Tissue Engineering
Julia Palmer
Undergraduate Researcher
University of Idaho
Cataldo, Idaho, United States
Colin R. Marchus, n/a
Graduate Researcher
University of Idaho, Idaho, United States
Nathan Schiele
Associate Professor
University of Idaho, Idaho, United States
Tendon injuries are common and often result in altered mechanical properties, affecting quality of life. In developing tendons, the collagen molecules within the collagen fibrils are crosslinked by lysyl oxidase (LOX), an enzyme produced by cells, to form a strong collagen network [1]. The amount of these crosslinks impacts the mechanical properties of the forming tendon [1]. A major challenge is the limited information on how LOX production is regulated by cells. To address this, our recent work found that LOX production may be increased by mesenchymal stem cells (MSCs) when treated with transforming growth factor (TGF)β2, but the cell signaling pathways involved in this regulation are unknown. TGFβ cell signaling may include the Akt pathway, and our prior work showed that Akt may regulate stem cell differentiation toward the tendon lineage (tenogenesis) [2]. Akt is a kinase that, once activated (phosphorylated), can have downstream effects on cell growth, protein synthesis, and apoptosis. Though previous studies have indicated that Akt signaling and TGFβ impact tendon, there remains a gap in knowledge on how Akt regulates the production of LOX by MSCs. Therefore, the objective of this study was to determine how TGFβ-induced LOX production by MSCs is regulated by Akt signaling. To test this, we treated MSCs with a chemical inhibitor of Akt (MK-2206) as well as a chemical activator (SC-79) and evaluated LOX protein production.
Murine C3H10T1/2 MSCs (ATCC, Manassas VA) were seeded into 12-well plates at 20,000 cells/cm2 in standard culture medium. The medium was then supplemented with 50 ng/mL TGFβ-2 along with Akt inhibitor, MK-2206 (MedChem Express, Monmouth Junction, NJ). MK-2206 was evaluated at 5 nM, 50 nM, and 500 nM, and dimethyl sulfoxide (DMSO) was used as the vehicle control. These experiments were completed in triplicate. In a pilot study, MSCs were treated with SC-79 (Millipore) at 1 µM, 10 µM, and 100 µM to test chemical activation of Akt. DMSO was used as the vehicle control. In both Akt inhibition and activation experiments, the culture medium was changed every three days. In inhibition, cells were imaged and collected on day 1, 3, and 7 after treatment. In activation cells were imaged and collected on day 1 and 3. Western blotting and band densitometry were used to measure phosphorylated-Akt (p-Akt) and LOX protein levels.
As expected, TGFβ2 treatment in the control groups resulted in fibroblastic cell morphology, which was found at all timepoints. Medium supplemented with TGFβ2 and MK-2206 appeared to impact the MSCs as a function of concentration. Cell morphology and proliferation did not appear significantly altered at 5 nM and 50 nM, but cells appeared less fibroblastic and lower in density at 500 nM (Fig 1A). This corresponded with the levels of p-Akt observed, which were significantly reduced beginning at day 3 (p=0.012) with 50 nM MK-2206 and 500 nM (p=0.013) MK-2206, compared to vehicle controls. Interestingly, despite the inhibition of Akt activation (e.g., reduced p-Akt) with MK-2206, LOX levels were not significantly altered compared to vehicle controls (p >0.05) at any timepoint or with any concentration of MK-2206 (Fig 1B). To further test this, we conducted a pilot test without using TGFβ2 and evaluated SC-79, which is known to activate Akt [3]. However, the highest concentration of SC-79 resulted in cell death, and at the lower concentrations of SC-79, Akt did not appear to be activated. Protein levels of LOX also were not impacted compared to vehicle controls. Further studies are ongoing to better understand SC-79 and its activation of Akt in C3H10T1/2 MSCs. Overall, our initial findings suggest that activation of the Akt cell signaling pathway by TGFβ2 in MSCs may have a limited role in regulating LOX production. This is an interesting finding that may result in the exploration of other potential pathways that are involved in TGFβ-induced LOX production. These findings enhance our understanding of how the Akt pathway plays a role in collagen crosslinking, which will improve tendon tissue engineering and treatments to restore tendon function.
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.