Vitamin K2: A Vitamin that Works like a Hormone, Impinging on Gene Expression

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Chapter 5
Vitamin K2: A Vitamin that Works like a Hormone,
Impinging on Gene Expression
Jan Oxholm Gordeladze
Additional information is available at the end of the chapter
http://dx.doi.org/10.5772/intechopen.80388
© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,
and reproduction in any medium, provided the original work is properly cited.
Vitamin K2: A Vitamin that Works like a Hormone,
Impinging on Gene Expression
Jan OxholmGordeladze
Additional information is available at the end of the chapter
Abstract
Vitamin K2 binds to the intranuclear receptor SXR and results in the activation of a pleth-
ora of genes, both directly and indirectly. Among these genes are important biological
markers of cellular characteristics or features (also known as cell phenotypes), as well as
a set of molecules known to be involved in both hormone-induced, G-protein-mediated
cell signalling, either directly or indirectly activating so-called sirtuins and/or histone
deacetylaces (HDACs), known as determinants of cell types and their specic functions
in a given tissue. Hence, vitamin K2 may be closely involved in or serving as a traditional
molecular ‘link’ between hormonal receptors and intracellular signalling pathways. It
has been stated that a true hormone is a product of living cells, which circulates in body
uids (such as blood) and elicits a specic and often stimulatory eect on the activity of
cells situated remotely from its point of origin. A large bulk of evidence published over
the past 10 years establishes vitamin K2 in this category of substances. Hence, vitamin K2
should be considered and consequently classied as a hormone.
Keywords: vitamin K2, SXR, G-proteins, vitamin A-D-K2 cascade, bioinformatics,
in vitro model systems
1. Introduction
Vitamin K has since more than 25 years been known to serve as a powerful nutrient factor
in the preservation of homeostatic bone turnover, along with blood cloing biochemistry. In
addition, vitamin K has been used as a therapeutic remedy in the clinic to treat and prevent
bone brileness (osteoporosis) in Japan and many other countries around the world. Moreover,
beyond its enzymatic character as a cofactor for the vitamin K-dependent GGCX (gamma-
glutamyl carboxylase), Professor Inoue and his co-workers have shown that the K2 variant is,
© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

in fact, a transcriptional modulator of osseous marker genes, also serving as an extracellular
matrix-related molecule, via the stimulation of the ‘steroid and xenobiotic receptor’ SXR [1]. A
microarray-based revelation of the present action of vitamin K2 in bone-derived osteoblastic cell
genes corroborated the notion that the K2 variant menaquinone-4 (MK-4), in fact, is a hormone.
Among the signicantly upregulated genes, both growth dierentiation factor 15 (GDF15) and
stanniocalcin 2 (STC2) were concluded to serve as novel target genes, which both circumvented
the traditional GGCX- and SXR-mediated pathways found in osteoblast-like cells.
2. Vitamin K2: vitamin and hormone
The induction of both GDF15 and STC2 genes is construed as specic to MK-4, since it was
shown not to be brought about by another vitamin K(2) isoform MK-7, vitamin K(1) or the
MK-4 side chain containing the geranylgeraniol group. A survey into the signalling pathways
in question indicated that MK-4 sustained phosphorylation of protein kinase A (PKA) and
that MK-4 mediated upregulation of genes, such as GDF15 and STC2, was diminished by the
exposure to a PKA inhibitor or by siRNA constructs against PKA. These observations were
in line with the concept that vitamin K(2) was capable of modulating its own target gene
expression in bone-derived (osteoblastic) cell entities through a PKA-driven pathway, which
in essence was dierent from the traditional vitamin K-dependent signalling pathways [2].
Vitamin K2 has been included as a member in a group of molecules, constituting the ‘require-
ment’ for blood to coagulate; however, it has been demonstrated to function or serve as a key in
the homeostasis of osseous tissue, thus showing eectiveness as one therapeutic agent, among
others, in the curative treatment portfolio of bone ailments and diseases, i.e. like bone loss or
brileness (osteomalacia and osteoporosis). Furthermore, it has, since several decades, been
acknowledged that vitamin K2 mediates transcriptional modulation of marker genes in osteo-
blastic cells, as well as reinforcing bone formation via the nuclear steroid and xenobiotic recep-
tor, SXR. In this context, Dr. Satoshi Inoue and his research team identied several genes, which
were upregulated by vitamin K2 (and a prototypical SXR like ligand, rifampicin) in osteoblastic
cells, through microarray analysis and PCR. A plethora of genes was upregulated, among which
collagen synthesis and accumulation in osteoblast-like MG63 cells were enhanced several times
over by vitamin K2 treatment. Therefore, the results of Dr. Inoue and his research group more
than suggested a novel function for vitamin K2 in the formation of osseous tissues, i.e. that K2
was a true transcriptional regulator of extracellular matrix-related genes, being involved in the
assembly of collagen. At present, we know that vitamin K2 (or menaquinone-7 = MK-7, among
other vitamin K2 metabolites) works through this nuclear receptor and consequently should be
classied as a hormone and not solely be construed as ‘a vitamin’.
Arterial stiness is always associated with an enhanced cardiovascular risk, morbidity and
mortality [3]. The present article reviews the main vitamins being involved in arterial sti-
ness and enabling destiening; their mechanism of action, providing a brief description of
the latest studies in the area; and their implications for primary cardiovascular prevention,
clinical practice and therapy. Despite inconsistent proof for ‘softening’ brought about by
vitamin supplementation in a plethora of clinical trials, promising results were observed in
Cell Signalling - Thermodynamics and Molecular Control66

selected populations. The chief mechanisms pertain to anti-atherogenic potential, substantial
augmentation of endothelial functionality (pertaining to vitamins A, C, D and E, respectively)
and general metabolic proling (pertaining to vitamins A, B12, C, D and K, respectively), sup-
pression of the renin-angiotensin-aldosterone (R-R-A) system (vitamin D), anti-inammatory
(vitamins A-D-E-K) and antioxidant eects (vitamins A-C-E), diminished homocysteine lev-
els (vitamin B12) and a reversal of the calcication of arteries (vitamin K). Vitamins A, B12,
C, D and E, as well as vitamin K status, are important in evaluating the risk of cardiovascular
events, and, nally, supplementation with vitamins may serve as an ecient, individually
based and less costly ‘destiening’ therapeutic mode.
Vitamin K is renowned for being an important (vital) nutrient, sustaining both bone homeo-
stasis and blood coagulation. Therefore, it is both clinically and ubiquitously used as a thera-
peutic agent or treatment for osteoporosis in Japan and western countries. Besides its powerful
enzymatic, cofactor role of the vitamin K-dependent g-glutamyl carboxylase (GGCX), it has
since long been known that vitamin K2 may serve as a transcriptional regulator of marker
osteoblastic genes, as well as in matrix-related, extracellular genes. In this context the activa-
tion of the so-called steroid and xenobiotic receptor (known as SXR) is mandatory.
Hence, genes known to be upregulated by vitamin K2 isoforms like menaquinone-4 (MK-4)
were applied using oligonucleotide-based microarray analyses. Among the MK-4 upregu-
lated gene species, the growth dierentiation factor 15 (GDF15) and stanniocalcin 2 (STC2)
were discovered as new MK-4 target genes, being independent of the GGCX and SXR path-
ways in osteoblastic cells. The observed induction of GDF15 and STC2 was construed as
specic to MK-4, since it was not seen with exposure to MK-7 or vitamin K1. Surprisingly, a
scrutiny of the main signalling pathways showed that MK-4 stimulated PKA (protein kinase
A) phosphorylation. Furthermore, the MK-4-dependent induction of both GDF15 and STC2
genes was obliterated subsequent to the treatment with PKA inhibitors or siRNAs against
PKA. Therefore, it was postulated that vitamin K2 (MK-7) could modulate target gene expres-
sion in osteoblastic cells via PKA-dependent mechanisms, which were distinct from any pre-
viously known vitamin K-mediated signalling pathway.
The paper found that vitamin K2 is recognised, along with calcium, vitamin D and magne-
sium, as essential in supporting strong bones and healthy arteries. In the paper, Nutritional
strategies for skeletal and cardiovascular health: hard bones, soft arteries, rather than vice versa, the
authors cite a US Surgeon General’s Report that states that one in two Americans over 50 is
expected to have or to be at risk of developing osteoporosis, which causes 8.9 million fractures
annually, with an estimated cumulative cost of incident fractures predicted at $474 billion
during the next 20 years in the USA.
Furthermore, a study conducted by the Mayo Clinic [4, 5] reported that ‘compared with 30
years ago, forearm fractures have risen more than 32% in boys and 56% in girls’. Meanwhile,
strong epidemiological associations exist between decreased bone mineral density (BMD)
and increased risk of cardiovascular (CV) disease. For example, individuals with osteoporosis
have a higher risk of coronary artery disease and vice versa. This problem will be magnied,
according to the paper, if the therapies for osteoporosis (calcium supplements) independently
increase risk of myocardial infarction. To that end, the authors conducted a comprehensive and
Vitamin K2: A Vitamin that Works like a Hormone, Impinging on Gene Expression
http://dx.doi.org/10.5772/intechopen.80388
67

systematic review of the scientic literature to determine the optimal dietary strategies and
nutritional supplements for long-term skeletal health and cardiovascular health. They sum-
marised what is helpful for building strong bones while maintaining soft and supple arteries:
• Obtain calcium from dietary sources (the best choice is a calcium hydroxyapatite) with the
adequate animal protein, fruit and vegetable intake.
• Concomitantly increase potassium consumption, while reducing sodium intake should be
taken into account.
• Maintain vitamin D levels in the normal range.
• Increase the intake of foods rich in vitamins K1 and K2.
The study notes: ‘A meta-analysis concluded that while supplementation with phytonadione
(vitamin K1) improved bone health, vitamin K2 was even more eective in this regard. This
large and statistically rigorous meta-analysis concluded that high vitamin K2 levels were
associated with reduced vertebral fractures by approximately 60%, hip fractures by 77% and
all non-vertebral fractures by approximately 81%. Supplementation with vitamin K2 as MK-7
increased bone strength in postmenopausal women in 3-year clinical study’.
‘Additionally, increased vitamin K2 intake has been associated with decreased arterial cal-
cium deposition and the ability to reverse vascular calcication in animal models. Moreover
clinical trial proved that vitamin K2 supplementation increases elasticity of the arteries (in
3 years)’, the paper stated. The authors recommend increasing the intake of foods rich in
vitamins K1 and K2 to secure skeletal and cardiovascular health. ‘The positive health poten-
tial of vitamin K2 is more eective than for vitamin K1, the paper reads. Yet, Dr. Hogne
Vik, Chief Medical Ocer with NaoPharma, world leader in vitamin K2 R&D, exclusive
global supplier of MenaQ7 vitamin K2 as MK-7, and sponsor of the 3-year studies cited in
the paper, explains that it is not possible to get sucient amounts of vitamin K2 through a
European or US diet’.
The only food that contains enough vitamin K2 is the Japanese dish Nao. ‘This means that
if you want to get enough vitamin K2 into your body, then you have to take dietary supple-
ments or functional foods containing vitamin K2’, he said. ‘We are gratied, but not surprised,
that our 3-year clinical studies were cited in this paper’, Dr. Vik continues. ‘NaoPharma has
driven the clinical research that has demonstrated vitamin K2’s benets for human health,
and our breakthrough studies provided the rst intervention data conrming the associations
that observational studies made previously: that vitamin K2 as MK-7 is available beyond the
liver to support bone and cardiovascular health. And it does this by activating proteins that
help the body to properly utilise calcium – there by simultaneously supporting both skeletal
and cardiovascular health’.
How are we going to interpret the above-described information about the mechanism of
action of vitamin K2? Well, according to the denition given in dictionaries and scientic
papers, vitamin K2 (as in the form or MK-4 or MK-7) ts this denition and should be classi-
ed as one.
Cell Signalling - Thermodynamics and Molecular Control68

3. What features characterise a true hormone?
To make the picture clearer, let us start all over again. According to Figure 1, the impact of
a hormone is one signal among dierentiation signals (also called epigenator factors), which
may be temperature variations, oxygen tension, mechano-stimulation or humoral factor/
hormones. The ‘hormone’ eventually activates a transcription factor or microRNA synthesis,
which may impinge upon DNA in terms of a certain or given spectrum of mRNAs appearing
in the cytosol of the cell. Of major interest here are eventually two classes of molecules utmost
important for the acquisition of the nal cell phenotype—the histones and the sirtuins (see
Figure 5).
The response elicited by a single hormone (epigenator) may look like the network of interact-
ing factors (mostly transcription factors), such as the network representing the closely cooper-
ating network (mostly represented by transcription factor), as seen in Figure 2a and b.
The reader is recommended to look up the remainder of the genes shown above and will be
amazed as to the plethora of biological eects being modulated (directly and/or indirectly)
by the nuclear receptor NR1/NR2 = SXR, to which vitamin K2 binds, exerting its multitude of
biological eects. The crucial question is then: what may be the immediate eect of vitamin
K2 (e.g. MK-7) on cell phenotype, for example, on bone chips harbouring live osteoblastic
cells? In Figure 3, bovine chips from young calves were incubated for 14 days in growth
medium, which were analysed for osteocalcin, IL-10, TGFβ, OPG and RANKL (osteoblast and
osteoclast markers), respectively. For detailed summary of results, see Figure 3.
Figure 1. The epigenator-initiator-maintainer model of hormonal impact on the phenotype of a given cell. The hormone
(binding to a given receptor—here represented by two dierent transmembrane proteins) will eventually elicit a
response determined by the joint eect of transcription factors and microRNAs determining the end-point eect of the
epigenator, such as cell dierentiation and/or eux of secretory products.
Vitamin K2: A Vitamin that Works like a Hormone, Impinging on Gene Expression
http://dx.doi.org/10.5772/intechopen.80388
69

Figure 3. Bovine chips from young calves incubated as stated above. The chips were (a) incubated for 7 or 21 days and
analysed for osteocalcin, IL-10, TGFβ, OPG and RANKL (all parameters’ characteristics for the osteoblast phenotype);
(b) incubated for 7 days and thereafter for 14 days in the presence of either vitamin K2 = MK-7; siRNA against the
vitamin K2 receptor SXR or with pre-mir-760. The nal measurements of secretory products were as indicated above
(osteocalcin, IL-10, TGFβ, OPG and RANKL).
Figure 2. (a) and (b) GeneCards-based emulation of molecules interacting with NR1/NR2 (which is identical to SXR,
identied/described by the Japanese researcher, Professor Satoshi Inoue). Interestingly, NR1/NR2 is integrated in a
large network of interacting molecules (genes), representing various classes of transcription factors (e.g. PPARG, RORC,
RARA, RXR) and not to forget the thyroid receptors A and B!
Cell Signalling - Thermodynamics and Molecular Control70





vitamin K2 was mediated solely through the action of the nuclear receptor SXR.



was able to substitute for insulin, as well as some growth factors (like growth hormone and a
few interleukins (not shown)) (Figure 4).
Figure 5 shows a strong relationship (network between a human osteoblast microRNA pro-
      
listed according to their sequential appearance as the osteoblast develops from a stem cell into
a ‘full-blown’, mature mineralizing osteoblast.
sirtuins (SIRTs), histone deacetylases (HDACs) and transcription factors (with special refer-
        
        1).
This was also the case for other tissues as well, e.g. lung, heart, brain, muscle, white/beige fat
tissues and many others (not mentioned here2).


Figure 4.        
growth factors.
Vitamin K2: A Vitamin that Works like a Hormone, Impinging on Gene Expression
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Author details
Jan Oxholm Gordeladze
Address all correspondence to: j.o.gordeladze@medisin.uio.no
Department of Molecular Medicine, Section for Biochemistry, Institute of Basic Medical
Science, Blindern, Norway
References
[1] Sultana H, Watanabe K, Md MR, Takashima R, Ai O, Komai M, et al. Eects of Vitamin
K2 on the Expression of Genes Involved in Bile Acid Synthesis and Glucose Homeostasis
in Mice with Humanized PXR. Sendai, Japan: Laboratory of Nutrition, Graduate School
of Agricultural Science, Tohoku University. 2018
[2] Ichikawa T, Horie-Inoue K, Ikeda K, Blumberg B, Inoue S. Vitamin K2 induces phos-
phorylation of protein kinase A and expression of novel target genes in osteoblastic cells.
Journal of Molecular Endocrinology. 2007;39(4):239-247
Figure 5. Network (based on the computerprogram Mir@nt@n) postulating the interaction between key microRNAs,
transcription factors, sirtuins, and histone deacetylases (HDACs) in the developing osteoblast from stem cells via
preosteoblasts. The compilation of genes was based on various searches on PubMed for genes appearing during the
course of preosteoblast to mature (mineralizing) osteoblasts from mammalian species, including humans.
Cell Signalling - Thermodynamics and Molecular Control72

[3] Mosos I. Review article—Crosstalk between vitamins A, B12, D, K, C, and E status and
arterial stiness. Disease Markers. 2017;2017:14. Article ID: 8784971. DOI: 10.1155/2017/
8784971
[4] hps://www.bodyscience.com.au/blog/dr-hogne-vik-vitamin-k2-interview/
[5] Gordeladze JO. General Reading on Vitamin K2: Vitamin K2—Vital for Health and Well-
Being. IntechOpen. 2017. DOI: 10.5772/61430
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