LEKTI proteolytic processing in human primary keratinocytes, tissue distribution and defective expression in Netherton syndrome

SPINK5, encoding the putative multi-domain serine protease inhibitor LEKTI, was recently identified as the defective gene in the severe autosomal recessive ichthyosiform skin condition, Netherton syndrome (NS). Using monoclonal and polyclonal antibodies, we show that LEKTI is a marker of epithelial differentiation, strongly expressed in the granular and uppermost spinous layers of the epidermis, and in differentiated layers of stratified epithelia. LEKTI expression was also demonstrated in normal differentiated human primary keratinocytes (HK) through detection of a 145 kDa full-length protein and a shorter isoform of 125 kDa. Both proteins are N-glycosylated and rapidly processed in a post-endoplasmic reticulum compartment into at least three C-terminal fragments of 42, 65 and 68 kDa, also identified in conditioned media. Processing of the 145 and 125 kDa precursors was prevented in HK by treatment with a furin inhibitor. In addition, in vitro cleavage of the recombinant 145 kDa precursor by furin generated C-terminal fragments of 65 and 68 kDa, further supporting the involvement of furin in LEKTI processing. In contrast, LEKTI precursors and proteolytic fragments were not detected in differentiated HK from NS patients. Defective expression of LEKTI in skin sections was a constant feature in NS patients, whilst an extended reactivity pattern was observed in samples from other keratinizing disorders, demonstrating that loss of LEKTI expression in the epidermis is a diagnostic feature of NS. The identification of novel processed forms of LEKTI provides the basis for future functional and structural studies of fragments with physiological relevanc

Hck contributes to bone homeostasis by controlling the recruitment of osteoclast precursors

ABSTRACT In osteoclasts, Src controls podosome organization and bone degradation, which leads to an osteopetrotic phenotype in src ؊/؊ mice. Since this phenotype was even more severe in src ؊/؊ hck ؊/؊ mice, we examined the individual contribution of Hck in bone homeostasis. Compared to wt mice, hck ؊/؊ mice exhibited an osteopetrotic phenotype characterized by an increased density of trabecular bone and decreased bone degradation, although osteoclastogenesis was not impaired. Podosome organization and matrix degradation were found to be defective in hck ؊/؊ osteoclast precursors (preosteoclast) but were normal in mature hck ؊/؊ osteoclasts, probably through compensation by Src, which was specifically overexpressed in mature osteoclasts. As a consequence of podosome defects, the 3-dimensional migration of hck ؊/؊ preosteoclasts was strongly affected in vitro. In vivo, this translated by altered bone homing of preosteoclasts in hck ؊/؊ mice: in metatarsals of 1-wk-old mice, when bone formation strongly depends on the recruitment of these cells, reduced numbers of osteoclasts and abnormal developing trabecular bone were observed. This phenotype was still detectable in adults. In summmary, Hck is one of the very few effectors of preosteoclast recruitment described to date and thereby plays a critical role in bone remodeling.-Vérollet, C., Gallois, A., Dacquin, R., Lastrucci, C., Pandruvada, S. M. N., Ortega, N., Poincloux, R., Behar, A., Cougoule, C., Lowell, C., Al Saati, T., Jurdic, P., Maridonneau-Parini, I. Hck contributes to bone homeostasis by controlling the recruitment of osteoclast precursors. FASEB J. 27, 3608 -3618 (2013). www.fasebj.org Key Words: osteopetrosis ⅐ cell migration ⅐ podosomes ⅐ Src tyrosine kinases Bone is renewed continuously by a process known as bone remodeling. Bone remodeling is accomplished by 3 cell types: osteocytes, osteoblasts, and osteoclasts (OCs). Osteocytes are the mechanical sensors of bone that regulate osteoclast formation. Osteoblasts synthetize the matrix and promote its mineralization, while OCs are responsible for degradation of bones during bone development, homeostasis, and repair. The formation and degradation of bone are tightly balanced in both time and space. A dysregulation of this tight balance between bone formation and bone degradation may result either in loss of bone mass, such as in osteoporosis, or in contrast, in a progressive increase in bone mass, such as in osteopetrosis. Degrading OCs are large multinucleated giant cells formed by the differentiation and fusion of mononuclear monocyte lineage precursors after stimulation by receptor activator of nuclear factor -B ligand (RANKL) and macrophage colony-stimulationg factor (M-CSF) (1-3). They are characterized by high levels of cathepsin K and tartrate resistant acidic phosphatase (TRAP) activities, whic

Tuberculosis is associated with expansion of a motile, permissive and immunomodulatory CD16(+) monocyte population via the IL-10/STAT3 axis

The human CD14+ monocyte compartment is composed by two subsets based on CD16 expression. We previously reported that this compartment is perturbed in tuberculosis (TB) patients, as reflected by the expansion of CD16+ monocytes along with disease severity. Whether this unbalance is beneficial or detrimental to host defense remains to be elucidated. Here in the context of active TB, we demonstrate that human monocytes are predisposed to differentiate towards an anti-inflammatory (M2-like) macrophage activation program characterized by theCD16+CD163+MerTK+pSTAT3+ phenotype and functional properties such as enhanced protease-dependent motility, pathogen permissivity and immunomodulation. This process is dependent on STAT3 activation, and loss-of-function experiments point towards a detrimental role in host defense against TB. Importantly, we provide a critical correlation between the abundance of the CD16+CD163+MerTK+pSTAT3+ cells and the progression of the disease either at the local level in a non-human primate tuberculous granuloma context, or at the systemic level through the detection of the soluble form of CD163 in human sera. Collectively, this study argues for the pathogenic role of the CD16+CD163+MerTK+pSTAT3+ monocyte-to-macrophage differentiation program and its potential as a target for TB therapy,and promotes the detection of circulating CD163 as a potential biomarker for disease progression and monitoringof treatment efficacy.Fil: Lastrucci, Claire. Centre National de la Recherche Scientifique; FranciaFil: Bénard, Alan. Centre National de la Recherche Scientifique; FranciaFil: Balboa, Luciana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Pingris, Karine. Centre National de la Recherche Scientifique; FranciaFil: Souriant, Shanti. Centre National de la Recherche Scientifique; FranciaFil: Poincloux, Renaud. Centre National de la Recherche Scientifique; FranciaFil: Al Saati, Talal. Inserm; FranciaFil: Rasolofo, Voahangy. Pasteur Institute in Antananarivo; MadagascarFil: González Montaner, Pablo. Gobierno de la Ciudad de Buenos Aires. Hospital de Infecciosas ; ArgentinaFil: Inwentarz, Sandra. Gobierno de la Ciudad de Buenos Aires. Hospital de Infecciosas ; ArgentinaFil: Moraña, Eduardo José. Gobierno de la Ciudad de Buenos Aires. Hospital de Infecciosas ; ArgentinaFil: Kondova, Ivanela. Biomedical Primate Research Centre; Países BajosFil: Verreck, Franck A. W.. Biomedical Primate Research Centre; Países BajosFil: Sasiain, María del Carmen. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Neyrolles, Olivier. Centre National de la Recherche Scientifique; FranciaFil: Maridonneau Parini, Isabel. Centre National de la Recherche Scientifique; FranciaFil: Lugo Villarino, Geanncarlo. Centre National de la Recherche Scientifique; FranciaFil: Cougoule, Celine. Centre National de la Recherche Scientifique; Franci

Production d'anticorps monoclonaux a l'aide de splenocytes de souris "nude" porteuses de tumeurs humaines

SIGLEINIST T 74126 / INIST-CNRS - Institut de l'Information Scientifique et TechniqueFRFranc

Etude des délétions géniques d'ATM (ataxia-telangectasia mutated) et de p53 dans les cellules de Reed-Sternberg dans la maladie de Hodgkin par technique de PCR sur cellules isolées par micromanipulation "single cell PCR

TOULOUSE3-BU Sciences (315552104) / SudocSudocFranceF

The oncogenic activity of the Src family kinase Hck requires the cooperative action of the plasma membrane- and lysosome-associated isoforms

International audienc