Yi-Ping Li, PhD

Bone formation and bone resorption are physiologically controlled by the activities of osteoblasts and osteoclasts. Imbalances in these activities can arise from a variety of hormonal or inflammatory perturbations, resulting in skeletal abnormalities characterized by decreased bone mass, as in osteoporosis, or increased bone mass, in osteopetrosis. Increased osteoclast activity is seen in many osteopenic disorders, including postmenopausal osteoporosis, Paget's disease, bone metastases, periodontitis, and rheumatoid arthritis. Osteoporosis is the most common human bone disease, leading to hip and vertebral fractures. Major objectives of research in the Li laboratory are to elucidate the molecular mechanisms that control bone formation and bone resorption. We are also studying mechanisms of head formation during embryonic development and identifying genes that are important for craniofacial development. Craniofacial abnormalities constitute a major proportion of human birth defects. There are over one hundred human genetic syndromes known to result in craniofacial abnormalities. Although hundreds of thousands of children are affected each year, the genes responsible for these inherited diseases remain largely unknown.  Research in our lab seeks to advance our understanding of these diseases and the underlying signaling pathways so that effective new therapies might be discovered to treat and prevent these debilitating defects.

Mechanisms of Bone Resorption

Many common lytic bone disorders such as osteoporosis, bone aseptic loosening, and tumor-induced bone destruction are correlated to elevated osteoclast activity. The main physiological function of osteoclasts is to degrade the mineralized bone matrix: a process involving the dissolution of the crystalline hydroxyapatite by the targeted secretion of hydrochloric acid via the osteoclast proton pump (V-ATPase) through the ruffled border into the resorption lacunae and then followed by the proteolytic cleavage of the collagen rich organic matrix. In collaboration with Dr. Stashenko's laboratory, we cloned and characterized the genes encoding ATP6i (Li Y-P et al Nature Genetics. 23:447-451) and cathepsin K, through differential screening of a human osteoclastoma cDNA library. Using a gene knockout approach, we have studied in vivo functions of these two genes. We demonstrated that ATP6i is an osteoclast-specific subunit of a proton pump, while cathepsin K is a key osteoclast-specific cysteine protease involved in the degradation of bone matrix proteins. Our work indicated that mutations of ATP6i and cathepsin K are responsible for some forms of osteopetrosis in humans.

We also characterized RANKL-induced signaling proteins by Microarray genome-wide screening. Regulator of G-Signaling Protein 10 (RGS10) was found to be prominently expressed in RANKL-induced mouse osteoclast-like cells (OLCs) and human osteoclasts. So, we generated RGS10 knockout mice. The mutant mice exhibit severe osteopetrosis due to impaired osteoclast differentiation. Ectopic expression of RGS10 dramatically increased the sensitivity of osteoclast differentiation to RANKL signaling. Thus, our results, for the first time, reveal the role of RGS10 in vivo as a critical regulator in the RANKL-evoked RANKL-PLCg-[Ca²⁺]_i-oscillation-NFAT2 signaling pathway for terminal differentiation of osteoclasts.  

Another goal of our research is to discover the role and mechanism of key subunits of the osteoclast proton pump (SOPP), such as a3, d2, C1 and ac45, in osteoclast functions (e.g., osteoclast-mediated extracellular acidification, membrane trafficking, exocytosis), and to elucidate their potential role in developing a means to cure or alleviate human osteolytic diseases. We found that the expression of Ac45, d2, and C1 are highly induced by RANKL and M-CSF during osteoclast differentiation and that Ac45, d2, and C1 are required for the functions of osteoclastic bone resorption and extracellular acidification. We will continue to investigate if Ac45 (ATP6AP1) is an essential regulator of osteoclast proton pump function and if it has multiple functions besides extracellular acidification, including roles in cell fusion, polarization, intracellular trafficking, exocytosis, cell signaling, and subcellular relocalization in osteoclast differentiation, activation, and bone resorption.

An additional facet of our research on bone resorption is an investigation of the mechanisms underlying transcription factors that specify osteoclast lineage commitment and differentiation. This is highly significant since elucidating osteoclast lineage commitment and differentiation has potential for defining new therapeutic targets for bone disorders that involve osteoclast generation and activation. We have established an osteoclast precursor cell line, MOCP-5, to facilitate the study of osteoclast gene transcriptional regulation and differentiation. We have also characterized the mouse cathepsin K gene promoter and expression in MOPC-5 cells. Based on its high and specific expression in osteoclasts and the physiological significance of cathepsin K, we chose cathepsin K as the gene model and MOCP-5 as the cell model to characterize how the cathepsin K gene is regulated during osteoclast differentiation. The critical cis-regulatory elements (CCREs) of the mouse cathepsin K gene are being mapped by mutagenesis of the promoter, and the CCRE-binding protein(s) are being characterized.  

Molecular Basis of Bone Formation
The long-term goal of this research is to elucidate the mechanism of bone formation and to develop a means to cure or alleviate bone abnormalities, such as osteoporosis. In the last 12 years we have benefited from the mouse knockout, conditional mouse knockout, and micro-arrays. However, the whole mechanism of osteoblast lineage commitment and differentiation, from the mesenchymal stem cell to the mature osteoblast, is still unclear; particularly, the progression in early stages of osteoblast progenitors to pre-osteoblasts and then to osteoblasts. Compared with transcriptional regulation in bone formation, the molecular mechanisms of regulation at the translational level is largely unknown. Our previous work shows that osteoblast transcription factor-1 (OBTF1) is expressed in all stages of osteoblast lineage. Furthermore, we have demonstrated that OBTF1 is located in both the nucleus and the cytoplasm, which indicates that it may be a dual regulator of transcription and translation. We are dedicated to the insightful investigation of the functions of OBTF1 in the early and late stages of osteoblast differentiation and in the regulation of bone formation at the translational and transcriptional levels.

In addition, we are examining the genes that control the differentiation of mesenchymal stem cells to osteoblasts. Using subtractive differential screening, we recently identified several novel genes that are specifically expressed in osteoblasts. Sequence analysis of one novel clone identified a basic helix-loop-helix (bHLH) DNA binding domain, indicating that it may be a novel transcription factor, which we have designated osteoblast transcription factor-2 (OBTF2). We confirmed its role as a transcription factor by cotransfection of an OBTF2 cDNA expression construct and an osteocalcin promoter-CAT reporter construct into two cell lines: the osteoblast cell line ROS 17/2.8 and the nonosteoblastic cell line C3H10T1/2. Constitutive expression of OBTF2 in chicken embryos enhances bone. We are characterizing the functions of these novel genes in vitro by the RNAi approach and in vivo by the mouse gene knockout approach in our laboratory.  

Transcriptional Regulation in Craniofacial Development
Another area of research addresses factors controlling craniofacial development. Although rapid progress has been made in the study of head formation and related diseases through the mouse model and genetic studies in the last decade, the underlying mechanisms and disease genes are largely unknown. Mutations in Six3 and Shh genes are frequently identified in holoprosencephaly (HPE) cases, however, more than 65% of HPE cases remain unexplained, suggesting the involvement of many other genes. To further advance our knowledge on this important topic, our lab uses the mouse, chicken and drosophila models to define other HPE disease genes. we have cloned genes that are specifically expressed at the anterior ends of developing mouse embryos. We are characterizing the functions of genes, which encode putative novel transcription factors in chicken and mouse models, in head formation. We are also studying mouse mutants with developmental defects that resemble the craniofacial abnormalities seen in a variety of human genetic disorders. Through misexpression, dominant negative inactivation, and targeted mutation, we are investigating the actions of a network of transcription factors in controlling craniofacial development in both the mouse and chicken.

We showed that targeted disruption of the gene encoding a cellular nucleic-acid binding protein (CNBP) in mice results in severe craniofacial defects. The defects include lack of anterior head structures, such as the mandible and eyes, and are reminiscent of the effects of otocephalic mutations reported in humans. Furthermore, we are characterizing the origin of these abnormalities in CNBP mutant embryos to facilitate dissection of the mechanisms governing craniofacial development. We are defining the role of CNBP in forebrain development using a tissue-specific targeted disruption (gene conditional knock-out) approach and the role of CNBP in craniofacial development using a neural crest cell (NCC)-specific targeted disruption approach. Cnbp silencing resulted in the absence of BF-1, Six3, and Hesx1 expression, suggesting a mechanism for the function of Cnbp.  Our data suggests that Cnbp may be a central member of the pathways controlling anterior patterning and craniofacial development.  

Additional research areas include:

Revealing the mechanisms directing musculoskeletal development and diseaseTo define the role of Znf9 in myotonic dystrophy type 2 (DM2) and muscle development, we characterized Znf9+/- mice and found that their phenotype reflects many of the features in myotonic dystrophy. Znf9 is highly expressed in skeletal and heart muscle. Our data demonstrated that Znf9 haploinsufficiency might contribute to the myotonic dystrophy phenotype in Znf9+/- mice.

Stem Cell reprogramming for tissue regeneration and organogenesis

We are currently investigating if gene profile reprogrammed human mesenchymal stem cells (hMSCs) seeded on scaffolding biomaterial will mimic natural tissue generation and enable improved tissue regeneration and maxillofacial wound repair.  We have already identified some key transcription factors that will be used. This concept is of vital importance since hMSCs are the major stem cells that can be realistically obtained from adults and since only autologous (patient's own) stem cells can be used therapeutically.

Characterizing a novel vascular disrupting agent

In recent years, vascular disrupting agents (VDAs) that exploit the differences between tumor and normal vessels, have been shown to not only inhibit angiogenesis (the formation of blood vessels which is critical to tumor growth and survival), but to also rapidly cause an irreversible and tumor-specific shutdown (or occlusion) of blood vessels. We have identified a remarkable new drug thatpowerfully acts against tumors (quickly destroys 95% of the cells in a solid tumor with a single dose in all the animal cancer models tested thus far), while circumventing the difficulties and adverse effects of conventional antitumor treatments. We are currently developing this drug and investigating the molecular mechanism of its antitumor effects.

Resource of animal Models in my lab: varied history of experience with animal models.  The use of animal models is pivotal as we seek to understand basic biological processes and translate that knowledge into new diagnostics, preventions, treatments and products.  My lab has a long and varied history of experience with animal models, including mouse, chicken, and fly (drosophila) models. These three Animal Models, maintained in independent facilities in my lab (Please see details on my lab Web page: http://bioinformatics.forsyth.org/ypli/index.html), act as a platform for our research and collaboration.) 

Selected Publications 

Soltanoff CS, Chen W, Yang S, Li Y-P. (2009) The signaling networks that control the lineage commitment and differentiation of bone cells. Crit. Rev. Eukaryot. Gene Expr.19:1-46.
 

Wu H, Xu G, Li YP. (2009)  Atp6vOd2 is an essential component of the osteoclast-specific proton pump that mediates extracellular acidification in bone resorption. J. Bone Miner. Res. 24(5):871-885.

Feng S, Deng, L, Chen W, Shao J, Xu G Li YP. (2009) Atp6v1c1 is an essential component of the osteoclast proton pump and in F-actin ring formation in osteoclasts. Biochem. J. 417(1):195-203.

Tu Q, Zhang J, Fix A, Brewer E, Li YP, Zhang ZY, Chen J. (2009) Targeted overexpression of BSP in osteociasts promotes bone metastasis of breast cance cells. J. Cell Physiol. 218(1):135-145.

Yang, S., Chen, W., Stashenko, P. and Li, Y-P (2007) RGS 10A is an essential factor in RANKL evoking signaling of osteoclast differentiation. J.Cell Sci. 120:3362-3371.
 

Yang S, Li Y-P. (2007) RGS10 null mutation impairs osteoclast differentiation resulting from the loss of [Ca2+]i oscillation regulation. Genes Dev. 21(14):1803-1816.

Chen W, Wang Y, Abe Y, Cheney L, Udd B, Li Y-P. (2007) Haploinsufficiency for Znf9 in Znf9+/- mice is associated with multiorgan abnormalities resembling myotonic dystrophy. J. Mol. Biol. 368:8-17.

Chen W, Yang S, Abe Y, Li M, Wang Y, Shao J, Li E, Li YP. (2007) Novel pycnodysostosis mouse model uncovers cathepsin K function as a potential regulator of osteoclast apoptosis and senescence. Hum. Mol. Genet. 16:410-423.

Yang S, Li YP. (2007) RGS12 is essential for RANKL: evoked signaling for terminal differentiation of osteo-clasts in vitro. J. Bone Miner. Res. 22:45-54.

Abe Y, Chen W, Huang W, Nishino M, Li YP. (2006) CNBP regulates forebrain formation at organogenesis stage in chick embryos. Dev. Biol. 295:116-127.

Staff

Assistant Reseach Investigator
Wei Chen, M.D.

Staff Associate
Wei Tang, Ph.D.

Research Assistant
Christie Taylor