PTPRJ promotes osteoclast maturation and activity by inhibiting Cbl-mediated ubiquitination of NFATc1 in late osteoclastogenesis.

Bone-resorbing osteoclasts (OCLs) are multinucleated phagocytes, whose central roles in regulating bone formation and homeostasis are critical for normal health and development. OCLs are produced from precursor monocytes in a multistage process that includes initial differentiation, cell-cell fusion, and subsequent functional and morphological maturation; the molecular regulation of osteoclastogenesis is not fully understood. Here, we identify the receptor-type protein tyrosine phosphatase PTPRJ as an essential regulator specifically of OCL maturation. Monocytes from PTPRJ-deficient (JKO) mice differentiate and fuse normally, but their maturation into functional OCLs and their ability to degrade bone are severely inhibited. In agreement, mice lacking PTPRJ throughout their bodies or only in OCLs exhibit increased bone mass due to reduced OCL-mediated bone resorption. We further show that PTPRJ promotes OCL maturation by dephosphorylating the M-CSF receptor (M-CSFR) and Cbl, thus reducing the ubiquitination and degradation of the key osteoclastogenic transcription factor NFATc1. Loss of PTPRJ increases ubiquitination of NFATc1 and reduces its amounts at later stages of osteoclastogenesis, thereby inhibiting OCL maturation. PTPRJ thus fulfills an essential and cell-autonomous role in promoting OCL maturation by balancing between the pro- and anti-osteoclastogenic activities of the M-CSFR and maintaining NFATc1 expression during late osteoclastogenesis.

Piezo2 expressed in proprioceptive neurons is essential for skeletal integrity.

In humans, mutations in the PIEZO2 gene, which encodes for a mechanosensitive ion channel, were found to result in skeletal abnormalities including scoliosis and hip dysplasia. Here, we show in mice that loss of Piezo2 expression in the proprioceptive system recapitulates several human skeletal abnormalities. While loss of Piezo2 in chondrogenic or osteogenic lineages does not lead to human-like skeletal abnormalities, its loss in proprioceptive neurons leads to spine malalignment and hip dysplasia. To validate the non-autonomous role of proprioception in hip joint morphogenesis, we studied this process in mice mutant for proprioceptive system regulators Runx3 or Egr3. Loss of Runx3 in the peripheral nervous system, but not in skeletal lineages, leads to similar joint abnormalities, as does Egr3 loss of function. These findings expand the range of known regulatory roles of the proprioception system on the skeleton and provide a central component of the underlying molecular mechanism, namely Piezo2.

Phosphorylation of the phosphatase PTPROt at Tyr399 is a molecular switch that controls osteoclast activity and bone mass in vivo.

Bone resorption by osteoclasts is essential for bone homeostasis. The kinase Src promotes osteoclast activity and is activated in osteoclasts by the receptor-type tyrosine phosphatase PTPROt. In other contexts, however, PTPROt can inhibit Src activity. Through in vivo and in vitro experiments, we show that PTPROt is bifunctional and can dephosphorylate Src both at its inhibitory residue Tyr527 and its activating residue Tyr416 Whereas wild-type and PTPROt knockout mice exhibited similar bone masses, mice in which a putative C-terminal phosphorylation site, Tyr399, in endogenous PTPROt was replaced with phenylalanine had increased bone mass and reduced osteoclast activity. Osteoclasts from the knock-in mice also showed reduced Src activity. Experiments in cultured cells and in osteoclasts derived from both mouse strains demonstrated that the absence of phosphorylation at Tyr399 caused PTPROt to dephosphorylate Src at the activating site pTyr416 In contrast, phosphorylation of PTPROt at Tyr399 enabled PTPROt to recruit Src through Grb2 and to dephosphorylate Src at the inhibitory site Tyr527, thus stimulating Src activity. We conclude that reversible phosphorylation of PTPROt at Tyr399 is a molecular switch that selects between its opposing activities toward Src and maintains a coherent signaling output, and that blocking this phosphorylation event can induce physiological effects in vivo. Because most receptor-type tyrosine phosphatases contain potential phosphorylation sites at their C termini, we propose that preventing phosphorylation at these sites or its consequences may offer an alternative to inhibiting their catalytic activity to achieve therapeutic benefit.

Mineral Deposits in Ficus Leaves: Morphologies and Locations in Relation to Function.

Ficus trees are adapted to diverse environments and have some of the highest rates of photosynthesis among trees. Ficus leaves can deposit one or more of the three major mineral types found in leaves: amorphous calcium carbonate cystoliths, calcium oxalates, and silica phytoliths. In order to better understand the functions of these minerals and the control that the leaf exerts over mineral deposition, we investigated leaves from 10 Ficus species from vastly different environments (Rehovot, Israel; Bologna, Italy; Issa Valley, Tanzania; and Ngogo, Uganda). We identified the mineral locations in the soft tissues, the relative distributions of the minerals, and mineral volume contents using microcomputed tomography. Each Ficus species is characterized by a unique 3D mineral distribution that is preserved in different environments. The mineral distribution patterns are generally different on the adaxial and abaxial sides of the leaf. All species examined have abundant calcium oxalate deposits around the veins. We used micromodulated fluorimetry to examine the effect of cystoliths on photosynthetic efficiency in two species having cystoliths abaxially and adaxially (Ficusmicrocarpa) or only abaxially (Ficuscarica). In F. microcarpa, both adaxial and abaxial cystoliths efficiently contributed to light redistribution inside the leaf and, hence, increased photosynthetic efficiency, whereas in F. carica, the abaxial cystoliths did not increase photosynthetic efficiency.

The mammalian lectin galectin-8 induces RANKL expression, osteoclastogenesis, and bone mass reduction in mice.

Skeletal integrity is maintained by the co-ordinated activity of osteoblasts, the bone-forming cells, and osteoclasts, the bone-resorbing cells. In this study, we show that mice overexpressing galectin-8, a secreted mammalian lectin of the galectins family, exhibit accelerated osteoclasts activity and bone turnover, which culminates in reduced bone mass, similar to cases of postmenopausal osteoporosis and cancerous osteolysis. This phenotype can be attributed to a direct action of galectin-8 on primary cultures of osteoblasts that secrete the osteoclastogenic factor RANKL upon binding of galectin-8. This results in enhanced differentiation into osteoclasts of the bone marrow cells co-cultured with galectin-8-treated osteoblasts. Secretion of RANKL by galectin-8-treated osteoblasts can be attributed to binding of galectin-8 to receptor complexes that positively (uPAR and MRC2) and negatively (LRP1) regulate galectin-8 function. Our findings identify galectins as new players in osteoclastogenesis and bone remodeling, and highlight a potential regulation of bone mass by animal lectins.

Chronic Akt1 deficiency attenuates adverse remodeling and enhances angiogenesis after myocardial infarction.

BACKGROUND: Akt1 is a key signaling molecule in multiple cell types, including endothelial cells. Accordingly, Akt1 was proposed as a therapeutic target for ischemic injury in the context of myocardial infarction (MI). The aim of this study was to use multimodal in vivo imaging to investigate the impact of systemic Akt1 deficiency on cardiac function and angiogenesis before and after MI. METHODS AND RESULTS: In vivo cardiac MRI was performed before and at days 1, 8, 15, and 29 to 30 after MI induction for wild-type, heterozygous, and Akt1-deficient mice. Noninfarcted hearts were imaged using ex vivo stereomicroscopy and microcomputed tomography. Histological examination was performed for noninfarcted hearts and for hearts at days 8 and 29 to 30 after MI. MRI revealed mildly decreased baseline cardiac function in Akt1 null mice, whereas ex vivo stereomicroscopy and microcomputed tomography revealed substantially reduced coronary macrovasculature. After MI, Akt1(-/-) mice demonstrated significantly attenuated ventricular remodeling and a smaller decrease in ejection fraction. At 8 days after MI, a larger functional capillary network at the remote and border zone, accompanied by reduced scar extension, preserved cardiac function, and enhanced border zone wall thickening, was observed in Akt1(-/-) mice when compared with littermate controls. CONCLUSIONS: Using multimodal imaging to probe the role of Akt1 in cardiac function and remodeling after MI, this study revealed reduced adverse remodeling in Akt1-deficient mice after MI. Augmented myocardial angiogenesis coupled with a more functional myocardial capillary network may facilitate revascularization and therefore be responsible for preservation of infarcted myocardium.

Certain biominerals in leaves function as light scatterers.

Cystoliths are amorphous calcium carbonate bodies that form in the leaves of some plant families. Cystoliths are regularly distributed in the epidermis and protrude into the photosynthetic tissue, the mesophyll. The photosynthetic pigments generate a steep light gradient in the leaf. Under most illumination regimes the outer mesophyll is light saturated, thus the photosynthetic apparatus is kinetically unable to use the excess light for photochemistry. Here we use micro-scale modulated fluorometry to demonstrate that light scattered by the cystoliths is distributed from the photosynthetically inefficient upper tissue to the efficient, but light deprived, lower tissue. The results prove that the presence of light scatterers reduces the steep light gradient, thus enabling the leaf to use the incoming light flux more efficiently. MicroCT and electron microscopy confirm that the spatial distribution of the minerals is compatible with their optical function. During the study we encountered large calcium oxalate druses in the same anatomical location as the cystoliths. These druses proved to have similar light scattering functions as the cystoliths. This study shows that certain minerals in the leaves of different plants distribute the light flux more evenly inside the leaf.

High resolution 3D imaging of ex-vivo biological samples by micro CT.

Non-destructive volume visualization can be achieved only by tomographic techniques, of which the most efficient is the x-ray micro computerized tomography (µCT). High resolution µCT is a very versatile yet accurate (1-2 microns of resolution) technique for 3D examination of ex-vivo biological samples(1, 2). As opposed to electron tomography, the µCT allows the examination of up to 4 cm thick samples. This technique requires only few hours of measurement as compared to weeks in histology. In addition, µCT does not rely on 2D stereologic models, thus it may complement and in some cases can even replace histological methods(3, 4), which are both time consuming and destructive. Sample conditioning and positioning in µCT is straightforward and does not require high vacuum or low temperatures, which may adversely affect the structure. The sample is positioned and rotated 180° or 360°between a microfocused x-ray source and a detector, which includes a scintillator and an accurate CCD camera, For each angle a 2D image is taken, and then the entire volume is reconstructed using one of the different available algorithms(5-7). The 3D resolution increases with the decrease of the rotation step. The present video protocol shows the main steps in preparation, immobilization and positioning of the sample followed by imaging at high resolution.

A C-terminal lobe of the beta subunit of Na,K-ATPase and H,K-ATPase resembles cell adhesion molecules.

The beta subunit of Na,K-ATPase is required for stabilization and maturation of the catalytic alpha subunits and is also involved in cell adhesion and establishing epithelial cell polarity. However, the mechanism of cell adhesion effects and protein partners of beta are unknown. We have applied fold recognition methods to predict that a C-terminal domain of the beta subunits of Na,K-ATPase and H,K-ATPase has an immunoglobulin-like fold, which resembles cell adhesion molecules. Comparison of the predicted C-terminal domain with a recently published structure of shark rectal gland Na,K-ATPase at 2.4 A in which alpha, beta, and FXYD subunits were resolved confirms that the beta subunit ectodomain contains an immunoglobulin-like structure. Expression in Escherichia coli of a sequence corresponding to the C-terminal domain, followed by its purification, refolding, and circular dichroism analysis, shows that the domain is independently stable with prominent beta sheet secondary structure, as predicted. Proteolytic digestion of the purified detergent-soluble recombinant Na,K-ATPase (alpha1beta1) is also indicative of a stable C-terminal domain of beta in the native complex. The major conclusion of this work is consistent with prior evidence for a role of the beta subunit in cell-cell adhesion, and it attributes that function largely to the C-terminal lobe of the beta ectodomain. In the light of these findings, we discuss its role in cell adhesion and recognition of the beta subunits of Na,K-ATPase, including potential protein partners.

The microenvironment effect on the generation of reactive oxygen species by Pd-bacteriopheophorbide.

Generation of reactive oxygen species (ROS) is the hallmark of important biological processes and photodynamic therapy (PDT), where ROS production results from in situ illumination of certain dyes. Here we test the hypothesis that the yield, fate, and efficacy of the species evolved highly depend on the dye's environment. We show that Pd-bacteriopheophorbide (Pd-Bpheid), a useful reagent for vascular targeted PDT (VTP) of solid tumors, which has recently entered into phase II clinical trials under the code name WST09 (trade name TOOKAD), forms appreciable amounts of hydroxyl radicals, superoxide radicals, and probably hydrogen peroxide in aqueous medium but not in organic solvents where singlet oxygen almost exclusively forms. Evidence is provided by pico- and nanosecond time-resolved spectroscopies, ESR spectroscopy with spin-traps, time-resolved singlet oxygen phosphorescence, and chemical product analysis. The quantum yield for singlet oxygen formation falls from approximately 1 in organic solvents to approximately 0.5 in membrane-like systems (micelles or liposomes), where superoxide and hydroxyl radicals form at a minimal quantum yield of 0.1%. Analysis of photochemical products suggests that the formation of oxygen radicals involves both electron and proton transfer from (3)Pd-Bpheid at the membrane/water interface to a colliding oxygen molecule, consequently forming superoxide, then hydrogen peroxide, and finally hydroxyl radicals, with no need for metal catalysis. The ability of bacteriochlorophyll (Bchl) derivatives to form such radicals upon excitation at the near infrared (NIR) domain opens new avenues in PDT and research of redox regulation in animals and plants.

Enhanced intracellular mobility and nuclear accumulation of DNA plasmids associated with a karyophilic protein.

The use of synthetic gene delivery systems in human gene transfer is hampered by poor transfection efficiencies, largely because of the inability of DNA to translocate across the nuclear pore complex. A means to overcome this barrier is to bind the DNA to nuclear localization signals (NLSs), which are recognized by shuttling receptors of the nuclear import machinery. Here, we studied the intracellular transport of plasmid DNA microinjected into HeLa cell cytoplasm, alone or as a complex with intact or NLS-deleted NFkappaB p50, using confocal microscopy imaging. We found that association of NLS-carrying p50 with DNA facilitated not only nuclear entry of the DNA but also its migration through the cytoplasm toward the nucleus. Facilitated transport of p50-DNA complexes in the cytoplasm proceeded along microtubules in a dynein-dependent manner and is mediated by the heterodimeric nuclear transport receptor that recognizes the p50-born NLS.

Polyproline type II conformation in the C-terminal domain of the nuclear pore complex protein gp210.

gp210 is a major constituent of the nuclear pore complex (NPC) with possible structural and regulatory roles. It interacts with components of the NPC via its C-terminal domain (CTD), which follows a transmembrane domain and a massive ( approximately 200 kDa) N-terminal region that resides in the lumen of the perinuclear space. Here, we report the solution structure of the human gp210 CTD as determined by various spectroscopic techniques. In water, the CTD adopts an extended, largely unordered conformation, which contains a significant amount of left-handed polyproline type II (PII) helical structure. The conformation of the CTD is altered by high pH, charged detergents, and the hydrogen bond-promoting reagent trifluoroethanol (TFE), which decrease the PII fraction of the fragment. TFE also induces a conformational change in a region containing an SPXX motif whose serine becomes specifically phosphorylated during mitosis. We propose that PII elements in the CTD may play a role in its interaction with the NPC and may serve as recognition sites for regulatory proteins bearing WW or other, unknown PII-binding motifs.

The PKA phosphorylation of vitronectin: effect on conformation and function.

Vitronectin (Vn) stabilizes the inhibitory form of plasminogen activator inhibitor-1 (PAI-1), an important modulator of fibrinolysis. We have previously reported that Vn is specifically phosphorylated by PKA (at Ser378), a kinase we have shown to be released from platelets upon their physiological activation. Here we describe the molecular consequences of this phosphorylation and show (by circular dichroism, and by phosphorylation with casein kinase II) that it acts by modulating the conformation of Vn. The PKA phosphorylation of Vn is enhanced in the presence of either PAI-1, or heparin, or both. This enhanced phosphorylation occurs exclusively on Ser378 as shown with the Vn mutants Ser378Ala and Ser378Glu. The binding of PKA phosphorylated Vn to immobilized PAI-1 and to immobilized plasminogen is shown to be lower than that of Vn. The evidence compiled here suggests that this phosphorylation of Vn can modulate plasminogen activation and consequently control fibrinolysis.