Crosstalk between extracellular matrix pathways in mesenchymal stromal cells

Mesenchymal stromal cells (MSCs) are renowned for their ability to differentiate into various cell types, their capacity to produce and remodel extracellular matrix (ECM) components, and their context-sensitive mode of action. These properties make them an ideal in vitro model for studying ECM-related signalling pathways, and pathology-dependent changes in pathway communication, e.g. in fibrosis. Understanding how biochemical signals or mechanical factors drive MSC fate is key to targeting them for therapeutic purposes. In this regard, the ERK and ROCK pathways are likely to play crucial roles, as they are involved in the mediation of fibrotic phenotypes. Yet there are no consistent data on their possible interplay. We hypothesised that ROCK displays inhibitory effects on ERK and that therefore, inhibition of ROCK together with non-canonical TGF-β signalling leads to increased ERK pathway activation. Primary adipose-derived MSCs, isolated and cultured from three human donors, were treated with either 10 µM Y-27632 (ROCK inhibitor), 10 ng/ml TGF-β3 or a combination of both. In order to observe the dynamic changes in ERK pathway activation, a time course experiment was set up. Cells were analysed after 0.5 h, 1 h, 4 h and 24 h, at which phospho- and total ERK expression were quantified by western blot. Current experimental set ups evaluate the influence of ECM components on these pathways. Our results indicate that ERK phosphorylation increases substantially upon inhibition of ROCK. In part of the donors, this was further pronounced by induction of MSCs with TGF-β3. No effect on ERK phosphorylation was seen when cells were treated with TGF-β3 alone. Data from the time course experiment showed that ERK phosphorylation and dephosphorylation occurs in less than 24 h. Preliminary data from MSCs seeded on plates coated with hyaluronic acid show a differential activation of the ERK pathway depending on the concentration of the coating. Our data demonstrate a non-canonical crosstalk between ROCK, ERK and TGF- β pathways, with an inhibitory effect of ROCK on ERK. They further suggest pathway regulation depending on ECM stiffness and/or biochemical signals. Thereby, they help towards understanding compensation as well as feedback mechanisms emerging from the interplay of ECM-regulated. Ultimately, this aids in better understanding mechanisms behind ECM remodelling by MSCs.