Signalling pathways for PDGF induced mesenchymal stem cell self-renewal

Mesenchymal stem cells (MSC) have 2 defining properties – self renewal during mitosis, and multiple differentiation potentials.  The mechanisms that regulate MSC fate (self-renewal vs. differentiation) may have important implications in regenerative medicine, tissue engineering and possibly for management of disease states such as osteoporosis.  Platelet-derived growth factor (PDGF) signalling greatly increases self-renewal of MSCs in vitro. However their underlying signal transduction pathways remain largely unknown.  Therefore the aim of this study was to test the hypothesis that PDGF-induced self renewal and differentiation of MSCs is mediated by distinct signalling pathways.

Methods:

MSCs were stimulated with PDGF-BB in the presence or absence of inhibitors of PDGFR-β, MEK/Erk, JAK/STAT and PI3K/Akt pathways.  Mitoses were assessed by flow cytometry for EdU DNA incorporation. Western blotting was used to assess phosphorylation status of signalling molecules and changes in the level of cell cycle proteins. Differentiation to osteoblast and adipocyte lineages was induced and assessed by qRT-PCR for expression of differentiation association genes and by alizarin red and oil red O staining.

Results:

PDGFRβ activation by PDGF-BB rapidly induced both MEK/Erk and PI3K/Akt signalling pathways.  Induction of PI3K/Akt activated downstream signal cascades, mTOR and its associated proteins p70S6K and 4E-BP1.  These pathways induced the expression of cyclin-D1, cyclin-D3 and CDK6 to promote cell cycle progression.  Inhibition of PI3K/Akt blocked MSC proliferation. In contrast, activation of Erk signalling did not alter proliferation, but potently inhibited the adipocytic differentiation by blocking PPARγ and CEBPα expression.

Conclusions:

The results demonstrate that PDGFRβ signalling simultaneously activates two major transduction pathways with opposing fate decisions of proliferation and differentiation.   PDGF maintains self-renewal by inducing proliferation and preventing cell differentiation. This suggests that activation of multiple intracellular cascades may be required for successful and sustainable self-renewal and may offer opportunities for therapeutic manipulation to increase stem cell expansion.