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Migration of the cells in osteoblastic lineage, including preosteoblasts and osteoblasts, has been postulated to influence bone formation. However, the molecular bases that link preosteoblastic/ osteoblastic cell migration and bone formation are incompletely understood. Nck (noncatalytic region of tyrosine kinase; collectively referred to Nck1 and Nck2) is a member of the signaling adaptors that regulate cell migration and cytoskeletal structures, but its function in cells in the osteoblastic lineage is not known. Therefore, we examined the role of Nck in migration of these cells. Nck is expressed in preosteoblasts/osteoblasts, and its knockdown suppresses migration as well as cell spreading and attachment to substrates. In contrast, Nck1 overexpression enhances spreading and increases migration and attachment. As for signaling, Nck double knockdown suppresses migration toward IGF1 (insulin-like growth factor 1). In these cells, Nck1 binds to IRS-1 (insulin receptor substrate 1) based on immunoprecipitation experiments using anti-Nck and anti-IRS-1 antibodies. In vivo, Nck knockdown suppresses enlargement of the pellet of DiI-labeled preosteoblasts/osteoblasts placed in the calvarial defects. Genetic experiments indicate that conditional double deletion of both Nck1 and Nck2 specifically in osteoblasts causes osteopenia. In these mice, Nck double deficiency suppresses the levels of boneformation parameters such as bone formation rate in vivo. Interestingly, bone-resorption parameters are not affected. Finally, Nck deficiency suppresses repair of bone injury after bone marrow ablation. These results reveal that Nck regulates preosteoblastic/ osteoblastic migration and bone mass. [ABSTRACT FROM AUTHOR]

Osteoclastogenesis is under the control of posttranscriptional and transcriptional events. However, posttranscriptional regulation of osteoclastogenesis is incompletely understood. CNOT3 is a component of the CCR4 family that regulates mRNA stability, but its function in bone is not known. Here, we show that Cnot3 deficiency by deletion of a single allele induces osteoporosis. Cnot3 deficiency causes an enhancement in bone resorption in association with an elevation in bone formation, resulting in high-turnover type bone loss. At the cellular level, Cnot3 deficiency enhances receptor activator of NF-κB ligand (RANKL) effects on osteoclastogenesis in a cell-autonomous manner. Conversely, Cnot3 deficiency does not affect osteoblasts directly. Cnot3 deficiency does not alter RANKL expression but enhances receptor activator of NF-κB (RANK) mRNA expression in bone in vivo. Cnot3 deficiency promotes RANK mRNA stability about twofold in bone marrow cells of mice. Cnot3 knockdown also increases RANK mRNA expression in the precursor cell line for osteoclasts. Anti-CNOT3 antibody immunoprecipitates RANK mRNA. Cnot3 deficiency stabilizes luciferase reporter expression linked to the 3'-UTR fragment of RANK mRNA. In contrast, Cnot3 overexpression destabilizes the luciferase reporter linked to RANK 3-UTR. In aged mice that exhibit severe osteoporosis, Cnot3 expression levels in bone are reduced about threefold in vivo. Surprisingly, Cnot3 deficiency in these aged mice further exacerbates osteoporosis, which also occurs via enhancement of osteoclastic activity. Our results reveal that CNOT3 is a critical regulator of bone mass acting on bone resorption through posttranscriptional down-regulation of RANK mRNA stability, at least in part, even in aging-induced osteoporosis. [ABSTRACT FROM AUTHOR]

Hyperpolarization-activated cation currents (Ih) contribute to various physiological properties and functions in the brain, including neuronal pacemaker activity, setting of resting membrane potential, and dendritic integration of synaptic input. Four subunits of the Hyperpolarization-activated and Cyclic-Nucleotide-gated nonselective cation channels (HCN1–4), which generate Ih, have been cloned recently. To better understand the functional diversity of Ih in the brain, we examined precise immunohistochemical localization of four HCNs in the rat brain. Immunoreactivity for HCN1 showed predominantly cortical distribution, being intense in the neocortex, hippocampus, superior colliculus, and cerebellum, whereas those for HCN3 and HCN4 exhibited subcortical distribution mainly concentrated in the hypothalamus and thalamus, respectively. Immunoreactivity for HCN2 had a widespread distribution throughout the brain. Double immunofluorescence revealed colocalization of immunoreactivity for HCN1 and HCN2 in distal dendrites of pyramidal cells in the hippocampus and neocortex. At the electron microscopic level, immunogold particles for HCN1 and HCN2 had similar distribution patterns along plasma membrane of dendritic shafts in layer I of the neocortex and stratum lacunosum moleculare of the hippocampal CA1 area, suggesting that these subunits could form heteromeric channels. Our results further indicate that HCNs are localized not only in somato-dendritic compartments but also in axonal compartments of neurons. Immunoreactivity for HCNs often occurred in preterminal rather than terminal portions of axons and in specific populations of myelinated axons. We also found HCN2-immunopositive oligodendrocytes including perineuronal oligodendrocytes throughout the brain. These results support previous electrophysiological findings and further suggest unexpected roles of Ih channels in the brain. J. Comp. Neurol. 471:241–276, 2004. © 2004 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]