Kalirin : novel role in osteocyte function

Indiana University-Purdue University Indianapolis (IUPUI)Communication between bone cells is important for the maintenance of bone mass. Although osteocytes are deeply embedded within the mineralized matrix, they are essential for the regulation of osteoblast and osteoclast functions. However, the intracellular proteins that control the morphology and function of osteocytes, and their ability to communicate with other bone cells are still unknown. Kalirin is a novel multi-domain GTP exchange factor (GEF) protein that activates the RhoGTPases. Recently, we found that 14 week old female Kalirin knockout (Kal-KO) mice exhibit a 45% decrease in trabecular bone density and have significantly lower cortical area, perimeter, thickness and polar cross-sectional moment of inertia (-12.6%, -7.2%, -7.6% and -21.9%, respectively) than WT mice. Kalirin was found to be expressed in osteoclasts and osteoblasts but its expression and function in osteocytes is currently unclear. We examined the role of Kalirin on the morphology and function of osteocytes. Primary osteocytes were isolated by sequential collagenase digestions from long bones (femurs and tibias) of 10-week old WT and Kal-KO mice. Immunofluorescent staining revealed Kalirin was localized to the perinuclear region of primary osteocytes and MLO-Y4 cells, and was detected along the cytoplasmic processes of primary osteocytes. We also examined primary osteocytes isolated from the long bones of Kal-KO and WT mice for changes in the length and number of cytoplasmic processes. Kal-KO osteocytes were found to express significantly fewer cytoplasmic processes per cell (3.3±0.21) than WT osteocytes (4.7±0.3). In addition, the cytoplasmic processes of Kal-KO osteocytes were shorter (79.5±4.6 µm) than those observed for WT osteocytes (85.4±3.6 µm) (p <0.01). Quantitative PCR revealed the expression of mRNA for the three major Kalirin isoforms (Kal-7, Kal-9, Kal-12) in primary osteocytes and in MLO-Y4 cells. Moreover, the mRNA levels of osteoprotegerin (OPG) and SOST, which are important for controlling osteoclast differentiation and Wnt signaling leading to bone formation, respectively, were reduced in Kal-KO osteocytes. Next, the role of Kalirin in osteocyte morphology and function was further examined. Treatment of MLO-Y4 cells for 5 days with nerve growth factor, which is known to activate Kalirin in neurons, or over-expression of the Ser-Thr kinase domain of Kal-12, promoted cytoplasmic process elongation and upregulated phosphorylated ERK and RhoA levels. Together, these results suggest that Kalirin controls osteocyte morphology and function in part by regulating cytoskeletal remodeling and the activity of ERK and RhoA. Furthermore, Kalirin may control the bone remodeling cycle by regulating osteocyte signaling to osteoclasts and osteoblasts

Osteoblast differentiation and migration are regulated by Dynamin GTPase activity

Bone formation is controlled by osteoblasts but the signaling proteins that control osteoblast differentiation and function are still unclear. We examined if the dynamin GTPase, which is associated with actin remodeling and migration in other cells, plays a role in osteoblast differentiation and migration. Dynamin mRNA was expressed in primary osteoblasts throughout differentiation (0–21 days). However, alkaline phosphatase (ALP) activity, a marker of osteoblast differentiation, was decreased in osteoblasts over-expressing dynamin. Conversely, ALP activity was increased following shRNA-mediated knockdown of dynamin and in osteoblasts treated with the dynamin inhibitor, dynasore. Dynasore also reduced c-fos and osterix expression, markers of early osteoblasts, suggesting a role for dynamin in pre-osteoblast to osteoblast differentiation. Since dynamin GTPase activity is regulated by tyrosine phosphorylation, we examined the mechanism of dynamin dephosphorylation in osteoblasts. Dynamin formed a protein complex with the tyrosine phosphatase PTP-PEST and inhibition of phosphatase activity increased the level of phosphorylated dynamin. Further, PTP-PEST blocked the Src-mediated increase in the phosphorylation and GTPase activity of wild-type dynamin but not the phosphorylation mutant dynY231F/Y597F. Although ALP activity was increased in osteoblasts expressing GTPase-defective dynK44A, and to a lesser extent dynY231F/Y597F, osteoblast migration was significantly inhibited by dynK44A and dynY231F/Y597F. These studies demonstrate a novel role for dynamin GTPase activity and phosphorylation in osteoblast differentiation and migration, which may be important for bone formation

The Rho-GEF Kalirin regulates bone mass and the function of osteoblasts and osteoclasts

Bone homeostasis is maintained by the balance between bone resorption by osteoclasts and bone formation by osteoblasts. Dysregulation in the activity of the bone cells can lead to osteoporosis, a disease characterized by low bone mass and an increase in bone fragility and risk of fracture. Kalirin is a novel GTP-exchange factor protein that has been shown to play a role in cytoskeletal remodeling and dendritic spine formation in neurons. We examined Kalirin expression in skeletal tissue and found that it was expressed in osteoclasts and osteoblasts. Furthermore, micro-CT analyses of the distal femur of global Kalirin knockout (Kal-KO) mice revealed significantly reduced trabecular and cortical bone parameters in Kal-KO mice, compared to WT mice, with significantly reduced bone mass in 8, 14 and 36 week-old female Kal-KO mice. Male mice also exhibited a decrease in bone parameters but not to the level seen in female mice. Histomorphometric analyses also revealed decreased bone formation rate in 14 week-old female Kal-KO mice, as well as decreased osteoblast number/bone surface and increased osteoclast surface/bone surface. Consistent with our in vivo findings, the bone resorbing activity and differentiation of Kal-KO osteoclasts was increased in vitro. Although alkaline phosphatase activity by Kal-KO osteoblasts was increased in vitro, Kal-KO osteoblasts showed decreased mineralizing activity, as well as decreased secretion of OPG, which was inversely correlated with ERK activity. Taken together, our findings suggest that deletion of Kalirin directly affects osteoclast and osteoblast activity, leading to decreased OPG secretion by osteoblasts which is likely to alter the RANKL/OPG ratio and promote osteoclastogenesis. Therefore, Kalirin may play a role in paracrine and/or endocrine signaling events that control skeletal bone remodeling and the maintenance of bone mass

Polymersome-mediated intracellular antibiotic delivery to treat Porphyromonas gingivalis-infected oral epithelial cells

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Surface modification for bonding between amalgam and orthodontic brackets

Objective: Testing of methods to enhance the shear bond strength (SBS) between orthodontic metal brackets and amalgam by sandblasting and different primers. Materials And Methods: Three hundred samples of amalgam restorations (KerrAlloy®) were prepared in self-cured acrylic blocks, polished, and divided into two groups: nonsandblasted and sandblasted. Each group was divided into five subgroups with different primers used in surface treatment methods, with a control group of bonded brackets on human mandibular incisors. Following the surface treatments, mandibular incisor brackets (Unitek®) were bonded on the amalgam with adhesive resin (Transbond XT®). The SBS of the samples was tested. The adhesive remnant index (ARI) and failure modes were then determined under a stereo-microscope. Two-way analysis of variance, Chi-square, and Kruskal–Wallis tests were performed to calculate the correlations between and among the SBS and ARI values, the failure modes, and surface roughness results. Results: There were statistically significant differences of SBS among the different adhesive primers and sandblasting methods (P 0.05). Conclusions: Using adhesive primers with sandblasting together effectively enhances the SBS between orthodontic metal brackets and amalgam. The two primers with the ingredient methacryloxydecyl dihydrogen phosphate (MDP) monomer, Alloy Primer® and Assure Plus®, were the most effective. Including sandblasting in the treatment is essential to achieve the bonding strength required

Effect of Prebiotics-Enhanced Probiotics on the Growth of Streptococcus mutans

Streptococcus mutans predominantly creates an acidic environment in an oral cavity. This results in dental demineralization and carious lesions. The probiotics are beneficial microorganisms that modulate the bacterial balance in the digestive system. Prebiotics are defined as nondigestible oligosaccharides that are utilized for the selective stimulation of the beneficial microorganisms. The objective of this study was to evaluate the efficacy of the prebiotics, galactooligosaccharides (GOS) and fructooligosaccharides (FOS), for enhancing the probiotic Lactobacillus acidophilus ATCC 4356, for inhibiting Streptococcus mutans (A32-2) for the prevention of dental caries. The growth rate of the S. mutans significantly decreased when cocultured with L. acidophilus in the GOS-supplemented medium at 3%, 4%, and 5%. In the FOS-supplemented medium, the growth rate of S. mutans significantly decreased in all concentrations when cocultured with L. acidophilus. There was no significant difference in the growth rate of L. acidophilus in all concentrations of either GOS or FOS. It can be concluded that the growth rate of S. mutans was significantly retarded when cocultured with L. acidophilus and the proper concentration of prebiotics. These prebiotics have potential for a clinical application to activate the function of the naturally intraoral L. acidophilus to inhibit S. mutans