Biomedical subjects

Roberta Faccio

Publications and source records attributed to Roberta Faccio.

12 recordsLinked to original sources

PubMed2006

The isozyme-specific effects of cyclooxygenase-deficiency on bone in mice.

Prostaglandin E(2) (PGE(2)) plays a critical role in skeletal physiology and bone loss. PGE(2) production is regulated in vivo by at least two cyclooxygenase (COX) isozymes, COX-1 and COX-2. The purpose of this study was to investigate the in vivo effects of the selective deletion of COX-1 or COX-2 on bone mineral density (BMD), bone microarchitecture and bone strength in wild type (WT), COX-1(-/-) and COX-2(-/-) mice. Using a LUNAR PIXImus, BMD was measured in 18 (WT), 18 COX-1(-/-) and 16 COX-2(-/-) mice. COX-1(-/-) mice exhibited significantly higher BMD (0.0506 g/cm(2) +/- 0.0014 g/cm(2)) than either WT (0.0493 g/cm(2) +/- 0.0019, P < or = 0.05) or COX-2(-/-) (0.0473 g/cm(2) +/- 0.0034, P < or = 0.01) mice. COX-2(-/-) mice had significantly lower BMD than WT (P < or = 0.01) or COX-1(-/-) (P < or = 0.01). Flexure stress of the femurs, determined by breaking the bones with three-point bending, correlated with bone density. Although plasma levels of both Ca(2+) and PTH were comparable in wild type and COX-1(-/-) mice, both were elevated in COX-2(-/-) mice consistent with primary hyperparathyroidism. These studies suggest that COX enzymes are important regulators of BMD and bone strength in mice. The beneficial effect of absence of the COX-1 enzyme on skeletal parameters may be secondary to decreases in PGE(2). On the other hand, primary hyperparathyroidism and lower bone magnesium content may account for the lower BMD and impairments in bone strength of COX-2(-/-) mice. Further elucidation of the effects of the COX pathway on bone remodeling may provide important information on potential therapeutic targets for preventing and/or treating osteoporosis.

Animals↗

PubMed2006

Cortactin has an essential and specific role in osteoclast actin assembly.

Osteoclasts are essential for bone dynamics and calcium homeostasis. The cells form a tight seal on the bone surface, onto which they secrete acid and proteases to resorb bone. The seal is associated with a ring of actin filaments. Cortactin, a c-Src substrate known to promote Arp2/3-mediated actin assembly in vitro, is expressed in osteoclasts and localizes to the sealing ring. To address the role of cortactin and actin assembly in osteoclasts, we depleted cortactin by RNA interference. Cortactin-depleted osteoclasts displayed a complete loss of bone resorption with no formation of sealing zones. On nonosteoid surfaces, osteoclasts flatten with a dynamic, actin-rich peripheral edge that contains podosomes, filopodia, and lamellipodia. Cortactin depletion led to a specific loss of podosomes, revealing a tight spatial compartmentalization of actin assembly. Podosome formation was restored in cortactin-depleted cells by expression of wild-type cortactin or a Src homology 3 point mutant of cortactin. In contrast, expression of a cortactin mutant lacking tyrosine residues phosphorylated by Src did not restore podosome formation. Cortactin was found to be an early component of the nascent podosome belt, along with dynamin, supporting a role for cortactin in actin assembly.

Actin-Related Protein 2↗

PubMed2006

Inhibition of IKK activation, through sequestering NEMO, blocks PMMA-induced osteoclastogenesis and calvarial inflammatory osteolysis.

Osteoclasts, the primary bone-resorbing cells, play a crucial role in periprosthetic bone loss in response to implant-derived wear debris. Differentiation and activation of osteoclasts at the implant-bone interface are fueled by elevated levels of locally secreted inflammatory cytokines that heighten the osteolytic response. Among these cytokines are members of the TNF superfamily, including TNF and RANKL, which primarily act through activation of the transcription factor NF-kappaB. Activation of NF-kappaB is required for osteoclast formation, and its inhibition hampers osteoclastogenesis and bone loss. Activation of NF-kappaB is permitted following its dissociation from the inhibitory protein IkappaBalpha, an event subsequent to phosphorylation of the latter protein by the upstream IkappaBalpha kinase (IKK) complex. Our recent findings show that attenuating IKK complex assembly, by using a short peptide termed NEMO-binding domain (NBD) peptide, that blocks binding of IKK2 and IKK1 to IKKgamma/NEMO, inhibits NF-kappaB activation, and arrests RANKL-induced osteoclastogenesis. In this study, we examined if NBD is capable of blocking inflammatory osteolysis by PMMA particles. Our findings indicate that NBD peptide inhibits PMMA-induced IKK2 and NF-kappaB activation. More importantly, this peptide potently arrests PMMA-stimulated osteoclastogenesis and alleviates PMMA-induced inflammatory and osteolytic responses in mice. Thus, NBD peptide is considered as a promising modality to regulate inflammatory osteolysis.

Animals↗

PubMed2005

Vav3 regulates osteoclast function and bone mass.

Osteoporosis, a leading cause of morbidity in the elderly, is characterized by progressive loss of bone mass resulting from excess osteoclastic bone resorption relative to osteoblastic bone formation. Here we identify Vav3, a Rho family guanine nucleotide exchange factor, as essential for stimulated osteoclast activation and bone density in vivo. Vav3-deficient osteoclasts show defective actin cytoskeleton organization, polarization, spreading and resorptive activity resulting from impaired signaling downstream of the M-CSF receptor and alpha(v)beta3 integrin. Vav3-deficient mice have increased bone mass and are protected from bone loss induced by systemic bone resorption stimuli such as parathyroid hormone or RANKL. Moreover, we provide genetic and biochemical evidence for the role of Syk tyrosine kinase as a crucial upstream regulator of Vav3 in osteoclasts. Thus, Vav3 is a potential new target for antiosteoporosis therapy.

Animals↗

PubMed2004

T cells support osteoclastogenesis in an in vitro model derived from human multiple myeloma bone disease: the role of the OPG/TRAIL interaction.

The development of multiple myeloma (MM) bone disease is mediated by increased number and activity of osteoclasts (OCs). Using an in vitro osteoclastogenesis model consisting of unstimulated and unfractionated peripheral blood mononuclear cells (PBMCs) from patients with MM, we showed that T cells support the formation of OCs with longer survival. Different from T-cell-depleted MM PBMC cultures, exogenous macrophage-colony stimulating factor (M-CSF) and receptor activator of nuclear factor-kappaB ligand (RANKL) were necessary for the formation of OCs; however, they did not exhibit longer survival. We found up-regulated production of RANKL, osteoprotegerin (OPG), and TNF-related apoptosis-inducing ligand (TRAIL) by fresh MM T cells. Despite high OPG levels, the persistence of osteoclastogenesis can be related to the formation of the OPG/TRAIL complex demonstrated by immunoprecipitation experiments and the addition of anti-TRAIL antibody which decreases OC formation. OCs overexpressed TRAIL decoy receptor DcR2 in the presence of MM T cells and death receptor DR4 in T-cell-depleted cultures. In addition, increased Bcl-2/Bax (B-cell lymphoma-2/Bcl2-associated protein X) ratio, following Bcl-2 up-regulation, was detected in OCs generated in the presence of T cells. Our results highlight that MM T cells support OC formation and survival, possibly involving OPG/TRAIL interaction and unbalanced OC expression of TRAIL death and decoy receptors.

Aged↗

PubMed2004

The HIV protease inhibitor ritonavir blocks osteoclastogenesis and function by impairing RANKL-induced signaling.

Highly active antiretroviral therapy (HAART), which includes HIV protease inhibitors (PIs), has been associated with bone demineralization. To determine if this complication reflects accelerated resorptive activity, we studied the impact of two common HIV PIs, ritonavir and indinavir, on osteoclast formation and function. Surprisingly, we find that ritonavir, but not indinavir, inhibits osteoclast differentiation in a reversible manner and also abrogates bone resorption by disrupting the osteoclast cytoskeleton, without affecting cell number. Ritonavir given in vivo completely blunts parathyroid hormone-induced osteoclastogenesis in mice, which confirms that the drug is bone sparing. In keeping with its antiresorptive properties, ritonavir impairs receptor activator of nuclear factor kappaB ligand-induced (RANKL-induced) activation of NF-kappaB and Akt signaling pathways, both critical to osteoclast formation and function. In particular, ritonavir is found to inhibit RANKL-induced Akt signaling by disrupting the recruitment of TNF receptor-associated factor 6/c-Src complex to lipid rafts. Thus, ritonavir may represent a bone-sparing PI capable of preventing development of osteopenia in patients currently on HAART.

Animals↗

PubMed2003

High dose M-CSF partially rescues the Dap12-/- osteoclast phenotype.

Osteoclasts are macrophage derived cells and as such are subject to regulation by molecules impacting other members of the immune system. Dap12 is an adaptor protein expressed by NK cells and B and T lymphocytes. Dap12 also mediates maturation of myeloid cells and is expressed by osteoclasts which are dysfunctional in its absence. We find Dap12-/- osteoclast precursors fail to differentiate, in vitro, and the abnormality is partially rescued by high dose M-CSF. The relative paucity of osteoclast number, even in presence of high dose cytokine, is attended by dampened proliferation of precursor cells and their failure to normally migrate towards the osteoclast-recognized matrix protein, osteopontin. Furthermore, Dap12-/- osteoclasts generated in high dose M-CSF fail to normally organize their cytoskeleton. The incapacity of Dap12 null cells to undergo normal osteoclast differentiation is not due to blunted stimulation of major RANK ligand (RANKL) or M-CSF induced signaling pathways. On the other hand, when plated on osteopontin, Dap12-/- pre-osteoclasts do not activate the tyrosine kinase, Syk, which normally binds to the adaptor protein and transmits downstream signals. Attesting to the importance of the Dap12/Syk complex, Syk deficient macrophages do not undergo normal osteoclastogenesis. Furthermore, the same cells plated onto osteopontin, adhere poorly and fail to phosphorylate c-Src or Pyk2, two kinases central to organization of the osteoclast cytoskeleton.

Adaptor Proteins, Signal Transducing↗

PubMed2003

Vav1/2/3-null mice define an essential role for Vav family proteins in lymphocyte development and activation but a differential requirement in MAPK signaling in T and B cells.

The Vav family of Rho guanine nucleotide exchange factors is thought to orchestrate signaling events downstream of lymphocyte antigen receptors. Elucidation of Vav function has been obscured thus far by the expression of three highly related family members. We generated mice lacking all Vav family proteins and show that Vav-null mice produce no functional T or B cells and completely fail to mount both T-dependent and T-independent humoral responses. Whereas T cell development is blocked at an early stage in the thymus, immature B lineage cells accumulate in the periphery but arrest at a late "transitional" stage. Mechanistically, we show that the Vav family is crucial for both TCR and B cell receptor (BCR)-induced Ca2+ signaling and, surprisingly, is only required for mitogen-activated protein kinase (MAPK) activation in developing and mature T cells but not in B cells. Thus, the abundance of immature B cells generated in Vav-null mice may be due to intact Ras/MAPK signaling in this lineage. Although the expression of Vav1 alone is sufficient for normal lymphocyte development, our data also reveal lineage-specific roles for Vav2 and Vav3, with the first demonstration that Vav3 plays a critical compensatory function in T cells. Together, we define an essential role for the entire Vav protein family in lymphocyte development and activation and establish the limits of functional redundancy both within this family and between Vav and other Rho-guanine nucleotide exchange factors.

Animals↗

PubMed2003

Dynamic changes in the osteoclast cytoskeleton in response to growth factors and cell attachment are controlled by beta3 integrin.

The beta3 integrin cytoplasmic domain, and specifically S752, is critical for integrin localization and osteoclast (OC) function. Because growth factors such as macrophage colony-stimulating factor and hepatocyte growth factor affect integrin activation and function via inside-out signaling, a process requiring the beta integrin cytoplasmic tail, we examined the effect of these growth factors on OC precursors. To this end, we retrovirally expressed various beta3 integrins with cytoplasmic tail mutations in beta3-deficient OC precursors. We find that S752 in the beta3 cytoplasmic tail is required for growth factor-induced integrin activation, cytoskeletal reorganization, and membrane protrusion, thereby affecting OC adhesion, migration, and bone resorption. The small GTPases Rho and Rac mediate cytoskeletal reorganization, and activation of each is defective in OC precursors lacking a functional beta3 subunit. Activation of the upstream mediators c-Src and c-Cbl is also dependent on beta3. Interestingly, although the FAK-related kinase Pyk2 interacts with c-Src and c-Cbl, its activation is not disrupted in the absence of functional beta3. Instead, its activation is dependent upon intracellular calcium, and on the beta2 integrin. Thus, the beta3 cytoplasmic domain is responsible for activation of specific intracellular signals leading to cytoskeletal reorganization critical for OC function.

Amino Acid Sequence↗

PubMed2003

c-Fms and the alphavbeta3 integrin collaborate during osteoclast differentiation.

beta(3) integrin-null osteoclasts are dysfunctional, but their numbers are increased in vivo. In vitro, however, the number of beta(3)(-/-) osteoclasts is reduced because of arrested differentiation. This paradox suggests cytokine regulation of beta(3)(-/-) osteoclastogenesis differs in vitro and in vivo. In vitro, additional MCSF, but not receptor activator of NF-kappaB ligand (RANKL), completely rescues beta(3)(-/-) osteoclastogenesis. Similarly, activation of extracellular signal-regulated kinases (ERKs) and expression of c-Fos, both essential for osteoclastogenesis, are attenuated in beta(3)(-/-) preosteoclasts, but completely restored by additional MCSF. In fact, circulating and bone marrow cell membrane-bound MCSFs are enhanced in beta(3)(-/-) mice, correlating with the increase in the osteoclast number. To identify components of the MCSF receptor that is critical for osteoclastogenesis in beta(3)(-/-) cells, we retrovirally transduced authentic osteoclast precursors with chimeric c-Fms constructs containing various cytoplasmic domain mutations. Normalization of osteoclastogenesis and ERK activation, in beta(3)(-/-) cells, uniquely requires c-Fms tyrosine 697. Finally, like high-dose MCSF, overexpression of c-Fos normalizes the number of beta(3)(-/-) osteoclasts in vitro, but not their ability to resorb dentin. Thus, while c-Fms and alpha(v)beta(3) collaborate in the osteoclastogenic process via shared activation of the ERK/c-Fos signaling pathway, the integrin is essential for matrix degradation.

Animals↗

PubMed2002

Localization and possible role of two different alpha v beta 3 integrin conformations in resting and resorbing osteoclasts.

Integrins are membrane receptors that mediate interactions between cells and the extracellular matrix. We recently showed that the osteoclast integrin alpha(v)beta(3) exists in two different conformations, so-called 'basal' and 'activated', with each exhibiting a distinct function. In this study we demonstrate that, in non-resorbing osteoclasts, the 'activated' form of alpha(v)beta(3) accumulates in the motile areas of the plasma membrane. During bone resorption this conformation is prevalent in the ruffled membrane, whereas the 'basal' form of alpha(v)beta(3) is also present in the sealing zone. Moreover, hepatocyte growth factor (HGF) and macrophage colony stimulating factor (M-CSF), two molecules involved in osteoclastogenesis and osteoclast survival, modulate alpha(v)beta(3) conformation in vitro. Preincubation with HGF or M-CSF induces a shift of conformation of alpha(v)beta(3) in primary human osteoclasts (OCs) and in the osteoclast-like cell line (GCT 23). Activated integrin promotes osteoclast migration to the alpha(v)beta(3) ligand osteopontin and enhances bone resorption. Thus, HGF and M-CSF modulate the alpha(v)beta(3) conformational states required for osteoclast polarization and resorption. The capacity of growth factors to alter the affinity of alpha(v)beta(3) toward its ligands offers a potential explanation for the diverse responses of osteoclasts to the same ligand.

Antibodies, Monoclonal↗

PubMed2001

Activated coagulation factor X: a novel mitogenic stimulus for human mesangial cells.

Intraglomerular activation of the coagulation cascade is a common feature of mesangioproliferative glomerulonephritis. Besides thrombin, very little is known about the cellular effects of other components of the coagulation system. This study investigated the effect of activated factor X (FXa) on cultured human mesangial cells. This serine protease induced a significant and dose-dependent increase in DNA synthesis. In addition to its mitogenic effect, FXa caused a striking upregulation of platelet-derived growth factor (PDGF) A and B chain gene expression. Next, the intracellular mitogenic signaling pathways activated by FXa were investigated. FXa induced a rapid spike in cytosolic calcium concentration followed by a sustained plateau. This response was not influenced by the downregulation of thrombin receptors. In addition, FXa stimulated a significant upregulation of different tyrosine-phosphorylated proteins. One of these phosphorylated cellular proteins was represented by the c-jun N-terminal kinase, a member of the mitogen-activated protein kinase family. To evaluate the role of FXa enzymatic activity and of PDGF autocrine secretion, FXa-induced DNA synthesis was studied in the presence of leupeptin, a specific serine protease inhibitor, and neutralizing anti-PDGF antibody. To investigate the role of tyrosine kinase (TK) activation on FXa mitogenic effect, FXa-stimulated thymidine uptake was evaluated in the presence of genistein and herbimycin A, two powerful and specific TK inhibitors. FXa-elicited DNA synthesis was also examined after protein kinase C (PKC) downregulation by prolonged incubation with phorbol-12-myristate-13-acetate to study the influence of the phospholipase C-PKC axis. The proliferative effect of FXa required its proteolytic activity, and the activation of TK was only partially dependent on PKC activation while it was PDGF independent. Finally, it was shown by reverse transcription-PCR that mesangial cells do not express the signaling splicing variant of the putative FXa receptor, effector protease receptor-1. In conclusion, the present study demonstrated that FXa is a powerful mitogenic factor for human mesangial cells, and it induces its cellular effect not through effector protease receptor-1, but most likely by binding a protease-activated receptor and activating phospholipase C-PKC and TK signaling pathways.

Base Sequence↗