COPB2 loss of function causes a coatopathy with osteoporosis and developmental delay.

Coatomer complexes function in the sorting and trafficking of proteins between subcellular organelles. Pathogenic variants in coatomer subunits or associated factors have been reported in multi-systemic disorders, i.e., coatopathies, that can affect the skeletal and central nervous systems. We have identified loss-of-function variants in COPB2, a component of the coatomer complex I (COPI), in individuals presenting with osteoporosis, fractures, and developmental delay of variable severity. Electron microscopy of COPB2-deficient subjects' fibroblasts showed dilated endoplasmic reticulum (ER) with granular material, prominent rough ER, and vacuoles, consistent with an intracellular trafficking defect. We studied the effect of COPB2 deficiency on collagen trafficking because of the critical role of collagen secretion in bone biology. COPB2 siRNA-treated fibroblasts showed delayed collagen secretion with retention of type I collagen in the ER and Golgi and altered distribution of Golgi markers. copb2-null zebrafish embryos showed retention of type II collagen, disorganization of the ER and Golgi, and early larval lethality. Copb2+/- mice exhibited low bone mass, and consistent with the findings in human cells and zebrafish, studies in Copb2+/- mouse fibroblasts suggest ER stress and a Golgi defect. Interestingly, ascorbic acid treatment partially rescued the zebrafish developmental phenotype and the cellular phenotype in Copb2+/- mouse fibroblasts. This work identifies a form of coatopathy due to COPB2 haploinsufficiency, explores a potential therapeutic approach for this disorder, and highlights the role of the COPI complex as a regulator of skeletal homeostasis.

Loading-induced bone formation is mediated by Wnt1 induction in osteoblast-lineage cells.

Mechanical loading on the skeleton stimulates bone formation. Although the exact mechanism underlying this process remains unknown, a growing body of evidence indicates that the Wnt signaling pathway is necessary for the skeletal response to loading. Recently, we showed that Wnts produced by osteoblast lineage cells mediate the osteo-anabolic response to tibial loading in adult mice. Here, we report that Wnt1 specifically plays a crucial role in mediating the mechano-adaptive response to loading. Independent of loading, short-term loss of Wnt1 in the Osx-lineage resulted in a decreased cortical bone area in the tibias of 5-month-old mice. In females, strain-matched loading enhanced periosteal bone formation in Wnt1F/F controls, but not in Wnt1F/F; OsxCreERT2 knockouts. In males, strain-matched loading increased periosteal bone formation in both control and knockout mice; however, the periosteal relative bone formation rate was 65% lower in Wnt1 knockouts versus controls. Together, these findings show that Wnt1 supports adult bone homeostasis and mediates the bone anabolic response to mechanical loading.

Rac1 activation controls nuclear localization of beta-catenin during canonical Wnt signaling.

Canonical Wnt signaling critically regulates cell fate and proliferation in development and disease. Nuclear localization of beta-catenin is indispensable for canonical Wnt signaling; however, the mechanisms governing beta-catenin nuclear localization are not well understood. Here we demonstrate that nuclear accumulation of beta-catenin in response to Wnt requires Rac1 activation. The role of Rac1 depends on phosphorylation of beta-catenin at Ser191 and Ser605, which is mediated by JNK2 kinase. Mutations of these residues significantly affect Wnt-induced beta-catenin nuclear accumulation. Genetic ablation of Rac1 in the mouse embryonic limb bud ectoderm disrupts canonical Wnt signaling and phenocopies deletion of beta-catenin in causing severe truncations of the limb. Finally, Rac1 interacts genetically with beta-catenin and Dkk1 in controlling limb outgrowth. Together these results uncover Rac1 activation and subsequent beta-catenin phosphorylation as a hitherto uncharacterized mechanism controlling canonical Wnt signaling and may provide additional targets for therapeutic intervention of this important pathway.

P3h3-null and Sc65-null Mice Phenocopy the Collagen Lysine Under-hydroxylation and Cross-linking Abnormality of Ehlers-Danlos Syndrome Type VIA.

Tandem mass spectrometry was applied to tissues from targeted mutant mouse models to explore the collagen substrate specificities of individual members of the prolyl 3-hydroxylase (P3H) gene family. Previous studies revealed that P3h1 preferentially 3-hydroxylates proline at a single site in collagen type I chains, whereas P3h2 is responsible for 3-hydroxylating multiple proline sites in collagen types I, II, IV, and V. In screening for collagen substrate sites for the remaining members of the vertebrate P3H family, P3h3 and Sc65 knock-out mice revealed a common lysine under-hydroxylation effect at helical domain cross-linking sites in skin, bone, tendon, aorta, and cornea. No effect on prolyl 3-hydroxylation was evident on screening the spectrum of known 3-hydroxyproline sites from all major tissue collagen types. However, collagen type I extracted from both Sc65-/- and P3h3-/- skin revealed the same abnormal chain pattern on SDS-PAGE with an overabundance of a γ112 cross-linked trimer. The latter proved to be from native molecules that had intramolecular aldol cross-links at each end. The lysine under-hydroxylation was shown to alter the divalent aldimine cross-link chemistry of mutant skin collagen. Furthermore, the ratio of mature HP/LP cross-links in bone of both P3h3-/- and Sc65-/- mice was reversed compared with wild type, consistent with the level of lysine under-hydroxylation seen in individual chains at cross-linking sites. The effect on cross-linking lysines was quantitatively very similar to that previously observed in EDS VIA human and Plod1-/- mouse tissues, suggesting that P3H3 and/or SC65 mutations may cause as yet undefined EDS variants.

Heterozygous WNT1 variant causing a variable bone phenotype.

Osteogenesis imperfecta (OI) is a family of heritable disorders of bone fragility. Most individuals with OI have mutations in the genes encoding type I collagen; at least 17 other genes have been associated with OI. Biallelic loss-of-function mutations in WNT1 cause severe OI. Heterozygous missense variants in WNT1 are responsible for early-onset osteoporosis with variable bone phenotypes. Herein, we report a third-generation family with four affected individuals, some presenting with multiple low-impact fractures in childhood and others presenting with early-onset osteoporosis without a striking fracture history. A WNT1 variant (c. 1051 > C; p.Trp351Arg) was identified in the proband and segregated with a bone phenotype in three additional family members, consistent with autosomal dominant inheritance. In the proband, whole genome sequencing also revealed a de novo duplication (434 kb) of 22q11.2 that involves 25 genes, 4 of which are associated with human disease when haploinsufficient. Though smaller than the typical (1.5 Mb) 22q11.2 duplication, the duplication in the proband may be responsible for additional nonosseous aspects of his phenotype (hypotonia, developmental delay, small genitalia, strabismus, and depression in preadolescence). This case demonstrates the variability of bone phenotype conferred by a WNT1 variant and extends the spectrum of bone phenotypes associated with heterozygous WNT1 mutations.

Up-regulation of glycolytic metabolism is required for HIF1α-driven bone formation.

The bone marrow environment is among the most hypoxic in the body, but how hypoxia affects bone formation is not known. Because low oxygen tension stabilizes hypoxia-inducible factor alpha (HIFα) proteins, we have investigated the effect of expressing a stabilized form of HIF1α in osteoblast precursors. Brief stabilization of HIF1α in SP7-positive cells in postnatal mice dramatically stimulated cancellous bone formation via marked expansion of the osteoblast population. Remarkably, concomitant deletion of vascular endothelial growth factor A (VEGFA) in the mouse did not diminish bone accrual caused by HIF1α stabilization. Thus, HIF1α-driven bone formation is independent of VEGFA up-regulation and increased angiogenesis. On the other hand, HIF1α stabilization stimulated glycolysis in bone through up-regulation of key glycolytic enzymes including pyruvate dehydrogenase kinase 1 (PDK1). Pharmacological inhibition of PDK1 completely reversed HIF1α-driven bone formation in vivo. Thus, HIF1α stimulates osteoblast formation through direct activation of glycolysis, and alterations in cellular metabolism may be a broadly applicable mechanism for regulating cell differentiation.

WNT7B promotes bone formation in part through mTORC1.

WNT signaling has been implicated in both embryonic and postnatal bone formation. However, the pertinent WNT ligands and their downstream signaling mechanisms are not well understood. To investigate the osteogenic capacity of WNT7B and WNT5A, both normally expressed in the developing bone, we engineered mouse strains to express either protein in a Cre-dependent manner. Targeted induction of WNT7B, but not WNT5A, in the osteoblast lineage dramatically enhanced bone mass due to increased osteoblast number and activity; this phenotype began in the late-stage embryo and intensified postnatally. Similarly, postnatal induction of WNT7B in Runx2-lineage cells greatly stimulated bone formation. WNT7B activated mTORC1 through PI3K-AKT signaling. Genetic disruption of mTORC1 signaling by deleting Raptor in the osteoblast lineage alleviated the WNT7B-induced high-bone-mass phenotype. Thus, WNT7B promotes bone formation in part through mTORC1 activation.

Post-translationally abnormal collagens of prolyl 3-hydroxylase-2 null mice offer a pathobiological mechanism for the high myopia linked to human LEPREL1 mutations.

Myopia, the leading cause of visual impairment worldwide, results from an increase in the axial length of the eyeball. Mutations in LEPREL1, the gene encoding prolyl 3-hydroxylase-2 (P3H2), have recently been identified in individuals with recessively inherited nonsyndromic severe myopia. P3H2 is a member of a family of genes that includes three isoenzymes of prolyl 3-hydroxylase (P3H), P3H1, P3H2, and P3H3. Fundamentally, it is understood that P3H1 is responsible for converting proline to 3-hydroxyproline. This limited additional knowledge also suggests that each isoenzyme has evolved different collagen sequence-preferred substrate specificities. In this study, differences in prolyl 3-hydroxylation were screened in eye tissues from P3h2-null (P3h2(n/n)) and wild-type mice to seek tissue-specific effects due the lack of P3H2 activity on post-translational collagen chemistry that could explain myopia. The mice were viable and had no gross musculoskeletal phenotypes. Tissues from sclera and cornea (type I collagen) and lens capsule (type IV collagen) were dissected from mouse eyes, and multiple sites of prolyl 3-hydroxylation were identified by mass spectrometry. The level of prolyl 3-hydroxylation at multiple substrate sites from type I collagen chains was high in sclera, similar to tendon. Almost every known site of prolyl 3-hydroxylation in types I and IV collagen from P3h2(n/n) mouse eye tissues was significantly under-hydroxylated compared with their wild-type littermates. We conclude that altered collagen prolyl 3-hydroxylation is caused by loss of P3H2. We hypothesize that this leads to structural abnormalities in multiple eye tissues, but particularly sclera, causing progressive myopia.

The swaying mouse as a model of osteogenesis imperfecta caused by WNT1 mutations.

Osteogenesis imperfecta (OI) is a heritable disorder of connective tissue characterized by bone fragility and low bone mass. Recently, our group and others reported that WNT1 recessive mutations cause OI, whereas WNT1 heterozygous mutations cause early onset osteoporosis. These findings support the hypothesis that WNT1 is an important WNT ligand regulating bone formation and bone homeostasis. While these studies provided strong human genetic and in vitro functional data, an in vivo animal model to study the mechanism of WNT1 function in bone is lacking. Here, we show that Swaying (Wnt1(sw/sw)) mice previously reported to carry a spontaneous mutation in Wnt1 share major features of OI including propensity to fractures and severe osteopenia. In addition, biomechanical and biochemical analyses showed that Wnt1(sw/sw) mice exhibit reduced bone strength with altered levels of mineral and collagen in the bone matrix that is also distinct from the type I collagen-related form of OI. Further histomorphometric analyses and gene expression studies demonstrate that the bone phenotype is associated with defects in osteoblast activity and function. Our study thus provides in vivo evidence that WNT1 mutations contribute to bone fragility in OI patients and demonstrates that the Wnt1(sw/sw) mouse is a murine model of OI caused by WNT1 mutations.

WNT1 mutations in early-onset osteoporosis and osteogenesis imperfecta.

This report identifies human skeletal diseases associated with mutations in WNT1. In 10 family members with dominantly inherited, early-onset osteoporosis, we identified a heterozygous missense mutation in WNT1, c.652T→G (p.Cys218Gly). In a separate family with 2 siblings affected by recessive osteogenesis imperfecta, we identified a homozygous nonsense mutation, c.884C→A, p.Ser295*. In vitro, aberrant forms of the WNT1 protein showed impaired capacity to induce canonical WNT signaling, their target genes, and mineralization. In mice, Wnt1 was clearly expressed in bone marrow, especially in B-cell lineage and hematopoietic progenitors; lineage tracing identified the expression of the gene in a subset of osteocytes, suggesting the presence of altered cross-talk in WNT signaling between the hematopoietic and osteoblastic lineage cells in these diseases.

Restoration of the serum level of SERPINF1 does not correct the bone phenotype in Serpinf1 null mice.

Osteogenesis imperfecta (OI) is a group of genetic disorders characterized by bone fragility and deformity. OI type VI is unique owing to the mineralization defects observed in patient biopsies. Furthermore, it has been reported to respond less well to standard therapy with bisphosphonates [1]. Others and we have previously identified SERPINF1 mutations in patients with OI type VI. SERPINF1 encodes pigment epithelium derived factor (PEDF), a secreted collagen-binding glycoprotein that is absent in the sera of patients with OI type VI. Serpinf1 null mice show increased osteoid and decreased bone mass, and thus recapitulate the OI type VI phenotype. We tested whether restoration of circulating PEDF in the blood could correct the phenotype of OI type VI in the context of protein replacement. To do so, we utilized a helper-dependent adenoviral vector (HDAd) to express human SERPINF1 in the mouse liver and assessed whether PEDF secreted from the liver was able to rescue the bone phenotype observed in Serpinf1(-/-) mice. We confirmed that expression of SERPINF1 in the liver restored the serum level of PEDF. We also demonstrated that PEDF secreted from the liver was biologically active by showing the expected metabolic effects of increased adiposity and impaired glucose tolerance in Serpinf1(-/-) mice. Interestingly, overexpression of PEDF in vitro increased mineralization with a concomitant increase in the expression of bone gamma-carboxyglutamate protein, alkaline phosphatase and collagen, type I, alpha I, but the increased serum PEDF level did not improve the bone phenotype of Serpinf1(-/-) mice. These results suggest that PEDF may function in a context-dependent and paracrine fashion in bone homeostasis.

Characterization of TGFß1-induced tendon-like structure in the scaffold-free three-dimensional tendon cell culture system.

The biological mechanisms regulating tenocyte differentiation and morphological maturation have not been well-established, partly due to the lack of reliable in vitro systems that produce highly aligned collagenous tissues. In this study, we developed a scaffold-free, three-dimensional (3D) tendon culture system using mouse tendon cells in a differentially adherent growth channel. Transforming Growth Factor-ß (TGFß) signaling is involved in various biological processes in the tendon, regulating tendon cell fate, recruitment and maintenance of tenocytes, and matrix organization. This known function of TGFß signaling in tendon prompted us to utilize TGFß1 to induce tendon-like structures in 3D tendon constructs. TGFß1 treatment promoted a tendon-like structure in the peripheral layer of the constructs characterized by increased thickness with a gradual decrease in cell density and highly aligned collagen matrix. TGFß1 also enhanced cell proliferation, matrix production, and morphological maturation of cells in the peripheral layer compared to vehicle treatment. TGFß1 treatment also induced early tenogenic differentiation and resulted in sufficient mechanical integrity, allowing biomechanical testing. The current study suggests that this scaffold-free 3D tendon cell culture system could be an in vitro platform to investigate underlying biological mechanisms that regulate tenogenic cell differentiation and matrix organization.

Indian hedgehog requires additional effectors besides Runx2 to induce osteoblast differentiation.

Indian hedgehog (Ihh) is indispensable for osteoblast differentiation during embryonic development of the endochondral skeleton. In the absence of Ihh, cells of the osteoblast lineage fail to activate the expression of Runx2, a transcription factor integral to osteoblast differentiation. However, it is hitherto unclear whether the lack of Runx2 expression is solely responsible for the failure of osteoblast formation in Ihh-null embryos. Here, by creating a mouse allele that expresses Runx2 in a Cre-dependent manner, we show that force-expression of Runx2 in the skeletogenic cells restores bone formation in the Runx2-null, but not in the Ihh-null embryo. Thus, the mechanism through which Ihh induces osteoblast differentiation requires other effectors in addition to Runx2.

Indian Hedgehog signalling triggers Nkx3.2 protein degradation during chondrocyte maturation.

The Ihh (Indian Hedgehog) pathway plays an essential role in facilitating chondrocyte hypertrophy and bone formation during skeletal development. Nkx3.2 (NK3 homeobox 2) is initially induced in chondrocyte precursor cells, maintained in early-stage chondrocytes and down-regulated in terminal-stage chondrocytes. Consistent with these expression patterns, Nkx3.2 has been shown to enhance chondrocyte differentiation and cell survival, while inhibiting chondrocyte hypertrophy and apoptosis. Thus, in the present study, we investigated whether Nkx3.2, an early-stage chondrogenic factor, can be regulated by Ihh, a key regulator for chondrocyte hypertrophy. We show that Ihh signalling can induce proteasomal degradation of Nkx3.2. In addition, we found that Ihh can suppress levels of Lrp (low-density-lipoprotein-receptor-related protein) (Wnt co-receptor) and Sfrp (secreted frizzled-related protein) (Wnt antagonist) expression, which, in turn, may selectively enhance Lrp-independent non-canonical Wnt pathways in chondrocytes. In agreement with these findings, Ihh-induced Nkx3.2 degradation requires Wnt5a, which is capable of triggering Nkx3.2 degradation. Finally, we found that Nkx3.2 protein levels in chondrocytes are remarkably elevated in mice defective in Ihh signalling by deletion of either Ihh or smoothened. Thus these results suggest that Ihh/Wnt5a signalling may play a role in negative regulation of Nkx3.2 for appropriate progression of chondrocyte hypertrophy during chondrogenesis.

Long lifespan in worms with long telomeric DNA.

Telomere length is a crucial factor in senescence, but it has not been determined whether animals with long telomeres live longer than those with normal-length telomeres in the isogenic background of a given species. Here we show the effect of long telomeres on lifespan in the nematode Caenorhabditis elegans. We examined the effect of telomere length on lifespan by overexpressing HRP-1, a telomere-binding protein, which gradually increased telomere length in worms. Worms with longer telomeres lived longer. We confirmed that the extension of lifespan was due to the increased telomere length, and not to the overexpression of HRP-1 per se, by examining the lifespans of nontransgenic progeny of the transgenic worms, who retained the longer telomeres. The lifespan-extending effect of long telomeres was dependent on daf-16. The number of germ stem cells was not affected in worms with long telomeres, indicating that the telomere effect on lifespan is independent of germ stem cell cycling. Worms with long telomeres were more resistant to heat stress. Taken together, our results suggest that signaling may be initiated in postmitotic somatic cells by telomere length to regulate organismal lifespan.

Lrp5 and Lrp6 redundantly control skeletal development in the mouse embryo.

The role of Wnt signaling in osteoblastogenesis in the embryo remains to be fully established. Although ß-catenin, a multifunctional protein also mediating canonical Wnt signaling, is indispensable for embryonic osteoblast differentiation, the roles of the key Wnt co-receptors Lrp5 and Lrp6 are unclear. Indeed, global deletion of either Lrp5 or Lrp6 did not overtly affect osteoblast differentiation in the mouse embryo. Here, we generated mice lacking both receptors specifically in the embryonic mesenchyme and observed an absence of osteoblasts in the embryo. In addition, the double-deficient embryos developed supernumerary cartilage elements in the zeugopod, revealing an important role for mesenchymal Lrp5/6 signaling in limb patterning. Importantly, the phenotypes of the Lrp5/6 mutant closely resembled those of the ß-catenin-deficient embryos. These phenotypes are likely independent of any effect on the adherens junction, as deletion of α-catenin, another component of the complex, did not cause similar defects. Thus, Lrp5 and 6 redundantly control embryonic skeletal development, likely through ß-catenin signaling.

Noncanonical Wnt signaling through G protein-linked PKCdelta activation promotes bone formation.

Wnt signaling regulates a variety of developmental processes in animals. Although the beta-catenin-dependent (canonical) pathway is known to control cell fate, a similar role for noncanonical Wnt signaling has not been established in mammals. Moreover, the intracellular cascades for noncanonical Wnt signaling remain to be elucidated. Here, we delineate a pathway in which Wnt3a signals through the Galpha(q/11) subunits of G proteins to activate phosphatidylinositol signaling and PKCdelta in the murine ST2 cells. Galpha(q/11)-PKCdelta signaling is required for Wnt3a-induced osteoblastogenesis in these cells, and PKCdelta homozygous mutant mice exhibit a deficit in embryonic bone formation. Furthermore, Wnt7b, expressed by osteogenic cells in vivo, induces osteoblast differentiation in vitro via the PKCdelta-mediated pathway; ablation of Wnt7b in skeletal progenitors results in less bone in the mouse embryo. Together, these results reveal a Wnt-dependent osteogenic mechanism, and they provide a potential target pathway for designing therapeutics to promote bone formation.

The Gli2 transcriptional activator is a crucial effector for Ihh signaling in osteoblast development and cartilage vascularization.

Indian hedgehog (Ihh) critically regulates multiple aspects of endochondral bone development. Although it is generally believed that all Ihh functions are mediated by the Gli family of transcription activators and repressors, formal genetic proof for this notion has not been provided. Moreover, the extent to which different Gli proteins contribute to Ihh functions is not fully understood. Previous work has shown that de-repression of the Gli3 repressor is the predominant mode through which Ihh controls chondrocyte proliferation and maturation, but that osteoblast differentiation and hypertrophic cartilage vascularization require additional mechanisms. To test the involvement of Gli2 activation in these processes, we have generated a mouse strain that expresses a constitutive Gli2 activator in a Cre-dependent manner, and have attempted to rescue the Ihh-null mouse with the Gli2 activator, either alone or in combination with Gli3 removal. Here, we report that the Gli2 activator alone is sufficient to induce vascularization of the hypertrophic cartilage in the absence of Ihh but requires simultaneous removal of Gli3 to restore osteoblast differentiation. These results therefore provide direct genetic evidence that Gli2 and Gli3 collectively mediate all major aspects of Ihh function during endochondral skeletal development.

Transient expansion and myofibroblast conversion of adipogenic lineage precursors mediate bone marrow repair after radiation.

Radiation causes a collapse of bone marrow cells and elimination of microvasculature. To understand how bone marrow recovers after radiation, we focused on mesenchymal lineage cells that provide a supportive microenvironment for hematopoiesis and angiogenesis in bone. We recently discovered a nonproliferative subpopulation of marrow adipogenic lineage precursors (MALPs) that express adipogenic markers with no lipid accumulation. Single-cell transcriptomic analysis revealed that MALPs acquire proliferation and myofibroblast features shortly after radiation. Using an adipocyte-specific Adipoq-Cre, we validated that MALPs rapidly and transiently expanded at day 3 after radiation, coinciding with marrow vessel dilation and diminished marrow cellularity. Concurrently, MALPs lost most of their cell processes, became more elongated, and highly expressed myofibroblast-related genes. Radiation activated mTOR signaling in MALPs that is essential for their myofibroblast conversion and subsequent bone marrow recovery at day 14. Ablation of MALPs blocked the recovery of bone marrow vasculature and cellularity, including hematopoietic stem and progenitors. Moreover, VEGFa deficiency in MALPs delayed bone marrow recovery after radiation. Taken together, our research demonstrates a critical role of MALPs in mediating bone marrow repair after radiation injury and sheds light on a cellular target for treating marrow suppression after radiotherapy.

Heterozygous variant in WNT1 gene in two brothers with early onset osteoporosis.

Osteoporosis is a multifactorial disorder characterized by low bone mass and strength, leading to increased risk of fracture. The WNT pathway plays a critical role in bone remodeling by enhancing osteoblastic differentiation, which promotes bone formation, and inhibiting osteoclastic differentiation, decreasing bone resorption. Therefore, genetic alterations of this pathway will lead to impaired bone homeostasis and could contribute to varying response to treatment. We present the case of two brothers with early osteoporosis who were found to have a heterozygous variant of unknown significance in the WNT1 gene, c.1060_1061delCAinsG (p.H354Afs*39). This finding demonstrates that frameshift variants in WNT1 may also act in a dominant fashion leading to decreased bone mass.