The silencing of Bcl‑xL in the non‑small cell lung cancer (NSCLC) cell line, A549, downregulates miR‑361‑5p expression. This study aimed to determine the biological effects of miR‑361‑5p on NSCLC, and to elucidate the molecular mechanisms through which apoptosis is regulated. MicroRNA (miRNA or miR) functional analyses were performed via transfection of miR‑361‑5p mimics and inhibitors, demonstrating that the inhibition of miR‑361‑5p induced the apoptosis of NSCLC cells. To elucidate the function of miR‑361‑5p in vivo, cells transfected with miR‑361‑5p inhibitors were microinjected into zebrafish embryos, and immunostained using antibodies to detect the active form of caspase‑3. Co-transfection with siBcl‑xL and miR‑361‑5p mimics illustrated the association between Bcl‑xL, miR‑361‑5p and apoptosis; miR‑361‑5p mimics blocked the apoptosis initiated by siBcl‑xL. Luciferase reporter assays identified mothers against decapentaplegic homolog 2 (SMAD2) as a novel target of miR‑361‑5p and the reduction of its protein level was validated by western blot analysis. To confirm the molecular mechanisms through which apoptosis is regulated, gene rescue experiments revealed that the ectopic expression of SMAD2 attenuated the inhibitory effects on apoptosis induced by miR‑361‑5p. In this study, to the best of our knowledge, we provide the first evidence that miR‑361‑5p functions as an oncomiR in A549 and SK‑LU‑1 cells through the regulation of SMAD2, suggesting that miR‑361‑5p may be employed as a potential therapeutic target for the miRNA-based therapy of NSCLC.
Transforming growth factor-β (TGF-β) is a pleiotropic growth factor implicated in the development of atherosclerosis for its role in mediating glycosaminoglycan (GAG) chain hyperelongation on the proteoglycan biglycan, a phenomenon that increases the binding of atherogenic lipoproteins in the vessel wall. Phosphorylation of the transcription factor Smad has emerged as a critical step in the signaling pathways that control the synthesis of biglycan, both the core protein and the GAG chains. We have used flavopiridol, a well-known cyclin-dependent kinase inhibitor, to study the role of linker region phosphorylation in the TGF-β-stimulated synthesis of biglycan. We used radiosulfate incorporation and SDS-PAGE to assess proteoglycan synthesis, real-time polymerase chain reaction to assess gene expression, and chromatin immunoprecipitation to assess the binding of Smads to the promoter region of GAG Synthesizing genes. Flavopiridol blocked TGF-β-stimulated synthesis of mRNA for the GAG synthesizing enzymes, and chondroitin 4-sulfotransferase (C4ST-1), chondroitin sulfate synthase-1 (ChSy-1) and TGF-β-mediated proteoglycans synthesis as well as GAG hyperelongation. Flavopiridol blocked TGF-β-stimulated Smad2 phosphorylation at both the serine triplet and the isolated threonine residue in the linker region. The binding of Smad to the promoter region of the C4ST-1 and ChSy-1 genes was stimulated by TGF-β, and this response was blocked by flavopiridol, demonstrating that linker region phosphorylated Smad can pass to the nucleus and positively regulate transcription. These results demonstrate the validity of the kinases, which phosphorylate the Smad linker region as potential therapeutic target(s) for the development of an agent to prevent atherosclerosis.
Hyperelongation of glycosaminoglycan chains on proteoglycans facilitates increased lipoprotein binding in the blood vessel wall and the development of atherosclerosis. Increased mRNA expression of glycosaminoglycan chain synthesizing enzymes in vivo is associated with the development of atherosclerosis. In human vascular smooth muscle, transforming growth factor-β (TGF-β) regulates glycosaminoglycan chain hyperelongation via ERK and p38 as well as Smad2 linker region (Smad2L) phosphorylation. In this study, we identified the involvement of TGF-β receptor, intracellular serine/threonine kinases and specific residues on transcription factor Smad2L that regulate glycosaminoglycan synthesizing enzymes. Of six glycosaminoglycan synthesizing enzymes, xylosyltransferase-1, chondroitin sulfate synthase-1, and chondroitin sulfotransferase-1 were regulated by TGF-β. In addition ERK, p38, PI3K and CDK were found to differentially regulate mRNA expression of each enzyme. Four individual residues in the TGF-β receptor mediator Smad2L can be phosphorylated by these kinases and in turn regulate the synthesis and activity of glycosaminoglycan synthesizing enzymes. Smad2L Thr220 was phosphorylated by CDKs and Smad2L Ser250 by ERK. p38 selectively signalled via Smad2L Ser245. Phosphorylation of Smad2L serine residues induced glycosaminoglycan synthesizing enzymes associated with glycosaminoglycan chain elongation. Phosphorylation of Smad2L Thr220 was associated with XT-1 enzyme regulation, a critical enzyme in chain initiation. These findings provide a deeper understanding of the complex signalling pathways that contribute to glycosaminoglycan chain modification that could be targeted using pharmacological agents to inhibit the development of atherosclerosis.
Transforming growth factor beta (TGFbeta) is secreted as a large latent precursor from both normal and transformed cells which needs to be activated for biological activity. The active TGFbeta binds either directly to TbetaR-II or indirectly by binding to beta-glycan which then presents the TGFbeta to TbetaR-II. Formation of the TGFbeta-TbetaR-II complex rapidly leads to phosphorylation of TbetaR-I. TbetaR-I, in turn, phosphorylates receptor-specific Smads and induces their translocation into the nucleus. TGFbeta is able to act as a growth stimulator or inhibitor and elicits a broad spectrum of biological effects on various cell types. However, these cells may lose their sensitivity and responsiveness to TGFbeta. Down-regulation or loss of functional receptors, aberrant signal transduction pathways due to Smad mutations, loss of the cell's ability to activate latent TGFbeta, loss of the peptide itself or functional genes that control the transcription and translation of TGFbeta may contribute to development of cancer.