Yonsei Med J.  2018 Nov;59(9):1131-1137. 10.3349/ymj.2018.59.9.1131.

Decreased Expression of TRPV4 Channels in HEI-OC1 Cells Induced by High Glucose Is Associated with Hearing Impairment

• Xing Y
• Ming J
• Liu T
• Zhang N
• Zha D
• Lin Y

• KMID: 2422499
• DOI: http://doi.org/10.3349/ymj.2018.59.9.1131

Abstract

PURPOSE
Previous reports have shown that hyperglycemia-induced inhibition of transient receptor potential vanilloid sub type 4 (TRPV4), a transient receptor potential ion channel, affects the severity of hearing impairment (HI). In this study, we explored the role of TRPV4 in HI using HEI-OC1 cells exposed to high glucose (HG).
MATERIALS AND METHODS
HEI-OC1 cells were cultured in a HG environment (25 mM D-glucose) for 48 hours, and qRT-PCR and Western blotting were used to analyze the expression of TRPV4 at the mRNA and protein level. TRPV4 agonist (GSK1016790A) or antagonist (HC-067047) in cultured HEI-OC1 cells was used to obtain abnormal TRPV4 expression. Functional TRPV4 activity was assessed in cultured HEI-OC1 cells using the MTT assay and a cell death detection ELISA.
RESULTS
TRPV4 agonists exerted protective effects against HG-induced HI, as evidenced by increased MTT levels and inhibition of apoptosis in HEI-OC1 cells. TRPV4 overexpression significantly increased protein levels of phosphorylated p38 mitogen-activated protein kinase (p-p38 MAPK), while TRPV4 antagonists had the opposite effect. Our results indicated that TRPV4 is a hyperglycemia-related factor that can inhibit cell proliferation and promote cell apoptosis by activating the MAPK signaling pathway in HEI-OC1 cells.
CONCLUSION
Our results show that the overexpression of TRPV4 can attenuate cell death in HEI-OC1 cells exposed to HG.

Keyword

TRPV4; high glucose; hearing impairment; HEI-OC1 cells

MeSH Terms

Apoptosis
Blotting, Western
Cell Death
Cell Proliferation
Enzyme-Linked Immunosorbent Assay
Glucose*
Hearing Loss*
Hearing*
Ion Channels
Protein Kinases
RNA, Messenger
Glucose
Ion Channels
Protein Kinases
RNA, Messenger

Figure

• 

  Fig. 1 HG reduces the expression of TRPV4 in HEI-OC1 cells. HEI-OC1 cells were cultured under euglycemic, hyperglycemia, and osmotic control conditions for 48 h, and qRT-PCR (A) and Western blot (B) were used to analyze the expression of TRPV4 mRNA and protein. Results are representative of three independent experiments. *Indicates differences from controls at p<0.05. TRPV4, transient receptor potential vanilloid sub type 4; HG, high glucose.

• 

  Fig. 2 The effect of reagents on the expression of TRPV4. (A) The relative expression of TRPV4 mRNA in high glucose cultured HEI-OC1 cells after treatment with HC-067047 (HC) or GSK1016790A (GSK), compared with the untreated control, was determined by qRT-PCR. (B) Western blotting was used to analyze the expression of TRPV4 protein in HEI-OC1 cells after treatment with HC-067047 or GSK1016790A. Data are representative of three experiments. *Indicates differences from euglycemic controls at p<0.05, †Indicates differences from euglycemic controls at p<0.01, ‡Indicates differences from untreated controls at p<0.05, respectively. TRPV4, transient receptor potential vanilloid sub type 4.

• 

  Fig. 3 TRPV4 directly affects proliferation and apoptosis in HEI-OC1 cells. (A) The effect of TRPV4 on cell proliferation of HEI-OC1 cells was detected using the MTT assay. (B) The effect of TRPV4 on cell apoptosis of HEI-OC1 cells was detected using the cell death detection ELISA-Plus apoptosis assay. Data are representative of three experiments. *indicates differences from euglycemic controls at p<0.05, †Indicates differences from euglycemic controls at p<0.01, ‡Indicates differences from untreated controls at p<0.05, respectively. TRPV4, transient receptor potential vanilloid sub type 4; HC, TRPV4 antagonist HC-067047; GSK, TRPV4 agonist GSK1016790A.

• 

  Fig. 4 Overexpression of TRPV4 triggered activation of the MAPK signaling pathway. (A) After pretreatment with the TRPV4 agonist GSK1016790A (GSK) or the TRPV4 antagonist HC067047 (HC), the p38 concentration in the culture medium of HEI-OC1 cells was assessed by ELISA. (B) The protein expression of p38 was analyzed by Western blotting. Data represent means±SD from three independent experiments. *Indicates differences from euglycemic controls at p<0.05, †Indicates differences from euglycemic controls at p<0.01, ‡Indicates differences from untreated controls at p<0.05, respectively. TRPV4, transient receptor potential vanilloid sub type 4; MSPK, mitogen-activated protein kinase.

• 

  Fig. 5 TRPV4 regulates cochlear hair cell development by targeting MAPK signaling pathways. After pretreatment with the TRPV4 agonist GSK1016790A (GSK) or a combination of GSK1016790A and the p38 inhibitor SB203580 (GSK+SB), the effect of TRPV4 on cell proliferation of HEI-OC1 cells was detected using the MTT assay, and the effect of TRPV4 on cell apoptosis of HEI-OC1 cells was detected using the cell death detection ELISA-Plus apoptosis assay. (A) Inhibition of MAPK signaling rescued cell proliferation induced by TRPV4 in HEI-OC1 cells. (B) Disruption of MAPK signaling promoted cell apoptosis induced by TRPV4 in HEI-OC1 cells. Data are represented as means±SD from three independent experiments. *Indicates differences from euglycemic controls at p<0.05, †Indicates differences from euglycemic controls at p<0.01, ‡Indicates differences from untreated controls at p<0.05, respectively. TRPV4, transient receptor potential vanilloid sub type 4; MSPK, mitogenactivated protein kinase.

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