Activation of CHK1 in Supporting Cells Indirectly Promotes Hair Cell Survival

Abstract

The sensory hair cells of the inner ear are exquisitely sensitive to ototoxic insults. Loss of hair cells after exposure to ototoxic agents causes hearing loss. Chemotherapeutic agents such as cisplatin causes hair cell loss. Cisplatin forms DNA mono-adducts as well as intra- and inter-strand DNA crosslinks. DNA cisplatin adducts are repaired through the DNA damage response. The decision between cell survival and cell death following DNA damage rests on factors that are involved in determining damage tolerance, cell survival and apoptosis. Cisplatin damage on hair cells has been the main focus of many ototoxic studies, yet the effect of cisplatin on supporting cells has been largely ignored. In this study, the effects of DNA damage response in cochlear supporting cells were interrogated. Supporting cells play a major role in the development, maintenance and oto-protection of hair cells. Loss of supporting cells may indirectly affect hair cell survival or maintenance. Activation of the Phosphoinositide 3-Kinase (PI3K) signaling was previously shown to promote hair cell survival. To test whether activating PI3K signaling promotes supporting cell survival after cisplatin damage, cochlear explants from the neural subset (NS) Cre Pten conditional knockout mice were employed. Deletion of Phosphatase and Tensin Homolog (PTEN) activates PI3K signaling in multiple cell types within the cochlea. Supporting cells lacking PTEN showed increased cell survival after cisplatin damage. Supporting cells lacking PTEN also showed increased phosphorylation of Checkpoint Kinase 1 (CHK1) levels after cisplatin damage. Nearest neighbor analysis showed increased numbers of supporting cells with activated PI3K signaling in close proximity to surviving hair cells in cisplatin damaged cochleae. We propose that increased PI3K signaling promotes supporting cell survival through phosphorylation of CHK1 and increased survival of supporting cells indirectly increases hair cell survival after cisplatin damage.

Keywords: AKT; CHK1; PI3 kinase signaling; cell survival; cisplatin; cochlea; ototoxicity; supporting cell.

Figure 1

Expression of hair cell and supporting cell markers in differentiating immortalized multipotent otic progenitor (iMOP) cells. Proliferating iMOP cells cultured in basic fibroblast growth factor (bFGF) were subjected to 5-ethynyl-2′-deoxyuridine (EdU) incorporation. (A) Hoechst labeled nuclei from proliferating iMOP cells show EdU incorporation in 37.9% of cells (n = 3). (B) Hoechst labeled nuclei from differentiating iMOP cells showed EdU incorporation in 3.3% of cells (n = 3). Differentiating iMOP cells express (C) MYO7A and (D) glial fibrillary acidic protein (GFAP) in (E) phalloidin marked otospheres. (F) Otospheres from differentiating iMOP cultures were used to test for the effects of cisplatin treatment.

Figure 2

Checkpoint Kinase 1 (CHK1) phosphorylation after activating phosphoinositide 3-kinase (PI3K) signaling. Propidium iodide (PI) and annexin V FACS analysis of (A) control dimethyl sulfoxide (DMSO), (B) cisplatin (C) cisplatin and bpV(HOpic) and (D) cisplatin and LY treated cells from representative plots. Average percentage of cells from viable (PI- annexin V-) and apoptotic (PI+ annexin V+) quandrants were displayed (n = 4). (E) Western blot of phospho-AKT (pAKT), total AKT and ACTB in control DMSO, 10 μM bpV(HOpic) or 25 μM LY294002 treated cells (n = 4). (F) Quantification of pAKT/AKT ratio from Western blots (n = 4). (G) Western blot of pCHK1, total phospho-CHK1 (CHK1), pAKT, total AKT and ACTB in control DMSO, 20 μM cisplatin, 20 μM cisplatin and 10 μM bpV(HOpic) treated cells (n = 4). (H) Quantification of pCHK1/CHK1 ratios from Western blots (n = 4). Statistical significance was determined using the Student’s t-test and error bars are in standard deviation (SD).

Figure 3

Effects of bpV(HOpic) on hair cell survival after cisplatin damage. (A) Timeline of cochlear explant cultures describing the addition of 10 μM bpV(HOpic) 1 day before 10 μM cisplatin treatment and harvesting of explants for immunostaining. Immunofluorescence of MYO7A, phalloidin and merged images from (B) control cochlear explant (n = 4), (C) cochlear explant treated with cisplatin (n = 4) and (D) cochlear explant pretreated with 10 μM bpV(HOpic) before cisplatin damage (n = 4). (E) Quantification of MYO7A inner hair cells (IHC) from the basal, middle and apical regions of cochlea explants in control (black), cisplatin (white) and bpV(HOpic)/cisplatin (gray; n = 4). (F) Average percent of IHC in control (black; n = 4), cisplatin (white; n = 4) and bpV(HOpic) and cisplatin (gray; n = 4) treated explants normalized to control. (G) Percentage of MYO7A outer hair cells (OHC) in control (black; n = 4), bpV(HOpic) and cisplatin (gray; n = 4) and cisplatin treated (white; n = 4) cochlear explants from the basal, middle and apical regions. (H) Average percent of OHC from control (black; n = 4), cisplatin (white; n = 4) and bpV(HOpic) and cisplatin (gray; n = 4) treated explants. Percentage of surviving IHC, OHC and undefined hair cells in (I) control, (J) cisplatin and (K) bpV(HOpic) and cisplatin treated cochlear explants. Statistical significance was determined using the Student’s t-test and error bars are in SD.

Figure 4

Labeling cochlear cells using a neural subset (NS) Cre tdTomato reporter mouse. (A) Diagram of the NS Cre recombinase transgene using a fragment of the human GFAP (hGFAP) promoter driving Cre recombinase and a STOP floxed tdTomato reporter inserted in the ROSA26 (R26) locus. (B) tdTomato fluorescence from NS Cre tdTomato cochlea. Confocal section of the cochlear sensory epithelium labeled with (C) MYO7A, (D) phalloidin, (E) tdTomato from the NS Cre tdTomato reporter mouse. (F) Merged image (n = 4).

Figure 5

Hair cell survival after genetic activation of PI3K signaling and cisplatin damage. (A) Timeline of cochlear explant cultures describing the addition of 10 μM cisplatin treatment for cochlear explants. MYO7A, phalloidin, tdTomato and merged fluorescent images from (B) NS Cre tdTomato (control) after cisplatin treatment (n = 4) and (C) NS Cre Pten cKO tdTomato (Pten cKO) cochleae after cisplatin treatment (n = 4). (D) pCHK1, phalloidin, tdTomato and merged fluorescent images from Pten cKO mouse after cisplatin damage. (E) Density (cells/0.1 mm²) of MYO7A- tdTomato+ supporting cells in control (n = 4) and Pten cKO cochleae (n = 4). (F) Density of MYO7A+ tdTomato+ hair cells in control (n = 4) and Pten cKO cochleae (n = 4). (G) Density of MYO7A+ tdTomato- hair cells in control (n = 4) and Pten cKO cochleae (n = 4). Statistical significance was determined using the Student’s t-test and error bars are in SD.

Figure 6

Nearest neighbor analysis of supporting cell to surviving hair cells. (A) 3D rendering of a confocal stack with MYO7A and phalloidin labeled hair cells along with tdTomato labeled supporting cell (B) 3D images were compressed into a maximal intensity projection to generate a 2D mask for hair cells (green) and tdTomato labeled supporting cells (red). (C) Using the 2D masks, percent of surviving hair cells (green) that reside adjacent to supporting cells (red) were determined in control (n = 4) and Pten cKO cochleae (n = 4). All remaining cells in the field of view were labeled blue.

Figure 7

Interaction strength of supporting cell and surviving hair cells. Region containing tdTomato+supporting cell and MYO7A+ tdTomato- hair cells were analyzed. (A) Supporting cell masks were generated from tdTomato labeled supporting cells (red). (B) Hair cell masks were generated from MYO7A labeled hair cells (green). (C) Merged image of supporting cell and hair cell masks. (D) Centroid of hair cell and supporting cells were generated from masks. Distance between an individual hair cell centroid to all supporting cell centroids in the field of view was determined. (E) Number of cell interactions within a field of view and the mean distance between the centroid of a hair cell to multiple supporting cells was displayed in the histogram. (F) The observed distances between a hair cell and supporting cells were fitted into probability density, p(d), and an interaction potential model, q(d). (G) Interaction strength between hair cell and supporting cells was calculated from the interaction potential q(d). (H) Comparison of the interaction strength between surviving hair cells and control supporting cells (n = 10) or surviving hair cells and Pten cKO supporting cells (n = 10) after cisplatin treatment. Statistical significance was determined using the Student’s t-test and error bars in SD.

Figure 8

Model of CHK1 phosphorylation through cisplatin damage and PI3K signaling. Activation of PI3K signaling either by bpV(HOpic) or Pten deletion results in increased AKT phosphorylation. DNA damage caused by cisplatin activates DNA damage response proteins that increase pCHK1 levels. Activated AKT can directly or indirectly increase pCHK1 levels by activating the DNA damage response proteins. Together, PI3K activation and cisplatin-induced DNA damage results in increased pCHK1 to promote supporting cell survival.