Gelsolin deficiency blocks podosome assembly and produces increased bone mass and strength.

Osteoclasts are unique cells that utilize podosomes instead of focal adhesions for matrix attachment and cytoskeletal remodeling during motility. We have shown that osteopontin (OP) binding to the alpha(v)beta(3) integrin of osteoclast podosomes stimulated cytoskeletal reorganization and bone resorption by activating a heteromultimeric signaling complex that includes gelsolin, pp(60c-src), and phosphatidylinositol 3'-kinase. Here we demonstrate that gelsolin deficiency blocks podosome assembly and alpha(v)beta(3)-stimulated signaling related to motility in gelsolin-null mice. Gelsolin-deficient osteoclasts were hypomotile due to retarded remodeling of the actin cytoskeleton. They failed to respond to the autocrine factor, OP, with stimulation of motility and bone resorption. Gelsolin deficiency was associated with normal skeletal development and endochondral bone growth. However, gelsolin-null mice had mildly abnormal epiphyseal structure, retained cartilage proteoglycans in metaphyseal trabeculae, and increased trabecular thickness. With age, the gelsolin-deficient mice expressed increased trabecular and cortical bone thickness producing mechanically stronger bones. These observations demonstrate the critical role of gelsolin in podosome assembly, rapid cell movements, and signal transduction through the alpha(v)beta(3) integrin.

Review of catastrophic hand diagnosis over 15 years in a tertiary hospital: do we make proper use of the term? / Revisión del diagnóstico de mano catastrófica a lo largo de 15 años en un hospital terciario: ¿hacemos un uso adecuado del término?

OBJECTIVE: To describe the characteristics of patients diagnosed with 'catastrophic hand' by the plastic surgery department in a tertiary hospital in the period between 2000-2015, analyse the evolution of the use of the term 'catastrophic hand' and conduct a review of the literature with a view to proposing the optimal definition for said expression. MATERIAL AND METHOD: We conducted a descriptive and retrospective study of patients diagnosed with 'catastrophic hand' who required hospital care by the plastic surgery department of a tertiary hospital in the period between 2000-2015. We conducted a literature review on the use of the term 'catastrophic hand' and we applied the classifications proposed in the publications consulted to our results. RESULTS: The number of 'catastrophic hand' diagnoses was 133. We observed a downward trend in the use of the term over the years. Applying classifications based on the impossibility of recovery of an acceptable hand (presence of three long fingers and thumb) by reconstructive procedures, only 9 out of 133 patients could be considered to have 'catastrophic hands', constituting 6.7% of the total cases. DISCUSSION: The term 'catastrophic hand' has different meanings, and this generates ambiguity. The use of a classification based on the possibility of obtaining an acceptable hand diminishes its use and improves the approach for such injuries. CONCLUSION: We advocate limiting the use of 'catastrophic hand' to cases that meet criteria of 'mutilated hand' and 'metacarpal hand' to avoid indiscriminate use of the term and optimise therapeutic management.

Asthma exacerbations: risk factors for hospital readmissions.

OBJECTIVE: The aim of our study is to analyse hospital readmissions due to asthma, as well as the factors associated with their increase. STUDY DESIGN: We carried out a retrospective study including all admissions of patients over 18 years old due to exacerbation of asthma occurring in our hospital between the years 2000 and 2010. METHODS: The data were gathered by two members of the research team, by reviewing the clinical records. The first hospital admission of each patient was included for this study. An early readmission (ER) was defined as that which occurred in the following 15 days after hospital discharge and late readmission (LR) to that occurring from 16 days after discharge. RESULTS: This study included 2166 hospital admissions and 1316 patients, with a mean age of 62.6 years. Of the 1316 patients analysed, 36 (2.7%) had one ER and 313 (23.8%) one LR. The only factor independently associated with a higher probability of an ER was poor lung function. A higher probability of LR was associated with a greater severity of the asthma (OR: 17.8, for severe asthma versus intermittent asthma), to have had any hospital admission in the previous year (OR: 3.5) and the use of a combination of ICS-LABA as maintenance treatment. CONCLUSIONS: About 25% of the patients in our area admitted to hospital due to asthma exacerbation had repeat episodes of hospitalisation.

Regulation of sodium-dependent phosphate transport in osteoclasts.

Osteoclasts are the primary cells responsible for bone resorption. They are exposed to high ambient concentrations of inorganic phosphate (Pi) during the process of bone resorption and they possess specific Pi-transport system(s) capable of taking up Pi released by bone resorption. By immunochemical studies and PCR, we confirmed previous studies suggesting the presence of an Na-dependent Pi transporter related to the renal tubular "NaPi" proteins in the osteoclast. Using polyclonal antibodies to NaPi-2 (the rat variant), an approximately 95-kD protein was detected, localized in discrete vesicles in unpolarized osteoclasts cultured on glass coverslips. However, in polarized osteoclasts cultured on bone, immunofluorescence studies demonstrated the protein to be localized exclusively on the basolateral membrane, where it colocalizes with an Na-H exchanger but opposite to localization of the vacuolar H-ATPase. An inhibitor of phosphatidylinositol 3-kinase, wortmannin, and an inhibitor of actin cytoskeletal organization, cytochalasin D, blocked the bone-stimulated increase in Pi uptake. Phosphonoformic acid (PFA), an inhibitor of the renal NaPi-cotransporter, reduced NaPi uptake in the osteoclast. PFA also elicited a dose-dependent inhibition of bone resorption. PFA limited ATP production in osteoclasts attached to bone particles. Our results suggest that Pi transport in the osteoclast is a process critical to the resorption of bone through provision of necessary energy substrates.

Osteopontin deficiency produces osteoclast dysfunction due to reduced CD44 surface expression.

Osteopontin (OPN) was expressed in murine wild-type osteoclasts, localized to the basolateral, clear zone, and ruffled border membranes, and deposited in the resorption pits during bone resorption. The lack of OPN secretion into the resorption bay of avian osteoclasts may be a component of their functional resorption deficiency in vitro. Osteoclasts deficient in OPN were hypomotile and exhibited decreased capacity for bone resorption in vitro. OPN stimulated CD44 expression on the osteoclast surface, and CD44 was shown to be required for osteoclast motility and bone resorption. Exogenous addition of OPN to OPN-/- osteoclasts increased the surface expression of CD44, and it rescued osteoclast motility due to activation of the alpha(v)beta(3) integrin. Exogenous OPN only partially restored bone resorption because addition of OPN failed to produce OPN secretion into resorption bays as seen in wild-type osteoclasts. As expected with these in vitro findings of osteoclast dysfunction, a bone phenotype, heretofore unappreciated, was characterized in OPN-deficient mice. Delayed bone resorption in metaphyseal trabeculae and diminished eroded perimeters despite an increase in osteoclast number were observed in histomorphometric measurements of tibiae isolated from OPN-deficient mice. The histomorphometric findings correlated with an increase in bone rigidity and moment of inertia revealed by load-to-failure testing of femurs. These findings demonstrate the role of OPN in osteoclast function and the requirement for OPN as an osteoclast autocrine factor during bone remodeling.

Receptor-operated osteoclast calcium sensing.

Osteoclasts "sense" elevated extracellular calcium, which leads to cytoskeletal changes that may be linked to phospholipase C (PLC) activation and the associated rise in intracellular calcium ([Ca(2+)](i)). Since PLC is linked to transient receptor potential channels (trp), we hypothesized that receptor activated calcium influx due to this channel type would be activated by osteoclasts sensing [Ca(2+)](e). We found that high [Ca(2+)](e) induced similar intracellular Ca(2+) rises in chicken osteoclasts with or without intracellular Ca(2+) store depletion by either TPEN or thapsigargin, thus defining store-insensitive Ca(2+) influx. This store-insensitive calcium sensing component was blocked by the PLC antagonist U73122. Also, the calcium channel inhibitor SKF 96365, a blocker of store-independent trp-like channels, was effective in inhibiting calcium sensing in the presence of thapsigargin. Thus, a store-independent component of calcium sensing was associated with ion channels linked to PLC. Since receptor activated transient receptor potential (trp) family cation channels open in a PLC-dependent and store-independent manner, we suggest that receptor operated channels are activated in osteoclasts stimulated by high extracellular Ca(2+).

Engagement of the osteoclast integrin alpha v beta 3 by osteopontin stimulates phosphatidylinositol 3-hydroxyl kinase activity.

Osteoclast adhesion via integrin alpha v beta 3 to the bone matrix protein osteopontin (OPN) results in stimulation of bone resorption. We characterized an immediate signaling event that takes place upon osteoclast interaction with OPN. OPN binding to alpha v beta 3 results in the rapid production of the phosphoinositides (PtdInsP), including phosphatidylinositol triphosphate (PtdIns 3,4,5P3). Stimulation of 3,4,5-PtdInsP3 production by OPN was produced by increased activity of PtdInsP 3-OH kinase, which was found in immunoprecipitates formed by antibodies to integrin alpha v beta 3. The association of PtdIns 3-OH kinase with integrin may have been through association with c-src. The latter was present in immunoprecipitates formed by the antibodies to integrin alpha v beta 3, and src kinase was activated by OPN. These findings demonstrate a mechanism for rapid generation of cell signals upon matrix protein binding to alpha v beta 3, which resembles the mechanisms used by tyrosine kinase growth factor receptors.

Volume regulatory decrease in UMR-106.01 cells is mediated by specific alpha1 subunits of L-type calcium channels.

An early cellular response of osteoblasts to swelling is plasma membrane depolarization, accompanied by a transient increase in intracellular calcium ([Ca2+]i), which initiates regulatory volume decrease (RVD). The authors have previously demonstrated a hypotonically induced depolarization of the osteoblast plasma membrane, sufficient to open L-type Ca channels and mediate Ca2+ influx. Herein is described the initiation of RVD in UMR-106.01 cells, mediated by hypotonically induced [Ca2+]i transients resulting from the activation of specific isoforms of L-type Ca channels. The authors further demonstrate that substrate interaction determines which specific alpha1 Ca channel subunit isoform predominates and mediates Ca2+ entry and RVD. Swelling-induced [Ca2+]i transients, and RVD in cells grown on a type I collagen matrix, are inhibited by removal of Ca from extracellular solutions, dihydropyridines, and antisense oligodeoxynucleotides directed exclusively to the alpha1C isoform of the L-type Ca channel. Ca2+ transients and RVD in cells grown on untreated glass cover slips were inhibited by similar maneuvers, but only by antisense oligodeoxynucleotides directed to the alpha1S isoform of the L-type Ca channel. This represents the first molecular identification of the Ca channels that transduce the initiation signal for RVD by osteoblastic cells.

Characterization of phospholipase C activity of the plasma membrane and cytosol of an osteoblast-like cell line.

The properties of phospholipase C (PL-C) in the plasma membranes (PM) and the cytosol of osteoblast-like osteosarcoma cells, UMR-106, were analyzed to see if separate enzymes or similar enzymes were involved in signalling, transduction, and arachidonate release. The cytosolic PL-C displayed substrate affinities in the order of phosphatidylinositol (PI) greater than phosphatidylinositol-4-phosphate (PIP) or phosphatidylinoisitol-4, 5-bisphosphate (PIP2). Hydrolysis of PI, PIP, and PIP2 by cytosolic PL-C was not affected by GTP or GTP gamma S and other nucleotides. PI hydrolysis by PM and cytosolic PL-C was undetectable in the presence of 500 microM EGTA and displayed two activity plateaus at various concentrations of Ca2+. The Km for Ca2+ in the PL-C activity of the first plateau was 0.08 microM. Significant hydrolysis of PIP2 by cytosolic PL-C was observed in the absence of Ca2+. In contrast to the enzyme(s) predominant in the cytosol, the order of substrate affinities for PM PL-C was PIP2 greater than PIP greater than PI. Only PIP2 hydrolysis by PM PL-C was stimulated by both GTP and GTP gamma S in a dose-dependent manner. PIP2 hydrolysis by PL-C of the PM was not observed in the absence of Ca2+, serving to further discriminate this enzyme activity from that of the cytosol. PIP2 hydrolysis by PL-C of the PM also was biphasic in the dependence on Ca2+. At resting cytosolic Ca2+ levels, the Vmax of the high affinity activity already had been achieved. Guanine nucleotide stimulation of PIP2 hydrolysis by PM PL-C was characterized by increased maximum activity with an unchanged Km for Ca2+ or for PIP2. The pH optimum of PIP2 hydrolysis was similar between cytosolic and PM forms of PL-C. PIP2 hydrolysis with production of IP3 (PL-C activity) in UMR-106 cells treated with [2-3H]-myoinositol was stimulated by PTH, and this stimulation was not inhibited by pertussis toxin. These data suggest that UMR-106 cells possess at least two distinct PL-C activities, one predominant in the cytosol and activated by increasing cytosolic Ca2+ with PI as the substrate. The second enzyme, a GTP-activated PIP2-specific PL-C in the plasma membranes may play an important role in hormone-induced PIP2 hydrolysis mediated through guanine nucleotide regulatory proteins and may participate in the hormonal regulation of osteoblast cytosolic Ca2+ and bone remodeling functions.

[Tuberculosis in Santiago de Compostela from 1999 to 2002. An epidemiological study]. / Tuberculosis en el área de Santiago de Compostela durante los años 1999, 2000, 2001 y 2002. Un estudio epidemiológico.

OBJECTIVE: The purpose of this study was to determine the epidemiological characteristics of tuberculosis (TB) in the Public Health System District of Santiago de Compostela (population : 386125) from 1999 to 2002. METHODS: Inclusion criteria were: 1). microbiological and/or pathological diagnosis of TB in any specimen, and 2). patient younger 35 years old with recent medical history of TB. Mantoux test positive, and pleural effusion with linfocitosis and adenosine deaminase >47 IU/ml. RESULTS: 946 patients were included (568 men and 378 women), with ages ranging from 2 months to 96 years. The incidence of TB was 60.9/100000 in 1999, 67.6/100000 in 2000, 61.9/100000 in 2001 and 54.6/100000 in 2002. The incidence rate of tuberculous meningitis was 1.03/100000 in 1999 and 2000, 0.77/100000 in 2001 and 0.51/100000 in 2002. The percentage of cases associated with HIV was 3.4% in 1999, 1.9% in 2000, 2.4% in 2001 and 2002. We found an increase in the rate of males over 55 years of age; with incidence per 100000 inhabitants of 122.4 in 1999, 142.8 in 2000, 115 in 2001 and 119 in 2002, whereas in females the incidence was 40.6 in 1999, 60.9 in 2000, 54.1 in 2001 and 39.1 in 2002. CONCLUSIONS: In last four years the incidence of tuberculosis has decreased but remains high in males over 55 years old.

[Reason for a medical visit in the diagnosis of lung cancer. A review of 481 cases]. / Motivo de consulta en el diagnóstico de cáncer de pulmón. Revisión de 481 casos.

INTRODUCTION: Lung cancer is the most frequent cancer death related cause in the world. Its clinical presentation usually corresponds to advanced stages. The indication of screening programs for the diagnosis in early phases has been debated for years. AIMS: To know the clinical characteristics in the presentation of the lung cancer in our health care area. MATERIAL AND METHODS: All of the incident lung cancer cases for 3 years (January 1, 1997 to December 31, 1999) among those patients with usual residence in the Santiago de Compostela health care area were retrospectively collected from the available information in the Codification Service. The histological type was graded according to the World Health Organization classification. Date of death was obtained from the patient's clinical history, mortality record, or telephonic calls to the patient's home. The rest of the information was obtained from the clinical records of the patient and Pathology Service. RESULTS: Four-hundred and eighty-one lung cancer cases were diagnosed (incident gross rate of 41.79 per 100,000 inhabitants and year). Median age was 66.9 years (interquartile range 60.5-74.4), 92.77% being males and 94.1% corresponding to smokers or former smokers. The most prevalent symptoms motivating the visit were general syndrome (20%), thoracic pain (19%) and hemoptisis (17%). A group of patients (56), fundamentally males, had an incidental diagnosis. CONCLUSIONS: The incidence of lung cancer seems to be high in our health care area. The standard patient with lung cancer in this health care area is a 67-year-old, male, who smokes, diagnosed in advanced stages, who consults due to a general syndrome and/or chest pain. Some of the symptoms appear with significant gender differences. Likewise, there are differences between histologies, the high frequency of chest pain in adenocarcinoma being outstanding, although it is also the first cause for consultation in small cell lung cancer. On the contrary, hemoptisis, the most frequent consultation cause in squamous cell carcinoma and the general syndrome in the big cells type or with clinical-radiological diagnosis.

Revisión del diagnóstico de mano catastrófica a lo largo de 15 años en un hospital terciario: ¿hacemos un uso adecuado del término? / Review of catastrophic hand diagnosis over 15 years in a tertiary hospital: do we make proper use of the term?

Objetivo: Describir las características de los pacientes diagnosticados de «mano catastrófica» por el Servicio de Cirugía Plástica en un hospital de tercer nivel en el periodo comprendido entre 2000 y 2015; analizar la evolución del uso del término «mano catastrófica», y realizar una revisión bibliográfica con el objetivo de proponer la definición más óptima de dicha expresión. Material y método: Realizamos un estudio descriptivo y retrospectivo de los pacientes diagnosticados de «mano catastrófica» que requirieron atención hospitalaria por el Servicio de Cirugía Plástica de un hospital terciario en el periodo comprendido entre 2000 y 2015. Efectuamos una revisión bibliográfica acerca del uso del término «mano catastrófica» y aplicamos a nuestros resultados las clasificaciones propuestas en las publicaciones consultadas. Resultados: El número de diagnósticos de «mano catastrófica» fue de 133. Objetivamos una tendencia descendente en el empleo del mencionado término con el transcurso de los años. Aplicando clasificaciones basadas en la imposibilidad de recuperación de una mano aceptable (presencia de 3 dedos largos y pulgar) mediante procedimientos reconstructivos, tan solo 9 de 133 pacientes podían ser considerados «manos catastróficas», constituyendo un 6,7% del total de casos. Discusión: El término «mano catastrófica» posee diferentes acepciones, generando ambigüedad. El uso de una clasificación basada en la posibilidad de obtener una mano aceptable disminuye su utilización y mejora el abordaje de dichas lesiones. Conclusión: Abogamos por limitar el uso de «mano catastrófica» a los casos que cumplan criterios de «mano mutilada» y «mano metacarpiana» para evitar su uso indiscriminado y optimizar su manejo terapéutico

Objective: To describe the characteristics of patients diagnosed with 'catastrophic hand' by the plastic surgery department in a tertiary hospital in the period between 2000-2015, analyse the evolution of the use of the term 'catastrophic hand' and conduct a review of the literature with a view to proposing the optimal definition for said expression. Material and method: We conducted a descriptive and retrospective study of patients diagnosed with 'catastrophic hand' who required hospital care by the plastic surgery department of a tertiary hospital in the period between 2000-2015. We conducted a literature review on the use of the term 'catastrophic hand' and we applied the classifications proposed in the publications consulted to our results. Results: The number of 'catastrophic hand' diagnoses was 133. We observed a downward trend in the use of the term over the years. Applying classifications based on the impossibility of recovery of an acceptable hand (presence of three long fingers and thumb) by reconstructive procedures, only 9 out of 133 patients could be considered to have 'catastrophic hands', constituting 6.7% of the total cases. Discussion: The term 'catastrophic hand' has different meanings, and this generates ambiguity. The use of a classification based on the possibility of obtaining an acceptable hand diminishes its use and improves the approach for such injuries. Conclusion: We advocate limiting the use of 'catastrophic hand' to cases that meet criteria of 'mutilated hand' and 'metacarpal hand' to avoid indiscriminate use of the term and optimise therapeutic management

Coeficiente intelectual de niños escolarizados en instituciones públicas de las zonas nororiental y noroccidental de Medellín según el nivel de seguridad alimentaria del hogar y condiciones socieconómicas / Intelligence quotient of school-aged children enrolled in public schools located in the northeast and northwest areas of Medellin based on the level of household food security and socioeconomic conditions

Objective: To analyze the association between school-aged children intelligence quotient and their household food security levels and socioeconomic conditions. Methodology: Cross-sectional analysis with a randomly selected group of 423 school-aged children enrolled in 41 public schools located in the northeast and northwest areas of Medellin, Colombia. The intelligence quotient (IQ) was assessed using the Wechsler Intelligence Scale for Children-IV (WISC-IV). Household food security was assessed using the Latin American and Caribbean Household Food Security Scale. Results: There were significant differences (p<0,001) between children IQ in household with good food security (mean: 93.6 ± 12.7) and severe household food insecurity (mean: 87.5 ± 15.4). The lower IQ scores were explained partly by diminished household food insecurity and socioeconomic conditions. Conclusions: To live under unfavorable environmental conditions predicts children cognitive problems and these effects are bigger if the families experiencing food insecurity or hunger.

Objetivo. AAnalizar la asociación entre el coeficiente intelectual de niños entre 6 y 8 años escolarizados en instituciones públicas de las zonas nororiental y nor-occidental de Medellín con los niveles de seguridad alimentaria de sus hogares y condiciones socioeconómicas. Metodología. Estudio observational analítico de corte transversal. La muestra estuvo constituida por 423 niños. El coeficiente intelectual fue valorado mediante la Escala de Inteligencia Wechsler para Niños WISC-IV. Para la medición de la seguridad alimentaria en el hogar se utilizó la Escala Latinoamericana y Caribeña de Seguridad Alimentaria. Las condiciones socioeconómicas fueron elegidas tomando como referencia antecedentes de investigación. Resultados. Se hallaron diferencias estadísticamente significativas (p<0,001) en el coeficiente intelectual total de los niños que pertenecían a hogares seguros (media: 93,6 DE: 12,7) con respecto a aquellos de hogares en inseguridad alimentaria severa (media: 87,5 DE: 15,4). El coeficiente intelectual limítrofe fue explicado en parte por la inseguridad alimentaria severa en el hogar (OR: 3,57 IC: 1,61-7,91) y las condiciones socioeconómicas. Conclusión. Vivir en medio de condiciones precarias predice la alteración cognitiva, estos efectos son mayores si las familias de los niños se encuentran en inseguridad alimentaria.

Activin A stimulates IkappaB-alpha/NFkappaB and RANK expression for osteoclast differentiation, but not AKT survival pathway in osteoclast precursors.

Recent studies have reported that activin A enhances osteoclastogenesis in cultures of mouse bone marrow cells stimulated with receptor activator of nuclear factor-kappaB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). However, the exact mechanisms by which activin A functions during osteoclastogenesis are not clear. RANKL stimulation of RANK/TRAF6 signaling increases nuclear factor-kappaB (NFkappaB) nuclear translocation and activates the Akt/PKB cell survival pathway. Here we report that activin A alone activates IkappaB-alpha, and stimulates nuclear translocation of NFkappaB and receptor activator of nuclear factor-kappaB (RANK) expression for osteoclastogenesis, but not Akt/PKB survival signal transduction including BAD and mammalian target of rapamycin (mTOR) for survival in osteoclast precursors in vitro. Activin A alone failed to activate Akt, BAD, and mTOR by immunoblotting, and it also failed to prevent apoptosis in osteoclast precursors. While activin A activated IkappaB-alpha and induced nuclear translocation of phosphorylated-NFkappaB, and it also enhanced RANK expression in osteoclast precursors. Moreover, activin A enhanced RANKL- and M-CSF-stimulated nuclear translocation of NFkappaB. Our data suggest that activin A enhances osteoclastogenesis treated with RANKL and M-CSF via stimulation of RANK, thereby increasing the RANKL stimulation. Activin A alone activated the NFkappaB pathway, but not survival in osteoclast precursors in vitro, but it is, thus, insufficient as a sole stimulus to osteoclastogenesis.

c-Src is required for stimulation of gelsolin-associated phosphatidylinositol 3-kinase.

We have shown that osteopontin binding to integrin alphav beta3 in osteoclasts stimulates gelsolin-associated phosphatidylinositol (PtdIns) 3-hydroxyl kinase (PI 3-kinase), leading to increased levels of gelsolin-bound PtdIns 3,4-P2, PtdIns 4,5-P2, and PtdIns 3, 4,5-P3, uncapping of barbed end actin, and actin filament formation. Inhibition of PI 3-kinase activity by wortmannin blocks osteopontin stimulation of actin filament formation, suggesting that activation of gelsolin-associated PI 3-kinase is an important pathway in cytoskeletal regulation. To study the mechanism of gelsolin-associated PI 3-kinase activation, we analyzed anti-gelsolin immunoprecipitates for the association of protein kinases. We demonstrated that c-Src co-immunoprecipitates with gelsolin, and that osteopontin stimulates its activity. Elimination of osteopontin-stimulated Src activity associated with gelsolin through antisense oligodeoxynucleotides blocked the stimulation of PI 3-kinase activity associated with gelsolin and the gelsolin-dependent cytoskeletal reorganization induced by osteopontin, including increased F-actin levels. In addition, treatment of osteoclasts with antisense oligonucleotides to Src reduced bone resorption. Our results demonstrate that osteopontin stimulates gelsolin-associated Src, leading to increased gelsolin-associated PI 3-kinase activity and PtdIns 3,4,5-P3 levels, which facilitate actin filament formation, osteoclast motility, and bone resorption.

Effect of 1,25-dihydroxyvitamin D3 on phospholipid composition of rat renal brush border membrane.

To analyze the effects of vitamin D on renal tubular cell membrane phospholipid metabolism, the effects of vitamin D depletion and repletion on renal brush border membrane (BBM) phosphatidylethanolamine (PE), phosphatidylcholine (PC), and phosphatidylinositol (PI) were studied. The PC content of BBM from kidneys of rats deprived of vitamin D for 5-6 wk was 33.5 +/- 2.2 nmol Pi/mg protein. This was significantly lower than the PC content of BBM from kidneys of rats supplemented with vitamin D2 for 2 wk, 41.0 +/- 0.4 nmol Pi/mg protein. Vitamin D depletion also decreased the content of BBM PE. The fatty acid composition of BBM PC was altered by vitamin D depletion. Vitamin D depletion increased palmitic acid and decreased stearic, linoleic, and arachidonic acid. Vitamin D repletion with a single physiological dose of 1,25(OH)2D3 (30 pmol), 16 h prior to study tended to increase membrane content of PC and significantly increased the linoleic acid content of the PC fraction. Single-dose vitamin D repletion with a pharmacological dose of 1,25(OH)2D3 (2.4 nmol) produced a significant increase in BBM content of PC and also significantly increased the linoleic acid content of PC. These results demonstrate that vitamin D deficiency affects PC and PE content of rat renal BBM and their fatty acid composition, and that vitamin D repletion with 1,25(OH)2D3 for 16 h partially normalizes the changes in PC.

Phosphatidylinositol 3,4,5-trisphosphate directs association of Src homology 2-containing signaling proteins with gelsolin.

Podosomes are adhesion structures in osteoclasts and are structurally related to focal adhesions mediating cell motility during bone resorption. Here we show that gelsolin coprecipitates some of the focal adhesion-associated proteins such as c-Src, phosphoinositide 3-kinase (PI3K), p130(Cas), focal adhesion kinase, integrin alpha(v)beta(3), vinculin, talin, and paxillin. These proteins were inducibly tyrosine-phosphorylated in response to integrin activation by osteopontin. Previous studies have defined unique biochemical properties of gelsolin related to phosphatidylinositol 3,4,5-trisphosphate in osteoclast podosomes, and here we demonstrate phosphatidylinositol 3,4,5-trisphosphate/gelsolin function in mediating organization of the podosome signaling complex. Overlay and GST pull-down assays demonstrated strong phosphatidylinositol 3,4,5-trisphosphate-PI3K interactions based on the Src homology 2 domains of PI3K. Furthermore, lipid extraction of lysates from activated osteoclasts eliminated interaction between gelsolin, c-Src, PI3K, and focal adhesion kinase despite equal amounts of gelsolin in both the lipid-extracted and unextracted experiment. The cytoplasmic protein tyrosine phosphatase (PTP)-proline-glutamic acid-serine-threonine amino acid sequences (PEST) was also found to be associated with gelsolin in osteoclast podosomes and with stimulation of alpha(v)beta(3)-regulated phosphorylation of PTP-PEST. We conclude that gelsolin plays a key role in recruitment of signaling proteins to the plasma membrane through phospholipid-protein interactions and by regulation of their phosphorylation status through its association with PTP-PEST. Because both gelsolin deficiency and PI3K inhibition impair bone resorption, we conclude that phosphatidylinositol 3,4,5-trisphosphate-based protein interactions are critical for osteoclast function.