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Hepatocyte growth factor mimetic protects lateral line hair cells from aminoglycoside exposure.

Uribe PM, Kawas LH, Harding JW, Coffin AB - Front Cell Neurosci (2015)

Bottom Line: We found that a Dihexa concentration of 1 μM confers optimal protection from acute treatment with either ototoxin.Pretreatment with Dihexa does not affect the amount of fluorescently tagged gentamicin that enters hair cells, indicating that Dihexa's protection is likely mediated by intracellular events and not by inhibiting aminoglycoside entry.Our data suggest that Dihexa confers protection of hair cells through an HGF-mediated mechanism and that Dihexa holds clinical potential for mitigating chemical ototoxicity.

View Article: PubMed Central - PubMed

Affiliation: Department of Integrative Physiology and Neuroscience, Washington State University Pullman, WA, USA.

ABSTRACT
Loss of sensory hair cells from exposure to certain licit drugs (e.g., aminoglycoside antibiotics, platinum-based chemotherapy agents) can result in permanent hearing loss. Here we ask if allosteric activation of the hepatocyte growth factor (HGF) cascade via Dihexa, a small molecule drug candidate, can protect hair cells from aminoglycoside toxicity. Unlike native HGF, Dihexa is chemically stable and blood-brain barrier permeable. As a synthetic HGF mimetic, it forms a functional ligand by dimerizing with endogenous HGF to activate the HGF receptor and downstream signaling cascades. To evaluate Dihexa as a potential hair cell protectant, we used the larval zebrafish lateral line, which possesses hair cells that are homologous to mammalian inner ear hair cells and show similar responses to toxins. A dose-response relationship for Dihexa protection was established using two ototoxins, neomycin and gentamicin. We found that a Dihexa concentration of 1 μM confers optimal protection from acute treatment with either ototoxin. Pretreatment with Dihexa does not affect the amount of fluorescently tagged gentamicin that enters hair cells, indicating that Dihexa's protection is likely mediated by intracellular events and not by inhibiting aminoglycoside entry. Dihexa-mediated protection is attenuated by co-treatment with the HGF antagonist 6-AH, further evidence that HGF activation is a component of the observed protection. Additionally, Dihexa's robust protection is partially attenuated by co-treatment with inhibitors of the downstream HGF targets Akt, TOR and MEK. Addition of an amino group to the N-terminal of Dihexa also attenuates the protective response, suggesting that even small substitutions greatly alter the specificity of Dihexa for its target. Our data suggest that Dihexa confers protection of hair cells through an HGF-mediated mechanism and that Dihexa holds clinical potential for mitigating chemical ototoxicity.

No MeSH data available.


Related in: MedlinePlus

Dihexa-mediated protection is inhibited by MEK, Akt, and TOR inhibitors. (A) Co-treatment with 1 μM MEK or Akt inhibitors (UO126 and Akt Inhibitor VIII, respectively) partially attenuates Dihexa-dependent protection (Two-way ANOVA; Dihexa: F(3,144) = 37.13 p < 0.001). When comparing the 1 μM Dihexa treatment vs. 1 μM Dihexa plus 1 μM UO126 p-values are as follows: 50 μM neomycin (p < 0.05), 100 μM neomycin (p < 0.05), 200 μM neomycin (p < 0.001), and 400 μM neomycin (p < 0.05). When comparing the 1 μM Dihexa treatment vs. 1 μM Dihexa plus 1 μM Akt inhibitor VIII p-values are as follows: 50 μM neomycin (p > 0.05), 100 μM neomycin (p < 0.05), 200 μM neomycin (p < 0.001), 400 μM neomycin (p > 0.05). (B) Co-treatment with 10 μM of the TOR inhibitor, rapamycin, also partially attenuates Dihexa-mediated protection from neomycin (Two-way ANOVA; Dihexa: F(3,120) = 70.41 p < 0.001). When comparing the 1 μM Dihexa treatment vs. 1 μM Dihexa plus 10 μM rapamycin p-values were as follows: 50 μM neomycin (p < 0.001), 100 μM neomycin (p < 0.001), 200 and 400 μM neomycin (p > 0.05). N = 6–9 animals per treatment, error bars represent ± s.e.m.
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Figure 6: Dihexa-mediated protection is inhibited by MEK, Akt, and TOR inhibitors. (A) Co-treatment with 1 μM MEK or Akt inhibitors (UO126 and Akt Inhibitor VIII, respectively) partially attenuates Dihexa-dependent protection (Two-way ANOVA; Dihexa: F(3,144) = 37.13 p < 0.001). When comparing the 1 μM Dihexa treatment vs. 1 μM Dihexa plus 1 μM UO126 p-values are as follows: 50 μM neomycin (p < 0.05), 100 μM neomycin (p < 0.05), 200 μM neomycin (p < 0.001), and 400 μM neomycin (p < 0.05). When comparing the 1 μM Dihexa treatment vs. 1 μM Dihexa plus 1 μM Akt inhibitor VIII p-values are as follows: 50 μM neomycin (p > 0.05), 100 μM neomycin (p < 0.05), 200 μM neomycin (p < 0.001), 400 μM neomycin (p > 0.05). (B) Co-treatment with 10 μM of the TOR inhibitor, rapamycin, also partially attenuates Dihexa-mediated protection from neomycin (Two-way ANOVA; Dihexa: F(3,120) = 70.41 p < 0.001). When comparing the 1 μM Dihexa treatment vs. 1 μM Dihexa plus 10 μM rapamycin p-values were as follows: 50 μM neomycin (p < 0.001), 100 μM neomycin (p < 0.001), 200 and 400 μM neomycin (p > 0.05). N = 6–9 animals per treatment, error bars represent ± s.e.m.

Mentions: Activation of the receptor tyrosine kinase c-Met leads to the recruitment of many signaling mediators and ultimately the activation of downstream signals, most notably Akt-TOR and Ras-ERK (Organ and Tsao, 2011). The MAPK and Akt inhibitors, UO126 and Akt inhibitor VIII respectively, were used to determine to what extent, if any, activation of their targets is required for Dihexa-mediated protection. Optimal concentrations of UO126 and Akt Inhibitor VIII (1 μM) were determined based on the highest concentration of each compound that did not shift the neomycin dose-response curve (data not shown). At neomycin concentrations of 100, 200, and 400 μM either inhibitor demonstrated partial attenuation of Dihexa otoprotection, indicating at least partial reliance on their associated signaling proteins for protective effects (Figure 6A). Interestingly, when both inhibitors are co-administered the result is similar to each administered independently, suggesting that other signaling proteins may also contribute to protection (data not shown). Phosphorylation of Akt leads to activation of TOR, a molecular sensor of metabolism and cellular homeostasis (Wullschleger et al., 2006). We used the TOR inhibitor rapamycin to determine the extent to which activation of TOR is required in Dihexa-mediated protection. 10 μM rapamycin alone did not affect the neomycin dose-response curve (Figure 6B). However, co-treatment with 1 μM Dihexa and 10 μM rapamycin showed significant attenuation of hair cell protection at 50 and 100 μM neomycin. From these results we hypothesize that Dihexa-mediated protection relies, at least in part, on the activation of multiple downstream targets of the HGF/c-Met system (Akt-TOR and Ras-ERK).


Hepatocyte growth factor mimetic protects lateral line hair cells from aminoglycoside exposure.

Uribe PM, Kawas LH, Harding JW, Coffin AB - Front Cell Neurosci (2015)

Dihexa-mediated protection is inhibited by MEK, Akt, and TOR inhibitors. (A) Co-treatment with 1 μM MEK or Akt inhibitors (UO126 and Akt Inhibitor VIII, respectively) partially attenuates Dihexa-dependent protection (Two-way ANOVA; Dihexa: F(3,144) = 37.13 p < 0.001). When comparing the 1 μM Dihexa treatment vs. 1 μM Dihexa plus 1 μM UO126 p-values are as follows: 50 μM neomycin (p < 0.05), 100 μM neomycin (p < 0.05), 200 μM neomycin (p < 0.001), and 400 μM neomycin (p < 0.05). When comparing the 1 μM Dihexa treatment vs. 1 μM Dihexa plus 1 μM Akt inhibitor VIII p-values are as follows: 50 μM neomycin (p > 0.05), 100 μM neomycin (p < 0.05), 200 μM neomycin (p < 0.001), 400 μM neomycin (p > 0.05). (B) Co-treatment with 10 μM of the TOR inhibitor, rapamycin, also partially attenuates Dihexa-mediated protection from neomycin (Two-way ANOVA; Dihexa: F(3,120) = 70.41 p < 0.001). When comparing the 1 μM Dihexa treatment vs. 1 μM Dihexa plus 10 μM rapamycin p-values were as follows: 50 μM neomycin (p < 0.001), 100 μM neomycin (p < 0.001), 200 and 400 μM neomycin (p > 0.05). N = 6–9 animals per treatment, error bars represent ± s.e.m.
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Figure 6: Dihexa-mediated protection is inhibited by MEK, Akt, and TOR inhibitors. (A) Co-treatment with 1 μM MEK or Akt inhibitors (UO126 and Akt Inhibitor VIII, respectively) partially attenuates Dihexa-dependent protection (Two-way ANOVA; Dihexa: F(3,144) = 37.13 p < 0.001). When comparing the 1 μM Dihexa treatment vs. 1 μM Dihexa plus 1 μM UO126 p-values are as follows: 50 μM neomycin (p < 0.05), 100 μM neomycin (p < 0.05), 200 μM neomycin (p < 0.001), and 400 μM neomycin (p < 0.05). When comparing the 1 μM Dihexa treatment vs. 1 μM Dihexa plus 1 μM Akt inhibitor VIII p-values are as follows: 50 μM neomycin (p > 0.05), 100 μM neomycin (p < 0.05), 200 μM neomycin (p < 0.001), 400 μM neomycin (p > 0.05). (B) Co-treatment with 10 μM of the TOR inhibitor, rapamycin, also partially attenuates Dihexa-mediated protection from neomycin (Two-way ANOVA; Dihexa: F(3,120) = 70.41 p < 0.001). When comparing the 1 μM Dihexa treatment vs. 1 μM Dihexa plus 10 μM rapamycin p-values were as follows: 50 μM neomycin (p < 0.001), 100 μM neomycin (p < 0.001), 200 and 400 μM neomycin (p > 0.05). N = 6–9 animals per treatment, error bars represent ± s.e.m.
Mentions: Activation of the receptor tyrosine kinase c-Met leads to the recruitment of many signaling mediators and ultimately the activation of downstream signals, most notably Akt-TOR and Ras-ERK (Organ and Tsao, 2011). The MAPK and Akt inhibitors, UO126 and Akt inhibitor VIII respectively, were used to determine to what extent, if any, activation of their targets is required for Dihexa-mediated protection. Optimal concentrations of UO126 and Akt Inhibitor VIII (1 μM) were determined based on the highest concentration of each compound that did not shift the neomycin dose-response curve (data not shown). At neomycin concentrations of 100, 200, and 400 μM either inhibitor demonstrated partial attenuation of Dihexa otoprotection, indicating at least partial reliance on their associated signaling proteins for protective effects (Figure 6A). Interestingly, when both inhibitors are co-administered the result is similar to each administered independently, suggesting that other signaling proteins may also contribute to protection (data not shown). Phosphorylation of Akt leads to activation of TOR, a molecular sensor of metabolism and cellular homeostasis (Wullschleger et al., 2006). We used the TOR inhibitor rapamycin to determine the extent to which activation of TOR is required in Dihexa-mediated protection. 10 μM rapamycin alone did not affect the neomycin dose-response curve (Figure 6B). However, co-treatment with 1 μM Dihexa and 10 μM rapamycin showed significant attenuation of hair cell protection at 50 and 100 μM neomycin. From these results we hypothesize that Dihexa-mediated protection relies, at least in part, on the activation of multiple downstream targets of the HGF/c-Met system (Akt-TOR and Ras-ERK).

Bottom Line: We found that a Dihexa concentration of 1 μM confers optimal protection from acute treatment with either ototoxin.Pretreatment with Dihexa does not affect the amount of fluorescently tagged gentamicin that enters hair cells, indicating that Dihexa's protection is likely mediated by intracellular events and not by inhibiting aminoglycoside entry.Our data suggest that Dihexa confers protection of hair cells through an HGF-mediated mechanism and that Dihexa holds clinical potential for mitigating chemical ototoxicity.

View Article: PubMed Central - PubMed

Affiliation: Department of Integrative Physiology and Neuroscience, Washington State University Pullman, WA, USA.

ABSTRACT
Loss of sensory hair cells from exposure to certain licit drugs (e.g., aminoglycoside antibiotics, platinum-based chemotherapy agents) can result in permanent hearing loss. Here we ask if allosteric activation of the hepatocyte growth factor (HGF) cascade via Dihexa, a small molecule drug candidate, can protect hair cells from aminoglycoside toxicity. Unlike native HGF, Dihexa is chemically stable and blood-brain barrier permeable. As a synthetic HGF mimetic, it forms a functional ligand by dimerizing with endogenous HGF to activate the HGF receptor and downstream signaling cascades. To evaluate Dihexa as a potential hair cell protectant, we used the larval zebrafish lateral line, which possesses hair cells that are homologous to mammalian inner ear hair cells and show similar responses to toxins. A dose-response relationship for Dihexa protection was established using two ototoxins, neomycin and gentamicin. We found that a Dihexa concentration of 1 μM confers optimal protection from acute treatment with either ototoxin. Pretreatment with Dihexa does not affect the amount of fluorescently tagged gentamicin that enters hair cells, indicating that Dihexa's protection is likely mediated by intracellular events and not by inhibiting aminoglycoside entry. Dihexa-mediated protection is attenuated by co-treatment with the HGF antagonist 6-AH, further evidence that HGF activation is a component of the observed protection. Additionally, Dihexa's robust protection is partially attenuated by co-treatment with inhibitors of the downstream HGF targets Akt, TOR and MEK. Addition of an amino group to the N-terminal of Dihexa also attenuates the protective response, suggesting that even small substitutions greatly alter the specificity of Dihexa for its target. Our data suggest that Dihexa confers protection of hair cells through an HGF-mediated mechanism and that Dihexa holds clinical potential for mitigating chemical ototoxicity.

No MeSH data available.


Related in: MedlinePlus