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.

Verteporfin treatment controls morphology, phenotype, and global gene expression for cells of the human nucleus pulposus

“…[26][27][28][29][30][31][32][33] Literature similarly supports that NP cells are mechanosensitive in vitro and in vivo, and as such, many groups have developed biomaterials designed to control the phenotype and behaviors of NP cells. 7,[34][35][36][37][38][39][40][41][42] Much of this work consistently shows that NP cell morphology responds to both ligand and stiffness, where soft, laminin-presenting substrates promote a juvenile, healthy rounded morphology, but stiff and/or non-laminin associated ligands allow a spread, fibroblastic-like shape observed in adult NP. 7,15,37,[42][43][44][45][46][47][48] These reports also support the idea that changes in NP cell shape are associated with concomitant increases in expression of phenotypic markers and matrix biosynthesis.…”

“…SRF and TEAD transcriptional activation was assessed as previously described. 42 Briefly, a lentivirus (HIV) vector containing firefly luciferase under the control of four SRF response elements (pGreenFire1-ELK1-SRF-EF1-Neo, Systems Biosciences Palo Alto, California) and a plasmid containing four TEAD binding sites upstream of a firefly luciferase reporter gene (8xGTIIC, Addgene, Watertown, Massachusetts) were transfected into HEK293T cells with the transfer vector, pMD2.G, and packaging gene vector, psPAX2 (Addgene). The vehicle control reference was taken from vehicle control cultures at each timepoint.…”

“…Prior studies to modify cell morphology in primary human NP cells employed methods such as controlled hydrogel substrate stiffness for culture. 15,36,37,42 use of genetic-or antibody-based inhibition. 42,43 or small molecule inhibitors.…”

“…15,36,37,42 use of genetic-or antibody-based inhibition. 42,43 or small molecule inhibitors. 42 The present study is the first to describe use of a clinically relevant pharmacological treatment to induce a rounded, clustered shape in primary human NP cells when cultured upon stiff substrates.…”

“…42,43 or small molecule inhibitors. 42 The present study is the first to describe use of a clinically relevant pharmacological treatment to induce a rounded, clustered shape in primary human NP cells when cultured upon stiff substrates. VP has a t ½ of 5 to 6 hours 75 and was accordingly replenished every 24 hours throughout the culture period.…”