α(1-3)-Fuc and/or β(1-2)-Xyl residues (Lerouge et al., 1998). While secreted glycoproteins contain terminal GlcNAc residues in addition to the core Fuc and Xyl, these terminal residues are trimmed off by enzymes either en route to the vacuole or within the vacuole (Lerouge et al., 1998). Thus the structure of N-glycans is a useful indicator for the intracellular pathway of a protein (Vitale and Hinz, 2005).Unfortunately, most seed storage proteins, particularly those in cereals, are not glycosylated. However, information on N-glycan structures can be obtained from recombinant glycoproteins. For example, a KDEL-tagged antibody, which was located primarily in ER-derived zein bodies, was predominantly made up of molecules with single GlcNAc residues lacking Fuc (Rademacher et al., 2008). In contrast, recombinant human lactoferrin isolated from maize seeds was reported to contain pauci-Man-type N-glycans with β(1,2)-Xyl and α(1,3)-linked core Fuc (Samyn-Petit et al., 2001). Interestingly, this glycan pattern suggests a vacuolar location of this recombinant protein, and provides a second strong evidence for the presence of PSVs in maize, although the actual subcellular localization of lactoferrin in maize endosperm cells has not been confirmed.In previous studies we have shown that recombinant glycoproteins can help to clarify questions about the intracellular trafficking of proteins in cereal endosperm, and we found that a recombinant fungal phytase, although secreted from leaf cells, is mainly localized in the PSVs of wheat and rice endosperm (Arcalis et al., 2004; Drakakaki et al., 2006). In this study we used recombinant phytase to facilitate the visualization and characterization of the PSVs in maize, and we followed the intracellular fate of recombinant phytase in developing endosperm using a combination of microscopy and N-glycan analysis, revealing that the trafficking of the protein does indeed change as the seed matures. This behavior is mirrored by the two endogenous (aglycosylated) globulins, CAG and CL-1, indicating that the diversion of storage proteins may be a common strategy in seed development.