Genome-wide identification of chromatin-enriched RNA reveals that unspliced dentin matrix protein-1 mRNA regulates cell proliferation in squamous cell carcinoma.

Chromatin-enriched noncoding RNAs (ncRNAs) have emerged as key molecules in epigenetic processes by interacting with chromatin-associated proteins. Recently, protein-coding mRNA genes have been reported to be chromatin-tethered, similar with ncRNA. However, very little is known about whether chromatin-enriched mRNA is involved in the chromatin modification process. Here, we comprehensively examined chromatin-enriched RNA in squamous cell carcinoma (SQCC) cells by RNA subcellular localization analysis, which was a combination of RNA fractionation and RNA-seq. We identified 11 mRNAs as highly chromatin-enriched RNAs. Among these, we focused on the dentin matrix protein-1 (DMP-1) gene because its expression in SQCC cells has not been reported. Furthermore, we clarified that DMP-1 mRNA was retained in chromatin in its unspliced form in SQCC in vitro and in vivo. As the inhibition of the unspliced DMP-1 mRNA (unspDMP-1) expression resulted in decreased cellular proliferation in SQCC cells, we performed ChIP-qPCR to identify cell cycle-related genes whose expression was epigenetically modified by unspDMP-1, and found that the CDKN1B promoter became active in SQCC cells by inhibiting unspDMP-1 expression. This result was further validated by the increased CDKN1B gene expression in the cells treated with siRNA for unspDMP-1 and by restoration of the decreased cellular proliferation rate by simultaneously inhibiting CDKN1B expression in SQCC cells. Further, to examine whether unspDMP-1 was able to associate with the CDKN1B promoter region, SQCC cells stably expressing PP7-mCherry fusion protein were transiently transfected with the unspDMP-1 fused to 24 repeats of the PP7 RNA stem loop (unspDMP-1-24xPP7) and we found that unspDMP-1-24xPP7 was efficiently precipitated with the antibody against mCherry and was significantly enriched in the CDKN1B promoter region. Thus, unspDMP-1 is a novel chromatin-enriched RNA that epigenetically regulates cellular proliferation of SQCC.

Lactacystin, a proteasome inhibitor, enhances BMP-induced osteoblastic differentiation by increasing active Smads.

Proteasome inhibitors enhance bone formation and osteoblastic differentiation in vivo and in vitro. In the present study, we examined whether the molecular mechanisms of lactacystin, one of many proteasome inhibitors, stimulated the osteoblastic differentiation of C2C12 cells that is induced by bone morphogenetic proteins (BMPs). Pretreatment with lactacystin enhanced the alkaline phosphatase (ALP) activity induced by BMP2, BMP4 or BMP7, but lactacystin did not induce ALP in the absence of BMPs. In addition, lactacystin-stimulated BMP2 induced mRNA expression of ALP, type I collagen, osteonectin, osteocalcin, Id1, Osterix, and Runx2. Lactacystin maintained BMP2-induced phosphorylation of Smad1/5/8 and increased the length of time that these Smads were bound to target DNA. Moreover, lactacystin prevented BMP receptor-induced Smad degradation. This enhancement of BMP2-induced ALP activity and Smad phosphorylation by lactacystin was also observed in primary osteoblasts. These findings suggest that pretreatment with lactacystin accelerates BMP-induced osteoblastic differentiation by increasing the levels of phosphorylated Smads, which are maintained because BMP receptor-induced degradation is inhibited. We propose that optimized stimulation by proteasome inhibitors in a clinical setting may facilitate autogenous or BMP-induced bone formation in areas of defective bone.

Low-level laser irradiation enhances BMP-induced osteoblast differentiation by stimulating the BMP/Smad signaling pathway.

Low-level laser irradiation (LLLI) has been shown to induce bone formation and osteoblast differentiation both in vivo and in vitro. However, the molecular mechanism by which LLLI stimulates osteoblast differentiation is still unclear. The aim of the present study was to examine whether Ga-Al-As laser irradiation could enhance BMP2-induced alkaline phosphatase (ALP) activity in C2C12 cells. Laser irradiation at 0.5 W for 20 min enhanced BMP2-induced ALP activity. Laser treatment alone did not affect ALP activity. To exclude the effect of pH or temperature changes during irradiation, we shortened the exposure time to 2 min, with various levels of laser power. At 2.5 W, irradiation stimulated BMP2-induced ALP activity but not cell proliferation, whereas 1 or 5 W laser power did not induce any significant effects. Irradiation stimulated BMP2-induced phosphorylation of Smad1/5/8 and BMP2 expression, but had no effect on the expression of inhibitory Smads 6 and 7, BMP4, or insulin-like growth factor 1. Laser irradiation enhanced Smad-induced Id1 reporter activity as well as expression of bone morphogenetic protein (BMP)-induced transcription factors such as Id1, Osterix, and Runx2. Laser irradiation also stimulated BMP-induced expressions of type I collagen, osteonectin, and osteocalcin mRNA, markers of osteoblasts. This enhancement of BMP2-induced ALP activity and Smad phosphorylation by laser irradiation was also observed in primary osteoblasts. These results suggest that LLLI accelerates the differentiation of BMP-induced osteoblasts by stimulating the BMP/Smad signaling pathway.

The current and future therapies of bone regeneration to repair bone defects.

Bone defects often result from tumor resection, congenital malformation, trauma, fractures, surgery, or periodontitis in dentistry. Although dental implants serve as an effective treatment to recover mouth function from tooth defects, many patients do not have the adequate bone volume to build an implant. The gold standard for the reconstruction of large bone defects is the use of autogenous bone grafts. While autogenous bone graft is the most effective clinical method, surgical stress to the part of the bone being extracted and the quantity of extractable bone limit this method. Recently mesenchymal stem cell-based therapies have the potential to provide an effective treatment of osseous defects. In this paper, we discuss both the current therapy for bone regeneration and the perspectives in the field of stem cell-based regenerative medicine, addressing the sources of stem cells and growth factors used to induce bone regeneration effectively and reproducibly.