Our skeletons do more than just hold us upright
Do what you love. Know your own bone; gnaw at it, bury it, unearth it, and gnaw it still.
— Henry David Thoreau
Most of us appreciate that if it weren’t for our skeletons, we’d be bags of protoplasm oozing on the ground. But beyond that, a common perception of bone is that it’s simply an inert mineralized tissue that does only a few things: protect delicate organs, help us to walk, act as a mineral store and house the blood-making machinery.
But that perception has started to shift over the past two decades. Thanks to advances in cellular and molecular biology tools, experts now say that bone is a dynamic tissue that sends out and receives messages from organs. It even tweaks the functions of organs and actively participates in maintaining mineral and energy homeostasis throughout the body.
Old to new
Bone constantly turns over. This process is called bone remodeling and rebuilds the skeleton bit by bit. Bone remodeling is the reason you don’t have the same skeleton today as you did 10 or so years ago.
Understanding how bone remodeling happens at the cellular and molecular levels was a challenge for decades because the mineralized matrix of bone, containing calcium and phosphate, had made culturing bone cells by conventional methods difficult. But now a clearer picture is starting to come into focus. There are thought to be three types of bone cells: osteoblasts, osteoclasts and osteocytes. Osteoblasts build bone by putting down the mineralized matrix. Osteoclasts chew down bone. They are unique in that they are the only cells in the body designed to destroy their host tissue. Both cell types sit on the surface of the bone.
The challenge of studying bone is most evident when it comes to the osteocytes, cells derived from osteoblasts that make up 90 percent of bone. Because they sit deep inside the bone, studying them had been especially hard, and the difficulty led a number of researchers to ignore the cells. As Henry Kronenberg at the Massachusetts General Hospital quips, the conventional thinking used to be that osteocytes were just “stupid osteoblasts that got buried and stuck in bone.”
Lynda Bonewald at the University of Missouri in Kansas City, an immunologist and hematologist by training, became intrigued by the osteocytes inside the bone matrix in the late 1980s because of their striking resemblance to neurons with dendritic protrusions. When she asked experts in the bone field what osteocytes did, “I was told they were just placeholders,” she says. “I couldn’t accept that explanation. I started thinking of ways to make cell lines.” Starting in 1997, Bonewald’s group began to report osteocyte lines, such as MLO-Y4, which gave researchers a better idea of what the cells actually do. Osteocytes act as the mechanosensors of bone, probably sensing changes in fluid flow and how the skeleton is weighted during rest or exercise, a hypothesis Bonewald says histomorphologists proposed decades ago. She says osteocytes are probably not important as mechanosensors in the embryonic skeleton or very active postnatally during growth. But they are extremely important in adults. Osteocytes direct osteoclasts and osteoblasts where to degrade old bone and set down new material. They also secrete hormones. “Instead of being thought of as pitiful cells that got confused and stuck inside bone, they are now thought of as the master cells,” says Kronenberg. “They are the brains of the outfit.”
Perhaps the biggest shift in how bone is perceived is in its function as an endocrine organ. Bone used to be thought of as a tissue that responded only to a couple of hormones, such as the parathyroid hormone sent out by the parathyroid glands and estrogen made by the ovaries. But findings in the past two decades have given indications that the bone doesn’t just passively take orders from other organs: It makes its own hormones to modulate mineral metabolism and energy expenditure.
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The role of bone in mineral metabolism came as a surprise less than 15 years ago when a hormone called fibroblast growth factor 23 was discovered. FGF23 “has potent effects on the proximal tubule of the kidney to regulate the reabsorption of phosphate,” says Kronenberg. “It was interesting and surprising when it was first realized that the major source of FGF23 was the osteocyte.” That osteocytes signaled to the kidneys when the body needed to hold onto phosphate alerted researchers to that fact that bone actively manages mineral metabolism.
The connection between bone and energy expenditure was first proposed by the group of Gerard Karsenty at Columbia University. His group used genetic approaches to show that leptin, the hormone released from fat tissue to regulate appetite and metabolism, inhibited bone formation through the nervous system. The work tied together appetite, energy metabolism and bone remodeling.
Osteocalcin was another surprise in the energy-expenditure picture. The protein has been cited in the literature for more than 40 years and is used as a marker for osteoblast activity. But “we didn’t know what osteocalcin did,” says Thomas Clemens at Johns Hopkins University.
In the 1990s, the Karsenty laboratory made a knockout mouse missing osteocalcin. The mouse was expected to have a bone phenotype, but, unexpectedly, it was a plump animal with only minor skeletal abnormalities. Around 2008, the Clemens group created a different mouse that lacked the insulin receptor on its osteoblasts. That mouse also became fat and looked just like the osteocalcin-null mouse that the Karsenty group had made a decade before. Like the Clemens group, the Karsenty group had made the mouse missing the insulin receptor in osteoblasts and had gotten the same phenotype. The mouse studies “linked insulin signaling in the osteoblasts to the production of osteocalcin,” says Clemens.
The thinking now goes that insulin stimulates osteocalcin production by osteoblasts. The osteocalcin molecule gets stored in the mineralized matrix. When osteoclasts dissolve bone, osteocalcin enters the bloodstream. From there, researchers have shown, one of the post-translationally modified forms of osteocalcin increases insulin secretion from the pancreas and enhances the ability of adipocytes to use glucose.
|Sclerostin inhibits osteoblast-mediated bone formation. Image credit: Amgen
Because bone remodeling demands a lot of energy, “this new paradigm really allows us to think about the skeleton as the sensor for metabolic activity and also as a fine-tuner for insulin sensitivity,” says Clifford Rosen at the Maine Medical Center Research Institute. “It takes a lot of energy to make bone. We don’t know anything about the dynamics of how these cells use their energy.”
The knockout mice have been critical in revealing osteocalcin’s purpose, but there is a question mark hanging over the extent to which osteocalcin influences the insulin pathway in humans, say Clemens and Rosen. “The mouse has given us tremendous insights, but moving to humans, it’s much more complicated,” says Rosen. “We need to get a better idea of how important is osteocalcin in fine-tuning insulin secretion.”
Clemens and Rosen explain that in some mouse models osteocalcin looks to be critical for regulating the insulin pathway. But mice aren’t metabolic equivalents of us, because their metabolic rates are 100 to 1,000 times faster than ours. Both Rosen and Clemens say the differences in metabolic rates raise the question of whether the osteocalcin effects seen in mice come about simply because of the peculiarities of mouse metabolism. “It’s a big challenge,” says Rosen. “How do we apply what we see in mice to humans?”
And that is exactly what the next research steps should answer, says Clemens. He says that, while association studies in humans seem to suggest osteocalcin has an effect on insulin secretion, there haven’t been any studies that show a clear cause-and-effect relationship. Those kinds of studies are begging to be done.
Understanding fundamental bone biology has had great repercussions for one of the most recognized diseases of bone: osteoporosis. Osteoporosis appears in postmenopausal women, the elderly and people suffering from some diseases, such as anemia. The bones become fragile and easily snap. According to the National Osteoporosis Foundation, about 34 million Americans are at risk for the disease. By 2025, the foundation projects, osteoporosis will cost the American healthcare system $25.3 billion per year.
Osteoporosis happens when osteoclasts outstrip osteoblasts in performance. The reason postmenopausal women are more at risk is thought to be that estrogen indirectly inhibits the activity of osteoclasts. But after menopause, estrogen’s protection disappears, and the osteoclasts start breaking down bone more quickly than osteoblasts can keep up. The speeding up of osteoclasts starts happening in the elderly for reasons yet to be deciphered. This causes elderly people to grow hunched, shrink in height and become more susceptible to broken bones.
But in the past 20 years, drugs have appeared to treat osteoporosis. Most of the ones on the market inhibit bone breakdown, or resorption, one way or another. One class of drugs is the bisphosphonates, which trigger apoptosis in osteoclasts. “The bisphosphonate category is probably about 80 percent of the osteoporosis drug use in the United States right now,” notes Art Santora of Merck.
Another drug is a monoclonal antibody called denosumab, which is produced by Amgen. It is an inhibitor of RANK ligand, which was shown in the 1990s to be the key stimulator of osteoclast development through the Wnt/β-catenin pathway. By inhibiting RANK ligand, the drug prevents osteoclasts from maturing and chewing away the bone.
All these drugs are catabolic agents in that they stop the breakdown of bone. Given their numbers, Scott Simonet of Amgen says, “That area of the market is pretty saturated.”
The excitement lies in drugs that can help build bone. The only anabolic agent on the market is a recombinant version of parathyroid hormone called teriparatide, marketed as Forteo by Lilly. The drug stimulates osteoblasts to put down new bone. Parathyroid hormone’s classical role is to stimulate bone breakdown so that calcium is released to maintain serum calcium levels. But, for reasons not yet known, the hormone does the opposite and builds bone when injected once daily. The drug is effective for only 12 to 18 months.
Experts are excited about a drug that Amgen, in partnership with a company called UCB, has in phase III clinical trials. Simonet says that the drug is being developed for osteoporosis and fracture repair. The anabolic drug AMG 785 is a monoclonal antibody that targets a molecule called sclerostin.
The story of sclerostin best illustrates how molecular biology has been pivotal for bone therapeutics. In 1958, sclerosteosis was first described in two South African girls of Dutch-Afrikaner descent. Sclerosteosis patients have heavy, thick bones with large jaws and protruding foreheads; their thick facial bones pinch their facial nerves. Several research groups established that the gene involved was SOST and that sclerosteosis was a loss-of-function mutation of that gene.
“We didn’t know where sclerostin was coming from, but, after several years of soul-searching, it became clear that it was coming from the skeleton,” says Rosen. Coincidentally, at the same time, the Bonewald group’s osteocyte lines were coming out. Those cell lines helped researchers establish in the mid-2000s that osteocytes were secreting sclerostin to stop bone formation. Amgen’s AMG 785 shuts down sclerostin by blocking its inhibitory activity on osteoblasts.
Clemens and others take delight in pointing out that researchers had known about sclerostin’s existence for many years. But once its molecular biology was established, it took less than a decade to get a drug against it in the pipeline. “It’s really remarkable,” says Clemens.
|Scanning electron micrograph of an osteocyte in resin. Click on the image to see in full resolution. Image credit: Lynda Bonewald
Lots more to do
Experts interviewed for this story were unified in their upbeat enthusiasm for the future of molecular biology research into bone simply because there are so many rich hunting grounds in both basic and clinical endeavors. Experts are unrestrained in their enthusiasm when they say that new anabolic drugs will be developed in the next decade to help patients with postmenopausal, age- or disease-induced osteoporosis and skeletal fragility.
For basic researchers, there are many directions to pursue. For one, they need to understand how bone cells communicate and respond to mechanical and biochemical signals both at local and systemic levels. For example, “bone is an incredibly locally focused tissue,” says Kronenberg. “If you break a leg, you want to fix that fracture right where it is. You don’t want a systemic response to a fracture.” How bone senses when to work locally and when to act globally is a question.
Another interesting idea that is emerging is that there is two-way communication between muscle and bone. “We always think of muscle affecting the skeleton” by pulling and pushing on the bones, says Rosen. “But there’s the converse side: How does the skeleton regulate muscle?”
This is work Bonewald has undertaken in collaboration with the groups of Marco Brotto and Mark Johnson, also at the University of Missouri in Kansas City. “We took some of our osteocyte-conditioned media and put it on muscle cells. [Brotto] was absolutely blown away to see that the osteocytes secrete factors that support myogenesis,” Bonewald says. “If you put the conditioned media on intact muscles that are contracting, it increases muscle force.” She says the collaborators are working on figuring out the factors secreted by the osteocytes and how they affect signal-transduction pathways in muscle.
Indeed, factors secreted by bones are high on the exploration list. This is especially true for the relatively new discovery that bone senses and possibly influences metabolism. In Rosen’s opinion, “the skeleton is secreting lots of endocrine factors. We know about FGF23, sclerostin and osteocalcin. We don’t know enough about those, and my guess is there are also other substances being produced.”
As schoolchildren, our earliest encounters with bone are with the jangling skeleton hanging in the back of the high-school biology laboratory. But with new findings emerging about the inner workings of the skeleton, bones can no longer be viewed as stiff and inert structures that simply hold us upright. Bone is truly a dynamic, living tissue that is constantly listening and responding to the way we live.
Rajendrani Mukhopadhyay (firstname.lastname@example.org) is the senior science writer for ASBMB Today and the technical editor for The Journal of Biological Chemistry. Follow her on Twitter (www.twitter.com/rajmukhop).