Nontransgenic genome modification in plant cells.

Zinc finger nucleases (ZFNs) are a powerful tool for genome editing in eukaryotic cells. ZFNs have been used for targeted mutagenesis in model and crop species. In animal and human cells, transient ZFN expression is often achieved by direct gene transfer into the target cells. Stable transformation, however, is the preferred method for gene expression in plant species, and ZFN-expressing transgenic plants have been used for recovery of mutants that are likely to be classified as transgenic due to the use of direct gene-transfer methods into the target cells. Here we present an alternative, nontransgenic approach for ZFN delivery and production of mutant plants using a novel Tobacco rattle virus (TRV)-based expression system for indirect transient delivery of ZFNs into a variety of tissues and cells of intact plants. TRV systemically infected its hosts and virus ZFN-mediated targeted mutagenesis could be clearly observed in newly developed infected tissues as measured by activation of a mutated reporter transgene in tobacco (Nicotiana tabacum) and petunia (Petunia hybrida) plants. The ability of TRV to move to developing buds and regenerating tissues enabled recovery of mutated tobacco and petunia plants. Sequence analysis and transmission of the mutations to the next generation confirmed the stability of the ZFN-induced genetic changes. Because TRV is an RNA virus that can infect a wide range of plant species, it provides a viable alternative to the production of ZFN-mediated mutants while avoiding the use of direct plant-transformation methods.

Halofuginone inhibits Smad3 phosphorylation via the PI3K/Akt and MAPK/ERK pathways in muscle cells: effect on myotube fusion.

Halofuginone, a novel inhibitor of Smad3 phosphorylation, has been shown to inhibit muscle fibrosis and to improve cardiac and skeletal muscle functions in the mdx mouse model of Duchenne muscular dystrophy. Here, we demonstrate that halofuginone promotes the phosphorylation of Akt and mitogen-activated protein kinase (MAPK) family members in a C2 muscle cell line and in primary myoblasts derived from wild-type and mdx mice diaphragms. Halofuginone enhanced the association of phosphorylated Akt and MAPK/extracellular signal-regulated protein kinase (ERK) with the non-phosphorylated form of Smad3, accompanied by a reduction in Smad3 phosphorylation levels. This reduction was reversed by inhibitors of the phosphoinositide 3'-kinase/Akt (PI3K/Akt) and MAPK/ERK pathways, suggesting their specific role in mediating halofuginone's inhibitory effect on Smad3 phosphorylation. Halofuginone enhanced Akt, MAPK/ERK and p38 MAPK phosphorylation and inhibited Smad3 phosphorylation in myotubes, all of which are crucial for myotube fusion. In addition, halofuginone increased the association Akt and MAPK/ERK with Smad3. As a consequence, halofuginone promoted myotube fusion, as reflected by an increased percentage of C2 and mdx myotubes containing high numbers of nuclei, and this was reversed by specific inhibitors of the PI3K and MAPK/ERK pathways. Together, the data suggest a role, either direct or via inhibition of Smad3 phosphorylation, for Akt or MAPK/ERK in halofuginone-enhanced myotube fusion, a feature which is crucial to improving muscle function in muscular dystrophies.

Inhibition of transforming growth factor beta signaling by halofuginone as a modality for pancreas fibrosis prevention.

OBJECTIVES: Chronic pancreatitis is characterized by inflammation and fibrosis. We evaluated the efficacy of halofuginone, an inhibitor of collagen synthesis and myofibroblast activation, in preventing cerulein-induced pancreas fibrosis. METHODS: Collagen synthesis was evaluated by in situ hybridization and staining. Levels of prolyl 4-hydroxylase beta (P4Hbeta), cytoglobin/stellate cell activation-associated protein (Cygb/STAP), transgelin, tissue inhibitors of metalloproteinases, serum response factor, transforming growth factor beta (TGFbeta), Smad3, and pancreatitis-associated protein 1 (PAP-1) were determined by immunohistochemistry. Metalloproteinase activity was evaluated by zymography. RESULTS: Halofuginone prevented cerulein-dependent increase in collagen synthesis, collagen cross-linking enzyme P4Hbeta, Cygb/STAP, and tissue inhibitors of metalloproteinase 2. Halofuginone did not affect TGFbeta levels in cerulein-treated mice but inhibited serum response factor synthesis and Smad3 phosphorylation. In culture, halofuginone inhibited pancreatic stellate cell (PSC) proliferation and TGFbeta-dependent increase in Cygb/STAP and transgelin synthesis and metalloproteinase 2 activity. Halofuginone increased c-Jun N-terminal kinase phosphorylation in PSCs derived from cerulein-treated mice. Halofuginone prevented the increase in acinar cell proliferation and further increased the cerulein-dependent PAP-1 synthesis. CONCLUSIONS: Halofuginone inhibits Smad3 phosphorylation and increases c-Jun N-terminal kinase phosphorylation, leading to the inhibition of PSC activation and consequent prevention of fibrosis. Halofuginone increased the synthesis of PAP-1, which further reduces pancreas fibrosis. Thus, halofuginone might serve as a novel therapy for pancreas fibrosis.