Involvement of YAP, TAZ and HSP90 in contact guidance and intercellular junction formation in corneal epithelial cells.

The extracellular environment possesses a rich milieu of biophysical and biochemical signaling cues that are simultaneously integrated by cells and influence cellular phenotype. Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (WWTR1; TAZ), two important signaling molecules of the Hippo pathway, have been recently implicated as nuclear relays of cytoskeletal changes mediated by substratum rigidity and topography. These proteins intersect with other important intracellular signaling pathways (e.g. Wnt and TGFß). In the cornea, epithelial cells adhere to the stroma through a 3-dimensional topography-rich basement membrane, with features in the nano-submicron size-scale that are capable of profoundly modulating a wide range of fundamental cell behaviors. The influences of substratum-topography, YAP/TAZ knockdown, and HSP90 inhibition on cell morphology, YAP/TAZ localization, and the expression of TGFß2 and CTGF, were investigated. The results demonstrate (a) that knockdown of TAZ enhances contact guidance in a YAP dependent manner, (b) that CTGF is predominantly regulated by YAP and not TAZ, and (c) that TGFß2 is regulated by both YAP and TAZ in these cells. Additionally, inhibition of HSP90 resulted in nuclear localization and subsequent transcriptional-activation of YAP, formation of cell-cell junctions and co-localization of E-cadherin and ß-catenin at adherens junctions. Results presented in this study reflect the complexities underlying the molecular relationships between the cytoskeleton, growth factors, heat shock proteins, and co-activators of transcription that impact mechanotransduction. The data reveal the importance of YAP/TAZ on the cell behaviors, and gene and protein expression.

Substratum compliance modulates corneal fibroblast to myofibroblast transformation.

PURPOSE: The transformation of fibroblasts to myofibroblasts is critical to corneal wound healing, stromal haze formation, and scarring. It has recently been demonstrated that the provision of biomimetic substratum topographic cues inhibits the progression toward the myofibroblast phenotype under the influence of transforming growth factor ß1 (TGF-ß1). The objective of this study was to determine the effect of another fundamental biophysical cue, substrate compliance, on TGF-ß1-induced myofibroblast transformation of primary corneal cells isolated from human and rabbit corneas. METHODS: Human and rabbit corneal fibroblasts were cultured on surfaces of varying substrate compliance (4-71 kPa) and tissue culture plastic (TCP) (> 1 gigapascal [GPa]). Cells were cultured in media containing TGF-ß1 at concentrations of 0, 1, or 10 ng/mL for 72 hours. RNA and protein were collected from cells cultured on polyacrylamide gels and TCP and were analyzed for the expression of α-smooth muscle actin (α-SMA), a key marker of myofibroblast transformation, using quantitative PCR, immunocytochemistry, and Western blot. RESULTS: Cells grown on more compliant substrates demonstrated significantly reduced amounts of α-SMA mRNA compared with TCP. Immunocytochemistry and Western blot analysis determining the presence of α-SMA corroborated this finding, thus confirming a reduced transformation to the myofibroblast phenotype on more compliant substrates compared with cells on TCP in the presence of TGF-ß1. CONCLUSIONS: These data indicate that substrate compliance modulates TGF-ß1-induced expression of α-SMA and thus influences myofibroblast transformation in the corneal stroma. This provides further evidence that biomimetic biophysical cues inhibit myofibroblast transformation and participate in stabilizing the native cellular phenotype.

Early responses of vascular endothelial cells to topographic cues.

Vascular endothelial cells in vivo are exposed to multiple biophysical cues provided by the basement membrane, a specialized extracellular matrix through which vascular endothelial cells are attached to the underlying stroma. The importance of biophysical cues has been widely reported, but the signaling pathways that mediate cellular recognition and response to these cues remain poorly understood. Anisotropic topographically patterned substrates with nano- through microscale feature dimensions were fabricated to investigate cellular responses to topographic cues. The present study focuses on early events following exposure of human umbilical vein endothelial cells (HUVECs) to these patterned substrates. In serum-free medium and on substrates without protein coating, HUVECs oriented parallel to the long axis of underlying ridges in as little as 30 min. Immunocytochemistry showed clear differences in the localization of the focal adhesion proteins Src, p130Cas, and focal adhesion kinase (FAK) in HUVECs cultured on topographically patterned surfaces and on planar surfaces, suggesting involvement of these proteins in mediating the response to topographic features. Knockdown experiments demonstrated that FAK was not necessary for HUVEC alignment in response to topographic cues, although FAK knockdown did modulate HUVEC migration. These data identify key events early in the cellular response to biophysical stimuli.

Role of substratum stiffness in modulating genes associated with extracellular matrix and mechanotransducers YAP and TAZ.

PURPOSE: Primary open-angle glaucoma is characterized by increased resistance to aqueous humor outflow and a stiffer human trabecular meshwork (HTM). Two Yorkie homologues, Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif, encoded by WWTR1 (TAZ), are mechanotransducers of the extracellular-microenvironment and coactivators of transcription. Here, we explore how substratum stiffness modulates the YAP/TAZ pathway and extracellular matrix genes in HTM cells and how this may be play a role in the onset and progression of glaucoma. METHODS: HTM cells from normal donors were cultured on hydrogels mimicking the stiffness of normal (5 kPa) and glaucomatous (75 kPa) HTM. Changes in expression of YAP/TAZ related genes and steroid responsiveness were determined. Additionally, transglutaminase-2 expression was determined after YAP silencing. RESULTS: YAP and TAZ are both expressed in human trabecular meshwork cells. In vitro, YAP and TAZ were inversely regulated by substratum stiffness. YAP and 14-3-3σ were downregulated to different extents on stiffer substrates; TAZ, tissue transglutaminase (TGM2), and soluble frizzled-related protein-1 (sFRP-1) were significantly upregulated. CTGF expression appeared to be altered differentially by both YAP and TAZ. Myocilin and angiopoietin-like 7 expression in response to dexamethasone was more pronounced on stiffer substrates. We demonstrated a direct effect by YAP on TGM2 when YAP was silenced by small interfering RNA. CONCLUSIONS: The expression of YAP/TAZ and ECM-related-genes is impacted on physiologically relevant substrates. YAP was upregulated in cells on softer substrates. Stiffer substrates resulted in upregulation of canonical Wnt modulators, TAZ and sFRP-1, and thus may influence the progression of glaucoma. These results demonstrate the importance of YAP/TAZ in the HTM and suggest their role in glaucoma.

Focal adhesion kinase knockdown modulates the response of human corneal epithelial cells to topographic cues.

A rapidly expanding literature broadly documents the impact of biophysical cues on cellular behaviors. In spite of increasing research efforts in this field, the underlying signaling processes are poorly understood. One of the candidate molecules for being involved in mechanotransduction is focal adhesion kinase (FAK). To examine the role of FAK in the response of immortalized human corneal epithelial (hTCEpi) cells to topographic cues, FAK was depleted by siRNA transfection. Contrary to expectations, FAK knockdown resulted in an enhanced response with a greater number of hTCEpi cells aligned to the long axis of anisotropically ordered surface ridges and grooves. Both underlying topographic features and FAK depletion modulated the migration of corneal epithelial cells. The impact of FAK knockdown on both migration and alignment varied depending on the topographic cues to which the cells were exposed, with the most significant change observed on the biologically relevant size scale (400nm). Additionally, a change in expression of genes encoding perinuclear Nesprins 1 and 2 (SYNE1, 2) was observed in response to topographic cues. SYNE1/2 expression was also altered by FAK depletion, suggesting that these proteins might represent a link between cytosolic and nuclear signaling processes. The data presented here have relevance to our understanding of the fundamental processes involved in corneal cell behavior to topographic cues. These results highlight the importance of incorporating biophysical cues in the conduction of in vitro studies and into the design and fabrication of implantable prosthetics.