Thrombospondin-1 inhibits osteogenic differentiation of human mesenchymal stem cells through latent TGF-β activation

Transforming growth factor-β (TGF-β) is a critical regulator of bone development and remodeling. TGF-β must be activated from its latent form in order to signal. Thrombospondin-1 (TSP1) is a major regulator of latent TGF-β activation and TSP1 control of TGF-β activation is critical for regulation of TGF-β activity in multiple diseases. Bone marrow-derived mesenchymal stem cells (MSCs) have osteogenic potential and they participate in bone remodeling in injury and in response to tumor metastasis. Since both TSP1 and TGF-β inhibit osteoblast differentiation, we asked whether TSP1 blocks osteoblast differentiation of MSCs through its ability to stimulate TGF-β activation. TSP1 added to human bone marrow-derived MSCs under growth conditions increases active TGF-β. Cultured MSCs express TSP1 and both TSP1 expression and TGF-β activity decrease during osteoblast differentiation. TSP1 and active TGF-β block osteoblast differentiation of MSCs grown in osteogenic media as measured by decreased Runx2 and alkaline phosphatase expression. The inhibitory effect of TSP1 on osteoblast differentiation is due to its ability to activate latent TGF-β, since a peptide which blocks TSP1 TGF-β activation reduced TGF-β activity and restored osteoblast differentiation as measured by increased Runx2 and alkaline phosphatase expression. Anti-TGF-β neutralizing antibody also increased alkaline phosphatase expression in the presence of TSP1. These studies show that TSP1 regulated TGF-β activity is a critical determinant of osteoblast differentiation.     

MiR-210-3p inhibits osteogenic differentiation and promotes adipogenic differentiation correlated with Wnt signaling in ERα-deficient rBMSCs

MicroRNAs (miRNAs) regulate activities in living organisms through various signaling pathways and play important roles in the development and progression of osteoporosis. The balance between osteogenic and adipogenic differentiation of rBMSCs is closely related to the occurrence of osteoporosis. ERα regulates bone metabolism in various tissues. However, the correlation among ERα, miRNAs, and the differentiation of rBMSCs is still unclear. In this study, we used lentivirus transfection into rBMSCs to construct an ERα-deficient model, analyzed the differences in expressed miRNAs between control and ERα-deficient rBMSCs. The results revealed that the expression of 25 miRNAs were upregulated, 164 miRNAs were downregulated, and some of the regulated miRNAs such as miR-210-3p and miR-214-3p were related to osteogenic or adipogenic differentiation, as well as to particular signaling pathways. Next, we overexpressed miR-210-3p to evaluate its effects on the osteogenic and adipogenic differentiation of rBMSCs, and identified the relationship among miR-210-3p, Wnt signaling pathway, and the differentiation of rBMSCs. The results indicated that ERα-deficient inhibited osteogenic differentiation, promoted adipogenic differentiation, and regulated the expression of some miRNAs. Meanwhile, overexpression of miR-210-3p promoted osteogenic differentiation and inhibited adipogenic differentiation of rBMSCs, processes likely to be related to the Wnt signaling pathway. In conclusion, we identified a group of upregulated and downregulated miRNAs in ERα-deficient rBMSCs that might play a vital role in regulating osteogenic or adipogenic differentiation. One of these, miR-210-3p, inhibited osteogenic differentiation and promoted adipogenic differentiation correlated with the Wnt signaling pathway in ERα-deficient rBMSCs, providing new insight into the regulation of bone metabolism.     

The flavonoid quercetin induces hypoxia-inducible factor-1α (HIF-1α) and inhibits cell proliferation by depleting intracellular iron

Quercetin, a flavonoid with anti-oxidant, metal chelating, kinase modulating and anti-proliferative properties, can induce hypoxia-inducible factor-1α (HIF-1α) in normoxia, but its mechanism of action has not been determined. In this study we characterized the induction of HIF-1α and the inhibition of cell proliferation caused by quercetin in HeLa and ASM (airway smooth muscle) cells and examined the effect of iron on these processes. Furthermore, we investigated the relevance of the intracellular levels of quercetin to HIF-1α expression and cell proliferation. Our data demonstrate that quercetin depletes intracellular calcein–chelatable iron and that supplying additional iron from extracellular or intracellular pools abrogates the induction of HIF-1α by quercetin. Moreover, addition of iron reverses the quercetin-induced inhibition of DNA synthesis, cell proliferation and cycle progression, but to different extents, depending on cell type. We propose that quercetin stabilises HIF-1α and inhibits cell proliferation predominantly by decreasing the concentration of intracellular iron through chelation.     

Tax1 binding protein 3 regulates osteogenic and adipogenic differentiation through inactivating Wnt/β-catenin signalling

Tax1 binding protein 3 (Tax1bp3) is a PDZ domain-containing protein that is overexpressed in cancer. Previous studies recognized Tax1bp3 as an inhibitor of β-catenin. Till now it is not known whether Tax1bp3 regulates osteogenic and adipogenic differentiation of mesenchymal progenitor cells. In the current study, the data showed that Tax1bp3 was expressed in bone and was increased in the progenitor cells when induced toward osteoblast and adipocyte differentiation. The overexpression of Tax1bp3 in the progenitor cells inhibited osteogenic differentiation and conversely stimulated adipogenic differentiation, and the knockdown of Tax1bp3 affected the differentiation of the progenitor cells oppositely. Ex vivo experiments using the primary calvarial osteoblasts from osteoblast-specific Tax1bp3 knock-in mice also demonstrated the anti-osteogenic and pro-adipogenic function of Tax1bp3. Mechanistic investigations revealed that Tax1bp3 inhibited the activation of canonical Wnt/β-catenin and bone morphogenetic proteins (BMPs)/Smads signalling pathways. Taken together, the current study has provided evidences demonstrating that Tax1bp3 inactivates Wnt/β-catenin and BMPs/Smads signalling pathways and reciprocally regulates osteogenic and adipogenic differentiation from mesenchymal progenitor cells. The inactivation of Wnt/β-catenin signalling may be involved in the reciprocal role of Tax1bp3.     

MicroRNA 107 partly inhibits endothelial progenitor cells differentiation via HIF-1β

Endothelial progenitor cells (EPCs) play an important role in tissue repair after ischemic heart disease. In particular, the recovery of endothelial function is reliant on the ability and rate of EPCs differentiate into mature endothelial cells. The present study evaluated the effect of microRNA 107 (miR-107) on the mechanism of EPCs differentiation. EPCs were isolated from rats' bone marrow and miR-107 expression of EPCs in hypoxic and normoxic conditions were measured by real-time qualitative PCR. CD31 was analyzed by flow cytometry and eNOS was examined by real-time qualitative PCR and western blotting and these were used as markers of EPC differentiation. In order to reveal the mechanism, we used miR107 inhibitor and lentiviral vector expressing a short hairpin RNA (shRNA) that targets miR-107 and hypoxia-inducible factor-1 β (HIF-1β) to alter miR107 and HIF-1β expression. MiR-107 expression were increased in EPCs under hypoxic conditions. Up-regulation of miR-107 partly suppressed the EPCs differentiation induced in hypoxia, while down-regulation of miR-107 promoted EPC differentiation. HIF-1β was the target. This study indicated that miR-107 was up-regulated in hypoxia to prevent EPCs differentiation via its target HIF-1β. The physiological mechanisms of miR-107 must be evaluated if it is to be used as a potential anti-ischemia therapeutic regime.     

EMILIN1-α4/α9 integrin interaction inhibits dermal fibroblast and keratinocyte proliferation

EMILIN1 promotes α4β1 integrin-dependent cell adhesion and migration and reduces pro-transforming growth factor-β processing. A knockout mouse model was used to unravel EMILIN1 functions in skin where the protein was abundantly expressed in the dermal stroma and where EMILIN1-positive fibrils reached the basal keratinocyte layer. Loss of EMILIN1 caused dermal and epidermal hyperproliferation and accelerated wound closure. We identified the direct engagement of EMILIN1 to α4β1 and α9β1 integrins as the mechanism underlying the homeostatic role exerted by EMILIN1. The lack of EMILIN1-α4/α9 integrin interaction was accompanied by activation of PI3K/Akt and Erk1/2 pathways as a result of the reduction of PTEN. The down-regulation of PTEN empowered Erk1/2 phosphorylation that in turn inhibited Smad2 signaling by phosphorylation of residues Ser245/250/255. These results highlight the important regulatory role of an extracellular matrix component in skin proliferation. In addition, EMILIN1 is identified as a novel ligand for keratinocyte α9β1 integrin, suggesting prospective roles for this receptor-ligand pair in skin homeostasis.     

Adrenomedullin promotes human endothelial cell proliferation via HIF-1α

Adrenomedullin (ADM) and hypoxia-inducible factor-1α (HIF-1α) are important pro-proliferation genes in response to hypoxic stress. Although it was reported that ADM is a target gene for HIF-1, recent studies also showed that ADM regulates HIF-1 expression and its activity; however, the mechanism of action remains unknown. Two stable human endothelial cell lines with HIF-1α knockdown by hy926-siHIF-1α or HMEC-siHIF-1α were established. mRNA and protein expression of ADM and HIF-1α in EA.hy926 and HMEC1 cells were examined under hypoxic stress. Upon ADM treatment, cell proliferation was investigated and the expression profiles of HIF-1α and its target genes (VEGF, PFKP, PGK1, and AK1) were examined. Furthermore, the proline hydroxylase (PHD) mRNA level and its activity were investigated. We observed that mRNA and protein expression of ADM in hypoxia are earlier events than HIF-1α in EA.hy926 and HMEC1 cells. ADM-promoted cell proliferation of endothelial cells, which was HIF-1α dependent. We also found that ADM up-regulated the mRNA and protein expressions of HIF-1α- and HIF-1-targeted genes, and ADM up-regulated the protein expressions of HIF-1α through down-regulation of PHD mRNA expression and PHD activity.     

Autologous blood transfusion promotes autophagy and inhibits hepatocellular carcinoma progression through HIF-1α signalling pathway

To explore the molecular mechanism of autologous blood transfusion promoting autophagy of hepatocellular carcinoma (HCC) cells and inhibiting the HCC progression through HIF-1α signalling pathway. This is a research paper. Rat hepatocellular carcinoma model and HepG2 cell model were built. The rats with HCC were conducted a surgery, and their blood was collected for detection to detect the recurrence and metastasis of the rats. Western blot was used to analysed the expression of HIF-1α, TP53, MDM2, ATG5 and ATG14 protein. The apoptosis rate of HepG2 cells was detected by flow cytometry, and autophagosomes were observed by transmission electron microscopy. HIF-1α expression was measured by immunofluorescence assay. The expressions of HIF-1α, TP53, MDM2, ATG5 and ATG14 protein were highest in model + autoblood group compared with the model group. HIF-1α content of model group was higher, but content of TP53, MDM2, ATG5 and ATG14 in the model group is the second. The highest apoptosis rate was found in HepG2 + autoblood group. The number of autophagosomes in HepG2 + autoblood was obviously larger than that of HepG2 + autoblood + inhibitor. HIF-1α expression of immunofluorescence assay showed that high expression of HIF-1α was clearly observed in HepG2 and HepG2 + autoblood group from confocal observation. However, there was no HIF-1α protein expression in HepG2 + autoblood + inhibitor group. The migration rate in HepG2 group, HepG2 + autoblood group and HepG2 + autoblood + inhibitor group was 85.71 ± 7.38%, 14.36 ± 6.54% and 61.25 ± 5.39%, respectively. Autologous blood transfusion promotes autophagy of HCC cells through HIF-1α signalling pathway, which further inhibits HCC migration and erosion.     

HOTAIRM1 promotes osteogenic differentiation and alleviates osteoclast differentiation by inactivating the NF-κB pathway

Osteoporosis (OP), one of the most prevalent chronic progressive bone diseases, is caused by deficiency in bone formation by osteoblasts or excessive bone resorption by osteoclasts and subsequently increases the risk of bone fractures. Emerging evidence has indicated that long noncoding RNAs (lncRNAs) play key roles in many biological processes and various disorders. However, the role and mechanism of HOX antisense intergenic RNA myeloid 1 (HOTAIRM1), a myeloid-specific lncRNA, in osteoclast differentiation, osteogenic differentiation, and OP remain unclear. In this study, we found that HOTAIRM1 was upregulated during ossification of ligamentum flavum and osteogenic differentiation, while it was downregulated in osteoclast differentiation and in the bone and serum of human and mouse with OP. Further investigation revealed that silencing Hotairm1 decreased the expression of the osteogenic markers and attenuated osteogenesis. Moreover, forced Hotairm1 expression inhibited the expressions of the osteoclastogenesis markers and alleviated receptor activator of nuclear factor kappa B (NF-κB) ligand (RANKL)-induced osteoclast differentiation. Mechanically, Hotairm1 repressed the phosphorylation of p65 and inhibitor of κBα (IκBα) and attenuated RANKL-mediated enhancement of phos-p65 and IκBα, suggesting that Hotairm1 inhibits RANKL-induced osteoclastogenesis through the NF-κB pathway. In conclusion, our data identified a crucial role of HOTAIRM1 in OP, providing a proof of this molecule as a potential diagnostic marker and a possible therapeutic target against OP.     

α-Tocopheryl succinate affects malignant cell viability,proliferation, and differentiation

The widespread occurrence of malignant tumors motivates great attention to finding and investigating effective new antitumor preparations. Such preparations include compounds of the vitamin E family. Among them, α-tocopheryl succinate (vitamin E succinate (VES)) has the most pronounced antitumor properties. In this review, various targets and mechanisms of the antitumor effect of vitamin E succinate are characterized. It has been shown that VES has multiple intracellular targets and effects, and as a result VES is able to induce apoptosis in tumor cells, inhibit their proliferation, induce differentiation, prevent metastasizing, and inhibit angiogenesis. However, VES has minimal effects on normal cells and tissues. Due to the variety of targets and selectivity of action, VES is a promising agent against malignant neoplasms. More detailed studies in this area can contribute to development of effective and safe chemotherapeutic preparations.     

α-Enolase inhibits apoptosis and promotes cell invasion and proliferation of skin cutaneous melanoma

Molecular Biology Reports - The glycolytic enzyme, α-Enolase (ENO1), catalyzes the production of phosphoenolpyruvate from 2-phosphoglycerate, thereby enhancing glycolysis and contributing to...     

Spleen tyrosine kinase suppresses osteoblastic differentiation through MAPK and PKCα

Spleen tyrosine kinase (Syk) is a non-receptor protein kinase present in abundance in a wide range of hematopoietic cells. Syk reportedly plays a crucial role in immune signaling in B cells and cells bearing Fcγ-activation receptors. The role of syk in osteoblastic differentiation has not been well elucidated. We report herein the role of syk in osteoblastic differentiation. We investigated the effects of two syk inhibitors on osteoblastic differentiation in mouse preosteoblastic MC3T3-E1 cells and bone marrow stromal ST2 cells. Expression of syk was detected in these two cell lines. Two syk inhibitors stimulated mRNA expression of osteoblastic markers (ALP, Runx2, Osterix). Mineralization of extracellular matrix was also promoted by treatment with syk inhibitors. Knockdown of Syk caused increased mRNA expression of osteoblastic markers. In addition, syk inhibitor and knockdown of Syk suppressed phosphorylation of mitogen-activated protein kinase (MAPK) and protein kinase Cα (PKCα). Our results indicate that syk might regulate osteoblastic differentiation through MAPK and PKCα.     

VPA inhibits renal cancer cell migration by targeting HDAC2 and down-regulating HIF-1α

Cell migration plays major roles in human renal cancer-related death, but the molecular mechanisms remain unclear. Valproic acid (VPA) is a broad-spectrum inhibitor of class I and II histone deacetylases and shows great anticancer activity in a variety of human cancers. In this study, we found that VPA significantly inhibited cell migration but not proliferation of human renal cancer ACHN cells. Mechanistic studies found that VPA significantly inhibited the expression of HIF-1α. Knockdown of HIF-1α could obviously inhibited cell migration, while over-expression of HIF-1α markedly rescued the inhibition of VPA on cell migration. Further studies found that knockdown of HDAC2 completely mimicked the effects of VPA on HIF-1α and cell migration, and over-expression of HIF-1α could also rescue the effects of HDAC2 knockdown on cell migration. Collectively, these results indicated that the potential of specific inhibition of HDAC2 by small molecular chemicals may lead to future therapeutic agents in human renal cancer treatment.