Poster BB5 - KASH-Disrupted Nuclear Mechanosensation in Tendon

Introduction: Mechanobiology regulates cellular differentiation, yet many of its biomechanical and biochemical pathways remain obscure. Furthering our understanding of these pathways is crucial for future applications in tissue regeneration and engineering especially for highly complex and mechanically active tissues such as tendon. Tendon injuries are one of the leading causes of disability, accounting for almost half of the 50 million musculoskeletal injuries that occur annually in the US^1. Nuclear mechanosensing is almost unexplored in tendon, but may play a key role in regulating tendon tissue’s mechanobiological response to injury. The linker of the nucleoskeleton and cytoskeleton (LINC) complex is a key component of nuclear mechanotransduction and connects the cytoskeleton to the outer nuclear membrane (ONM), while anchoring itself into the inner nuclear membrane (INM). The KASH domain nuclear proteins make up the tether between the ONM and the cytoskeleton^2. Disruptions to this complex and associated proteins disrupt the cellular response to mechanical loading via nuclear expression/signaling and transcriptional activity disruption^3. Through utilizing a dominant-negative KASH cell line with disabled nuclear mechanosensing, we aim to further understand the changes in expression and localization of β-catenin, a multirole protein involved in tenogenesis that may be a key molecule in translating mechanical signals to tenogenic differentiation in stem cells^4,5. We hypothesized that disrupting nuclear mechanosensing would prevent tenogenesis in stem cells by preventing β-catenin localization to the nucleus, thus showing a causal link between β-catenin and nuclear mechanosensing in tendon development for the first time.

Materials and

Methods: Mouse primary cells were isolated from a dominant negative KASH mouse line^6. These cells were split into two treatments groups: one received 3 ug/mL of Doxycycline Hyclate(DOX) in 10% FBS IMDM media(K-), and another that had DOX and 0.05 ug/mL of TGF β2 (K+). The DOX addition drove expression of m-Cherry with dominant negative KASH expression to validate the inactivation of the LINC complex in the dominant negative KASH cells. Cells were cultured for up to 3 weeks and collected at days 1,3,7,14 and 21. Cells were stained with DAPI, Phalloidin and m-Cherry and imaged on a LSM 900 confocal microscope. Additionally, Western Blots quantified β-catenin level differences between TGFβ2 (tenogenesis) and non-TGFβ2 (control) groups. ImageJ was used to calculate the protein densitometry and to evaluate the β-catenin localization and density in cells.

Results, Conclusions, and Discussions: m-Cherry expression was observed during fluorescence imaging, suggesting that the dominant negative KASH cell lineage was expressing a disrupted KASH protein tether and that the LINC complex and nuclear mechanosensing of the cell were sufficiently disrupted (Fig 1A.). It was observed from both fluorescence imaging and densitometry measurements that β-catenin production peaks at the 7-day time point. However, the addition of TGFβ2 in early timepoints (D1) aided in β-catenin local cellular expression for K+ treatment groups. At longer time points (D3 - 14), the inactivation of LINC reduces β-catenin production over the two week period to levels below those seen even in the non TGFβ2 addition groups (K-)(Fig D-E.). Image analysis did not find differences in β-catenin localization in either the nucleus or cytoplasm between treatment groups, and will be explored further in future experiments (Fig B-C.). Additionally, the tendon marker Scleraxis will be quantified to assess the effects of KASH protein disruption and TGFβ2 supplementation on tenogenesis in the absence of nuclear mechanosensing capability. Future work will continue to untangle mechanotransductive pathways that can be exploited to regenerate tendon from stem cells using mechanical signals.

Further exploration of the effects of KASH disruption and TGF β2 supplementation of β-catenin localization and expression is needed. While our initial results show alterations in β-catenin production intensities between treatment groups, they confirm a connection between nuclear mechanosensing and β-catenin production only at early (7 day) timepoints. Future studies will further illuminate the biochemical effects of disrupted mechanobiological sensing pathways and suggest small molecules to selectively stimulate or inhibit these pathways towards tendon regeneration from stem cells.

Acknowledgements (Optional): References.
1. Wu, F., Nerlich, M., & Docheva, D. Tendon injuries: Basic science and new repair proposals. EFORT open reviews, 2(7), 332–342. (2017).
2. Kim, D. I., Birendra, K. & Roux, K. J. Making the LINC: SUN and KASH protein interactions. Biol. Chem. 396, 295–310 (2015).
3. Alam, S. G. et al. The mammalian LINC complex regulates genome transcriptional responses to substrate rigidity. Sci. Rep. 6, 38063 (2016).
4. Uzer, G. et al. Sun-mediated mechanical LINC between nucleus and cytoskeleton regulates βcatenin nuclear access. J. Biomech. 74, 32–40 (2018).
5. Wang, T. et al. Reduction of mechanical loading in tendons induces heterotopic ossification and activation of the β-catenin signaling pathway. J. Orthop. Transl. 29, 42–50 (2021).
6. Pancheri, N. M. et al. The LINC complex regulates Achilles tendon elastic modulus, Achilles and tail tendon collagen crimp, and Achilles and tail tendon lateral expansion during early postnatal development. BioRxiv Prepr. Serv. Biol. 2023.11.13.566892 (2023).