c-Src Control of Chloride Channel Support for Osteoclast HCl Transport and Bone Resorption

Abstract

Bone degradation by osteoclasts depends upon active transport of hydrogen ions to solubilize bone mineral. This transport is supported by the parallel actions of a proton ATPase and a chloride channel located in the osteoclast ruffled membrane. We have previously identified a novel chloride channel, p62, which appears to be the avian counterpart to CLIC-5b and is expressed coincident with the appearance of acid secretion as avian osteoclasts differentiate in culture. In this article, we show that suppression of CLIC-5b in differentiating avian osteoclasts results in decreased acidification by vesicles derived from these cells and decreased ability of the cells to resorb bone. Acidification is rescued by the presence of valinomycin, consistent with a selective loss of chloride channel but not proton pump activity. Osteoclast bone resorption is known to be dependent on the expression of the tyrosine kinase, c-Src. We show that CLIC-5b from osteoclasts has affinity for both Src SH2 and SH3 domains. We find that suppression of expression of Src in developing osteoclasts results in decreased vesicular acidification, which is rescued by valinomycin, consistent with the loss of chloride conductance in the proton pump-containing vesicles. Suppression of c-Src causes no change in the steady state level of CLIC-5b expression, but does result in failure of proton pump and CLIC-5b to colocalize in cultured osteoclast precursors. We conclude that suppression of c-Src interferes with osteoclast bone resorption by disrupting functional co-localization of proton pump and CLIC-5b.Skeleton integrity requires that osteoclast-mediated bone resorption be intact and regulated (1). Osteoclasts can remodel bone because these multinucleated cells secret sufficient acid into the resorption compartment to solubilize bone mineral (2, 3), an alkaline salt that becomes increasingly soluble at pH values below 6 (4, 5). The resorption compartment is delineated by a circumferential tightly adherent sealing zone (6). The osteoclast plasma membrane enclosed within the sealing zone differentiates into a highly specialized structure, the ruffled border. Across this membrane the cell actively transports HCl, which dissolves bone mineral and activates acid hydrolases required for bone resorption (7,8). The transport of HCl occurs in two steps. An electrogenic ATP-dependent proton pump (9 -12), inserts H ϩ into the resorption compartment. Chloride ions follow passively through a parallel chloride conductance, short circuiting the electrogenic pump and allowing the massive HCl secretion necessary during bone resorption (10).We had previously identified a 62-kDa protein, p62, from avian osteoclast ruffled border that could be reconstituted to form a DNDS 2 -sensitive chloride channel (13). This protein is antigenically related to bovine CLIC-5b, a chloride channel of bovine kidney microsomal membranes (14). Expression of avian p62 in differentiating avian osteoclasts is coincident with the appearance of outwardly rectifying chloride conductance, valinom...

S-Nitrosylation Regulates Nuclear Translocation of Chloride Intracellular Channel Protein CLIC4

“…Various members have been implicated in cell signaling cascades, ranging from stimulation of chloride channel activity by CLIC3 following association with extracellular signal-regulated kinase (ERK) 7 (1) to CLIC5A that is implicated in bone resorption and HCl transport in osteoclasts, in response to phosphorylation by c-Src (2). However, a number of functions of CLIC proteins are independent of channel activity (3,4).…”

“…The best characterized of the CLIC family members is CLIC4, identified in our laboratory as a p53 and TNF␣ 2 response gene that is up-regulated during keratinocyte differentiation. CLIC4 has emerged as a crucial player in many physiological processes, including tubular morphogenesis during angiogenesis (5-7), transdifferentiation of mammary fibroblasts to myofibroblasts in response to TGF-␤ (8), and adipocyte differentiation (9).…”

Targeting Src signaling in metastatic bone disease

“…[16][17][18] Suppression of Src also interferes with ion transport, which is required to solubilize bone mineral during bone resorption by osteoclasts. 19 Although Src activity is not essential for osteoclast survival, Src and other SFKs are involved in antiapoptotic signaling in osteoclasts induced by receptor activator for nuclear factor j b ligand (RANKL) and other tumor necrosis factor family members. [20][21][22] Disruption of Src signaling reduces the number of osteoclasts both in vitro and in vivo.…”

Calcium Signalling and Calcium Transport in Bone Disease

“…The actindirected disposition of CLIC protein has also been observed in microvilli of placental cells [Berryman et al, 2004]. In osteoclasts the coordinated disposition of V-ATPase and CLIC required for full expression of the bone resorption phenotype [Edwards et al, 2006]. It is clear that much of the osteoclasts organization exists to support the massive acid secretion for bone calcium solubilization.…”

“…The CLIC family of intracellular proteins, which are chloride channels, have been identified with acidification in osteoclasts for some time [Blair & Schlesinger, 1990. Recently, CLIC5, has been directly implicated in osteoclast bone resorption and H + transport [Edwards et al, 2006]. In combination the CLCN7 exchanger and CLIC5 provide a H + leak and charge neutralization that are important in acidification [Grabe & Oster, 2001].…”