**1. Role of microRNAs in the determination of cellular phenotype**

MicroRNAs (miRNAs or miRs) are conserved, small non‐coding RNAs (18–25 nucleotides long) instrumental in the regulation of gene expression, and serve as a part of a network of factors, including transcription factors determining the phenotype of a certain cell in the body (ref). The transcription factors, such as the SXR = PXR = NR1I2 serve as receptors, much the same way as the receptors for vitamin A (RXR) and D (VDR), and may modulate gene transcription to determine the cell phenotype with its defined phenotypic characteristics (e.g. mineralizing osteoblast or non‐mineralizing vessel‐lining epithelial cells or heart valve fibroblast). It is well known that vitamin K2 (MK‐7) stabilizes the two phenotypes (also in the presence of an inflammatory environment), thus preserving 'correct' inter‐organ cross‐talk, as would be expected in a healthy organism.

First, it should be emphasized that vitamin K2 binds to a nuclear receptor, which is part of a regulatory network consisting of microRNAs and other transcription factors. Second, this network represents a minimal lattice of regulatory factors, which may be manipulated in order to breach the stability of a certain cell phenotype (e.g. a cancer cell), or doing the opposite: reinforcing the phenotype in question (e.g. mineralizing osteoblast and non‐mineralizing fibroblast during renal failure, uremia, for instance). Without going into detail, it is asserted that the regulatory networks presented here bargain for: (1) how vitamin K2 is involved in the stabilization of the osteoblastic phenotype, and (2) which are the major players (microRNAs and genes) determining whether a cell will adapt mineralizing properties or not (osteoblast or fibroblast).

question and/or their severity. MicroRNA species and putative target genes related to liver disease (NAFLD & NASH), as well as cardio (vascular) affection were pooled from three dif-

Introductory Chapter: Vitamin K2 http://dx.doi.org/10.5772/66384 15

At highest stringency applied, there are but a few genes and microRNAs emerging as 'connected': hsa‐mir‐122 with FBXO32 (F‐Box protein 32; involved in FOXO‐mediated signalling) and STAT4 (transcription factor); hsa‐mir‐144 with ABCA1 (ATP binding cassette subfamily A member 1, involved in cholesterol and sphingolipids transport from golgi and ER to the apical membrane and regulation of lipid metabolism by PPARα), hsa‐mir‐33b with ABCA1 and SLC25A25 (solute carrier family 25 member 25), and finally hsa‐mir‐145 with TFAM (transcription factor A, mitochondrial). Without going into details, all three articles [21–23], emphasize microRNA species 122, 144, and 33b, as instrumental in regulating hepatic lipid metabolism, with emphasis on hsa‐mir‐122. This microRNA‐species is instrumental in the optimization of fatty acid oxidation vs synthesis, cholesterol production, as well as VLDL secretion to the circulation, and thus determining the health status of any individual in terms of risk of incurring atherosclerosis. The fact that this regulatory system, shown in **Figure 4**, lacks reciprocal regulatory loops makes it more vulnerable and

**Figure 4.** Hierarchical regulatory system consisting of microRNAs, transcription factors and genes involved in the mineralization phenotype of osteoblastic cells, but also present in fibroblasts having attained untoward/unwanted mineralizing properties during exposure to inflammatory cytokines and/or co‐cultured with Th‐1 and Th‐17 cells.

ferent articles (**Figure 5**).

Suffice to say (with reference to **Figure 3**), the master transcription factor JUN, impinges on a set of microRNAs (let‐7 species) in a hierarchical structure of traditional genes and other microRNAs, which are well known in the literature, as being part of the WNT‐Notch, the TGFβ and the BMP pathways (determining the osteoblast phenotype) (see KEGGs pathways), where specific markers like WNT6, DKK1, and CTNNB1 (β‐catenin, activator of Runx2, the most referred marker of osteoblastic cells), are represented, and where some of the major microRNA‐species, like miR‐125, miR‐21, miR‐221, miR‐27 and miR‐23, known to be important in the differentiation and stabilization of the osteoblast phenotype is ensured.

**Figure 3.** The involvement of vitamin K2 (MK‐4 and MK‐7), binding to NR1I2 = SXR = PXR, exerting its effect via hsa‐ miri‐760 on regulatory loops in osteoblasts (according to the **Mir@nt@n** algorithm). This chart shows how vitamin K2 may affect the regulatory system determining the phenotype of osteoblast as a mineralizing cell in the body, involving microRNAs, transcription factors (e.g. FOS, JUN, SP1 & SP3) and 'functional' or 'marker' genes, like RUNX1*.*

In very much the same way, one may analyse the gene‐transcription factor—microRNA axis in conditions like non‐alcoholic fatty liver disease (NAFLD) or non‐alcoholic steatohepatitis (NASH), and hopefully arrive at blood‐born microRNAs, representing the diseases in question and/or their severity. MicroRNA species and putative target genes related to liver disease (NAFLD & NASH), as well as cardio (vascular) affection were pooled from three different articles (**Figure 5**).

regulatory networks presented here bargain for: (1) how vitamin K2 is involved in the stabilization of the osteoblastic phenotype, and (2) which are the major players (microRNAs and genes) determining whether a cell will adapt mineralizing properties or not (osteoblast or fibroblast).

14 Vitamin K2 - Vital for Health and Wellbeing

Suffice to say (with reference to **Figure 3**), the master transcription factor JUN, impinges on a set of microRNAs (let‐7 species) in a hierarchical structure of traditional genes and other microRNAs, which are well known in the literature, as being part of the WNT‐Notch, the TGFβ and the BMP pathways (determining the osteoblast phenotype) (see KEGGs pathways), where specific markers like WNT6, DKK1, and CTNNB1 (β‐catenin, activator of Runx2, the most referred marker of osteoblastic cells), are represented, and where some of the major microRNA‐species, like miR‐125, miR‐21, miR‐221, miR‐27 and miR‐23, known to be impor-

In very much the same way, one may analyse the gene‐transcription factor—microRNA axis in conditions like non‐alcoholic fatty liver disease (NAFLD) or non‐alcoholic steatohepatitis (NASH), and hopefully arrive at blood‐born microRNAs, representing the diseases in

**Figure 3.** The involvement of vitamin K2 (MK‐4 and MK‐7), binding to NR1I2 = SXR = PXR, exerting its effect via hsa‐ miri‐760 on regulatory loops in osteoblasts (according to the **Mir@nt@n** algorithm). This chart shows how vitamin K2 may affect the regulatory system determining the phenotype of osteoblast as a mineralizing cell in the body, involving

microRNAs, transcription factors (e.g. FOS, JUN, SP1 & SP3) and 'functional' or 'marker' genes, like RUNX1*.*

tant in the differentiation and stabilization of the osteoblast phenotype is ensured.

**Figure 4.** Hierarchical regulatory system consisting of microRNAs, transcription factors and genes involved in the mineralization phenotype of osteoblastic cells, but also present in fibroblasts having attained untoward/unwanted mineralizing properties during exposure to inflammatory cytokines and/or co‐cultured with Th‐1 and Th‐17 cells.

At highest stringency applied, there are but a few genes and microRNAs emerging as 'connected': hsa‐mir‐122 with FBXO32 (F‐Box protein 32; involved in FOXO‐mediated signalling) and STAT4 (transcription factor); hsa‐mir‐144 with ABCA1 (ATP binding cassette subfamily A member 1, involved in cholesterol and sphingolipids transport from golgi and ER to the apical membrane and regulation of lipid metabolism by PPARα), hsa‐mir‐33b with ABCA1 and SLC25A25 (solute carrier family 25 member 25), and finally hsa‐mir‐145 with TFAM (transcription factor A, mitochondrial). Without going into details, all three articles [21–23], emphasize microRNA species 122, 144, and 33b, as instrumental in regulating hepatic lipid metabolism, with emphasis on hsa‐mir‐122. This microRNA‐species is instrumental in the optimization of fatty acid oxidation vs synthesis, cholesterol production, as well as VLDL secretion to the circulation, and thus determining the health status of any individual in terms of risk of incurring atherosclerosis. The fact that this regulatory system, shown in **Figure 4**, lacks reciprocal regulatory loops makes it more vulnerable and unstable, when threatened by 'disease states', like NAFLD/NASH, than systems found in the osteoblast, which apparently appears more resilient to change, when exposed to conditions where inflammation prevails.

**Addendum 1**

**gram to 'retrieve' regulatory networks**

**List of microRNAs and genes used as 'input' into the Mir@nt@n‐algorithm, asking the pro‐**

Introductory Chapter: Vitamin K2 http://dx.doi.org/10.5772/66384 17

**Figure 5.** MicroRNAs, transcription factors and 'functional' genes related to liver function in patients with NAFLD/ NASH with or without metabolic cardiovascular disease (see Refs. [21–23]). The three charts represent decreasing stringency/from top to bottom), and it should be emphasized that the regulatory system does not contain any reciprocal regulatory feedback systems, as was shown for the 'stabilization' of the osteoblast (or mineralizing phenotype).

Finally, it should be emphasized that bioinformatics analyses of the 'NRI2‐relative' NR1I3, the constitutive androstane receptor (CAR), which interacts with NR1I2 = SXR = PXR, is also biologically interfering with many of the same factors (e.g. PPARα, CEPBα, STATs and T3) [24], thus linking them together in a very tight regulatory network, affecting lipid metabolism. Understanding the impact of vitamin K2 on these regulatory systems seems to be mandatory to grasp and acknowledge the idea that this fat‐soluble molecule exerts such a tremendous effect on biological processes compatible with organ health, disease free old age, and thus 'longevity'.
