ABSTRACT
BACKGROUND: Afamin was recently identified as a novel osteoclast-derived coupling factor that can stimulate the in vitro and in vivo migration of preosteoblasts. AIM: In order to understand in more detail the biological roles of afamin in bone metabolism, we investigated its effects on osteoclastic differentiation and bone resorption. METHODS: Osteoclasts were differentiated from mouse bone marrow cells. Tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells were considered as osteoclasts, and the resorption area was determined by incubating the cells on dentine discs. The intracellular cAMP level was determined using a direct enzyme immunoassay. Signaling pathways were investigated using western blot and RT-PCR. Recombinant afamin was administered exogenously to bone cell cultures. RESULTS: Afamin stimulated both osteoclastogenesis and in vitro bone resorption. Consistently, the expressions of osteoclast differentiation markers were significantly increased by afamin. Although afamin mainly affected the late-differentiation stages of osteoclastogenesis, the expression levels of receptor activator of nuclear factor-κB ligand (RANKL)-dependent signals were not changed. Afamin markedly decreased the levels of intracellular cAMP with reversal by pretreatment with pertussis toxin (PTX), a specific inhibitor of Gi-coupled receptor signaling. In addition, PTX almost completely blocked afamin-stimulated osteoclastogenesis. Furthermore, pretreatment with KN93 and STO609 - Ca2+/cal - mo dulin-dependent protein kinase (CaMK) and CaMK kinase inhibitors, respectively - significantly prevented decreases in the intracellular cAMP level by afamin while attenuating afamin-stimulated osteoclastogenesis. CONCLUSION: Afamin enhances osteoclastogenesis by decreasing intracellular cAMP levels via Gi-coupled receptor and CaMK pathways.
Subject(s)
Albumins/pharmacology , Bone Resorption/drug therapy , Calcium-Calmodulin-Dependent Protein Kinase Type 1/metabolism , Glycoproteins/pharmacology , Osteoclasts/drug effects , Osteogenesis/drug effects , Receptors, G-Protein-Coupled/metabolism , Recombinant Proteins/pharmacology , Acid Phosphatase/genetics , Acid Phosphatase/metabolism , Animals , Blotting, Western , Bone Resorption/metabolism , Calcium-Calmodulin-Dependent Protein Kinase Type 1/genetics , Cell Differentiation/drug effects , Cells, Cultured , Cyclic AMP/metabolism , Isoenzymes/genetics , Isoenzymes/metabolism , Mice , Mice, Inbred C57BL , NF-kappa B/genetics , NF-kappa B/metabolism , Osteoclasts/cytology , Osteoclasts/metabolism , RANK Ligand/genetics , RANK Ligand/metabolism , RNA, Messenger/genetics , Real-Time Polymerase Chain Reaction , Receptors, G-Protein-Coupled/genetics , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction , Tartrate-Resistant Acid PhosphataseABSTRACT
Proton beams generated from thin aluminum and Mylar foil targets that are irradiated by a 30fs Ti:sapphire laser pulse with an intensity of 2.2x10;{18}Wcm;{2} were investigated. Protons from the Mylar targets were observed to have an energy higher by a factor of 2 and were higher in number by an order of magnitude as compared with those generated from the aluminum targets. The maximum proton energy of 1.3+/-0.12MeV obtained from the Mylar target was found to be similar with previous observations that used laser pulses with different intensities. To address the anomalous behavior of the maximum proton energy for plastic targets, an acceleration model is proposed. In this model, the protons are accelerated by a resistively induced electric field in the front of the target, which can account for the experimental observations.
ABSTRACT
Epidermal growth factor (EGF) is synthesized in the pancreas and diabetic animals have low levels of EGF. However, the role of EGF in regulating the major function of the pancreas, insulin secretion, has not been studied. Here, we show that EGF rapidly increased insulin secretion in mouse pancreatic islets, as well as in a pancreatic beta-cell line. These events were dependent on a Ca(2+) influx and phospholipase D (PLD) activity, particularly PLD2, as determined using pharmacological blockers and molecular manipulations such as over-expression and siRNA of PLD isozymes. In addition, EGF also increased plasma insulin levels and mediated glucose lowering in normal and diabetic mice. Here, for the first time, we provide evidence that EGF is a novel secretagogue that regulates plasma glucose levels and a candidate for the development of therapeutics for diabetes.