S P E C I A L
P e r s p e c t i v e s
i n
F E A T U R E
E n d o c r i n o l o g y
Bone and Mineral Metabolism: Where Are We, Where
Are We Going, and How Will We Get There?
Henry M. Kronenberg
Endocrine Unit, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts
02114
Context: Advances in diagnosing and treating metabolic bone diseases will require ways to assess
cellular signaling within human bones, ideally noninvasively. Only then will we be able to fully
harness the increased molecular understanding of bone that derives from human genetics and
model organisms, primarily rodents. New hormones regulating mineral ion homeostasis surely
remain to be discovered, probably through advances in the study of human genetic disease.
ones are pretty amazing. Evolution designed them to
last a few decades and now they are expected to last
for as long as a century while leveraging our muscles, protecting our organs, regulating our calcium and phosphorus homeostasis, and supporting hematopoiesis. It is not
surprising that, despite their impressive design, bones fail
to protect us as we age: two million osteoporotic fractures
occur in the United States each year. We have found ways
to identify weak bones and strengthen them enough to
prevent a fraction of those fractures, but our understanding, diagnostic tools, and available therapies remain rudimentary. In the first part of this essay, I will briefly summarize current approaches to dealing with osteoporosis,
their limitations, and how we might progress. Then I will
discuss diseases of mineral metabolism.
B
Osteoporosis
We have fairly good ways to predict who might fracture by
combining measurements of bone mass with assessment of
epidemiological risk factors, most importantly age (1), but
we need new approaches to choose people for therapy
more appropriately.
ISSN Print 0021-972X ISSN Online 1945-7197
Printed in USA
Copyright © 2016 by the Endocrine Society
Received October 4, 2015. Accepted January 28, 2016.
First Published Online February 23, 2016
doi: 10.1210/jc.2015-3607
A major predictor of fractures is bone mass. We measure bone mass using dual-energy x-ray absorptiometry
(DXA), a tool with a precision unrivaled in clinical endocrinology. Nevertheless, most people who fracture have
bone density measured by DXA that is outside of the socalled “osteoporosis” range (T score ⬍ ⫺2.5). Part of the
explanation for this paradox is that most hip and forearm
fractures occur after falls, and some people fall a lot more
than others. But the limitations of DXA measurements
probably explain much of why some people fracture at
higher bone densities than others. What we really want to
measure is bone strength, not bone mass, so that we can
compare that strength to plausible stresses caused by falls.
Bone strength reflects the amount of bone (bone mass) but
also architectural parameters (for example, how the struts
of trabeculae connect together) and material properties of
bone (for example, how strongly collagen is cross-linked).
We are getting better at measuring architectural parameters now with the few high-resolution peripheral quantitative computed tomography (HR-pQCT) machines that
can identify trabeculae and cortical pores. But these machines too have limitations: the resolution is not yet great
enough to identify individual trabeculae satisfactorily and
Abbreviations: DXA, dual-energy x-ray absorptiometry; FGF, fibroblast growth factor; HRpQCT, high-resolution peripheral quantitative computed tomography.
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to measure degrees of cortical porosity accurately. Secondgeneration scanners may provide more useful information. Equally importantly, HR-pQCT today can only image peripheral bones: the tibia and radius, not the hip or
spine in which most fractures occur. And HR-pQCT, as an
x-ray technique, cannot begin to identify important material properties of bone. Minimally invasive techniques,
such as microindentation of bone (2), are beginning to
develop indices of bone’s material properties, but much
needs to be done to develop noninvasive ways to assess the
molecular parameters crucial to bone strength. It would be
wonderful to be able to distinguish the mineral phase from
the bone matrix and thereby diagnose osteomalacia noninvasively, for example. That is currently impossible, although investigations using magnetic resonance imaging
suggest that this may become feasible (3). Finite element
analysis technology, originally invented to predict the
strength of bridges and airplanes, has been applied to the
analysis of bone strength with some success, but this has
been limited by the available structural data used for the
calculations (4). One can hope that over the next several
years, continued progress in imaging will allow bioengineers to come up with better predictive estimates of bone
strength in the clinical context.
As impressive as the new imaging methods have been,
they look at bone matrix and ignore the complicated cell
biology responsible for laying down and destroying that
matrix. At the moment, the only way to get at those cells
is with bone biopsy, a technique sufficiently invasive that
patients do not flock to studies that involve biopsies. Bone
biopsies can teach us a lot about the cells buried in bone
(osteocytes) and on the bone surface (osteoblasts, lining
cells, and osteoclasts), but they also reveal our profound
ignorance of the mesenchymal cells even a few microns
away from the bone surface. Where do osteoblasts, the
cells that lay down the bone matrix on the bone surface, in
adult humans come from? At the moment, that’s anybody’s guess; no one knows for sure. With marrow aspiration, we can isolate cells that, when grown on plastic or
injected sc in mice, can become osteoblasts, chondrocytes,
and adipocytes. These cells can self-renew, so they are
called mesenchymal stem cells by some. They may prove
useful as reagents in regenerative medicine, but their roles,
if any, in actually forming bone in normal skeletal homeostasis has not been established. As yet, no one can identify
precursors of osteoblasts unambiguously on bone biopsies, and this means that no one has yet been able to study,
even in vitro and in animal models, the hormonal and
paracrine signals that regulate the proliferation, differentiation, and lifespans of osteoblast precursors. A number
of groups have used lineage-tracing technology and cell
surface markers to identify precursors of osteoblasts in
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vivo in mice, but these are all preliminary studies (5–9).
The lineage-tracing experiments identify groups of cells
that include the precursors of osteoblasts, but all of the
marked populations are genetically heterogeneous, probably with similarly heterogeneous fates. Still, the iterative
process of using multiple approaches to identify precursors of osteoblasts at differing stages of differentiation is
likely, in the next few years, to take us to where hematologists, who identified hematopoietic stem cells and their
progeny years ago, have been for some time. An important
goal would be to mark key osteoblast precursors in vivo in
humans in a way that would allow the tracking of the fates
of these cells and the effects of hormonal/paracrine manipulation and the effects of specific drugs and genetic
polymorphisms on those responses. Right now, therapies
for osteoporosis, although they target varying molecules
and pathways, do not distinguish patients on the basis of
the cellular basis of their disease. Only when we begin to
understand the variation in the cellular basis of disease
pathogenesis will we be able to devise and tailor therapies
to specific kinds of pathophysiology.
We have a few serological markers that reflect roughly
total rates of bone formation (for example, procollagen
I-amino-terminal peptide) or of bone resorption (for example, immunogenic fragments of collagen 1 containing
characteristic cross-link moieties [NTX and CTX]). These
assays that all measure portions of collagen I, the major
protein of bone and its precursor, procollagen, reflect the
action of osteoblasts to deposit bone matrix and osteoclasts to resorb the matrix, but they cannot get at the
mechanisms regulating the numbers and activities of the
bone-forming and -resorbing cells. Investigators are beginning to assess blood levels of local bone regulators,
such as the wnt antagonist, sclerostin (10), but the meaning of the levels in the blood of such proteins, which act
only a few cells away from the cells that make them, is not
known.
To understand the enormous variation in bone strength
between individuals and within individuals over time, I
suspect we will need new ways of assessing, in humans in
vivo, the activities of pathways known to regulate bone
cells, such as the wnt, notch, fibroblast growth factor
(FGF), bone morphogenetic protein, IGF, and TNF pathways and their molecular components, on cells in living
bone. We are not close to attaining that goal, but advances
in marking cells with antibodies and detecting metabolites, combined with new imaging modalities, may make
that goal attainable.
When we can identify relevant bone cells and their precursors in vivo, as well as the pathways controlling their
fates, one can imagine then being able to apply the endocrinologist’s tools, stimulation and suppression tests, to
doi: 10.1210/jc.2015-3607
bring out abnormalities in these cells and pathways. In the
case of bone, of course, the stimuli that are relevant include
not just chemical activators or inhibitors of pathways, but
also mechanical forces that can be applied to elicit important responses. When we find ways to assess the cellular
responses within bone, I am guessing that we may then be
able to exploit more fully the already substantial data from
genome-wide association studies regarding bone mass and
fractures. The goal would be to develop ways of assessing
the biology behind an individual’s bone mass/strength and
perhaps develop and identify individual therapies adapted
toward the specific biology of each individual. Right now,
the idea of personalized medicine in the osteoporosis
world has no real meaning. Therapies are designed for
broad epidemiologically defined groups and result in variable effectiveness. Our most powerful therapies, the anabolic agents, PTH (1–34) and antibodies to sclerostin,
work well but lose their effectiveness rather quickly for, at
the moment, completely unknown reasons. Some answers
will come from studies of genetically manipulated mice,
but we will need equivalently revealing studies in humans
to understand how these agents work and how to develop
better agents for strengthening bones.
Mineral Metabolism
The most exciting advance in the understanding of mineral
metabolism in the last decade was the discovery of the
phosphate-regulating hormone, FGF23. FGF23 acts on
the renal proximal tubule to decrease the resorption of
phosphate and to suppress the activity of the 25-hydroxyvitamin D 1-␣ hydroxylase. New hormones do not
come along all that often any more, so it is worth reflecting
upon how the actions of FGF23 were discovered and why
they were not discovered sooner. The latter question is
easy to answer; there is no FGF23 gland, so that the classic
approaches of endocrinology, gland ablation and hormone purification from glands, was not possible. FGF23
is made primarily by osteocytes, the major cells in bone,
surrounded by bone matrix (containing most of the body’s
phosphate, of course), so that traditional gland ablation
experiments could not be contemplated. In fact, the actions of FGF23 were discovered serendipitously when examining the causes of inherited forms of rickets. Most
revealingly, in autosomal dominant hypophosphatemic
rickets, the mineralization disorder was caused by point
mutations in the coding region of FGF23. This result was
surprising because, before the discovery of FGF23, there
were no obvious physiological mysteries that demanded
the presence of a new important hormone. Nevertheless,
we now know, largely through the studies of FGF23
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knockout mice, that normal levels of FGF23 are required
for normal phosphate and vitamin D homeostasis. It took
human genetic studies and studies of a cancer syndrome,
tumor-associated osteomalacia, to demonstrate potential
roles for FGF23 in humans.
The serendipitous path to discovery of the roles of
FGF23 in mammals has an important implication regarding the completeness of our list of important regulators of
calcium and phosphate homeostasis. We have to anticipate that the list is incomplete. Further discoveries of such
hormones are likely to come from further delineation of
genetic causes of mineral disorders. It seems likely that the
use of exome and whole genome deep DNA sequencing
will reveal such new hormones in the next few years, as all
the Mendelian diseases yield their mysteries.
That FGF23 was discovered only a few years ago also
explains why we really know very little yet about FGF23
regulation and action. We know that very low levels of
blood and total body phosphate are associated with low
levels of FGF23 and that high levels of phosphate are associated with high FGF23 levels. But the changes in FGF23
levels in response to acute changes in blood phosphate
occur slowly, and it seems clear that the regulation of
FGF23 by phosphate is not a simple relationship analogous to the way that calcium regulates PTH secretion. It is
not yet clear, for example, whether osteocytes directly
sense changes in blood levels of phosphate and change the
secretion of FGF23 accordingly. And, if phosphate does
regulate FGF23 production and secretion in osteocytes
directly, the intracellular mechanisms that phosphate regulates are not at all clear. Cells use sodium-phosphate
transporters to move phosphate into cells, and some cells
respond to the increase of intracellular phosphate by
changing activation of kinases such as ERK1 and -2. But
whether these kinase pathways or others in osteocytes regulate FGF23 synthesis and secretion in response to
changes in extracellular phosphate remains to be
determined.
We are only beginning to understand how FGF23 acts.
FGF23 is a member of the large FGF family of ligands,
most of which work by activating FGF receptors 1– 4. The
activation of this receptor family by FGF23 is weak and
requires the presence of an FGF23-binding coreceptor,
klotho. The genetic evidence that klotho mediates the
specificity and actions of FGF23 is substantial, but many
mysteries about klotho remain. Soluble forms of klotho
circulate, and their roles in mediating actions of FGF23
and/or independent actions should be clarified in the next
few years. FGF23 not only regulates phosphate levels and
1,25-dihydroxyvitamin D levels through actions on the
proximal tubule, but also increases calcium and sodium
reabsorption by activating the TRPV5 channel and by in-
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creasing the abundance of the sodium– chloride cotransporter, respectively, in the distal tubule. These actions of
FGF23, and doubtless others, do not fit easily into a tidy
role for the hormone primarily in phosphate metabolism.
We need to be prepared for a more encompassing paradigm to explain the physiological roles and regulation of
FGF23.
Not all of the actions of FGF23 require the presence of
klotho. When klotho is removed from parathyroid cells in
vivo, FGF23 can still decrease PTH secretion (11). Strikingly, high levels of FGF23, like those seen in renal failure,
can increase cardiac hypertrophy in an apparently klothoindependent fashion (12). Because FGF23 is a better predictor of mortality in renal failure than measures of phosphate burden, this direct klotho-independent role of
FGF23 in the heart will continue to receive much deserved
attention.
The excitement about FGF23 should not cause us to
forget how little we understand about PTH. Perhaps the
largest mystery about PTH secretion is still our ignorance
of the mechanisms whereby high extracellular calcium
suppresses PTH secretion. The efforts of Brown (13) and
others have shown that the calcium-sensing receptor
somehow responds to changes in extracellular calcium levels by activating a number of intracellular signaling pathways that mediate the suppression of PTH secretion. But
how these changes in intracellular signaling lead to suppression of PTH secretion remains a complete mystery.
Undoubtedly, the continued absence of a cell line that
mimics the properties of parathyroid chief cells has held
back progress. Perhaps the use of three-dimensional scaffolds or other approaches successful with culture of other
epithelial cell types will provide a suitable model to allow
advances in solving this remaining mystery.
The last part of the title of this essay is “how we will get
there.” Readers may have noticed that I’ve had an easier
time talking about what we need to learn than how we will
learn it. The good news, of course, is that the methods for
discovery in human physiology and disease have never
been stronger. Powerful animal models, undoubtedly using CRISPR-Cas technology not just to change genes in
mice and cells but also in nonmurine animal models, will
allow the generation of relevant animal models. Whole
genome sequencing should lead to the identification of the
causes of all diseases inherited in simple Mendelian fashion and greater understanding of polygenic diseases. But,
undoubtedly, new technologies, undreamt of yet, will allow further progress in the next decade. Mechanisms for
the design of sophisticated clinical trials, along with increasingly sophisticated ways of extracting important
clues from the “big data” sources made possible through
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the introduction of computerized databases for managing
patient care will provide ways to apply the insights of
discovery science to the cure and treatment of disease. I’m
not worried about a scarcity of good ideas if we can convince our government and other funding sources to support this effort.
Acknowledgments
Address all correspondence and requests for reprints to: Henry
M. Kronenberg, Massachusetts General Hospital, 50 Blossom
St, Endocrine Unit, Thier 1101, Boston, MA 02114. E-mail:
[email protected].
This work was supported by National Institutes of Health
Grant DK011794.
Disclosure Summary: H.M.K. consulted for Novartis and
performed research sponsored by Amgen.
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