Immune Regulator Tied to Bone Building

Protein Points Toward New Therapeutic Target for Osteoporosis

Usually scientists can predict which organ system a genetic knockout will affect. But when Laurie Glimcher, the Irene Heinz Given professor of immunology at HSPH and an HMS professor of medicine, created a knockout mouse to see how eliminating an immune-system signaling protein would affect lymphocytes, she and her colleagues were taken by surprise.

In the first analysis, research fellow Dallas Jones reported that the mice looked normal. Still, he was having trouble getting a bone marrow sample. When Glimcher suggested that an X-ray might help, he just rolled his eyes. “I thought, ‘Yeah, right, like there’s going to be a skeletal phenotype in this mouse,’” recalled Jones.

Glimcher had made leaps from immunology to skeletal biology before, however. In three earlier immune-system knockouts, she had uncovered cartilage abnormalities, in collaboration with her father Melvin Glimcher, the Harriet M. Peabody professor of orthopedic surgery at Children’s Hospital Boston.

Molecular biologists specializing in immunology Laurie Glimcher (below left), Dallas Jones, and Marc Wein (above left to right), with the help of skeletal biologist Melvin Glimcher (below right), who is Laurie’s father, found a key molecular mechanism regulating bone formation. This is the fourth paper published by the father–daughter team making connections between the fields of immunology and bone biology.

Photos by Graham Ramsay

Jones ran the X-ray and examined bone sections using light microscopy. The images showed that bone mass was elevated and bone had obliterated the marrow cavity. He later determined that these mice were forming bone more than four times as fast as wild-type mice throughout their skeletal system. Of all the connections she had made between the immune and skeletal systems, said Glimcher, “this one is without a doubt the most exciting.”

Immunology to Bone Biology
The study may have implications for the estimated 10 million Americans over age 50 who suffer from osteoporosis—and the many more who will face the disease as they age. Glimcher and colleagues have not only discovered that the protein, Schnurri-3 (Shn3), inhibits bone growth, they have identified the molecular mechanisms behind this remarkable phenotype. Their results, reported in the May 26 Science, have inspired them to launch a search for small molecules that disrupt the Shn3 mechanism. If identified, these would have the potential to therapeutically unleash the body’s capacity to grow more bone.

“The next generation of advancements in the treatment of osteoporosis, everyone thinks, will come from finding ways to actively increase bone mass,” said Hank Kronenberg, chief of the Endocrine Unit at Massachusetts General Hospital, who is not an author on the paper.

In addition to the clinical implications, the study is an example of work in the emerging field of osteoimmunology. Scientists have been documenting links between immune and bone cells for decades, but the fields have largely remained separate and collaborations have been the exception. “Interdisciplinary work like this, in which immunology people make a major contribution to the bone field, is something everybody is striving for, but it doesn’t happen as often as we’d like,” said Kronenberg. “It’s not easy to make those leaps.”

The Ubiquitin Tag Team
The rapid bone growth in the Shn3 knockout forced co–first authors Jones and MD–PhD candidate Marc Wein to venture outside their field of immunology. “We did lots of background reading to learn about the skeletal system,” said Wein, who, with Jones, drew on the expertise of Glimcher’s father, one of the paper’s co-authors.

Image courtesy of Laurie Glimcher Hidden architecture. Though born with normally formed bones, within one to two weeks, Schnurri-3– knockout mice (Shn3-/-) show signs of increased bone mass compared with wild types (WT). By six weeks, as shown in these X-ray microcomputed tomography images, the marrow cavity starts to fill in. By seven months, it is almost completely full.

They learned that the skeleton is a dynamic structure that completely remodels itself every 10 years or so. Bone-forming osteoblasts and bone-resorbing osteoclasts keep the reformation process in constant balance.

The investigators determined that the osteoclasts in their mouse model were functioning normally because the numbers and behaviors of these cells essentially matched those in wild-type mice. Zeroing in on the osteoblasts led them to Runx2, a transcription factor that controls osteoblast differentiation and activates key bone-formation genes—the very genes the researchers detected to be upregulated in the absence of Shn3. Based on these observations, the researchers hypothesized that Shn3 interacts with Runx2 to inhibit its ability to stimulate bone formation.

Using standard techniques, Wein confirmed their hypothesis. He found that Shn3 interacts with Runx2 and promotes its degradation. At first, Jones and Wein thought Shn3 must be a ubiquitin ligase, an enzyme that marks proteins for degradation in the proteasome by tagging them with ubiquitin, yet the researchers found no biochemical evidence to support this idea. They eventually started to think of Shn3 as an “adapter protein” that forms a complex with another enzyme to carry out Runx2 degradation.

They embarked on what Glimcher called a “treasure hunt” for Shn3’s ubiquitin ligase partner. They tested a family of ligases and found that a protein called WWP1 interacts with Shn3 and, in doing so, becomes more efficient at tagging Runx2. To determine WWP1’s role in osteoblasts, they used RNA interference to reduce its level in the cells. Cell culture tests showed the same behavior as Shn3-knockout osteoblasts: cells with reduced levels of WWP1 clumped to form an excess amount of mineralized nodules. This evidence suggests that without either Shn3 or WWP1, Runx2 fails to degrade and bone formation kicks into overdrive.

Because many targeted drug therapies work by inhibiting the function of an enzyme, the investigators suspect that WWP1 may be a valuable molecular target for therapies that can help treat osteoporosis and the bone--wasting effects of some cancers. Glimcher’s lab is collaborating with the lab of biochemist Gregory Petsko at Brandeis University to identify the small molecules that inhibit WWP1. Though the highest hurdles of drug discovery lie in the distant future, said Petsko, “as early-stage targets go, this looks promising. It just smells like a good target.”

A glimpse into the complexity of bone, however, reveals just how much research remains to be done. “Many scientists think bone is bone is bone,” said Melvin Glimcher, “but without the right structural organization, bones can easily break.” Different types of bone, such as spongy, cancellous bone and hard, cortical bone, serve different purposes. Together they form an architecture that works—the same way different construction materials work together to form a building that is strong enough to support its weight but flexible enough to withstand an earthquake. “That architecture repeats itself down to the molecular level,” said Glimcher, whose lab is using biophysics techniques to examine the composition of the mutant mouse bones.

Laurie Glimcher’s lab is collaborating with researchers at Beth Israel Deaconess Medical Center to test the strength of the bones, much the same way one would test a cookie for crunch. “It will take the work of a lot of different labs to really understand what’s different about these bones,” said Wein.

—Elizabeth Dougherty

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