Cell Cycle,Differentiation and Regeneration: Where to Begin?

Hair cells, the sensory cells of inner ear, perform essential functions in hearing and balance. However, mammalian hair cells, like most of the CNS neurons, lack the capacity to regenerate. This is in sharp contrast to lower vertebrates in which hair cell regeneration occurs spontaneously through cell division of supporting cells, which leads to hearing restoration. It is believed that the lack of regeneration in mammals is, to a large degree, due to the block of cell cycle re-entry imposed by negative cell growth genes in the inner ear. Recent studies have identified retinoblastoma gene, a well-known tumor suppressor, as the key gene involved in cell cycle exit of inner ear sensory cells. In the inner ear of pRb conditional knockout mice, hair cells undergo continuous cell division, and at the same time differentiate and become functional. Cell division continues in early postnatal cochlea and adult vestibule. Remarkably, the vestibular hair cells without pRb survive, and function at both the cellular and system levels. The time course and effects of pRb inhibition shows that there is a separation between the roles of pRb in cell cycle exit, and subsequent maturation and apoptosis. Those studies reveal distinctly different roles of pRb in the cochlear and vestibular sensory epithelia. The review discusses additional areas to be studied for regeneration of mature hair cells, and highlights the importance of transient and reversible block of pRb function as one of the routes to be explored for regeneration.     

Cell cycle regulation in the inner ear sensory epithelia: Role of cyclin D1 and cyclin-dependent kinase inhibitors

Sensory hair cells and supporting cells of the mammalian cochlea and vestibular (balance) organs exit the cell cycle during embryogenesis and do not proliferate thereafter. Here, we have studied the mechanisms underlying the maintenance of the postmitotic state and the proliferative capacity of these cells. We provide the first evidence of the role of cyclin D1 in cell cycle regulation in these cells. Cyclin D1 expression disappeared from embryonic hair cells as differentiation started. The expression was transiently upregulated in cochlear hair cells early postnatally, paralleling the spatiotemporal pattern of unscheduled cell cycle re-entry of cochlear hair cells from the p19^Ink4d/p21^Cip1 compound mutant mice. Cyclin D1 misexpression in vitro in neonatal vestibular HCs from these mutant mice triggered S-phase re-entry. Thus, cyclin D1 suppression is important for hair cell's quiescence, together with the maintained expression of cyclin-dependent kinase inhibitors. In contrast to hair cells, cyclin D1 expression was maintained in supporting cells when differentiation started. The expression continued during the neonatal period when supporting cells have been shown to re-enter the cell cycle upon stimulation with exogenous mitogens. Thereafter, the steep decline in supporting cell's proliferative activity paralleled with cyclin D1 downregulation. Thus, cyclin D1 critically contributes to the proliferative plasticity of supporting cells. These data suggest that targeted cyclin D1 induction in supporting cells might be an avenue for proliferative regeneration in the inner ear.     

Disrupting the Interaction between Retinoblastoma Protein and Raf-1 Leads to Defects in Progenitor Cell Proliferation and Survival during Early Inner Ear Development

The retinoblastoma protein (pRb) is required for cell-cycle exit of embryonic mammalian hair cells but is not required for hair cell fate determination and early differentiation, and this provides a strategy for hair cell regeneration by manipulating the pRb pathway. To reveal the mechanism of pRb functional modification in the inner ear, we compared the effects of attenuated pRb phosphorylation by an inhibitor of the Mitogen-Activated Protein (MAP) kinase pathway and an inhibitor of the Rb–Raf-1 interaction on cultured chicken otocysts. We demonstrated that the activity of pRb is correlated with its phosphorylation state, which is regulated by a newly established cell cycle-independent pathway mediated by the physical interaction between Raf-1 and pRb. The phosphorylation of pRb plays an important role during the early stage of inner ear development, and attenuated phosphorylation in progenitor cells leads to cell cycle arrest and increased apoptosis along with a global down-regulation of the genes involved in cell cycle progression. Our study provides novel routes to modulate pRb function for hair cell regeneration.     

EGFR signaling is required for regenerative proliferation in the cochlea: conservation in birds and mammals

Proliferation and transdifferentiaton of supporting cells in the damaged auditory organ of birds lead to robust regeneration of sensory hair cells. In contrast, regeneration of lost auditory hair cells does not occur in deafened mammals, resulting in permanent hearing loss. In spite of this failure of regeneration in mammals, we have previously shown that the perinatal mouse supporting cells harbor a latent potential for cell division. Here we show that in a subset of supporting cells marked by p75, EGFR signaling is required for proliferation, and this requirement is conserved between birds and mammals. Purified p75+ mouse supporting cells express receptors and ligands for the EGF signaling pathway, and their proliferation in culture can be blocked with the EGFR inhibitor AG1478. Similarly, in cultured chicken basilar papillae, supporting cell proliferation in response to hair cell ablation requires EGFR signaling. In addition, we show that EGFR signaling in p75+ mouse supporting cells is required for the down-regulation of the cell cycle inhibitor p27(Kip1) (CDKN1b) to enable cell cycle re-entry. Taken together, our data suggest that a conserved mechanism involving EGFR signaling governs proliferation of auditory supporting cells in birds and mammals and may represent a target for future hair cell regeneration strategies.     

Cell cycle regulation in hair cell development and regeneration in the mouse cochlea

Cell cycle inhibitors play important roles in the development of mammalian cochleae. Loss of function of those factors in mice at various developmental stages results in distinct phenotypes characterized by overproduction or loss of cochlear sensory cells. Our recent study showed that acute deletion of the retinoblastoma protein (Rb) induces rapid cell cycle reentry and subsequent loss of postnatal cochlear hair cells in mice. Clearly, these regulators play multiple roles in cell cycle exit and differentiation of hair cell and supporting cell progenitors. They are also crucial in maintenance of postmitotic states and survival of differentiated hair cells and supporting cells. In mammals, lost hair cells cannot be spontaneously replaced, leading to permanent deafness. However, lower vertebrates such as birds and fish can naturally regenerate damaged hair cells from the underlying supporting cells through proliferation and transdifferentiation. Thus, manipulating cell cycle inhibitors in mammalian cochleae could provide a new avenue to restore hearing in deaf people caused by a variety of genetic mutations and environmental insults.     

The retinoblastoma gene pathway regulates the postmitotic state of hair cells of the mouse inner ear

Precursors of cochlear and vestibular hair cells of the inner ear exit the cell cycle at midgestation. Hair cells are mitotically quiescent during late-embryonic differentiation stages and postnatally. We show here that the retinoblastoma gene Rb and the encoded protein pRb are expressed in differentiating and mature hair cells. In addition to Rb, the cyclin dependent kinase inhibitor (CKI) p21 is expressed in developing hair cells, suggesting that p21 is an upstream effector of pRb activity. p21 apparently cooperates with other CKIs, as p21-null mice exhibited an unaltered inner ear phenotype. By contrast, Rb inactivation led to aberrant hair cell proliferation, as analysed at birth in a loss-of-function/transgenic mouse model. Supernumerary hair cells expressed various cell type-specific differentiation markers, including components of stereocilia. The extent of alterations in stereociliary bundle morphology ranged from near-normal to severe disorganization. Apoptosis contributed to the mutant phenotype, but did not compensate for the production of supernumerary hair cells, resulting in hyperplastic sensory epithelia. The Rb-null-mediated proliferation led to a distinct pathological phenotype, including multinucleated and enlarged hair cells, and infiltration of hair cells into the mesenchyme. Our findings demonstrate that the pRb pathway is required for hair cell quiescence and that manipulation of the cell cycle machinery disrupts the coordinated development within the inner ear sensory epithelia.     

Multiple roles of Notch signaling in cochlear development

Notch signaling inhibits hair cell differentiation, based on studies on mice deficient in Notch signaling-related genes and its downstream genes. However, the precise mechanisms of this inhibition are unknown because it is difficult to control the timing and duration of the suppression of Notch signaling. Here, we developed a novel in vitro culture and analysis method for mouse fetal cochleae and examined the roles of Notch signaling by its reversible inhibition through the use of Notch signaling inhibitors of gamma-secretase and TNF-alpha-converting enzyme. Notch inhibition with Notch signaling inhibitor treatment increases the number of cochlear hair cells, as observed in gene deletion experiments. We elucidated that this increase is regulated by the dichotomy between hair cells and supporting cells from common progenitors. We also propose other roles of Notch signaling in cochlear development. First, Notch signaling arrests the cell cycle of the cochlear epithelium containing putative hair cells and supporting cell progenitors because Notch inhibition with inhibitor treatment increases the number of 5-bromo-2'-deoxyuridine (BrdU)-positive cells that can differentiate into hair cells or supporting cells. Second, Notch signaling is required for the induction of Prox1-positive supporting cells. Third, Notch signaling is required for the maintenance of supporting cells.     

LncRNA-XIST promotes dermal papilla induced hair follicle regeneration by targeting miR-424 to activate hedgehog signaling

BackgroundAlopecia is a highly prevalent disease characterizing by the loss of hair. Dermal papilla (DP) cells are the inducer of hair follicle regeneration, and in vitro three-dimensional (3D) culturing DP cells have been proven to induce hair follicle regeneration. However, the molecular mechanisms behind the regulation of 3D culturing DP cells remain unclear.Methods3D-cultivated DP cells were used as in vitro cell model. DP sphere xenograft to nude mice was performed for in vivo study of hair follicle regeneration. qRT-PCR, Western blotting, and immunofluorescence were used for detecting the level of XIST, miR-424 and Hedgehog pathway-related proteins, respectively. H&E staining was used to examine hair neogenesis. Cell viability, proliferation and ALP activity were measured by MTT, CCK-8 and ELISA assays, respectively. Luciferase assays were used for studying molecular regulation between XIST, miR-424 and Shh 3′UTR.ResultsXIST and Shh were up-regulated, and miR-424 was down-regulated in 3D DP cells. Molecular regulation studies suggested that XIST sponged miR-424 to promote Shh expression. Knockdown of XIST suppressed DP cell activity, cell proliferation, ALP activity and the expression of other DP markers by sponging miR-424. Knockdown of XIST suppressed Shh mediated hedgehog signaling by targeting miR-424. Moreover, the knockdown of XIST inhibited DP sphere induced in vivo hair follicle regeneration and hair development.ConclusionXIST sponges miR-424 to promote Shh expression, thereby activating hedgehog signaling and facilitating DP mediated hair follicle regeneration.     

Restrictions in cell cycle progression of adult vestibular supporting cells in response to ectopic cyclin D1 expression

Sensory hair cells and supporting cells of the mammalian inner ear are quiescent cells, which do not regenerate. In contrast, non-mammalian supporting cells have the ability to re-enter the cell cycle and produce replacement hair cells. Earlier studies have demonstrated cyclin D1 expression in the developing mouse supporting cells and its downregulation along maturation. In explant cultures of the mouse utricle, we have here focused on the cell cycle control mechanisms and proliferative potential of adult supporting cells. These cells were forced into the cell cycle through adenoviral-mediated cyclin D1 overexpression. Ectopic cyclin D1 triggered robust cell cycle re-entry of supporting cells, accompanied by changes in p27(Kip1) and p21(Cip1) expressions. Main part of cell cycle reactivated supporting cells were DNA damaged and arrested at the G2/M boundary. Only small numbers of mitotic supporting cells and rare cells with signs of two successive replications were found. Ectopic cyclin D1-triggered cell cycle reactivation did not lead to hyperplasia of the sensory epithelium. In addition, a part of ectopic cyclin D1 was sequestered in the cytoplasm, reflecting its ineffective nuclear import. Combined, our data reveal intrinsic barriers that limit proliferative capacity of utricular supporting cells.     

In vitro growth and differentiation of mammalian sensory hair cell progenitors: a requirement for EGF and periotic mesenchyme

The sensory hair cells and supporting cells of the organ of Corti are generated by a precise program of coordinated cell division and differentiation. Since no regeneration occurs in the mature organ of Corti, loss of hair cells leads to deafness. To investigate the molecular basis of hair cell differentiation and their lack of regeneration, we have established a dissociated cell culture system in which sensory hair cells and supporting cells can be generated from mitotic precursors. By incorporating a Math1-GFP transgene expressed exclusively in hair cells, we have used this system to characterize the conditions required for the growth and differentiation of hair cells in culture. These conditions include a requirement for epidermal growth factor, as well as the presence of periotic mesenchymal cells. Lastly, we show that early postnatal cochlear tissue also contains cells that can divide and generate new sensory hair cells in vitro.     

Notch Signaling Limits Supporting Cell Plasticity in the Hair Cell-Damaged Early Postnatal Murine Cochlea

In mammals, auditory hair cells are generated only during embryonic development and loss or damage to hair cells is permanent. However, in non-mammalian vertebrate species, such as birds, neighboring glia-like supporting cells regenerate auditory hair cells by both mitotic and non-mitotic mechanisms. Based on work in intact cochlear tissue, it is thought that Notch signaling might restrict supporting cell plasticity in the mammalian cochlea. However, it is unresolved how Notch signaling functions in the hair cell-damaged cochlea and the molecular and cellular changes induced in supporting cells in response to hair cell trauma are poorly understood. Here we show that gentamicin-induced hair cell loss in early postnatal mouse cochlear tissue induces rapid morphological changes in supporting cells, which facilitate the sealing of gaps left by dying hair cells. Moreover, we provide evidence that Notch signaling is active in the hair cell damaged cochlea and identify Hes1, Hey1, Hey2, HeyL, and Sox2 as targets and potential Notch effectors of this hair cell-independent mechanism of Notch signaling. Using Cre/loxP based labeling system we demonstrate that inhibition of Notch signaling with a γ- secretase inhibitor (GSI) results in the trans-differentiation of supporting cells into hair cell-like cells. Moreover, we show that these hair cell-like cells, generated by supporting cells have molecular, cellular, and basic electrophysiological properties similar to immature hair cells rather than supporting cells. Lastly, we show that the vast majority of these newly generated hair cell-like cells express the outer hair cell specific motor protein prestin.     

Regulation of retinoblastoma protein functions by ectopic expression of human cyclins.

The retinoblastoma susceptibility gene (RB) product, the retinoblastoma protein (pRb), functions as a regulator of cell proliferation. Introduction of the RB gene into SAOS-2 osteosarcoma cells, which lack functional pRb, prevents cell cycle progression. Such growth-suppressive functions can be modulated by phosphorylation of pRb, which occurs via cell cycle-regulated kinases. We show that constitutively expressed cyclins A and E can overcome pRb-mediated suppression of proliferation. pRb becomes hyperphosphorylated in cells overexpressing these cyclins, and this phosphorylation is essential for cyclin A- and cyclin E-mediated rescue of pRb-blocked cells. This suggests that G1 and S phase cyclins can act as regulators of pRb function in the cell cycle by promoting pRb phosphorylation.     

Cyclin D1/Cdk4 regulates retinoblastoma protein-mediated cell cycle arrest by site-specific phosphorylation.

The retinoblastoma protein (pRb) inhibits progression through the cell cycle. Although pRb is phosphorylated when G1 cyclin-dependent kinases (Cdks) are active, the mechanisms underlying pRb regulation are unknown. In vitro phosphorylation by cyclin D1/Cdk4 leads to inactivation of pRb in a microinjection-based in vivo cell cycle assay. In contrast, phosphorylation of pRb by Cdk2 or Cdk3 in complexes with A- or E-type cyclins is not sufficient to inactivate pRb function in this assay, despite extensive phosphorylation and conversion to a slowly migrating "hyperphosphorylated form." The differential effects of phosphorylation on pRb function coincide with modification of distinct sets of sites. Serine 795 is phosphorylated efficiently by Cdk4, even in the absence of an intact LXCXE motif in cyclin D, but not by Cdk2 or Cdk3. Mutation of serine 795 to alanine prevents pRb inactivation by Cdk4 phosphorylation in the microinjection assay. This study identifies a residue whose phosphorylation is critical for inactivation of pRb-mediated growth suppression, and it indicates that hyperphosphorylation and inactivation of pRb are not necessarily synonymous.     

Functional interactions between the estrogen receptor coactivator PELP1/MNAR and retinoblastoma protein

PELP1 (proline-, glutamic acid-, and leucine-rich protein-1 (also referred to as MNAR, or modulator of nongenomic activity of estrogen receptor)), a recently identified novel coactivator of estrogen receptors, is widely expressed in a variety of 17 beta-estradiol (E2)-responsive reproductive tissues and is developmentally regulated in mammary glands. pRb (retinoblastoma protein), a cell cycle switch protein, plays a fundamental role in the proliferation, development, and differentiation of eukaryotic cells. To study the putative function of PELP1, we established stable MCF-7 breast cancer cell lines overexpressing PELP1. PELP1 overexpression hypersensitized breast cancer cells to E2 signaling, enhanced progression of breast cancer cells to S phase, and led to persistent hyperphosphorylation of pRb in an E2-dependent manner. Using phosphorylation site-specific pRb antibodies, we identified Ser-807/Ser-811 of pRb as a potential target site of PELP1. Interestingly, PELP1 was discovered to be physiologically associated with pRb and interacted via its C-terminal pocket domain, and PELP1/pRb interaction could be modulated by antiestrogen agents. Using mutant pRb cells, we demonstrated an essential role for PELP1/pRb interactions in the maximal coactivation functions of PELP1 using cyclin D1 as one of the targets. Taken together, these findings suggest that PELP1, a steroid coactivator, plays a permissive role in E2-mediated cell cycle progression, presumably via its regulatory interaction with the pRb pathway.     

Rb1 gene inactivation expands satellite cell and postnatal myoblast pools

Satellite cells are well known as a postnatal skeletal muscle stem cell reservoir that under injury conditions participate in repair. However, mechanisms controlling satellite cell quiescence and activation are the topic of ongoing inquiry by many laboratories. In this study, we investigated whether loss of the cell cycle regulatory factor, pRb, is associated with the re-entry of quiescent satellite cells into replication and subsequent stem cell expansion. By ablation of Rb1 using a Pax7CreER,Rb1 conditional mouse line, satellite cell number was increased 5-fold over 6 months. Furthermore, myoblasts originating from satellite cells lacking Rb1 were also increased 3-fold over 6 months, while terminal differentiation was greatly diminished. Similarly, Pax7CreER,Rb1 mice exhibited muscle fiber hypotrophy in vivo under steady state conditions as well as a delay of muscle regeneration following cardiotoxin-mediated injury. These results suggest that cell cycle re-entry of quiescent satellite cells is accelerated by lack of Rb1, resulting in the expansion of both satellite cells and their progeny in adolescent muscle. Conversely, that sustained Rb1 loss in the satellite cell lineage causes a deficit of muscle fiber formation. However, we also show that pharmacological inhibition of protein phosphatase 1 activity, which will result in pRb inactivation accelerates satellite cell activation and/or expansion in a transient manner. Together, our results raise the possibility that reversible pRb inactivation in satellite cells and inhibition of protein phosphorylation may provide a new therapeutic tool for muscle atrophy by short term expansion of the muscle stem cells and myoblast pool.     

Hair cell regeneration in the avian auditory epithelium

Regeneration of sensory hair cells in the mature avian inner ear was first described just over 20 years ago. Since then, it has been shown that many other non-mammalian species either continually produce new hair cells or regenerate them in response to trauma. However, mammals exhibit limited hair cell regeneration, particularly in the auditory epithelium. In birds and other non-mammals, regenerated hair cells arise from adjacent non-sensory (supporting) cells. Hair cell regeneration was initially described as a proliferative response whereby supporting cells re-enter the mitotic cycle, forming daughter cells that differentiate into either hair cells or supporting cells and thereby restore cytoarchitecture and function in the sensory epithelium. However, further analyses of the avian auditory epithelium (and amphibian vestibular epithelium) revealed a second regenerative mechanism, direct transdifferentiation, during which supporting cells change their gene expression and convert into hair cells without dividing. In the chicken auditory epithelium, these two distinct mechanisms show unique spatial and temporal patterns, suggesting they are differentially regulated. Current efforts are aimed at identifying signals that maintain supporting cells in a quiescent state or direct them to undergo direct transdifferentiation or cell division. Here, we review current knowledge about supporting cell properties and discuss candidate signaling molecules for regulating supporting cell behavior, in quiescence and after damage. While significant advances have been made in understanding regeneration in non-mammals over the last 20 years, we have yet to determine why the mammalian auditory epithelium lacks the ability to regenerate hair cells spontaneously and whether it is even capable of significant regeneration under additional circumstances. The continued study of mechanisms controlling regeneration in the avian auditory epithelium may lead to strategies for inducing significant and functional regeneration in mammals.     

Intracellular signaling pathways involved in post-mitotic dopaminergic PC12 cell death induced by 6-hydroxydopamine

Oxidative stress has been shown to mediate neuron damage in Parkinson's disease (PD). In the present report, we intend to clarify the intracellular pathways mediating dopaminergic neuron death after oxidative stress production using post-mitotic PC12 cells treated with the neurotoxin 6-hydroxydopamine (6-OHDA). The use of post-mitotic cells is crucial, because one of the suggested intracellular pathways implicated in neuron death relates to the re-entry of neurons (post-mitotic cells) in the cell cycle. We find that 6-OHDA sequentially increases intracellular oxidants, functional cell damage and caspase-3 activation, leading to cell death after 12 h of incubation. Prevention of cell damage by different antioxidants supports the implication of oxidative stress in the observed neurotoxicity. Oxidative stress-dependent phosphorylation of the MAPK JNK and oxidative stress-independent PKB/Akt dephosphorylation are involved in 6-OHDA neurotoxicity. Decrease in p21(WAF1/CIP1) and cyclin-D1 expression, disappearance of the non-phosphorylated band of retinoblastoma protein (pRb), and expression of proliferating cell nuclear antigen, not present in PC12 post-mitotic cells, suggest a re-entry of differentiated cells into cell cycle. Our results indicate that such a re-entry is mediated by oxidative stress and is involved in 6-OHDA-induced cell death. We conclude that at least three intracellular pathways are involved in 6-OHDA-induced cell death in differentiated PC12 cells: JNK activation, cell cycle progression (both oxidative stress-dependent), and Akt dephosphorylation (not related to the increase of oxidants); the three pathways are necessary for the cells to die, since blocking one of them is sufficient to keep the cells alive.