Yap activation in irradiated parotid salivary glands is regulated by ROCK activity

Abstract

Radiotherapy plays a major role in the curative treatment of head and neck cancer, either as a single modality therapy, or in combination with surgery or chemotherapy, or both. Despite advances to limit radiation-induced side-effects, the major salivary glands are often affected. This frequently leads to hyposalivation which causes an increased risk for xerostomia, dental caries, mucositis, and malnutrition culminating in a significant impact on patients’ quality of life. Previous research demonstrated that loss of salivary function is associated with a decrease in polarity regulators and an increase in nuclear Yap localization in a putative stem and progenitor cell (SPC) population. Yap activation has been shown to be essential for regeneration in intestinal injury models; however, the highest levels of nuclear Yap are observed in irradiated salivary SPCs that do not regenerate the gland. Thus, elucidating the inputs that regulate nuclear Yap localization and determining the role that Yap plays within the entire tissue following radiation damage and during regeneration is critical. In this study, we demonstrate that radiation treatment increases nuclear Yap localization in acinar cells and Yap-regulated genes in parotid salivary tissues. Conversely, administration of insulin-like growth factor 1 (IGF1), known to restore salivary function in mouse models, reduces nuclear Yap localization and Yap transcriptional targets to levels similar to untreated tissues. Activation of Rho-associated protein kinase (ROCK) using calpeptin results in increased Yap-regulated genes in primary acinar cells while inhibition of ROCK activity (Y-27632) leads to decreased Yap transcriptional targets. These results suggest that Yap activity is dependent on ROCK activity and provides new mechanistic insights into the regulation of radiation-induced hyposalivation.

Radiation-Induced Salivary Gland Dysfunction: Mechanisms, Therapeutics and Future Directions

“…A reduction in the interaction between E-cadherin and β-catenin leads to actin filament fragmentation in mouse parotid glands 7–30 days post-IR due to increased rho-associated kinase (ROCK) signaling that can be reversed by post-IR IGF-1 treatment [ 50 ]. Further evaluation of this pathway showed that ROCK signaling leads to activation and nuclear translocation of the transcriptional regulator Yes-associated protein (Yap) that is modulated by ROCK inhibition or IGF-1 treatment [ 64 ]. Yap activity is typically beneficial in injury models, although in salivary glands increased activation of Yap is seen in subsets of stem and progenitor cells, as well as the entire acinar compartment in parotid glands at days 5–30 post-IR in models that do not restore salivary function [ 55 , 64 ].…”

“…Further evaluation of this pathway showed that ROCK signaling leads to activation and nuclear translocation of the transcriptional regulator Yes-associated protein (Yap) that is modulated by ROCK inhibition or IGF-1 treatment [ 64 ]. Yap activity is typically beneficial in injury models, although in salivary glands increased activation of Yap is seen in subsets of stem and progenitor cells, as well as the entire acinar compartment in parotid glands at days 5–30 post-IR in models that do not restore salivary function [ 55 , 64 ]. In contrast, post-IR IGF-1 treatment reduces Yap activity and improves salivary gland function in a PKCζ-dependent manner [ 55 , 64 ].…”

“…Yap activity is typically beneficial in injury models, although in salivary glands increased activation of Yap is seen in subsets of stem and progenitor cells, as well as the entire acinar compartment in parotid glands at days 5–30 post-IR in models that do not restore salivary function [ 55 , 64 ]. In contrast, post-IR IGF-1 treatment reduces Yap activity and improves salivary gland function in a PKCζ-dependent manner [ 55 , 64 ]. Together, these data support the mechanism whereby IR induces dissociation of tight junction proteins to promote loss of PKCζ-mediated apical/basolateral polarity, ROCK-dependent actin cytoskeletal rearrangements and loss of salivary gland function, responses that can be reversed by IGF-1 to restore salivary function of irradiated parotid glands.…”

“…This period is also marked by release of ATP, which activates the P2X7 receptor (P2X7R), and P2X7R-dependent release of prostaglandin E 2 (PGE 2 ) in murine parotid cells [ 48 ]. During the transition phase, loss of apical/basolateral polarity as a result of PKCζ inactivation [ 55 , 62 , 63 ], increases nuclear Yes-associated protein (Yap) levels [ 55 , 64 ], compensatory proliferation [ 62 , 65 , 66 ], cellular senescence [ 60 , 67 , 68 ], and cytoskeletal rearrangements [ 50 , 69 ], which contribute to long-term dysfunction. Changes in innervation and vasculature have been reported as early as 24 h post-IR [ 53 ], as well as at chronic time points [ 54 , 70 ].…”

The Radiation-Induced Regenerative Response of Adult Tissue-Specific Stem Cells: Models and Signaling Pathways

“…The functional recovery of saliva production normally achieved by the administration of IGF-1 after IR [ 91 ] was absent under Pkcζ ablation. Similarly, 5 Gy of head/neck-IR of FVB mice resulted in higher nuclear Yap1 localization in the acinar compartment while no changes were reported in cells of major ducts [ 92 ]. IGF-1 treatment of irradiated mice restored Yap1 nuclear localization to normal, suggesting that the SG functional recovery promoted by IGF-1 is a consequence of the reduction of Yap1 nuclear translocation.…”