Guochun Zhu

C/EBPα regulates osteoclast lineage commitment

Despite recent insights gained from the effects of targeted deletion of the Finkel-Biskis-Jinkins osteosarcoma oncogene (c-fos), Spleen focus-forming virus (SFFV) proviral integration 1 (PU.1), microphthalmia-associated transcription factor, NF-κB, and nuclear factor of activated cells cytoplasmic 1 (NFATc1) transcription factor genes, the mechanism underlying transcription factors specifying osteoclast (OC) lineage commitment from monocyte/macrophage remains unclear. To characterize the mechanism by which transcription factors regulate OC lineage commitment, we mapped the critical cis-regulatory element in the promoter of cathepsin K (Ctsk), which is expressed specifically in OCs, and found that CCAAT/enhancer binding protein α (C/EBPα) is the critical cis-regulatory element binding protein. Our results indicate that C/EBPα is highly expressed in pre- OCs and OCs. The combined presence of macrophage colony-stimulating factor and receptor activator of NF-κB ligand significantly induces high C/EBPα expression. Furthermore, C/EBPα−/− newborn mice exhibited impaired osteoclastogenesis, and a severe osteopetrotic phenotype, but unaffected monocyte/macrophage development. Impaired osteoclastogenesis of C/EBPα−/− mouse bone marrow cells can be rescued by c-fos overexpression. Ectopic expression of C/EBPα in mouse bone marrow cells and monocyte/macrophage cells, in the absence of receptor activator of NF-κB ligand, induces expression of receptor activator of NF-κB, c-fos, Nfatc1, and Ctsk, and it reprograms monocyte/macrophage cells to OC-like cells. Our results demonstrate that C/EBPα directly up-regulates c-fos expression. C/EBPα+/− mice exhibit an increase in bone density compared with C/EBPα+/+ controls. These discoveries establish C/EBPα as the key transcriptional regulator of OC lineage commitment, providing a unique therapeutic target for diseases of excessive bone resorption, such as osteoporosis and arthritis.

Cbfβ deletion in mice recapitulates cleidocranial dysplasia and reveals multiple functions of Cbfβ required for skeletal development

Significance Cleidocranial dysplasia (CCD) is a hereditary human skeletal disease. Mutations in Runt-related transcription factor 2, which functions as a heterodimer with core binding factor β (Cbfβ), are found in most individuals with CCD. It has been suspected that Cbfβ may be responsible for other CCD cases. The pathogenesis of CCD and the role of Cbfβ in postnatal skeletogenesis remain unclear. There has been no animal model to study this disease. We demonstrate that ablation of Cbfβ in various skeletal cells results in severe craniofacial and skeletal dysplasia with the phenotype recapitulating clinical features of CCD. The findings from this study of Cbfβ in the skeleton provide insight into the role of Cbfβ in postnatal skeletogenesis and pathogenesis of CCD, which may assist in developing new therapies for CCD and osteoporosis. The pathogenesis of cleidocranial dysplasia (CCD) as well as the specific role of core binding factor β (Cbfβ) and the Runt-related transcription factor (RUNX)/Cbfβ complex in postnatal skeletogenesis remain unclear. We demonstrate that Cbfβ ablation in osteoblast precursors, differentiating chondrocytes, osteoblasts, and odontoblasts via Osterix-Cre, results in severe craniofacial dysplasia, skeletal dysplasia, abnormal teeth, and a phenotype recapitulating the clinical features of CCD. Cbfβf/fOsterix-Cre mice have fewer proliferative and hypertrophic chondrocytes, fewer osteoblasts, and almost absent trabecular bone, indicating that Cbfβ may maintain trabecular bone formation through its function in hypertrophic chondrocytes and osteoblasts. Cbfβf/fCollagen, type 1, alpha 1 (Col1α1)–Cre mice show decreased bone mineralization and skeletal deformities, but no radical deformities in teeth, mandibles, or cartilage, indicating that osteoblast lineage-specific ablation of Cbfβ results in milder bone defects and less resemblance to CCD. Activating transcription factor 4 (Atf4) and Osterix protein levels in both mutant mice are dramatically reduced. ChIP assays show that Cbfβ directly associates with the promoter regions of Atf4 and Osterix. Our data further demonstrate that Cbfβ highly up-regulates the expression of Atf4 at the transcriptional regulation level. Overall, our genetic dissection approach revealed that Cbfβ plays an indispensable role in postnatal skeletal development and homeostasis in various skeletal cell types, at least partially by up-regulating the expression of Atf4 and Osterix. It also revealed that CCD may result from functional defects of the Runx2/Cbfβ heterodimeric complex in various skeletal cells. These insights into the role of Cbfβ in postnatal skeletogenesis and CCD pathogenesis may assist in the development of new therapies for CCD and osteoporosis.

Inhibiting Periapical Lesions through AAV-RNAi Silencing of Cathepsin K

Dental caries, one of the most prevalent infectious diseases worldwide, affects approximately 80% of children and the majority of adults. Dental caries may result in endodontic disease, leading to dental pulp necrosis, periapical inflammation and bone resorption, severe pain, and tooth loss. Periapical inflammation may also increase inflammation in other parts of the body. Although many studies have attempted to develop therapies for this disease, there is still an urgent need for effective treatments. In this study, we applied a novel gene therapeutic approach using recombinant adeno-associated virus (AAV)-mediated RNAi knockdown of Cathepsin K (Ctsk) gene expression, to target osteoclasts and periapical bone resorption in a mouse model. We found that AAV-sh-Cathepsin K (AAV-sh-Ctsk) impaired osteoclast function in vivo and furthermore reduced bacterial infection-stimulated bone resorption by 88%. Reduced periapical lesion size was accompanied by decreases in mononuclear leukocyte infiltration and inflammatory cytokine expression. Our study shows that AAV-RNAi silencing of Cathepsin K in periapical tissues can significantly reduce endodontic disease development, bone destruction, and inflammation in the periapical lesion. This is the first demonstration that AAV-mediated RNAi knockdown gene therapy may significantly reduce the severity of endodontic disease.

Core Binding Factor Beta (Cbfβ) Controls the Balance of Chondrocyte Proliferation and Differentiation by Upregulating Indian hedgehog (Ihh) Expression and Inhibiting Parathyroid Hormone-Related Protein Receptor (PPR) Expression in Postnatal Cartilage and Bone Formation

Core binding factor beta (Cbfβ) is essential for embryonic bone morphogenesis. Yet the mechanisms by which Cbfβ regulates chondrocyte proliferation and differentiation as well as postnatal cartilage and bone formation remain unclear. Hence, using paired‐related homeobox transcription factor 1‐Cre (Prx1‐Cre) mice, mesenchymal stem cell–specific Cbfβ‐deficient (Cbfβf/fPrx1‐Cre) mice were generated to study the role of Cbfβ in postnatal cartilage and bone development. These mutant mice survived to adulthood but exhibited severe sternum and limb malformations. Sternum ossification was largely delayed in the Cbfβf/fPrx1‐Cre mice and the xiphoid process was noncalcified and enlarged. In newborn and 7‐day‐old Cbfβf/fPrx1‐Cre mice, the resting zone was dramatically elongated, the proliferation zone and hypertrophic zone of the growth plates were drastically shortened and disorganized, and trabecular bone formation was reduced. Moreover, in 1‐month‐old Cbfβf/fPrx1‐Cre mice, the growth plates were severely deformed and trabecular bone was almost absent. In addition, Cbfβ deficiency impaired intramembranous bone formation both in vivo and in vitro. Interestingly, although the expression of Indian hedgehog (Ihh) was largely reduced, the expression of parathyroid hormone‐related protein (PTHrP) receptor (PPR) was dramatically increased in the Cbfβf/fPrx1‐Cre growth plate, indicating that that Cbfβ deficiency disrupted the Ihh‐PTHrP negative regulatory loop. Chromatin immunoprecipitation (ChIP) analysis and promoter luciferase assay demonstrated that the Runx/Cbfβ complex binds putative Runx‐binding sites of the Ihh promoter regions, and also the Runx/Cbfβ complex directly upregulates Ihh expression at the transcriptional level. Consistently, the expressions of Ihh target genes, including CyclinD1, Ptc, and Pthlh, were downregulated in Cbfβ‐deficient chondrocytes. Taken together, our study reveals not only that Cbfβ is essential for chondrocyte proliferation and differentiation for the growth and maintenance of the skeleton in postnatal mice, but also that it functions in upregulating Ihh expression to promoter chondrocyte proliferation and osteoblast differentiation, and inhibiting PPR expression to enhance chondrocyte differentiation.

Deletion of core-binding factor β (Cbfβ) in mesenchymal progenitor cells provides new insights into Cbfβ/Runxs complex function in cartilage and bone development.

Core-binding factor β (Cbfβ) is a subunit of the Cbf family of heterodimeric transcription factors, which plays a critical role in skeletal development through its interaction with the Cbfα subunits, also known as Runt-related transcription factors (Runxs). However, the mechanism by which Cbfβ regulates cartilage and bone development remains unclear. Existing Cbfβ-deficient mouse models cannot specify the role of Cbfβ in skeletal cell lineage. Herein, we sought to specifically address the role of Cbfβ in cartilage and bone development by using a conditional knockout (CKO) approach. A mesenchymal-specific Cbfβ CKO mouse model was generated by using the Dermo1-Cre mouse line to specifically delete Cbfβ in mesenchymal stem cells, which give rise to osteoblasts and chondrocytes. Surprisingly, the mutant mice had under-developed larynx and tracheal cartilage, causing alveolus defects that led to death shortly after birth from suffocation. Also, the mutant mice exhibited severe skeletal deformities from defective intramembranous and endochondral ossification, owing to delayed chondrocyte maturation and impaired osteoblast differentiation. Almost all bones of the mutant mice, including the calvariae, vertebrae, tibiae, femurs, ribs, limbs and sternums were defective. Importantly, we showed that Cbfβ was expressed throughout the skeleton during both embryonic and postnatal development, which explains the multiple-skeletal defects observed in the mutant mice. Consistently, Cbfβ deficiency impaired both chondrocyte proliferation and hypertrophy zone hypertrophy during growth-plate development in the long bones of mutant mice. Notably, Cbfβ, Runx1 and Runx2 displayed different expression patterns in the growth plates of the wild-type mice, indicating that Cbfβ/Runx1 complex and Cbfβ/Runx2 complex may regulate chondrocyte proliferation and hypertrophy, respectively, in a spatial and temporal manner. Cbfβ deletion in the mesenchymal progenitors affected bone development by dramatically down-regulating Collagen X (Col X) and Osterix (Osx) but had a dispensable effect on osteoclast development. Collectively, the results demonstrate that Cbfβ mediates cartilage and bone development by interacting with Runx1 and Runx2 to regulate the expressions of Col X and Osx for chondrocyte and osteoblast development. These findings not only reveal a critical role for Cbfβ in cartilage and bone development but also facilitate the design of novel therapeutic approaches for skeletal diseases.

RNA Interference-Mediated Silencing of Atp6i Prevents Both Periapical Bone Erosion and Inflammation in the Mouse Model of Endodontic Disease

ABSTRACT Dental caries is one of the most prevalent infectious diseases in the United States, affecting approximately 80% of children and the majority of adults. Dental caries may lead to endodontic disease, where the bacterial infection progresses to the root canal system of the tooth, leading to periapical inflammation, bone erosion, severe pain, and tooth loss. Periapical inflammation may also exacerbate inflammation in other parts of the body. Although conventional clinical therapies for this disease are successful in approximately 80% of cases, there is still an urgent need for increased efficacy of treatment. In this study, we applied a novel gene-therapeutic approach using recombinant adeno-associated virus (AAV)-mediated Atp6i RNA interference (RNAi) knockdown of Atp6i/TIRC7 gene expression to simultaneously target periapical bone resorption and periapical inflammation. We found that Atp6i inhibition impaired osteoclast function in vitro and in vivo and decreased the number of T cells in the periapical lesion. Notably, AAV-mediated Atp6i/TIRC7 knockdown gene therapy reduced bacterial infection-stimulated bone resorption by 80% in the mouse model of endodontic disease. Importantly, Atp6i +/− mice with haploinsufficiency of Atp6i exhibited protection similar to that in mice with bacterial infection-stimulated bone erosion and periapical inflammation, which confirms the potential therapeutic effect of AAV-small hairpin RNA (shRNA)-Atp6i/TIRC7. Our results demonstrate that AAV-mediated Atp6i/TIRC7 knockdown in periapical tissues can inhibit endodontic disease development, bone resorption, and inflammation, indicating for the first time that this potential gene therapy may significantly improve the health of those who suffer from endodontic disease.

RNAi-Mediated Silencing of Atp6i and Atp6i Haploinsufficiency Prevents Both Bone Loss and Inflammation in a Mouse Model of Periodontal Disease

Periodontal disease affects about 80% of adults in America, and is characterized by oral bacterial infection-induced gingival inflammation, oral bone resorption, and tooth loss. Periodontitis is also associated with other diseases such as rheumatoid arthritis, diabetes, and heart disease. Although many efforts have been made to develop effective therapies for this disease, none have been very effective and there is still an urgent need for better treatments and preventative strategies. Herein we explored for the first time the possibility that adeno-associated virus (AAV)-mediated RNAi knockdown could be used to treat periodontal disease with improved efficacy. For this purpose, we used AAV-mediated RNAi knockdown of Atp6i/TIRC7 gene expression to target bone resorption and gingival inflammation simultaneously. Mice were infected with the oral pathogen Porphyromonas gingivalis W50 (P. gingivalis) in the maxillary periodontium to induce periodontitis. We found that Atp6i depletion impaired extracellular acidification and osteoclast-mediated bone resorption. Furthermore, local injection of AAV-shRNA-Atp6i/TIRC7 into the periodontal tissues in vivo protected mice from P. gingivalis infection-stimulated bone resorption by >85% and decreased the T-cell number in periodontal tissues. Notably, AAV-mediated Atp6i/TIRC7 knockdown also reduced the expression of osteoclast marker genes and inflammation-induced cytokine genes. Atp6i+/− mice with haploinsufficiency were similarly protected from P. gingivalis infection-stimulated bone loss and gingival inflammation. This suggests that AAV-shRNA-Atp6i/TIRC7 therapeutic treatment may significantly improve the health of millions who suffer from P. gingivalis-mediated periodontal disease.

Silencing of Atp6v1c1 Prevents Breast Cancer Growth and Bone Metastasis

Previous studies have shown that Atp6v1c1, a regulator of the assembly of the V0 and V1 domains of the V-ATPase complex, is up-regulated in metastatic oral tumors. Despite these studies, the function of Atp6v1c1 in tumor growth and metastasis is still unknown. Atp6v1c1s expression in metastatic oral squamous cell carcinoma indicates that Atp6v1c1 has an important function in cancer growth and metastasis. We hypothesized that elevated expression of Atp6v1c1 is essential to cancer growth and metastasis and that Atp6v1c1 promotes cancer growth and metastasis through activation of V-ATPase activity. To test this hypothesis, a Lentivirus-mediated RNAi knockdown approach was used to study the function of Atp6v1c1 in mouse 4T1 mammary tumor cell proliferation and migration in vitro and cancer growth and metastasis in vivo. Our data revealed that silencing of Atp6v1c1 in 4T1 cancer cells inhibited lysosomal acidification and severely impaired 4T1 cell growth, migration, and invasion through Matrigel in vitro. We also show that Atp6v1c1 knockdown with Lenti-c1s3, a lentivirus targeting Atp6v1c1 for shRNA mediated knockdown, can significantly inhibit 4T1 xenograft tumor growth, metastasis, and osteolytic lesions in vivo. Our study demonstrates that Atp6v1c1 may promote breast cancer growth and bone metastasis through regulation of lysosomal V-ATPase activity, indicating that Atp6v1c1 may be a viable target for breast cancer therapy and silencing of Atp6v1c1 may be an innovative therapeutic approach for the treatment and prevention of breast cancer growth and metastasis.

Chondrocyte-specific Knockout of Cbfβ Reveals the Indispensable Function of Cbfβ in Chondrocyte Maturation, Growth Plate Development and Trabecular Bone Formation in Mice

Despite years of research into bone formation, the mechanisms by which transcription factors specify growth plate development and trabecular bone formation remain unclear and the role of hypertrophic chondrocytes in trabeculae morphogenesis is controversial. To study the role of Core binding factor beta (Cbfβ) in postnatal cartilage development and endochondral bone formation, we generated chondrocyte-specific Cbfβ-deficient mice (Cbfβf/fCol2α1-Cre mice) using floxed alleles of Cbfβ (Cbfβf/f) and Cre driven by the Collagen 2α1 promoter (Col2α1-Cre). Cbfβf/fCol2α1-Cre mice evaded developmental and newborn lethality to survive to adulthood and displayed severe skeletal malformation. Cbfβf/fCol2α1-Cre mice had dwarfism, hypoplastic skeletons, defective bone mineralization, shortened limbs, shortened sternum bodies, and un-calcified occipital bones and hyoid bones. In the long bone cartilage, the resting zone was elongated, and chondrocyte proliferation and hypertrophy were impaired in Cbfβf/fCol2α1-Cre mice, which led to deformation of the growth plates. Primary spongiosa formation was delayed, diaphysis was shortened and trabecular bone formation was almost absent in the mutant mice. In addition, lamellar bone formation in the secondary spongiosa was also impaired. However, osteoclast formation in the trabecular bone was not affected. Cbfβ deficiency led to down-regulation of chondrocyte-regulating genes [i.e, patched (Ptc1), Cyclin D1 and Indian hedgehog (Ihh)] in the cartilage. Interestingly, the expression of Runx2 and Runx3 was not changed in the cartilage of the mutants. Collectively, the results revealed that Cbfβ is crucial for postnatal skeletal development and endochondral bone formation through its function in growth plate development and chondrocyte proliferation and differentiation. This study also revealed that chondrocyte maturation, mediated by Cbfβ, was critical to trabecular bone morphogenesis. Significantly, these findings provide insight into the role of Cbfβ in postnatal skeletogenesis, which may assist in the development of new therapies for osteoporosis.

G Protein and its signaling pathway in bone development and disease.

G protein signaling is comprised of G protein coupled receptors (GPCRs) that detect ligands or sense cations, heterotrimeric G proteins, and downstream effectors and regulators. G protein signaling plays important roles in bone development, remodeling, and disease. In human cases, mutations of certain GPCRs and G proteins impair bone development and metabolism, resulting in bone diseases. This review focuses on the functions of G proteins and GPCRs in osteoblasts and osteoclasts, their signaling pathways, and their gene mutations in mouse models and human diseases. We have discussed the roles of all four types of G proteins (i.e. Gs, Gq/11, Gi/o, and G12/13) and assessed the roles of the GPCRs, such as type 1 Parathyroid hormone receptor (PTH1R), calcitonin receptor, cation sensing receptor (CaSR), relaxin family peptides, cannabinoid receptor, frizzleds, and proton sensing receptor in normal bone formation and remodeling. The roles of regulators of G protein signaling (RGS) and GTPase-activating proteins (GAP) in G-protein signaling pathways are also reviewed. Lastly, we give perspective for the research of G protein signaling in bone development and disease.