The Mechanism Of Regulation The Inner Ear Precursor Cell Proliferation By Signaling Pathways

Inner ear sensory hair cells (HCs) are vulnerable to acoustic trauma, ototoxic drugs, or genetic defects, which can all lead to permanent hearing and balance dysfunctions. Permanent hearing and balance dysfunctions are due to the fact that mammals, including humans, lack of the ability to regenerate sensory HCs spontaneously after damage. Activation of HC regeneration, therefore, might be an ideal way to reverse hearing and balance dysfunction. Non-mammalian vertebrates such as birds, however, can spontaneously regenerate sensory HCs following acoustic trauma or ototoxic insult through the proliferation of SCs within the sensory epithelium.Generally considere that the inner ear hair cells and supporting cells have a common progenitor cells. Some researchers even believe that supporting cells are the hair cells in a particular stage of differentiation, and they can continue differenting to hair cells when their environment changed. Therefore, supporting cells were recognized as the progenitor cells in the processor of hair cells regeneration. To study the mechanism of non-mammalian vertebrates supporting cells proliferation, would be helpful to study the mammalian hair cells regeneration.Biological cells accept various signals from intracellular and extracellular. Cell signal transduction is a basic and important life activities. The gene mutation or abnormal expression of signal molecule would lead to uncontrolled cellular response to extracellular signals, and resulting to diseases. The occurrence and development of many diseases (particularly cancer) have direct or indirect relationship with abnormal cell signal transduction. Many signal pathways are involved in the inner ear development and sensory cells regeneration. There also have the crosstalk between signal pathways. They transfer and recognize signal with each other, and collaborativly control the inner ear sensory progenitor cells division and differentiation.Sensory progenitor cells can differentiate into HCs or SCs. Studying and clarifying the mechanism of signaling pathways regulating the sensory progenitor cells proliferation, then regulating the signaling pathway involved in the sensory progenitor cells proliferation and differentitation is one approach to the regeneration of sensory HCs.The protein pRb encoded by retinoblastoma gene Rbl forces sensory progenitor cells to exit cell cycle and keep differentiated HCs and supporting cells (SCs) in a quiescent state. pRb function is regulated by phosphorylation through MEK/ERK or Rb/Raf-1 signaling pathway. In addition, Notch and Wnt signaling pathways are highly conserved signaling pathways, existing in various organisms. Notch signaling pathway controls cells fate by interaction between the local cells. Notch signaling pathway controls inner ear development by lateral inhibition, and also induces proliferation and differentiation in the early development of inner ear.Wnt signaling plays a dual role in inner ear stem cells, stimulating both expansion and differentiation. Wnt signaling pathway not only control the development of inner ear, but also promote the development of sensory progenitor cells. And there exists the crosstalk between Notch and Wnt signal pathways.pRb phosphorylation is crucial for progenitor cell proliferation and survival during the early embryonic stage of avian otocysts sensory epithelium development. However in damaged avian utricle, the role of pRb in regulating the cell cycling of SCs or HC regeneration still remains unclear. And the role of Notch and Wnt signaling pathway and its crosstalk in regulating the proliferation of sensory progenitor cells is also unknown. To further elucidate the function of these signaling pathways associated with inner ear development and its mechanism in sensory progenitor cells proliferation, we study the function of pRb phosphorylation on SCs re-entering the cell cycle triggered by gentamycin induced sensory cell damage, and the role of Notch and Wnt signaling pathways in the proliferation of mammalian sensory progenitor cells. Together these studying improve our understanding in regulating these singaling pathways in proliferation of mammalian sensory progenitor cells, and may shed light on the mammalian hair cell regeneration from SCs in damaged organ.Part1 pRb phosphoralation regulates the proliferation of sensory progenitor cells in gentamicin damaged neonatal avian utricleObjectives To study the function of pRb phosphorylation on sensory progenitor cells re-entering the cell cycle triggered by gentamycin induced sensory cell damage, and the role of MEK/ERK and Rb/Raf-1 signaling pathway in regulating the phosphorylation of pRb. Which might help us to further explore mammalian HC regeneration by manipulating the pRb pathway.Methods We cultured the neonatal (1 to 3-day-old) chicken utricles with gentamycin, which is an aminoglycosides sensitive to utricle HCs, to damage the HC. Then we used BrdU (bromo-deoxyuridine) and MEK inhibitor U0126, which is a commonly used inhibitor of the MAP kinase pathway and specifically blocks ERK phosphorylationto inhibit the MEK/ERK pathway; we also used the Rb-Raf-1 inhibitor RRD-251, which is a small molecule disruptor of the Rb-Raf-1 interaction that significantly inhibits angiogenesis and tumor growth both in vitro and in vivo in a pRb-dependent manner, to inhibit Rb/Raf-1 pathways. And then we investigated the expression of the genes and protein that regulate pRb and cell cycle progression after treatment with U0126 and RRD-251 by using realtime PCR and western-blot.Results After gentamycin induced sensory cell damage, pRb phosphorylation is important for the quiescent SCs re-entering the cell cycle in the neonatal chicken utricle. The proliferation of SCs is decreased in a dose-dependent manner in response to both U0126 and RRD-251.The expression of the genes regulating pRb function and cell cycle progression, and the protein expression levels of phospho-p44/42 ERK, p44/42 ERK, pRb in damaged neonatal chicken utricle also decreased.Conclusions The neonatal chicken sensory progenitor cells can spontaneously re-enter the cell cycle following ototoxic insult. The high proliferation rate of avian SCs makes it as a useful model to study the regeneration of inner ear hair cells. This study revealed an essential role of pRb in S-phase entry of the SCs in the damaged neonatal chicken utricle and have shown that the proliferation of these cells requires the phosphorylation of pRb. We have shown that the MEK/ERK signaling pathway and the Rb/Raf-1 signaling pathway both play roles in phosphorylating pRb. Understanding the function of pRb in the proliferation of inner ear SCs might help us to further explore mammalian HC regeneration by manipulating the pRb pathway.Part2 The function of Notch and Wnt signaling pathways regulating the proliferation of sensory progenitor cells in neonatal mouse cochleaObjectives To study the role of Notch and Wnt signaling pathways in the process of proliferation of mammalian sensory progenitor cells in neonatal mouse cochlea. Improving our understanding in regulating these singaling pathways in proliferation of mammalian sensory progenitor cells, and shedding light on the mammalian hair cell regeneration from SCs in damaged organ.Methods We cultured the neonatal (PO to 3-day-old) mouse cochlea and used EdU (5-Ethynyl-2’-deoxyuridine) and γ-secretase inhibitor (DAPT) to inhibit Notch signaling pathway; we also used QS11 which was a agonist of Wnt signaling pathway to upregulate β-catenin nuclear translocation; And then we investigated the expression of the genes and protein that regulate progenitor cells and cell cycle progression after treatment with DAPT and QS11 by using realtime PCR and western-blot.Results After given a small dose of DAPT to inhibit Notch signaling pathway, the hair cells increased and lots of the proliferation cells in the normal or damaged cochlea sensory epithelium. Just given QS11 to activate Wnt signal pathway, the hair cells and supporting cells did not change significantly. While given DAPT and QS11 together, the hair cells increased obviously and many more proliferation cells in the normal or damaged cochlea sensory epithelium.Investigating the expression of the genes and protein that regulate progenitor cells and cell cycle progression showed:The gene expression level of Hesl decreased after using small dose of DAPT. While the Hes1 expression level increased after using DAPT and QS11 together. The Math1、c-myc-2 expression levels increased after given the DAPT alone or given DAPT and QS11 together. And the protein expression level of β-catenin all increased after given the DAPT alone or given DAPT and QS11 together.Conclusions This suggests that inhibitting the Notch signaling pathway by small doses of y-secretase inhibitors can promote the proliferation of sensory progenitor cells. And activating the Wnt signaling pathway could further collaborate with DAPT to promote the proliferation of sensory progenitor cells. The proliferation and regeneration of inner ear sensory progenitor cells presumably due to the upregulating expression of Wnt/β-catenin signaling pathway by genes including Hes1.