Osteocrin (Ostn) is a recently discovered secreted protein produced by cells of the osteoblast lineage that shows a well conserved homology with members of the natriuretic peptide (NP) family. We hypothesized that Ostn could interact with the NP receptors, thereby modulating NP actions on the skeleton. Ostn binds specifically and saturably to the NP peptide receptor-C (NPR-C) receptor with a K d of ϳ5 nM with no binding to the GC-A or GC-B receptors. Deletion of several of the residues deemed important for NP binding to NPR-C led to abolition of Ostn binding, confirming the presence of a "natriuretic motif." Functionally, Ostn was able to augment C-type natriuretic peptide-stimulated cGMP production in both pre-chondrocytic (ATDC5) and osteoblastic (UMR106) cells, suggesting increased NP levels due to attenuation of NPR-C associated NP clearance. Ostn-transgenic mice displayed elongated bones and a marked kyphosis associated with elevated bone cGMP levels, suggesting that elevated natriuretic peptide activity contributed to the increased bone length possibly through an increase in growth plate chondrocyte proliferation. Thus, we have demonstrated that Ostn is a naturally occurring ligand of the NPR-C clearance receptor and may act to locally modulate the actions of the natriuretic system in bone by blocking the clearance action of NPR-C, thus locally elevating levels of C-type natriuretic peptide. Osteocrin (Ostn)3 is a recently discovered novel small secreted protein with prohormone-like characteristics (1). To date, the exact role of Ostn has not been elucidated. Limited homology, however, was observed between Ostn and members of the natriuretic peptide family, suggesting a possible functional link. The natriuretic system, key in the maintenance of vascular tone and cardiovascular homeostasis, consists of three related natriuretic peptides (NPs), ANP, BNP, and CNP (2) and three receptors (NPRs) mediating the biological activity of these peptides. The GC-A receptor, which preferentially binds ANP and BNP, and the GC-B receptor, whose cognate ligand is CNP, are coupled to guanylyl cyclase, producing cGMP as a secondary messenger (3, 4). The third receptor, NPR-C, has no guanylyl cyclase activity and binds all three NPs with similar affinity (5). To date, no specific endogenous ligand has been identified for NPR-C, and it is thought to act mainly as a clearance receptor (6). However, other biological functions have been postulated for this receptor (6).A number of recent reports have demonstrated that the NPs also play a key role in regulation of the skeleton (7). Mice overexpressing either BNP (8) or CNP (9) or lacking NPR-C (10) display elongated bones, whereas mice lacking CNP (11) or a functional GC-B receptor exhibit dwarfism (12). Interestingly, we initially identified Ostn in cells of the osteoblast lineage, the bone-producing cells, which together with its regulatory effects on these cells suggested a role in bone activity. Given the homology of Ostn to the NP family and its putative bone regulatory actio...
Nascent-polypeptide-associated complex and coactivator alpha (␣NAC) is a protein shuttling between the nucleus and the cytoplasm. Upon phosphorylation at residue serine 43 by integrin-linked kinase, ␣NAC is translocated to the nuclei of osteoblasts, where it acts as an AP-1 coactivator to increase osteocalcin gene transcription. To determine the physiological role of nuclear ␣NAC, we engineered a knock-in mouse model with a serine-to-alanine mutation at position 43 (S43A). The S43A mutation resulted in a decrease in the amount of nuclear ␣NAC with reduced osteocalcin gene promoter occupancy, leading to a significant decrease in osteocalcin gene transcription. The S43A mutant bones also expressed decreased levels of ␣ 1 (I) collagen mRNA and as a consequence had significantly less osteoid tissue. Transient transfection assays and chromatin immunoprecipitation confirmed the ␣ 1 (I) collagen gene as a novel ␣NAC target. The reduced quantity of bone matrix in S43A mutant bones was mineralized faster, as demonstrated by the significantly reduced mineralization lag time, producing a lower volume of immature, woven-type bone characterized by poor lamellation and an increase in the number of osteocytes. Accordingly, the expression of the osteocyte differentiation marker genes DMP-1 (dentin matrix protein 1), E11, and SOST (sclerostin) was increased. The accelerated mineralization phenotype was cell autonomous, as osteoblasts isolated from the calvaria of S43A mutant mice mineralized their matrix faster than did wild-type cells. Thus, inhibition of ␣NAC nuclear translocation results in an osteopenic phenotype caused by reduced expression of osteocalcin and type I collagen, accelerated mineralization, and immature woven-bone formation.
Since the c-Jun coactivator ␣NAC was initially identified in a differential screen for genes expressed in differentiated osteoblasts, we examined whether the osteocalcin gene, a specific marker of terminal osteoblastic differentiation, could be a natural target for the coactivating function of ␣NAC. We had also previously shown that ␣NAC can specifically bind DNA in vitro, but it remained unclear whether the DNA-binding function of ␣NAC is expressed in vivo or if it is required for coactivation. We have identified an ␣NAC binding site within the murine osteocalcin gene proximal promoter region and demonstrated that recombinant ␣NAC or ␣NAC from ROS17/2.8 nuclear extracts can specifically bind this element. Using transient transfection assays, we have shown that ␣NAC specifically potentiated the c-Jun-dependent transcription of the osteocalcin promoter and that this activity specifically required the DNA-binding domain of ␣NAC. Chromatin immunoprecipitation confirmed that ␣NAC occupies its binding site on the osteocalcin promoter in living osteoblastic cells expressing osteocalcin. Inhibition of the expression of endogenous ␣NAC in osteoblastic cells by use of RNA interference provoked a decrease in osteocalcin gene transcription. Our results show that the osteocalcin gene is a target for the ␣NAC coactivating function, and we propose that ␣NAC is specifically targeted to the osteocalcin promoter through its DNA-binding activity as a means to achieve increased specificity in gene transcription.
The biological activity of 24R,25-dihydroxyvitamin D3 [24R,25(OH)2D3] remains controversial, but it has been suggested that it contributes to fracture healing. Cyp24a1-/- mice, synthesizing no 24R,25(OH)2D3, show suboptimal endochondral ossification during fracture repair, with smaller callus and reduced stiffness. These defects were corrected by 24R,25(OH)2D3 treatment, but not by 1,25-dihydroxyvitamin D3. Microarrays with Cyp24a1-/- callus mRNA identified FAM57B2 as a mediator of the 24R,25(OH)2D3 effect. FAM57B2 produced lactosylceramide (LacCer) upon specific binding of 24R,25(OH)2D3. Fam57b inactivation in chondrocytes (Col2-Cre Fam57bfl/fl) phenocopied the callus formation defect of Cyp24a1-/- mice. LacCer or 24R,25(OH)2D3 injections restored callus volume, stiffness, and mineralized cartilage area in Cyp24a1-null mice, but only LacCer rescued Col2-Cre Fam57bfl/fl mice. Gene expression in callus tissue suggested that the 24R,25(OH)2D3/FAM57B2 cascade affects cartilage maturation. We describe a previously unrecognized pathway influencing endochondral ossification during bone repair through LacCer production upon binding of 24R,25(OH)2D3 to FAM57B2. Our results identify potential new approaches to ameliorate fracture healing.
c-Jun is an immediate-early gene whose degradation by the proteasome pathway is required for an efficient transactivation. In this report, we demonstrated that the c-Jun coactivator, nascent polypeptide associated complex and coactivator alpha (alphaNAC) was also a target for degradation by the 26S proteasome. The proteasome inhibitor lactacystin increased the metabolic stability of alphaNAC in vivo, and lactacystin, MG-132, or epoxomicin treatment of cells induced nuclear translocation of alphaNAC. We have shown that the ubiquitous kinase glycogen synthase kinase 3beta (GSK3beta) directly phosphorylated alphaNAC in vitro and in vivo. Inhibition of the endogenous GSKappa3beta activity resulted in the stabilization of this coactivator in vivo. We identified the phosphoacceptor site in the C-terminal end of the coactivator, on position threonine 159. We demonstrated that the inhibition of GSK3beta activity by treatment of cells with the inhibitor 5-iodo-indirubin-3'-monoxime, as well as with a dominant-negative GSK3beta mutant, induced the accumulation of alphaNAC in the nuclei of cells. Mutation of the GSK3beta phosphoacceptor site on alphaNAC induced a significant increase of its coactivation potency. We conclude that GSK3beta-dependent phosphorylation of alphaNAC was the signal that directed the protein to the proteasome. The accumulation of alphaNAC caused by the inhibition of the proteasome pathway or the activity of GSK3beta contributes to its nuclear translocation and impacts on its coactivating function.
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