Recently our laboratory identified cell-cell adhesion as an important factor determining mesenchymal stem cell (MSC) differentiation. More specifically, we discovered that Cadherin-11 (CDH11) was necessary for MSC differentiation into smooth muscle cells (SMC) through activation of key myogenic transcription factors such as serum response factor (SRF) via the ROCK pathway. Others, showed that differentiation of fibroblast to myofibroblasts during wound healing and production of extracellular matrix (ECM) synthesis that was associated with a shift in cadherin profile from cadherin-2 (CDH2) to CDH11. However, the role of CDH11 in ECM production and deposition is unknown. This is the focus of the present study.
Tissues collected from CDH11 null mice (Cdh11-/-) and wide type (WT) such as aorta, bladder and skin were tested and compared for their mechanical properties. Human dermal fibroblasts and mice dermal fibroblasts were isolated and subjected to experiments. Western blot and real time RT-PCR were employed to measure the level of protein and gene expression, including the main ECM components as collagen and elastin and some relevant transcriptional factors. An engineered surface with immobilized the fusion protein cadherin-11 with the Fc-domain (Cad-Fc) was used as a tool of CDH11 engagement without the complications of high cell densities.
Surprisingly, we observed a significant reduction in mechanical properties of aorta, bladder and skin of Cdh11-/- as compared to WT tissues. The change in mechanical properties was accompanied by reduced collagen and elastin content. At a cellular level, RT-PCR and western blot analyses both confirmed an attenuated expression of collagens and elastin in Cdh11-/- dermal fibroblasts compared to WT. Further, knocking down CDH11 in human dermal fibroblasts significantly reduced the ability of cells to generate force, as evidenced by the extent of hydrogel compaction. In contrast, engagement of CDH11 on CDH11-Fc surface significantly promoted collagen and elastin synthesis, which was accompanied by significant upregulation of key transcriptional factors such as MYOCD, SRF, MRTF-A/B. Surprisingly, CDH11 engagement promoted nuclear localization of MRTF-A, a key event for subsequent transcription. Interestingly, knocking down anyone of these transcriptional factors down-regulated the ECM synthesis. Further experiments demonstrated that both the TGF-β and Rho/ROCK pathways mediated the effects of CDH11 engagement on collagen and elastin synthesis.
Our study identified CDH11 as a critical player in mediating collagen and elastin synthesis both in vivo and in vitro. Engagement of CDH11 enhanced expression of transcriptional factors MRTFA/B, SRF and MYOCD through activation of TGF-β and ROCK pathways. Nuclear localization of MRTF-A was a key event following CDH11 contact. Taken together, our results demonstrate an important role of CDH11-mediated adherens junctions in ECM synthesis with implications in mechanical properties of tissues and wound healing.