Associate Professor of Biological Chemistry, Harvard Medical School, Boston
Senior Research Associate, Department of Orthopaedic Surgery, Children's Hospital Medical Center, Boston
Post-doctoral training: National Institutes of Health; Harvard Medical School, Children's Hospital.
Ph.D., Boston University School of Medicine
Molecular Mechanisms Regulating Skeletal Development and Metastasis of Cancer Cells to Bone
Bone tissue functions as a mechanically responsive structural component of the body and as a major organ essential for maintaining calcium and phosphate homeostasis. The skeleton is the target of numerous human genetic disorders and recently mouse models have identified new regulatory pathways that affect the skeleton. As a normal process of aging and hormonal changes after the menopause, skeletal mass can decrease by as much as 30% leading to bone fracture and compromised quality of life in the elderly population. Historically the laboratory has addressed molecular mechanisms regulating formation and mineralization of bone by osteoblasts and turnover of bone tissue by osteoclasts, the bone resorbing cells. We are defining the key regulatory events for the progressive differentiation of osteoprogenitor stem cells to osteogenic cells by identifying transcription factor complexes that control expression of tissue-specific genes. Our studies are showing that early events of skeletal development are recapitulated in the adult skeleton for the normal maintenance of bone mass.
The skeleton is also a target of metastatic cancers and recent studies from our laboratory and others are demonstrating that signaling pathways which mediate responsiveness of the bone forming and bone resorbing cells of the skeleton for organogenesis are also pathways that are activated in cancer cells which metastasize to bone.
Areas of current investigation for graduate and MD/PhD students and postdoctoral fellows include:
1. Combinatorial Control Mechanisms for Skeletal Development
This laboratory is defining a regulatory network of developmental factors with a focus on three major pathways critical for skeletal pattern formation, early embryonic bone development, and osteoblast differentiation: the bone morphogenetic protein (BMP) family, the Wnt signaling pathway and Hox homeodomain factors. The integration of these signaling pathways for tissue-specific gene expression is being characterized for two genes, the transcription factor Runx2/Cbfa1 established as essential for skeletal development and a bone-specific Runx2 target gene, osteocalcin, that represents one of the major non-collagenous bone matrix proteins (Choi et al., 2001; Lengner et al., 2002). The Runx2/Cbfa transcription factor is essential for osteogenic differentiation and functions as a master regulatory gene through multiple properties (Fig. 1). Runx2 can change the phenotype of a cell, e.g., from a non-osseous adipocyte to an osteoblast, as well as control recruitment of stem cells into the chondrogenic and osteogenic phenotype (Lengner et al., 2005). Characterizing the functional activities of this protein has provided new paradigms for understanding gene regulation. First, Runx2 expression is regulated in stem cells by early development cues including Hox genes, BMP/TGFb, and Wnt proteins (Balint et al., 2003, Zaidi et al., 2002; Gaur et al., 2005, 2006; Bodine et al., 2004). Secondly, this transcription factor is a scaffolding protein that is targeted to specific subnuclear domains for the assembly of multimeric complexes on target genes (reviewed in Lian et al., 2004). Third, Runx2 recruits chromatin remodeling proteins and assembles complexes at Runx2 regulatory elements in genes to either activate or repress gene transcription (Javed et al., 1999; Young et al., 2005). By mutational analyses of different protein interacting domains of Runx2, we are identifying the coregulatory proteins essential for osteoblast differentiation in vitro and for skeletal development in vivo (Zaidi et al., 2004; Afzal et al., 2005). Experimental approaches include cell culture models for chondrocyte and osteoblast differentiation using human embryonic and mesenchymal stem cells, gene regulation studies and characterization of mouse phenotypes.
2. Tissue Specific Gene Regulatory Mechanisms
The osteocalcin gene encodes a calcium binding ECM protein that is developmentally regulated during bone formation by a plethora of hormones, growth factors, and cell signaling proteins involved in calcium homeostasis and bone remodeling. The integration of independent signals must converge on the promoter to account for complex physiologic control of the gene during bone formation and turnover. Thus, the promoter of the osteocalcin gene provides a molecular blueprint for understanding developmental and hormonal regulation of gene expression required for bone formation (Hassan et al., 2004; Javed et al., 1999; Gutierrez et al., 2004). Identification of tissue-specific regulatory sequences and their cognate binding factors have allowed us to develop strategies for cell based therapy to target therapeutic genes specifically to bone. Examples of osteocalcin tissue-specific regulatory elements undergoing characterization include the steroid response elements, runt homology (Runx/Cbfa), homeodomain (HD), Hox, C/EBP, ATF and AP-1 protein binding sites. The HD proteins (Msx, Dlx) and Hoxa10 regulate osteocalcin at different stages of cell differentiation (Fig. 2). Transcriptional control of the gene is evaluated at multiple levels including chromatin modifications and recruitment of transcription factors and their coregulatory proteins by chromatin immunoprecipitation studies. Enforced expression and siRNA knockdown studies of regulatory factors strategies are used to establish their function in vitro and in vivo using transgenic mouse models. Such analyses have revealed new insights into understanding the complex interplay of physiologic mediators of tissue differentiation. This program of research provides a student with fundamental techniques requisite for characterizing gene regulation and expression in a biological context.
3. Cancer Cell Biology in the Bone Microenvironment
The end stage of breast and prostate cancer is metastasis to bone, with very poor prognosis with nearly 70% mortality within a year. Cancer cells cause destruction of the bone, resulting in fractures and severe pain. Understanding the mechanisms which induce metastasis of the primary cancer cell to the bone environment needs to be addressed. We have identified high expression levels of the Runx2 transcription factor in metastatic breast and prostate cancer cell lines. Runx target genes in the cancer cell include the entire class of matrix metalloproteinases characterized for their role in tissue invasion, the vascular endothelial growth factor, a potent angiogenic factor involved as a primary event in tumor growth and several cell growth and osteoblastic genes expressed in the bone environment that allow for tumor growth (Fig. 3) (Pratap et al., 2005). The cancer cell responds to TGFb and BMP growth factors in the bone extracellular matrix and stimulates bone resorbing cells. In recent studies, we have shown metastatic cancer cell lines in which Runx2 activity has been blocked through genetic mutations, that the osteolytic disease of breast cancer cells can be prevented in the mouse (Barnes et al., 2004; Javed et al., 2005). The presence of mutant Runx2 protein in metastatic cells inhibits cell invasion (in vitro assays) and genes associated with tumor growth (Fig. 3). We are now turning our attention to mechanisms responsible for activation of Runx2 in the primary tumor that would lead to the metastatic event in vivo. Experimental approaches include generation of human cancer cell lines with mutants of Runx2, assessing tumor growth by in vivo imaging of tumors in breast, prostate and bone tissues, and examining gene expression profiles of the tumors are assayed.
4. Gene Therapy and Tissue Engineering for Skeletal Diseases
Genetic disorders of the skeleton require expression of the normal proteins, specifically in bone cells. Here the osteocalcin gene promoter provides the appropriate method for targeting expressed genes to mature osteoblasts. Using OC-EGFP mice, we are characterizing a population of mesenchymal stem cells from these mice that differentiate into bone cells expressing GFP in donor mice. Numerous donor mice carrying mutated genes that mimic human genetic disorders are available for addressing the critical number of mesenchymal stem cells differentiated to osteoblasts that can provide a sufficient level of normal protein to correct the skeletal disorder. A second example where tissue engineering is being developed relates to the problem of non-union fractures where scar tissue rather than new bone formation occurs. By using autologous cells expressing osteogenic factors, bone formation can be induced. Lastly, we are culturing human embryonic stem cells and promoting their differentiation to chondrogenic and osteogenic lineages and testing these in mouse models. These powerful properties are being exploited for treatment of skeletal diseases that require rebuilding of skeletal tissue by promoting lineage allocation of stem cells or marrow progenitor cells.
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