Poster AA5 - Fibronectin Synergy Site Enhances Activation of YAP and NF-κB in Nucleus Pulposus Cells

Introduction: During intervertebral disc degeneration, the innermost nucleus pulposus (NP) loses its phenotypic characteristics, driving inflammation and extracellular matrix (ECM) reorganization that stiffens the tissue as hydration and proteoglycan are lost [1]. The mechanosensitive transcription factor YAP and the inflammatory transcription factor NF-κB are both influenced by these changes, translocating to the nucleus when activated. Previous work has shown that manipulation of integrin interactions can regulate NP cell phenotype [5,6] and that fibronectin can influence both fibrotic and inflammatory signaling in the disc [2, 3]. Our group has recently shown that fibronectin sensitizes the activation of contractility, YAP, and NF-κB in NP cells, when compared to collagen or laminin [7]. Here, we implement a peptide modified hydrogel system to identify specific domains of fibronectin that drive this fibrotic and inflammatory signaling. We identify a role for the fibronectin synergy domain in regulation of NP fibrotic and inflammatory signaling. This was assessed through quantification of activation of the mechanosensitive transcription factor YAP, the inflammatory transcription factor NF-κB (p65), expression of sulfated glycosaminoglycan (sGAG) production, and gene expression (rt-PCR).

Materials and

Methods: NP cells were isolated from fresh bovine caudal discs the same day of slaughter (Westville Meats, FL) and cultured in high glucose DMEM with 10% FBS and 1% pen-strep for 2 passages. Primary human NP cells were also tested (ScienCell). Dextran vinyl sulfone (DexVS) was synthesized as previously described [8,9] and used to generate hydrogels with 8% w/v by crosslinking with 20mM DTT. Gels were modified with RGD peptide (CGRGDS, 2mM), synergy site peptide (Syn: CGGRPREDRVPPSRNSITLTN, 2mM) [10], both (RGD-Syn, 2mM of each), or laminin mimetic peptide (LM: CSRARKQAASIKVAVSADR, 2mM) by overnight incubation in triethanolamine buffer (pH 8.0, sigma). Peptide modified gels were rinsed with PBS and NP cells were seeded and allowed to spread for one day. Cells were fixed with 4% PFA and stained with DAPI, 488-Phalloidin, YAP/Alexa-568-α-mouse and p65/Alexa-647-α-rabbit antibodies then imaged at 20x 0.8NA. Cell area (ImageJ) and nuclear to cytoplasmic ratio for YAP and p65 was quantified in MATLAB by taking the ratio of average nuclear signal to average cytoplasmic signal based on edge detected nuclear masks. Sulfated glycosaminoglycans (sGAG) production was measured by the DMMB assay and aggrecan gene expression normalized to GAPDH was measured using qPCR for samples isolated from cells grown on 2D gels for 3 days (Aurum total RNA kit, iScript and iTaq, Bio-Rad).

Results, Conclusions, and Discussions: Results and

Discussion: YAP and p65 nuclear localization were highest for the RGD-Syn combination group (Fig1 A-C), in consensus with previous observations for full length fibronectin [5]. Cells also spread the most on RGD and RGD-Syn gels (Fig 1C). Synergy site alone did not allow cell spreading and YAP activation, likely because it acts in combination with RGD to enhace integrin binding (Fig1 A,C). Laminin mimetic peptide allowed for a moderate cell spreading, but showed reduced activation of YAP, consistent with previous work on full length laminin. sGAG production was similar between groups, but aggrecan gene expression was significantly higher on laminin peptide compared to RGD or RGD-Syn (Fig 1D, E). This may indicate a longer experiment is needed to detect changes in sGAG produced. Increased YAP activation was also observed in human NP cells on RGD and RGD-Syn gels (Fig. 1F, G).

Conclusion: Fibronectin expression has been shown to increase in degenerate discs [4]. Here, we show that the synergy domain of fibronectin which enhances binding of α5β1 to RGD [10], also activates YAP and NF-κB in NP cells. This indicates that α5β1 integrin and the synergy site in fibronectin are potential regulators of fibrotic remodeling responses in the intervertebral disc. This may provide a therapeutic target for slowing or reversing intervertebral disc degeneration.

References: [1] Iatridis+ Spine Journal (2012), [2] Garcia+ Cells (2019), [3] Anderson+ Spine (2003), [4] Oegema+ Spine (2000), [5] Tan+ Biomaterials (2021), [6] Fearing+ FASEB (2019), [7] Naha & Driscoll, JOR (2023), [8] Matera+ ACS Biomater Sci Eng (2019), [9] Yu+ Biomacromolecules (2012), [10] Jardon+ eLife (2017)

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