Mutant Osteoblasts Research Articles

Melatonin receptor 1A variants as genetic cause of idiopathic osteoporosis.

Idiopathic osteoporosis (IOP) is a rare form of early-onset osteoporosis diagnosed in patients with no known metabolic or hormonal cause of bone loss and unknown pathogenesis. Patients with IOP commonly report both childhood fractures and family history of osteoporosis, raising the possibility of genetic etiologies of IOP. Whole-exome sequencing analyses of different IOP cohorts identified multiple variants in melatonin receptor 1A (MTNR1A) with a potential pathogenic outcome. A rare MTNR1A variant (rs374152717) was found in members of an Ashkenazi Jewish family with IOP, and an MTNR1A variant (rs28383653) was found in a nonrelated female IOP cohort (4%). Both variants occur at a substantially higher frequency in Ashkenazi Jewish individuals than in the general population. We investigated consequences of the heterozygous (rs374152717) variant [MTNR1Ac.184+1G>T (MTNR1Ac.184+1G>T)] on bone physiology. A mouse model of the human rs374152717 variant reproduced the low bone mass (BM) phenotype of young-adult patients with IOP. Low BM occurred because of induction of senescence in mutant osteoblasts followed by compromised differentiation and function. In human cells, introduction of rs374152717 led to translation of a nonfunctional protein and subsequent dysregulation of melatonin signaling. These studies provide evidence that MTNR1A mutations entail a genetic etiology of IOP and establish the rs374152717 variant as a loss-of-function allele that impairs bone turnover by inducing senescence in osteoblasts. The higher prevalence of the MTNR1A variants identified in IOP cohorts versus the general population indicates a greater risk of IOP in those carrying these variants, especially Ashkenazi Jewish individuals bearing the rs374152717 variant.

Excessive osteoclast activation by osteoblast paracrine factor RANKL is a major cause of the abnormal long bone phenotype in Apert syndrome model mice.

The fibroblast growth factor (FGF)/FGF receptor (FGFR) signaling pathway plays important roles in the development and growth of the skeleton. Apert syndrome caused by gain‐of‐function mutations of FGFR2 results in aberrant phenotypes of the skull, midface, and limbs. Although short limbs are representative features in patients with Apert syndrome, the causative mechanism for this limb defect has not been elucidated. Here we quantitatively confirmed decreases in the bone length, bone mineral density, and bone thickness in the Apert syndrome model of gene knock‐in Fgfr2 S252W/+ (EIIA‐Fgfr2 S252W/+ ) mice. Interestingly, despite these bone defects, histological analysis showed that the endochondral ossification process in the mutant mice was similar to that in wild‐type mice. Tartrate‐resistant acid phosphatase staining revealed that trabecular bone loss in mutant mice was associated with excessive osteoclast activity despite accelerated osteogenic differentiation. We investigated the osteoblast–osteoclast interaction and found that the increase in osteoclast activity was due to an increase in the Rankl level of osteoblasts in mutant mice and not enhanced osteoclastogenesis driven by the activation of FGFR2 signaling in bone marrow‐derived macrophages. Consistently, Col1a1‐Fgfr2 S252W/+ mice, which had osteoblast‐specific expression of Fgfr2 S252W, showed significant bone loss with a reduction of the bone length and excessive activity of osteoclasts was observed in the mutant mice. Taken together, the present study demonstrates that the imbalance in osteoblast and osteoclast coupling by abnormally increased Rankl expression in Fgfr2 S252W/+ mutant osteoblasts is a major causative mechanism for bone loss and short long bones in Fgfr2 S252W/+ mice.

Accelerated osteoblastic differentiation in patient-derived dental pulp stem cells carrying a gain-of-function mutation of TRPV4 associated with metatropic dysplasia

Metatropic dysplasia (MD) is a congenital skeletal dysplasia characterized by severe platyspondyly and dumbbell-like long-bone deformities. These skeletal phenotypes are predominantly caused by autosomal dominant gain-of-function (GOF) mutations in transient receptor potential vanilloid 4 (TRPV4), which encodes a nonselective Ca2+-permeable cation channel. Previous studies have shown that constitutive TRPV4 channel activation leads to irregular chondrogenic proliferation and differentiation, and thus to the disorganized endochondral ossification seen in MD. Therefore, the present study investigated the role of TRPV4 in osteoblast differentiation and MD pathogenesis. Specifically, the behavior of osteoblasts differentiated from patient-derived dental pulp stem cells carrying a heterozygous single base TRPV4 mutation, c.1855C > T (p.L619F) was compared to that of osteoblasts differentiated from isogenic control cells (in which the mutation was corrected using the CRISPR/Cas9 system). The mutant osteoblasts exhibited enhanced calcification (indicated by intense Alizarin Red S staining), increased intracellular Ca2+ levels, strongly upregulated runt-related transcription factor 2 and osteocalcin expression, and increased expression and nuclear translocation of nuclear factor-activated T cell c1 (NFATc1) compared to control cells. These results suggest that the analyzed TRPV4 GOF mutation disrupts osteoblastic differentiation and induces MD-associated disorganized endochondral ossification by increasing Ca2+/NFATc1 pathway activity. Thus, inhibiting the NFATc1 pathway may be a promising potential therapeutic strategy to attenuate skeletal deformities in MD.

Disruption of Dhcr7 and Insig1/2 in cholesterol metabolism causes defects in bone formation and homeostasis through primary cilium formation

Human linkage studies suggest that craniofacial deformities result from either genetic mutations related to cholesterol metabolism or high-cholesterol maternal diets. However, little is known about the precise roles of intracellular cholesterol metabolism in the development of craniofacial bones, the majority of which are formed through intramembranous ossification. Here, we show that an altered cholesterol metabolic status results in abnormal osteogenesis through dysregulation of primary cilium formation during bone formation. We found that cholesterol metabolic aberrations, induced through disruption of either Dhcr7 (which encodes an enzyme involved in cholesterol synthesis) or Insig1 and Insig2 (which provide a negative feedback mechanism for cholesterol biosynthesis), result in osteoblast differentiation abnormalities. Notably, the primary cilia responsible for sensing extracellular cues were altered in number and length through dysregulated ciliary vesicle fusion in Dhcr7 and Insig1/2 mutant osteoblasts. As a consequence, WNT/β-catenin and hedgehog signaling activities were altered through dysregulated primary cilium formation. Strikingly, the normalization of defective cholesterol metabolism by simvastatin, a drug used in the treatment of cholesterol metabolic aberrations, rescued the abnormalities in both ciliogenesis and osteogenesis in vitro and in vivo. Thus, our results indicate that proper intracellular cholesterol status is crucial for primary cilium formation during skull formation and homeostasis.

Dchs1-Fat4 regulation of osteogenic differentiation in mouse.

In human, mutations of the protocadherins FAT4 and DCHS1 result in Van Maldergem syndrome, which is characterised, in part, by craniofacial abnormalities. Here, we analyse the role of Dchs1-Fat4 signalling during osteoblast differentiation in mouse. We show that Fat4 and Dchs1 mutants mimic the craniofacial phenotype of the human syndrome and that Dchs1-Fat4 signalling is essential for osteoblast differentiation. In Dchs1/Fat4 mutants, proliferation of osteoprogenitors is increased and osteoblast differentiation is delayed. We show that loss of Dchs1-Fat4 signalling is linked to increased Yap-Tead activity and that Yap is expressed and required for proliferation in osteoprogenitors. In contrast, Taz is expressed in more-committed Runx2-expressing osteoblasts, Taz does not regulate osteoblast proliferation and Taz-Tead activity is unaffected in Dchs1/Fat4 mutants. Finally, we show that Yap and Taz differentially regulate the transcriptional activity of Runx2, and that the activity of Yap-Runx2 and Taz-Runx2 complexes is altered in Dchs1/Fat4 mutant osteoblasts. In conclusion, these data identify Dchs1-Fat4 as a signalling pathway in osteoblast differentiation, reveal its crucial role within the early Runx2 progenitors, and identify distinct requirements for Yap and Taz during osteoblast differentiation.

Gaucher disease iPSC-derived osteoblasts have developmental and lysosomal defects that impair bone matrix deposition

Gaucher disease (GD) is caused by bi-allelic mutations in GBA1, the gene that encodes acid β-glucocerebrosidase (GCase). Individuals affected by GD have hematologic, visceral and bone abnormalities, and in severe cases there is also neurodegeneration. To shed light on the mechanisms by which mutant GBA1 causes bone disease, we examined the ability of human induced pluripotent stem cells (iPSC) derived from patients with Types 1, 2 and 3 GD, to differentiate to osteoblasts and carry out bone deposition. Differentiation of GD iPSC to osteoblasts revealed that these cells had developmental defects and lysosomal abnormalities that interfered with bone matrix deposition. Compared with controls, GD iPSC-derived osteoblasts exhibited reduced expression of osteoblast differentiation markers, and bone matrix protein and mineral deposition were defective. Concomitantly, canonical Wnt/β catenin signaling in the mutant osteoblasts was downregulated, whereas pharmacological Wnt activation with the GSK3β inhibitor CHIR99021 rescued GD osteoblast differentiation and bone matrix deposition. Importantly, incubation with recombinant GCase (rGCase) rescued the differentiation and bone-forming ability of GD osteoblasts, demonstrating that the abnormal GD phenotype was caused by GCase deficiency. GD osteoblasts were also defective in their ability to carry out Ca2+-dependent exocytosis, a lysosomal function that is necessary for bone matrix deposition. We conclude that normal GCase enzymatic activity is required for the differentiation and bone-forming activity of osteoblasts. Furthermore, the rescue of bone matrix deposition by pharmacological activation of Wnt/β catenin in GD osteoblasts uncovers a new therapeutic target for the treatment of bone abnormalities in GD.

Lack of Lrp5 Signaling in Osteoblasts Sensitizes Male Mice to Diet-Induced Disturbances in Glucose Metabolism

Wnt signaling through the low-density lipoprotein-related receptor 5 (Lrp5) coreceptor regulates osteoblast maturation, matrix mineralization, and intermediary metabolism. In the mature osteoblast, signals downstream of Lrp5 are required for normal long-chain fatty acid β-oxidation. Mice rendered deficient for this coreceptor in osteoblasts and osteocytes accumulate body fat with elevated serum lipid levels but retain normal insulin sensitivity. In the present study, we challenged Lrp5-mutant mice with a high-fat diet (HFD) to determine whether they were more susceptible to diet-induced disturbances in glucose homeostasis. The HFD-fed Lrp5 mutant mice maintained a low bone mass phenotype with an increase in adipose tissue mass and hypertriglyceridemia and hypercholesterolemia. Examination of glucose metabolism revealed that Lrp5 deficiency in the osteoblast also resulted in hyperglycemia and hyperinsulinemia, with reductions in glucose tolerance, insulin sensitivity, and serum undercarboxylated osteocalcin. The results from in vivo genetic epistasis and in vitro studies suggest that this phenotype proceeds via the accumulation of diacylglycerol species and impaired insulin signaling in Lrp5-deficient osteoblasts. In turn, glucose uptake and osteocalcin production are diminished in mutant osteoblasts. Taken together, these data identify a link between Wnt-Lrp5 signaling and insulin signaling in the osteoblast that has the potential to influence energy balance and compound the detrimental effects of a HFD on whole-body metabolism.

Increased NF-κB Activity and Decreased Wnt/β-Catenin Signaling Mediate Reduced Osteoblast Differentiation and Function in ΔF508 Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Mice

The prevalent human ΔF508 mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) is associated with reduced bone formation and bone loss in mice. The molecular mechanisms by which the ΔF508-CFTR mutation causes alterations in bone formation are poorly known. In this study, we analyzed the osteoblast phenotype in ΔF508-CFTR mice and characterized the signaling mechanisms underlying this phenotype. Ex vivo studies showed that the ΔF508-CFTR mutation negatively impacted the differentiation of bone marrow stromal cells into osteoblasts and the activity of osteoblasts, demonstrating that the ΔF508-CFTR mutation alters both osteoblast differentiation and function. Treatment with a CFTR corrector rescued the abnormal collagen gene expression in ΔF508-CFTR osteoblasts. Mechanistic analysis revealed that NF-κB signaling and transcriptional activity were increased in mutant osteoblasts. Functional studies showed that the activation of NF-κB transcriptional activity in mutant osteoblasts resulted in increased β-catenin phosphorylation, reduced osteoblast β-catenin expression, and altered expression of Wnt/β-catenin target genes. Pharmacological inhibition of NF-κB activity or activation of canonical Wnt signaling rescued Wnt target gene expression and corrected osteoblast differentiation and function in bone marrow stromal cells and osteoblasts from ΔF508-CFTR mice. Overall, the results show that the ΔF508-CFTR mutation impairs osteoblast differentiation and function as a result of overactive NF-κB and reduced Wnt/β-catenin signaling. Moreover, the data indicate that pharmacological inhibition of NF-κB or activation of Wnt/β-catenin signaling can rescue the abnormal osteoblast differentiation and function induced by the prevalent ΔF508-CFTR mutation, suggesting novel therapeutic strategies to correct the osteoblast dysfunctions in cystic fibrosis.

A Novel IFITM5 Mutation in Severe Atypical Osteogenesis Imperfecta Type VI Impairs Osteoblast Production of Pigment Epithelium-Derived Factor

Osteogenesis imperfecta (OI) types V and VI are caused, respectively, by a unique dominant mutation in IFITM5, encoding BRIL, a transmembrane ifitm-like protein most strongly expressed in the skeletal system, and recessive null mutations in SERPINF1, encoding pigment epithelium-derived factor (PEDF). We identified a 25-year-old woman with severe OI whose dermal fibroblasts and cultured osteoblasts displayed minimal secretion of PEDF, but whose serum PEDF level was in the normal range. SERPINF1 sequences were normal despite bone histomorphometry consistent with type VI OI and elevated childhood serum alkaline phosphatase. We performed exome sequencing on the proband, both parents, and an unaffected sibling. IFITM5 emerged as the candidate gene from bioinformatics analysis, and was corroborated by membership in a murine bone co-expression network module containing all currently known OI genes. The de novo IFITM5 mutation was confirmed in one allele of the proband, resulting in a p.S40L substitution in the intracellular domain of BRIL but was absent in unaffected family members. IFITM5 expression was normal in proband fibroblasts and osteoblasts, and BRIL protein level was similar to control in differentiated proband osteoblasts on Western blot and in permeabilized mutant osteoblasts by microscopy. In contrast, SERPINF1 expression was decreased in proband osteoblasts; PEDF was barely detectable in conditioned media of proband cells. Expression and secretion of type I collagen was similarly decreased in proband osteoblasts; the expression pattern of several osteoblast markers largely overlapped reported values from cells with a primary PEDF defect. In contrast, osteoblasts from a typical case of type V OI, with an activating mutation at the 5'-terminus of BRIL, have increased SERPINF1 expression and PEDF secretion during osteoblast differentiation. Together, these data suggest that BRIL and PEDF have a relationship that connects the genes for types V and VI OI and their roles in bone mineralization.

Abstract 2471: The tumor suppressor gene Rb1 controls telomeric length and genomic instability that predisposes to osteosarcoma development in irradiated mice

Abstract Osteosarcomas are capable of exhibiting both alternative lengthening of telomeres (ALT) and genomic instability, and are commonly associated with inactivation of the Rb1 gene implicated in both, cell cycle checkpoint regulation and telomere maintainance. We demonstrate a functional relationship between the loss of Rb1, genomic instability and the shortening of telomeres in radiation-induced osteosarcoma. Conditional somatic deletion of a floxed-Rb1 allele in the osteoblastic lineage produces an increased sensitivity to the osteosarcomagenic effects of radiation (Gonzalez-Vasconcellos et al, 2010). In Rb1 +/- osteoblast cell lines established from non-irradiated Rb1fl/+ Col1a1-Cre mice we show that after irradiation there was the expected impaired cell cycle arrest accompanied by an increase in genomic instability and reduced radiation induced senescence. The DNAmicronucleus assay showed an increase of 1.53 fold in Rb +/- primary cells as compared to Rb+/+ after 2 Gy irradiation (p=0.0032). We could prove that the reduced Rb1 gene expression in primary osteoblasts leads to a reduction in telomeric length of up to 50% that increases in a passage dependent manner and was shown to be independent of radiation exposure. These mutant cells showed an inclination towards tetraploidy and radiation induced anaphase bridges formation was increased in 2.6 fold in the mutant cells after 2 Gy irradiation (p = 0.001). These studies suggest the tumor suppressor gene Rb1 functioning not only as a gatekeeper if not also as a caretaker of the genome controlling the telomere dynamics and genomic stability. This maintenance of the genome by Rb1 was proven to be driven by epigenetic changes on the Rb+/- primary osteoblasts involving histone methylation. Lower levels of 3metLys20-H4 fluorescence intensity (p = 0.0079) were detected in Rb1 mutant osteoblasts as compared to wt. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2471. doi:10.1158/1538-7445.AM2011-2471

Differential effects of alendronate and losartan therapy on osteopenia and aortic aneurysm in mice with severe Marfan syndrome

Reduced bone mineral density (osteopenia) is a poorly characterized manifestation of pediatric and adult patients afflicted with Marfan syndrome (MFS), a multisystem disorder caused by structural or quantitative defects in fibrillin-1 that perturb tissue integrity and TGFβ bioavailability. Here we report that mice with progressively severe MFS (Fbn1(mgR/mgR) mice) develop osteopenia associated with normal osteoblast differentiation and bone formation. In vivo and ex vivo experiments, respectively, revealed that adult Fbn1(mgR/mgR) mice respond more strongly to locally induced osteolysis and that Fbn1(mgR/mgR) osteoblasts stimulate pre-osteoclast differentiation more than wild-type cells. Greater osteoclastogenic potential of mutant osteoblasts was largely attributed to Rankl up-regulation secondary to improper TGFβ activation and signaling. Losartan treatment, which lowers TGFβ signaling and restores aortic wall integrity in mice with mild MFS, did not mitigate bone loss in Fbn1(mgR/mgR) mice even though it ameliorated vascular disease. Conversely, alendronate treatment, which restricts osteoclast activity, improved bone quality but not aneurysm progression in Fbn1(mgR/mgR) mice. Taken together, our findings shed new light on the pathogenesis of osteopenia in MFS, in addition to arguing for a multifaceted treatment strategy in this congenital disorder of the connective tissue.

OA/Gpnmb Mutation Impairs Osteoblast and Osteoclast Differentiation and Function

Osteoactivin/Gpnmb (OA/Gpnmb) is a glycoprotein identified in bone, and later found to be strongly localized to bone‐forming osteoblasts. There exists in mice a naturally occurring mutation for OA/Gpnmb, which is attributable to premature stop codon resulting in a truncated 150 amino acid OA/Gpnmb protein. Micro‐CT analysis of aged mice show decreased bone volume (BV/TV), trabecular number (Tb.N), and trabecular thickness (Tb. Th.) in OA/Gpnmb mutant mice when compared to wild‐type, implying that OA/Gpnmb mutant mice are more osteoporotic. Immunoflourescent analysis has shown OA/Gpnmb protein in mutant osteoblasts (OB) is retained in the peri‐nuclear/cis‐Golgi area. Electron microscopy analysis of osteoblasts showed no morphological differences in mutant osteoblasts versus control, suggesting OA/Gpnmb retention is not cytotoxic. qPCR mRNA and western blot analyses showed increased RANKL expression in OB from OA/Gpnmb mutant. Co‐culture of bone marrow cells showed marked increase in osteoclast (OC) numbers and size in mutant cultures when compared to WT. Treatment of WT and Mutant osteoblasts with recombinant OA/Gpnmb protein resulted in enhanced osteoclast number and size in vitro in WT and but not in mutant OB, suggesting an inherent defect to in mutant compared to WT OC. Collectively, these data suggest OA/Gpnmb acts as a regulator of osteoblast‐osteoclast differentiation and function.Grant Funding Source: NIAMS

Osteoblastic molecular scaffold Gab1 is required for maintaining bone homeostasis

The Grb2-associated binder 1 (Gab1), which serves as a scaffolding adaptor protein, plays a crucial role in transmitting key signals that control cell growth, differentiation and function from multiple receptors. However, its biological role in osteoblast activity and postnatal bone metabolism remains unclear. To elucidate the in vivo function of Gab1 in postnatal bone remodeling, we generated osteoblast-specific Gab1 knockout mice. Disruption of Gab1 expression in osteoblasts led to decreased trabecular bone mass with a reduced bone formation rate and a decreased bone resorption. Bones from Gab1 mutants also exhibited inferior mechanical properties. Moreover, primary osteoblasts from Gab1 mutant mice demonstrated markedly suppressed osteoblast mineralization, increased susceptibility to apoptosis and decreased expression of receptor activator of NF-kappaB ligand (RANKL). Activation of serine-threonine Akt kinase and extracellular signal-regulated kinase in response to insulin and insulin-like growth factor 1 was attenuated in Gab1 mutant osteoblasts. Our results show that Gab1-mediated signals in osteoblasts are crucial for normal postnatal bone homeostasis.

Pro416Arg cherubism mutation in Sh3bp2 knock-in mice affects osteoblasts and alters bone mineral and matrix properties

Cherubism is an autosomal dominant disorder in children characterized by unwarranted symmetrical bone resorption of the jaws with fibrous tissue deposition. Mutations causing cherubism have been identified in the adaptor protein SH3BP2. Knock-in mice with a Pro416Arg mutation in Sh3bp2 exhibit a generalized osteoporotic bone phenotype. In this study, we examined the effects of this “cherubism” mutation on spectroscopic indices of “bone quality” and on osteoblast differentiation. Fourier-transform infrared imaging (FTIRI) analysis of femurs from wild-type and Sh3bp2 knock-in mice showed decreased mineral content, decreased mineral crystallinity/crystal size, and increased collagen maturity in homozygous mutants. To assess osteoblast maturation in vivo, knock-in mice were crossed with transgenic mice over-expressing GFP driven by 3.6-kb or 2.3-kb Col1a1 promoter fragments. Reduced numbers of mature osteoblasts were observed in homozygous mice. Neonatal calvarial cultures, which were enriched for osteoblasts by depletion of hematopoietic cells (negative selection for Ter119- and CD45-positive cells) were investigated for osteoblast-specific gene expression and differentiation, which demonstrated that differentiation and mineralization in homozygous osteoblast cultures was impaired. Co-cultures with calvarial osteoblasts and bone marrow macrophages showed that mutant osteoblasts appear to increase osteoclastogenesis resulting in increased bone resorption on bone chips. In summary, the Sh3bp2 mutation in cherubism mice alters bone quality, reduces osteoblast function, and may contribute to excessive bone resorption by osteoclasts. Our data, together with previous osteoclast studies, demonstrate a critical role of Sh3bp2 in bone remodeling and osteoblast differentiation.

Medical treatment of craniosynostosis: recombinant Noggin inhibits coronal suture closure in the rat craniosynostosis model

INTRODUCTION - The mechanisms underlying craniosynostosis remains unknown. However, mutations in FGFR2 are associated with craniosynostotic syndromes. We previously compared gene expression patterns of patent and synostosing coronal sutures in the nude rat and demonstrated down regulation of Noggin in synostosing sutures. Noggin expression is also suppressed by FGF2 and constitutive FGFR2 signaling [Warren et al. (2003) Nature, vol. 422, pp. 625-9; McMahon et al. (1998) Genes Dev, vol. 12, pp. 1438-52]. Thus, we therefore hypothesized that the addition of rhNoggin to prematurely fusing sutures should prevent synostosis. MATERIALS AND METHODS - Cohorts of nude rats were subjected to: 1) surgical elevation of the coronal suture (shams); 2) surgical elevation and placement of normal or FGFR2 mutant human osteoblasts onto the underlying dura (xenotransplants); or 3) xenotransplantation with co-application of heparin acrylic beads soaked with recombinant human (rh) Noggin. Eleven days post-surgery the sutures were harvested, stained, and histologically examined. RESULTS - Animals that received control osteoblasts, sham surgery, or no surgery demonstrated normal skull growth and coronal suture histology, whereas animals transplanted only with FGFR2 mutant osteoblasts showed evidence of bridging synostosis on the calvarial dural surface. Sutures treated with FGFR2 mutant osteoblasts and rhNoggin remained patent. CONCLUSION - The chimeric nude rate model is a viable model of craniosynostosis. FGFR2 mutations in osteoblasts induce bridging osteosynthesis demonstrating one of the mechanisms for premature suture fusion. Topical application of rhNoggin protein prevents craniosynostosis in the weanling nude rat xenotransplantation model of syndromic craniosynostosis.

Dlx5, a Positive Regulator of Osteoblastogenesis, is Essential for Osteoblast-Osteoclast Coupling

The homeodomain protein Dlx5 is an activator of Runx2 (a key regulator of osteogenesis) and is thought to be an important regulator of bone formation. At present, however, the perinatal lethality of Dlx5-null mice has hampered the elucidation of its function in osteogenesis. Here we provide the first analysis of the effects of Dlx5 inactivation on bone development. Femurs of Dlx5-null mouse embryos at the end of gestation exhibit a reduction in both total and trabecular bone volume associated with increased trabecular separation and reduced trabecular number. These parameters are often associated with pathological conditions characterized by reduced osteoblast activity and increased bone resorption. Dlx5(-/-) osteoblasts in culture display reduced proliferation and differentiation rate and reduction of Runx2, Osx, Osteocalcin and Bone Sialoprotein expression. In addition to impaired osteoblast function, Dlx5(-/-) femurs exhibit significant increases in osteoclast number. As Dlx5 is not expressed by osteoclasts, we suggest that its osteoblastic expression might control osteoblast/osteoclast coupling. Cultured Dlx5(-/-) osteoblasts displayed a higher RANKL/OPG ratio. Furthermore, Dlx5(-/-) osteoblasts induced a higher number of TRAP-positive multinucleated cells in normal spleen cultures with a globally increased resorption activity. These findings suggest that Dlx5 is a central regulator of bone turnover as it activates bone formation directly and bone resorption indirectly.

PERK is essential for neonatal skeletal development to regulate osteoblast proliferation and differentiation

Loss of function mutations of Perk (eukaryotic translation initiation factor 2 alpha kinase 3) in humans and mice cause severe neonatal developmental defects, including diabetes, growth retardation and multiple skeletal dysplasias. Comprehensive analyses on bone tissue, at the cellular and molecular level in PERK-deficient mice demonstrated that neonatal Perk-/- mice are severely osteopenic, which is caused by a deficiency in the number of mature osteoblasts, impaired osteoblast differentiation, and reduced type I collagen secretion. Impaired differentiation of osteoblasts in Perk KO mice was associated with decreased expression of Runx2 and Osterix, key regulators of osteoblast development. Reduced cell proliferation and reduced expression of key cell cycle factors including cyclin D, cyclin E, cyclin A, Cdc2, and CDK2 occur in parallel with the differentiation defect in mutant osteoblasts. In addition, the trafficking and secretion of type I collagen is compromised as manifested by abnormal retention of procollagen I in the endoplasmic reticulum, and reduced mature collagen production and mineralization. Taken together, these studies identify PERK as a novel regulator of skeletal development and osteoblast biology.

Colony-stimulating factor-1 (CSF-1) rescues osteoblast attachment, survival and sorting of beta-actin mRNA in the toothless (tl-osteopetrotic) mutation in the rat.

We have shown that in the osteopetrotic rat mutation toothless (tl) osteoblasts are absent from older bone surfaces in mutants and that mutant osteoblasts in vivo lack the prominent stress fiber bundles polarized along bone surfaces in osteoblasts from normal littermates. Our recent data demonstrate that in normal osteoblasts in vitro beta- and gamma-actin mRNAs have different, characteristic intracellular distributions and that tl (mutant) osteoblasts fail to differentially sort these mRNAs. Because bone resorption and formation are highly interdependent and injections of CSF-1, a growth factor, increase bone resorption and growth in tl rats, we examined the effects of CSF-1 treatment on osteoblast survival and ultrastructure in vivo and ability to sort actin mRNAs in vitro. Neonatal CSF-1 treatment of mutants restores osteoblasts on older bone surfaces, normalizes the intracellular distribution of stress fibers in osteoblasts in vivo and promotes normal sorting of beta-actin mRNA in mutant osteoblasts in vitro without normalizing gamma-actin distribution. These data suggest the beta- and gamma-actin mRNAs in osteoblasts are sorted by different mechanisms and that the differential sorting of beta-actin mRNA is related to the characteristic polarization of stress fibers in osteoblasts and their survival on bone surfaces. This experimental system can be used to explore the relationships and regulation of these aspects of cell and tissue biology.

Osteoactivin, an anabolic factor that regulates osteoblast differentiation and function

Osteoactivin (OA) is a novel glycoprotein that is highly expressed during osteoblast differentiation. Using Western blot analysis, our data show that OA protein has two isoforms, one is transmembranous and the other is secreted into the conditioned medium of primary osteoblasts cultures. Fractionation of osteoblast cell compartments showed that the mature, glycosylated OA isoform of 115 kDa is found in the membranous fraction. Both OA isoforms (secreted and transmembrane) are found in the cytoplasmic fraction of osteoblasts. Overexpression of EGFP-tagged OA in osteoblasts showed that OA protein accumulates into vesicles for transportation to the cell membrane. We examined OA protein production in primary osteoblast cultures and found that OA is maximally expressed during the third week of culture (last stage of osteoblast differentiation). Glycosylation studies showed that OA isoform of 115 kDa is highly glycosylated. We also showed that retinoic acid (RA) stimulates the mannosylation of OA protein. In contrast, tunicamycin (TM) strongly inhibited N-glycans incorporation into OA protein. The functional role of the secreted OA isoform was revealed when cultures treated with anti-OA antibody, showed decreased osteoblast differentiation compared to untreated control cultures. Gain-of-function in osteoblasts using the pBABE viral system showed that OA overexpression in osteoblast stimulated their differentiation and function. The availability of a naturally occurring mutant mouse with a truncated OA protein provided further evidence that OA is an important factor for terminal osteoblast differentiation and mineralization. Using bone marrow mesenchymal cells derived from OA mutant and wild-type mice and testing their ability to differentiate into osteoblasts showed that differentiation of OA mutant osteoblasts was significantly reduced compared to wild-type osteoblasts. Collectively, our data suggest that OA acts as a positive regulator of osteoblastogenesis.

Disruption of the Insulin-like Growth Factor Type 1 Receptor in Osteoblasts Enhances Insulin Signaling and Action

Defective bone formation is common in patients with diabetes, suggesting that insulin normally exerts anabolic actions in bone. However, because insulin can cross-activate the insulin-like growth factor type 1 receptor (IGF-1R), which also functions in bone, it has been difficult to establish the direct (IGF-1-independent) actions of insulin in osteoblasts. To overcome this problem, we examined insulin signaling and action in primary osteoblasts engineered for conditional disruption of the IGF-1 receptor (DeltaIGF-1R). Calvarial osteoblasts from mice carrying floxed IGF-1R alleles were infected with adenoviral vectors expressing the Cre recombinase (Ad-Cre) or green fluorescent protein (Ad-GFP) as control. Disruption of IGF-1R mRNA (>90%) eliminated IGF-1R without affecting insulin receptor (IR) mRNA and protein expression and eliminated IGF-1R/IR hybrids. In DeltaIGF-1R osteoblasts, insulin signaling was markedly increased as evidenced by increased phosphorylation of insulin receptor substrate 1/2 and enhanced ERK/Akt activation. Microarray analysis of RNA samples from insulin-treated, DeltaIGF-1R osteoblasts revealed striking changes in several genes known to be downstream of ERK including Glut-1 and c-fos. Treatment of osteoblasts with insulin induced Glut-1 mRNA, increased 2-[1,2-(3)H]-deoxy-d-glucose uptake, and enhanced proliferation. Moreover, insulin treatment rescued the defective differentiation and mineralization of DeltaIGF-1R osteoblasts, suggesting that IR signaling can compensate, at least in part, for loss of IGF-1R signaling. We conclude that insulin exerts direct anabolic actions in osteoblasts by activation of its cognate receptor and that the strength of insulin-generated signals is tempered through interactions with the IGF-1R.