Inhibition of RhoA-Subfamily GTPases Suppresses Schwann Cell Proliferation Through Regulating AKT Pathway Rather Than ROCK Pathway.

Dandan Tan,Muhua Lai,Mengjie Pan,Jiasong Guo,Jinkun Wen,Haowen Zhang,Junyao Deng,Lixia Li,Xiaofang Hu,Changhui Qian,Xianghai Wang

doi:10.3389/fncel.2018.00437

Abstract

Inhibiting RhoA-subfamily GTPases by C3 transferase is widely recognized as a prospective strategy to enhance axonal regeneration. When C3 transferase is administered for treating the injured peripheral nerves, Schwann cells (SCs, important glial cells in peripheral nerve) are inevitably impacted and therefore SC bioeffects on nerve regeneration might be influenced. However, the potential role of C3 transferase on SCs remains elusive. Assessed by cell counting, EdU and water-soluble tetrazolium salt-1 (WST-1) assays as well as western blotting with PCNA antibody, herein we first found that CT04 (a cell permeable C3 transferase) treatment could significantly suppress SC proliferation. Unexpectedly, using Y27632 to inhibit ROCK (the well-accepted downstream signal molecule of RhoA subfamily) did not impact SC proliferation. Further studies indicated that CT04 could inactivate AKT pathway by altering the expression levels of phosphorylated AKT (p-AKT), PI3K and PTEN, while activating AKT pathway by IGF-1 or SC79 could reverse the inhibitory effect of CT04 on SC proliferation. Based on present data, we concluded that inhibition of RhoA-subfamily GTPases could suppress SC proliferation, and this effect is independent of conventional ROCK pathway but involves inactivation of AKT pathway.

Highlights

Rho family small guanosine triphosphatases (Rho-GTPases) are essential in the regulation of diverse cellular functions such as regulation of actin cytoskeleton, vesicular trafficking and transcriptome dynamics (Hu and Selzer, 2017; Nomikou et al, 2017)
Overall data of the present study demonstrate that: (1) data got from the assessments of cell density, EdU, FIGURE 4 | Inhibition of ROCK does not affect SC proliferation. (A–G) EdU assay showed that EdU positive ratio was not affected by Y27632 treatment (n = 15). (H) Statistical diagram of cell density suggested that the number of total cells was not altered in the presence of Y27632 (n = 15). (I) water-soluble tetrazolium salt-1 (WST-1) measurement revealed that there was no significant difference in the absorbance value between the control group and Y27632 group (n = 4). (J,K) Western blotting indicated that the expression of PCNA was unaffected in the Y27632 treated cells (n = 6)
WST-1 and PCNA expression indicate that the inhibition of RhoA-subfamily GTPases by C3 transferase (CT04) can suppress the SC proliferation; (2) Live/Dead cell staining assay indicates CT04 does not induce cell death in the SCs cultures, which means the effect of CT04 on SC proliferation was not related to the cytotoxicity; (3) Y27632 does not affect the SC proliferation

Summary

Introduction

Rho family small guanosine triphosphatases (Rho-GTPases) are essential in the regulation of diverse cellular functions such as regulation of actin cytoskeleton, vesicular trafficking and transcriptome dynamics (Hu and Selzer, 2017; Nomikou et al, 2017). RhoA subfamily is widely recognized as a crucial molecular switch to initiate growth cone collapse and inhibit axonal regrowth in the nervous system (Antoine-Bertrand et al, 2011; Matsukawa et al, 2018). Inhibiting RhoA subfamily has been accepted as a prospective strategy to facilitate axonal regrowth and functional recovery after PNI (Hiraga et al, 2006; Auer et al, 2012; Hynds, 2015; Joshi et al, 2015). Due to C3 transferase is able to selectively inactivate RhoA-subfamily GTPases, it is widely used to promote neural regeneration (Auer et al, 2012, 2013; Zhou et al, 2012; Forgione and Fehlings, 2014; Gutekunst et al, 2016)

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