Staphylococcus aureus protein A plays a critical role in mediating bone destruction and bone loss in osteomyelitis

Staphylococcus aureus is the most frequent causative organism of osteomyelitis. It is characterised by widespread bone loss and bone destruction. Previously we demonstrated that S. aureus protein A (SpA) is capable of binding to tumour necrosis factor receptor-1 expressed on pre-osteoblastic cells, which results in signal generation that leads to cell apoptosis resulting in bone loss. In the current report we demonstrate that upon S. aureus binding to osteoblasts it also inhibits de novo bone formation by preventing expression of key markers of osteoblast growth and division such as alkaline phosphatase, collagen type I, osteocalcin, osteopontin and osteocalcin. In addition, S. aureus induces secretion of soluble RANKL from osteoblasts which in turn recruits and activates the bone resorbing cells, osteoclasts. A strain of S. aureus defective in SpA failed to affect osteoblast growth or proliferation and most importantly failed to recruit or activate osteoclasts. These results suggest that S. aureus SpA binding to osteoblasts provides multiple coordinated signals that accounts for bone loss and bone destruction seen in osteomyelitis cases. A better understanding of the mechanisms through which S. aureus leads to bone infection may improve treatment or lead to the development of better therapeutic agents to treat this notoriously difficult disease.     

TRAIL expression is induced in both osteoblasts containing intracellular Staphylococcus aureus and uninfected osteoblasts in infected cultures

Staphylococcus aureus is the principal etiological agent of osteomyelitis (bone infection), which is characterized by a progressive inflammatory response resulting in extensive damage to bone tissue. Recent studies have demonstrated the ability of S. aureus to invade and persist inside osteoblasts (bone matrix-forming cells) and other eukaryotic cells. The presence of intracellular S. aureus in bone tissue may be relevant to the pathology of osteomyelitis, a disease often refractory to antibiotic treatment and subject to recurrence months and even years after apparently successful therapy. The present study examined the production of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) following S. aureus infection, and whether expression of the molecule was induced by those osteoblasts containing intracellular S. aureus. Results from this study suggest that osteoblasts containing intracellular S. aureus induce TRAIL expression in uninfected osteoblasts present in infected cultures.     

Staphylococcus epidermidis serine–aspartate repeat protein G (SdrG) binds to osteoblast integrin alpha V beta 3

Staphylococcus epidermidis is the leading etiologic agent of orthopaedic implant infection. Contamination of the implanted device during insertion allows bacteria gain entry into the sterile bone environment leading to condition known as osteomyelitis. Osteomyelitis is characterised by weakened bones associated with progressive bone loss. The mechanism through which S. epidermidis interacts with bone cells to cause osteomyelitis is poorly understood. We demonstrate here that S. epidermidis can bind to osteoblasts in the absence of matrix proteins. S. epidermidis strains lacking the cell wall protein SdrG had a significantly reduced ability to bind to osteoblasts. Consistent with this, expression of SdrG in Lactococcus lactis resulted in significantly increased binding to the osteoblasts. Protein analysis identified that SdrG contains a potential integrin recognition motif. αVβ3 is a major integrin expressed on osteoblasts and typically recognises RGD motifs in its ligands. Our results demonstrate that S. epidermidis binds to recombinant purified αVβ3, and that a mutant lacking SdrG failed to bind. Blocking αVβ3 on osteoblasts significantly reduced binding to S. epidermidis. These studies are the first to identify a mechanism through which S. epidermidis binds to osteoblasts and potentially offers a mechanism through which implant infection caused by S. epidermidis leads to osteomyelitis.     

Staphylococcus aureus protein A induced inflammatory response in human corneal epithelial cells

In the present study, we examined the role of Staphylococcus aureus protein A (SpA) in inducing inflammatory response in human corneal epithelial cells (HCECs). Exposure of HCECs to SpA induces rapid NF-kappaB activation and secretion of proinflammatory cytokine/chemokines (TNF-alpha and IL-8) in both concentration and time-dependent manner. Challenge of HCECs with live SpA(-/-) mutant S. aureus strains resulted in significantly reduced production of the cytokines when compared to the wild-type S. aureus strain. SpA also elicited the activation of MAP Kinases P38, ERK, but not JNK, in HCECs. SpA-induced production of proinflammatory cytokine were completely blocked by the NF-kappaB and p38 inhibitors and partially inhibited by the Jnk inhibitor. Pretreatment with anti-TLR2 neutralizing antibody had no effect on SpA-induced inflammatory response in HCECs, suggesting that this response is independent of TLR2 signaling. Moreover, unlike TLR2 ligands, SpA failed to induce the expression of antimicrobial peptides (hBD2 and LL-37) in HCECs. These studies indicate that SpA is a S. aureus virulence factor that stimulates HCEC inflammatory response through a pathway distinct from TLR2 in HCECs.     

Some improved versions of methods for the indirect detection of staphylococcal protein A gene expressed in Escherichia coli.

Several versions of methods for the indirect detection of expression of staphylococcal protein A gene (spa) in Escherichia coli (E. coli) were devised by making use of biological properties of staphylococcal protein A (SpA). i) Hemagglutination of sheep red blood cells (SRBC) sensitized with anti-SRBC-antibodies using heat-treated spa-transformed E. coli organisms; Native spa-transformed E. coli organisms did not agglutinate the sensitized SRBC. The heat-treatment (60 C, 4 hr) of the transformants, however, caused positive hemagglutination like SpA-positive Staphylococcus aureus (S. aureus) organisms. ii) Halo formation around colonies on agar plates containing normal dog serum, which is originally used for the detection of SpA of S. aureus. A mutant strain NMJ was isolated, which showed formation of the halo of precipitate due to interaction between immunoglobulin and SpA. iii) A new version of immunodetection; After lysis of the transformants grown on a nitrocellulose membrane by alkali, SpA could be directly detected by immuno-detection procedures after inactivation of endogenous peroxidase in bacteria by phenylhydrazine and hydrogen peroxide.     

Local osteogenic expression of cyclooxygenase-2 and systemic response in porcine models of osteomyelitis

It is suggested that cyclooxygenase 2 (COX-2) derived prostaglandins contributes to the progressive bone loss seen in osteomyelitis lesions. In the present study we examined the expression of COX-2 in bones from 23 pigs with experimental osteomyelitis. Osteomyelitis was induced with Staphylococcus aureus and groups of animals were euthanized following 6 h, 12 h, 24 h, 2 days, 5 days, 11 days and 15 days, respectively. Expression of COX-2 was evaluated immunohistochemically and combined with characterization of morphological changes in bone tissue. Furthermore, the serum concentrations of alkaline phosphatase and haptoglobin were measured. Extensive COX-2 expression by osteoblasts was present 2 days after inoculation together with many activated osteoclasts. Simultaneously, the serum concentration of alkaline phosphatase decreased whereas the haptoglobin concentration increased. This is the first in vivo study showing an early wave of COX-2 mediated bone resorption during osteomyelitis. Therefore, treatment aiming to reduce the break down of bone tissue directed by the COX-2 pathway might be suggested early in the course of the disease.     

EGFR/FAK and c‐Src signalling pathways mediate the internalisation of Staphylococcus aureus by osteoblasts

Internalisation of Staphylococcus aureus in osteoblasts plays a critical role in the persistence and recurrence of osteomyelitis, the mechanisms involved in this process remain largely unknown. In the present study, evidence of internalised S. aureus in osteoblasts was found in long bone of haematogenous osteomyelitis in mice after 2 weeks of infection. Meanwhile, eliminating extracellular S. aureus by gentamicin can partially rescue bone loss, whereas the remaining intracellular S. aureus in osteoblasts may be associated with continuous bone destruction. In osteoblastic MC3T3 cells, intracellular S. aureus was detectable as early as 15 min after infection, and the internalisation rates increased with the extension of infection time. Additionally, S. aureus invasion stimulated the expression of phosphor‐focal adhesion kinase (FAK), phosphor‐epidermal growth factor receptor (EGFR) and phosphor‐c‐Src in a time‐dependent way, and blocking EGFR/FAK or c‐Src signalling significantly reduced the internalisation rate of S. aureus in osteoblasts. Our findings provide new insights into the mechanism of S. aureus internalisation in osteoblast and raise the potential of targeting EGFR/FAK and c‐Src as adjunctive therapeutics for treating chronic S. aureus osteomyelitis.     

Staphylococcus aureus isolated from Kawasaki disease patients hyper-releases extracellular protein A.

S. aureus isolates from patients with Kawasaki disease (KD) release high levels of extracellular protein A (SpA), as compared to S. aureus in other diseases. The molecular weight of this released protein A is about 70 kDa. Extracellular KD SpA purified by affinity chromatography possessed the same amino acid sequence at the NH2-terminal IgG binding region and the same antigenic specificity as recombinant and cell-wall-bound SpA preparations. The size of DNA fragments containing the spa gene from S. aureus KD strains was 160-165 kb. All of these DNA fragments contained the igb portion encoding the IgG-binding region of KD SpA. Significantly higher molecular size of the SpA molecules hyper-released in the stationary-phase culture and the lack of production of other exo-proteins allow us to speculate that S. aureus isolated from patients with KD have mutations occurring in the agr locus.     

Staphylococcus aureus protein A mediates invasion across airway epithelial cells through activation of RhoA GTPase signaling and proteolytic activity

Staphyococcus aureus and especially the epidemic methicillin-resistant S. aureus strains cause severe necrotizing pneumonia. The mechanisms whereby these organisms invade across the mucosal epithelial barrier to initiate invasive infection are not well understood. Protein A (SpA), a highly conserved and abundant surface protein of S. aureus, activates TNF receptor 1 and EGF receptor (EGFR) signaling cascades that can perturb the cytoskeleton. We demonstrate that wild-type S. aureus, but not spa mutants, invade across polarized airway epithelial cell monolayers via the paracellular junctions. SpA stimulated a RhoA/ROCK/MLC cascade, resulting in the contraction of the cytoskeleton. SpA(+) but not SpA(-) mutants stimulated activation of EGFR and along with subsequent calpain activity cleaved the membrane-spanning junctional proteins occludin and E-cadherin, facilitating staphylococcal transmigration through the cell-cell junctions. Treatment of polarized human airway epithelial monolayers with inhibitors of ROCK, EGFR, MAPKs, or calpain prevented staphylococcal penetration through the monolayers. In vivo, blocking calpain activity impeded bacterial invasion into the lung parenchyma. Thus, S. aureus exploits multiple receptors available on the airway mucosal surface to facilitate invasion across epithelial barriers.     

Recurrent infections and immune evasion strategies of Staphylococcus aureus

Staphylococcus aureus causes purulent skin and soft tissue infections (SSTIs) that frequently reoccur. Staphylococal SSTIs can lead to invasive disease and sepsis, which are among the most significant causes of infectious disease mortality in both developed and developing countries. Human or animal infections with S. aureus do not elicit protective immunity against staphylococcal diseases. Here we review what is known about the immune evasive strategies of S. aureus that enable the pathogen's escape from protective immune responses. Three secreted products are discussed in detail, staphylococcal protein A (SpA), staphylococcal binder of immunoglobulin (Sbi) and adenosine synthase A (AdsA). By forming a complex with V(H)3-type IgM on the surface of B cells, SpA functions as a superantigen to modulate antibody responses to staphylococcal infection. SpA also captures pathogen-specific antibodies by binding their Fcγ portion. The latter activity of SpA is shared by Sbi, which also associates with complement factors 3d and factor H to promote the depletion of complement. AdsA synthesizes the immune signaling molecule adenosine, thereby dampening innate and adaptive immune responses during infection. We discuss strategies how the three secreted products of staphylococci may be exploited for the development of vaccines and therapeutics.     

The adhesion of protein A-bearing Staphylococcus aureus organisms to soluble-staphylococcal-antigens-coated HeLa cells mediated by specific antibodies

The adhesion of staphylococcal protein A (SpA)-bearing Staphylococcus aureus Cowan I organisms to HeLa cells was enhanced by pretreatment of HeLa cells with staphylococcal extracellular antigens and antibodies to them. The adhesion of HLj, an SpA-poor mutant derived from Cowan I, to HeLa cells was not enhanced by the same pretreatment of HeLa cells. Furthermore, the enhanced staphylococcal adhesion was inhibited by soluble SpA. The antigen(s) responsible for the enhanced staphylococcal adhesion was(were) heat stable. Pretreatment of HeLa cells with the mixture of staphylococcal extracellular antigens and antibodies to them also enhanced the adhesion of Cowan I. Similarly the adhesion of Cowan I was enhanced by pretreatment of HeLa cells with extracellular antigens of Pseudomonas aeruginosa and antibodies to them. These results indicated that cell-bound SpA mediated the binding of S. aureus to immune complexes composed of extracellular bacterial products and antibodies to them bound to the surface of HeLa cells, and suggested another role of cell-bound SpA as a co-adhesin with other factors in infections due to S. aureus.     

Attachment of Staphylococcus aureus is required for activation of nuclear factor kappa B in human osteoblasts

Nuclear factor kappa B (NF-κB) plays a prominent role in the pathogenesis of infectious diseases. Staphylococcus aureus (S. aureus), which can attach to and invade human osteoblasts, is the most common causative agent of osteomyelitis. To determine whether S. aureus can activate NF-κB in human osteoblasts and explore the possible factors of activation in response to infection, we used flow cytometry, enzyme-linked immunosorbent assay, immunoblots, and electrophoretic mobility shift assays to quantify the invasion of bacteria, to measure the interleukin-6 (IL-6) of culture supernatants, and to investigate the IκBα degradation and NF-κB activation in human osteoblasts. Moreover, we explored the possible factors responsible for the activation of NF-κB by preventing S. aureus from physically touching human osteoblasts or inhibiting the invasion of S. aureus into human osteoblasts under co-culture conditions, by incubating proteinase K-treated or ultraviolet-killed S. aureus with human osteoblasts and by treating human osteoblasts with peptidoglycan (PGN) or lipoteichoic acid (LTA). We found that S. aureus induced the IκBα degradation and NF-κB activation, which could regulate IL-6 secretion in the culture supernatants of human osteoblasts in response to infection. In addition, the maximal IκBα degradation and NF-κB activation in human osteoblasts occurred prior to the maximal invasion of S. aureus. It was the attachment not invasion or the secreted soluble factor(s), PGN, LTA of S. aureus, that could induce the IκBα degradation and NF-κB activation in human osteoblasts. These results indicated that S. aureus can activate NF-κB in human osteoblasts and that the attachment of S. aureus is required for this activation in response to infection.     

Staphylococcus aureus protein A triggers T cell-independent B cell proliferation by sensitizing B cells for TLR2 ligands

B cells possess functional characteristics of innate immune cells, as they can present Ag to T cells and can be stimulated with microbial molecules such as TLR ligands. Because crude preparations of Staphylococcus aureus are frequently used as polyclonal B cell activators and contain potent TLR2 activity, the scope of this study was to analyze the impact of S. aureus-derived TLR2-active substances on human B cell activation. Peripheral B cells stimulated with chemically modified S. aureus cell wall preparations proliferated in response to stimulation with crude cell wall preparations but failed to be activated with pure peptidoglycan, indicating that cell wall molecules other than peptidoglycan are responsible for B cell proliferation. Subsequent analysis revealed that surface protein A (SpA), similar to BCR cross-linking with anti-human Ig, sensitizes B cells for the recognition of cell wall-associated TLR2-active lipopeptides (LP). In marked contrast to TLR7- and TLR9-triggered B cell stimulation, stimulation with TLR2-active LP and SpA or with crude cell wall preparations failed to induce IgM secretion, thereby revealing qualitative differences in TLR2 signaling compared with TLR7/9 signaling. Notably, combined stimulation with SpA plus TLR2 ligands induced vigorous proliferation of a defined B cell subset that expressed intracellular IgM in the presence of IL-2. Conclusion: S. aureus triggers B cell activation via SpA-induced sensitization of B cells for TLR2-active LP. Combined SpA and TLR2-mediated B cell activation promotes B cell proliferation but fails to induce polyclonal IgM secretion as seen after TLR7 and TLR9 ligation.     

An acute osteomyelitis model in traumatized rat tibiae involving sand as a foreign body, thermal injury, and bimicrobial contamination

The multfactorial nature of bone injuries in modern warfare and emergency trauma patients warrants enhancement of existing models. To develop a more appropriate model, rat tibiae (n = 195) were mechanically injured, divided into 2 groups (with or without thermal injury), and contaminated with a range of Staphylococcus aureus (Cowan 1) inocula. In some experiments, S. aureus inocula also contained Escherichia coli or foreign bodies (sand or soil). The primary outcome measure was the amount of S. aureus remaining in the tibia (tibial bacterial load) 24 h after contamination, reported as log10 cfu/g bone. S. aureus showed ID50 and ID95 values of 72 and 977 cfu, respectively. Values were lower than seen previously by using S. aureus strain SMH. S. aureus tibial bacterial loads were higher in tibiae with mechanical and thermal injury (log10 4.15 +/- 0.27 cfu/g) versus mechanical injury alone (log10 3.1 +/- 0.47 cfu/g, P = 0.028). The addition of E. coli to the S. aureus inoculum had no effect on tibial bacterial loads (S. aureus only, log10 4.24 +/- 0.92 cfu/g; S. aureus + E. coli, log10 4.1 +/- 1.0 cfu/g, P = 0.74). Sand, added as a foreign body, increased tibial bacterial load. Combined mechanical and thermal trauma of the tibia is associated with increased S. aureus tibial bacterial loads, increasing the risk of acute osteomyelitis. Understanding the interplay of mechanical and thermal injuries, bimicrobial contamination, and foreign bodies may improve our understanding of traumatic bone injuries and the pathogenesis of osteomyelitis.     

Distinction of two subtypes of human leukocyte interferon (IFN-alpha) on B cell activation. B cell proliferation by two subtypes of IFN-alpha

We examined the effect of interferon (IFN), with particular emphasis on the effects of the two subtypes of IFN-alpha (IFN-alpha A and IFN-alpha B) on the B cell proliferation induced by Staphylococcus aureus Cowan I bacterium (SpA Col). An increase of SpA Col-induced proliferation was observed in the presence of 100 to 1000 U/ml of IFN-alpha, but a decrease of SpA Col-induced proliferation was observed in the presence of 1000 to 10,000 U/ml of IFN-beta. The two subtypes of IFN-alpha had different effects on cell proliferation; a significant enhancement was shown in the presence of 1000 to 10,000 U/ml of IFN-alpha A, but inhibition was shown in the presence of 1000 to 10,000 U/ml of IFN-alpha B. In the reconstitution test of the two subtypes of IFN-alpha, the boundary between enhancement and inhibition of SpA Col-induced proliferation was revealed when the proportion of IFN-alpha A and IFN-alpha B (IFN-alpha A:IFN-alpha B) ranged between 8:2 and 9:1. Toward the SpA Col-induced responses, the above IFN were all found to act on B cells directly, independent of the presence of T cells. Proliferative responses by IFN-alpha and IFN-alpha A, however, were shown to be slightly dependent on the presence of monocytes. The lymphocyte proliferation induced by other mitogens (phytohemagglutinin, concanavalin A, pokeweed mitogen, and protein A of S. aureus) were all inhibited by the above IFN.     

Suppressive effect of dexamethasone on TIMP-1 production involves murine osteoblastic MC3T3-E1 cell apoptosis

High dose glucocorticoid (GC) treatment induces osteoporosis partly via increasing osteoblast apoptosis. However, the mechanism of GC-induced apoptosis has not been fully elucidated. Osteoblast-derived tissue inhibitor of metalloproteinase-1 (TIMP-1) was recently reported to be involved in bone metabolism. Our previous study demonstrated that TIMP-1 suppressed apoptosis of the mouse bone marrow stromal cell line MBA-1 (pre-osteoblast) induced by serum deprivation. Therefore, we tested the effect of the GC dexamethasone (Dex) on TIMP-1 production in murine osteoblastic MC3T3-E1 cells and further determined whether this action is associated with Dex-induced osteoblast apoptosis. Dex decreased TIMP-1 production in MC3T3-E1 cells, and this effect was blocked by the glucocorticoid receptor (GR) antagonists, RU486 and RU40555. Recombinant TIMP-1 protein reduced caspase-3 activation and apoptosis induced by Dex in MC3T3-E1 cells. In addition, the pro-apoptotic effect of the Dex was augmented by suppression of TIMP-1 with siRNA. Furthermore, mutant TIMP-1, which has no inhibitory effects on MMPs, yet protects MC3T3-E1 cells against Dex-induced apoptosis. Our study demonstrates that Dex suppresses TIMP-1 production in osteoblasts through GR, and this effect is associated with its induction of osteoblast apoptosis. The anti-apoptotic action of TIMP-1 is independent of its inhibitory effects on MMPs activities. The decrease in TIMP-1 production caused by Dex may contribute to the mechanisms of Dex-induced bone loss.     

Insights into the pathogenesis of axial spondyloarthropathy from network and pathway analysis

Background

Complex chronic diseases are usually not caused by changes in a single causal gene but by an unbalanced regulating network resulting from the dysfunctions of multiple genes or their products. Therefore, network based systems approach can be helpful for the identification of candidate genes related to complex diseases and their relationships. Axial spondyloarthropathy (SpA) is a group of chronic inflammatory joint diseases that mainly affect the spine and the sacroiliac joints. The pathogenesis of SpA remains largely unknown.

Results

In this paper, we conducted a network study of the pathogenesis of SpA. We integrated data related to SpA, from the OMIM database, proteomics and microarray experiments of SpA, to prioritize SpA candidate disease genes in the context of human protein interactome. Based on the top ranked SpA related genes, we constructed a SpA specific PPI network, identified potential pathways associated with SpA, and finally sketched an overview of biological processes involved in the development of SpA.

Conclusions

The protein-protein interaction (PPI) network and pathways reflect the link between the two pathological processes of SpA, i.e., immune mediated inflammation, as well as imbalanced bone modelling caused new boneformation and bone loss. We found that some known disease causative genes, such as TNFand ILs, play pivotal roles in this interaction.

     

NELL-1 binds to APR3 affecting human osteoblast proliferation and differentiation

Nel-like protein 1 (NELL-1) is an osteoinductive molecule associated with premature calvarial suture closure. Here we identified apoptosis related protein 3 (APR3), a membrane protein known as a proliferation suppressor, as a binding protein of NELL-1 by biopanning. NELL-1 and APR3 colocalized on the nuclear envelope of human osteoblasts. NELL-1 significantly inhibited proliferation of osteoblasts co-transfected with APR3 through further down-regulation of Cyclin D1. The co-expression of NELL-1 and APR3 enhanced Ocn and Bsp expression and mineralization. RNAi of APR3 significantly reduced the differentiation effect of NELL-1. These findings suggest that the effects of NELL-1 on osteoblastic differentiation and proliferation are partly through binding to APR3.     

miR‐181c‐5p mediates simulated microgravity‐induced impaired osteoblast proliferation by promoting cell cycle arrested in the G2 phase

Impaired osteoblast proliferation plays fundamental roles in microgravity‐induced bone loss, and cell cycle imbalance may result in abnormal osteoblast proliferation. However, whether microgravity exerts an influence on the cell cycle in osteoblasts or what mechanisms may underlie such an effect remains to be fully elucidated. Herein, we confirmed that simulated microgravity inhibits osteoblast proliferation. Then, we investigated the effect of mechanical unloading on the osteoblast cell cycle and found that simulated microgravity arrested the osteoblast cell cycle in the G₂ phase. In addition, our data showed that cell cycle arrest in osteoblasts from simulated microgravity was mainly because of decreased cyclin B1 expression. Furthermore, miR‐181c‐5p directly inhibited cyclin B1 protein translation by binding to a target site in the 3′UTR. Lastly, we demonstrated that inhibition of miR‐181c‐5p partially counteracted cell cycle arrest and decreased the osteoblast proliferation induced by simulated microgravity. In conclusion, our study demonstrates that simulated microgravity inhibits cell proliferation and induces cell cycle arrest in the G₂ phase in primary mouse osteoblasts partially through the miR‐181c‐5p/cyclin B1 pathway. This work may provide a novel mechanism of microgravity‐induced detrimental effects on osteoblasts and offer a new avenue to further investigate bone loss induced by mechanical unloading.     

Fluid shear stress inhibits TNF-alpha-induced apoptosis in osteoblasts: a role for fluid shear stress-induced activation of PI3-kinase and inhibition of caspase-3

In bone, a large proportion of osteoblasts, the cells responsible for deposition of new bone, normally undergo programmed cell death (apoptosis). Because mechanical loading of bone increases the rate of new bone formation, we hypothesized that mechanical stimulation of osteoblasts might increase their survival. To test this hypothesis, we investigated the effects of fluid shear stress (FSS) on osteoblast apoptosis using three osteoblast cell types: primary rat calvarial osteoblasts (RCOB), MC3T3-E1 osteoblastic cells, and UMR106 osteosarcoma cells. Cells were treated with TNF-alpha in the presence of cyclohexamide (CHX) to rapidly induce apoptosis. Osteoblasts showed significant signs of apoptosis within 4-6 h of exposure to TNF-alpha and CHX, and application of FSS (12 dyne/cm(2)) significantly attenuated this TNF-alpha-induced apoptosis. FSS activated PI3-kinase signaling, induced phosphorylation of Akt, and inhibited TNF-alpha-induced activation of caspase-3. Inhibition of PI3-kinase, using LY294002, blocked the ability of FSS to rescue osteoblasts from TNF-alpha-induced apoptosis and blocked FSS-induced inhibition of caspase-3 activation in osteoblasts treated with TNF-alpha. LY294002 did not, however, prevent FSS-induced phosphorylation of Akt suggesting that activation of Akt alone is not sufficient to rescue cells from apoptosis. This result also suggests that FSS can activate Akt via a PI3-kinase-independent pathway. These studies demonstrate for the first time that application of FSS to osteoblasts in vitro results in inhibition of TNF-alpha-induced apoptosis through a mechanism involving activation of PI3-kinase signaling and inhibition of caspases. FSS-induced activation of PI3-kinase may promote cell survival through a mechanism that is distinct from the Akt-mediated survival pathway.