REVIEW
ol
n1
A, 6
net
Introduction
The first of these has been the complete change in our
Chapuy et al. (1997) took a different approach by assessing the
these two factors at concentrations of 25OHD up to 78 nM.
This indicated that optimal vitamin D status was much higher
than originally thought and prompted the introduction of a
defining the key genes involved in mediating innate immune
enzyme that catalyzes the synthesis of active 1,25-dihydroxy-
intracrine responses to 25OHD, most notably the induction of
261Journal of Endocrinology (2008) 198, 261–269 DOI: 10.1677/JOE-08-0170relationship between serum 25OHD and serum parathyroid
hormone. They described an inverse correlation between
vitamin D (1,25(OH)2D) from 25OHD (Liu et al. 2006). As a
consequence, the resulting macrophages demonstrated strongunderstanding of what constitutes normal vitamin D status.
For many years, vitamin D deficiency in humans was defined
by the serum concentration of 25-hydroxyvitamin D
(25OHD, the main circulating form of vitamin D) associated
with rickets (!8 ng/ml or 20 nM). However, in 1997
responses to Mycobacterium tuberculosis (Liu et al. 2006). The
DNA array analysis of macrophages activated with an
M tuberculosis lipoprotein highlighted the induction of
mRNA for both the vitamin D receptor (VDR) and
25-hydroxyvitamin D-1a-hydroxylase (CYP27b1), the‘vitamin D’ and ‘thyroid hormone’ were more or less equal at
w6300 entries each.
Two key concepts have fuelled the vitamin D renaissance.
impaired.
The second seminal development in vitamin D research
arose from the studies by Robert Modlin et al. aimed atVitamin D’ as one of its top ten ‘medical breakthroughs’ of
the year. The reason for this they stated has been the recent
remarkable increase in studies documenting new actions for
vitamin D and the potential significance this may have for
human health (http://www.time.com/time/specials/2007/
top10/). A simple investigation using PubMed shows that
since March 2003, there have been w9500 entries for the
keyword ‘vitamin D’, whereas ‘thyroid hormone’ generated
almost 6800 entries over the same period of time. This
contrasts with the preceding 5 years (1998–2003) where0022–0795/08/0198–261 q 2008 Society for Endocrinology Printed in Greatwith serum 25OHD concentrations that are higher than
‘deficient’ (i.e. adequate to prevent rickets) but lower than
optimal (Holick 2008). Although there is still a continuing
discussion concerning the precise levels of 25OHD, which
define these different categories of vitamin D status, there is
broad agreement that vitamin D insufficiency is prevalent in
populations across the globe (Holick 2007). This is
particularly true for individuals with dark skin pigmentation
or those living in Northern latitudes, where the capacity for
u.v. light-induced epidermal synthesis of vitamin D isAt the end of 2007, Time magazine listed the ‘Benefits of new term, ‘vitamin D insufficiency’, to describe individualsBack to the future: a new look at ‘
Rene F Chun1, John S Adams1,2 and Martin Hewiso
1Department of Orthopaedic Surgery, David Geffen School of Medicine, UCL
2Molecular Biology Institute, UCLA, Los Angeles, California 90095, USA
(Correspondence should be addressed to M Hewison; Email: mhewison@med
Abstract
Our perception of the vitamin D system continues to evolve.
Recent studies have re-evaluated the parameters for adequate
vitamin D status in humans, revealing a high prevalence of
insufficiency in many populations throughout the world.
Other reports have highlighted the potential consequences of
vitamin D insufficiency beyond established effects on bone
homeostasis. Most notably, there is now strong evidence of a
role for vitamin D in modulating innate and adaptive
immunities, with insufficiency being linked to infectious
disease and other immune disorders. To date, signalingd’ vitamin D
,2
15 Charles E Young Drive South, Los Angeles, California 90095, USA
.ucla.edu)
pathways for these new responses to vitamin D have been
based on established endocrine models for active 1,25-
dihydroxyvitamin D, despite present evidence for more
localized, intracrine modes of action. In the following review,
we provide a fresh perspective on vitamin D signaling in non-
classical target cells such as macrophages by highlighting novel
factors associated with the transport and action of this
pluripotent secosteroid.
Journal of Endocrinology (2008) 198, 261–269Britain Online version via http://www.endocrinology-journals.org

R F CHUN and others . Vitamin D262the antibacterial peptide cathelicidin (Liu et al. 2006).
Subsequent studies have confirmed that this is a pivotal step
in the innate immune response to infection by pathogens such
as M tuberculosis (Liu et al. 2007b, Martineau et al. 2007).
Crucially, ex vivo experiments showed that the induction of
macrophage cathelicidin is significantly compromised when
using serum from25OHD-insufficient donors (Liu et al. 2006),
indicating that similar responses in vivo may also be dependent
on vitamin D status.
The induction ofmacrophage function by locally synthesized
1,25(OH)2D has highlighted a link between vitamin D
insufficiency and tuberculosis, as well as a potential role for
vitamin D supplementation in preventing and/or treating this
disease (Liu et al. 2007a). However, in view of the pluripotent
nature of 1,25(OH)2D as an inducer of non-classical responses
(Holick 2006, 2007, Johnson et al. 2006, Norman 2006, Spina
et al. 2006, Adams & Hewison 2008) and the widespread
expression of CYP27b1 in cells other than macrophages
(Townsend et al. 2005, Hewison et al. 2007), it is likely that
vitamin D insufficiency will have broad-ranging biological
effects that extend beyond innate immunity. Thus, presentwork
within thevitaminDfieldhas expanded the analysis of the effects
of vitamin D status on disease epidemiology and clinical
outcomes (Holick 2007), while other studies have investigated
novel targets for locally synthesized 1,25(OH)2D (Adams &
Hewison 2008) or potential therapeutic uses for vitamin D
metabolites (Trump et al. 2006).
Significantly, this new perspective on vitamin D physiology
has also prompted a re-evaluation of the cellular and
molecular mechanisms associated with this secosteroid. In
particular, the broader acceptance of localized tissue-specific
metabolism as a key component of non-classical vitamin D
physiology suggests that established models for vitamin D
signal transduction based on endocrine mechanisms are
incomplete. For example, although there has been a
remarkable increase in our understanding of how liganded
VDR interacts with accessory proteins to facilitate chromatin
remodeling and the recruitment of transcriptional machinery
(Kato et al. 2004, Fujiki et al. 2005, Jurutka et al. 2007,
Yamaoka et al. 2007), much less is known concerning the
pathways that lead up to these nuclear events. In the following
review, we have addressed this by providing a new perspective
on vitamin D signal transduction, which goes beyond the
already well-documented actions of CYP27b1 and VDR.
The proposed model (see Fig. 1) has been designed
specifically to illustrate novel factors that may be integrated
to facilitate key intracrine effects of vitamin D, in particular
the induction of antibacterial innate immune responses.
Reflecting its title, the review also aims to illustrate the
importance of comparative and evolutionary biology as a tool
for better understanding the way in which vitamin D works.
Specifically, the review focuses on how the analysis of the
changes in vitamin D function, which have accompanied
alterations in environmental exposure to u.v. light, has helped
to identify new biological targets and modes of action for
vitamin D.Journal of Endocrinology (2008) 198, 261–269Vitamin D-binding protein (DBP): passive carrier
or active facilitator
In common with other steroid hormones, circulating vitamin
D metabolites are bound to serum carrier proteins in the
serum. Although this facet of vitamin D is well documented,
relatively little is known about its impact on the tissue
availability of vitamin D metabolites. Serum albumin and
serum DBP are the principle carriers of vitamin D. The
affinity of albumin for 25OHD (KaZ6!10
K5 M) and
1,25(OH)2D (KaZ5.4!10
K4 M) is substantially lower than
that of DBP (7!10K8 M and 4!10K7 M respectively)
(Bikle et al. 1985, 1986). The vast majority of vitamin D
ligands are bound to DBP but, because of its relative
abundance (650 mM compared with 5 mM for DBP), some
vitamin D metabolites are also carried by albumin.
Additionally, the vast majority of vitamin D carrier proteins
are empty because of their comparatively high levels in
relation to typical circulating levels of vitamin D metabolites
(in humans 25OHDZ50 nM and 1,25(OH)2DZ0.1 nM).
Despite the fact that almost all the available ligands are
likely to be bound to DBP, it has generally been assumed
that the relatively small free fraction of vitamin D metabolites
is the fraction that enters target cells (White & Cooke 2000,
Speeckaert et al. 2006). However, in some circumstances,
binding to DBP may be a more active feature of vitamin D
physiology. Nykjaer et al. (1999) showed that in the proximal
tubules of the kidney megalin, a cell surface receptor binds and
internalizes DBP via an endocytic pathway. Mice with the
ablation of the megalin gene are unable to recover DBP in this
fashion and thus lose vitamin D via the urine, with resulting
rachitic bone disease (Nykjaer et al. 1999). Subsequent studies
have further refined this mechanism to include cubilin
(Nykjaer et al. 2001) and disabled-2 (Nagai et al. 2005) as
facilitators of megalin action in the kidney.
Unlike the endocrine synthesis of 1,25(OH)2D in the
kidney, autocrine responses to vitamin D appear to be highly
dependent on the availability of substrate 25OHD. Thus, a
key question concerns whether target cell delivery of 25OHD
occurs as a consequence of receptor-mediated membrane
transfer as is the case in the kidney or whether it simply
involves passive transfer of the free metabolite. Two recent
studies have supported both models. In the first of these,
megalin-mediated uptake of DBP was described in T47D
breast cancer cells (Rowling et al. 2006). Further studies
confirmed that this mechanism was able to facilitate the
uptake of 25OHD by these cells and subsequent intracrine
activation to 1,25(OH)2D (Rowling et al. 2006). Although
the general applicability of this mechanism for cell-specific
acquisition of vitamin D metabolites has yet to be fully
defined, it is interesting to note that the expression of megalin
and cubilin has also been linked to intracrine synthesis of
1,25(OH)2D by bone-forming osteoblastic cells (van Driel
et al. 2006, Atkins et al. 2007).www.endocrinology-journals.org