Unveiling novel genes upregulated by both rhBMP2 and rhBMP7 during early osteoblastic transdifferentiation of C2C12 cells

Abstract

FindingsWe set out to analyse the gene expression profile of pre-osteoblastic C2C12 cells during osteodifferentiation induced by both rhBMP2 and rhBMP7 using DNA microarrays. Induced and repressed genes were intercepted, resulting in 1,318 induced genes and 704 repressed genes by both rhBMP2 and rhBMP7. We selected and validated, by RT-qPCR, 24 genes which were upregulated by rhBMP2 and rhBMP7; of these, 13 are related to transcription (Runx2, Dlx1, Dlx2, Dlx5, Id1, Id2, Id3, Fkhr1, Osx, Hoxc8, Glis1, Glis3 and Cfdp1), four are associated with cell signalling pathways (Lrp6, Dvl1, Ecsit and PKCδ) and seven are associated with the extracellular matrix (Ltbp2, Grn, Postn, Plod1, BMP1, Htra1 and IGFBP-rP10). The novel identified genes include: Hoxc8, Glis1, Glis3, Ecsit, PKCδ, LrP6, Dvl1, Grn, BMP1, Ltbp2, Plod1, Htra1 and IGFBP-rP10.BackgroundBMPs (bone morphogenetic proteins) are members of the TGFβ (transforming growth factor-β) super-family of proteins, which regulate growth and differentiation of different cell types in various tissues, and play a critical role in the differentiation of mesenchymal cells into osteoblasts. In particular, rhBMP2 and rhBMP7 promote osteoinduction in vitro and in vivo, and both proteins are therapeutically applied in orthopaedics and dentistry.ConclusionUsing DNA microarrays and RT-qPCR, we identified both previously known and novel genes which are upregulated by rhBMP2 and rhBMP7 during the onset of osteoblastic transdifferentiation of pre-myoblastic C2C12 cells. Subsequent studies of these genes in C2C12 and mesenchymal or pre-osteoblastic cells should reveal more details about their role during this type of cellular differentiation induced by BMP2 or BMP7. These studies are relevant to better understanding the molecular mechanisms underlying osteoblastic differentiation and bone repair.

HtrA1 is upregulated during RANKL‐induced osteoclastogenesis, and negatively regulates osteoblast differentiation and BMP2‐induced Smad1/5/8, ERK and p38 phosphorylation

“…This discrepancy might be due to two different cell types being used. Consistent to our observations, a more recent study has also shown that HtrA1 is up-regulated by BMP2 and BMP7 during osteoblastic differentiation of C2C12 cells [15]. Nevertheless, our studies extended previous findings [7] to show that osteoclast-derived HtrA1 has an inhibitory effect on osteoblast differentiation and gene expression, in addition to its effect on osteoblast mineralization process.…”

Systemic analysis of osteoblast-specific DNA methylation marks reveals novel epigenetic basis of osteoblast differentiation

“…GLIS1 encodes for a GLI-related Kruppel-like zinc finger transcription factor and can effectively promote the reprogramming of somatic cells during induced pluripotent stem cells (iPSC) generation (Maekawa and Yamanaka, 2011, Maekawa et al, 2011). Importantly, GLIS1 is upregulated during the osteoblastic differentiation (Bustos-Valenzuela et al, 2011) and has been linked to coronary artery calcified plaque (Divers et al, 2013), which is closely related to osteoblastic differentiation and activity (Doherty et al, 2003). Together, these results suggest that GLIS1 expression is likely to be tightly regulated in osteoblasts and cell type-specific DNA methylation may help to achieve this fine-tuning of expression.

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Yeti, aDrosophila melanogasteressential gene, encodes a protein required for chromatin organization

“…Thus, the identification of new factors required for chromatin dynamics and organization can expand our understanding of mechanisms underpinning developmental diseases. Several studies implicate CFDP1, the human ortholog of YETI, in osteogenesis and craniofacial development (Diekwisch et al, 1999;Diekwisch et al, 2002;Thisse, 2004;Wu et al, 2008;Bustos-Valenzuela et al, 2011;Makeyev and Bayarsaihan, 2011). Future research can provide a better understanding of BCNT protein functions in chromatin organization and yield more insight into their role in developmental disorders.…”

“…First, the mouse BCNT protein, called CP27 or CFDP1, plays essential cellular functions and is required for teeth and bone development (Diekwisch et al, 1999;Diekwisch et al, 2002). Second, in mouse, downregulation of the Cfdp1 gene correlates with the induction of cleft palate due to persistent expression of the PAX3-encoding gene in neural crest cells (Wu et al, 2008); upregulation of Cfdp1 was also observed after experimentally induced osteodifferentiation (Bustos-Valenzuela et al, 2011). Third, the Zebrafish BCNT gene, called Rltpr, is developmentally expressed in the branchial arches that will give rise to the craniofacial structures (Thisse, 2004).…”