Vitamin K was discovered in 1929 as a substance essential for blood coagulation and had been clinically utilized before the precise mechanism of action became aware in 1970s. The function as a cofactor of γ-glutamyl carboxylase (GGCX) was the mechanism firstly discovered with the identification of several substrate proteins including blood coagulation factors and osteocalcin. Recently, we and others have shown that vitamin K has other modes of function, such as ligand of nuclear receptor SXR (steroid and xenobiotic receptor) and its murine ortholog PXR (pregnane X receptor) and modulator of protein kinase A (PKA) activity. Besides its importance in blood coagulation, involvement of vitamin K has been shown in two major aging-related diseases, osteoporosis and osteoarthritis. Based on clinical and epidemiological studies, vitamin K is shown to have protective roles for both of them. Interestingly, clinical studies concerning single nucleotide polymorphisms (SNPs) of GGCX and γ-carboxylated status of osteocalcin suggested relationship between GGCX activity and bone-protective effect, while recent findings from basic research indicated that vitamin K functions mediated by SXR/PXR as well as GGCX are important in the bone metabolism. We also suggested that cartilage-protective effect is mediated by SXR/PXR signaling by animal experiments using Pxr knockout mice.
- γ-glutamyl carboxylase (GGCX)
- steroid and xenobiotic receptor (SXR)
- pregnane X receptor (PXR)
- protein kinase A (PKA)
In 1929, a Danish biochemist, Dr. Henrik Dam predicted a fat-soluble diet substance which is essential for blood coagulation. The substance was referred as “Koagulationsvitamin” in German; thus it is called vitamin K in English named after the initial letter of its German word. He shared the Nobel Prize in Physiology or Medicine in 1943 with an American biochemist Dr. Edward A. Doisy who later identified the structure of vitamin K. During the 1970s, the mechanism of vitamin K began to be revealed with the discovery, namely, vitamin K was necessary for γ-carboxylation of some coagulation factors which is catalyzed by an enzyme called γ-glutamyl carboxylase (GGCX) [1, 2]. Interestingly, warfarin, which inhibits vitamin K function, was in medical use since 1954, and vitamin K administration to newborn babies for preventing intracranial hemorrhage started in many countries in the 1960s before the enzymatic mechanisms of vitamin K function had been clarified.
Recently, epidemiological and clinical studies suggested that vitamin K is related to various physiological and pathological processes besides coagulation. Based on these studies, vitamin K was approved to be used as a drug preventing osteoporotic fracture in several Asian countries. Moreover, for these two decades, another mode of vitamin K action has been elucidated. We discovered vitamin K functions as a ligand for a nuclear receptor, SXR (steroid and xenobiotic receptor), and its murine ortholog, PXR (pregnane X receptor) , which have physiological or pathological significance. Summing up, vitamin K plays important roles in wide variety of biological process in various modes of actions.
In this chapter we are going to introduce novel mechanism of vitamin K action mediated by SXR/PXR as well as recent findings concerning classical vitamin K action mediated by GGCX. Then we would like to discuss the functions of vitamin K in some aging-related diseases based on recent discoveries.
2. Multiple mechanisms of vitamin K function
The classical function of vitamin K is a cofactor of GGCX which was clarified in the 1970s [1, 2]. GGCX catalyzes the addition of a carboxyl group to glutamate residues in the substrate proteins, which is coupled by oxidization of vitamin K hydroquinone to vitamin K epoxide. Vitamin K-dependent coagulation factors (II, VII, IX, and X) are well known substrates for GGCX. They become active when several glutamate residues are γ-carboxylated. So far, 18 human proteins are reported to be γ-carboxylated and their functions are regulated by γ-carboxylation status in most of them. It is known that cyclic use of vitamin K is necessary for its function as a cofactor for GGCX . To be recycled, vitamin K epoxide should be reduced by an enzyme called vitamin K epoxide reductase (VKOR). Warfarin, which has an anticoagulant activity, inhibits VKOR, causing a subsequent decrease in GGCX activity (Figure 1).
Recently, another mode of GGCX-dependent vitamin K function was reported in the study of proapoptotic effect of vitamin K. Handa et al. found proapoptotic protein Bak was covalently modified by vitamin K epoxide and regulated by its modification . This function is dependent on GGCX-mediated vitamin K function since GGCX activity is required to generate vitamin K epoxide (Figure 1).
On the other hand, we discovered GGCX-independent mode of vitamin K function mediated by transcriptional regulation  as compared to posttranscriptional modifications explained above. Vitamin K was found to be one of the ligands of the nuclear receptor, SXR, and its murine ortholog, PXR. This receptor is also called NR1I2 according to standardized nomenclature designated by the nuclear receptor committee. In 1998, SXR/PXR was cloned as a novel nuclear receptor that is mainly expressed in the liver and intestine . At first, its functions were characterized as a ligand-dependent transcription factor which is activated by various pharmaceutical agents and xenobiotic compounds . It was originally classified as an orphan receptor since the endogenous ligand was not known when it was cloned. It was later shown that some kinds of secondary bile acids (such as lithocholic acid) could be endogenous ligands for this receptor [8, 9]. It forms a heterodimer with 9-cis-retinoid acid receptor (RXR) on ligand stimulation. This complex then binds to SXR-responsive elements (SXRE) in the promoter or enhancer regions of target genes (Figure 1). Some of its target genes are the drug-metabolizing enzyme, such as
There is another mode of vitamin K function which modulates activation of signal transduction pathway. This is inferred by existence of some genes induced by vitamin K, not by SXR agonist, rifampicin . This induction was not affected by knocking down of GGCX suggesting that this is γ-carboxylation-independent pathway. Expression of those genes was suppressed by protein kinase A (PKA) inhibitor, showing the novel vitamin K function as a modulator of PKA activity (Figure 1).
Inhibition of another protein kinase, protein kinase C (PKC) α and ε, by vitamin K was also reported . Inhibition of IKK (inhibitor of nuclear factor kappa B kinase) and subsequent inhibition of NFkB (nuclear factor kappa B) were observed. Whether this function of vitamin K is independent of mechanisms described above remains to be elucidated.
3. Epidemiological and clinical studies on vitamin K and aging-related skeletal diseases
A traditional Japanese food, “natto” (fermented soybeans) contains high concentrations of MK-7, a form of vitamin K2 (menaquinone), synthesized by microorganisms. Epidemiological study conducted in Japan revealed negative correlation of Natto intake and incidence of hip fracture , which drew attention toward possible link between vitamin K and osteoporosis. Later, among several nutrients including vitamin D and calcium, vitamin K was shown to be the only nutrient that is significantly correlated with hip fracture incidence in Japanese population . Furthermore, the fracture-preventing effect of vitamin K was observed in several clinical studies in Japan, which was confirmed by meta-analysis . Based on these results, vitamin K2 is used for treatment of osteoporosis in several Asian countries. We previously reported a functional single nucleotide polymorphism (SNP) in
Vitamin K also has some epidemiological evidences in relationship with another skeletal disease, osteoarthritis. Low vitamin K intake was correlated to the prevalence of osteoarthritis both in North America and in Japan [18–20]. Unfortunately, therapeutic effect of vitamin K for established osteoarthritis was not proven by a trial , suggesting that the study period was too short or vitamin K has only preventive effect.
4. Paradoxical GGCX-mediated vitamin K functions on bone metabolism
It is difficult to evaluate vitamin K function on bone tissue mediated by GGCX
5. SXR-mediated vitamin K functions on bone and cartilage
As described above, we proposed another mode of vitamin K function as a ligand of a nuclear receptor, SXR, and its murine ortholog, PXR. We showed that SXR is also expressed in osteoblastic cell lines and is activated by vitamin K2 . We further identified SXR-dependent vitamin K-responsive genes by microarray analysis using human osteoblastic cell line, MG63 cells stably overexpressing SXR . The identified genes included
The involvement of SXR/PXR signaling in bone metabolism
We also proposed SXR/PXR-dependent mechanism concerning vitamin K effect on articular cartilage . We found that systemic
In this chapter, we described multiple mechanisms of vitamin K functions clarified so far and their involvement in aging-related skeletal diseases as examples for their biological significance. Besides blood coagulation, osteoporosis, and osteoarthritis, it became gradually aware that many physiological and pathological phenomena, such as fertility , atherosclerosis [37–39], brain development , dementia , and glucose metabolism [42–44], are related to the status of vitamin K sufficiency. We sincerely hope that vitamin K study leads to discoveries of new biological mechanisms and targets for disease prevention and treatment and eventually contributes to human culture and welfare.