Bone is formed in a unique, highly complex balancing act. Essentially, old bone makes way for new. Old bone cells get dismantled and absorbed into the body, while new bone cells – called osteoblasts – begin as special stem cells in the bone marrow.
In the balancing act, adult bone size, shape, strength, and structural integrity have to be maintained. Too much old bone cannot be removed, and too much new bone cannot crowd into the space.
The specialized cells that dismantle old bone are called osteoclasts. When the body needs more old bone to be dismantled, it makes new osteoclasts. The “birth” of new osteoclasts is actually called osteoclastgenesis.
What has not been fully known are the exact molecular processes the body uses to stop osteoclastgenesis and prevent too many new osteoclasts from being made. Now, an HSS-led collaboration of rheumatologists and scientists has made key new discoveries about specific cellular signaling that can halt osteoclastgenesis.
The study revealed that an integrin pair of the Beta 2 family known as CD11b/CD18 is able to use two complementary molecular mechanisms that can stop osteoclastgenesis. Reacting to changing environmental conditions in the body, the integrin signaling can prevent too many new osteoclasts from being make, maintaining the integrity of bone.
Without the CD11b/CD18 beta 2 integrin, osteoclastgenesis is not signaled to stop, new osteoclasts keep getting reproduced, and dismantle too much bone.
The study found that the beta 2 integrin can signal either of two proteins that can halt important processes that lead to more new osteoclasts. Those proteins are B-cell lymphoma 6 protein – or BCL6 – and receptor activator of nuclear factor kappa-B ligand – or RANKL.
CD11b can signal either of those proteins to stop osteoclastgenesis by preventing the molecular action of another protein called NFATc1, which stands for nuclear factor of activated T-cells, cytoplasmic 1. NFATc1 is known as the master transcription factor of osteoclastgenesis. Until now, this key role of CD11b/CD18 Beta 2 signaling in the end cycle of bone life was not known.
The study was recently published in the Journal of Bone Mineral Research. HSS Authors include Chief Scientific Officer and Richard L. Menschel Research Chair Steven R. Goldring, MD; Associate Chief Scientific Officer and David M. Koch Chair Lionel B. Ivashkiv, MD; and Associate Scientist and Director of the HSS Osteolysis Lab Edward Purdue, PhD.
The research team studied special mice that were bred without the CD11b integrin. As the mice grew, they showed decreased bone mass that was associated with increased numbers of osteoclasts not offset by increased new bone formation.
If too many osteoclasts are getting made, and the mice have no CD11b integrin, that means some action normally taken by CD11b must be necessary to stopping osteoclastgenesis.
New osteoclasts are formed in an intricate process of molecular fusion involving immune system proteins called monocytes and macrophages. These earliest molecular “raw materials” of new osteoclasts are called precursors. At a certain point in development, new osteoclasts are said to “differentiate” themselves from the precursors. Osteoclastgenesis is like any process requiring “raw materials”, when the body needs new osteoclasts, it has to first make more precursors.
On a very basic level, when an adult body needs any new cell, it makes an exact copy of an existing one. The genes of the DNA inside the existing cell’s nucleus have their genetic information precisely transcribed so those genes can be reproduced. This gene transcription is conducted by proteins known, appropriately, as transcription factors. The most important transcription factor in osteoclastgenesis is NFATc1.
To make sure that NFATc1, or any transcription factor, can only transcribe the genes of specifically needed cells, many things have to happen. Importantly, the genes inside the cell the body needs to reproduce have to be made open to transcription.
During gene transcription, proteins called transcriptional coregulators can be recruited to insure only the proper genes are open. There are, essentially, two kinds of coregulators. Their names describe what they do. Co-activators activate gene transcription by making genes open to transcription. Co-repressors repress transcription by making genes chemically unavailable, so no transcription can happen.
BLC6 is a corepressor. Its job is to stop transcription of genes of cells like osteoclast precursors whose reproduction would lead to more osteoclasts.
Like any protein, NFATc1, itself, has genes. As changing conditions demand, the body can recruit other necessary proteins to act on the genes of NFATc1. When its needed, BLC6 can be recruited to act on NFATc1‘s genes so that NFATc1 finds that genes inside a particular cell are not open to transcription. No transcription, no reproduction, no new osteoclasts.
What the study found is signaling from CD11b does this BLC6 recruitment. Mice who had no CD11b, lacked the integrin signaling necessary to recruit BLC6 to act on NFATc1 to shut genes to transcription. Without the beta integrin signaling, BLC6 did not act on NFATc1, so the transcription factor kept on transcribing. More transcription, more reproduction, too many new osteoclasts.
Because BLC6’s normal actions of preventing transcription would hold down the number of osteoclasts that get made, in scientific terms, BLC6 is described as “negatively regulating” osteoclastgenesis. The study is the first discovery that CD11b beta integrin signaling is required for BLC6 to properly do its job of negative regulation.
The study also found how the integrin signaling was required for another protein to be triggered into proper action. That protein is known as RANKL.
RANKL is involved in cellular activities concerning both old and new bone. RANKL on the surface of osteoblasts can activate receptors on the surface of osteoclasts, thus new bones cells can be involved in regulating activities of the cells that dismantle old bone. The receptors on the osteoclasts that RANKL activates are called, appropriately, RANK, which stands for receptor activator of nuclear factor kappa-B.
The HSS study found that CD11b is required to stop RANKL from activating RANK to trigger a chain of cellular processes necessary to make new osteoclasts. That chain includes RANK inducing NFATc1 into action to do the necessary genetic transcribing that will ultimately result in new osteoclasts.
The CD11b beta integrin signaling is needed to stop the entire chain of RANKL to RANK to NFATc1 molecular mechanisms. Without that stop signal from CD11b, the genetic transcription that leads to new osteoclasts keeps happening.
For the first time, CD11b is now revealed as a negative regulator of the earliest stages of osteoclast differentiation. Using two complementary mechanisms, both of which affect NFATc1 behavior, CD11b can react to changing environmental conditions in the body and signal for osteoclastgenesis to stop, preventing new osteoclasts from being made, and maintaining the integrity of bone.
Because CD11b is an integrin it can well respond to changing conditions in the body. Integrins are receptors on a cell’s membrane that receive information from both the immediate environment surrounding a cell and internal conditions within the cell. Integrins use this inside/outside information to regulate aspects of cell life including cell shape, size, motility, life cycle, reproduction, and attachment to other cells and tissue,
Integrins mediate activity between cells and can send signals that trigger actions by other cells, regulating the life of the cell as conditions in the body require.
Integrins can come in pairs, clusters and sub-units. There are hundreds of them inside the body. Integrins are also classified into families, further specifying their cellular location. The CD11b/CD18 pair in the study are part of the Beta 2 family, also written as ß2. Integrins of the ß2 family are only expressed on the membranes of leukocytes, for example.
Like any citizen of the U.S. might be referred to as “the American”, integrins are sometimes described using only their family name, like “the beta 2 integrin”. No matter the name used, the ß2 integrin CD11b/CD18 has now been revealed to play an essential role in osteoclast reproduction.
The study has implications across the HSS specialties of orthopedics and rheumatology. The chronic inflammation of autoimmune conditions can prematurely degrade bone, harming joints. The thinning of bone in osteoporosis is a result of osteoclast activity not being compensated by osteoblasts building new bone.
There is an unwelcome inflammatory reaction to a joint replacement called osteolysis that affects how bone and implant interact. The inflammation of osteolysis can trigger osteoclasts into action, damaging the bone. Osteolysis can ultimately result in implant failure and revision surgery. HSS is one of the few academic institutions with a dedicated, interdisciplinary osteolysis research program.
Knowing key cellular components of osteoclast activity provides new ways to monitor patient condition and new targets for developing therapies to prevent premature reabsorption of bone in many conditions including arthritis, inflammatory arthritis, autoimmune conditions and osteolysis. Further research will help refine and expand the avenues of therapeutic opportunity opened by these discoveries.
HSS and Seoul National University
College of Medicine:
Kyung-Hyun Park-Min, MD
Eun Young Lee
Weill Cornel Medical College:
Chuanxin Huang, PhD
Ari M Melnick, MD
University of Connecticut Health Center:
Sun-Kyeong Lee, PhD
Joseph A Lorenzo, MD