Tejbeer Kaur

Transtympanic Administration of Short Interfering (si)RNA for the NOX3 Isoform of NADPH Oxidase Protects Against Cisplatin-Induced Hearing Loss in the Rat

Cisplatin produces hearing loss in cancer patients. Reactive oxygen species (ROS) in the cochlea leads to lipid peroxidation, death of outer hair cells (OHCs), and hearing loss. The cochlea expresses a unique isoform of NADPH oxidase, NOX3, which serves as the primary source of ROS generation in the cochlea. Inhibition of NOX3 could offer a unique protective target against cisplatin ototoxicity. Here, we document that knockdown of NOX3 using short interfering (si) RNA abrogated cisplatin ototoxicity, as evidenced by protection of OHCs from damage and reduced threshold shifts in auditory brainstem responses (ABRs). Transtympanic NOX3 siRNA reduced the expression of NOX3 in OHCs, spiral ganglion (SG) cells, and stria vascularis (SV) in the rat. NOX3 siRNA also reduced the expression of transient receptor potential vanilloid 1 (TRPV1) channel and kidney injury molecule-1 (KIM-1), biomarkers of cochlear damage. Also, transtympanic NOX3 siRNA reduced the expression of Bax, abolished the decrease in expression of Bcl2, and reduced apoptosis induced by cisplatin in the cochlea. These data suggest that NOX3 regulates stress-related genes in the cochlea, such as TRPV1 and KIM-1, and initiates apoptosis in the cochlea. This appears to be the first study of the efficacy of transtympanic delivery of siRNA attenuating cisplatin ototoxicity.

Short interfering RNA against STAT1 attenuates cisplatin-induced ototoxicity in the rat by suppressing inflammation.

Cisplatin is widely used for treating various solid tumors. However, this drug produces dose-limiting ototoxicity and nephrotoxicity, which significantly reduce the quality of life of cancer patients. While nephrotoxicity could be alleviated by diuresis, there is currently no approved treatment for hearing loss. Previous studies show that the ROS and inflammation are major contributors to cisplatin-induced hearing loss. In this study, we show that ROS trigger the inflammatory process in the cochlea by activating signal transducer and activator of transcription-1 (STAT1). Activation of STAT1 activation was dependent on ROS generation through NOX3 NADPH oxidase, knockdown of which by siRNA reduced STAT1 activation. Moreover, STAT1 siRNA protected against activation of p53, reduced apoptosis, reduced damage to OHCs and preserved hearing in rats. STAT1 siRNA attenuated the increase in inflammatory mediators, such as TNF-α, inhibition of which protected cells from cisplatin-mediated apoptosis. Finally, we showed that trans-tympanic administration of etanercept, a TNF-α antagonist, protected against OHC damage and cisplatin-induced hearing loss. These studies suggest that controlling inflammation by inhibition of STAT1-dependent pathways in the cochlea could serve as an effective approach to treat cisplatin ototoxicity and improve the overall quality of life for cancer patients.

NOX3 NADPH Oxidase Couples Transient Receptor Potential Vanilloid 1 to Signal Transducer and Activator of Transcription 1-Mediated Inflammation and Hearing Loss

Transient receptor potential vanilloid 1 (TRPV1) is implicated in cisplatin ototoxicity. Activation of this channel by cisplatin increases reactive oxygen species generation, which contribute to loss of outer hair cells in the cochlea. Knockdown of TRPV1 by short interfering RNA protected against cisplatin ototoxicity. In this study, we examined the mechanism underlying TRPV1-mediated ototoxicity using cultured organ of Corti transformed cells (UB/OC-1) and rats. Trans-tympanic injections of capsaicin produced transient hearing loss within 24 h, which recovered by 72 h. In UB/OC-1 cells, capsaicin increased NOX3 NADPH oxidase activity and activation of signal transducer and activator of transcription 1 (STAT1). Intratympanic administration of capsaicin transiently increased STAT1 activity and expression of downstream proinflammatory molecules. Capsaicin produced a transient increase in CD14-positive inflammatory cells into the cochlea, which mimicked the temporal course of STAT1 activation but did not alter the expression of apoptotic genes or damage to outer hair cells. In addition, trans-tympanic administration of STAT1 short interfering RNA protected against capsaicin-induced hearing loss. These data suggest that activation of TRPV1 mediates temporary hearing loss by initiating an inflammatory process in the cochlea via activation of NOX3 and STAT1. Thus, these proteins represent reasonable targets for ameliorating hearing loss.

Fractalkine Signaling Regulates Macrophage Recruitment into the Cochlea and Promotes the Survival of Spiral Ganglion Neurons after Selective Hair Cell Lesion.

Macrophages are recruited into the cochlea in response to injury caused by acoustic trauma or ototoxicity, but the nature of the interaction between macrophages and the sensory structures of the inner ear remains unclear. The present study examined the role of fractalkine signaling in regulating the injury-evoked behavior of macrophages following the selective ablation of cochlear hair cells. We used a novel transgenic mouse model in which the human diphtheria toxin receptor (huDTR) is selectively expressed under the control of Pou4f3, a hair cell-specific transcription factor. Administration of diphtheria toxin (DT) to these mice resulted in nearly complete ablation of cochlear hair cells, with no evident pathology among supporting cells, spiral ganglion neurons, or cells of the cochlear lateral wall. Hair cell death led to an increase in macrophages associated with the sensory epithelium of the cochlea. Their numbers peaked at 14 days after DT and then declined at later survival times. Increased macrophages were also observed within the spiral ganglion, but their numbers remained elevated for (at least) 56 d after DT. To investigate the role of fractalkine signaling in macrophage recruitment, we crossed huDTR mice to a mouse line that lacks expression of the fractalkine receptor (CX3CR1). Disruption of fractalkine signaling reduced macrophage recruitment into both the sensory epithelium and spiral ganglion and also resulted in diminished survival of spiral ganglion neurons after hair cell death. Our results suggest a fractalkine-mediated interaction between macrophages and the neurons of the cochlea. SIGNIFICANCE STATEMENT It is known that damage to the inner ear leads to recruitment of inflammatory cells (macrophages), but the chemical signals that initiate this recruitment and the functions of macrophages in the damaged ear are unclear. Here we show that fractalkine signaling regulates macrophage recruitment into the cochlea and also promotes the survival of cochlear afferents after selective hair cell lesion. Because these afferent neurons carry sound information from the cochlea to the auditory brainstem, their survival is a key determinant of the success of cochlear prosthetics. Our data suggest that fractalkine signaling in the cochlea is neuroprotective, and reveal a previously uncharacterized interaction between cells of the cochlea and the innate immune system.

Adenosine A1 Receptor Protects Against Cisplatin Ototoxicity by Suppressing the NOX3/STAT1 Inflammatory Pathway in the Cochlea

Cisplatin is a commonly used antineoplastic agent that produces ototoxicity that is mediated in part by increasing levels of reactive oxygen species (ROS) via the NOX3 NADPH oxidase pathway in the cochlea. Recent studies implicate ROS generation in mediating inflammatory and apoptotic processes and hearing loss by activating signal transducer and activator of transcription (STAT1). In this study, we show that the adenosine A1 receptor (A1AR) protects against cisplatin ototoxicity by suppressing an inflammatory response initiated by ROS generation via NOX3 NADPH oxidase, leading to inhibition of STAT1. Trans-tympanic administration of the A1AR agonist R-phenylisopropyladenosine (R-PIA) inhibited cisplatin-induced ototoxicity, as measured by auditory brainstem responses and scanning electron microscopy in male Wistar rats. This was associated with reduced NOX3 expression, STAT1 activation, tumor necrosis factor-α (TNF-α) levels, and apoptosis in the cochlea. In vitro studies in UB/OC-1 cells, an organ of Corti immortalized cell line, showed that R-PIA reduced cisplatin-induced phosphorylation of STAT1 Ser727 (but not Tyr701) and STAT1 luciferase activity by suppressing the ERK1/2, p38, and JNK mitogen-activated protein kinase (MAPK) pathways. R-PIA also decreased the expression of STAT1 target genes, such as TNF-α, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) and reduced cisplatin-mediated apoptosis. These data suggest that the A1AR provides otoprotection by suppressing NOX3 and inflammation in the cochlea and could serve as an ideal target for otoprotective drug therapy. SIGNIFICANCE STATEMENT Cisplatin is a widely used chemotherapeutic agent for the treatment of solid tumors. Its use results in significant and permanent hearing loss, for which no US Food and Drug Administration-approved treatment is currently available. In this study, we targeted the cochlear adenosine A1 receptor (A1AR) by trans-tympanic injections of the agonist R-phenylisopropyladenosine (R-PIA) and showed that it reduced cisplatin-induced inflammation and apoptosis in the rat cochlea and preserved hearing. The mechanism of protection involves suppression of the NOX3 NADPH oxidase enzyme, a major target of cisplatin-induced reactive oxygen species (ROS) generation in the cochlea. ROS initiates an inflammatory and apoptotic cascade in the cochlea by activating STAT1 transcription factor, which is attenuated by R-PIA. Therefore, trans-tympanic delivery of A1AR agonists could effectively treat cisplatin ototoxicity.

Essential Role of NADPH Oxidase-Dependent Reactive Oxygen Species Generation in Regulating MicroRNA-21 Expression and Function in Prostate Cancer

AIMS Oncogenic microRNAs (miRs) promote tumor growth and invasiveness. One of these, miR-21, contributes to carcinogenesis in prostate and other cancers. In the present study, we tested the hypothesis that NADPH oxidase-dependent reactive oxygen species (ROS) regulate the expression and function of miR-21 and its target proteins, maspin and programmed cell death 4 (PDCD4), in prostate cancer cells. RESULTS The highly aggressive androgen receptor negative PC-3M-MM2 prostate cancer cells demonstrated high expression of miR-21 and p47(phox) (an essential subunit of NADPH oxidase). Using loss-of-function strategy, we showed that transfection of PC-3M-MM2 cells with anti-miR-21- and p47(phox) siRNA (si-p47(phox)) led to reduced expression of miR-21 with concurrent increase in maspin and PDCD4, and decreased the invasiveness of the cells. Tail-vein injections of anti-miR-21- and si-p47(phox)-transfected PC-3M-MM2 cells in severe combined immunodeficient mice reduced lung metastases. Clinical samples from patients with advanced prostate cancer expressed high levels of miR-21 and p47(phox), and low expression of maspin and PDCD4. Finally, ROS activated Akt in these cells, the inhibition of which reduced miR-21 expression. INNOVATION The levels of NADPH oxidase-derived ROS are high in prostate cancer cells, which have been shown to be involved in their growth and migration. This study demonstrates that ROS produced by this pathway is essential for the expression and function of an onco-miR, miR-21, in androgen receptor-negative prostate cancer cells. CONCLUSION These data demonstrate that miR-21 is an important target of ROS, which contributes to the highly invasive and metastatic phenotype of prostate cancer cells.

Role of β-arrestin1/ERK MAP kinase pathway in regulating adenosine A1 receptor desensitization and recovery

Exposure of cells to adenosine receptor (AR) agonists leads to receptor uncoupling from G proteins and downregulation of the A(1)AR. The receptor levels on the cell surface generally recover on withdrawal of the agonist, because of either translocation of the sequestered A(1)AR back to plasma membrane or de novo synthesis of A(1)AR. To examine the mechanism(s) underlying A(1)AR downregulation and recovery, we treated ductus deferens tumor (DDT(1) MF-2) cells with the agonist R-phenylisopropyladenosine (R-PIA) and showed a decrease in membrane A(1)AR levels by 24 h, which was associated with an unexpected 11-fold increase in A(1)AR mRNA. Acute exposure of these cells to R-PIA resulted in a rapid translocation of beta-arrestin1 to the plasma membrane. Knockdown of beta-arrestin1 by short interfering RNA (siRNA) blocked R-PIA-mediated downregulation of the A(1)AR, suppressed R-PIA-dependent ERK1/2 and activator protein-1 (AP-1) activity, and reduced the induction of A(1)AR mRNA. Withdrawal of the agonist after a 24-h exposure resulted in rapid recovery of plasma membrane A(1)AR. This was dependent on the de novo protein synthesis and on the activity of ERK1/2 but independent of beta-arrestin1 and nuclear factor-kappaB. Together, these data suggest that exposure to A(1)AR agonist stimulates ERK1/2 activity via beta-arrestin1, which subserves receptor uncoupling and downregulation, in addition to the induction of A(1)AR expression. We propose that such a pathway ensures both the termination of the agonist signal and recovery by priming the cell for rapid de novo synthesis of A(1)AR once the drug is terminated.

Selective Deletion of Cochlear Hair Cells Causes Rapid Age-Dependent Changes in Spiral Ganglion and Cochlear Nucleus Neurons

During nervous system development, critical periods are usually defined as early periods during which manipulations dramatically change neuronal structure or function, whereas the same manipulations in mature animals have little or no effect on the same property. Neurons in the ventral cochlear nucleus (CN) are dependent on excitatory afferent input for survival during a critical period of development. Cochlear removal in young mammals and birds results in rapid death of target neurons in the CN. Cochlear removal in older animals results in little or no neuron death. However, the extent to which hair-cell-specific afferent activity prevents neuronal death in the neonatal brain is unknown. We further explore this phenomenon using a new mouse model that allows temporal control of cochlear hair cell deletion. Hair cells express the human diphtheria toxin (DT) receptor behind the Pou4f3 promoter. Injections of DT resulted in nearly complete loss of organ of Corti hair cells within 1 week of injection regardless of the age of injection. Injection of DT did not influence surrounding supporting cells directly in the sensory epithelium or spiral ganglion neurons (SGNs). Loss of hair cells in neonates resulted in rapid and profound neuronal loss in the ventral CN, but not when hair cells were eliminated at a more mature age. In addition, normal survival of SGNs was dependent on hair cell integrity early in development and less so in mature animals. This defines a previously undocumented critical period for SGN survival.

Macrophage recruitment and epithelial repair following hair cell injury in the mouse utricle

The sensory organs of the inner ear possess resident populations of macrophages, but the function of those cells is poorly understood. In many tissues, macrophages participate in the removal of cellular debris after injury and can also promote tissue repair. The present study examined injury-evoked macrophage activity in the mouse utricle. Experiments used transgenic mice in which the gene for the human diphtheria toxin receptor (huDTR) was inserted under regulation of the Pou4f3 promoter. Hair cells in such mice can be selectively lesioned by systemic treatment with diphtheria toxin (DT). In order to visualize macrophages, Pou4f3–huDTR mice were crossed with a second transgenic line, in which one or both copies of the gene for the fractalkine receptor CX3CR1 were replaced with a gene for GFP. Such mice expressed GFP in all macrophages, and mice that were CX3CR1GFP/GFP lacked the necessary receptor for fractalkine signaling. Treatment with DT resulted in the death of ∼70% of utricular hair cells within 7 days, which was accompanied by increased numbers of macrophages within the utricular sensory epithelium. Many of these macrophages appeared to be actively engulfing hair cell debris, indicating that macrophages participate in the process of ‘corpse removal’ in the mammalian vestibular organs. However, we observed no apparent differences in injury-evoked macrophage numbers in the utricles of CX3CR1+/GFP mice vs. CX3CR1GFP/GFP mice, suggesting that fractalkine signaling is not necessary for macrophage recruitment in these sensory organs. Finally, we found that repair of sensory epithelia at short times after DT-induced hair cell lesions was mediated by relatively thin cables of F-actin. After 56 days recovery, however, all cell-cell junctions were characterized by very thick actin cables.

Epigallocatechin-3-gallate, a prototypic chemopreventative agent for protection against cisplatin-based ototoxicity

Cisplatin-induced ototoxicity is one of the major factors limiting cisplatin chemotherapy. Ototoxicity results from damage to outer hair cells (OHCs) and other regions of the cochlea. At the cellular level, cisplatin increases reactive oxygen species (ROS) leading to cochlear inflammation and apoptosis. Thus, ideal otoprotective drugs should target oxidative stress and inflammatory mechanisms without interfering with cisplatins chemotherapeutic efficacy. In this study, we show that epigallocatechin-3-gallate (EGCG) is a prototypic agent exhibiting these properties of an effect otoprotective agent. Rats administered oral EGCG demonstrate reduced cisplatin-induced hearing loss, reduced loss of OHCs in the basal region of the cochlea and reduced oxidative stress and apoptotic markers. EGCG also protected against the loss of ribbon synapses associated with inner hair cells and Na+/K+ ATPase α1 in the stria vascularis and spiral ligament. In vitro studies showed that EGCG reduced cisplatin-induced ROS generation and ERK1/2 and signal transducer and activator of transcription-1 (STAT1) activity, but preserved the activity of STAT3 and Bcl-xL. The increase in STAT3/STAT1 ratio appears critical for mediating its otoprotection. EGCG did not alter cisplatin-induced apoptosis of human-derived cancer cells or cisplatin antitumor efficacy in a xenograft tumor model in mice because of its inability to rescue the downregulation of STAT3 in these cells. These data suggest that EGCG is an ideal otoprotective agent for treating cisplatin-induced hearing loss without compromising its antitumor efficacy.