TGFβ signaling promotes matrix assembly during mechanosensitive embryonic salivary gland restoration

Abstract

Mechanical properties of the microenvironment regulate cell morphology and differentiation within complex organs. However, methods to restore morphogenesis and differentiation in organs in which compliance is suboptimal are poorly understood. We used mechanosensitive mouse salivary gland organ explants grown at different compliance levels together with deoxycholate extraction and immunocytochemistry of the intact, assembled matrices to examine the compliance-dependent assembly and distribution of the extracellular matrix and basement membrane in explants grown at permissive or non-permissive compliance. Extracellular matrix and basement membrane assembly were disrupted in the glands grown at low compliance compared to those grown at high compliance, correlating with defective morphogenesis and decreased myoepithelial cell differentiation. Extracellular matrix and basement membrane assembly as well as myoepithelial differentiation were restored by addition of TGFβ1 and by mechanical rescue, and mechanical rescue was prevented by inhibition of TGFβ signaling during the rescue. We detected a basal accumulation of active integrin β1 in the differentiating myoepithelial cells that formed a continuous peripheral localization around the proacini and in clefts within active sites of morphogenesis in explants that were grown at high compliance. The pattern and levels of integrin β1 activation together with myoepithelial differentiation were interrupted in explants grown at low compliance but were restored upon mechanical rescue or with application of exogenous TGFβ1. These data suggest that therapeutic application of TGFβ1 to tissues disrupted by mechanical signaling should be examined as a method to promote organ remodeling and regeneration.

Heterotypic control of basement membrane dynamics during branching morphogenesis

“…Loss of activated integrin β1 was detected at the periphery of the salivary gland epithelium in developing glands grown at aberrantly low compliance that was associated with a decrease in myoepithelial differentiation, consistent with integrin β1 functioning as a compliance sensor in branching morphogenesis (Peters et al, 2015). Recent work reveals that integrin endocytosis and the recycling of integrins back to the cell surface can be controlled by microenvironmental stiffness (Du et al, 2011; Huebsch et al, 2010) to regulate the number of available integrins, which may occur in branching organs.…”

“…In the developing salivary gland, branching morphogenesis and epithelial differentiation were shown to be inhibited by culture of submandibular gland organ explants on gels of aberrantly high stiffness/low compliance (Miyajima et al, 2011; Peters et al, 2014a). The mechanisms driving these compliance-mediated differences in glandular development are unknown, but significant differences in the organization of the ECM and basement membrane were observed in glands cultured at low vs high compliance (Peters et al, 2015). The hippo effector protein, Yap, which is known to be mechanosensitive (Low et al, 2014), is critical in forming airway epithelium in the developing lung (Mahoney et al, 2014), whereas activation of the hippo effector, Taz, is required for ductal morphogenesis in the salivary gland (Enger et al, 2013).…”

“…With the recognition that multiple cooperating cell types are required for organogenesis and epithelial basement membrane assembly and remodeling, engineered scaffolds must promote survival and self-organization of both epithelial and mesenchymal cell populations, including the mesenchymal fibroblasts that support the epithelial-stromal interface and the integrated vasculature and innervation. Additionally, engineered scaffolds must provide an appropriate environmental compliance or stiffness to allow tensional homeostasis and regulation of integrin-based cell adhesions (Du et al, 2011; Huebsch et al, 2010) and support functional differentiation and retention of complex interacting cell populations (Bissell and Bilder, 2003; Hu et al, 2008; Peters et al, 2015). Ultimately, scaffolds that best support dynamic homotypic and heterotypic cell interactions and encourage the cells to deposit and remodel their own extracellular matrices and basement membranes may be required to engineer functional and sustainable organs.…”

Elastin-PLGA hybrid electrospun nanofiber scaffolds for salivary epithelial cell self-organization and polarization

“…To understand the contribution of compliance to morphogenesis and differentiation of developing submandibular salivary glands, we previously engineered artificial environments of differing compliance [20–22]. Embryonic salivary gland organ explants underwent morphogenesis and differentiation most similar to the in vivo processes in a compliant environment.…”

Endothelial cell regulation of salivary gland epithelial patterning

“…Immunocytochemistry (ICC) was performed as previously described (Daley et al, 2009;Peters et al, 2015). Briefly, SMG organ explants or SMG cell fractionation/reconstitutions glands were fixed with 4% paraformaldehyde (w/v) (Electron Microscopy Sciences) containing 5% sucrose (w/v) for 20 min at room temperature (RT) and permeablized with 0.1% Triton X-100 (Sigma-Aldrich) or fixed/permeablized in 100% cold MeOH at −20°C.…”