The PTH-Gαs-protein kinase A cascade controls αNAC localization to regulate bone mass.

The binding of PTH to its receptor induces Gα(s)-dependent cyclic AMP (cAMP) accumulation to turn on effector kinases, including protein kinase A (PKA). The phenotype of mice with osteoblasts specifically deficient for Gα(s) is mimicked by a mutation leading to cytoplasmic retention of the transcriptional coregulator αNAC, suggesting that Gαs and αNAC form part of a common genetic pathway. We show that treatment of osteoblasts with PTH(1-34) or the PKA-selective activator N(6)-benzoyladenosine cAMP (6Bnz-cAMP) leads to translocation of αNAC to the nucleus. αNAC was phosphorylated by PKA at serine 99 in vitro. Phospho-S99-αNAC accumulated in osteoblasts exposed to PTH(1-34) or 6Bnz-cAMP but not in treated cells expressing dominant-negative PKA. Nuclear accumulation was abrogated by an S99A mutation but enhanced by a phosphomimetic residue (S99D). Chromatin immunoprecipitation (ChIP) analysis showed that PTH(1-34) or 6Bnz-cAMP treatment leads to accumulation of αNAC at the Osteocalcin (Ocn) promoter. Altered gene dosages for Gα(s) and αNAC in compound heterozygous mice result in reduced bone mass, increased numbers of osteocytes, and enhanced expression of Sost. Our results show that αNAC is a substrate of PKA following PTH signaling. This enhances αNAC translocation to the nucleus and leads to its accumulation at target promoters to regulate transcription and affect bone mass.

Altered gene dosage confirms the genetic interaction between FIAT and αNAC.

Factor inhibiting ATF4-mediated transcription (FIAT) interacts with Nascent polypeptide associated complex and coregulator alpha (αNAC). In cultured osteoblastic cells, this interaction contributes to maximal FIAT-mediated inhibition of Osteocalcin (Ocn) gene transcription. We set out to demonstrate the physiological relevance of this interaction by altering gene dosage in compound Fiat and Naca (encoding αNAC) heterozygous mice. Compound Naca(+/-); Fiat(+/-) heterozygous animals were viable, developed normally, and exhibited no significant difference in body weight compared with control littermate genotypes. Animals with a single Fiat allele had reduced Fiat mRNA expression without changes in the expression of related family members. Expression of the osteocyte differentiation marker Dmp1 was elevated in compound heterozygotes. Static histomorphometry parameters were assessed at 8weeks of age using microcomputed tomography (µCT). Trabecular measurements were not different between genotypes. Cortical thickness and area were not affected by gene dosage, but we measured a significant increase in cortical porosity in compound heterozygous mice, without changes in biomechanical parameters. The bone phenotype of compound Naca(+/-); Fiat(+/-) heterozygotes confirms that FIAT and αNAC are part of a common genetic pathway and support a role for the FIAT/αNAC interaction in normal bone physiology.

SUMOylated αNAC potentiates transcriptional repression by FIAT.

The transcriptional coregulator αNAC (Nascent polypeptide associated complex And Coregulator alpha) and the transcriptional repressor FIAT (Factor Inhibiting ATF4-mediated Transcription) interact but the biological relevance of this interaction remains unclear. The activity of αNAC is extensively modulated by post-translational modifications (PTMs). We identified a novel αNAC PTM through covalent attachment of the Small Ubiquitin-like MOdifier (SUMO1). Recombinant αNAC was a SUMO1 target in in vitro SUMOylation assays and we confirmed that αNAC is conjugated to SUMO1 in cultured osteoblasts and in calvarial tissue. The amino acid sequence of αNAC contains one copy of the composite "phospho-sumoyl switch" motif that couples sequential phosphorylation and SUMOylation. We found that αNAC is selectively SUMOylated at lysine residue 127 within the motif and that SUMOylation is enhanced when a phosphomimetic mutation is introduced at the nearby serine residue 132. SUMOylation did not alter the DNA-binding capacity of αNAC. The S132D, hyper-SUMOylated αNAC mutant specifically interacted with histone deacetylase-2 (HDAC2) and enhanced the inhibitory activity of FIAT on ATF4-mediated transcription from the Osteocalcin gene promoter. This effect required binding of SUMOylated αNAC to the target promoter. We propose that maximal transcriptional repression by FIAT requires its interaction with SUMOylated, HDAC2-interacting αNAC.

Differential effects of oral doxercalciferol (Hectorol) or paricalcitol (Zemplar) in the Cyp27b1-null mouse model of uremia.

BACKGROUND/AIMS: Kidney disease patients experience declining calcitriol levels and develop secondary hyperparathyroidism (SHPT). Animal models of uremia based on 5/6 nephrectomy (NTX) do not consistently reproduce this calcitriol deficiency. We developed an animal model, the NTX Cyp27b1-null mouse, which completely lacks endogenous calcitriol, and examined the suitability of this model for evaluation of treatment with vitamin D analogs in uremia. METHODS: NTX was performed at 2 months of age. One week post-NTX, animals were treated for 4 weeks with vehicle; doxercalciferol at 30, 100 or 300 pg/g body weight (b.w.); or paricalcitol at 100, 300 or 1,000 pg/g b.w. by gavage 3 times per week. RESULTS: Serum blood urea nitrogen and creatinine were elevated. Vehicle-treated NTX null mice had hypocalcemia and SHPT. Doxercalciferol at 100 or 300 pg/g b.w. normalized serum calcium and parathyroid hormone (PTH) levels. Paricalcitol at 300 or 1,000 pg/g normalized serum calcium, but PTH levels remained elevated. Osteomalacia was corrected by 100 pg/g b.w. of doxercalciferol or 1,000 pg/g b.w. of paricalcitol. The highest dose of doxercalciferol, but not of paricalcitol, significantly reduced osteitis fibrosa. CONCLUSION: Our results reveal the differential efficacy of doxercalciferol and paricalcitol in this novel animal model incorporating both calcitriol deficiency and renal insufficiency.

Nuclear alpha NAC influences bone matrix mineralization and osteoblast maturation in vivo.

Nascent-polypeptide-associated complex and coactivator alpha (alpha NAC) is a protein shuttling between the nucleus and the cytoplasm. Upon phosphorylation at residue serine 43 by integrin-linked kinase, alpha 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 alpha 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 alpha 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 alpha(1)(I) collagen mRNA and as a consequence had significantly less osteoid tissue. Transient transfection assays and chromatin immunoprecipitation confirmed the alpha(1)(I) collagen gene as a novel alpha 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 alpha 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.

Osteocrin is a specific ligand of the natriuretic Peptide clearance receptor that modulates bone growth.

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 Kd of approximately 5 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.

Sequence-specific DNA binding by the alphaNAC coactivator is required for potentiation of c-Jun-dependent transcription of the osteocalcin gene.

Since the c-Jun coactivator alphaNAC 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 alphaNAC. We had also previously shown that alphaNAC can specifically bind DNA in vitro, but it remained unclear whether the DNA-binding function of alphaNAC is expressed in vivo or if it is required for coactivation. We have identified an alphaNAC binding site within the murine osteocalcin gene proximal promoter region and demonstrated that recombinant alphaNAC or alphaNAC from ROS17/2.8 nuclear extracts can specifically bind this element. Using transient transfection assays, we have shown that alphaNAC specifically potentiated the c-Jun-dependent transcription of the osteocalcin promoter and that this activity specifically required the DNA-binding domain of alphaNAC. Chromatin immunoprecipitation confirmed that alphaNAC occupies its binding site on the osteocalcin promoter in living osteoblastic cells expressing osteocalcin. Inhibition of the expression of endogenous alphaNAC 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 alphaNAC coactivating function, and we propose that alphaNAC is specifically targeted to the osteocalcin promoter through its DNA-binding activity as a means to achieve increased specificity in gene transcription.

GSK3 beta-dependent phosphorylation of the alpha NAC coactivator regulates its nuclear translocation and proteasome-mediated degradation.

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.

Focal contact clustering in osteoblastic cells under mechanical stresses: microgravity and cyclic deformation.

We quantitatively compared vinculin-related adhesion parameters in osteoblastic cells submitted to two opposing mechanical stresses: low deformation and frequency strain regimens (stretch conditions) and microgravity exposure (relaxed conditions). In both ROS 17/2.8 cells and rat primary osteoblastic cells, 1% cyclic deformations at 0.05 Hz for 10 min per day for seven days stimulated cell growth compared to static culture conditions, while relaxed ROS cells proliferated in a similar way to static cultures (BC). We studied the short-term (up to 24 h) adaptation of focal contact reorganization under these two conditions. Cyclic deformation induced a biphasic response comprising the formation of new focal contacts followed by clustering of these focal contacts in both ROS cells and primary osteoblasts. Microgravity exposure induced a reduction in focal contact number and clustering in ROS cells. To evaluate whether the proliferation (stretch) or survival (relaxed) status of ROS cells influences focal contact organization, we inhibited the ERK proliferative-dependent pathway. Inhibition of proliferation by PD98059 was partially reversed, but not fully restored by stretch. Stretch-induced clustering of vinculin-positive contacts also persisted in the presence of PD98059, whereas the increase in focal contact number was abolished. In conclusion, we show that focal contacts are mechanoeffectors, and we suggest that their morphologic organization might serve as a discriminant functional parameter between survival and proliferation status in ROS 17/2.8 osteoblastic cells.

Focal contacts organization in osteoblastic cells under microgravity and cyclic deformation conditions.

We compared quantitatively vinculin-related adhesion parameters in osteoblastic cells submitted to opposite mechanical stresses, i.e., low deformation and frequency strain regimens (stretch condition) and microgravity exposure (relaxed condition). Cyclic deformation induced a biphasic response comprising new focal contacts formation followed by their clustering in ROS cells. Microgravity exposure induced a reduction in focal contact number and clustering in ROS cells. We previously demonstrated that 1% cyclic deformations at 0.05 Hz during a daily 10 min episode over 7 days stimulated ROS 17/2.8 growth as compared to static culture whereas relaxed ROS proliferated similarly to static culture (BC). To evaluate whether the proliferation (stretch) or the survival (relaxed) status of ROS cells influences focal contact organization, we inhibited ERKs proliferative-dependent pathway. Inhibition of proliferation by PD98059 was overcome although not fully restored by stretch. Furthermore stretch-induced clustering of vinculin-positive contacts still occurs in the presence of ERKs inhibitor, whereas the increase in focal contact number is abolished. In conclusion, we showed that focal contacts are mechanoeffectors and that hyper-mechanical stimulation could up regulate focal contacts size as compared to hypo-mechanical that down regulate clusterization.