Microtopographical Features Recruit RhoA/ROCK through TRPV1 Channels to Direct Cell and Neurite Growth

Abstract

Objectives:

(1) Identify intracellular signaling pathways required for translating biophysical cues into directed neurite growth. (2) Describe the role of TRPVI channels as mediators of cell response to microtopographical features.

Methods:

Microscopic parallel grooves were generated on methacrylate polymers using photopolymerization. Spiral ganglion neurites (SGNs) were cultured on the polymers, and alignment to grooves was quantified using a ratio of aligned length to total neurite length. Control alignment was compared with that of SGNs treated with RhoA activator, RhoA inhibitor, ROCK inhibitors, cAMP and cGMP analogs, and TRPV channel inhibitors. RNA interference was used to reduce expression of ROCK and TRPVI to confirm that activity of each was required for alignment. Alignment of NIH3T3 fibroblasts was compared to fibroblasts transfected with TRPVI.

Results:

Neurites aligned preferentially to micropattern grooves. RhoA activator and cGMP analogs increased alignment, while RhoA inhibitor and cAMP analogs decreased alignment. ROCK inhibitors decreased neurite alignment, and SGNs transfected with ROCK-targeted oligonucleotides also displayed reduction in alignment. Neurites exposed to TRPV inhibitors exhibited poor alignment, as did TRPVI knockdown cells. TRPVI-transfected fibroblasts aligned more strongly than NIH3T3 fibroblasts. For all alignment comparisons, P < .05.

Conclusions:

Microtopographic features generated via photopolymerization of methacrylate polymers activate RhoA and ROCK through TRPVI channels to direct cell and neurite alignment.