Osteoporosis is a condition in which the body’s bones become weak and brittle. There are more than 10 million cases of osteoporosis every year in India, and it disproportionately affects ageing women more than men. The hormone oestrogen plays a crucial role in this condition because it stimulates the growth and formation of new bone. After menopause, the decreased function of ovaries leads to oestrogen being depleted in the body, resulting in the loss of bone mass.
In a recent study published in the journal Nature, researchers at the Universities of California in San Francisco and Davis reported uncovering a novel brain-derived hormone that they say is responsible for increased bone mass in postpartum lactating mothers. The hormone is called CCN3.
A ‘secret’ path
Oestrogen plays a crucial osteoanabolic role: it stimulates the growth and formation of new bone. Consider oestrogen as a manager who tells (or signals) the members of her bone construction crew when to start and finish their jobs. During breastfeeding, the body signals to suppress oestrogen production in the ovaries, diverting energy away from the reproductive system to focus on milk production. This drop in oestrogen should lead to weaker bones.
But surprisingly, mothers’ bones become stronger in this time to meet the high calcium demands of their babies and to make up for bone loss during pregnancy.
As a result, scientists have suspected that there is another way in which the body strengthens bones, independent of oestrogen.
For their studies, the researchers started with mice genetically modified to not produce a protein called oestrogen receptor alpha in the hypothalamus. Through systematic studies with these mice, they found that specific neurons, called KISS1 neurons, used the CCN3 hormone to maintain bone mineralisation during lactation.
CCN3 belongs to the CCN family of proteins. They are involved in several biological processes, including embryonic development, tissue repair, wound healing, and cancer progression.
Previous work has shown that KISS1 neurons are located in the arcuate nucleus (ARC), a critical part of the hypothalamus that regulates metabolism, reproduction, and bone health. Scientists also know KISS1 neurons are key to regulating bone mass in females.
A brain-derived factor?
The body maintains its bone mass — not gaining too much, not losing too much — by maintaining a balance between skeletal stem cell-based bone formation and osteoclast-based bone resorption. Skeletal stem cells give rise to bone tissue, while osteoclasts are cells that break down bone tissue.
The researchers isolated a specific skeletal stem cell population from wild-type mice and transplanted them into mutant mice. The result was higher bone mineralisation in the mutant female mice.
The researchers also delivered such skeletal stem cells near the ARC of the mutant mice using minimally invasive surgery, and recorded the appearance of mineralised bone-like structures in the hypothalamus.
These structures didn’t appear in wild-type mice, however, implying the presence of a brain-derived factor that promoted bone formation in the mutant mice.
Mounting a unique response
To identify the factor driving these changes, the researchers put the mutant mice on a high-fat diet known to affect the function of KISS1 neurons, including the production of the suspected factor.
As expected, the mutant mice lost bone mass on this diet. The researchers extracted RNA from the ARC of these mice and sequenced it. They found that two genes, called Ccn3 and Penk,had been upregulated earlier but whose effects were lost after the high-fat diet began.
A gene is said to be upregulated when a cell expresses it more because it needs to produce more of the protein the gene encodes for. (The opposite goes for downregulation.)
Of the two genes, only the hormone produced by the Ccn3 gene — called CCN3 — was found to be located in all the KISS1 neurons in the mutant mice.
Importantly, the researchers found that CCN3 had been explicitly upregulated in mutant female mice but not in wild-type female and male mice or in mutant male mice. This suggested that the mutant female mice, which were unable to sense oestrogen because they lacked oestrogen receptor alpha, mounted a unique response. It highlighted a potential sex-specific regulatory mechanism of bone formation involving CCN3.
The team also found CCN3 increased the frequency with which (or the absolute number of) skeletal stem cells matured into cells that form bone and cartilage. CCN3 enhanced the potential of skeletal stem cells to mature into these bone- and cartilage-forming cells.
Thus, the hormone helped produce more cells that built bones and cartilage and made these cells better at doing their job, leading to stronger and healthier bones.
A dose-dependent response
Finally, the researchers conducted a series of experiments to establish the role of CCN3 as an osteoanabolic hormone, i.e. that it is involved in making bone. When they extracted skeletal stem cells from wild-type mice and cultured the cells with the CCN3 hormone, they recorded a 200% increase in mineralisation.
They were able to replicate this result in male and female human skeletal stem cells as well, confirming its broad applicability.
Ex vivo and in vivo experiments further validated these findings. Low doses of mouse CCN3 significantly increased the bone mass of young and aged femurs and of adult wild-type mice. In two-year-old male mice with bone fractures, treatment with CCN3 accelerated bone repair — and higher the dose, the faster was the repair.
In mice, the researchers also reported that CCN3 is absent during early and late pregnancy, appears within seven days after birth, and drops again as lactation decreases. Deliberately reducing the amount of CCN3 hormone in KISS1 neurons before pregnancy didn’t affect a mouse mother’s fertility or ability to produce milk.
But when mothers with low CCN3 levels were lactating and had a low-calcium diet, they had lower bone density, which negatively affected the survival of their offspring.
A new treatment choice?
These findings prove the CCN3 hormone secreted by KISS1 neurons in the ARC part of the hypothalamus is crucial to maintain maternal bone mass, at least in mice.
The groundbreaking study also revealed a pathway in which hypothalamic neurons sidestep conventional routes to directly release hormones into the bloodstream, providing fresh perspectives on the communication between the brain and the body in mice. It also emphasises the need to allocate funds for research on female health and biology.
The identification of CCN3 presents new opportunities to investigate its potential as a therapeutic agent for hereditary and chronic skeletal disorders, broadening the range of treatment choices for osteoporosis.
Adhiraj Goel is joining the biology department at the Massachusetts Institute of Technology as a post-baccalaureate scholar in 2024. He has a keen interest in science communication.
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