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.

Fig 1. Yap activation is increased in irradiated salivary glands.

FVB mice were either untreated (UT) or irradiated (IR) with 5Gy and dissected on days 4, 5, 7, and 30 following radiation treatment. Levels of (A-B) phosphorylated Yap on serine 127 (pYap S127) and (C-D) Taz were evaluated following radiation treatment. Immunoblots were re-probed with total Yap (A) or ERK (C) as a loading control. (E) Immunofluorescent staining was used to determine the percentage of cells that displayed nuclear Yap (red) in the acinar compartment (denoted by an asterisk). Composite images with DAPI (blue) are presented in both high and low magnification (scale bar for high magnification = 30 μm, low magnification = 100 μm). Quantification of the percentage of Yap positive cells with nuclear Yap staining from E was determined in the acinar enriched (F) and ductal (G) compartments separately. Relative mRNA levels of Cyr61 (H), Ccn2 (I) and Arhgef17 (J) were determined by qRT-PCR and normalized to Gapdh. Results are presented from at least four mice per condition (each data point graphed in B, D, F-J represents individual mice); E- G used 10–15 images per mouse; error bars denote mean ± SEM. Multiple comparisons were conducted using a Tukey-Kramer test and significant differences (p<0.05) between treatment groups are denoted with different letters above the bar graphs. Treatment groups with different letters are significantly different from each other.

Fig 2. Post-therapeutic IGF1 treatment reduces Yap activation.

FVB mice (4–6 weeks old) were either untreated (UT) or irradiated (IR) with 5Gy and given post-therapeutic IGF1 (IR+IGF) on days 4–7. Parotid salivary glands were dissected on days 5 and 30 following radiation treatment. Levels of (A-B) phosphorylated Yap on serine 127 (pYap S127) and (C-D) Taz were evaluated following IR+IGF treatment. Immunoblots were re-probed with total Yap or ERK as a loading control. (E) Immunofluorescent staining was used to determine the percentage of cells that displayed nuclear Yap (red) in the acinar compartment. Composite images with DAPI (blue) are presented in both high and low magnification (scale bar for high magnification = 30 μm, low magnification = 100 μm). Quantification of the percentage of Yap positive cells with nuclear Yap staining from E was determined in the acinar enriched (F) and ductal (G) compartments separately. Relative mRNA levels of Cyr61 (H), Ccn2 (I) and Arhgef17 (J) were determined by qRT-PCR and normalized to Gapdh. Results are presented from at least four mice per condition (each data point graphed in B, D, F-J represents individual mice); E-F used 10–15 images per mouse; error bars denote mean ± SEM. Multiple comparisons were conducted using a Tukey-Kramer test and significant differences (p<0.05) between treatment groups are denoted with different letters above the bar graphs. Treatment groups with different letters are significantly different from each other.

Fig 3. Activation of ROCK leads to Yap transcriptional activity.

Parotid salivary glands from FVB mice were dissected and cultured as primary acinar cell cultures. At sub-confluency, the cells were treated 10 μM calpeptin (ROCK activator) or DMSO vehicle control for two hours. (A) Levels of phosphorylated ROCK were determined by immunoblot following calpeptin treatment. Immunoblots were reprobed for total ROCK as a loading control. (B) Quantification of A. (C) Levels of phosphorylated LIMK2 were determined by immunoblot following calpeptin treatment. Immunoblots were reprobed for total LIMK2 as a loading control. (D) Quantification of C. (E) Levels of phosphorylated MLC were determined by immunoblot following calpeptin treatment. Immunoblots were reprobed for total MLC as a loading control. (F) Quantification of E. (G) Levels of phosphorylated Yap on serine 127 (pYAP S127) were determined following calpeptin treatment. Immunoblots were reprobed for total Yap as a loading control. (H) Quantification of G. (E) Relative mRNA levels of Cyr61 (I), Ccn2 (J) and Arhgef17 (K) were determined by qRT-PCR and normalized to Gapdh. (L) Immunofluorescent staining with Phalloidin (green) was used to visualize F-actin structures after calpeptin treatment. Indications of F-actin fragmentation are noted with asterisks (scale bar for magnification = 20 μm). Results are presented from at least three independent in vitro primary cell culture experiments per condition (each data point graphed in B, D, F, and H-K represents an independent cell preparation); error bars denote mean ± SEM. Significant difference (p<0.05) between vehicle control and inhibitor treated cells was determined by Student’s t-test.

Fig 4. Inhibition of ROCK activity reduces Yap transcriptional activity following radiation treatment.

Parotid salivary glands from FVB mice were dissected and cultured as primary acinar cell cultures. One day after dissection, the primary cells were irradiated with 5Gy and cell lysates or RNA were collected on Day 5 after radiation treatment. On Day 4 after radiation treatment, the cells were either treated with 20 μM Y-27632 (ROCK inhibitor) or vehicle control. Effects of ROCK inhibition on phosphorylated LIMK (A) or phosphorylated MLC (C) in primary salivary cells were evaluated by immunoblotting. Blots were reprobed for total levels of LIMK or MLC. (B) Quantification by densitometry of A normalized to vehicle control. (D) Quantification by densitometry of C normalized to vehicle control. (E) Effects of ROCK inhibition on phosphorylated Yap on serine 127 (pYap S127) were evaluated by immunoblotting. Blots were reprobed for total levels of Yap. (F) Quantification by densitometry of E normalized to vehicle control. (E) Relative mRNA levels of Cyr61 (G), Ccn2 (H) and Arhgef17 (I) were determined by qRT-PCR and normalized to Gapdh. (J) Immunofluorescent staining with Phalloidin (green) was used to visualize F-actin structures after radiation with and without Y-27632 treatment (scale bar for magnification = 20 μm). Results are presented from at least three independent in vitro primary cell culture experiments per condition (each data point graphed in B, D, and F-I represents an independent cell preparation); error bars denote mean ± SEM. Multiple comparisons were conducted using a Tukey-Kramer test and significant differences (p<0.05) between treatment groups are denoted with different letters above the bar graphs. Treatment groups with different letters are significantly different from each other.