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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online September 4, 2003 as doi:10.1096/fj.03-0035fje. |
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* Center for Cellular and Molecular Neurobiology,
Department of Neurology, University of Liège, B-4000, Liège, Belgium; and
CNRS, Station Biologique, Roscoff, Bretagne, France
2Correspondence: Center for Cellular and Molecular Neurobiology, University of Liège, 17 Place Delcour, B-4020 Liège, Belgium. E-mail: bmalgrange{at}ulg.ac.be
SPECIFIC AIMS
Molecular cues that drive the generation of the highly organized organ of Corti with intricated hair cells (HCs) and supporting cells (SCs) remain broadly unknown. We wanted to assess whether the activity of cyclin-dependent kinases (CDKs) could control cell fate, differentiation, or proliferation in embryonic and neonatal organs of Corti.
PRINCIPAL FINDINGS
1. Induction of supernumerary hair cells by roscovitine and other pharmacological inhibitors of CDKs
Cochlear explants from E19 rat embryos developed a normal sensory epithelium after 5 days in culture with a single row of inner hair cells and three rows of outer hair cells supported by one row of inner phalangeal cells (IPC) and three rows of Deiters’ cells, respectively. Cultures treated during 5 days with the CDK inhibitor roscovitine developed a higher number of so-called supernumerary hair cells and corresponding SCs (i.e., Deiters’ cells and IPC) organized in rows as in control conditions.
To ascertain whether the effect of roscovitine was mediated by the inhibition of specific kinases, we tested a series of chemically related and unrelated CDK inhibitors. The 2,6,9 -tri-substituted purines were all able to induce the generation of supernumerary HCs and SCs. Their efficiency to induce this effect on isolated organs of Corti correlated with their potency to inhibit CDKs (aminopurvalanol >roscovitine >olomoucine). The kinase inhibitors’ inactive analogs methyl-aminopurvalanol and iso-olomoucine had no effect on the isolated organs of Corti. Chemically unrelated CDK1/2/5 inhibitors such as indirubin-3'-monoxime and alsterpaullone also induced supernumerary HCs and SCs whereas the CDK4/6 inhibitor, fascaplysin had no effect.
Roscovitine and other CDK inhibitory purines have been shown to inhibit the MAP kinases ERK1 and ERK2 in vitro and in vivo. We therefore used the MAPK pathway inhibitor U0126, which did not trigger supernumerary HCs and SCs (even at concentrations as high as 100 µM), thus confirming that the induction of supernumerary HCs and SCs by roscovitine and its purine analogs was specifically mediated by an inhibition of CDKs.
The effect of roscovitine on the development of supernumerary HCs and SCs, was directly related to the age of the animals and duration of treatment. The effect of roscovitine on the induction of supernumerary HCs and SCs culminated at E17, slightly decreased at E19, and disappeared at P0. The average length of supernumerary zones in E19 rat organ of Corti explants treated with roscovitine increased progressively with time in culture, reaching a maximum effect at 5 DIV.
2. Induction of supernumerary HCs by pharmacological blockade of CDKs activity is not related to regulation of cell proliferation and apoptotic cell death in the organ of Corti
Roscovitine, like other CDK inhibitors, has potent anti-mitotic properties. Therefore, we investigated cell proliferation in E19 explants of organ of Corti. Explants were incubated for either 2 or 5 DIV in the presence of 10 µM BrdU. No epithelial cells were labeled with BrdU in the area of HCs and SCs in E19 organs of Corti treated or not with roscovitine.
To determine whether apoptosis was implicated in HC and SC supernumerary differentiation induced by the inhibition of CDKs, E19 rat organs of Corti explants were double-stained for DNA fragmentation (TUNEL assay) and for f-actin in the stereocilia bundles and cuticular plate. After 1, 2, or 5 DIV, no TUNEL-positive nuclei were identified in either control or roscovitine-treated cultures.
3. Immunohistochemical analysis of the anatomical distribution of CDKs expression in the different cell types of E19 rat organ of Corti
To characterize the molecular components of the transduction cascade underlying the effect of roscovitine on supernumerary cells, we first studied the expression of CDK1, CDK2, CDK5, CDK7, and ERK1/2 by immunohistochemistry using sections obtained from E19 rat organ of Corti (Fig. 1
).CDK1, CDK5, CDK7, and ERK1/2 were shown to be ubiquitously expressed throughout the organ of Corti, including the HCs (Fig. 1P
). The only cell type where all the CDKs were expressed is the Hensen’s cells. CDK2 was found to be specifically present in Hensen’s cells (see h in Fig. 1F
) and to a lesser extent in pillar cells in E19 explants of organs of Corti (Fig. 1D-F
). The specific expression pattern of CDK2 persisted throughout the early postnatal period and cells immunopositive for myosin VIIA (i.e., HCs) or for jagged1 (i.e., Deiters’ cells and IPC) were never immunoreactive for CDK2.
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4. Immunoblot quantification of CDKs expression in the organ of Corti ex vivo and in vitro
We next investigated the levels of expression of CDKs in the organ of Corti after affinity purification (Fig. 2
).We also assessed ERK1 and ERK2, which were used as irrelevant internal controls. We wanted to compare levels of protein expression in organs of Corti between two developmental stages, E19 and P0, which are respectively sensitive and insensitive to the inhibition of CDKs for the induction of supernumerary HCs and SCs. As shown in Fig. 2A
, expression of CDK1/2, assessed with a PSTAIRE antibody, diminished between E19 and P0 for each condition (ex vivo explants and cultures with or without roscovitine). In contrast, the level of CDK2 remained more constant (Fig. 2B
), suggesting that the expression of CDK1 was lower at P0 ex vivo and that CDK1 was drastically down-regulated in vitro, especially in the presence of roscovitine with E19 and P0 organs of Corti.
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We used affinity chromatography on immobilized purvalanol, which sensitizes the recovery of CDK5 and CDK7, but also ERK1 and ERK2, specific targets of the 2,6,9-tri-substituted purines. The expression of CDK5 was slightly higher at E19 ex vivo and increased after culturing E19 explants with or without roscovitine (Fig. 2C
). In contrast, CDK7 expression was much higher in P0 organs of Corti ex vivo and roscovitine had no effect on supernumerary cells at this stage, suggesting that CDK7 may not be involved in the transduction of this effect (Fig. 2D
). Consistently, the expression of ERK1 and ERK2 was not different between E19 and P0 ex vivo and remained stable in culture (Fig. 2E, F
).
5. Inhibition of CDK1/2 activities in E19 rat organs of Corti after treatment with pharmacological CDK inhibitors
We examined the changes in CDK1/2 activity occurring in lysates of E19 and P0 organs of Corti during roscovitine-induced HCs and SCs differentiation. p9CKShs1-Sepharose affinity-purified lysates were assessed for their ability to phosphorylate histone H1. In control conditions, kinase activity in cultures from P0 organs of Corti was decreased by 93% compared with E19 explants. Treatment with roscovitine decreased CDK1/2 activity by 73% in the E19 cultured organs of Corti.
CONCLUSIONS AND SIGNIFICANCE
Our results provide clear evidence that pharmacological inhibition of CDK stimulates the production of a large number of supernumerary HCs and corresponding SCs in E19 rat organ of Corti. The low expression of CDKs and the lack of CDK1/2 activity at birth underlie the absence of supernumerary cells at this later stage.
Our immunocytochemical data revealed that potential molecular targets of roscovitine and derivatives (CDK1, 2, and 5) are expressed in the organ of Corti and specifically in Hensen’s cells which have been shown to be possible progenitors for supernumerary HCs and corresponding SCs. In p27KIP1 knockout animals in which supernumerary HCs are produced, S-phase positive cells appear in the region of Hensen’s cells and in the greater epithelial ridge (GER), and are never observed in Deiters’ cells or inner phalangeal cells area (i.e., the SCs underneath HCs). Altogether, the present work strengthens the idea that Hensen’s cells are suitable candidates to represent the cellular source of supernumerary HCs and corresponding SCs triggered by the inhibition of CDK1, 2, and 5 in the organ of Corti. As an alternative to Hensen’s cells, GER area represents a good candidate region that may contain specific progenitors, since this area has been shown to be a potential source of new HCs. We could speculate that pharmacological inhibitors of CDKs may inhibit CDK1, 5, or 7, which are detected immunohistochemically in the GER, and trigger GER cells to differentiate into inner HCs and inner phalangeal cells. In conclusion, we showed that cells in the GER and/or Hensen’s cells are good candidates to be progenitors for supernumerary HCs in our culture model, consistent with previous demonstrations in which they are also the only cells able to generate new HCs.
Several studies provide evidence that the decision to become either a HC or a SC involves positive and negative regulators, in particular, bHLH transcription factors and the Notch pathway. The balance between these positive and negative regulators plays an essential role during the development of the organ of Corti, and the absence or inhibition of one of these factors may break an equilibrium and give rise to the absence of HCs, as in Math1 null mice, or to the overproduction of HCs in the case of Jagged-2, HES1, or HES5 knockout animals. We propose that the inhibition of a subset of CDKs thereby blocks the phosphorylation of yet to be identified factors that either need to be phosphorylated to repress the differentiation of HCs or to be unphosphorylated to activate HC differentiation. Furthermore, our data emphasize that a tonic activation of CDKs may be an essential repressive mechanism by which the highly defined normal number of HCs and SCs appears to be tightly controlled during the development of the organ of Corti.
The demonstration that the inhibition of CDKs induces supernumerary HCs might open prospects for using this apparently well-tolerated drug to devise new strategies to stimulate HC regeneration in the mammalian organ of Corti.
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0035fje; doi: 10.1096/fj.03-0035fje 
This article has been cited by other articles:
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  J. M. Jones, M. Montcouquiol, A. Dabdoub, C. Woods, and M. W. Kelley Inhibitors of Differentiation and DNA Binding (Ids) Regulate Math1 and Hair Cell Formation during the Development of the Organ of Corti J. Neurosci., January 11, 2006; 26(2): 550 - 558. [Abstract] [Full Text] [PDF]
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