TGFβ2-induced tenogenesis impacts cadherin and connexin cell-cell junction proteins in mesenchymal stem cells

Abstract

Tenogenic differentiation of stem cells is needed for tendon tissue engineering approaches. A current challenge is the limited information on the cellular-level changes during tenogenic induction. Tendon cells in embryonic and adult tendons possess an array of cell-cell junction proteins that include cadherins and connexins, but how these proteins are impacted by tenogenic differentiation is unknown. Our objective was to explore how tenogenic induction of mesenchymal stem cells (MSCs) using the transforming growth factor (TGF)β2 impacted protein markers of tendon differentiation and protein levels of N-cadherin, cadherin-11 and connexin-43. MSCs were treated with TGFβ2 for 21 days. At 3 days, TGFβ2-treated MSCs developed a fibroblastic morphology and significantly decreased levels of N-cadherin protein, which were maintained through 21 days. Similar decreases in protein levels were found for cadherin-11. Connexin-43 protein levels significantly increased at 3 days, but then decreased below control levels, though not significantly. Protein levels of scleraxis and tenomodulin were significantly increased at day 14 and 21, respectively. Taken together, our results indicate that TGFβ2 is an inducer of tendon marker proteins (scleraxis and tenomodulin) in MSCs and that tenogenesis alters the protein levels of N-cadherin, cadherin-11 and connexin-43. These findings suggest a role for connexin-43 early in tenogenesis, and show that early-onset and sustained decreases in N-cadherin and cadherin-11 may be novel markers of tenogenesis in MSCs.

Keywords: Cadherin; Connexin; Stem cells; Tendon; Tenogenesis; Tissue engineering.

Figure 1.

Representative images (20x) of MSCs. Actin cytoskeleton (green) and cell nuclei (blue) are shown in MSCs at 3, 7, 14, and 21d (A-H). TGFβ2-treated MSCs (B, D, F, H) had increased proliferation, and appeared more fibroblastic and elongated, compared to controls (A, C, E, G).

Figure 2.

Representative WB images of tendon marker proteins produced by MSCs. WB showed increases in Scx, Tnmd, and TNC following 3d, 7d (A), 14d, and 21d (B) of TGFβ2 treatment. Quantified band densitometry showed significant increases in Scx (C) and Tnmd (D), while TNC (E) levels were similar to controls. All bands were normalized to β-actin and to their respective timepoint controls. * = p<0.05, ** = p≤0.01, *** = p≤0.001, **** = p≤0.0001. Mean ± standard deviation.

Figure 3.

Representative WB images of cell-cell junction proteins produced by MSCs. WB showed decreases in N-cad and cad-11, and changes in cxn-43 following 3d, 7d (A), 14d, and 21d (B) of TGFβ2 treatment. Quantified band densitometry showed significant decreases in N-cad (C) and cad-11 (D), while cxn-43 increased at 3d (E). All bands were normalized to β-actin and to their respective timepoint controls. * = p<0.05, ** = p≤0.01, *** = p≤0.001, **** = p≤0.0001. Mean ± standard deviation.