A robust in vitro screening assay to identify NF-κB inhibitors for inflammatory muscle diseases

https://doi.org/10.1016/j.intimp.2009.07.001Get rights and content

Abstract

Specific therapies are not available for inflammatory muscle diseases. We and others have shown that the pro-inflammatory NF-κB pathway is highly activated in these conditions. Since NF-κB is an important therapeutic target, we decided to utilize an in vitro screening assay to identify potential inhibitors that block TNF-α induced NF-κB activation in a C2C12 muscle line stably expressing an NF-κB luciferase reporter gene. Upon evaluation of multiple anti-inflammatory agents in undifferentiated myoblasts as well as differentiated myotubes , we found different levels of inhibition depending on the state of differentiation. Interestingly, we found that some drugs that are known to inhibit NF-κB in immune cells were not effective in muscle cells. Drug toxicity was assessed for using an MTT cell viability assay, and the validity of the luciferase assay was verified by immunostaining for NF-κB nuclear translocation in myoblasts. In conclusion, we have determined the optimal assay conditions for detecting potentially valuable NF-κB inhibitors for the first time in a muscle cell line that may have significant therapeutic potential for inflammatory muscle diseases.

Introduction

Corticosteroids have revolutionized treatment of several chronic inflammatory conditions including autoimmune and genetic muscle diseases. However, despite their therapeutic benefits, their use is limited due to significant side effects including steroid induced myopathy, fat deposition, adrenal deficiency, hyperglycemia and osteoporosis. The NF-κB signaling pathway is the primary activator of inflammation in inflammatory myopathies, and therefore is an attractive therapeutic target for these diseases [1], [2], [3].

The NF-κB family of proteins are dimeric transcription factors that regulate the expression of large a number of genes involved in inflammation, immune response and cell survival. In non-stimulated cells, NF-κB remains bound to the inhibitory protein I-κB and is sequestered in the cytoplasm. However, upon stimulation with cytokines such as TNF-α and infectious agents, the cytosolic kinase, IKK phosphorylates I-κB, at which point it becomes susceptible to ubiquitination and degradation. The activated NF-κB dimer is then able to translocate to the nucleus to bind NF-κB promoter elements and drive the expression of a large number of pro-inflammatory genes.

Transient activation of the NF-κB pathway is part of the normal regenerative process in muscle, but persistent over-activation has been shown to be detrimental, leading to large amounts of muscle wasting [4]. Studies by us and others have demonstrated that NF-κB is strongly upregulated in the biopsies of Duchenne Muscular Dystrophy (DMD) patients, localized to both myofibers and within infiltrating monocytes, a feature which also persists in inflammatory myopathy patients [2], [3].

A problem with the use of anti-inflammatory drugs in patients suffering from inflammatory myopathies is that while the agents being administered have been shown to be effective in immune cells, they have not been shown to be effective in myocytes and this could result in suboptimal effectiveness at improving muscle pathology [5]. Therefore, the purpose of this study was to design and evaluate an in vitro system to identify inhibitors that block cytokine induced NF-κB activation in skeletal muscle cells; such inhibitors would likely be beneficial for inflammatory myopathy patients.

Section snippets

Cell culture

Commercially available C2C12 cells that have an integrated chromosomal reporter consisting of six tandem NF-κB response elements coupled to luciferase were cultured with DMEM containing penicillin (100 U/ml) and streptomycin (100 µg/ml), hygromycin (100 µg/ml) and 10% Fetal Bovine Serum (ATCC). These myoblasts were differentiated to myotubes by reducing serum concentrations (replacing 10% FBS with 2% Horse Serum) and allowing them to remain in culture for a minimum of 48 h [6].

Drug treatment

Myoblasts and

Results

A range of TNF-α (10 ng/ml) treatment time points was initially tested in the NF-κB C2C12 myoblast reporter cell line to determine when the optimal induction of luciferase would occur. It was found that a minimum of 2.5 h of TNF-α treatment was required in order to detect induction, which lead to a 5 fold increase in luciferase output (Supplementary Fig. 1), whereas a TNF-α treatment time of 24 h resulted in a 20 fold increase. Using this latter time of treatment, we then tested prednisolone's

Discussion

The NF-kB pathway can be activated by multiple pro-inflammatory molecules such as IL-1 and LPS, but the in vitro system we used specifically monitors only TNF-α induced NF-κB activation. It is important to acknowledge that some agents using this assay could inhibit luciferase readout by blocking the TNF-α pathway. Therefore, the type of NF-κB inhibition that is desired to screen for is dependent on the choice of pro-inflammatory molecule used for its induction.

Prednisolone, one of the most

Acknowledgements

Andreas R. Baudy is a pre-doctoral student in the Molecular Medicine Program of the Institute for Biomedical Sciences at the George Washington University. This work is from a dissertation to be presented to the above program in partial fulfillment of the requirements for the Ph.D. degree.

Dr. Nagaraju is supported by National Institute of Health (RO1-AR050478 and 5U54HD053177), Foundation to Eradicate Dystrophy, Muscular Dystrophy Association, The Myositis Association and US Department of

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