Expression Of LIM Proteins CRP2 And CRIP2 In The Rat Inner Ear And Subcellular Localizations In Olfactory Precursor Cells

The mammalian inner ear possesses a very limited ability for self-repair and regeneration. For this reason, the loss of sensory hair cells from the ears of mature mammalians usually leads to permanent deficits in hearing as consequence of acoustic trauma, treatment with ototoxic drugs, infections or autoimmune pathologies, or as part of the aging process. In contrast, the ears of most non-mammalian vertebrates (fish, amphibians and avian) can regenerate hair cells after injury. The process of hair cell regeneration has been characterized most completely in the avian inner ear, where lost sensory cells are quickly replaced via a process of regenerative cell proliferation. Thus, the factors that permit sensory regeneration in the ears of non-mammals but inhibit such regeneration in mammals are of great biological and clinical interest.LIM protein family is shown to play myriad roles in cell fate specification, differentiation, and cytoskeletal organization. They likely act to promote the formation of multimeric transcription regulatory complexes with bridging factors such as basic belic-loop-helix (bHLH) and GATA proteins. Additionally, they could function antagonistically toward LIM homeodomain proteins by competing for binding to the essential cofactor LIM-domain-binding protein.CRP2 and CRIP2 are encoded by csrp2 and crip2, respectively. CRP2 contains two tandemLy arranged LIM domains and is implicated in diverse processes linked to cellular differentiation and growth control. Conditional cell transformation reveals that CRP2 is essential to the maintenance of normal cell function. CRIP2 is a novel LIM protein and was first characterized in 2003. It consists of two LIM domains spaced by a short amino acid stretch. It serves as a novel LIM-only protein that is highly homologous to rat ESP1 and CRP2 sequences. CRIP2 display a wide, overlapping tissue distribution with protein tyrosine phosphatase which have a potential role in the dynamics of the actin cytoskeleton. The CRIP2 displays amino sequence identities of 50% compared with the sequence of CRP2. On the protein level, the two proteins share 42% similarity. It has been reported that these two proteins have distinct tissue distribution in various tissues, suggesting that each might serve related but specific roles in tissue organization or function. Although LIM proteins have been found to play essential role in cell proliferation and differentiation, there was no report showing the expression of Group 2 LIM proteins in the inner earAIMSystematically examined the expression of CRIP2 and CRP2 in the rat inner ear and olfactory precursor cells and investigate the relationship between LPS stimulation and CRIP2 expression in the cultured MC.Methods and Results1.Preparation and identification of anti-CRP2 and anti-CRIP2 sera Cloning of csrp2 and crip2 and Construction of expression vector. Complete sequences of csrp2 and crip2 were amplified using cDNA synthesized from undifferentiated olfactory precursor cells. BamHI and HindIII sites were designed in the primers to facilitate cloning into pRSET A expression vector. The PCR products were first cloned into pGEM-T-Easy vector and the BamHI-HindIII fragments were then subcloned in the pRSET A expression vector, respectively. The insert sizes were confirmed by digestion and further verified by sequencing. Expression and purification of the recombinant protein. Under the control of IPTG-inducible phage T7 promoter, csrp2 cDNA in pRSET A is predicted to encode a recombinant protein of 193 aa with a molecular weight of ~24 kDa. The crip2 cDNA in pRSET A is predicted to encode a recombinant protein of 208 aa with a molecular weight of ~27 kDa. Small-scale cultures of the positive clones were subjected to IPTG induction to identify clones capable of expressing the predicted recombinant proteins. Recombinant proteins migrated at apparent molecular weight that matched the prediction. The results confirmed that the CRP2 and CRIP2 could be efficiently expressed in E. coli host cells. Expression was not detected in uninduced recombinant strains. Western Blot results confirmed the expressed proteins were the target proteins.To examine the relative distribution of the expressed recombinant protein in the soluble and insoluble fractions, both the supernatant and the pellet of the cell lysate were examined to detect the recombinant proteins. The expression of both recombinant proteins was detected predominantly in the soluble fraction.After purifying the recombinant proteins by affinity chromatography, predominantly one band of the recombinant protein could be seen in the SDS-PAGE and Western Blot, indicating that the recombinant proteins were highly purified. The protein yields were 0.7 mg/mL and 0.6 mg/mL for CRP2 and CRIP2, respectively.Characterization of the antisera. After immunizing rabbits and mice with His-tagged CRP2 and CRIP2, respectively, anti-CRP2 and anti-CRIP2 sera were purified by protein G affinity chromatography. Titers of both antibodies were analyzed by ELISA using purified CRP2 or CRIP2 as antigen and were showed to be approximately 1:1600. There was no cross-reactivity between two antibodies. Titers of mice antibody were approximately 1:6400.It is known that antibodies that recognize proteins in their denatured form (SDS-PAGE gel) are not always able to detect the same proteins in a native form, and vice versa. To determine if the prepared antibodies detect endogenic CRP2 and CRIP2 in eukaryotic cells, the antibodies were used in lysate of Cos-7 cells transfected with pcDNA3.1(-)-csrp2.and Cos-7 cells transfected with pcDNA3.1(-)-crip2. The results presented a protein of ~21 kDa was detected by Western Blot analysis using rabbit anti-CRP2 antibody. Similarly, a protein of ~24 kDa was blotted with the rabbit anti-CRIP2 antibody. In contrast, no band was visualized in the Cos-7 cells transfected with pcDNA3.1(-) using either antibody. The results indicated that these two antibodies are specific to CRP2 and CRIP2, respectively. Same results were achieved using mice antibodies that we prepared (Data not shown).2.Expression of CRP2 and CRIP2 in rat inner earAnalysis CRIP2 and CRP2 mRNA by RT-PCR. We analyzed the expression of crip2 and csrp2 genes at several key developmental stages, RT-PCR analysis of mRNA extracted from cochleae showed that crip2 are expressed in rat inner ears of different age while the expression of csrp2 can be detected in the embryonic rat inner ear. The nucleic acid sequences of all these PCR products were verified by sequencing and are identical to the CRIP2 sequence previous reported. The RT-PCR products were compared and all 6 groups'cochlea specimens expressed CRIP2 mRNA at similar levels.CRIP2 protein expression. The expression levels of CRIP2 and CRP2 were further determined at protein level. CRIP2 and CRP2 protein expression within the inner ear were initially determined in the rat using Western Blot analysis. Both 6 groups'cochlea expressed CRIP2 protein (Fig. 2). Anti-CRIP2 antibody detected a single band of ~25kDa within the protein extract from the cochlea. Compared withβ-actin expression, the expression of CRIP2 in different cochlea has no significant difference, indicating no significant correlation between the level of CRIP2 and ages tested. The expression of CRP2 can be detected in the embryonic rat inner ear. This result is consistent with mRNA levels at different development stages.Immunofluorescent localization. In order to understand the functional relationship between CRIP2 and development of hearing organs, we tried to determine the presence and expression pattern of CRIP2 in the cochlea of rats. The immunofluorescence results showed that CRIP2 was detected in the spiral ganglion cells, basal membrane and stria vascularis in rat cochlea tissue. Moreover, the expression of CRIP2 was found in hair cells and supporting cells of the organ of Corti. In contrast, CRIP2 expression was not detected at the peripheral auditory nerve. A similar CRIP2 expression pattern was found in the cochlea of newborn rat (data not shown). No specific fluorescence was detected in negative controls.3. LPS upregulates the expression of CRIP2 in the cultured MC.In order to understand the functional relationship between CRIP2 and immunity of inner ear, we tried to determine the presence and expression pattern of CRIP2 in the cultured MC. Hochest 33258 was used as a marker for the nuclei. The immunofluorescence results showed that CRIP2 was localized predominantly in the cytoplasm. The expression of CRIP2 is upregualted by the stimulation of LPS. And as the concentration of LPS increases, the stimulation became stronger. RT-PCR and Western Blot analysis showed the similar results.4. Sub-cellular localization and expression analysis of CRP2 and CRIP2 proteins in rat olfactory precursor cells.With the prepared CRP2 and CRIP2 antibodies, we detected the subcellular localization of CRIP2 and CRP2 in the undifferentiated olfactory precursor cells and in the differentiated end cells by immunofluoence staining. Hochest 33258 was used as a marker for the nuclei. The results show that in the undifferentiated olfactory precursor cells, CRP2 was localized both in the nucleus and cytoplasm, which is consistent with previous reports that CRP2 are localized in the cytoplasm along the cytoskeleton and in the nucleus. In contrast, CRIP2 was localized predominantly in the cytoplasm, which is consistent with a previous report that CRIP2 is present in the cytoplasm of epithelial cells. In the differentiated end cells, only the expression of CRIP2 in the cytoplasm can be detected and anti-CRP2 does not detect any protein. Western Blot analysis showed the similar results.ConclusionIn this study we confirmed the expression of CRIP2 and CRP2 in the rat inner ear and olfactory precursor cells by RT-PCR, Western Blot and immunofluorescence analysis. The expression profile of CRIP2, which has the characteristic of important transcriptional regulator, suggests that CRIP2 plays a role in the development and formation of cochlea. The expression profile of CRP2 suggests that CRP2 may play a role in the inner ear development. This study is the first report confirming the expression of CRIP2 and CRP2 in rat inner ear and olfactory precursor cells other than hearts and other organs.