Research topic: Studying the role calreticulin protein plays in regulating osteoclast formation and its therapeutic potential to inhibit bone loss
Research results: Found that calreticulin inhibits osteoclast formation in an animal model
Patient care application of results: Potential use of calreticulin as a new, naturally occurring inhibitor of bone resorption.
Simplified patient care application: New treatment protocols that prevent bone loss
OREF grant recipient investigates a natural inhibitor of osteoclast formation
Jay D. Lenn
The process of bone remodeling is a balancing act between two cell types: osteoclasts that resorb bone and osteoblasts that form new bone. The molecules primarily responsible for regulating this balance have been well characterized in research. And the mechanism of age-associated bone loss—when osteoclasts outpace the work of osteoblasts—is also generally well-defined.
Filling in the remaining gaps in our understanding—such as defining more accurately the signaling pathways that regulate osteoclast formation and function—may suggest new targets for therapy. Charla R. Fischer, MD, assistant professor of orthopaedic surgery at Columbia University, is studying the role of the calreticulin protein in regulating osteoclast formation and the protein’s therapeutic potential to inhibit bone loss in women.
Dr. Fischer specializes in the surgical treatment of the spine, including interventions for treating osteoporosis-associated vertebral compression fractures.
“I started out with a question based on my patients’ needs,” Dr. Fischer said. “Now I’m looking at the question in the lab, and ultimately, I would like to use the answers to treat my patients more effectively. I’m particularly interested in how osteoporosis affects spine surgery. If the bone is weak, the results of surgery are not as good. If we can better understand how bone loss occurs, we may improve outcomes.”
Dr. Fischer’s work was supported by a 2014 Orthopaedic Research and Education Foundation (OREF) New Investigator Grant, a $50,000 award to advance the scientific training of next-generation orthopaedic surgeons by providing seed and start-up funding for promising research projects.
The basics of osteoclast formation and function
There are three primary players in regulating osteoclast formation and function:
• Receptor activator of nuclear factor κB (RANK), a receptor on the surface of osteoclasts, as well as the surface of macrophages, which are the precursors to osteoclasts
• RANK ligand (RANKL), a signaling protein released by osteoblasts that binds to RANK
• Osteoprotegerin (OPG), a decoy receptor for RANKL, that is also released by osteoblasts
When RANKL binds to RANK receptors on the surface of macrophages, clusters of these cells fuse to form a multinucleated osteoclast. RANKL also binds to RANK on the surface of mature osteoclasts—a signal that sets in motion the cell’s bone-resorbing activity. OPB regulates osteoclast activity by intercepting some RANKL proteins.
Estrogen also plays a role by limiting the expression of RANKL by osteoblasts. In women, the decline in estrogen after menopause results in less regulation of RANKL and subsequently more osteoclast formation and activity.
Inhibiting RANKL-induced osteoclastogenesis
In previous research, Dr. Fischer and her colleagues identified the protein calreticulin as a potential regulator of osteoclastogenesis. In subsequent experiments, they observed that calreticulin inhibited the RANKL-induced activation of a master transcription factor of osteoclast formation.
A transcription factor, by binding to a segment of DNA, moderates the rate at which genetic information is transcribed into the protein-building instructions of messenger-RNA. By inhibiting a primary transcription factor, calreticulin may have a negative effect on osteoclast formation by blocking the expression of one or more genes necessary for osteoclastogenesis.
Investigating calreticulin in an osteoporosis model
In the OREF-funded study, Dr. Fischer had two goals: to characterize calreticulin’s effect on osteoclastogenesis signaling pathways and to assess calreticulin’s therapeutic effect in a mouse model of osteoporosis.
She explained, “If we better understand steps along the way, we can further explore mechanism-based treatments. The results may introduce calreticulin as a new, naturally occurring inhibitor of bone resorption.”
In a series of in vitro experiments, the researchers introduced recombinant human calreticulin (rhCRG) to mouse macrophages. They assessed the effect of rhCRG on the expression of genes known to be involved in osteoclastogenesis, as well as its potential interaction with other transcription factors and associated proteins. They also tested in vitro whether rhCRG has an inhibitory effect on the function of mature osteoclasts.
The in vivo experiments were conducted with 60 adult female mice in three groups:
• In one test group, each subject underwent ovariectomy to model menopause-induced osteoporosis.
• In another test group, each mouse underwent ovariectomy and received a subcutaneous injection of a controlled-release hydrogel with rhCRG.
• The control group had a sham surgery with only an abdominal incision.
At six weeks, the researchers harvested the fifth lumbar vertebrae of all subjects. Using a series of tests, they compared among the three groups the total bone mass, trabecular bone volume, cortical bone thickness, compressive strength, and energy absorption.
“The results showed that calreticulin inhibits osteoclast formation in the mouse model,” stated Dr. Fischer. “Some of the next steps will be taking actual bone marrow-derived cells from patients and looking at calreticulin’s effect on osteoclastogenesis in these cells.”
Supporting emerging clinician scientists
Dr. Fischer credits her mentor and research colleague Francis Y. Lee, MD, PhD, for supporting her career as a clinician scientist. “Dr. Lee has been doing research on the osteoclastogenesis pathway for years,” she said. “He has not only helped me understand the science but also encouraged me to apply for grants, introduced me to other researchers in the field and collaborated with me on experiments.”
Dr. Fischer also views OREF as a supporting partner in her research career.
“OREF is an advocate for the clinician scientist. It encourages clinician scientists to pursue their research ideas and dreams. It has allowed me to do the research, helped me understand the grant writing process and enabled me to apply for more funding from different sources. And through the science that’s being researched, OREF is also an advocate for patients.”