Retinoblastoma protein family in cell cycle and cancer: A review

Two genes, p107 and Rb2/p130, are strictly related to RB, the most investigated tumor suppressor gene, responsible for susceptibility to retinoblastoma. The products of these three genes, namely pRb, p107, and pRb2/p130 are characterized by a peculiar steric confirmation, called “pocket,” responsible for most of the functional interactions characterizing the activity of these proteins in the homeostasis of the cell cycle. The interest in these genes and proteins springs from their ability to regulate cell cycle processes negatively, being able, for example, to dramatically slow down neoplastic growth. So far, among these genes, only RB is firmly established to act as a tumor suppressor, because its lack-of-function is clearly involved in tumor onset and progression. It has been found deleted or mutated in most retinoblastomas and sarcomas, but its inactivation is likely to play a crucial role in other types of human cancers. The two other members of the family have been discovered more recently and are currently under extensive investigation. We review analogies and differences among the pocket protein family members, in an attempt to understand their functions in normal and cancer cells. © 1996 Wiley-Liss, Inc.     

Overlapping and distinct pRb pathways in the mammalian auditory and vestibular organs

Retinoblastoma gene (Rb1) is required for proper cell cycle exit in the developing mouse inner ear and its deletion in the embryo leads to proliferation of sensory progenitor cells that differentiate into hair cells and supporting cells. In a conditional hair cell Rb1 knockout mouse, Pou4f3-Cre-pRb^-/-, pRb^-/- utricular hair cells differentiate and survive into adulthood whereas differentiation and survival of pRb^-/- cochlear hair cells are impaired. To comprehensively survey the pRb pathway in the mammalian inner ear, we performed microarray analysis of ^pRb-/- cochlea and utricle. The comparative analysis shows that the core pathway shared between pRb^-/- cochlea and utricle is centered on E2F, the key pathway that mediates pRb function. A majority of differentially expressed genes and enriched pathways are not shared but uniquely associated with pRb^-/-cochlea or utricle. In pRb^-/- cochlea, pathways involved in early inner ear development such as Wnt/β-catenin and Notch were enriched, whereas pathways involving in proliferation and survival are enriched in pRb^-/-utricle. Clustering analysis showed that the pRb^-/- inner ear has characteristics of a younger control inner ear, an indication of delayed differentiation. We created a transgenic mouse model (ER-Cre-pRb^flox/flox) in which Rb1 can be acutely deleted postnatally. Acute Rb1 deletion in the adult mouse fails to induce proliferation or cell death in inner ear, strongly indicating that Rb1 loss in these postmitotic tissues can be effectively compensated for, or that pRb-mediated changes in the postmitotic compartment result in events that are functionally irreversible once enacted. This study thus supports the concept that pRb-regulated pathways relevant to hair cell development, encompassing proliferation, differentiation and survival, act predominantly during early development.     

Identification of stage-specific markers during differentiation of hair cells from mouse inner ear stem cells or progenitor cells in vitro

The induction of inner ear hair cells from stem cells or progenitor cells in the inner ear proceeds through a committed inner ear sensory progenitor cell stage prior to hair cell differentiation. To increase the efficacy of inducing inner ear hair cell differentiation from the stem cells or progenitor cells, it is essential to identify comprehensive markers for the stem cells/progenitor cells from the inner ear, the committed inner ear sensory progenitor cells and the differentiating hair cells to optimize induction conditions. Here, we report that we efficiently isolated and expanded the stem cells or progenitor cells from postnatal mouse cochleae, and induced the generation of inner ear progenitor cells and subsequent differentiation of hair cells. We profiled the gene expression of the stem cells or progenitor cells, the inner ear progenitor cells, and hair cells using aRNA microarray analysis. The pathway and gene ontology (GO) analysis of differentially expressed genes was performed. Analysis of genes exclusively detected in one particular cellular population revealed 30, 38, and 31 genes specific for inner ear stem cells, inner ear progenitor cells, and hair cells, respectively. We further examined the expression of these genes in vivo and determined that Gdf10+Ccdc121, Tmprss9+Orm1, and Chrna9+Espnl are marker genes specific for inner ear stem cells, inner ear progenitor cells, and differentiating hair cells, respectively. The identification of these marker genes will likely help the effort to increase the efficacy of hair cell induction from the stem cells or progenitor cells.     

G1 cyclin/cyclin-dependent kinase-coordinated phosphorylation of endogenous pocket proteins differentially regulates their interactions with E2F4 and E2F1 and gene expression

Mitogenic stimulation leads to activation of G(1) cyclin-dependent kinases (CDKs), which phosphorylate pocket proteins and trigger progression through the G(0)/G(1) and G(1)/S transitions of the cell cycle. However, the individual role of G(1) cyclin-CDK complexes in the coordinated regulation of pocket proteins and their interaction with E2F family members is not fully understood. Here we report that individually or in concert cyclin D1-CDK and cyclin E-CDK complexes induce distinct and coordinated phosphorylation of endogenous pocket proteins, which also has distinct consequences in the regulation of pocket protein interactions with E2F4 and the expression of p107 and E2F1, both E2F-regulated genes. The up-regulation of these two proteins and the release of p130 and pRB from E2F4 complexes allows formation of E2F1 complexes not only with pRB but also with p130 and p107 as well as the formation of p107-E2F4 complexes. The formation of these complexes occurs in the presence of active cyclin D1-CDK and cyclin E-CDK complexes, indicating that whereas phosphorylation plays a role in the abrogation of certain pocket protein/E2F interactions, these same activities induce the formation of other complexes in the context of a cell expressing endogenous levels of pocket and E2F proteins. Of note, phosphorylated p130 "form 3," which does not interact with E2F4, readily interacts with E2F1. Our data also demonstrate that ectopic overexpression of either cyclin is sufficient to induce mitogen-independent growth in human T98G and Rat-1 cells, although the effects of cyclin D1 require downstream activation of cyclin E-CDK2 activity. Interestingly, in T98G cells, cyclin D1 induces cell cycle progression more potently than cyclin E. This suggests that cyclin D1 activates pathways independently of cyclin E that ensure timely progression through the cell cycle.     

Molecular control of brain size: Regulators of neural stem cell life, death and beyond

The proper development of the brain and other organs depends on multiple parameters, including strictly controlled expansion of specific progenitor pools. The regulation of such expansion events includes enzymatic activities that govern the correct number of specific cells to be generated via an orchestrated control of cell proliferation, cell cycle exit, differentiation, cell death etc. Certain proteins in turn exert direct control of these enzymatic activities and thus progenitor pool expansion and organ size. The members of the Cip/Kip family (p21Cip1/p27Kip1/p57Kip2) are well-known regulators of cell cycle exit that interact with and inhibit the activity of cyclin-CDK complexes, whereas members of the p53/p63/p73 family are traditionally associated with regulation of cell death. It has however become clear that the roles for these proteins are not as clear-cut as initially thought. In this review, we discuss the roles for proteins of the Cip/Kip and p53/p63/p73 families in the regulation of cell cycle control, differentiation, and death of neural stem cells. We suggest that these proteins act as molecular interfaces, or “pilots”, to assure the correct assembly of protein complexes with enzymatic activities at the right place at the right time, thereby regulating essential decisions in multiple cellular events.     

Overlapping and distinct pRb pathways in the mammalian auditory and vestibular organs

Retinoblastoma gene (Rb1) is required for proper cell cycle exit in the developing mouse inner ear and its deletion in the embryo leads to proliferation of sensory progenitor cells that differentiate into hair cells and supporting cells. In a conditional hair cell Rb1 knockout mouse, Pou4f3-Cre-pRb^™/™, pRb^™/™ utricular hair cells differentiate and survive into adulthood whereas differentiation and survival of pRb^™/™ cochlear hair cells are impaired. To comprehensively survey the pRb pathway in the mammalian inner ear, we performed microarray analysis of pRb^™/™ cochlea and utricle. The comparative analysis shows that the core pathway shared between pRb^™/™ cochlea and utricle is centered on e2F, the key pathway that mediates pRb function. A majority of differentially expressed genes and enriched pathways are not shared but uniquely associated with pRb^™/™ cochlea or utricle. In pRb^™/™ cochlea, pathways involved in early inner ear development such as Wnt/β-catenin and Notch were enriched, whereas pathways involved in proliferation and survival are enriched in pRb^™/™ utricle. Clustering analysis showed that the pRb^™/™ inner ear has characteristics of a younger control inner ear, an indication of delayed differentiation. We created a transgenic mouse model (ER-Cre-pRb^flox/flox) in which Rb1 can be acutely deleted postnatally. Acute Rb1 deletion in the adult mouse fails to induce proliferation or cell death in inner ear, strongly indicating that Rb1 loss in these postmitotic tissues can be effectively compensated for, or that pRb-mediated changes in the postmitotic compartment result in events that are functionally irreversible once enacted. This study thus supports the concept that pRb-regulated pathways relevant to hair cell development, encompassing proliferation, differentiation and survival, act predominantly during early development.Key words: hair cells, retinoblastoma, Rb1, proliferation, regeneration, apoptosis, inner ear     

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.     

Hes1 is a negative regulator of inner ear hair cell differentiation

Hair cell fate determination in the inner ear has been shown to be controlled by specific genes. Recent loss-of-function and gain-of-function experiments have demonstrated that Math1, a mouse homolog of the Drosophila gene atonal, is essential for the production of hair cells. To identify genes that may interact with Math1 and inhibit hair cell differentiation, we have focused on Hes1, a mammalian hairy and enhancer of split homolog, which is a negative regulator of neurogenesis. We report here that targeted deletion of Hes1 leads to formation of supernumerary hair cells in the cochlea and utricle of the inner ear. RT-PCR analysis shows that Hes1 is expressed in inner ear during hair cell differentiation and its expression is maintained in adulthood. In situ hybridization with late embryonic inner ear tissue reveals that Hes1 is expressed in supporting cells, but not hair cells, of the vestibular sensory epithelium. In the cochlea, Hes1 is selectively expressed in the greater epithelial ridge and lesser epithelial ridge regions which are adjacent to inner and outer hair cells. Co-transfection experiments in postnatal rat explant cultures show that overexpression of Hes1 prevents hair cell differentiation induced by Math1. Therefore Hes1 can negatively regulate hair cell differentiation by antagonizing Math1. These results suggest that a balance between Math1 and negative regulators such as Hes1 is crucial for the production of an appropriate number of inner ear hair cells.     

LEK1 is a potential inhibitor of pocket protein-mediated cellular processes

LEK1, a member of the LEK family of proteins, is ubiquitously expressed in developing murine tissues. Our current studies are aimed at identifying the role of LEK1 during cell growth and differentiation. Little is known about the function of LEK proteins. Recent studies in our laboratory have focused on the characterization of the LEK1 atypical Rb-binding domain that is conserved among all LEK proteins. Our findings suggest that LEK1 potentially functions as a universal regulator of pocket protein activity. Pocket proteins exhibit distinct expression patterns during development and function to regulate cell cycle, apoptosis, and tissue-specific gene expression. We show that LEK1 interacts with all three pocket proteins, p107, p130, and pRb. Additionally, this interaction occurs specifically between the LEK1 Rb-binding motif and the "pocket domain" of Rb proteins responsible for Rb association with other targets. Analyses of the effects of disruption of LEK1 protein expression by morpholino oligomers demonstrate that LEK1 depletion decreases cell proliferation, disrupts cell cycle progression, and induces apoptosis. Given its expression in developing cells, its association with pocket proteins, and its effects on proliferation, cell cycle, and viability of cells, we suggest that LEK1 functions in a similar manner to phosphorylation to disrupt association of Rb proteins with appropriate binding targets. Thus, the LEK1/Rb interaction serves to retain cells in a pre-differentiative, actively proliferative state despite the presence of Rb proteins during development. Our data suggest that LEK1 is unique among LEK family members in that it specifically functions during murine development to regulate the activity of Rb proteins during cell division and proliferation. Furthermore, we discuss the distinct possibility that a yet unidentified splice variant of the closely related human CENP-F, serves a similar function to LEK1 in humans.     

KRAB型锌指蛋白(KZNF)的研究进展

KRAB型锌指蛋白是哺乳动物中最大的转录调控因子家族, 它的多数成员在基因组上成簇分布。其结构特征是N端含有KRAB结构域, C端含有多个C2H2型锌指结构。KRAB结构域为一蛋白质-蛋白质相互作用区, 可以与多种协同转录抑制因子和转录因子结合, 使KRAB型锌指蛋白作为转录因子和/或转录调控因子发挥依赖于DNA结合的转录抑制功能, 在胚胎发育、细胞分化、细胞转化及细胞周期的调控中发挥重要功能。