Mechanosensitive transcriptional coactivators MRTF-A and YAP/TAZ regulate nucleus pulposus cell phenotype through cell shape

Abstract

Cells of the adult nucleus pulposus (NP) are critically important in maintaining overall disc health and function. NP cells reside in a soft, gelatinous matrix that dehydrates and becomes increasingly fibrotic with age. Such changes result in physical cues of matrix stiffness that may be potent regulators of NP cell phenotype and may contribute to a transition toward a senescent and fibroblastic NP cell with a limited capacity for repair. Here, we investigate the mechanosignaling cues generated from changes in matrix stiffness in directing NP cell phenotype and identify mechanisms that can potentially preserve a biosynthetically active, juvenile NP cell phenotype. Using a laminin-functionalized polyethylene glycol hydrogel, we show that when NP cells form rounded, multicell clusters, they are able to maintain cytosolic localization of myocardin-related transcription factor (MRTF)-A, a coactivator of serum-response factor (SRF), known to promote fibroblast-like behaviors in many cells. Upon preservation of a rounded shape, human NP cells similarly showed cytosolic retention of transcriptional coactivator Yes-associated protein (YAP) and its paralogue PDZ-binding motif (TAZ) with associated decline in activation of its transcription factor TEA domain family member-binding domain (TEAD). When changes in cell shape occur, leading to a more spread, fibrotic morphology associated with stronger F-actin alignment, SRF and TEAD are up-regulated. However, targeted deletion of either cofactor was not sufficient to overcome shape-mediated changes observed in transcriptional activation of SRF or TEAD. Findings show that substrate stiffness-induced promotion of F-actin alignment occurs concomitantly with a flattened, spread morphology, decreased NP marker expression, and reduced biosynthetic activity. This work indicates cell shape is a stronger indicator of SRF and TEAD mechanosignaling pathways than coactivators MRTF-A and YAP/TAZ, respectively, and may play a role in the degeneration-associated loss of NP cellularity and phenotype.-Fearing, B. V., Jing, L., Barcellona, M. N., Witte, S. E., Buchowski, J. M., Zebala, L. P., Kelly, M. P., Luhmann, S., Gupta, M. C., Pathak, A., Setton, L. A. Mechanosensitive transcriptional coactivators MRTF-A and YAP/TAZ regulate nucleus pulposus cell phenotype through cell shape.

Keywords: F-actin; SRF; TEAD; intervertebral disc; mechanotransduction.

Figure 1

A) Schema of the IVD and progression of human discs from young to old (left to right). B) The healthy disc undergoes major changes in stiffness as it progresses to a pathologic state. C) Schema depicting PEG-LM hydrogel synthesis with culture of isolated primary human NP cells. NHS, N-hydroxysuccinimide. D) 20% PEG-LM gels exhibit stiffness values resembling that of degenerative tissue, whereas 4% PEG-LM is more similar to healthy tissue. An unpaired, 2-tailed t test was used to test for evidence of differences between hydrogels of differing stiffness. ***P < 0.05.

Figure 2

A) Schema depicting the culture of primary human NP cells on top of soft and stiff PEG-LM hydrogels and LM-coated glass substrates. B) Representative morphology of human NP cells grown on the respective substrates, where a spread, fibroblast-like morphology dominates on the stiffest substrates and rounded, multicell clusters are more commonly observed on soft PEG-LM hydrogels. C) Panel of NP-specific phenotype markers are increased in cells that are cultured atop soft PEG-LM hydrogels. D) Biosynthetic activity, as measured through sGAG and DNA, is significantly increased in NP cells cultured on soft substrates. E) Actin orientation of human NP cells shows highly aligned (0 = nonaligned, 1 = aligned) actin in stiff culture conditions compared with radial alignment in soft PEG-LM. F) Transcriptional activity of SRF is significantly decreased in primary human NP cells on soft PEG-LM, as shown through an SRE luciferase reporter assay. G) Human NP cells demonstrate significantly higher nuclear MRTF-A localization when cultured on stiff PEG-LM hydrogels. H) Representative images of the subcellular localization of MRTF-A in human NP cells cultured upon different substrates. I) Transcriptional activity of TEAD assessed using the 8xGTIIC luciferase reporter shows a significant decrease in transactivation in cells cultured on soft PEG-LM compared with those on stiff PEG-LM and glass substrates. J) Ratiometric analysis of the nuclear staining intensity to the cytosolic intensity shows primarily cytosolic YAP/TAZ in primary human NP cells cultured on soft PEG-LM. K) Representative images of primary human NP cells cultured on different substrates show strong nuclear intensity of YAP/TAZ for cells on stiff PEG-LM– and LM-coated glass. Scale bar, 50 μm; green = F actin (phalloidin-488); blue, nucleus (DAPI). *P < 0.05, **P < 0.001, ***P < 0.0001 [significant differences between soft and stiff PEG-LM); ^#P < 0.05, ^##P < 0.001. ^###P < 0.0001 [significant differences between soft and glass substrates means (tp <0.0001 significant differences between stiff and glass substrates)]. B–E) Data are taken from replicates of 3 different human NP cell isolations and shown as means ± sd. F–K) Data are taken from replicates for 4 different human cell isolations and presented as means ± sd.

Figure 3

A) Fold-change normalized to the scramble control showing efficiency of siRNA knockdown of MRTF and YAP. B) YAP and MRTF knockdown do not promote changes in NP phenotype expression of AGC, COL2, and GLUT1 (fold-change normalized to scramble control). C) SRF transcriptional activation does not change when MRTF or YAP are knocked down; a significant decrease in transactivation was observed with a double knockdown of both MRTF and YAP on LM-coated glass compared with the scramble control. D) TEAD transactivation is not affected by the absence of YAP or MRTF and although it did not reach significance, there was a slight downward trend on LM-coated glass in double-knockdown cells. Data shown from replicates for 3 human cell isolations and presented as means ± sd. *P < 0.05 [significant difference between siRNA knockdown cells and scramble control (Dunnet’s test)].

Figure 4

A) Activation of SRF transcription factor is not significantly altered with treatment of Y27 compared to the vehicle (PBS) control or across time. B, C) The ratiometric quantification of the SRF coactivator MRTF-A remains predominantly nuclear (B), and may also be observed in the subcellular localization of MRTF in representative images (C), indicating no difference between Y27 and vehicle control treatments. D) TEAD transcriptional activation is not significantly different in human NP cells treated with Y27 compared to the vehicle control (PBS). E, F) Nuclear localization of the TEAD coactivator YAP/TAZ occurs with Y27 treatment (E), and indicates minimal cytosolic sequestration consistently between Y27 and vehicle control groups (F). G) F-actin alignment indicates no change in actin cytoskeletal organization and thus cell shape. Scale bars, 45 μm; green, coactivator; blue, DAPI/nucleus; n.s., not significant. Data presented are taken from replicates of three human cell isolations and presented as means ± sd.

Figure 5

A) SRF reporter shows a significant loss of transactivation with Lat B treatment. B) TEAD (8xGTIIC) reporter also indicates decreased TEAD activation with Lat B treatment. Human NP cells show marked differences in morphology upon disruption of F actin with Lat B treatment (C) along with less alignment, suggesting more cortical actin (D). E) Transcript levels of common NP markers are increased in human NP cells with Lat B changes in cell shape. Data presented are from 4 separate human cell isolations and shown as means ± sd. *P < 0.05, **P < 0.01 [significant differences between Lat B treatment and vehicle control (DMSO)].