The Gram-negative bacterium Vibrio harveyi produces and responds to three autoinducers, AI-1, AI-2, and CAI-1 to regulate cell density dependent gene expression by a process referred to as quorum sensing. The concentration of the autoinducers is sensed by three cognate hybrid sensor kinases, and information is channeled via the HPt protein LuxU to the response regulator LuxO. Here, a detailed biochemical study on the enzymatic activities of the membrane-integrated hybrid sensor kinase LuxN, the sensor for N-(D-3-hydroxybutanoyl)homoserine lactone (AI-1), is provided. LuxN was heterologously overproduced as the full-length protein in Escherichia coli. LuxN activities were characterized in vitro and are an autophosphorylation activity with an unusually high ATP turnover rate, stable LuxU phosphorylation, and a slow phosphatase activity with LuxUϳP as substrate. The presence of AI-1 affected the kinase but not the phosphatase activity of LuxN. The influence of AI-1 on the LuxN3 LuxU signaling step was monitored, and in the presence of AI-1, the kinase activity of LuxN, and hence the amount of LuxUϳP produced, were significantly reduced. Half-maximal inhibition of kinase activity by AI-1 occurred at 20 M. Together, these results indicate that AI-1 directly interacts with LuxN to down-regulate its autokinase activity and suggest that the key regulatory step of the AI-1 quorum sensing system of Vibrio harveyi is AI-1-mediated repression of the LuxN kinase activity.Vibrio harveyi is a free-living marine bacterium that controls bioluminescence, exopolysaccharide and siderophore production, type III secretion, and other processes through quorum sensing. V. harveyi has three quorum sensing systems. AI-1, an acyl homoserine lactone, sensed by LuxN is employed for intraspecies signaling (1). AI-2, a furanosyl borate diester, sensed by LuxPQ is involved in inter-species signaling (2). CAI-1, whose chemical nature is unknown, is sensed by CqsS and is proposed to be responsible for Vibrio-specific signaling (3). Information from all three hybrid sensor kinases is transduced via phosphorelay to the response regulator LuxO with the HPt protein LuxU acting as intermediate (Fig. 1). Phosphorylated LuxO, a 54 -dependent response regulator, activates transcription of genes encoding four small regulatory RNAs, which together with Hfq, destabilize the transcript for the LuxR protein, the master transcriptional regulator required for quorum sensing gene expression (4 -6). Bioluminescence is used as the canonical readout for V. harveyi quorum sensing controlled gene expression. Genetic studies indicated that LuxO is phosphorylated at low cell density, causing the luciferase operon to be repressed, and the cells are dark. When a threshold concentration of autoinducer is reached, LuxO is dephosphorylated, leading to induction of the luciferase operon and a bright phenotype (Fig. 1). It has been postulated that the hybrid sensor kinases switch from kinases to phosphatases at high autoinducer concentration and hence high cell density (3-5, ...

Objective. Syndecan 4, a heparan sulfate proteoglycan, has been associated with osteoarthritis. The present study was undertaken to analyze the functional role of syndecan 4 in endochondral ossification of mouse embryos and in adult fracture repair, which, like osteoarthritis, involves an inflammatory component.Methods. Sdc4 promoter activity was analyzed in Sdc4 ؊/؊ lacZ-knockin mice, using ␤-galactosidase staining. Endochondral ossification in embryos from embryonic day 16.5 was assessed by histologic and immunohistologic staining. Bone fracture repair was analyzed in femora of adult mice on days 7 and 14 postfracture. To evaluate Sdc2 and Sdc4 gene expression with and without tumor necrosis factor ␣ (TNF␣) and Wnt-3a stimulation, quantitative real-time polymerase chain reaction was performed.Results. In Sdc4 ؊/؊ lacZ-knockin animals, syndecan 4 promoter activity was detectable at all stages of chondrocyte differentiation, and Sdc4 deficiency inhibited chondrocyte proliferation. Aggrecan turnover in the uncalcified cartilage of the epiphysis was decreased transiently in vivo, but this did not lead to a growth phenotype at birth. In contrast, among adult mice, fracture healing was markedly delayed in Sdc4 ؊/؊ animals and was accompanied by increased callus formation. Blocking of inflammation via anti-TNF␣ treatment during fracture healing reduced these changes in Sdc4 ؊/؊ mice to levels observed in wild-type controls. We analyzed the differences between the mild embryonic and the severe adult phenotype, and found a compensatory up-regulation of syndecan 2 in the developing cartilage of Sdc4 ؊/؊ mice that was absent in adult tissue. Stimulation of chondrocytes with Wnt-3a in vitro led to increased expression of syndecan 2, while stimulation with TNF␣ resulted in up-regulation of syndecan 4 but decreased expression of syndecan 2. TNF␣ stimulation reduced syndecan 2 expression and increased syndecan 4 expression even in the presence of Wnt-3a, suggesting that inflammation has a strong effect on the regulation of syndecan expression.Conclusion. Our results demonstrate that syndecan 4 is functionally involved in endochondral ossification and that its loss impairs fracture healing, due to inhibition of compensatory mechanisms under inflammatory conditions.

Developmental osteogenesis, physiological bone remodelling and fracture healing require removal of matrix and cellular debris. Osteoclasts generated by the fusion of circulating monocytes degrade bone, whereas the identity of the cells responsible for cartilage resorption is a long-standing and controversial question. Here we show that matrix degradation and chondrocyte phagocytosis are mediated by fatty acid binding protein 5-expressing cells representing septoclasts, which have a mesenchymal origin and are not derived from haematopoietic cells. The Notch ligand Delta-like 4, provided by endothelial cells, is necessary for septoclast specification and developmental bone growth. Consistent with the termination of growth, septoclasts disappear in adult and ageing bone, but re-emerge in association with growing vessels during fracture healing. We propose that cartilage degradation is mediated by rare, specialized cells distinct from osteoclasts. Our findings have implications for fracture healing, which is frequently impaired in aging humans.

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BackgroundThe overexpression of tumor necrosis factor (TNF)-α leads to systemic as well as local loss of bone and cartilage and is also an important regulator during fracture healing. In this study, we investigate how TNF-α inhibition using a targeted monoclonal antibody affects fracture healing in a TNF-α driven animal model of human rheumatoid arthritis (RA) and elucidate the question whether enduring the anti TNF-α therapy after trauma is beneficial or not.MethodsA standardized femur fracture was applied to wild type and human TNF-α transgenic mice (hTNFtg mice), which develop an RA-like chronic polyarthritis. hTNFtg animals were treated with anti-TNF antibody (Infliximab) during the fracture repair. Untreated animals served as controls. Fracture healing was evaluated after 14 and 28 days of treatment by clinical assessment, biomechanical testing and histomorphometry.ResultsHigh levels of TNF-α influence fracture healing negatively, lead to reduced cartilage and more soft tissue in the callus as well as decreased biomechanical bone stability. Blocking TNF-α in hTNFtg mice lead to similar biomechanical and histomorphometrical properties as in wild type.ConclusionsHigh levels of TNF-α during chronic inflammation have a negative impact on fracture healing. Our data suggest that TNF-α inhibition by an anti-TNF antibody does not interfere with fracture healing.