Stimulation of ATF3 interaction with Smad4 via TGF-ß1 for matrix metalloproteinase 13 gene activation in human breast cancer cells.

We previously reported that transforming growth factor-ß1 (TGF-ß1) stimulated the sustained and prolonged expression of activating transcription factor 3 (ATF3) in highly metastatic and invasive human breast cancer cells (MDA-MB231), in contrast to normal human mammary epithelial cells. However, the mechanism behind the stability of ATF3 expression is not yet known. Based on an in silico approach with co-immunoprecipitation and mass spectrometric analyses, we identified a number of proteins, including Smad4, that interacted with ATF3 after TGF-ß1 treatment in MDA-MB231 cells. The knockdown of Smad4 using the siRNA technique resulted in a significant loss of ATF3 expression in these cells. Chromatin immunoprecipitation was then used to identify the formation of an ATF3 and Smad4 complex at the matrix metalloproteinase 13 (MMP13) promoter upon TGF-ß1-treatment, and the knockdown of Smad4 decreased MMP13 promoter activity in MDA-MB231 cells. Our findings indicate that Smad4 is a pre-requisite for providing stability to ATF3 via TGF-ß1 in human breast cancer cells. The targeting of Smad4 may thus provide the sustainable loss of ATF3 expression that is needed to control breast cancer progression.

TGF-ß1-stimulation of matrix metalloproteinase-13 expression by down-regulation of miR-203a-5p in rat osteoblasts.

Transforming growth factor-beta1 (TGF-ß1) is a pleiotropic and ubiquitous cytokine involved in bone development and bone remodeling. Matrix metalloproteinase-13 (MMP13) plays a role in the degradation of the extracellular matrix (ECM), and the regulation of this gene is critical in bone remodeling. We previously reported that TGF-ß1 stimulates MMP13 expression in rat osteoblasts. Recently, studies have examined the regulation of bone metabolism by microRNAs (miRNAs) to determine their therapeutic potential in osteogenesis. Here, we assessed the effect of TGF-ß1 on down-regulation of miRNAs that target MMP13 and stimulation of MMP13 expression in osteoblasts. We used in silico analysis and identified 11 specific miRNAs which directly target rat MMP13. Among these miRNAs, miR-203a-5p expression was significantly decreased by TGF-ß1-treatment in rat osteoblasts. Transient transfection of a miR-203a-5p mimic into rat osteoblasts reduced MMP13 expression. A luciferase reporter assay confirmed a direct targeting of miR-miR-203a-5p with the 3' untranslated regions of the MMP13 gene. Hence, we suggest that TGF-ß1 stimulated down-regulation of miR-203a-5p, resulting in the stimulation of MMP13 expression in rat osteoblasts. Thus, identification of the role of miR-203a-5p via TGF-ß1 and MMP13 in bone remodeling indicated its potential as a biomarker or therapeutic agent for treating bone and bone-related diseases.

Parathyroid hormone-stimulation of Runx2 during osteoblast differentiation via the regulation of lnc-SUPT3H-1:16 (RUNX2-AS1:32) and miR-6797-5p.

Parathyroid hormone (PTH) acts as a regulator of calcium homeostasis and bone remodeling. Runx2, an essential transcription factor in bone, is required for osteoblast differentiation. Noncoding RNAs such as long noncoding RNAs (lncRNAs) and microRNAs (miRNAs) play crucial roles in regulating gene expression in osteoblasts. In this study, we investigated the effects of PTH on osteoblast differentiation via Runx2, lncRNA, and miRNA expression in human bone marrow stromal cells (hBMSCs) and human osteoblastic cells (MG63). PTH-treatment of hBMSCs for 24 h, 7 days, and 14 days stimulated Runx2 mRNA expression. Using bioinformatics tools, we identified 17 lncRNAs originating from human Runx2 gene. Among these, lnc-SUPT3H-1:16 (RUNX2-AS1:32) expression was highly up-regulated by the 7 d PTH-treatment in hBMSCs. We also identified miR-6797-5p as the putative target of lnc-SUPT3H-1:16 and Runx2 using bioinformatics tools. PTH-treatment increased the expression of miR-6797-5p in hBMSCs, and overexpression of miR-6797-5p decreased osteoblast differentiation in MG63 cells, suggesting a role for lnc-SUPT3H-1:16 as sponge molecule. A luciferase gene reporter assay identified direct targeting of miR-6797-5p with lnc-SUPT3H-1:16 and 3'UTR Runx2 in MG63 cells. Thus, PTH stimulated the expression of lnc-SUPT3H-1:16, miR-6797-5p and Runx2, and due to the sponging mechanism of lnc- SUPT3H-1:16 towards miR-6797-5p, Runx2 was protected, resulting in the promotion of osteoblast differentiation.

Chitosan/nano-hydroxyapatite/nano-zirconium dioxide scaffolds with miR-590-5p for bone regeneration.

Bone tissue engineering (BTE) relies on biocomposite scaffolds and bioactive molecules for bone regeneration. The present study was aimed to synthesize and characterize biocomposite scaffolds containing chitosan (CS), nano-hydroxyapatite (nHAp) and nano­zirconium dioxide (nZrO2) along with microRNA (miRNA) for BTE applications. miRNAs act as post-transcriptional regulator of gene expression. The fabricated biocomposite scaffolds were characterized using SEM, FT-IR and XRD analyses. The effect of a bioactive molecule (miR-590-5p) with scaffolds was tested for osteoblast differentiation at the cellular and molecular levels using mouse mesenchymal stem cells (C3H10T1/2). The results showed that CS/nHAp/nZrO2 scaffolds promoted osteoblast differentiation, and this effect was further increased in the presence of miR-590-5p in C3H10T1/2 cells. Thus, we suggested that CS/nHAp/nZrO2 scaffolds with miR-590-5p would have potential towards the treatment of bone defects.

Syringic acid, a phenolic acid, promotes osteoblast differentiation by stimulation of Runx2 expression and targeting of Smad7 by miR-21 in mouse mesenchymal stem cells.

Syringic acid (SA), a phenolic acid, has been used in Chinese and Indian medicine for treating diabetes but its role in osteogenesis has not yet been investigated. In the present study, at the molecular and cellular levels, we evaluated the effects of SA on osteoblast differentiation. At the cellular level, there was increased alkaline phosphatase (ALP) activity and calcium deposition by SA treatment in mouse mesenchymal stem cells (mMSCs). At the molecular level, SA treatment of these cells stimulated expression of Runx2, a bone transcription factor, and of osteoblast differentiation marker genes such as ALP, type I collagen, and osteocalcin. It is known that Smad7 is an antagonist of TGF-ß/Smad signaling and is a negative regulator of Runx2. microRNAs (miRNAs) play a key role in the regulation of osteogenesis genes at the post-transcriptional level and studies have reported that Smad7 is one of the target genes of miR-21. We found that there was down regulation of Smad7 and up regulation of miR-21 in SA-treated mMSCs. We further identified that the 3'-untranslated region (UTR) of Smad7 was directly targeted by miR-21 in these cells. Thus, our results suggested that SA promotes osteoblast differentiation via increased expression of Runx2 by miR-21-mediated down regulation of Smad7. Hence, SA may have potential in orthopedic applications.

Role of Runx2 in breast cancer-mediated bone metastasis.

Breast cancer is one of the most prevalent forms of cancer in women. The currently available treatment for breast cancer is mostly curative except when it becomes metastatic. One of the major sites for metastasis of breast cancer is the bone. Homing of the circulating tumor cells is tightly regulated including a number of factors present in the cells and their microenvironment. Runx2, a transcription factor plays an important role in osteogenesis and breast cancer mediated bone metastases. One of the recent advances in molecular therapy includes the discovery of the small, non-coding microRNAs (miRNAs) and they target specific genes to reduce their expression at the post-transcriptional level. This review provides an outline of breast cancer mediated bone metastasis and summarizes the recent development on the regulation of Runx2 expression by miRNAs which can lead to novel molecular therapeutics for the same.

Runx2: Structure, function, and phosphorylation in osteoblast differentiation.

Runx2 is a master transcription factor for osteogenesis. The most important phenomenon that makes this protein a master regulator for osteogenesis is its structural integrity. In response to various stimuli, the domains in Runx2 interact with several proteins and regulate a number of cellular events via posttranslational modifications. Hence, in this review we summarized the structural integrity of Runx2 and its posttranslational modifications, especially the phosphorylation responsible for either stimulation or inhibition of its regulatory role in osteogenesis.

Visual regulation of refractive development: insights from animal studies.

Investigations employing animal models have demonstrated that ocular growth and refractive development are regulated by visual feedback. In particular, lens compensation experiments in which treatment lenses are used to manipulate the eye's effective refractive state have shown that emmetropization is actively regulated by signals produced by optical defocus. These observations in animals are significant because they indicate that it should be possible to use optical treatment strategies to influence refractive development in children, specifically to slow the rate of myopia progression. This review highlights some of the optical performance properties of the vision-dependent mechanisms that regulate refractive error development, especially those that are likely to influence the efficacy of optical treatment strategies for myopia. In this respect, the results from animal studies have been very consistent across species; however, to facilitate extrapolation to clinical settings, results are presented primarily for nonhuman primates. In agreement with preliminary clinical trials, the experimental data show that imposed myopic defocus can slow ocular growth and that treatment strategies that influence visual signals over a large area of the retina are likely to be most effective.