RESUMO
We have evaluated the role of the ADP-ribosyl cyclase, CD38, in bone remodeling, a process by which the skeleton is being renewed constantly through the coordinated activity of osteoclasts and osteoblasts. CD38 catalyzes the cyclization of its substrate, NAD+, to the Ca2+-releasing second messenger, cyclic ADP-ribose (cADPr). We have shown previously that CD38 is expressed both in osteoblasts and osteoclasts. Its activation in the osteoclast triggers Ca2+ release through ryanodine receptors (RyRs), stimulation of interleukin-6 (IL-6), and an inhibition of bone resorption. Here, we have examined the consequences of deleting the CD38 gene in mice on skeletal remodeling. We report that CD38-/- mice displayed a markedly reduced bone mineral density (BMD) at the femur, tibia, and lumbar spine at 3 months and at the lumbar spine at 4 months, with full normalization of the BMD at all sites at 5 months. The osteoporosis at 3 months was accompanied by a reduction in primary spongiosa and increased osteoclast surfaces on histomorphometric analysis. Hematopoetic stem cells isolated ex vivo from CD38-/- mice showed a dramatic approximately fourfold increase in osteoclast formation in response to incubation for 6 days with RANK-L and M-CSF. The osteoclasts so formed in these cultures showed a approximately 2.5-fold increase in resorptive activity compared with wild-type cells. However, when adherent bone marrow stromal cells were allowed to mature into alkaline phosphatase-positive colony-forming units (CFU-Fs), those derived from CD38-/- mice showed a significant reduction in differentiation compared with wild-type cells. Real-time RT-PCR on mRNA isolated from osteoclasts at day 6 showed a significant reduction in IL-6 and IL-6 receptor mRNA, together with significant decreases in the expression of all calcineurin A isoforms, alpha, beta, and gamma. These findings establish a critical role for CD38 in osteoclast formation and bone resorption. We speculate that CD38 functions as a cellular NAD+ "sensor," particularly during periods of active motility and secretion.
Assuntos
ADP-Ribosil Ciclase/fisiologia , Antígenos CD/fisiologia , Reabsorção Óssea , Osteoclastos/fisiologia , ADP-Ribosil Ciclase/genética , ADP-Ribosil Ciclase 1 , Animais , Antígenos CD/genética , Densidade Óssea , Osso e Ossos/anatomia & histologia , Diferenciação Celular , Células Cultivadas , Células-Tronco Hematopoéticas/fisiologia , Glicoproteínas de Membrana , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Osteoblastos/citologia , OsteogêneseRESUMO
We predict that the type 2 ryanodine receptor isoform (RyR-2) located in the osteoclastic membrane functions as a Ca(2+) influx channel and as a divalent cation (Ca(2+)) sensor. Cytosolic Ca(2+) measurements revealed Ca(2+) influx in osteoclasts at depolarized membrane potentials. The cytosolic Ca(2+) change was, as expected, not seen in Ca(2+)-free medium and was blocked by the RyR modulator ryanodine. In contrast, at basal membrane potentials (approximately 25 mV) ryanodine triggered extracellular Ca(2+) influx that was blocked by Ni(2+). In parallel, single-channel recordings obtained from inside-out excised patches revealed a divalent cation-selective approximately 60-pS conductance in symmetric solutions of Ba-aspartate [Ba-Asp; reversal potential (E(rev)) approximately 0 mV]. In the presence of a Ba(2+) gradient, i.e., with Ba-Asp in the pipette and Na-Asp in the bath, channel conductance increased to approximately 120 pS and E(rev) shifted to 21 mV. The conductance was tentatively classified as a RyR-gated Ca(2+) channel as it displayed characteristic metastable states and was sensitive to ruthenium red and a specific anti-RyR antibody, Ab(34). To demonstrate that extracellular Ca(2+) sensing occurred at the osteoclastic surface rather than intracellularly, we performed protease protection assays using pronase. Preincubation with pronase resulted in markedly attenuated cytosolic Ca(2+) signals triggered by either Ni(2+) (5 mM) or Cd(2+) (50 microM). Finally, intracellular application of antiserum Ab(34) potently inhibited divalent cation sensing. Together, these results strongly suggest the existence of 1) a membrane-resident Ca(2+) influx channel sensitive to RyR modulators; 2) an extracellular, as opposed to intracellular, divalent cation activation site; and 3) a cytosolic CaM-binding regulatory site for RyR. It is likely therefore that the surface RyR-2 not only gates Ca(2+) influx but also functions as a sensor for extracellular divalent cations.
Assuntos
Cálcio/metabolismo , Membrana Celular/química , Osteoclastos/ultraestrutura , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Animais , Animais Recém-Nascidos , Sítios de Ligação , Canais de Cálcio/fisiologia , Calmodulina/metabolismo , Cátions Bivalentes , Citosol/metabolismo , Ácido Egtázico/farmacologia , Condutividade Elétrica , Eletrofisiologia , Fura-2 , Ativação do Canal Iônico/fisiologia , Potenciais da Membrana , Níquel/farmacologia , Potássio/metabolismo , Potássio/farmacologia , Coelhos , Ratos , Ratos Wistar , Valinomicina/farmacologiaRESUMO
CD38 is an ectocyclase that converts NAD+ to the Ca2+-releasing second messenger cyclic ADP-ribose (cADPr). Here we report that in addition to CD38 ecto-catalysis, intracellularly expressed CD38 may catalyze NAD+-->cADPr conversion to cause cytosolic Ca2+ release. High levels of CD38 were found in the plasma membranes, endoplasmic reticulum, and nuclear membranes of osteoblastic MC3T3-E1 cells. More important, intracellular CD38 was colocalized with target ryanodine receptors. The cyclase also converted a NAD+ surrogate, NGD+, to its fluorescent product, cGDPr (Km approximately 5.13 microM). NAD+ also triggered a cytosolic Ca2+ signal. Similar results were obtained with NIH3T3 cells, which overexpressed a CD38-EGFP fusion protein. The Delta(-49)-CD38-EGFP mutant with a deleted amino-terminal tail and transmembrane domain appeared mainly in the mitochondria with an expected loss of its membrane localization, but the NAD+-induced cytosolic Ca2+ signal was preserved. Likewise, Ca2+ release persisted in cells transfected with the Myr-Delta(-49)-CD38-EGFP or Delta(-49)-CD38-EGFP-Fan mutants, both directed to the plasma membrane but in an opposite topology to the full-length CD38-EGFP. Finally, ryanodine inhibited Ca2+ signaling, indicating the downstream activation of ryanodine receptors by cADPr. We conclude that intracellularly expressed CD38 might link cellular NAD+ production to cytosolic Ca2+ signaling.