Absence of Gli1 Decreases PDGFRβ Expression Following Ductal Ligation Injury

Abstract

Fibrosis results from excess extracellular matrix deposition, which alters normal tissue architecture and impedes function. Salivary gland fibrosis can be caused by irradiation therapy, as a treatment for head and neck cancer, as well as by the autoimmune disease Sjögren’s Syndrome. The mechanisms that contribute to fibrosis are complex and poorly understood; however, defining the cell types and signaling pathways involved could reveal potential therapeutic targets. Stromal cell subpopulations have been shown to contribute to fibrosis. Specifically, Gli1 is a transcription factor expressed by mesenchymal stem cell‐like cells, which can contribute directly to fibrosis by transdifferentiating into myofibroblasts following injury. In the salivary gland, it is unknown which stromal cells expand following fibrotic injury or if the Gli1+ cells contribute to fibrotic injury. We hypothesize that Gli1+ cells contribute to salivary gland fibrosis by undergoing a transdifferentiation into myofibroblasts. To induce salivary gland fibrosis, we used a ductal ligation model, where a ligation is placed on the main ducts feeding from the submandibular and sublingual glands to the mouth and performed immunohistochemistry. Following ligation injury, there is a progressive fibrotic response where both collagen deposition and actively remodeled collagen increase. There is also an expansion of the PDGFRα+ stroma and F4/80+ macrophage populations. Using single‐cell RNA‐sequencing data, we found that a majority of Gli1+ cells at embryonic day 16 also express PDGFRα, suggesting that Gli1+ cells are a subset of both the PDGFRα and PDGFRβ stromal cell subpopulations, as in other organs. After performing a ductal ligation surgery on glands of mice genetically null for Gli1, we found that trichrome and remodeled collagen area trend downwards and there is a significant decrease in PDGFRβ area. We used a lineage tracing strategy to track Gli1‐expressing cells in salivary glands of Gli1CreERT2;(CAG)ROSA26TdTomato mice. In response to injury, there was an expansion of Gli1+ cells with a subset of these cells beginning to express vimentin and PDGFRβ. As there was a significant decrease in PDGFRβ area following ligation injury in the absence of Gli1 function, Gli1 signaling may stimulate PDGFRβ expression. Our results suggest that although Gli1 signaling and Gli1+ cells do not significantly contribute to injury‐induced fibrosis in the salivary gland; PDGFRα+ and PDGRFβ+ stromal cells may be potential therapeutic targets.