**7. Conclusions**

The results are based on the measurements from independent biological triplicates and therefore suggestive of effects of MK-7 on gene expression during the tooth development. A clear effect on gene expression was apparent also at a dosage of 0.2 mg/kg body weight. The results indicate increased transcription of genes involved in development of bone (increased biosynthesis of important carbohydrates) and of enamel/dentin.

Further investigations are, however, required to elucidate these findings. Such experiments will likely entail the establishment of a clear dose-response relationship as well as of a timecourse of action. Also, effects of oral administration should be studied.

Summary of information obtained from the microarray analyses



Interestingly, also some genes related to enamel/dentine biosynthesis exhibited differential expression (e.g., Amelx, Ambn but not Enam). Microarray results were validated by realtime RT-PCR. Transcription factor analysis (using Ingenuity Pathways Analysis) suggested significant associations to the increased transcriptional activity of Myc (measured as changes in expression of Hspd1, Ctnnb1, Dkk1, and Psmb8 being in line with the predicted change). The downregulation of Dkk1 is interesting as denosumab treatment results in reduced Dkk1

The data also suggested highly significant associations to decreased apoptosis. This is likely mediated via altered significant associations to polyamine regulation (three genes), altered retinoic, acid apoptotic regulation (three genes), and altered osteoclast and osteoblast signaling (six genes).

The results are based on the measurements from independent biological triplicates and therefore suggestive of effects of MK-7 on gene expression during the tooth development. A clear effect on gene expression was apparent also at a dosage of 0.2 mg/kg body weight. The results indicate increased transcription of genes involved in development of bone (increased biosyn-

Further investigations are, however, required to elucidate these findings. Such experiments will likely entail the establishment of a clear dose-response relationship as well as of a time-

> *Int J Biochem Cell Biol. 2013 Mar;45(3):696–705. J Bone Miner Res. 2014 Dec;29(12):2676-87. J Cell Physiol. 2013 Jul;228(7):1594–600.*

> *J Biol Chem. 2010 Nov 5;285(45):34757–64. J Clin Invest. 2016 Mar 7. pii: 80672.*

*Bone. 2013 Aug;55(2):501–11. doi: 10.1016/j. bone.2013.03.012.*

level. Further, high Dkk1 levels have been associated with increased bone loss.

thesis of important carbohydrates) and of enamel/dentin.

*DNA-binding*:

*pathways, such as Notch signaling:*

*involved in a plethora of liver functions:*

course of action. Also, effects of oral administration should be studied.

**Lmcd1** *Transcriptional cofactor restricting GATA6 and GATA4 functioning by inhibiting* 

mineralization to hamper the development of brittleness.

**Dmxl2** *Protein involved in many functions including participation in signal transduction* 

for fracture repair performed by mature osteoblastic cells.

osteocalcin, and osteopontin, as well as vitamin D metabolism.

**Abcb4** *The membrane-associated protein encoded by this gene is a member of the* 

**Gene name Description of function:** *General* **and bone-related References**

Gata6 and Gata4 are transcription factors shown to be involved in TGFβ- and estrogen-mediated regulation of gene expression in osteoblasts, and thus bone

NOTCH signaling in BMSCs (bone marrow stromal/stem cells) is required

*superfamily of ATP-binding cassette (ABC) transporters. ABC proteins transport various molecules across extra- and intra-cellular membranes. The gene is heavily* 

In experimental animals, i.e., Abcb4 (-/-) mice with hepatic osteodystrophy, serum RANKL and TGFβ-levels were augmented, resulting in an excess bone resorption rate, probably due to a dysregulation of genes like osteoprotegerin,

Summary of information obtained from the microarray analyses

**7. Conclusions**

134 Vitamin K2 - Vital for Health and Wellbeing


**Gene name Description of function:** *General* **and bone-related References**

*such as the Notch proteins. Signaling by Notch receptors mediates a wide range of* 

*Clin Oral Implants Res. 2014 Apr;25(4):475–86. doi: 10.1111/clr.12178. Epub 2013 Apr 21.*

http://dx.doi.org/10.5772/66383

137

Vitamin K2 and Its Impact on Tooth Epigenetics

*Int J Mol Med. 2014 Sep;34(3):856–62. doi: 10.3892/ ijmm.2014.1846. Epub 2014 Jul 10.*

*J Bone Miner Res. 2015 Oct;30(10):1896–904. doi: 10.1002/jbmr.2540. Epub 2015 May 14 Exp Cell Res. 2015 May 1;333(2):289–302. doi: 10.1016/j. yexcr.2015.02.009. Epub 2015 Feb 20.*

*Titanium implant surfaces with modified topographies improve osteogenic properties in vivo. The activation of signaling stem cell pathways (such as TGFβ/BMP, Wnt, FGF, Hedgehog, and Notch) was characterized subsequent to incubations (24 and 72 h) with BCs to SLA and modSLA surfaces in the absence of osteogenic cell culture* 

*Key regulatory genes belonging to the TGFβ/BMP (TGFBRs, BMPRs, ACVRs, SMADs, Wnts, FZD1, FZDs, LRP5, NFATCs, PYGO2, LEF1) and Notch species (including PSENEN) pathways were upregulated on the modified surfaces. These data correlate with an increased expression of osteogenic markers (e.g. BSP and* 

*These finding indicate that activation of proosteogenic cell signaling pathways by* 

*associated with RHOB include oculo auricular syndrome and sertoli cell-only syndrome. Among its related pathways are Signaling by GPCR and Developmental Biology. GO annotations related to this gene include GTP binding and GDP* 

*Defects (mild affection to complete destruction) in the sealing zone were observed in the OPG-deficient animals. Resorption lacunae were not detected, indicating the loss of osteoclast-mediated bone resorption activity. Treatment with OPG resulted in a significant decrease in the expression of a cluster of instrumental genes (like for instance) Rho guanine nucleotide exchange factors (RhoGEFs), RhoGTPases, ROCK1 and ROCK2. This resulted in damage to or destruction of the sealing zone, thus* 

*organizational role in the retina. The protein encoded is assembled and secreted as a homooligomeric complex. Mutations in the present gene are lead to X-linked* 

*G-protein-coupled receptors (GPCRs) are key regulators of skeletal homeostasis and important in fracture healing. It was earlier shown that blockade of G(i) signaling in maturing osteoblasts enhanced cortical and trabecular bone formation and prevented age-related bone loss in female mice. Furthermore, activation of G(s) signaling induced massive trabecular bone formation, but a concomitant cortical bone loss. Here, "labile" tibial fractures, where endogenous G(i) signaling are blocked by PTX,* 

*Inhibition of endogenous G(i) activity gave a smaller callus, but enhanced net bone* 

**PTX treatment lowered the expression of Dkk1 and upregulated Lef1 mRNA upon fracture healing, indicating endogenous G(i) signaling in maintaining Dkk1 expression, while suppressing Wnt signaling. On the contrary, mice with activated Gs signaling demonstrated an increase in the initial callus size with enhanced callus bone production***. These results indicate that G(i) blockade and* 

*It was previously asserted that Rs1 constitutively activated Gs-coupled GPCR, under the control of the 2.3 kb Col I promoter, enhancing the steady state level mineral mass in trabecular bone of femurs. In this article, it was further concluded that Gs-signaling in OBs on enhanced intramembranous bone formation in calvariae of Col1(2.3)/Rs1 mice. Rs1 calvariae displayed a dramatic increase in bone volume with partial loss of cortical structure. Gene expression analysis of calvarial OBs showed that genes were affected by Rs1 signaling, featuring processes like: (a) differentiation, (b) synthesis of cytokine/growth factors, (c) angiogenesis, (d)* 

**Psenen** *Presenilins are required for intramembranous processing of transmembrane proteins,* 

*developmental cell fates*.

*osteocalcin, as well as BMP2 and BMP6*.

*modSLA and SLA surfaces leads to enhanced osteogenic*

*binding. An important paralog of this gene is RHOA*.

*inhibiting osteoclast-mediated bone resorption*.

*retinoschisis with ensuing severe loss in vision*.

*or G(s) signaling activated by Rs1, were achieved*.

*G(s) activation are important for proper fracture healing*.

*coagulation, as well as (e) energy metabolism*.

*formation in both mice, irrespective of age.*

**Rs1** *The present gene encodes an extracellular protein serving an important* 

**Rhob** *RHOB (Ras Homolog Family Member B) is a Protein Coding gene. Diseases* 

*supplements*.

the G(2) phase arrest observed.


**Gene name Description of function:** *General* **and bone-related References**

*phosphatase 2 functions as one of four main Ser/Thr phosphatases, and is involved in* 

*Bahl A1, Pöllänen E, Ismail K, Sipilä S, Mikkola TM, Berglund E, Lindqvist CM, Syvänen AC, Rantanen T, Kaprio J, Kovanen V, Ollikainen M.*

*Bahl A1, Pöllänen E2, Ismail K1, Sipilä S2, Mikkola TM2, Berglund E3, Lindqvist CM3, Syvänen AC3, Rantanen T2, Kaprio J1, Kovanen V2, Ollikainen* 

*Scand J Immunol. 2010 Mar;71(3):134-45. doi: 10.1111/j.1365– 3083.2009.02361.x.*

*Cell Signal. 2012 Aug;24(8):1713- 21. doi: 10.1016/j. cellsig.2012.04.011. Epub 2012 Apr 25.*

*M1.*

Loss of estrogen during menopause causes changes in the female body. Hence, it is expected that HRT-associated gene expression is due to the changes in the DNA methylation profile (DMP). Of the DMP genes, ACBA1, CCL5, FASLG, PPP2R2B, and UHRF1 were differentially expressed, all of which are associated with HRT or estrogenic regulation. All genes were also associated with bone mineral content (BMC), while ABCA1, FASLG, and

*proteins (SREBPs) and by liver X receptors (LXRAs). When sterols are depleted, LXRs are inactive and SREBPs bind promoter SREs and activate genes involved in cholesterol turnover. The protein STAR is involved in this process, and it is* 

Estrogen-containing hormone replacement therapy (HRT) leads to a relief of typical menopausal symptoms, benefits bone and muscle health, and is associated with tissue-specific gene expression profiles. Hence, It is plausible that part of the HRT-associated gene expression is due to changes in the

The gene expression patterns of white blood cells (WBCs) and their associations with body composition, including muscle and bone measures of monozygotic (MZ) female twin pairs discordant for HRT were assessed. Of genes with differentially methylated regions (DMRs), five (ACBA1, CCL5, FASLG, PPP2R2B, and UHRF1) were also differentially expressed. These have been associated with HRT or estrogenic regulation, but were also associated with bone mineral content (BMC). Additionally, ABCA1, FASLG, and UHRF1 were also

*and activate cytokine receptors with ensuing phosphorylation (activation) of receptor subunits. It is also a component of the interferon signaling pathways. It may* 

Interleukin-23 (IL-23) belonging to the IL-6/IL-12 family that plays a key role in autoimmune and inflammatory disorders. IL-23 binding to dendritic cells, macrophages and monocytes triggers the activation of Jak2 and Tyk2 which in turn phosphorylates STAT1, STAT3, STAT4, and STAT5 as well as induce formation of STAT3-STAT4 heterodimers. IL-23 is essential for the survival and/or expansion of inflammatory Th17 cells which, when activated by IL-23, sustain osteoclasto-genesis via the production of IL-17 (stimulator of the NF-kappa B) of mesenchymal cells. As a group, the IL-17 - IL-23 "axis" includes Th17 cells that play a major role in the development and maintenance

*protein is a regulator of K(lysine) acetyltransferase-5, involved in transcription, DNA damage response and the cell cycle control, by blocking its degradation:* The programmed cell death gene (PDCD5) was overexpressed in an

osteosarcoma (OS) cell line, MG-63. The results indicate that PDCD5 can induce apoptosis and G(2) phase arrest in MG-63 cells. Furthermore PDCD5 expression in established xenografted tumors was associated with a decrease in tumor size and weight. Furthermore, it was found that the Ras/Raf/MEK/ERK signaling pathway was hampered, leading to the inhibition of cyclin B and CDK1, and to the activation of caspases 3 and 9, respectively. These results are consistent with

**Ppp2r2b** *This gene encodes the family of phosphatase 2 regulatory B subunits. Protein* 

*the inhibitory control system of cellular division:*

136 Vitamin K2 - Vital for Health and Wellbeing

UHRF1 were also associated with body adiposity.

*homologous to a family of proteins STARD4:*

DNA methylation profile.

related to the body adiposity.

*therefore play a role in anti-viral immunity:*

of autoimmune arthritic inflammation.

the G(2) phase arrest observed.

**Pdcd5** *This gene encodes a protein that is upregulated during apoptosis. The encoded* 

**Stard4** *Cholesterol homeostasis is regulated by sterol regulatory element (SRE)-binding* 

**Tyk2** *This gene encodes the Janus protein kinases (JAKs). These proteins associate with* 


*Lmcd1, Dmxl2, Abcb4, Slc12a6, Mta1, Pou3f3, Dio2, Camk4, Ppp2r2b, Stard4, Tyk2, Pdcd5, Psenen, Rhob, Rs1, Trpm5, Amelx, DKK1, SP1, SP3, SP7, Runx2, Runx1, NR1/2, ADRB3, Foxc2, PGC1α, PPARA, PPARG, Dio2, UCP1, Adipoq, LEP, BETA3AR/ADRB3R/B3AR, hsa-mir-155,* 

Vitamin K2 and Its Impact on Tooth Epigenetics

http://dx.doi.org/10.5772/66383

139

*Hsa-mir-196a, hsa-mir-16, hsa-mir-455, hsa-mir-339, hsa-mir-125b, hsa-mir-328, hsa-mir-16, hsamir-149, hsa-mir-125b, hsa-mir-760, hsa-mir-133, hsa-mir-29, hsa-mir-27, hsa-mir-23, hsa-mir-320,* 

The genes, significantly modulated (directly or indirectly) by vitamin K2, are presented in

Of major interest here, from a regulatory point of view, and as a minimal "cluster" of necessary and sufficient genes, are probably the following species: RUNX1, RUNX2, SP1, SP3, and DIO2, along with the microRNA-species 149, 328, 339, and 760 (see **Figure 1**). It is well known that the osteoblast and odontoblast phenotypes are "determined" and "stabilized" by the RUNX- and SP-families of transcription factors (upregulated), as well as the

*hsa-mir-26b, hsa-mir-21, hsa-mir-302, hsa-mir-132, and hsa-mir-223*.

**Table 1.** "Dental" genes affected directly or indirectly by exposure to vitamin K2 (MK-7).

*and c/EBPB*.

**Table 1**.

**9. Major findings**
