STK33 alleviates gentamicin-induced ototoxicity in cochlear hair cells and House Ear Institute-Organ of Corti 1 cells

Abstract

Serine/threonine kinase 33 (STK33), a member of the calcium/calmodulin-dependent kinase (CAMK), plays vital roles in a wide spectrum of cell processes. The present study was designed to investigate whether STK33 expressed in the mammalian cochlea and, if so, what effect STK33 exerted on aminoglycoside-induced ototoxicity in House Ear Institute-Organ of Corti 1 (HEI-OC1) cells. Immunofluorescence staining and western blotting were performed to investigate STK33 expression in cochlear hair cells (HCs) and HEI-OC1 cells with or without gentamicin treatment. CCK8, flow cytometry, immunofluorescence staining and western blotting were employed to detect the effects of STK33 knockdown, and/or U0126, and/or N-acetyl-L-cysteine (NAC) on the sensitivity to gentamicin-induced ototoxicity in HEI-OC1 cells. We found that STK33 was expressed in both mice cochlear HCs and HEI-OC1 cells, and the expression of STK33 was significantly decreased in cochlear HCs and HEI-OC1 cells after gentamicin exposure. STK33 knockdown resulted in an increase in the cleaved caspase-3 and Bax expressions as well as cell apoptosis after gentamicin damage in HEI-OC1 cells. Mechanistic studies revealed that knockdown of STK33 led to activated mitochondrial apoptosis pathway as well as augmented reactive oxygen species (ROS) accumulation after gentamicin damage. Moreover, STK33 was involved in extracellular signal-regulated kinase 1/2 pathway in primary culture of HCs and HEI-OC1 cells in response to gentamicin insult. The findings from this work indicate that STK33 decreases the sensitivity to the apoptosis dependent on mitochondrial apoptotic pathway by regulating ROS generation after gentamicin treatment, which provides a new potential target for protection from the aminoglycoside-induced ototoxicity.

Keywords: apoptosis; extracellular signal-regulated kinase 1/2; gentamicin; reactive oxygen species; serine/threonine kinase 33.

Figure 1

STK33 Expression in the Cochlear Hair Cells (HCs) and HEI‐OC1 Cells. A, Positive control. Immunofluorescence staining showed STK33 expression in the cells of testis (white arrow). B, Representative images of STK33 (green) expression in P30 cochlear HCs by immunofluorescence staining (IHCs, yellow arrow, and OHCs, white arrow). Myosin 7a (red) was used as HC marker. C, Western blotting results showed that STK33 expression in CBA cochlea was consistent with that in testis. D, Western blotting results showed that STK33 was expressed in HEI‐OC1 cells. E, Immunofluorescence staining showed STK33 expression in HEI‐OC1 cells. F, Immunofluorescence staining showed the expression pattern of STK33 in the middle turn of mouse cochlea. At P4, STK33 (green) was expressed in IHCs and the intercellular space of OHCs. At P15, STK33 (green) expression was found in OHCs and IHCs. From P30, STK33 (green) was highly expressed in OHCs and IHCs. Myosin 7a (red) was used as HEI‐OC1 cells marker. Scale bars = 30 μm. IHCs, inner hair cells; OHCs, outer hair cells; HEI‐OC1, House Ear Institute‐Organ of Corti 1; STK33, serine/threonine kinase 33

Figure 2

STK33 Expression was Down‐Regulated in Cochlear Hair Cells (HCs) and HEI‐OC1 Cells After Gentamicin Treatment. CBA mice were subcutaneously injected gentamicin (200 mg/kg) from P7 to P14. The control group was injected sterile saline. A, ABR thresholds were increased after gentamicin treatment compared to the control ones at 5‐6 wk. **P < 0.01, n = 3. B‐C, Western blotting results confirmed that the expression level of STK33 was decreased in cochlear HCs after gentamicin treatment compared to the control group at 5‐6 wk. **P < 0.01, n = 3. D, STK33 expression in cochlear HCs was decreased after gentamicin treatment compared to the control ones by immunofluorescence staining at 5‐6 wk. Scale bars = 30 μm. E‐F, Western blotting results demonstrated that the expression of STK33 protein was significantly reduced in HEI‐OC1 cells with 3 mmol/L gentamicin treatment for 24 h compared to the untreated controls. GAPDH was served as the control. *P < 0.05, n = 3. G, STK33 (green) expression was decreased in HEI‐OC1 cells after gentamicin treatment compared to the untreated controls by immunofluorescence staining. Myosin 7a (red) was used as the marker in HEI‐OC1 cells. Scale bar = 30 μm. ABR, auditory brainstem response; HEI‐OC1, House Ear Institute‐Organ of Corti 1; STK33, serine/threonine kinase 33

Figure 3

Mitochondrial Apoptosis was Activated in Cochlear HCs After Gentamicin Treatment. A, Immunofluorescence staining results showed that TUNEL‐positive nuclei (white arrow) were increased in gentamicin‐induced hearing loss group compared to the control group in vivo experiment. Scale bars = 30 μm. B‐C, Western blotting results confirmed that the expression levels of Bax and cleaved caspase‐3 were increased in gentamicin‐induced hearing loss group compared to the control group. *P < 0.05, **P < 0.01, n = 3. D‐E, Immunofluorescence staining results showed that in vitro experiment, the expressions of cleaved caspase‐3 and TUNEL‐positive nuclei were increased after gentamicin damage compared to the untreated controls in primary culture of cochlear HCs. Scale bars = 30 μm. F‐G, Western blotting results confirmed that in primary culture of cochlear HCs, the expression levels of Bax and cleaved caspase‐3 protein were increased after gentamicin treatment compared to the untreated controls. **P < 0.01, n = 3. HC, hair cell; HEI‐OC1, House Ear Institute‐Organ of Corti 1; STK33, serine/threonine kinase 33

Figure 4

Knockdown of STK33 Increased the Apoptosis in HEI‐OC1 Cells After Gentamicin Treatment. A, Representative images of the transfection efficiency were measured by using nonsense siRNA conjugated with 6′‐carboxyfluorescein (FAM). Scale bars = 30 μm. B‐C, Western blotting results confirmed that transfection of siRNA‐STK33 was successful. **P < 0.01, n = 3. D, Knockdown of STK33 had little decrease in the cell viability in HEI‐OC1 cells of siRNA‐STK33 transfection compared to the siRNA‐Control ones by CCK8 assay. E‐F, After gentamicin exposure, the apoptotic cells were increased in HEI‐OC1 cells transfected with siRNA‐STK33 compared to siRNA‐Control ones by flow cytometry. G, Immunofluorescence staining result demonstrated that the TUNEL‐positive cells were increased in the siRNA‐STK33 transfection group compared to the siRNA‐Control transfection group after gentamicin damage. Scale bars = 30 μm. H‐I, Western blotting results showed that the protein expression levels of Bax and cleaved caspase‐3 were increased in the siRNA‐STK33 transfection group compared to the siRNA‐Control transfection group after gentamicin treatment. **P < 0.01, n = 3. HEI‐OC1, House Ear Institute‐Organ of Corti 1; STK33, serine/threonine kinase 33

Figure 5

p‐ERK1/2 Expression was Increased in Cochlear HCs and HEI‐OC1 Cells After Gentamicin Damage. Expression of p‐ERK1/2 was increased with gentamicin exposure compared to the untreated control by immunofluorescence staining and western blotting in cochlear HCs (A‐C), the primary culture of cochlear HCs (D‐F) and HEI‐OC1 cells (G‐I). *P < 0.05, **P < 0.01, n = 3. Scale bars = 30 μm. ERK1/2, extracellular signal‐regulated kinase 1/2; HC, hair cell; HEI‐OC1, House Ear Institute‐Organ of Corti 1

Figure 6

STK33 Expression was Decreased by U0126 Treatment in Primary Culture of Cochlear HCs and HEI‐OC1 Cells After Gentamicin Damage. Hair cells (HCs) were pre‐incubated with 10 μmol/L U0126 for 2 h, followed by treating with 3 mmol/L gentamicin for 24 h. The expression of STK33 was significantly reduced by U0126 which inhibited p‐ERK1/2 expression after gentamicin treatment in the primary culture of cochlear HCs by immunofluorescence staining (A‐B) and western blotting (C‐D). **P < 0.01, n = 3. Scale bars = 30 μm. E‐F, Western blotting results showed that knockdown STK33 by transfected with siRNA‐STK33 had no effect on p‐ERK1/2 expression compared to the siRNA‐Control transfection one after gentamicin treatment in HEI‐OC1 cells. G‐H, The expression level of STK33 was significantly decreased in HEI‐OC1 cells by U0126 which inhibited p‐ERK1/2 expression after gentamicin treatment by western blotting. **P < 0.01, n = 3. ERK1/2, extracellular signal‐regulated kinase 1/2; HEI‐OC1, House Ear Institute‐Organ of Corti 1; STK33, serine/threonine kinase 33

Figure 7

NAC Treatment Successfully Rescues Cell Apoptosis Induced by STK33 Down‐Regulation in Cochlear HCs and HEI‐OC1 Cells After Gentamicin Co‐Stimulus. Hair cells (HCs) and transfection of siRNA‐STK33 HEI‐OC1 cells were pre‐incubated with 2 mmol/L NAC for 2 h, followed by treating with 3 mmol/L gentamicin for 24 h. A, The intracellular ROS level was increased after gentamicin treatment by DCFH‐DA staining compared to the untreated control. But the intracellular ROS level was decreased by NAC treatment after gentamicin damage in cochlear HCs by DCFH‐DA staining. Scale bars = 30 μm. B‐C, Western blotting results demonstrated that the expression levels of Bax and cleaved caspase‐3 were decreased by NAC co‐treatment with gentamicin compared to the gentamicin treatment in cochlear HCs. *P < 0.05, n = 3. D‐F, The ROS levels were increased in HEI‐OC1 cells transfected of siRNA‐STK33 compared to transfected of siRNA‐control ones after gentamicin treatment by DCFH‐DA staining and flow cytometry, but NAC had the opposite role in the ROS levels. **P < 0.01, ^## P < 0.01, n = 3. Scale bars = 50 μm. G‐H, Western blotting results demonstrated that the expression levels of Bax and cleaved caspase‐3 were significantly decreased in HEI‐OC1 cells transfection of siRNA‐STK33 in combination with NAC pre‐treatment after gentamicin treatment compared to transfection of siRNA‐STK33. *P < 0.05, n = 3. HEI‐OC1, House Ear Institute‐Organ of Corti 1; NAC, N‐acetyl‐L‐cysteine; ROS, reactive oxygen species; STK33, serine/threonine kinase 33