As summarized in , the comprehensive, collated data of MAP kinase expression delineated upregulation of the phospho-MEK1/phospho-ERK1/2/phospho-90 RSK cascade within the early phase of 0–24 h after the noise exposure, which coincided with a TTS. This process did not involve the temporal surge in the levels of total-MEK1, total-ERK1/2 and total-p90 RSK; hence, the upregulation of phospho-MEK1/phospho-ERK1/2/phospho-90 RSK was largely due to transient phosphorylation of the proteins and did not involve de novo synthesis of the proteins. Immunohistochemical data showed that the expression of phospho-p90 RSK occurred in the lateral wall (spiral ligament) and in the sensory and supporting cells of the cochlea at 3 h post-noise exposure, at the time of the peak surge of phospho-p90 RSK.
Summary of upregulation of MEK1/ERK1/2/p90 RSK, JNK/c-Jun and p38 MAPK in the cochlea after exposure to the intense noise.
Phospho-JNK and phospho c-Jun, as well as total c-Jun, also showed surges beginning as early as 3 h after the noise exposure. The localization of phospho-JNK was demonstrated in the lateral wall (the spiral ligament), in the sensory and supporting cells of the organ of Corti, and in the spiral neurons at 3 h. The upregulation of a downstream effector, phospho-c-Jun, was dependent on the de novo synthesis of c-Jun.
In contrast to the upregulation of the phospho-MEK1/phospho-ERK1/2/phospho-90 RSK cascade within 0–24 h, the levels of phospho-JNK and phospho-p38 MAPK also demonstrated significant increases at the late phase of 48 h post-noise exposure. Total-p38 MAPK showed a significant and corresponding increase at 48 h; therefore, the upregulation of phospho-p38 MAPK at 48 h involved de novo synthesis of the protein. Immunohistochemical results showed that the expression of phospho-JNK and phospho-p38 MAPK occurred in the spiral neurons of the cochlea at this time point. ABR threshold testing indicated partial recovery of the TTS during the preceding 12–24 h and significant PTS at 14 days post-noise exposure.
Upstream from the MEK1/ERK1/2/p90 RSK signaling pathway, growth factor binding to receptor protein tyrosine kinases in the cell membrane triggers activation of a G-protein, Ras, by exchange of its guanosine diphosphate (GDP) to guanosine triphosphate (GTP) 
. Activated Ras phosphorylates Raf, and in turn, activated Raf phosphorylates MEK1, leading to the sequential phosphorylation of ERK 1/2 and p90 RSK 
. p90 RSK is a major downstream effector of the MEK1/ERK1/2 signaling pathway and mediates biological processes such as cell survival, protein synthesis, cell-proliferation, cell growth, and migration through the regulation of transcription factors, c-Fos, CREB (cAMP response element binding protein) and NF-kappa B (nuclear factor-kappa B) 
. p90 RSK activates CREB kinase, which in turn phosphorylates and activates CREB. CREB initiates transcription of survival-promoting genes, including Bcl-2 (B-cell lymphoma 2), Bcl-xL (B-cell lymphoma-extra large) and Mcl1 (myeloid cell leukemia sequence 1), and promotes survival of cultured primary neurons in vitro
. The physiological roles of this signaling pathway suggest that the upregulation of phospho-MEK1/phospho-ERK 1/2/phospho-p90 RSK with the peak surges during 3–6 h, as revealed in the present data, is a protective response to the noise trauma involving the sensory epithelium and the spiral ligament of the cochlea.
Activation of JNK by the upstream kinases MKK4 (MAP kinase kinase 4)/MKK7 
can be induced by acoustic trauma 
, ototoxic drugs, and electrode insertion 
to the inner ear. Phosphorylated JNK binds and phosphorylates downstream effectors such as a transcription factor, c-Jun, ATF2 (activating transcription factor 2), Elk1 (E-twenty six-like transcription factor 1) and p53 (tumor protein 53) 
. It is reported that these effectors mediate apoptosis in the sensory epithelium and in the lateral wall of the cochlea and neurons 
. In the present experiments, phospho-JNK was upregulated in the sensory epithelium, in the lateral wall (the spiral ligament), and in the spiral neurons as early as 3 h post-noise trauma, which is consistent with the previous reports suggesting that the peak expression of phospho-JNK occurred at 0–12 h after the noise exposure that can induce PTS 
. The present data also provides a novel finding of the second, late surge of phospho-JNK in the spiral neurons at 48 h post-noise trauma.
AM111 peptide (which is an equivalent term to D-JNKI-1 peptide) is a cell-permeable compound that inhibits phospho-JNK activity. Intratympanically applied AM111 onto the round window protects hearing from acoustic trauma and prevents ischemic damage to the cochlea 
. AM111 is currently under investigation as a potential therapeutic reagent to rescue acute sensorineural hearing loss (http://www.aurismedical.com/
, 2013). The present data of the time course of phospho-JNK expression provides significant insight into the design of appropriate therapeutic protocols using the JNK inhibitor.
Phosphorylation of p38 MAPK by the upstream regulators MMK3/MMK6 
can be induced by acoustic trauma and aminoglycoside to the cochlea 
. Activated p38 MAPK phosphorylates a number of substrates such as MSK1(mitogen- and stress-activated protein kinase 1)/MSK2 and MNK1(MAP kinase interacting serine/threonine kinase 1) 
. The p38 MAPK signaling pathway shares the downstream effectors, including ATF2 and Elk1, with the JNK pathway and is also involved in apoptosis 
The p38 MAPK inhibitors SB202190 and SB203580 dose-dependently decreased hair cell loss and protected hearing after acoustic overexposure of the mouse cochlea 
. In previous animal studies, the expression of phospho-p38 MAPK was observed in the sensory epithelium at 2–4 h post-noise exposure and the p38 MAPK inhibitors were injected into the mice immediately before the noise exposure 
. These studies have not addressed the cochlear expression of phospho-p38 MAPK for a longer time period than 4 h after noise exposure. The present data for the first time demonstrated the late upregulation of phospho-p38 MAPK in the spiral neurons at 48 h, which is dependent on de novo
synthesis of the p38 MAPK protein. The phospho-p38MAPK level showed a tendency to increase at 3 h, but was not significant at this time point. We assume that in the present experiments, the sensitivity for detecting phospho-p38 MAPK did not reach the level of significance to show its upregulation in the cochlea at 3 h after noise exposure. The level of total-p38 MAPK demonstrated significant, early upregulation with a peak at 3 h, followed by 6 h and 12 h, which is consistent with previous reports 
and suggestive of the de novo
The present data demonstrated activation of the MEK1/ERK1/2/p90 RSK signaling pathway in the spiral ligament and in the sensory and supporting cells of the organ of Corti, with the peaks occurring at 3–6 h and coinciding with the observed TTS after noise exposure. This process is independent of de novo protein synthesis and thought to be a protective response to noise trauma. It is generally accepted that JNK and p38 MAPK act as stress-induced kinases involved in apoptosis. In addition to the early upregulation, with the peak at 3 h after the noise exposure, the present data demonstrated the late upregulations of JNK and p38 MAPK pathways in the spiral neurons at 48 h after the noise trauma. The p38 MAPK activation is dependent on de novo protein synthesis. The comprehensive analysis of MAP kinase expression will be critical to understanding the molecular mechanism of NIHL and for developing therapeutic models for acute SNHL.