Cbfb Regulates Bone Development by Stabilizing Runx Family Proteins

Runx family proteins, Runx1, Runx2, and Runx3, play important roles in skeletal development. Runx2 is required for osteoblast differentiation and chondrocyte maturation, and haplodeficiency of RUNX2 causes cleidocranial dysplasia, which is characterized by open fontanelles and sutures and hypoplastic clavicles. Cbfb forms a heterodimer with Runx family proteins and enhances their DNA‐binding capacity. Cbfb‐deficient (Cbfb^?/?) mice die at midgestation because of the lack of fetal liver hematopoiesis. We previously reported that the partial rescue of hematopoiesis in Cbfb^?/? mice revealed the requirement of Cbfb in skeletal development. However, the precise functions of Cbfb in skeletal development still remain to be clarified. We deleted Cbfb in mesenchymal cells giving rise to both chondrocyte and osteoblast lineages by mating Cbfb^fl/fl mice with Dermo1 Cre knock‐in mice. Cbfb^fl/fl/Cre mice showed dwarfism, both intramembranous and endochondral ossifications were retarded, and chondrocyte maturation and proliferation and osteoblast differentiation were inhibited. The differentiation of chondrocytes and osteoblasts were severely inhibited in vitro, and the reporter activities of Ihh, Col10a1, and Bglap2 promoter constructs were reduced in Cbfb^fl/fl/Cre chondrocytes or osteoblasts. The proteins of Runx1, Runx2, and Runx3 were reduced in the cartilaginous limb skeletons and calvariae of Cbfb^fl/fl/Cre embryos compared with the respective protein in the respective tissue of Cbfb^fl/fl embryos at E15.5, although the reduction of Runx2 protein in calvariae was much milder than that in cartilaginous limb skeletons. All of the Runx family proteins were severely reduced in Cbfb^fl/fl/Cre primary osteoblasts, and Runx2 protein was less stable in Cbfb^fl/fl/Cre osteoblasts than Cbfb^fl/fl osteoblasts. These findings indicate that Cbfb is required for skeletal development by regulating chondrocyte differentiation and proliferation and osteoblast differentiation; that Cbfb plays an important role in the stabilization of Runx family proteins; and that Runx2 protein stability is less dependent on Cbfb in calvariae than in cartilaginous limb skeletons. © 2014 American Society for Bone and Mineral Research.     

Smad4‐Shh‐Nfic signaling cascade–mediated epithelial‐mesenchymal interaction is crucial in regulating tooth root development

Transforming growth factor β (TGF‐β)/bone morphogenetic protein (BMP) signaling is crucial for regulating epithelial‐mesenchymal interaction during organogenesis, and the canonical Smad pathway–mediated TGF‐β/BMP signaling plays important roles during development and disease. During tooth development, dental epithelial cells, known as Hertwig's epithelial root sheath (HERS), participate in root formation following crown development. However, the functional significance of HERS in regulating root development remains unknown. In this study we investigated the signaling mechanism of Smad4, the common Smad for TGF‐β/BMP signaling, in HERS in regulating root development. Tissue‐specific inactivation of Smad4 in HERS results in abnormal enamel and dentin formation in K14‐Cre;Smad4^fl/fl mice. HERS enlarges but cannot elongate to guide root development without Smad4. At the molecular level, Smad4‐mediated TGF‐β/BMP signaling is required for Shh expression in HERS and Nfic (nuclear factor Ic) expression in the cranial neural crest (CNC)‐derived dental mesenchyme. Nfic is crucial for root development, and loss of Nfic results in a CNC‐derived dentin defect similar to the one of K14‐Cre;Smad4^fl/fl mice. Significantly, we show that ectopic Shh induces Nfic expression in dental mesenchyme and partially rescues root development in K14‐Cre;Smad4^fl/fl mice. Taken together, our study has revealed an important signaling mechanism in which TGF‐β/BMP signaling relies on a Smad‐dependent mechanism in regulating Nfic expression via Shh signaling to control root development. The interaction between HERS and the CNC‐derived dental mesenchyme may guide the size, shape, and number of tooth roots. © 2010 American Society for Bone and Mineral Research     

Wnt-mediated reciprocal regulation between cartilage and bone development during endochondral ossification

The role of Wnt signaling is extensively studied in skeletal development and postnatal bone remodeling, mostly based on the genetic approaches of β-catenin manipulation. However, given their independent function, a requirement for β-catenin is not the same as that for Wnt. Here, we investigated the effect of Wnt proteins in both tissues through generating cartilage- or bone-specific Wls null mice, respectively. Depletion of Wls by Col2-Cre, which would block Wnt secretion in the chondrocytes and perichondrium, delayed chondrocyte hypertrophy in the growth plate and impaired perichondrial osteogenesis. Loss of Wls in chondrocytes also disturbed the proliferating chondrocyte morphology and division orientation, which was similar to the defect observed in Wnt5a null mice. On the other hand, inactivation of Wls in osteoblasts by Col1-Cre resulted in a shorter hypertrophic zone and an increase of TRAP positive cell number in the chondro-osseous junction of growth plate, coupled with a decrease in bone mass. Taken together, our studies reveal that Wnt proteins not only modulate differentiation and cellular communication within populations of chondrocytes, but also mediate the cross regulation between the chondrocytes and osteoblasts in growth plate.     

Macrophage subpopulations and their impact on chronic allograft rejection versus graft acceptance in a mouse heart transplant model

Macrophages infiltrating the allografts are heterogeneous, consisting of proinflammatory (M1 cells) as well as antiinflammatory and fibrogenic phenotypes (M2 cells); they affect transplantation outcomes via diverse mechanisms. Here we found that macrophage polarization into M1 and M2 subsets was critically dependent on tumor necrosis factor receptor–associated factor 6 (TRAF6) and mammalian target of rapamycin (mTOR), respectively. In a heart transplant model we showed that macrophage‐specific deletion of TRAF6 (LysM^CreTraf6 ^fl/fl) or mTOR (LysM^CreMtor^fl/fl) did not affect acute allograft rejection. However, treatment of LysM^CreMtor^fl/fl recipients with CTLA4‐Ig induced long‐term allograft survival (>100 days) without histological signs of chronic rejection, whereas the similarly treated LysM^CreTraf6 ^fl/fl recipients developed severe transplant vasculopathy (chronic rejection). The presentation of chronic rejection in CTLA4‐Ig–treated LysM^CreTraf6 ^fl/fl mice was similar to that of CTLA4‐Ig–treated wild‐type B6 recipients. Mechanistically, we found that the graft‐infiltrating macrophages in LysM^CreMtor^fl/fl recipients expressed high levels of PD‐L1, and that PD‐L1 blockade readily induced rejection of otherwise survival grafts in the LysM^CreMtor^fl/fl recipients. Our findings demonstrate that targeting mTOR‐dependent M2 cells is critical for preventing chronic allograft rejection, and that graft survival under such conditions is dependent on the PD‐1/PD‐L1 coinhibitory pathway.     

Effects of Deletion of ERα in Osteoblast‐Lineage Cells on Bone Mass and Adaptation to Mechanical Loading Differ in Female and Male Mice

Estrogen receptor alpha (ERα) has been implicated in bone's response to mechanical loading in both males and females. ERα in osteoblast lineage cells is important for determining bone mass, but results depend on animal sex and the cellular stage at which ERα is deleted. We demonstrated previously that when ERα is deleted from mature osteoblasts and osteocytes in mixed‐background female mice, bone mass and strength are decreased. However, few studies exist examining the skeletal response to loading in bone cell–specific ERαKO mice. Therefore, we crossed ERα floxed (ERα^fl/fl) and osteocalcin‐Cre (OC‐Cre) mice to generate animals lacking ERα in mature osteoblasts and osteocytes (pOC‐ERαKO) and littermate controls (LC). At 10 weeks of age, the left tibia was loaded in vivo for 2 weeks. We analyzed bone mass through micro‐CT, bone formation rate by dynamic histomorphometry, bone strength from mechanical testing, and osteoblast and osteoclast activity by serum chemistry and immunohistochemistry. ERα in mature osteoblasts differentially regulated bone mass in males and females. Compared with LC, female pOC‐ERαKO mice had decreased cortical and cancellous bone mass, whereas male pOC‐ERαKO mice had equal or greater bone mass than LC. Bone mass results correlated with decreased compressive strength in pOC‐ERαKO female L₅ vertebrae and with increased maximum moment in pOC‐ERαKO male femora. Female pOC‐ERαKO mice responded more to mechanical loading, whereas the response of pOC‐ERαKO male animals was similar to their littermate controls. © 2015 American Society for Bone and Mineral Research. © 2015 American Society for Bone and Mineral Research.     

Conditional Loss of Nmp4 in Mesenchymal Stem Progenitor Cells Enhances PTH-Induced Bone Formation

Activation of bone anabolic pathways is a fruitful approach for treating severe osteoporosis, yet FDA-approved osteoanabolics, eg, parathyroid hormone (PTH), have limited efficacy. Improving their potency is a promising strategy for maximizing bone anabolic output. Nmp4 (Nuclear Matrix Protein 4) global knockout mice exhibit enhanced PTH-induced increases in trabecular bone but display no overt baseline skeletal phenotype. Nmp4 is expressed in all tissues; therefore, to determine which cell type is responsible for driving the beneficial effects of Nmp4 inhibition, we conditionally removed this gene from cells at distinct stages of osteogenic differentiation. Nmp4-floxed (Nmp4^fl/fl) mice were crossed with mice bearing one of three Cre drivers including (i) Prx1Cre⁺ to remove Nmp4 from mesenchymal stem/progenitor cells (MSPCs) in long bones; (ii) BglapCre⁺ targeting mature osteoblasts, and (iii) Dmp1Cre⁺ to disable Nmp4 in osteocytes. Virgin female Cre⁺ and Cre⁻ mice (10 weeks of age) were sorted into cohorts by weight and genotype. Mice were administered daily injections of either human PTH 1-34 at 30 μg/kg or vehicle for 4 weeks or 7 weeks. Skeletal response was assessed using dual-energy X-ray absorptiometry, micro-computed tomography, bone histomorphometry, and serum analysis for remodeling markers. Nmp4^fl/fl;Prx1Cre⁺ mice virtually phenocopied the global Nmp4^−/− skeleton in the femur, ie, a mild baseline phenotype but significantly enhanced PTH-induced increase in femur trabecular bone volume/total volume (BV/TV) compared with their Nmp4^fl/fl;Prx1Cre⁻ controls. This was not observed in the spine, where Prrx1 is not expressed. Heightened response to PTH was coincident with enhanced bone formation. Conditional loss of Nmp4 from the mature osteoblasts (Nmp4^fl/fl;BglapCre⁺) failed to increase BV/TV or enhance PTH response. However, conditional disabling of Nmp4 in osteocytes (Nmp4^fl/fl;Dmp1Cre⁺) increased BV/TV without boosting response to hormone under our experimental regimen. We conclude that Nmp4^−/− Prx1-expressing MSPCs drive the improved response to PTH therapy and that this gene has stage-specific effects on osteoanabolism. © 2022 American Society for Bone and Mineral Research (ASBMR).     

RECQL4 Regulates p53 Function In Vivo During Skeletogenesis

RECQ DNA helicases play critical roles in maintaining genomic stability, but their role in development has been less well studied. Rothmund‐Thomson syndrome, RAPADILINO, and Baller‐Gerold syndrome are rare genetic disorders caused by mutations in the RECQL4 gene. These patients have significant skeletal developmental abnormalities including radial ray, limb and craniofacial defects. To investigate the role of Recql4 in the developing skeletal system, we generated Recql4 conditional knockout mice targeting the skeletal lineage. Inactivation of Recql4 using the Prx1‐Cre transgene led to limb abnormalities and craniosynostosis mimicking the major bone findings in human RECQL4 patients. These Prx1‐Cre⁺;Recql4^fl/fl mice as well as Col2a1‐Cre⁺;Recql4^fl/fl mice exhibited growth plate defects and an increased p53 response in affected tissues. Inactivation of Trp53 in these Recql4 mutants resulted in genetic rescue of the skeletal phenotypes, indicating an in vivo interaction between Recql4 and Trp53, and p53 activation as an underlying mechanism for the developmental bone abnormalities in RECQL4 disorders. Our findings show that RECQL4 is critical for skeletal development by modulating p53 activity in vivo. © 2015 American Society for Bone and Mineral Research     

Nedd4 Deficiency in Vascular Smooth Muscle Promotes Vascular Calcification by Stabilizing pSmad1

The nonosseous calcification process such as atherosclerosis is one of the major complications in several types of metabolic diseases. In a previous study, we uncovered that aberrant activity of transforming growth factor β (TGF‐β) signaling pathway could contribute to the vascular smooth muscle cells’ (VSMCs) calcification process. Also, we identified NEDD4 E3 ligase as a key suppressor of bone morphogenetic protein (BMP)/Smad pathway via a polyubiquitination‐dependent selective degradation of C‐terminal phosphorylated Smad1 (pSmad1) activated by TGF‐β. Here, we further validated and confirmed the role of Nedd4 in in vivo vascular calcification progression. First, Nedd4 deletion in SM22α‐positive mouse tissues (Nedd4^fl/fl;SM22α‐Cre) showed deformed aortic structures with disarranged elastin fibers at 24 weeks after birth. Second, vitamin D–induced aorta vascular calcification rate in Nedd4^fl/fl;SM22α‐Cre mice was significantly higher than their wild‐type littermates. Nedd4^fl/fl;SM22α‐Cre mice showed a development of vascular calcification even at very low‐level injection of vitamin D, but this was not exhibited in wild‐type littermates. Third, we confirmed that TGF‐β1–induced pSmad1 levels were elevated in Nedd4‐deficient primary VSMCs isolated from Nedd4^fl/fl;SM22α‐Cre mice. Fourth, we further found that Nedd4^fl/fl;SM22α‐Cre mVSMCs gained mesenchymal cell properties toward osteoblast‐like differentiation by a stable isotope labeling in cell culture (SILAC)‐based proteomics analysis. Finally, epigenetic analysis revealed that methylation levels of human NEDD4 gene promoter were significantly increased in atherosclerosis patients. Collectively, abnormal expression or dysfunction of Nedd4 E3 ligase could be involved in vascular calcification of VSMCs by activating bone‐forming signals during atherosclerosis progression. © 2016 American Society for Bone and Mineral Research.     

Wnt inhibitors Dkk1 and Sost are downstream targets of BMP signaling through the type IA receptor (BMPRIA) in osteoblasts

The bone morphogenetic protein (BMP) and Wnt signaling pathways both contribute essential roles in regulating bone mass. However, the molecular interactions between these pathways in osteoblasts are poorly understood. We recently reported that osteoblast‐targeted conditional knockout (cKO) of BMP receptor type IA (BMPRIA) resulted in increased bone mass during embryonic development, where diminished expression of Sost as a downstream effector of BMPRIA resulted in increased Wnt/β‐catenin signaling. Here, we report that Bmpr1a cKO mice exhibit increased bone mass during weanling stages, again with evidence of enhanced Wnt/β‐catenin signaling as assessed by Wnt reporter TOPGAL mice and TOPFLASH luciferase. Consistent with negative regulation of the Wnt pathway by BMPRIA signaling, treatment of osteoblasts with dorsomorphin, an inhibitor of Smad‐dependent BMP signaling, enhanced Wnt signaling. In addition to Sost, Wnt inhibitor Dkk1 also was downregulated in cKO bone. Expression levels of Dkk1and Sost were upregulated by BMP2 treatment and downregulated by Noggin. Moreover, expression of a constitutively active Bmpr1a transgene in mice resulted in the upregulation of both Dkk1 and Sost and partially rescued the Bmpr1a cKO bone phenotype. These effectors are differentially regulated by mitogen‐activated protein kinase (MAPK) p38 because pretreatment of osteoblasts with SB202190 blocked BMP2‐induced Dkk1 expression but not Sost. These results demonstrate that BMPRIA in osteoblasts negatively regulates endogenous bone mass and Wnt/β‐catenin signaling and that this regulation may be mediated by the activities of Sost and Dkk1. This study highlights several interactions between BMP and Wnt signaling cascades in osteoblasts that may be amenable to therapeutic intervention for the modification of bone mass density. © 2010 American Society for Bone and Mineral Research     

The Development of Dentin Microstructure Is Controlled by the Type of Adjacent Epithelium

Considerable amount of research has been focused on dentin mineralization, odontoblast differentiation, and their application in dental tissue engineering. However, very little is known about the differential role of functionally and spatially distinct types of dental epithelium during odontoblast development. Here we show morphological and functional differences in dentin located in the crown and roots of mouse molar and analogous parts of continuously growing incisors. Using a reporter (DSPP-cerulean/DMP1-cherry) mouse strain and mice with ectopic enamel (Spry2^+/−;Spry4^−/−), we show that the different microstructure of dentin is initiated in the very beginning of dentin matrix production and is maintained throughout the whole duration of dentin growth. This phenomenon is regulated by the different inductive role of the adjacent epithelium. Thus, based on the type of interacting epithelium, we introduce more generalized terms for two distinct types of dentins: cementum versus enamel-facing dentin. In the odontoblasts, which produce enamel-facing dentin, we identified uniquely expressed genes (Dkk1, Wisp1, and Sall1) that were either absent or downregulated in odontoblasts, which form cementum-facing dentin. This suggests the potential role of Wnt signalling on the dentin structure patterning. Finally, we show the distribution of calcium and magnesium composition in the two developmentally different types of dentins by utilizing spatial element composition analysis (LIBS). Therefore, variations in dentin inner structure and element composition are the outcome of different developmental history initiated from the very beginning of tooth development. Taken together, our results elucidate the different effects of dental epithelium, during crown and root formation on adjacent odontoblasts and the possible role of Wnt signalling which together results in formation of dentin of different quality. © 2021 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).     

Connexin43 and Runx2 Interact to Affect Cortical Bone Geometry,Skeletal Development,and Osteoblast and Osteoclast Function

The coupling of osteoblasts and osteocytes by connexin43 (Cx43) gap junctions permits the sharing of second messengers that coordinate bone cell function and cortical bone acquisition. However, details of how Cx43 converts shared second messengers into signals that converge onto essential osteogenic processes are incomplete. Here, we use in vitro and in vivo methods to show that Cx43 and Runx2 functionally interact to regulate osteoblast gene expression and proliferation, ultimately affecting cortical bone properties. Using compound hemizygous mice for the Gja1 (Cx43) and Runx2 genes, we observed a skeletal phenotype not visible in wild‐type or singly hemizygous animals. Cortical bone analysis by micro–computed tomography (μCT) revealed that 8‐week‐old male, compound Gja1^+/– Runx2^+/– mice have a marked increase in cross‐sectional area, endosteal and periosteal bone perimeter, and an increase in porosity compared to controls. These compound Gja1^+/– Runx2^+/– mice closely approximate the cortical bone phenotypes seen in osteoblast‐specific Gja1‐conditional knockout models. Furthermore, μCT analysis of skulls revealed an altered interparietal bone geometry in compound hemizygotes. Consistent with this finding, Alizarin red/Alcian blue staining of 2‐day‐old Gja1^+/– Runx2^+/– neonates showed a hypomorphic interparietal bone, an exacerbation of the open fontanelles, and a further reduction in the hypoplastic clavicles compared to Runx2^+/– neonates. Expression of osteoblast genes, including osteocalcin, osterix, periostin, and Hsp47, was markedly reduced in tibial RNA extracts from compound hemizygous mice, and osteoblasts from compound hemizygous mice exhibited increased proliferative capacity. Further, the reduced osteocalcin expression and hyperproliferative nature of osteoblasts from Cx43 deficient mice was rescued by Runx2 expression. In summary, these findings provide evidence that Cx43 and Runx2 functionally intersect in vivo to regulate cortical bone properties and affect osteoblast differentiation and proliferation, and likely contributes to aspects of the skeletal phenotype of Cx43 conditional knockout mice. © 2017 American Society for Bone and Mineral Research     

C5aR1 regulates migration of suppressive myeloid cells required for costimulatory blockade‐induced murine allograft survival

Costimulatory blockade‐induced murine cardiac allograft survival requires intragraft accumulation of CD11b⁺Ly6C^loLy6G^? regulatory myeloid cells (Mregs) that expand regulatory T cells (Tregs) and suppress effector T cells (Teffs). We previously showed that C5a receptor (C5aR1) signaling on T cells activates Teffs and inhibits Tregs, but whether and/or how C5aR1 affects Mregs required for transplant survival is unknown. Although BALB/c hearts survived >60 days in anti‐CD154 (MR1)‐treated or cytotoxic T‐lymphocyte associated protein 4 (CTLA4)‐Ig–treated wild‐type (WT) recipients, they were rejected at ~30 days in MR1‐treated or CTLA4‐Ig–treated recipients selectively deficient in C5aR1 restricted to myeloid cells (C5ar1^fl/flxLysM‐Cre). This accelerated rejection was associated with ~2‐fold more donor‐reactive T cells and ~40% less expansion of donor‐reactive Tregs. Analysis of graft‐infiltrating mononuclear cells on posttransplant day 6 revealed fewer Ly6C^lo monocytes in C5ar1^fl/flxLysM‐Cre recipients. Expression profiling of intragraft Ly6C^lo monocytes showed that C5aR1 deficiency downregulated genes related to migration/locomotion without changes in genes associated with suppressive function. Cotransfer of C5ar1^fl/fl and C5ar1^fl/flxLysM‐Cre myeloid cells into MR1‐treated allograft recipients resulted in less accumulation of C5ar1^?/? cells within the allografts, and in vitro assays confirmed that Ly6C^hi myeloid cells migrate to C5a/C5aR1‐initiated signals. Together, our results newly link myeloid cell–expressed C5aR1 to intragraft accumulation of myeloid cells required for prolongation of heart transplant survival induced by costimulatory blockade.     

Conditional deletion of Bmpr1a in differentiated osteoclasts increases osteoblastic bone formation,increasing volume of remodeling bone in mice

Bone undergoes remodeling consisting of osteoclastic bone resorption followed by osteoblastic bone formation throughout life. Although the effects of bone morphogenetic protein (BMP) signals on osteoblasts have been studied extensively, the function of BMP signals in osteoclasts has not been fully elucidated. To delineate the function of BMP signals in osteoclasts during bone remodeling, we deleted BMP receptor type IA (Bmpr1a) in an osteoclast‐specific manner using a knock‐in Cre mouse line to the cathepsin K locus (Ctsk^Cre/+;Bmpr1a^flox/flox, designated as Bmpr1a^ΔOc/ΔOc). Cre was specifically expressed in multinucleated osteoclasts in vivo. Cre‐dependent deletion of the Bmpr1a gene occurred at 4 days after cultivation of bone marrow macrophages obtained from Bmpr1a^ΔOc/ΔOc with RANKL. These results suggested that Bmpr1a was deleted after formation of osteoclasts in Bmpr1a^ΔOc/ΔOc mice. Expression of bone‐resorption markers increased, thus suggesting that BMPRIA signaling negatively regulates osteoclast differentiation. Trabeculae in tibia and femurs were thickened in 3.5‐, 8‐, and 12‐week‐old Bmpr1a^ΔOc/ΔOc mice. Bone histomorphometry revealed increased bone volume associated with increased osteoblastic bone‐formation rates (BFR) in the remodeling bone of the secondary spongiosa in Bmpr1a^ΔOc/ΔOc tibias at 8 weeks of age. For comparison, we also induced an osteoblast‐specific deletion of Bmpr1a using Col1a1‐Cre. The resulting mice showed increased bone volume with marked decreases in BFR in tibias at 8 weeks of age. These results indicate that deletion of Bmpr1a in differentiated osteoclasts increases osteoblastic bone formation, thus suggesting that BMPR1A signaling in osteoclasts regulates coupling to osteoblasts by reducing bone‐formation activity during bone remodeling. © 2011 American Society for Bone and Mineral Research