**6. Results**

Results from all dosages used suggested that numerous genes exhibited significantly altered expression. At this stage, results from pups given 2mg/kg have been more extensively analyzed.

These data results suggested that 281 genes were differentially expressed (p < 0.05), with changes in expression ranging from about 5- (Minpp1, Pdzd2) to about 0.05-fold (Zfp485, Slc2a5). Bioinformatics analysis (using Ingenuity Pathways Analysis) suggested that a major fraction of the observed changes in gene expression was associated with "cell death," the data suggesting a highly significant association to decreased apoptosis. This is likely mediated via altered expression of genes associated with regulation of metabolic substrates being converted to polyamine, retinoic acid-dependent regulation of apoptosis regulation (three genes), and altered osteoclast and osteoblast signaling (six genes). Several genes involved in synthesis of complex carbohydrates e.g., proteoglycans, heparin sulphate of chondroitin sulphate b, were upregulated.

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

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

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Vitamin K2 and Its Impact on Tooth Epigenetics

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*Development. 2008 Sep;135(17):2905–16.*

*J Bone Miner Res. 2013 Sep;28(9):1885-97. doi: 10.1002/jbmr.1943. Bone. 2009 Jul;45(1):27–41. doi: 10.1016/j. bone.2009.03.656. Epub 2009 Mar 21. Endocrinology. 2005 Jan;146(1):195–200. Epub 2004 Oct 7*

*Bone. 2008 Oct;43(4):700–7. Nat Med. 2006 Dec;12(12):1410–6. Epub 2006 Nov 26 J Endod. 2011 Dec;37(12):1647–52. doi: 10.1016/j. joen.2011.08.023. Epub* 

*2011 Oct 6.*

*are integral membrane proteins that lower intracellular chloride concentrations* 

Human osteoblasts express functional K-Cl cotransporters in their cell membrane that seems to be able to induce activation of volume-sensitive channels by KCl, necessary for normal osteoblast membrane currents, and

*coactivator, and stimulates the expression of WNT1 by inhibiting the expression of* 

Hypoxia-induced MTA1 (via HIF-1α) stimulates growth (and inhibits differentiation) of osteoblastic (MC3T3) cells which is deemed important in

*transcription factor. The encoded protein recognizes an octamer sequence in the DNA of target genes. This protein may play a role in development of the nervous* 

However, it was recently shown that this gene normally upregulates the genes of the Dlx-familiy (Dlx1, 2, 5, 6), as well as downstream genes like

*hormone by converting the prohormone thyroxine (T4) by deiodination to bioactive 3,3',5-triiodothyronine (T3). It is highly expressed in the thyroid, but is known to be* 

Cold exposure (in Misty mice) compensates for BAT (brown adipose tissue) dysfunction by increasing the expression of Acadl, Pgc1a, Dio2, and other thermogenic genes, by altering the expression of osseous Runx2

Genes upregulated by BMP-7 showed a strong enrichment for established osteogenic marker genes, and several others (MFI2, HAS3, ADAMTS9, HEY1, DIO2 and FGFR3) in osteoblasts. Furthermore, for DIO2 seems to

Outer ring deiodination (ORD) activity was seen in bone extracts of whole skeleton, bone marrow, and MC3T3-E1 osteoblasts. [1,25(OH)2VD]-treatment induced D2 activity, while estradiol, PTH, forskolin, leptin, TNFα, TGFβ,

*calmodulin-dependent protein kinase subfamily. It serves as a multifunctional serine-threonine protein kinase, and has been implicated in transcriptional control of* 

Silencing of CaMK1β obliterates the proliferation ability of osteoblasts, as well as expression of c-Fos. However, this does not influence the skeleton

CaMKs activate pathways mediated by CREB and NFATc1. Inhibition of CaMKs obliterates CREB phosphorylation, lowering c-Fos, and NFATc1 expression, and thus osteoclastogenesis activated by NF-κB ligand

Finally, BMP-receptor signaling in stem cells from human exfoliated deciduous (SHED) teeth enhances the expression of genes like BMP-4,

**Camk4** *Camk4 is a member of he serine/threonine protein kinase (PK) family, and the Ca2+*

*lymphocytes, neurons, as well as male germ cells:*

markersRunx2, Osterix, and/or Osteocalcin.

**Slc12a6** *This gene is a member of the K-Cl cotransporter (KCC) family. K-Cl cotransporters* 

**Mta1** *Transcriptional coregulator that can act as both a transcriptional corepressor and* 

**Pou3f3** *This gene encodes a POU-domain containing protein that functions as a* 

Gbx2, is involved in the patterning of the mammalian jaw.

**Dio2** *This gene belongs to the iodothyronine deiodinase family. It activates thyroid* 

*expressed many other peripheral tissues:*

impact osteoblastic differentiation.

and dexamethasone did not.

*below the electrochemical equilibrium potential:*

thus secretory functions.

*its transcriptional corepressor, SIX3:*

the process of fracture healing.

*system:*

and Rankl.

(RANKL).

Runx2, as well as DSPP.

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).
