(I-333) TGFβ Type I Receptor Regulates TGFβ-induced Lysyl Oxidase Production by Mesenchymal Stem Cells

Tendons are highly collagenous tissues that connect muscle to bone. Due to poor healing properties and a limited understanding of initial tendon formation, current treatments for tendon injury are ineffective. Specifically, there is limited information on the mechanisms which control collagen crosslinking and the collagen crosslinking enzyme, lysyl oxidase (LOX), production by mesenchymal stem cells (MSCs), which are commonly explored for developing new tendon therapies². LOX plays a crucial role in developing collagen crosslinks and impacts the mechanical properties and function of tendon^1,5. A potential mechanism for regulating LOX production involves the transforming growth factor (TGF) β signaling pathway. TGFβ signaling may be activated by both TGFβ1, a growth factor prevalent in tendon injury, and TGFβ2, found during normal tendon development and a known tenogenic factor ^3–5,7.  However, the impact of TGFβ1 and TGFβ2 on LOX production by MSCs is less clear. Though it is known that TGFβ1 and TGFβ2 can signal through TGFβ type I receptor (RI), TGFβRI’s role in the regulation of LOX has yet to be characterized⁶. Therefore, the objective of this study was to evaluate how TGFβ1 and TGFβ2 impact LOX production and determine what role TGFβRI plays in LOX regulation.   

Murine MSCs (C3H10T1/2, ATCC, Manassas VA) were cultured in standard media (Dulbecco’s Modified Eagle’s Medium (DMEM), 10% fetal bovine serum (FBS), and 1% Penicillin/Streptomycin) and seeded into 12-well plates at a concentration of 20,000 cells/cm². Prior to treatment cells were cultured for 24 hours in standard media and then switched to a low serum media (DMEM, 1% FBS, and 1% Penicillin/Streptomycin) for 24 hours. MSCs were then treated with TGFβ1 (0.1 ng/mL, 1 ng/mL, 10 ng/mL, 50 ng/mL, and 100 ng/mL), or TGFβ2 (1 ng/mL, 10 ng/mL, 50 ng/mL, and 100 ng/mL) with vehicle controls being either citric acid at 10 mM or sterile water, respectively in low serum media. Media was changed every 72 hours. To evaluate the role of TGFβRI, MSCs were treated with SB431542 (R&D Systems), a TGFβRI inhibitor. MSCs were treated with TGFβ1 (1 ng/mL, 10 ng/mL, 50 ng/mL) and 10 µM SB431542 (SB), or TGFβ2 (1 ng/mL, 10 ng/mL, 50 ng/mL) and 10 µM SB, with vehicle controls for SB treatments being DMSO. Cells were then imaged and collected for western blotting on days 1, 3, and 7. LOX protein levels were analyzed and normalized to β-actin and their respective time point-matched vehicle controls. TGFβRI inhibited cells were also cultured on coverslips, fixed, and stained after 3 days of treatment with DAPI and phalloidin. Statistical analysis was conducted through multiple unpaired t-tests, with statistical significance being p < 0.05.

TGFβ1 at 10 ng/mL increased LOX in MSCs at day 1 (p=0.011) and day 3 (p= 0.027). TGFβ1 at 50 ng/mL increased LOX at day 1 (p=0.0005) and levels trended up at day 3 (p=0.052) (Fig 1 A, B, C, D). At day 7, TGFβ1 did not impact LOX levels (Fig 1 E, F), suggesting that TGFβ1 may not maintain increased levels of LOX for extended periods. TGFβ2 at 10 ng/mL (p= 0.025) and 100 ng/mL (p= 0.025) increased LOX production at day 3 (Fig 2). TGFβ2 at 50 ng/mL increased LOX at day 3 (p=0.025), and day 7 (p=0.039). LOX levels trended up at day 1 with TGFβ2 treatments. The inhibition of TGFβRI with SB resulted in LOX levels trending down in MSCs treated with 10 ng/mL of TGFβ2 and 10 µM SB at day 3 (p= 0.069) and a significant decrease in LOX levels for MSCs treated with 50 ng/mL of TGFβ2 and 10 µM SB at day 3 (p= 0.033) (Fig 3 A, B). This shows that TGFβRI may play a role in regulating TGFβ2-induced LOX production by MSCs, which also supports the TGFβ signaling pathway as a potential regulator of collagen crosslinking in tendon development. In the case of TGFβ1, the impact of SB was more muted, and no differences were detected (p > 0.05 for each concentration). TGFβRI may not be the primary mechanism for TGFβ1 to regulate collagen crosslinking in tendon, but this will need further study. Interestingly, MSCs treated with SB and TGFβ1 or TGFβ2 showed substantial changes in cell morphology, compared to control groups (Fig 4), suggesting that TGFβRI may also play a role in controlling other aspects of cell behavior. Overall, TGFβ1 and TGFβ2 induce LOX production in MSCs, and TGFβ2 may be more impacted by TGFβR1 signaling. Future studies will investigate the role of other TGFβ receptors on LOX production. Elucidating the mechanism behind how collagen crosslinking is regulated can aid in developing more effective tendon tissue engineering and regenerative strategies.

This project was supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under Grant #P20GM103408 and NSF Grant #2145004

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