Partial Blindness to Submicron Topography in <b><i>NF1</i></b> Haploinsufficient Cultured Fibroblasts Indicates a New Function of Neurofibromin in Regulation of Mechanosensoric

Abstract

Cells sense physical properties of their extracellular environment and translate them into biochemical signals. In this study, cell responses to surfaces with submicron topographies were investigated in cultured human NF1 haploinsufficient fibroblasts. Age-matched fibroblasts from 8 patients with neurofibromatosis type 1 (NF1^+/–) and 9 controls (NF1^+/+) were cultured on surfaces with grooves of 200 nm height and lateral distance of 2 µm. As cellular response indicator, the mean cell orientation along microstructured grooves was systematically examined. The tested NF1 haploinsufficient fibroblasts were significantly less affected by the topography than those from healthy donors. Incubation of the NF1^+/– fibroblasts with the farnesyltransferase inhibitor FTI-277 and other inhibitors of the neurofibromin pathway ameliorates significantly the cell orientation. These data indicate that NF1 haploinsufficiency results in an altered response to specific surface topography in fibroblasts. We suggest a new function of neurofibromin in the sensoric mechanism to topographies and a partial mechanosensoric blindness by NF1 haploinsufficiency.

Restoring functional neurofibromin by protein transduction

“…The median deviation angle of the fibroblasts rose to about 1.4 fold (+/−0.21; p < 0.01) compared to fibroblasts transfected with control siRNA. These findings fit to experiments with primary NF1 +/− and age matched NF1+/+ fibroblasts 9 . These data demonstrate that the NF1 siRNA silencing in fibroblasts results in the cellular phenotype of partial mechanosensoric blindness.…”

“…We have previously described a reduced orientation capability of NF1 +/− fibroblasts cultured on PDMS nano-micro structures of parallel groves at a height of 200 nm and a width and interspace of 2 µm 9 . To develop an optimal experimental time scale for the following transduction experiments fibroblasts of 3 healthy donors ( NF1 +/+) and 3 NF1 patients ( NF1 +/−) were seeded on the structured gels for 24, 48 and 96 hours.…”

“…The partial blindness to micro-nano-structured PDMS gels was characterized in primary NF1 +/− fibroblasts derived from NF1 patients 9 . To ensure that this cellular phenotype is related to the expression of NF1 , siRNA-based NF1 knockdown experiments were performed in fibroblasts of a healthy human donor ( NF1 +/+; KF3).…”

“…Cellular effects of neurofibromin reduction were also demonstrated in vitro in several cell types such as fibroblasts, melanocytes, keratinocytes, osteoblasts, astrocytes, and hematopoietic cells 5 – 8 . NF1 +/− fibroblasts have a reduced capability of orientating themselves on nano-microstrucured surfaces indicating a function of neurofibromin in mechanosensoric regulation of such cells 9 . There is no systemic therapy for the whole clinical disease pattern of NF1.…”

“…We investigated whether synNF1 replacement could revert cellular effects of NF1 haploinsuffiency in vitro . One effect of NF1 haploinsuffiency used as first read out is the relative mechanosensoric blindness to micro-nano-structures measured as reduced capability to orientate themselves on polydimethysiloxane (PDMS) gels structured with parallel elevations of 200 nm height and a width of 2 µm, as described for fibroblasts derived from NF1 patients 9 . We tested if this effect could be provoked in normal fibroblasts by reducing the NF1 message with NF1 siRNAs and rescuing the induced effects by neurofibromin transduction.…”