Monday, September 24, 2012

LSJL increases DNA Methylation

Since DNA Methylation markers tend to be upregulated during cellular differentiation, this is further evidence that LSJL induces chondrogenic differentiation.

Endothelin is upregulated 2.258 fold by LSJL.

Localized methylation in the key regulator gene endothelin-1 is associated with cell type-specific transcriptional silencing.

"To identify key developmental regulator genes whose expression in terminally differentiated cells may be inhibited by DNA methylation, mouse dermal fibroblasts were demethylated with 5-aza-2'-deoxycytidine, and changes in gene expression monitored. Endothelin-1 (Et1 or Edn1), which encodes a cytokine with diverse regulatory functions, was among the genes upregulated following demethylation. CpG dinucleotides within a short region in intron 1 of the gene have dramatically higher levels of methylation in Et1-non-expressing fibroblasts and chondrocytes as compared to the Et1-expressing mouse cell line, mIMCD-3. Strong evolutionary conservation of this region implies its role in the cis-regulation of Et1 transcription. To confirm that should Et1 in dermal fibroblasts become aberrantly activated, it could indeed lead to the dysregulation of many downstream genes, we exposed fibroblasts to exogenous ET1 peptide and assayed for transcriptional changes by microarray. ET1 treatment resulted in significant expression changes - primarily downregulation - of a significant number of genes. In particular, Tgfbeta2 and Tgfbeta3 were among the downregulated genes, which in turn alter the expression status of their many target genes."

"inhibition of Et1 in fibroblasts and chondrocytes may involve targeted methylation of the Sp1 binding site (among other sites) in intron 1, which block Sp1 binding and contribute to Et1 silencing."

New born mice were used.

Bold genes were also altered in demethylated fibroblasts.

Genes upregulated in Fibroblasts that were treated with exogenous et1 that also upregulated(or downregulated) in LSJL:
Slc7a3(downregulated in LSJL)

Genes downregulated in exogenous et1 fibroblasts:
Asporin(up in LSJL)
cnn1(up in LSJL)
col6a2(up in one DNA region down in another)
Prrx2(up in LSJL)
col6a1(up in LSJL)
c1qtnf3(up in LSJL)
slc38a4(up in LSJL)
Ccl2(up in LSJL)
ccl7(up in LSJL)
il6(up in LSJL)
9930013L23Rik(up in LSJL)

Lrrc15(up in LSJL)

A large portion of differentially regulated genes were genes that were altered in demethylated fibroblasts.  A large portion of the similarly regulated genes were those only effected by Et1 which corresponds to the data as knee loading upregulated Et1.  So LSJL likely involves cellular methylation.

Epigenomic and microRNA-mediated regulation in cartilage development, homeostasis, and osteoarthritis.

"epigenetic derepression [IS] associated with DNA methylation loss on chondrocyte genes, including MMP3[up in LSJL], MMP9, MMP13, ADAMTS4[up in LSJL], IL-1β, and LEP"

5-Azacytidine makes human preadipocytes able to differentiate into mesoderm-derived cell lineages.

"In the present study we have evaluated whether (i) 5-azacytidine (AZA), a well-known demethylating agent, could be able to modify the phenotype of human preadipocytes and (ii) the modified cells could possess multilineage differentiation potential. Human preadipocytes at the 3rd passage were treated for 48 or 96 h with 10 μM AZA and then expanded up to passage 5. Stem cell markers, such as OCT-4, Nanog, and Sox2, were upregulated after 96 h of treatment with the demethylating treatment. Further, decreases in the expression of genes, such as adipose differentiation-related protein, characterizing the preadipocytes were noted. AZA-treated preadipocytes differentiated into cell lineages derived from mesoderm. Indeed, after incubation with inductive media for 3 weeks, osteblast-, chondrocyte-, and myoblast-like cells were detected in the cultures. Interestingly, both upregulation of stem cell markers and differentiation potential were maintained by the treated cultures expanded until the 5th passage. AZA, without the use of transduction methods, convert preadipocytes to a less differentiated state that can be induced, under suitable stimuli, to the formation of mesoderm-derived cell lineages."

Instead of trying to induce methylation you may want to induce demethylation to induce chondrogenesis.  Since LSJL increases methylation, decreasing methylation in the bone before LSJL and only then(as decreasing methylation will reduce differentiation as well) may allow for more available stem cells to differentiate chondrocytes.  However, the next study suggests that the methylation status of chondrogenic genes is already low in adult human cells.

"DNA methylation is one mechanism that regulates human chondrogenesis.  [What's] the CpG methylation status in human synovium-derived MSCs during experimental chondrogenesis?
Human synovium-derived MSCs were subjected to chondrogenic pellet culture for 3 weeks. The methylation status of 12 regions in the promoters of 10 candidate genes (SOX9, RUNX2, CHM1, FGFR3, CHAD, MATN4, SOX4, GREM1, GPR39, and SDF1) was analyzed before and after differentiation. The expression levels of these genes were analyzed. Methylation status was also examined in human articular cartilage.
10 of the 11 CpG-rich regions analyzed were hypomethylated in human progenitor cells before and after 3 weeks of pellet culture, regardless of the expression levels of the genes. The methylation status was consistently low in SOX9{up in LSJL}, RUNX2, CHM1, CHAD, and FGFR3 following an increase in expression upon differentiation and was low in GREM1 and GPR39 following a decrease in expression upon chondrogenesis. One exceptional instance of a differentially methylated CpG-rich region was in a 1-kb upstream sequence of SDF1, the expression of which decreased upon differentiation. Paradoxically, the hypermethylation status of this region was reduced after 3 weeks of pellet culture.
The DNA methylation levels of CpG-rich promoters of genes related to chondrocyte phenotypes are largely kept low during chondrogenesis in human synovium-derived MSCs."

"chondromodulin 1 (CHM1), fibroblast growth factor receptor 3 (FGFR3), [MATN4]{up in LSJL}, and chondroadherin (CHAD) for genes up-regulated in chondrogenic pellet cultures, and for genes down-regulated in chondrogenic pellet cultures, these were SOX4, Gremlin 1 (GREM1), G protein–coupled receptor 39 (GPR39), and stromal cell–derived factor 1 (SDF1)."

"DNA methylation conditions in immature mesenchymal cells are permissive of the expression of chondrocyte phenotype"

Although one study suggests that loss of DNA methylation is associated with cellular senescence.  However this may have been only a correlational association and not a causal association.  Also, DNA methylation may be a marker of cellular differentiation state which corresponds to other data rather than a cell cycle differentiation counter.

It's possible that the upregulation of DNA Methylation related genes is due to fibroblast differentiation.  However the presence of chondrogenic genes like Sox9 confirms condrogenic differentiation. 

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