microRNA-665 promotes the proliferation and matrix degradation of nucleus pulposus through targeting GDF5 in intervertebral disc degeneration.

Growing evidences suggested that microRNAs (miRNAs) played important roles in the development of intervertebral disc degeneration (IDD). However, the expression level and function of miR-665 in IDD remain unknown. In this study, we showed that the expression level of miR-665 was upregulated in degenerative human NP samples. In addition, miR-665 expression level gradually increased with the exacerbation of disc degeneration grade. Moreover, miR-665 expression level was positively associated with the Pfirrmann grade. Ectopic expression of miR-665 promoted NP cell growth. Furthermore, miR-665 overexpression decreased aggrecan and Col II expression and ectopic expression of miR-665 increased MMP-3 and MMP-13 expression in NP cell. We identified growth differentiation factor 5 (GDF5) was a direct target gene of miR-665 in NP cell and enforced expression of miR-665 decreased GDF5 expression. Elevated expression of miR-665 enhanced NP cell proliferation and decreased aggrecan and Col II expression. In addition, ectopic expression of miR-665 increased MMP-3 and MMP-13 expression through inhibiting GDF5 expression in NP cells. These results suggested that dysregulated miR-665 expression might act an important role in the development of IDD.

MiR-615-3p inhibits the osteogenic differentiation of human lumbar ligamentum flavum cells via suppression of osteogenic regulators GDF5 and FOXO1.

Ossification of the ligamentum flavum (OLF) is a disease of heterotopic ossification in spinal ligaments. The key of the OLF pathogenesis is the differentiation of fibroblasts into osteoblasts. In this study, we explored the role of miR-615-3p in the osteogenic differentiation of human LF cells. The expression of miR-615-3p was detected during the osteogenic differentiation of hFOB1.19 human osteoblasts, human BMSCs, and human LF cells. The qPCR results showed that miR-615-3p was being decreased during the osteogenic differentiation of these cell lineages. Then, both gain- and loss-function experiments, respectively performed by single-strand miR-615-3p mimic and antagomir, revealed that miR-615-3p negatively regulated the osteogenesis of hLF cells, manifested by a lighter staining degree with Alizarin Red and a decreased level of osteogenic marker genes, including alkaline phosphatase (ALP), RUNX2, osterix (ostx), osteocalcin (OCN), and osteopontin (OPN). Subsequently, our data on bioinformatic analysis, 3'-UTR luciferase activity assay, and protein level detection indicated that miR-615-3p directly targeted and suppressed the expression of FOXO1 and GDF5. Furthermore, knockdown of either FOXO1 or GDF5 could inhibit the osteogenic differentiation of hLF cells, which displayed a similar effect with the miR-615-3p mimic. In conclusion, miR-615-3p negatively regulates the osteogenic differentiation of hLF cells through post-transcriptionally suppressing osteogenic regulators GDF5 and FOXO1. It can be regarded as a potential target for human OLF therapy.

Canonical BMP-Smad signalling promotes neurite growth in rat midbrain dopaminergic neurons.

Ventral midbrain (VM) dopaminergic (DA) neurons project to the dorsal striatum via the nigrostriatal pathway to regulate voluntary movements, and their loss leads to the motor dysfunction seen in Parkinson's disease (PD). Despite recent progress in the understanding of VM DA neurogenesis, the factors regulating nigrostriatal pathway development remain largely unknown. The bone morphogenetic protein (BMP) family regulates neurite growth in the developing nervous system and may contribute to nigrostriatal pathway development. Two related members of this family, BMP2 and growth differentiation factor (GDF)5, have neurotrophic effects, including promotion of neurite growth, on cultured VM DA neurons. However, the molecular mechanisms regulating their effects on DA neurons are unknown. By characterising the temporal expression profiles of endogenous BMP receptors (BMPRs) in the developing and adult rat VM and striatum, this study identified BMP2 and GDF5 as potential regulators of nigrostriatal pathway development. Furthermore, through the use of noggin, dorsomorphin and BMPR/Smad plasmids, this study demonstrated that GDF5- and BMP2-induced neurite outgrowth from cultured VM DA neurons is dependent on BMP type I receptor activation of the Smad 1/5/8 signalling pathway.

GDF5 regulates TGFß-dependent angiogenesis in breast carcinoma MCF-7 cells: in vitro and in vivo control by anti-TGFß peptides.

BACKGROUND: TGFß overproduction in cancer cells is one of the main characteristics of late tumor progression being implicated in metastasis, tumor growth, angiogenesis and immune response. We investigated the therapeutic efficacy of anti-TGFß peptides in the control of angiogenesis elicited by conditional over-expression of TGFß. METHODS: We have inserted in human MCF7 mammary-cancer cells a mutated TGFß gene in a tetracycline-repressible vector to obtain conditional expression of mature TGFß upon transient transfection, evaluated the signaling pathways involved in TGFß-dependent endothelial cells activation and the efficacy of anti-TGFß peptides in the control of MCF7-TGFß-dependent angiogenesis. RESULTS: TGFß over-expression induced in MCF7 several markers of the epithelial-to-mesenchymal transition. Conditioned-medium of TGFß-transfected MCF7 stimulated angiogenesis in vivo and in vitro by subsequent activation of SMAD2/3 and SMAD1/5 signaling in endothelial cells, as well as SMAD4 nuclear translocation, resulting in over-expression of the pro-angiogenic growth and differentiation factor-5 (GDF5). Inhibition or silencing of GDF5 in TGFß-stimulated EC resulted in impairment of GDF5 expression and of TGFß-dependent urokinase-plasminogen activator receptor (uPAR) overproduction, leading to angiogenesis impairment. Two different TGFß antagonist peptides inhibited all the angiogenesis-related properties elicited in EC by exogenous and conditionally-expressed TGFß in vivo and in vitro, including SMAD1/5 phosphorylation, SMAD4 nuclear translocation, GDF5 and uPAR overexpression. Antagonist peptides and anti-GDF5 antibodies efficiently inhibited in vitro and in vivo angiogenesis. CONCLUSIONS: TGFß produced by breast cancer cells induces in endothelial cells expression of GDF5, which in turn stimulates angiogenesis both in vitro and in vivo. Angiogenesis activation is rapid and the involved mechanism is totally opposed to the old and controversial dogma about the AKL5/ALK1 balance. The GDF-dependent pro-angiogenic effects of TGFß are controlled by anti-TGFß peptides and anti-GDF5 antibodies, providing a basis to develop targeted clinical studies.