Poster L1 - Impact of TGFβ and Akt on lysyl oxidase production in mesenchymal stem cells

Friday, October 25, 2024

10:00 AM - 11:00 AM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

JP

Julia Palmer

Undergraduate Researcher
University of Idaho
Moscow, Idaho, United States

Co-Author(s)

HF

Hailey Faith

Undergraduate Researcher
University of Idaho, United States

Primary Investigator(s)

NS

Nathan R. Schiele, PhD

Associate Professor
University of Idaho
Moscow, Idaho, United States

Introduction: Tendon injuries are common and result in altered mechanical properties, affecting quality of life. In developing tendons, collagen molecules within collagen fibrils are crosslinked by the enzyme lysyl oxidase (LOX), establishing a strong collagen network and impacting the mechanical properties of tendon (Marturano et al 2013). A challenge for developing therapies to control LOX and tendon function is the limited information on the mechanisms that regulate LOX production 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)β1 or (TGF)β2 (Pancheri et al 2024), but the cell signaling pathways involved in this regulation are unknown. TGFβ cell signaling may include the Akt pathway, and our prior work suggests Akt may regulate stem cell differentiation toward the tendon lineage (tenogenesis) (Theodossiou et al 2021). Akt is a kinase that, once activated (phosphorylated), can influence various cellular processes including cell growth, protein synthesis, and apoptosis (Gopallawa et al 2021). Though previous studies suggest Akt signaling and TGFβ impact tendon, the cell signaling cascade that results in the regulation of LOX by MSCs remains unknown. Therefore, the objective of this study was to determine how TGFβ-induced LOX production by MSCs is regulated by Akt signaling. To assess this, we treated MSCs with TGFβ1 or TGFβ2 and a chemical inhibitor of Akt (MK-2206) or chemical activator of Akt (SC-79) and evaluated LOX protein production.

Materials and

Methods: 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 TGFβ2 along with Akt inhibitor, MK-2206 (MedChem Express, Monmouth Junction, NJ) at 500 nM, and sterile water and dimethyl sulfoxide (DMSO) were used as vehicle controls, respectively. These experiments were completed in triplicate. To evaluate if Akt plays a role in TGFβ1-mediated LOX production, a pilot study treated MSCs with 10 ng/mL of TGFβ1 and MK-2206 (500 nM), with citric acid and DMSO as respective vehicle controls (n=2). The culture medium was changed every three days, and the cells were imaged and collected on day 1, 3, and 7 after treatment. An additional pilot study treated MSCs with Akt activator, SC-79 (Millipore, St. Louis MO) at 1 µM, and 10 µM, with DMSO used as the vehicle control and MSCs were imaged and collected 30 min and 1 hr after treatment. Western blotting and band densitometry were used to assess phosphorylated-Akt (p-Akt), pan-Akt, and LOX protein levels. GAPDH was used a loading control. Data was analyzed using a one-way ANOVA or t-test with p < 0.05.

Results, Conclusions, and Discussions: TGFβ2 treatment resulted in a fibroblastic cell morphology at all timepoints. MK-2206 treatment appeared to impact the MSCs resulting in less fibroblastic morphology and lower cell density, beginning at day 1 (Fig 1A). TGFβ2-induced p-Akt was also found to trend down (p = 0.09) with MK-2206 treatment at day 1, and p-Akt remained reduced at days 3 and 7. LOX levels were significantly increased with MK-2206 at day 1 (p < 0.05), and continue to trend upward at days 3 and 7, compared to all control groups (Fig 1B). To determine if TGFβ1 impacts LOX production through Akt, we explored how LOX levels are impacted by MK-2206 and TGFβ1. Our initial findings suggest that MK-2206 altered TGFβ1-induced cell morphology and Akt activation (Fig 2A&B), similar to our prior findings with TGFβ2. However, at day 3, MK-2206 treatment resulted in a trending (p = 0.09) decrease in TGFβ1-induced LOX production (Fig 2C). This is an interesting finding and suggests that Akt may play a unique role in LOX production depending on the TGFβ isoform. This is particularly intriguing as TGFβ1 is typically associated with fibrosis, while TGFβ2 is typically associated with tenogenesis. Further studies are ongoing to better understand the role of Akt activation in LOX production by C3H10T1/2 MSCs. For example, our preliminary findings showed at SC-79 resulted in a transient Akt activation after 1 hr, and LOX appeared to be decreased, compared to vehicle controls. However, this will need to be studied further. In summary, activation of Akt in MSCs may have a regulatory role in TGFβ-induced LOX production. These findings enhance our understanding of how the Akt pathway is involved in LOX-mediated collagen crosslinking, which will improve tendon tissue engineering and treatments to restore tendon function.

Acknowledgements (Optional): 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, NSF Grant #2145004, and University of Idaho Office of Undergraduate Research – Summer Undergraduate Research Fellowship.