Uromodulin (Tamm-Horsfall glycoprotein/uromucoid) is a phosphatidylinositol-linked membrane protein

Uromodulin, originally identified as an immunosuppressive glycoprotein in the urine of pregnant women, has been previously shown to be identical to human Tamm-Horsfall glycoprotein (THP). THP is synthesized by the kidney and localizes to the renal thick ascending limb and early distal tubule. It is released into the urine in large quantities and thus represents a potential candidate for a protein secreted in a polarized fashion from the apical plasma membrane of epithelial cells in vivo. After introduction of the full-length cDNA encoding uromodulin/THP into HeLa, Caco-2, and Madin-Darby canine kidney cells by transfection, however, the expressed glycoprotein was almost exclusively cell-associated, as determined by immunoprecipitation after radioactive labeling of the cells. By immunofluorescence, THP was localized to the plasma membranes of transfected cells. In transfected cell extracts, THP also remained primarily in the detergent phase in a Triton X-114 partitioning assay, indicating that it has a hydrophobic character, in contrast to its behavior after isolation from human urine. Triton X-114 detergent-associated THP was redistributed to the aqueous phase after treatment of cell extracts with phosphatidylinositol-specific phospholipase C. Treatment of intact transfected HeLa cells with phosphatidylinositol-specific phospholipase C also resulted in the release of THP into the medium, suggesting that it is a glycosylphosphatidylinositol (GPI)-linked membrane protein. Similar to other known GPI-linked proteins, uromodulin/THP contains a stretch of 16 hydrophobic amino acids at its extreme carboxyl terminus which could function as a GPI addition signal and was shown to label with [3H]ethanolamine. The results indicate that THP is a member of this class of lipid-linked membrane proteins and is released into the urine after the loss of its hydrophobic anchor, probably by the action of a phospholipase or protease.     

New Short Tandem Repeat-Based Molecular Typing Method for Pneumocystis jirovecii Reveals Intrahospital Transmission between Patients from Different Wards

Pneumocystis pneumonia is a severe opportunistic infection in immunocompromised patients caused by the unusual fungus Pneumocystis jirovecii. Transmission is airborne, with both immunocompromised and immunocompetent individuals acting as a reservoir for the fungus. Numerous reports of outbreaks in renal transplant units demonstrate the need for valid genotyping methods to detect transmission of a given genotype. Here, we developed a short tandem repeat (STR)-based molecular typing method for P. jirovecii. We analyzed the P. jirovecii genome and selected six genomic STR markers located on different contigs of the genome. We then tested these markers in 106 P. jirovecii PCR-positive respiratory samples collected between October 2010 and November 2013 from 91 patients with various underlying medical conditions. Unique (one allele per marker) and multiple (more than one allele per marker) genotypes were observed in 34 (32%) and 72 (68%) samples, respectively. A genotype could be assigned to 55 samples (54 patients) and 61 different genotypes were identified in total with a discriminatory power of 0.992. Analysis of the allelic distribution of the six markers and minimum spanning tree analysis of the 61 genotypes identified a specific genotype (Gt21) in our hospital, which may have been transmitted between 10 patients including six renal transplant recipients. Our STR-based molecular typing method is a quick, cheap and reliable approach to genotype Pneumocystis jirovecii in hospital settings and is sensitive enough to detect minor genotypes, thus enabling the study of the transmission and pathophysiology of Pneumocystis pneumonia.     

Regulated in Development and DNA Damage Responses -1 (REDD1) Protein Contributes to Insulin Signaling Pathway in Adipocytes

REDD1 (Regulated in development and DNA damage response 1) is a hypoxia and stress response gene and is a negative regulator of mTORC1. Since mTORC1 is involved in the negative feedback loop of insulin signaling, we have studied the role of REDD1 on insulin signaling pathway and its regulation by insulin. In human and murine adipocytes, insulin transiently stimulates REDD1 expression through a MEK dependent pathway. In HEK-293 cells, expression of a constitutive active form of MEK stabilizes REDD1 and protects REDD1 from proteasomal degradation mediated by CUL4A-DDB1 ubiquitin ligase complex. In 3T3-L1 adipocytes, silencing of REDD1 with siRNA induces an increase of mTORC1 activity as well as an inhibition of insulin signaling pathway and lipogenesis. Rapamycin, a mTORC1 inhibitor, restores the insulin signaling after downregulation of REDD1 expression. This observation suggests that REDD1 positively regulates insulin signaling through the inhibition of mTORC1 activity. In conclusion, our results demonstrate that insulin increases REDD1 expression, and that REDD1 participates in the biological response to insulin.     

Thrombin signal transduction mechanisms in human glomerular epithelial cells.

We have previously shown that alpha-thrombin exerted a mitogenic effect on human glomerular epithelial cells and stimulated the synthesis of urokinase-type (u-PA) and tissue-type plasminogen activator (t-PA) and of their inhibitor, plasminogen activator inhibitor 1 (PAI-1). In the present study, we investigate the signal transduction mechanisms of thrombin in these cultured cells. Thrombin induced an increase in intracellular free calcium concentrations ([Ca2+]i) in a dose-dependent manner, a plateau being reached at 1 U/ml thrombin. A 60% inhibition of this effect was produced by 300 nM nicardipine, a dihydroperidine agent, or by 4 mM EGTA, indicating that increase in [Ca2+]i was due in part to extracellular Ca2+ entry through L-type voltage-sensitive calcium channels. Thrombin also induced an increase in inositol trisphosphate (IP3), suggesting that phospholipase C activation and phosphatidylinositides breakdown were stimulated. Interestingly thrombin-stimulated cell proliferation measured by 3H thymidine incorporation was inhibited by 300 nM nicardipine, and restored by addition of 10(-8) M ionomycin, indicating that calcium entry was critical for the mitogenic signal of thrombin. Conversely, nicardipine did not modify thrombin-stimulated synthesis of u-PA, t-PA, and PAI-1. Both thrombin-stimulated cell proliferation and protein synthesis required protein kinase C activation since these effects were blocked by 10 microM H7, an inhibitor of protein kinases, and by desensitization of protein kinase C by phorbol ester pretreatment of the cells. Interestingly, DFP-inactivated thrombin which binds the thrombin receptor and gamma-thrombin, which has some enzymatic activity but does not bind to thrombin receptor, had no effect when used alone. Simultaneous addition of these two thrombin derivatives had no effect on [Ca2+]i, and 3H thymidine incorporation but stimulated u-PA, t-PA, and PAI-1 synthesis although to a lesser extent than alpha-thrombin. This effect also required protein kinase C activation to occur, presumably by a pathway distinct from phosphoinositoside turnover since it was not associated with IP3 generation. In conclusion, multiple signalling pathways can be activated by alpha-thrombin in glomerular epithelial cells: 1) Ca2+ influx through a dihydroperidine-sensitive calcium channel, which seems critical for mitogenesis; 2) protein kinase C activation by phosphoinositide breakdown, which stimulates both mitogenesis and synthesis of u-PA, t-PA, and PAI-1; 3) protein kinase C activation by other phospholipid breakdown can stimulate u-PA, t-PA, and PAI-1 synthesis but not mitogenesis.     

Growth factor-like effect of urokinase type plasminogen activator in human renal cells.

Human renal glomerular epithelial cells possess membrane urokinase receptors. Addition of purified active urokinase to these cells in serum free minimum medium induced a dose-dependent increase in 3H-thymidine incorporation and a doubling of cell number after 48 hours of incubation. Both receptor occupancy and enzymatic activity of u-PA were required to stimulate cell proliferation. This effect was inhibited by down regulation of protein kinase C (PKC) or by H7, an inhibitor of PKC. It involved a pertussis toxin-sensitive pathway. This effect of urokinase was additive with EGF but not with thrombin growth factor activity and was not inhibited by aprotinin, an inhibitor of plasmin.     

T-DNA mutagenesis in Brachypodium distachyon

During the past decade, Brachypodium distachyon has emerged as an attractive experimental system and genomics model for grass research. Numerous molecular tools and genomics resources have already been developed. Functional genomics resources, including mutant collections, expression/tiling microarray, mapping populations, and genome re-sequencing for natural accessions, are rapidly being developed and made available to the community. In this article, the focus is on the current status of systematic T-DNA mutagenesis in Brachypodium. Large collections of T-DNA-tagged lines are being generated by a community of laboratories in the context of the International Brachypodium Tagging Consortium. To date, >13?000 lines produced by the BrachyTAG programme and USDA-ARS Western Regional Research Center are available by online request. The utility of these mutant collections is illustrated with some examples from the BrachyTAG collection at the John Innes Centre-such as those in the eukaryotic initiation factor 4A (eIF4A) and brassinosteroid insensitive-1 (BRI1) genes. A series of other mutants exhibiting growth phenotypes is also presented. These examples highlight the value of Brachypodium as a model for grass functional genomics.     

Characterization of adipocytes derived from fibro/adipogenic progenitors resident in human skeletal muscle

A population of fibro/adipogenic but non-myogenic progenitors located between skeletal muscle fibers was recently discovered. The aim of this study was to determine the extent to which these progenitors differentiate into fully functional adipocytes. The characterization of muscle progenitor-derived adipocytes is a central issue in understanding muscle homeostasis. They are considered as being the cellular origin of intermuscular adipose tissue that develops in several pathophysiological situations. Here fibro/adipogenic progenitors were isolated from a panel of 15 human muscle biopsies on the basis of the specific cell-surface immunophenotype CD15⁺/PDGFRα⁺CD56⁻. This allowed investigations of their differentiation into adipocytes and the cellular functions of terminally differentiated adipocytes. Adipogenic differentiation was found to be regulated by the same effectors as those regulating differentiation of progenitors derived from white subcutaneous adipose tissue. Similarly, basic adipocyte functions, such as triglyceride synthesis and lipolysis occurred at levels similar to those observed with subcutaneous adipose tissue progenitor-derived adipocytes. However, muscle progenitor-derived adipocytes were found to be insensitive to insulin-induced glucose uptake, in association with the impairment of phosphorylation of key insulin-signaling effectors. Our findings indicate that muscle adipogenic progenitors give rise to bona fide white adipocytes that have the unexpected feature of being insulin-resistant.Adipose tissue consists of several distinct anatomical compartments. It is not completely clear how all of them are formed despite the current high interest in understanding adipose tissue specificities. One intriguing compartment is the so-called intermuscular adipose tissue (IMAT), which can be found between muscle fibers.^{1, 2} Adipocytes accumulate and replace a large proportion of muscle fibers in muscular dystrophies,³ and muscle adiposity was even shown to be an accurate measurement of the severity of Duchenne muscular dystrophy.⁴ IMAT accumulation has also been reported in type II diabetes,^{5, 6} aged muscles,^{7, 8, 9} denervation,¹⁰ and in chronic disuse-induced muscle atrophy.^{11, 12} IMAT accumulation also occurs in muscles of healthy younger individuals after only 4 weeks of immobilization.¹¹ In pathological and nonpathological models, IMAT accumulation is linked to insulin resistance.^{5, 13, 14} The fat infiltration of muscle has not been markedly investigated for many years, whereas this process likely has deep impacts on muscle function because of the profound alterations induced in muscle structure and the important interplay between muscle and adipose tissues – which are both known to be very active factor-secreting tissues.¹⁵Muscle regeneration is supported by the extensively characterized satellite cells, which are myogenic progenitors laying along muscle fibers.¹⁶ In addition, a few groups have recently identified adipogenic progenitors resident in skeletal muscle. Among them are progenitors identified on the basis of specific cell-surface marker expressions, which can thus be physically separated by cell sorting. In humans, muscle adipogenic progenitors have been separated by flow cytometry as a CD15⁺CD56⁻ subpopulation by us and others.^{17, 18, 19, 20} CD56, the neural cell adhesion molecule 1, is known to be expressed by muscle satellite cells (which have the CD15⁻CD56⁺ immunophenotype). CD15 is an antigenic carbohydrate molecule found in several glycoproteins. Before its implication in the muscle adipogenic lineage, it was essentially known to be present in hematopoietic and neural cells. The CD15⁺CD56⁻ adipogenic progenitors express the mesenchymal stem or progenitor cell markers CD13, CD34, CD44, CD49, CD90, and CD105. They are negative for the lineage markers CD31, CD45, CD106, CD117, CD133, and STRO-1.^{18, 19, 20}In parallel in mice, muscle fibro/adipogenic progenitors (FAPs) have been identified as lin⁻(α7 integrin)⁻Sca-1⁺CD34⁺ cells²¹ and muscle mesenchymal progenitors with the immunophenotype CD31⁻CD45⁻SM/C-2.6⁻PDGFRα⁺ have been shown to contribute to fat cell formation in skeletal muscle.²² Further studies indicated that the two mouse immunophenotypes in fact specifically label the same progenitors that should be recognized as skeletal muscle-resident mesenchymal progenitors.²³ Finally, PDGFRα has also been used very recently in human to isolate muscle mesenchymal progenitors, which are equivalent to the mouse FAPs.^{24, 25}Despite the physiological importance of adipocytes derived from human or mouse skeletal muscle, characterization of these terminally differentiated cells is essentially limited to the expression of adipogenic markers. No comprehensive analyses have been reported, and the extent to which muscle adipogenic progenitors differentiate into fully functional adipocytes is unknown.Here we benefited from the recent identification of these progenitors to investigate their differentiation, as well as the functional characteristics and specificities of the derived adipocytes. The whole study has been performed in humans considering the functional importance of human IMAT. Muscle biopsies were taken from a panel of 15 donors. Canonical adipose stroma cells (ASCs) prepared from subcutaneous adipose tissue depots, and their derived adipocytes were used as references. In this study, we established first that the PDGFRα⁺CD56⁻ muscle progenitors are identical to the CD15⁺CD56⁻ progenitors, which therefore, can be also considered as the human counterparts of the FAPs isolated in mice. Then, our cellular, molecular, and biochemical data showed that bona fide white adipocytes are derived from human muscle-resident progenitors. However, these adipocytes have an unexpected impairment in insulin signaling associated with insulin resistance with reduced glucose uptake.     

Inhibition of hedgehog signaling decreases proliferation and clonogenicity of human mesenchymal stem cells

Human mesenchymal stem cells (hMSC) have the ability to differentiate into osteoblasts, adipocytes and chondrocytes. We have previously shown that hMSC were endowed with a basal level of Hedgehog signaling that decreased after differentiation of these cells. Since hMSC differentiation is associated with growth-arrest we investigated the function of Hh signaling on cell proliferation. Here, we show that inhibition of Hh signaling, using the classical inhibitor cyclopamine, or a siRNA directed against Gli-2, leads to a decrease in hMSC proliferation. This phenomenon is not linked to apoptosis but to a block of the cells in the G0/G1 phases of the cell cycle. At the molecular level, it is associated with an increase in the active form of pRB, and a decrease in cyclin A expression and MAP kinase phosphorylation. Inhibition of Hh signaling is also associated with a decrease in the ability of the cells to form clones. By contrast, inhibition of Hh signaling during hMSC proliferation does not affect their ability to differentiate. This study demonstrates that hMSC are endowed with a basal Hedgehog signaling activity that is necessary for efficient proliferation and clonogenicity of hMSC. This observation unravels an unexpected new function for Hedgehog signaling in the regulation of human mesenchymal stem cells and highlights the critical function of this morphogen in hMSC biology.     

Natural Killer Cell Function,an Important Target for Infection and Tumor Protection,Is Impaired in Type 2 Diabetes

Patients with Type 2 diabetes (T2D) are highly susceptible to infection and have an increased incidence of some tumors, possibly due to immune system dysfunction. In the innate cellular immune system, Natural Killer (NK) lymphocytes are important effectors responsible for controlling infections and combating tumor development. We analyzed NK cell subsets in 51 patients with long-standing T2D. Compared with healthy blood donors, diabetic patients showed a profound decrease in both NKG2D-positive NK cells (44% vs. 55.5%, P<0.01) and NKp46-positive cells (26% vs. 50%, P<0.01). Decreased expression of these receptors was associated with functional defects, such as reduced NK degranulation capacity when challenged with the tumor target cell line K562 (10.3 vs. 15.8%, P<0.05). This defect could be restored in vitro by stimulating NK cells from T2D patients with IL-15 (P<0.05). NKG2D expression was found to be negatively correlated with HBA1c level (r = −0.50; P = 0.009), suggesting that sustained hyperglycemia could directly influence NK cell defects. We demonstrated that endoplasmic reticulum (ER) stress, an important mediator in diabetes-associated complications, was inducible in vitro in normal NK cells and that tunicamycin treatment resulted in a significant decrease in NKG2D expression (P<0.05). Furthermore, markers of the Unfolded Protein Response (UPR) BiP, PDI and sXBP1 mRNAs were significantly increased in NK cells from T2D patients (P<0.05, P<0.01, P<0.05, respectively), indicating that ER stress is activated in vivo through both PERK and IRE1 sensors. These results demonstate for the first time defects in NK cell-activating receptors NKG2D and NKp46 in T2D patients, and implicate the UPR pathway as a potential mechanism. These defects may contribute to susceptibility to infections and malignancies and could be targetted therapeutically.