ABSTRACT
Radiation-induced fibrosis (RIF) is thought to involve the excessive accumulation of collagen and other extracellular matrix components; previously, we reported that ionizing radiation increased the type I collagen expression and that transforming growth factor (TGF)-ß was involved in this increase through activating its downstream mediator, Smad3. A recent study found that microRNAs (miRNAs)-small, noncoding sequences approximately 20 nucleotides long-negatively regulate the gene expression posttranscriptionally, and it has been suggested that miRNAs play essential roles in cellular processes, including fibrosis. However, their role in the development of RIF remains unexplored. In the present study, we examined the effects of miRNA on the expression of type I collagen induced by ionizing radiation and the mechanisms underlying the miRNA expression observed following ionizing radiation. We analyzed the regulation of miRNA following ionizing radiation by an miRNA real-time PCR, and found that miR-29 family members were downregulated in irradiated mouse fibroblasts and directly targeted type I collagen genes by specifically binding to the 3' untranslated region. We also found that the overexpression of miR-29 inhibited the ionizing radiation-induced expression of type I collagen, whereas the knockdown of miR-29 enhanced it. In addition, TGF-ß/Smad-signaling significantly decreased the transcription of miR-29, whereas the inhibition of this signaling pathway cancelled this decrease. In conclusion, miR-29 was involved in the regulation of type I collagen expression through the TGF-ß/Smad-signaling pathway in irradiated cells, suggesting that miR-29 may be an important regulator of RIF.
Subject(s)
Collagen Type I/genetics , Gene Expression Regulation/genetics , Gene Expression Regulation/radiation effects , MicroRNAs/genetics , Animals , Base Sequence , Down-Regulation/genetics , Down-Regulation/radiation effects , Fibrosis , Mice , NIH 3T3 Cells , Signal Transduction/genetics , Signal Transduction/radiation effects , Smad Proteins/metabolism , Transforming Growth Factor beta/metabolismABSTRACT
Bone is essentially composed of two components, hydroxyapatite and extracellular matrix proteins. The extracellular matrix of bone is primary composed of collagen, mostly type I collagen, with lesser amounts of other types of collagen such as type V collagen. Osteoblast differentiation is a multi-step process in which many classes of factors function in a coordinated manner. Sp7/Osterix, which binds to G/C-rich sequences, is a transcription factor that contributes to osteoblast differentiation. The present study aimed to clarify the involvement of Sp7/Osterix with the proximal promoter region of the mouse Col1a2 gene containing multiple G/C-rich sequences exist. Consequently, a functional analysis of the proximal mouse Col1a2 promoter showed that a substitution mutation of the second G/C-rich sequence from the transcription site specifically decreased the activity of osteoblastic cells. In addition, the experiments of overexpression of Sp7/Osterix and treatment with its specific siRNA showed that this G/C-rich sequence is responsible for the specific expression in osteoblastic cells. Consistent with these data, Sp7/Osterix bound to the region and increased the expression of the Col1a2 gene in association with osteoblast differentiation in the culture system.
Subject(s)
Collagen Type I/genetics , Gene Expression Regulation , Osteoblasts/metabolism , Promoter Regions, Genetic , Transcription Factors/genetics , Animals , Base Composition , Binding Sites , Cell Differentiation , Cell Line , Collagen Type I/metabolism , Genes, Reporter , Luciferases/genetics , Luciferases/metabolism , Mice , Mutation , NIH 3T3 Cells , Osteoblasts/cytology , Protein Binding , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Signal Transduction , Sp7 Transcription Factor , Transcription Factors/antagonists & inhibitors , Transcription Factors/metabolismABSTRACT
The murine preosteoblastic cell line, MC3T3-E1, is widely used to study bone formation and differentiation in vitro. However, this cell line is unstable in culture. The current study was designed to establish a stable osteoblastic cell line. A mammalian expression vector carrying the SV 40 large T antigen was introduced into a primary culture of cells isolated from the calvaria of newborn mice. Among isolated cell lines, the MN16 cell line was selected for further characterization. The MN16 cell line was cultured for 28 days, and compared with the MC3T3-E1 cell line with or without induction. The expression of bone-related genes was examined using the real-time RT-PCR technique. Alizarin red and von Kossa staining were used to detect mineralization of nodules in the cultures. The cell line showed the characteristics of osteoblastic cells in term of gene expression patterns of various molecular markers and calcium deposition in the cell layer after induction. Furthermore, the MN16 cells showed strong adhesion to the basic domain of collagen, a result that is specific for bone-derived cells. The MN16 cell line was found to be stably differentiated into bone formation cells in vitro and should be useful for studying bone biology.
