The genes for heat shock proteins (Hsps) can be upregulated in response to cellular trauma, resulting in enhanced cell survival and protection. Hsp32, also known as heme oxygenase 1, catalyzes the ...degradation of heme to produce carbon monoxide and bilirubin, which play a variety of cytoprotective functions at physiological concentrations, and iron, which is rapidly sequestered by the iron-binding protein ferritin. In the present study we examined the expression and localization of Hsp32 in the rat cochlea after heat shock using semi-quantitative reverse transcription polymerase chain reaction (RT-PCR), Western blot, and immunocytochemistry. Low levels of constitutive Hsp32 expression were observed in the normal rat cochlea by RT-PCR and Western blot.
Hsp32 mRNA (messenger RNA) was present at higher levels in a subfraction containing sensorineural epithelium and lateral wall than in a subfraction containing modiolus. Western blot revealed that Hsp32 protein levels increase in the rat cochlea following heat shock. Immunocytochemistry showed scattered staining of outer hair cells in the organ of Corti of normal untreated rats. Following heat shock Hsp32 is upregulated in outer hair cells and the cells of the stria vascularis. These results suggest a potential role for Hsp32 as a component of the oxidative stress response pathway in the rat cochlea.
Activation of heat shock factors (Hsfs) is one of the potential mechanisms for regulating the transcription of the heat shock proteins (Hsps) and certain other stress-responsive genes. Reverse ...transcription polymerase chain reaction (RT-PCR), Western blot and immunocytochemistry were used to examine the expression and localization of Hsf1, the stress-responsive member of the Hsf family, in the rat and mouse cochlea. Cerebellum was used as a positive control. Semi-quantitative RT-PCR of cochlear RNA revealed that
Hsf1 was more highly expressed in a subfraction containing sensorineural epithelium and lateral wall than in a subfraction containing modiolus, with the α splice form predominant over the β in both subfractions. Immunocytochemistry showed selective staining in the rodent cochlea. Hsf1 immunostaining was found in the nuclei of inner and outer hair cells in the organ of Corti, spiral ganglion cells in the modiolus, and cells in the marginal and intermediate layers of the stria vascularis. This is largely consistent with where Hsp70 induction is reported. Hsf1 activation following heat shock was examined by Western blot. Hyperthermia resulted in stress-induced Hsf1 hyperphosphorylation in cochlea as well as cerebellum. This hyperphosphorylation as well as the correlation of its localization with Hsp70 induction supports a role for Hsf1 in the cochlear stress response.
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•Slc44a2 is required for strong adhesion of cultured lung mesenchymal cells.•Absent or reduced Slc44a2 diminishes CD9 membrane foci in mesenchymal cells.•Loss of Slc44a2 causes tight ...cytoplasmic clustering of CD81 in mesenchymal cells.•Loss of Slc44a2 causes redistribution of N-cadherin in mesenchymal cells.•Mesenchymal cells with absent or reduced Slc44a2 proliferate more rapidly.
Slc44a2 is reported to interact with tetraspanins CD9 and CD81. To investigate how Slc44a2 affects adhesion protein expression, cells from wild-type (WT) Slc44a2+/+, heterozygous (HET) Slc44a2+/-, and knockout (KO) Slc44a2-/- mice were cultured from lung tissue. The cultured cells expressed vimentin, N-cadherin, p120 catenin, beta-catenin, actin, CD9, and CD81, but not E-cadherin. Vimentin expression with lack of E-cadherin indicated that the cultured cells were of mesenchymal origin. Slc44a2 KO cells and HET cells demonstrated lower adherence and faster proliferation than the WT cells. All three groups displayed dramatically altered intracellular distribution of N-cadherin, CD9, and CD81. The CD9 membrane foci observed in WT cell membranes were less frequent and diminished in size in HET cells and KO cells. N-cadherin was dispersed throughout both the cytoplasm and membrane in WT cells, with similar yet weaker distribution in HET cells; however, in KO cells, N-cadherin was densely aggregated in the perinuclear cytoplasm. CD81 had a distribution pattern in WT, HET, and KO cells similar to that of N-cadherin with dense cytoplasmic clusters in the cells. KO cells also exhibited reduced filamentous actin as compared to WT cells. These results suggest that Slc44a2 is necessary for proper cellular localization of adhesion proteins and growth regulation that may be related to altered adhesion signals.
In mammals, exposure to intense noise produces a permanent hearing loss called permanent threshold shift (PTS), whereas a moderate noise produces only a temporary threshold shift (TTS). Little is ...known about the molecular responses to such high intensity noise exposures. In this study we used gene arrays to examine the early response to acoustic overstimulation in the rat cochlea. We compared cochlear RNA from noise-exposed rats with RNA from unexposed controls. The intense PTS noise induced several immediate early genes encoding both transcription factors (c-FOS, EGR1, NUR77/TR3) and cytokines (PC3/BTG2, LIF and IP10). In contrast, the TTS noise down-regulated the gene for growth hormone. The response of these genes to different noise intensities was examined by quantitative RT-PCR 2.5 h after the 90-min noise exposure. For most genes, the extent of induction correlates with the intensity of the noise exposure. Three proteins (EGR1, NUR77/TR3, and IP10) were detected in many regions of the unexposed cochlea. After exposure to 120 dB noise, these proteins were present at higher levels or showed extended expression in additional regions of the cochlea. LIF was undetectable in the cochlea of unexposed rats, but could be seen in the organ of Corti and spiral ganglion neurons following noise. NUR77/TR3 was a nuclear protein before noise, but following noise translocated to the cytoplasm. These studies provide new insights into the molecular response to noise overstimulation in the mammalian cochlea.
SLC44A2 (
s
o
l
ute
c
arrier
44a2
), also known as CTL2 (
c
holine
t
ransporter-
l
ike protein
2
), is expressed in many supporting cell types in the cochlea and is implicated in hair cell survival ...and antibody-induced hearing loss. In mice with the mixed C57BL/6-129 background, homozygous deletion of
Slc44a2
exons 3–10 (
Slc44a2
Δ/Δ
) resulted in high-frequency hearing loss and hair cell death. To reduce effects associated with
a
ge-
r
elated
h
earing
l
oss (ARHL) in these strains, mice carrying the
Slc44a2
Δ
allele were backcrossed to the ARHL-resistant FVB/NJ strain and evaluated after backcross seven (N7) (99 % FVB).
Slc44a2
Δ/Δ
mice produced abnormally spliced
Slc44a2
transcripts that contain a frameshift and premature stop codons. Neither full-length SLC44A2 nor a putative truncated protein could be detected in
Slc44a2
Δ/Δ
mice, suggesting a likely null allele. Auditory brain stem responses (ABRs) of mice carrying the
Slc44a2
Δ
allele on an FVB/NJ genetic background were tested longitudinally between the ages of 2 and 10 months. By 6 months of age,
Slc44a2
Δ/Δ
mice exhibited hearing loss at 32 kHz, but at 12 and 24 kHz had sound thresholds similar to those of wild-type
Slc44a2
+/+
and heterozygous
+/Slc44a2
Δ
mice. After 6 months of age,
Slc44a2
Δ/Δ
mutants exhibited progressive hearing loss at all frequencies and
+/Slc44a2
Δ
mice exhibited moderate threshold elevations at high frequency. Histologic evaluation of
Slc44a2
Δ/Δ
mice revealed extensive hair cell and spiral ganglion cell loss, especially in the basal turn of the cochlea. We conclude that
Slc44a2
function is required for long-term hair cell survival and maintenance of hearing.
Cyclodextrins are sugar compounds that are increasingly finding medicinal uses due to their ability to complex with hydrophobic molecules. One cyclodextrin in particular, ...2-hydroxypropyl-beta-cyclodextrin (HPbetaCD), is used as a carrier to solubilize lipophilic drugs and is itself being considered as a therapeutic agent for treatment of Niemann-Pick Type C disease, due to its ability to mobilize cholesterol. Results from toxicological studies suggest that HPbetaCD is generally safe, but a recent study has found that it causes hearing loss in cats. Whether the hearing loss occurred via death of cochlear hair cells, rendering it permanent, was unexplored. In the present study, we examined peripheral auditory function and cochlear histology in mice after subcutaneous injection of HPbetaCD to test for hearing loss and correlate any observed auditory deficits with histological findings. On average, auditory brainstem response thresholds were elevated at 4, 16, and 32 kHz in mice one week after treatment with 8,000 mg/kg. In severely affected mice all outer hair cells were missing in the basal half of the cochlea. In many cases, surviving hair cells in the cochlear apex exhibited abnormal punctate distribution of the motor protein prestin, suggesting long term changes to membrane composition and integrity. Mice given a lower dose of 4,000 mg/kg exhibited hearing loss only after repeated doses, but these threshold shifts were temporary. Therefore, cyclodextrin-induced hearing loss was complex, involving cell death and other more subtle influences on cochlear physiology.
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