2B). Open in a separate window Fig 2 The a3-subunit is expressed in the N9 microglia cell line but not in other glial cells. RANKL triggered increased expression of a3. V-ATPases in microglia were shown to bind microfilaments, and stimulation with RANKL increased the proportion of V-ATPase associated with the detergent-insoluble cytoskeletal TNFSF8 Bilobalide fraction and with actin. In summary, microglia express the a3-subunit of V-ATPase. The expression of a3 and the interaction between V-ATPases and microfilaments was modulated by RANKL. These data suggest a novel molecular pathway for regulating microglia. for 30 minutes and the upper flocculent layer was collected and washed by resuspending in homogenization buffer and repelleting the membranes. The samples were then subjected to SDS-PAGE, blotted to nitrocellulose and probed with antibodies as described in Figure Legends. Brain-derived cell types The murine microglial cell line N9 [21] was grown in Iscoves Modified Dulbeccos Medium with 25 mm HEPES and l-glutamine, supplemented with 5% fetal calf serum (Hyclone, Logan UT), 100 IU/mL penicillin, 100 g/mL streptomycin and 50 nm -mercaptoethanol. Cells were cultured in a humidified 5% CO2 atmosphere at 37 C. CG-4 cells are a rat cell line that can by stimulated to differentiate into oligodendrocytes and astrocytes depending on the culture conditions [22]. To differentiate CG-4 cells into oligodendrocytes, tissue culture plates were pre-coated sequentially with poly-ornithine solution, and fibronectin in DME-N2 biotin plus 30% B104-conditioned media (CM). (B104 cells are a neuronal cell line which produce soluble factors required for CG-4 growth). CG-4 cells were grown and expanded under these conditions in a serum free medium relying on mitogens produced by the B104 cells. To differentiate CG-4 cells into oligodendrocytes, the conditioned medium was withdrawn. During a period of 48 hours the cells differentiated into oligodendrocyte-like cells. CG-4 cells were also induced to differentiate into astrocytes. The cells are grown and passaged as described above, and then induced to differentiate by withdrawing the B104 conditioned medium and replacing it with fetal bovine serum. The rat cell line rtSc95.1was used as a model for Schwann cells. RtSc95.1 cells were grown for 3 days in Dulbeccos Modified Eagle Medium (dMEM) plus 10% fetal bovine serum. Glia were obtained from Swiss Webster mouse pups using the Bilobalide methods described previously[23]. Briefly, cortices were removed, cleaned of meninges and trypsinized, and dissociated by trituration. Cells were plated in culture flasks at 50,000 cells cm2, and grown in modified eagles medium (MEM), 10% fetal calf serum, penicillin and streptomycin, essential amino acids and nonessential amino acids. Microglia were harvested from astrocytes that become confluent prior to 3-weeks in culture. Passaging microglia was accomplished by shaking and slapping the flask on a table several times and vigorously swirling the flasks to dislodge the microglia that were attached to the monolayer of astrocytes. The growth medium containing the dislodged microglia cells was centrifuged at 800 rpm for 5 minutes, most of the supernatant was removed and the cells in the pellet were resuspended in the remaining 2C3 ml, yielding a density of ~85,000 cells ml?1. The density of cells was 20,000 cells cm2 after plating. Stimulation of N9 microglia and primary mouse microglia was performed using recombinant GST-RANKL which contains amino acids 158C316 of the mouse RANKL gene [24]. Expression of GST-RANKL and isolation from bacterial extracts was performed by standard methods. PCR RT-PCR was performed on mRNA isolated from N9 microglia and RAW 264.7 osteoclast-like cells. Cells were scraped and lysed in TRIZOL reagent (Invitrogen), and total RNA was extracted according to the manufacturers instructions. RNA was quantified spectrophotometrically, and 1 g was reversely transcribed. The standard PCR conditions were 95C (10 min), and then 30 cycles of 94C (1 min), Bilobalide 54C (1 min), and 72C (2 min). In pilot experiments, this Bilobalide number of cycles did not reach saturation of the PCR reaction. The primer sequences used were as follows: RANK forward 5GGGTGGGGCGCAGACTTCAC 3; RANK reverse 5ATGCCAGCAGCCTGCACCAG 3; Bilobalide GAPDH forward 5AAATTCCATGGCACCGTCAA 3; GAPDH reverse 5AGGGATCTCGCTCCTGGAA 3. Primers were obtained from Invitrogen. For each run, water was used.