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Nitric Oxide Activates Signaling by c-Raf, MEK, p-JNK, p38 MAPK and p53 in Human Mesenchymal Stromal Cells inhibits their Osteogenic Differentiation by Blocking Expression of Runx2

Tino Felka, Christine Ulrich, Bernd Rolauffs, Falk Mittag, Torsten Kluba, Peter DeZwart, Gunnar Ochs, Michael Bonin, Kay Nieselt, Melanie L Hart and Wilhelm K Aicher

Introduction: Mesenchymal stromal cells (MSC) are a promising therapy for wound healing and regeneration of inflamed tissues. They are used clinically for different symptoms and diseases and are being investigated in clinical trials world wide at an increasing rate. However, depending on the application protocol and site of treatment, MSC may face an inflammatory environment. Objective: Nitric oxide (NO) is one of the soluble factors produced in acute and chronic inflammation and influences growth, apoptosis, proliferation and differentiation of cells. NO therefore my have an influence on MSC injected into inflamed sites. Thus we investigated the effects of NO radicals on human MSC. Methods: Human MSC were expanded and characterized. Expression of the mesenchymal linage markers was determined by flow cytometry and their tri-lineage differentiation was explored in vitro. MSC were incubated with the NO-donor sodium nitroprusside (SNP) at different concentrations (5 μM-5 mM) and over different periods of time (15 min–24 hrs), and analyzed for their respiratory activity, gene expression responses, cell signalling pathways, and differentiation potential. Results: Human MSC expressed the mesenchymal marker proteins CD73, CD90, CD105, CD146, but failed to express the hematopoietic markers CD11b, CD14, CD34, and CD45. Activation of the MSC in vitro by nitric oxide activated c-Raf-, p-38-MAPK, and p-JNK- mediated signalling in a dose dependent manner, and also significantly regulated genes involved in cellular proliferation (cyclin D1, GAS1), apoptotis (p53), and induced an intense nuclear factor E2-related factor (NRF2)-associated stress response. Moreover, NO inhibited the entry of MSC in the osteogenic differentiation pathway and NO-treated MSC expressed less of the transcription factor Runx2. In contrast, the expression of the adipogenic marker gene PPARγ2 remained unchanged. Conclusion: We conclude that NO modulates the metabolism of MSC and compromises their osteogenic differentiation potential, which may have detrimental consequences for bone remodelling or bone regeneration.