Analysis of Cochlear Protein Profiles of Wistar, Sprague-Dawley, and Fischer 344 Rats with Normal Hearing Function Samson Jamesdaniel, Dalian Ding, Mohammad Habiby Kermany, Haiyan Jiang, Richard Salvi, and Donald Coling* Center for Hearing and Deafness, University at Buffalo, The State University of New York, Buffalo, NY 14214 Received March 6, 2009
Differences in the expression of cochlear proteins are likely to affect the susceptibility of different animal models to specific types of auditory pathology. However, little is currently known about proteins that are abundantly expressed in inner ear. Identification of these proteins may facilitate the search for biomarkers of susceptibility and intervention targets. To begin to address this issue, we analyzed cochlear protein profiles of three strains of rats, Wistar, Sprague-Dawley, and Fischer 344, using a broad spectrum antibody microarray. Normal hearing function of the animals was ascertained using distortion product otoacoustic emissions (DPOAE). Of 725 proteins screened in whole cochlea, more than 80% were detected in all three strains. However, there were striking differences in the levels at which they occur. Among 213 proteins expressed at levels g2 fold of actin, only 7.5% were detected at these levels in all three strains. Myosin light chain kinase (MLCK) was immunolocalized in cuticular plate of outer hair cells (OHC) while mitogen activated protein (MAP) kinase-extracellular-signal regulated kinase1/2 (ERK1/2) was detected as foci in OHC, pillar cells, strial marginal cells, and fibroblasts of spiral ligament. A review of literature indicated that the expression of 7 (44%) of these 16 proteins were detected for the first time in the inner ear, although there were implications of the presence of some of these proteins. One of these abundant, but unstudied, proteins, MAP kinase activated protein kinase2 (MAPKAPK2), shows strong immunolabeling in pillar cells and inner hair cells (IHC). There was moderate MAPKAPK2 labeling in OHC, supporting cells, neurons, and marginal, intermediate, and basal cells. The current study provides the first, large cochlear protein profile of multiple rat strains. The diversity in expression of abundant proteins in these strains may contribute to differences in susceptibility of these strains to aging, noise, or ototoxic drugs. Keywords: proteomics • antibody microarray • cochlea • hearing • myosin light chain kinase
Introduction Different strains of rats are used as experimental models for studying various types of hearing loss. Out of the 39 different rat strains that are commercially available for scientific research, Wistar, Sprague-Dawley and Fischer 344 are commonly used in hearing research. Wistar rats have been widely used for evaluating drug induced hearing loss.1-3 SpragueDawley have been used to study both acoustic trauma4,5 and drug induced hearing loss.6,7 Fisher 344 has been the preferred model for age related hearing loss.8,9 Inherent differences between strains may make certain strains well suited to particular experimental conditions. Genomic and proteomic characteristics are crucial factors in establishing this specificity or disparity, which implies that gene and protein profiles can be vital resources for deciding appropriate animal models for different studies. * To whom correspondence should be addressed. Donald Coling, 137 Cary Hall, Center for Hearing and Deafness, University at Buffalo, The State University of New York, Buffalo, NY 14214. E-mail:
[email protected]. Phone: 716 829 5299. Fax: 716 829 2980.
3520 Journal of Proteome Research 2009, 8, 3520–3528 Published on Web 05/11/2009
Gene profiling has contributed immensely to understanding the molecular mechanisms involved in hearing function and pathology. For example, several genes have been implicated in hereditary hearing loss.10 With current advances in exon profiling of alternate splice isoforms, it is possible to gain insights about specialized functions of encoded proteins.11 There are, however, limitations to the type of information that can be gathered from gene profiling. Some genes, for example, may not be essential to the physiological processes in which they participate because their function may be compensated by other genes. It has been predicted that two-thirds of the approximately 20 000 genes in a cell are expressed at low levels.12 Although many of these may have vital functional roles, many are still poorly understood. To surmount these limitations or complement genetic studies, research can focus on proteins. Unlike gene profiles, translational and post-translational regulation can be studied in protein profiles. Proteins control and define the functional state of the cell and hence give further credence to the interpretation and application of genetic findings. 10.1021/pr900222c CCC: $40.75
2009 American Chemical Society
research articles
Analysis of Cochlear Protein Profiles of Rats So far, very few reports are available on the protein profile of the inner ear and the number of proteins identified in these studies is limited.13-18 These studies used two-dimensional electrophoresis methods or tandem mass spectrometry to explore the protein profile in various inner ear tissues. Recently, we demonstrated the application of antibody microarrays for studying the rat cochlear protein profile in cisplatin ototoxicity.19 The commercial availability of the antibodies that have been selected for developing the array and their functional importance facilitates the rapid translation of antibody microarrays into useful biological studies aimed at determining the protein location and abundance. In this study, we investigated the inner ear protein profile of three different rat strains with normal hearing function using a broad spectrum antibody microarray. Selection of the strain and sex of rats were based on animal models routinely used in our laboratory and not for the purpose of investigating effects of gender. We identified 590 proteins commonly expressed among the three strains and 213 proteins with higher abundance in specific strains. We believe the analysis may be useful for selection of an appropriate model and perhaps for the determination of strain-specific susceptibility to otopathologies.
(Molecular Devices Corporation, Sunnyvale, CA). Spots that showed a uniform distribution with a positive signal-to-noise ratio were taken for analysis. Immunocytochemistry. As described in detail in our previous report,21 the cochlea was removed from the temporal bone after cardiac perfusion with PBS followed by 10% buffered formalin and immediately fixed in 10% buffered formalin for 3 h. Cochlear tissue was dissected out and permeabilized in 1% (v/v) Triton X-100 for 30 min. The samples were blocked using 5% v/v Goat serum with 2% w/v BSA in PBS for 1 h and incubated at 4 °C overnight in primary antibody (ERK1/2, MAPKAPK2 or MLCK). Then the tissue were washed with PBS and incubated in antirabbit or antimouse secondary antibody conjugated to Cy3 or rhodamine (Sigma) for 1 h at room temperature. Phalloidin was used for labeling f-actin and TOPRO-3 for nuclei. ProLong Gold antifade reagent (P36934, Invitrogen Molecular Probes) was used to mount the specimen. Carl Zeiss Laser Scanning Systems LSM 510 was used to examine the immunofluorescence and software from Zeiss LSM Image Examiner (version 4,0,0,91, Carl Zeiss GmbH Jena) was used to analyze the images.
Experimental Procedures
Results
Animals. Male Wistar, female Sprague-Dawley and female Fischer 344 rats, 3-4 months old, were obtained from Charles River Laboratories (Wilmington, MA). We chose the strain and sex of rat models to match those routinely used in our laboratory.19,20 The animals, housed in the Lab Animal Facility of the University at Buffalo, with free access to food and water, were maintained in a temperature controlled room with 12 h light/dark cycle. The experimental protocol was approved by the Institutional Animal Care and Use Committee of University at Buffalo and all efforts were made to avoid or minimize stress to animals during the experimental procedure. DPOAE Measurement. DPOAEs at 2f1-f2 were measured in all the animals after anesthetizing them with isoflurane (4% induction, 1.5% maintenance with 1 L/min O2). HIS-3738 high frequency transducers (Intelligent Hearing System, Miami, FL) were used to deliver the two primary tones at an f2/f1 ratio of 1.2, with sound levels at L2 ) L1 - 10 dB. Ear canal sound pressure was averaged over 32 sweeps from the output of a ER10B+ probe microphone (Etymotics Research, Inc., Elk Grove Village, IL); the data were sampled at 40 kHz over a period of 204 ms using a Smart Distortion Product Otoacoustic Emission System (Intelligent Hearing Systems, Miami, FL, version 4.53). The noise floor was measured in a 24 Hz band surrounding 2f1-f2. Antibody Microarray. Rats were euthanized by CO2 inspiration and decapitation. As described in detail in our previous paper,19 the whole cochlea was dissected out from 2 animals in each of the strains. The cochlear proteins were extracted by homogenizing the cochlear tissue, which included the bony shell, lateral wall, basilar membrane and modiolus, with 150 µL lysis buffer supplemented with protease and phosphatase inhibitors (XPRESS Profiler725 kit, Sigma-Aldrich Corporation, St. Louis, MO). The Bradford assay was used to measure the protein concentration. Cy3 and Cy5 dyes (GE Healthcare, Buckinghamshire, UK) were used to label the cochlear proteins following the manufacturer’s protocol. The microarray slides were then incubated with the labeled proteins for 30 min and the fluorescent signal intensities of the antibodies were measured using a GenePix Professional 4200A Microarray Scanner
Audiometric Testing. All of the animals used in this study had normal cochlear function as assessed by otoacoustic emissions irrespective of strain (Figure 1). Otoacoustic emission amplitudes were in the range of 25-35 dB SPL in all three rat strains for a 70 dB (L1) stimuli at both 8 and 16 kHz (f2). The pattern in the distribution of hearing function, as reflected by the DPOAE for stimuli at sound intensities between 25 to 70 dB SPL at 8 and 16 kHz were reasonably similar across these strains. The maximum difference observed between Wistar, Sprague-Dawley and Fischer 344 rats at any given stimulus point was 1 fold) and 187/ 267/303 were expressed at lower levels (2 Fold of Actin) in All Three Rat Strains
a
fold expression (relative to actin) gene symbol
protein
Smad4 Extracellular-signal Regulated Kinase1/2 Myosin Light Chain Kinase
Smad4 Mapk3/1 Mylk
Substance P Receptor Proliferating Cell Protein Ki67 MAP Kinase Activated Protein Kinase2 Estrogen receptor1b
Tacr1 Mki67 Mapkapk2 Esr1
Ubiquitin
Ubc, Ubb
Phospholipase A2 group v
Pla2g5
Inner Centromere Protein Tryptophane Hydroxylase
Incenp Tph2,Tph1
Peripherin
Prph
Son Of Sevenless1 Protein arginine methyl transferase6
Sos1 Prmt6
Caspase11, caspase 4 gene product Neurofilament 200
Casp4 Nefh
function
Wistar
SpragueDawley
Fischer 344
mean
Transduces signal from TGF-beta Kinase involved in intracellular signaling Phosphorylates myosin regulatory light chains to facilitate myosin interaction with actin filaments to produce contractile activity Mediates neuropeptide signaling Associated with cellular proliferation Phosphorylates small heat shock proteins Acts as a transcriptional activator when bound to estrogen Signals intracellular translocation (e.g., to proteasome) Catalyzes calcium ion dependent hydrolysis of phospholipids Centromere-interacting passenger proteins Rate limiting enzyme in seratonin biosynthesis Contributes to the formation of intermediate filament networks Guanine nucleotide exchange factor S-adenosylmethionine-dependent methyltransferase Activator of caspases 1 and 3 Intermediate filament protein found primarily in neurons
5.2 3.3 4.7
4.5 10.1 3.4
7.3 2.4 6.9
5.7 5.3 5.0
2.3 2.2 5.6 2.1
10.2 5.4 5.7 9.7
2.4 6.7 3.1 2.5
5.0 4.8 4.8 4.8
2.3
5.8
5.2
4.4
2.4
5.1
4.4
4.0
2.0 2.5
6.1 2.9
2.4 4.8
3.5 3.4
2.0
4.0
3.9
3.3
2.2 2.0
4.3 3.0
2.2 3.2
2.9 2.7
2.1 2.3
2.3 2.7
3.8 2.0
2.7 2.3
a Among the 590 proteins that were detected to be commonly expressed in the cochlea of Wistar, Sprague-Dawley and Fischer 344 rats, only 16 were abundantly expressed at levels of g2 fold relative to actin, in all the three strains. b Estrogen receptor 1 antibody against amino acids 154-171.
Table 2. Cochlear Proteins Expressed at Abundant Levels (>2 Fold of Actin) in Two Strainsa
Cofilin SMAC Diablo Tumor Necrosis Factor R
MAG12 SNAP23
Wistar and Sprague-Dawley Protein Kinase BR Survivin
RActinin Nitric Oxide Synthase Endothelial
Wistar and Fischer 344 Caspase 7 Connexin43 Protein Phosphatase 1R
RCatenin BID Daxx E2F2 Focal Adhesion Kinase pp125 Glial Fibrillary Acidic Protein HDAC1 LIM Kinase1 Nitric Oxide Synthase, Brain Protein Kinase CR SNAP29
Sprague-Dawley and Fischer 344 ATF2 pThr69 71 RTubulin Caspase10 Connexin32 DNMT1 DP2 E2F4 ERK5 BIG MAPKBMK1 FRS2 GAD 65 Glutamic Acid Decarboxylase 65 Glutamic Acid Decarboxylase 65 67 HDAC2 JAB 1* MBD4 Mcl1 PARP PCAF Protein Phosphatase 2AR Protein S Tal Ubiquitin Cterminal Hydrolase L1
PUMA bbc3 Synaptopodin
LIN7
βCatenin pSer33 Cyclin D1 E2F1 Estrogen Receptorb GADD153 HAT1 JAB1* Myosin VI PRMT4 PTEN Urotensin II
a Among the antibodies that detected proteins at abundant levels in any two strains, 9 were found in Wistar and Sprague-Dawley, 6 in Wistar and Fischer 344 and 44 in Sprague-Dawley and Fischer 344. * indicates proteins that were detected by more than one antibody and hence mentioned two or more times in the list. b Estrogen receptor 1 was detected with an antibody against amino acids 18-32 in Sprague-Dawley and Fischer 344.
www.ncbi.nlm.nih.gov/UniGene/library.cgi?LID)371).36 The increased cochlear expression of caspase-11 in rats is an unexpected finding as reports indicate that it is hardly detectable in most tissues of normal mice.37 Caspase-11 has been implicated as a regulator of cell migration, cytokine maturation and apoptosis.38 Hence, it could be speculated that caspase11 may have a similar functional activity in the cochlea. Receptors have a vital role in enzyme mediated biological activity. Detection of substance P receptor (NK1) and estrogen 3524
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receptor (ER) among the top 16 proteins expressed in rat inner ear enhances their utility in cochlear interventional studies. NK1 are G-protein-coupled transmembrane receptors, differentially expressed in the brain and peripheral tissues, and play an important role in the transmission of nociceptive stimuli.39 Expression of NK1 has been immunolocalized in the spiral ganglion of mouse cochlea and application of substance P modulated the activity of spiral ganglion neurons.40 ER exists in the hormone-responsive target cells in a nonactivated state
research articles
Analysis of Cochlear Protein Profiles of Rats Table 3. Cochlear Proteins Expressed at Abundant Levels (>2 Fold of Actin) in Only One Strain
a
Wistar Csk Par4
βTubulin III I-Afadin
Caspase 3 Active Ngfr p75
Dimethyl Histone H3 diMeLys9
14-3-3 θ/τ Acetyl phospho Histone H3 AL9 S10 AuroraB Caspase 2 CrkL DNASE II ε Tubulin Fibroblast Growth Factor9 F1A γTubulin KCNK9 MAGI1 MCH MSK1 Nedd8 Nitric Oxide Synthase Endothelial* p120ctn PAD14 Peroxiredoxin 3 Presenilin1 Ran SNAP25
Sprague-Dawley R/β SNAP R E Catenin RNCatenin ASAP1 Centaurin b4 Bcl-10 βCOP Caspase 8 Cdc6 Cyclin D1 Desmin DNMT1 DR5 E2F1 Endothelin FOXP2 GATA1 FANCD2 Grb2 HDAC3 HDAC6 LDS1 LKB1 MKP1 MBD2a MDM2 MeCP2 MyosinIX, Myr5 Myosin VI Neurofibromin Nicastrin Nitric Oxide Synthase Endothelial* Nitric Oxide Synthase, Inducibile p130CAS p35 Cdk5 Regulator Parkin PDK1 PERP phosphoHistone H2AX pSer139 Protein phosphatase 1R PTEN RbAp48/RbAp46 Retinoblastoma Tau Vanilloid Receptor1
Annexin VII CalbindinD28K Chondroitin Sulfate DR4 GSK3β Histone H3 pSer28 MAP1 Myosin VI Protein Kinase C* RIP SUMO1
AP1 Caspase6 Collagen type IV Fas Ligand GRANZYME B IKKa MTA2 MTA1L Nitrotyrosine # Protein Kinase C* Rsk1 Transportin 1
Fischer 344 AP2a Caspase 9 Cyclin H Fas Ligand HDAC3 MAP Kinase Erk1Erk2 Myosin Va* p21WAF1 Cip1 Protein Kinase Cε S6 Kinase VEGFR1
R1 Syntrophin RSynuclein βTubulin I Connexin 32 Dimethyl Histone H3 diMeLys4 Dystrophin EGF Glutamine Synthetase GRP78 BiP ILP2 MAFF MBDin XAB1 Melanocortin3 Receptor Nck2 Nitric Oxide Synthase Endothelial* OP18 Stathmin p38 MAPK Pen2 PKR Raf1 S100 VDAC Porin BACE 1 Centrin Destrin ADF Fibrone ctin HDRP MITR MAP Kinase Kinase MEK MAPKK Myosin Va* PI 3Kinase p85a Pyk2 SLIPR MAGI3
a Out of the 213 antibodies that detected cochlear proteins at levels >2 fold of actin in the three strains, 7 were found only in Wistar rats, 88 in Sprague-Dawley and 43 in Fischer 344, at these abundant levels. * indicates proteins that were detected by more than one antibody and hence mentioned two or more times in the list. # denotes proteins that were detected due to specific post-translational modification of tyrosine.19
while activated receptors bind to DNA as a homo- and heterodimer. In the mammalian inner ear, ER have been found to be expressed in endolymph producing cells such as strial marginal cells in the cochlea and vestibular dark cells in the ampullae and utricle.41 ER has been reported to protect the auditory system from acoustic trauma42 as well as ototoxic injury in vitro.43 However, it is still unclear if ER have any sex specific functional role in the auditory system. Apart from these, ubiquitin is also being used as a drug target in interventional studies.44 Ubiquitin signals transport of its substrates to the 26S proteasome for degradation and regulates signal transduction cascades through inhibitory proteins IκBR and p27.45 Expression of the Uba52 gene has been found in the cochlea and vestibule.46 These possibilities suggest that some of the 16 abundantly expressed proteins could be potential targets for intervention based on their individual functional roles. Biological Markers. Review of the literature indicates that 3 of the abundantly expressed proteins are known anatomical or functional markers of the inner ear. One, proliferating cell protein Ki67 is a cell cycle related nuclear protein whose expression increases in proliferating cells. Interestingly, Ki67 has been reported in spiral ganglion neurons during in vitro propagation of adult-dissociated spiral ganglion cells of guinea
pig.47 The other two inner ear markers, peripherin and neurofilament 200, are intermediate filament (IF) proteins of the cytoskeleton. Peripherins are neuronal IF proteins of the peripheral as well as central nervous system. In the cochlea, peripherin expression has been used as a differentiation marker for type II neurons of the spiral ganglion.48 Apart from this, its expression has been correlated with axonal regeneration.49 Neurofilaments are one of the five major groups of IF, that are important components in the cytoskeleton of neurons. Neurofilament 200 has been detected in type II spiral ganglion cells, auditory nerve, afferent and efferent fibers to the hair cells.50 Novel Cochlear Proteins with Varied Functional Roles. The protein expression of Smad4, INCENP and Sos1 are yet to be reported in the cochlea. However, the Morton fetal cochlea cDNA library has reported Smad4 genes in the cochlea.36 Smad4 has a central role in transforming growth factor β signaling. Activated type I receptors phosphorylate receptorregulated Smads which form complexes with Smad4. These Smad complexes accumulate in the nucleus and binds to promoter elements causing transcriptional activation or repression of target genes.51 INCENP is a chromosomal passenger protein that binds with aurora-B kinase and survivin, and acts at multiple points during mitosis. INCENP levels have been found to increase in human cancer lines.52 Sos proteins are Journal of Proteome Research • Vol. 8, No. 7, 2009 3525
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Figure 5. Immunolocalization of MAP Kinase Activated Protein Kinase2. Immunostaining for MAPKAPK2 (red) was detected in (A) organ of Corti, (B) stria vascularis and (C) spiral ganglion of Sprague-Dawley rats. Strong signals were detected in inner hair cells (IHC) and pillar cells (PC), while outer hair cells (OHC) and supporting cells showed moderate staining (A). MAPKAPK2 was detected in marginal (SMC), intermediate (SIC) and basal cells (SBC) of the stria (B). Strong MAPKAPK2 signal was also detected in spiral ganglion neurons (SGC) dissected from Rosenthal’s canal (C). SGC’s are easily distinguished from surrounding glial cells. SGC’s have large and rounded nuclei while glial nuclei are smaller and irregularly shaped. Cell types and their orientation in the cochlea are shown in the schematic (D). Green indicates f-actin staining by FITC-phalloidin while blue indicates nuclear DNA staining by TO-PRO-3.
Figure 6. Immunolocalization of MAP Kinase ERK1. Extra cellular signal-regulated kinase1 has been detected in the (A) organ of Corti and (B) stria vascularis of Sprague-Dawley rats. Presence of ERK1 in the outer hair cells (OHC), pillar cells (PC) and certain foci in inner sulcus (IS) and outer sulcus (OS) is reflected by red immunostaining, while green and blue indicate f-actin and nuclear DNA staining respectively. In stria vascularis tissue, ERK1 immunostaining is detected in the strial marginal cells (SMC) as well as the blood vessels (BV).
guanine nucleotide exchange factors found on the cytoplasmic face of the plasma membrane. They activate both Ras and Rac in response to growth factor stimulation. Sos1 bound to GRB2 related adaptor protein displays Ras specific guanine nucleotide exchange factor activity, which hints at its possible role in cytoskeleton reorganization.53 A complex containing Sos1 and adaptor proteins Abi1 and Eps8 is required for Rac activity. In 3526
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Figure 7. Immunolocalization of Myosin Light Chain Kinase. Strong signals for myosin light chain kinase was detected in the cuticular plate of all three rows of outer hair cells (OHC) with no signals from inner hair cells (IHC) and pillar cells (PC) in Sprague-Dawley rats with normal hearing. Red staining indicates the presence of MLCK, green indicates f-actin while blue indicates the nuclei.
this way, Sos helps to coordinate the Ras and Rac activities in response to growth factor stimulation.54 Another member of
Analysis of Cochlear Protein Profiles of Rats
Figure 8. Immunolabeling of control tissue. Cochlear tissue labeled with red fluorescent dye tagged secondary antibodies with out any primary antibody, indicate the absence of non specific staining by A) anti rabbit secondary antibody in outer hair cells (OHC) and pillar cells (PC) of organ of Corti and B) anti mouse secondary antibody in strial marginal cells (SMC).
the Sos family, Sos2, has been reported to be expressed in a human fetal cochlear library.36 Given that there is a high degree homology between Sos1 and Sos2 rat proteins, it is possible that the Sos1 antibody from the microarray used in this study may have bound either Sos1 or Sos2 protein. The known functional activities attributed to many of these 16 proteins suggest interesting prospects due to their utility as interventional targets or biological markers. However, it has to be noted that these proteins constitute just 7.5% of the proteins found to be expressed at higher levels in the 3 strains (g2 fold actin). Although the detection of 590 proteins in all three strains shows the similarity in Wistar, Sprague-Dawley and Fischer 344 cochleae, the findings indicate that many of the proteins that were found to be expressed at higher levels differ in their level of expression in the inner ear of these three strains.
Conclusions Taken together, the results suggest the importance of strain specific selection of appropriate antibodies for experimental evaluations of the inner ear. The commonality in protein expression observed in this study can be linked to the common functions of the cochlear tissue displayed by the three rat strains. Conversely, the diversity in protein expression observed in this study is likely linked to differences in physiological responses of the three strains to sound, aging, noise and ototoxic drugs. The identification of abundantly expressed proteins, many of which have important biological applications, will greatly enhance the scope of inner ear research.
Acknowledgment. We acknowledge support from the National Organization for Hearing Research Foundation (DC), Deafness Research Foundation (DC), and NIH (R01DC006630, R01DC00909101, RS). Supporting Information Available: Tabulated microarray data. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Rybak, L. P.; Somani, S. Ototoxicity. Amelioration by protective agents. Ann. N.Y. Acad. Sci. 1999, 884, 143–51. (2) Lopez-Gonzalez, M. A.; Guerrero, J. M.; Rojas, F.; Delgado, F. Ototoxicity caused by cisplatin is ameliorated by melatonin and other antioxidants. J. Pineal Res. 2000, 28 (2), 73–80.
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