Human cells must be able to send signals that switch life processes on and off as they form the foetus, and later, to maintain the integrity of adult tissue. Notch proteins have been recognised for many years as part of signalling cascades that drive the development of the foetal brain, nerves and blood vessels. What had remained a mystery was whether Notch has any role in bone formation and health in adults.
The current results demonstrate for the first time in live, adult animals that genetic changes made to increase Notch signalling specifically in bone-making cells (osteoblasts) resulted in thickened, abnormal bone similar in some ways to that seen in osteosarcomas, a type of bone cancer. Conversely, eliminating notch resulted over the long term in the weaker bones seen in osteoporosis. The studies confirm that Notch plays a role in bone development, and suggest it also maintains bone strength with ageing. The data also provide the first evidence that Notch, connected in the past with leukaemia and intestinal tumours, may also play a role in the development of osteosarcoma.
"The findings are important because without Notch signalling in osteoblasts, the cells that build bone, you get inadequate new bone formation along with aggressive bone destruction by bone-degrading cells, both typical of osteoporosis," said Brendan Boyce, M.D. professor of Pathology at the University of Rochester Medical Center, and a study author. "In addition, normal Notch signalling appears to make bones stronger, but too much of it could result in osteosarcoma, the most common primary malignant bone tumour in children and teenagers. These studies suggest that well timed manipulations of a single process may represent new ways to fight two major bone diseases."
Brendan Lee, M.D., Ph.D., associate professor of Molecular and Human Genetics at the Baylor College of Medicine led the first study along with Boyce. The second study was led by Matthew Hilton, Ph.D., now assistant professor of Orthopaedics and Rehabilitation at the Medical Center, with Fanxin Long, Ph.D., principle investigator of this study. Long is assistant professor of Medicine at Washington University, where, until recently, Hilton was a post-doctoral fellow. They also partnered with the Endocrine Unit at Massachusetts General Hospital. Both studies, supported by grants from the National Institutes of Health, were published on-line on Feb. 24 and in hard copy on March 6.
Having been around since early in evolution, Notch proteins are named for notches in the wings of the flies in which Notch-related genes were discovered. Such protein receptors span a cell's outer membrane, enabling external biochemical messages to penetrate cells.
Part of the receptor is exposed to the cell's outside and designed to react with a specific signalling molecule (ligand). When a ligand docks into the receptor, like a ship coming into port, it changes the shape of the dock such that it sets off chain reactions inside the cell. When a ligand binds to Notch in particular, part of the protein, the notch intracellular domain (NICD), breaks away inside of the cell, travels to the cell's nucleus and influences gene expression there. Gene expression is the process whereby genetic instructions encoded in genes are converted into protein workhorses that make up the body's structures and carry its signals. In the current case, the NICD signal was shown to influence the decision made by stem cells in bone marrow about whether or not to become bone-making cells.
As we develop in the womb, successive generations of stem cells specialise (differentiate), with each group able to differentiate into fewer and fewer cell types. Many tissues maintain pools of stem cells into adulthood in case replacement cells are needed for healing or maintenance. Among theses are mesenchymal stem cells, which reside in adult bone marrow and can differentiate into, among other things, bone-making cells called osteoblasts. Osteoblasts are one of two cell types, which coupled together, enable bone to continuously recycle itself and stay strong. Where osteoblasts make new bone, osteoclasts "eat" ageing bone to make way for new bone in a careful balance with osteoblasts.
The current study suggests for the first time that Notch signaling influences the process by which stem cells "decide" whether to become bone-making osteoblasts. The data also argue that Notch regulates the process by which osteoblasts signal to osteoclasts, regulating their ability to eat bone.
In recent years, bone biologists have constructed a theoretical model that they believe represents the stages involved in the differentiation of mesenchymal cells into mature osteoblasts. The model holds that intermediates exist between stem cell and mature osteoblast and that signalling processes, including Notch, control the transition from one to the next. In the first step, mesenchymal stem cells commit to the osteoblast pathway or lineage. Once that decision is made, they become, in distinct stages, osteoblast precursors then immature osteoblasts and then mature osteoblasts. Notch signalling has different roles at each stage, inhibiting some transitions while encouraging others, researchers found. In short, Notch "escorts" the stem cells through the process until they form a pool of immature osteoblasts, then maintains that pool until the body calls for more bone-making cells.
In both studies, researchers manipulated the osteoblast differentiation process at different stages in study mice. The Long-Hilton study used genetic tools to shut down Notch early in the differentiation process, at the point where the signals would have enabled mesenchymal stem cells to commit to the osteoblast pathway. By interfering early, the Hilton team demonstrated that such signalling normally maintains a pool of mesenchymal stem cells, inhibiting their ability to differentiate into osteoblasts. Thus, shutting down Notch led to an initial increase in osteoblast differentiation and related bone formation. The initial spurt of bone making, however, was followed by decreased production by osteoblasts of osteoprotegerin, a protein that protects the skeleton by inhibiting osteoclast formation. In the absence of Notch, osteoclast formation increased over time, resulting in long-term, age-related bone loss.
While the Hilton team only shut down Notch signalling, the Lee-Boyce study used genetic engineering to greatly increase it in one set of experiments and to shut it down in another. Both sets of changes were made later in the differentiation process than in the Hilton study, near the last step in the pathway where immature osteoblasts mature into active bone-making cells. In the Boyce study, mice genetically engineered to have more Notch signalling in osteoblast precursors had a striking increase in the number of immature osteoblasts, which led to abnormally thickened bones. When they shut down notch signalling, the mice lost bone like those in the Hilton experiments.
After discovering that Notch signaling promotes bone-making cell proliferation, the Lee-Boyce team decided to see if it was involved in bone cancer, given that many cancers results from such uncontrolled growth. The team found that expression of Notch and its signalling partners was significantly higher in samples of primary untreated and post-treatment osteosarcomas compared to that in samples of normal bone.
Specifically, the team observed higher gene expression in cancer samples of Cyclin D1, a Notch signaling partner that drives cell proliferation to create the pool of immature osteoblasts necessary for bone formation. Past human research has observed that 10 percent of osteosarcomas show increased Cyclin D1 gene expression, and the current study suggests that, in a live animal, the proliferative effect of Notch signalling might contribute to bone cancer by triggering cyclin D1. Better understanding of this mechanism may guide future treatment design efforts, researchers said.
In further tests, researchers found that two secretase inhibitors, which shut down Notch signalling, also decreased pathogenic bone cell growth (proliferation) in osteosarcoma cell samples. One of the compounds, DAPT (N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl 10 ester), is a gamma secretase inhibitor. Gamma secretase snips off the Notch protein's tail (NICD) inside the cell, freeing it to travel to the cell nucleus and influence gene expression, unless DAPT interferes.
Interestingly, DAPT also blocks the action of secretases that snip into fragments the amyloid precursor protein (APP) in brain cells. Some of these cleaved APP fragments clump together to form "plaques" in the brains of patients with Alzheimer's disease that contribute to the death of brain cells. DAPT is currently in Phase II human clinical trials for the treatment of Alzheimer' disease.
"One theory is that Notch signalling normally maintains the mesenchymal stem cell pool in our bone marrow and inhibits their differentiation into osteoblasts," said Hilton. "So for some patients with oteoporosis, we may be able to briefly inhibit Notch signalling to allow more mesenchymal stem cells to differentiate into osteoblasts, creating a larger pool of bone-building cells. The obstacle to this strategy - that generating more bone building cells inevitably activates more bone degrading cells - could be overcome by combining a transient Notch inhibitor like DAPT with any of several already approved osteoporosis treatments that inhibit osteoclast formation or activity."