(759d) Investigating the Role of the Extracellular Matrix in Schwann Cell Phenotype

Introduction: Schwann cells (SCs) are capable of dedifferentiating to promote peripheral nervous system (PNS) regeneration following traumatic injury through upregulation of regenerative markers such as c-Jun, p75NTR and Sox-2. We have previously reported that extracellular matrix (ECM) cues such as ligand type and cell spreading area impact phenotype¹. However, a mechanistic understanding of the individual cues impacting the dedifferentiation process such as ECM stiffness and cell morphology remain poorly understood in SCs. Therefore, we sought to exam the interplay between SC regenerative phenotype and multiple ECM cues as regulated by mechanotransducers YAP/TAZ and RhoA to gain an understanding of SCs following traumatic PNS injury.

Materials and Methods: SC spreading area and morphology were controlled by cell density or microcontact printing laminin in shape or line patterns. Proteins were detected and quantified via immunofluorescent staining (IF) and Western blot (WB). To inhibit RhoA and YAP/TAZ activity, Y-27632 (Y)(20µM) and Verteporfin (V)(1µM) were utilized. Rac1 and MKK7 were inhibited using small interfering RNA (siRNA) treatment. Rho GTPases and YAP/TAZ-related transcriptional factors were examined by microarray assay using a Rat Clariom S Array. Statistics were performed via One-way ANOVA and Tukey’s post hoc test with P<0.005 considered significant.

Results: As SC spreading area increases, RhoA and YAP/TAZ are activated with a downregulation of Rac1 and SC regenerative markers. Further, as SC morphology is elongated, expression of Rac1 and SC regenerative markers are higher while similar levels of RhoA and YAP/TAZ are seen as compared to non-elongated cells (Fig 1A). When cell spreading area is maintained constant using micropatterning, both RhoA and YAP/TAZ inhibition significantly enhance c-Jun expression, signifying an upregulation in regenerative phenotype. These results suggest cell spreading inhibits SC regenerative phenotype by activation of RhoA and YAP/TAZ (Fig 1B). Additionally, elongated SCs show a higher activity of Rac1/MKK7/JNK signaling (Fig 1C). Interestingly, when Rac1 is inhibited via siRNA treatment, SC elongation does not promote expression of regenerative markers (c-Jun, Sox-2 and p75NTR), indicating cell elongation promotes SC regenerative phenotype by activation of Rac1 signaling.

Conclusion: Here, we have begun to illustrate the complex interplay between SC morphological cues and the highly plastic SCs. Cell spreading activates RhoA and YAP/TAZ while downregulating Rac1 signaling to inhibit a SC regenerative phenotype. Furthermore, SC elongation activates Rac1 signaling to upregulate SC regenerative phenotype. These findings show the dynamic nature of SCs and can be utilized in future PNS therapies.

References:

1. Xu, Z, Orkwis, JA, DeVine, BM, Harris, GM. Extracellular matrix cues modulate Schwann cell morphology, proliferation, and protein expression. J Tissue Eng Regen Med. 2020; 14: 229– 242.