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1.  Alterations in Size, Number, and Morphology of Gustatory Papillae and Taste Buds in BDNF Null Mutant Mice Demonstrate Neural Dependence of Developing Taste Organs 
Sensory ganglia that innervate taste buds and gustatory papillae (geniculate and petrosal) are reduced in volume by about 40% in mice with a targeted deletion of the gene for brain-derived neurotrophic factor (BDNF). In contrast, the trigeminal ganglion, which innervates papillae but not taste buds on the anterior tongue, is reduced by only about 18%. These specific alterations in ganglia that innervate taste organs make possible a test for roles of lingual innervation in the development of appropriate number, morphology, and spatial pattern of fungiform and circumvallate papillae and associated taste buds. We studied tongues of BDNF null mutant and wild-type littermates and made quantitative analyses of all fungiform papillae on the anterior tongue, the single circumvallate papilla on the posterior tongue, and all taste buds in both papilla types. Fungiform papillae and taste buds were reduced in number by about 60% and were substantially smaller in diameter in mutant mice 15–25 days postnatal. Remaining fungiform papillae were selectively concentrated in the tongue tip region. The circumvallate papilla was reduced in diameter and length by about 40%, and papilla morphology was disrupted. Taste bud number in the circumvallate was reduced by about 70% in mutant tongues, and the remaining taste buds were smaller than those on wild-type tongues. Our results demonstrate a selective dependence of taste organs on a full complement of appropriate innervation for normal growth and morphogenesis. Effects on papillae are not random but are more pronounced in specific lingual regions. Although the geniculate and petrosal ganglia sustain at least half of their normal complement of cell number in BDNF −/− mice, remaining ganglion cells do not substitute for lost neurons to rescue taste organs at control numbers. Whereas gustatory ganglia and the taste papillae initially form independently, our results suggest interdependence in later development because ganglia derive BDNF support from target organs and papillae require sensory innervation for morphogenesis.
PMCID: PMC2710125  PMID: 10363708
fungiform papilla; circumvallate papilla; geniculate ganglion; petrosal ganglion; trigeminal ganglion
2.  Mice lacking the p75 receptor fail to acquire a normal complement of taste buds and geniculate ganglion neurons by adulthood 
Brain derived neurotrophic factor and neurotrophin-4 are required for normal taste bud development. Although these neurotrophins normally function via the tyrosine kinase receptor, trkB, they also bind to the pan-neurotrophin receptor, p75. The goal of the present study was to determine whether the p75 receptor is required for the development or maintenance of a full complement of adult taste buds. Mice with p75 null mutations lose 34% of their circumvallate taste buds, 36% of their fungiform papillae, and 26% of their fungiform taste buds by adulthood. The reduction of taste buds in the adult circumvallate papilla was similar to that observed previously at postnatal day 7 ((Fan et al., 2004). Taken together, these findings indicate that the p75 receptor is critical for the development of a full complement of taste buds, but is not required for maintenance of circumvallate taste buds in adulthood. Immunolabeling for p75 was not observed in taste buds, indicating that p75 signaling influences taste bud number indirectly. However, geniculate ganglion neurons, which provides innervation to fungiform taste buds, express the p75 receptor. Mice with p75-null mutations also have fewer neurons in the geniculate ganglion. Together, these results suggest that survival of geniculate neurons is essential for the development of a full complement of taste buds.
PMCID: PMC2559943  PMID: 17083122
taste buds; p75 receptor; neurotrophin; geniculate ganglion
3.  A single-nucleotide polymorphism in the human p27kip1 gene (-838C>A) affects basal promoter activity and the risk of myocardial infarction 
BMC Biology  2004;2:5.
Excessive proliferation of vascular smooth muscle cells and leukocytes within the artery wall is a major event in the development of atherosclerosis. The growth suppressor p27kip1 associates with several cyclin-dependent kinase/cyclin complexes, thereby abrogating their capacity to induce progression through the cell cycle. Recent studies have implicated p27kip1 in the control of neointimal hyperplasia. For instance, p27kip1 ablation in apolipoprotein-E-null mice enhanced arterial cell proliferation and accelerated atherogenesis induced by dietary cholesterol. Therefore, p27kip1 is a candidate gene to modify the risk of developing atherosclerosis and associated ischaemic events (i.e., myocardial infarction and stroke).
In this study we found three common single-nucleotide polymorphisms in the human p27kip1 gene (+326T>G [V109G], -79C>T, and -838C>A). The frequency of -838A carriers was significantly increased in myocardial infarction patients compared to healthy controls (odds ratio [OR] = 1.73, 95% confidence interval [95%CI] = 1.12–2.70). In addition, luciferase reporter constructs driven by the human p27kip1 gene promoter containing A at position -838 had decreased basal transcriptional activity when transiently transfected in Jurkat cells, compared with constructs bearing C in -838 (P = 0.04).
These data suggest that -838A is associated with reduced p27kip1 promoter activity and increased risk of myocardial infarction.
PMCID: PMC400507  PMID: 15061869
myocardial infarction; p27kip1; single-nucleotide polymorphisms
4.  Fungiform Taste Bud Degeneration in C57BL/6J Mice Following Chorda-Lingual Nerve Transection 
Taste buds are dependent on innervation for normal morphology and function. Fungiform taste bud degeneration after chorda tympani nerve injury has been well documented in rats, hamsters, and gerbils. The current study examines fungiform taste bud distribution and structure in adult C57BL/6J mice from both intact taste systems and after unilateral chorda-lingual nerve transection. Fungiform taste buds were visualized and measured with the aid of cytokeratin 8. In control mice, taste buds were smaller and more abundant on the anterior tip (<1 mm) of the tongue. By 5 days after nerve transection taste buds were smaller and fewer on the side of the tongue ipsilateral to the transection and continued to decrease in both size and number until 15 days posttransection. Degenerating fungiform taste buds were smaller due to a loss of taste bud cells rather than changes in taste bud morphology. While almost all taste buds disappeared in more posterior fungiform papillae by 15 days posttransection, the anterior tip of the tongue retained nearly half of its taste buds compared to intact mice. Surviving taste buds could not be explained by an apparent innervation from the remaining intact nerves. Contralateral effects of nerve transection were also observed; taste buds were larger due to an increase in the number of taste bud cells. These data are the first to characterize adult mouse fungiform taste buds and subsequent degeneration after unilateral nerve transection. They provide the basis for more mechanistic studies in which genetically engineered mice can be used.
PMCID: PMC2811721  PMID: 17626272
receptor cells; gustation; axotomy; cyokeratin; neurofilament
5.  Analysis of Cyclin D3-cdk4 Complexes in Fibroblasts Expressing and Lacking p27kip1 and p21cip1 
Molecular and Cellular Biology  2000;20(23):8748-8757.
Our studies examined the effects of p27kip1 and p21cip1 on the assembly and activity of cyclin D3-cdk4 complexes and determined the composition of the cyclin D3 pool in cells containing and lacking these cyclin-dependent kinase inhibitors. We found that catalytically active cyclin D3-cdk4 complexes were present in fibroblasts derived from p27kip1-p21cip1-null mice and that immunodepletion of extracts of wild-type cells with antibody to p27kip1 and/or p21cip1 removed cyclin D3 protein but not cyclin D3-associated activity. Similar results were observed in experiments assaying cyclin D1-cdk4 activity. Data obtained using mixed cell extracts demonstrated that p27kip1 interacted with cyclin D3-cdk4 complexes in vitro and that this interaction was paralleled by a loss of cyclin D3-cdk4 activity. In p27kip1-p21cip1-deficient cells, the cyclin D3 pool consisted primarily of cyclin D3 monomers, whereas in wild-type cells, the majority of cyclin D3 molecules were complexed to cdk4 and either p27kip1 or p21cip1 or were monomeric. We conclude that neither p27kip1 nor p21cip1 is required for the formation of cyclin D3-cdk4 complexes and that cyclin D3-cdk4 complexes containing p27kip1 or p21cip1 are inactive. We suggest that only a minor portion of the total cyclin D3 pool accounts for all of the cyclin D3-cdk4 activity in the cell regardless of whether the cell contains p27kip1 and p21cip1.
PMCID: PMC86501  PMID: 11073976
6.  Gustatory stimuli representing different perceptual qualities elicit distinct patterns of neuropeptide secretion from taste buds 
Taste stimuli that evoke different perceptual qualities (e.g., sweet, umami, bitter, sour, salty) are detected by dedicated subpopulations of taste bud cells that employ distinct combinations of sensory receptors and transduction molecules. Here, we report that taste stimuli also elicit unique patterns of neuropeptide secretion from taste buds that are correlated with those perceptual qualities. We measured tastant-dependent secretion of glucagon-like peptide-1 (GLP-1), glucagon and neuropeptide Y (NPY) from circumvallate papillae of Tas1r3+/+, +/− and −/− mice. Isolated tongue epithelia were mounted in modified Ussing chambers, permitting apical stimulation of taste buds; secreted peptides were collected from the basal side and measured by specific ELISAs. Appetitive stimuli (sweet: glucose, sucralose; umami: monosodium glutamate; polysaccharide: Polycose) elicited GLP-1 and NPY secretion and inhibited basal glucagon secretion. Sweet and umami stimuli were ineffective in Tas1r3−/− mice, indicating an obligatory role for the T1R3 subunit common to the sweet and umami taste receptors. Polycose responses were unaffected by T1R3 deletion, consistent with the presence of a distinct polysaccharide taste receptor. The effects of sweet stimuli on peptide secretion also required the closing of ATP-sensitive K+ (KATP) channels, as the KATP channel activator diazoxide inhibited the effects of glucose and sucralose on both GLP-1 and glucagon release. Both sour citric acid and salty NaCl increased NPY secretion but had no effects on GLP-1 or glucagon. Bitter denatonium showed no effects on these peptides. Together, these results suggest that taste stimuli of different perceptual qualities elicit unique patterns of neuropeptide secretion from taste buds.
PMCID: PMC3687534  PMID: 23616560
7.  Differential expression of a BMP4 reporter allele in anterior fungiform versus posterior circumvallate taste buds of mice 
BMC Neuroscience  2010;11:129.
Bone Morphogenetic Protein 4 (BMP4) is a diffusible factor which regulates embryonic taste organ development. However, the role of BMP4 in taste buds of adult mice is unknown. We utilized transgenic mice with LacZ under the control of the BMP4 promoter to reveal the expression of BMP4 in the tongues of adult mice. Further we evaluate the pattern of BMP4 expression with that of markers of specific taste bud cell types and cell proliferation to define and compare the cell populations expressing BMP4 in anterior (fungiform papillae) and posterior (circumvallate papilla) tongue.
BMP4 is expressed in adult fungiform and circumvallate papillae, i.e., lingual structures composed of non-taste epithelium and taste buds. Unexpectedly, we find both differences and similarities with respect to expression of BMP4-driven ß-galactosidase. In circumvallate papillae, many fusiform cells within taste buds are BMP4-ß-gal positive. Further, a low percentage of BMP4-expressing cells within circumvallate taste buds is immunopositive for markers of each of the three differentiated taste cell types (I, II and III). BMP4-positive intragemmal cells also expressed a putative marker of immature taste cells, Sox2, and consistent with this finding, intragemmal cells expressed BMP4-ß-gal within 24 hours after their final mitosis, as determined by BrdU birthdating. By contrast, in fungiform papillae, BMP4-ß-gal positive cells are never encountered within taste buds. However, in both circumvallate and fungiform papillae, BMP4-ß-gal expressing cells are located in the perigemmal region, comprising basal and edge epithelial cells adjacent to taste buds proper. This region houses the proliferative cell population that gives rise to adult taste cells. However, perigemmal BMP4-ß-gal cells appear mitotically silent in both fungiform and circumvallate taste papillae, as we do not find evidence of their active proliferation using cell cycle immunomarkers and BrdU birthdating.
Our data suggest that intragemmal BMP4-ß-gal cells in circumvallate papillae are immature taste cells which eventually differentiate into each of the 3 taste cell types, whereas perigemmal BMP4-ß-gal cells in both circumvallate and fungiform papillae may be slow cycling stem cells, or belong to the stem cell niche to regulate taste cell renewal from the proliferative cell population.
PMCID: PMC2966460  PMID: 20942907
8.  MAD1 and p27KIP1 Cooperate To Promote Terminal Differentiation of Granulocytes and To Inhibit Myc Expression and Cyclin E-CDK2 Activity 
Molecular and Cellular Biology  2002;22(9):3014-3023.
To understand how cellular differentiation is coupled to withdrawal from the cell cycle, we have focused on two negative regulators of the cell cycle, the MYC antagonist MAD1 and the cyclin-dependent kinase inhibitor p27KIP1. Generation of Mad1/p27KIP1 double-null mice revealed a number of synthetic effects between the null alleles of Mad1 and p27KIP1, including embryonic lethality, increased proliferation, and impaired differentiation of granulocyte precursors. Furthermore, with granulocyte cell lines derived from the Mad1/p27KIP1 double-null mice, we observed constitutive Myc expression and cyclin E-CDK2 kinase activity as well as impaired differentiation following treatment with an inducer of differentiation. By contrast, similar treatment of granulocytes from Mad1 or p27KIP1 single-null mice resulted in differentiation accompanied by downregulation of both Myc expression and cyclin E-CDK2 kinase activity. In the double-null granulocytic cells, addition of a CDK2 inhibitor in the presence of differentiation inducer was sufficient to restore differentiation and reduce Myc levels. We conclude that Mad1 and p27KIP1 operate, at least in part, by distinct mechanisms to downregulate CDK2 activity and Myc expression in order to promote cell cycle exit during differentiation.
PMCID: PMC133749  PMID: 11940659
9.  Genome-Wide Analysis of Gene Expression in Primate Taste Buds Reveals Links to Diverse Processes 
PLoS ONE  2009;4(7):e6395.
Efforts to unravel the mechanisms underlying taste sensation (gustation) have largely focused on rodents. Here we present the first comprehensive characterization of gene expression in primate taste buds. Our findings reveal unique new insights into the biology of taste buds. We generated a taste bud gene expression database using laser capture microdissection (LCM) procured fungiform (FG) and circumvallate (CV) taste buds from primates. We also used LCM to collect the top and bottom portions of CV taste buds. Affymetrix genome wide arrays were used to analyze gene expression in all samples. Known taste receptors are preferentially expressed in the top portion of taste buds. Genes associated with the cell cycle and stem cells are preferentially expressed in the bottom portion of taste buds, suggesting that precursor cells are located there. Several chemokines including CXCL14 and CXCL8 are among the highest expressed genes in taste buds, indicating that immune system related processes are active in taste buds. Several genes expressed specifically in endocrine glands including growth hormone releasing hormone and its receptor are also strongly expressed in taste buds, suggesting a link between metabolism and taste. Cell type-specific expression of transcription factors and signaling molecules involved in cell fate, including KIT, reveals the taste bud as an active site of cell regeneration, differentiation, and development. IKBKAP, a gene mutated in familial dysautonomia, a disease that results in loss of taste buds, is expressed in taste cells that communicate with afferent nerve fibers via synaptic transmission. This database highlights the power of LCM coupled with transcriptional profiling to dissect the molecular composition of normal tissues, represents the most comprehensive molecular analysis of primate taste buds to date, and provides a foundation for further studies in diverse aspects of taste biology.
PMCID: PMC2712080  PMID: 19636377
10.  FGF Signaling Regulates the Number of Posterior Taste Papillae by Controlling Progenitor Field Size 
PLoS Genetics  2011;7(6):e1002098.
The sense of taste is fundamental to our ability to ingest nutritious substances and to detect and avoid potentially toxic ones. Sensory taste buds are housed in papillae that develop from epithelial placodes. Three distinct types of gustatory papillae reside on the rodent tongue: small fungiform papillae are found in the anterior tongue, whereas the posterior tongue contains the larger foliate papillae and a single midline circumvallate papilla (CVP). Despite the great variation in the number of CVPs in mammals, its importance in taste function, and its status as the largest of the taste papillae, very little is known about the development of this structure. Here, we report that a balance between Sprouty (Spry) genes and Fgf10, which respectively antagonize and activate receptor tyrosine kinase (RTK) signaling, regulates the number of CVPs. Deletion of Spry2 alone resulted in duplication of the CVP as a result of an increase in the size of the placode progenitor field, and Spry1−/−;Spry2−/− embryos had multiple CVPs, demonstrating the redundancy of Sprouty genes in regulating the progenitor field size. By contrast, deletion of Fgf10 led to absence of the CVP, identifying FGF10 as the first inductive, mesenchyme-derived factor for taste papillae. Our results provide the first demonstration of the role of epithelial-mesenchymal FGF signaling in taste papilla development, indicate that regulation of the progenitor field size by FGF signaling is a critical determinant of papilla number, and suggest that the great variation in CVP number among mammalian species may be linked to levels of signaling by the FGF pathway.
Author Summary
The sense of taste is important for an animal's ability to survive and thrive, because it enables discrimination between nutritious substances and toxins. Taste buds are housed largely on the tongue in structures called papillae; of the three types of gustatory papillae, the circumvallate papilla (CVP) is the largest. In rodents, a single CVP is located in the posterior midline of the tongue housing hundreds of taste buds, whereas in other mammals up to dozens of CVPs can be found. However, despite the great variation in the number of CVPs in mammals, its status as the largest of the taste papillae, and its importance in taste function, very little is known about its development. We identified members of the FGF signaling pathway as determinants of CVP number. We propose that perturbations to the FGF signaling pathway may have been involved in the dramatic differences in CVP number that arose during mammalian evolution.
PMCID: PMC3107195  PMID: 21655085
11.  Functional Cell Types in Taste Buds Have Distinct Longevities 
PLoS ONE  2013;8(1):e53399.
Taste buds are clusters of polarized sensory cells embedded in stratified oral epithelium. In adult mammals, taste buds turn over continuously and are replenished through the birth of new cells in the basal layer of the surrounding non-sensory epithelium. The half-life of cells in mammalian taste buds has been estimated as 8–12 days on average. Yet, earlier studies did not address whether the now well-defined functional taste bud cell types all exhibit the same lifetime. We employed a recently developed thymidine analog, 5-ethynil-2′-deoxyuridine (EdU) to re-evaluate the incorporation of newly born cells into circumvallate taste buds of adult mice. By combining EdU-labeling with immunostaining for selected markers, we tracked the differentiation and lifespan of the constituent cell types of taste buds. EdU was primarily incorporated into basal extragemmal cells, the principal source for replenishing taste bud cells. Undifferentiated EdU-labeled cells began migrating into circumvallate taste buds within 1 day of their birth. Type II (Receptor) taste cells began to differentiate from EdU-labeled precursors beginning 2 days after birth and then were eliminated with a half-life of 8 days. Type III (Presynaptic) taste cells began differentiating after a delay of 3 days after EdU-labeling, and they survived much longer, with a half-life of 22 days. We also scored taste bud cells that belong to neither Type II nor Type III, a heterogeneous group that includes mostly Type I cells, and also undifferentiated or immature cells. A non-linear decay fit described these cells as two sub-populations with half-lives of 8 and 24 days respectively. Our data suggest that many post-mitotic cells may remain quiescent within taste buds before differentiating into mature taste cells. A small number of slow-cycling cells may also exist within the perimeter of the taste bud. Based on their incidence, we hypothesize that these may be progenitors for Type III cells.
PMCID: PMC3540047  PMID: 23320081
12.  Distribution and characterization of functional amiloride-sensitive sodium channels in rat tongue 
The Journal of General Physiology  1996;107(4):545-554.
The role of amiloride-sensitive Na+ channels (ASSCs) in the transduction of salty taste stimuli in rat fungiform taste buds has been well established. Evidence for the involvement of ASSCs in salt transduction in circumvallate and foliate taste buds is, at best, contradictory. In an attempt to resolve this apparent controversy, we have begun to look for functional ASSCs in taste buds isolated from fungiform, foliate, and circumvallate papillae of male Sprague-Dawley rats. By use of a combination of whole-cell and nystatin-perforated patch-clamp recording, cells within the taste bud that exhibited voltage-dependent currents, reflective of taste receptor cells (TRCs), were subsequently tested for amiloride sensitivity. TRCs were held at - 70 mV, and steady-state current and input resistance were monitored during superfusion of Na(+)-free saline and salines containing amiloride (0.1 microM to 1 mM). Greater than 90% of all TRCs from each of the papillae responded to Na+ replacement with a decrease in current and an increase in input resistance, reflective of a reduction in electrogenic Na+ movement into the cell. ASSCs were found in two thirds of fungiform and in one third of foliate TRCs, whereas none of the circumvallate TRCs was amiloride sensitive. These findings indicate that the mechanism for Na+ influx differs among taste bud types. All amiloride-sensitive currents had apparent inhibition constants in the submicromolar range. These results agree with afferent nerve recordings and raise the possibility that the extensive labeling of the ASSC protein and mRNA in the circumvallate papillae may reflect a pool of nonfunctional channels or a pool of channels that lacks sensitivity to amiloride.
PMCID: PMC2217005  PMID: 8722566
13.  Two tumor suppressors, p27Kip1 and Patched-1, collaborate to prevent medulloblastoma 
Two cyclin-dependent kinase inhibitors, p18Ink4c and p27Kip1, are required for proper cerebellar development. Loss of either of these proteins conferred a proliferative advantage to granule neuron progenitors, although inactivation of Kip1 exerted a greater effect. Mice heterozygous for Patched1 (Ptc1+/−) that are either heterozygous or nullizygous for Kip1 developed medulloblastoma (MB) rapidly and with high penetrance. All tumors from Ptc1+/−;Kip1+/− or Ptc1+/−;Kip1−/− mice failed to express the wild type Ptc1 allele, consistent with its role as a canonical “two-hit” tumor suppressor. In contrast, expression of the wild type p27Kip1 protein was invariably maintained in MBs arising in Ptc1+/−;Kip1+/− mice, indicating that Kip1 is haploinsufficient for tumor suppression. Although MBs occurring in Ptc1+/− mice were histopathologically heterogeneous and contained intermixed regions of both rapidly proliferating and nondividing more differentiated cells, tumors that also lacked Kip1 were uniformly less differentiated, more highly proliferative, and invasive. Molecular analysis showed that the latter MBs exhibited constitutive activation of the Sonic hedgehog signaling pathway without loss of functional p53. Apart from gains or losses of single chromosomes, with gain of chromosome 6 being the most frequent, no other chromosomal anomalies were identified by spectral karyotyping, and half of the MBs so examined retained a normal karyotype. In this respect, this mouse MB model recapitulates the vast majority of human MBs that do not sustain TP53 mutations and are not aneuploid.
PMCID: PMC2637533  PMID: 19147535
p27Kip1; p18Ink4c; Patched-1; medulloblastoma; cerebellum
14.  The Cyclin-Dependent Kinase Inhibitor p57Kip2 Regulates Cell Cycle Exit, Differentiation, and Migration of Embryonic Cerebral Cortical Precursors 
Cerebral Cortex (New York, NY)  2011;21(8):1840-1856.
Mounting evidence indicates cyclin-dependent kinase (CDK) inhibitors (CKIs) of the Cip/Kip family, including p57Kip2 and p27Kip1, control not only cell cycle exit but also corticogenesis. Nevertheless, distinct activities of p57Kip2 remain poorly defined. Using in vivo and culture approaches, we show p57Kip2 overexpression at E14.5–15.5 elicits precursor cell cycle exit, promotes transition from proliferation to neuronal differentiation, and enhances process outgrowth, while opposite effects occur in p57Kip2-deficient precursors. Studies at later ages indicate p57Kip2 overexpression also induces precocious glial differentiation, suggesting stage-dependent effects. In embryonic cortex, p57Kip2 overexpression advances cell radial migration and alters postnatal laminar positioning. While both CKIs induce differentiation, p57Kip2 was twice as effective as p27Kip1 in inducing neuronal differentiation and was not permissive to astrogliogenic effects of ciliary neurotrophic factor, suggesting that the CKIs differentially modulate cell fate decisions. At molecular levels, although highly conserved N-terminal regions of both CKIs elicit cycle withdrawal and differentiation, the C-terminal region of p57Kip2 alone inhibits in vivo migration. Furthermore, p57Kip2 effects on neurogenesis and gliogenesis require the N-terminal cyclin/CDK binding/inhibitory domains, while previous p27Kip1 studies report cell cycle-independent functions. These observations suggest p57Kip2 coordinates multiple stages of corticogenesis and exhibits distinct and common activities compared with related family member p27Kip1.
PMCID: PMC3138513  PMID: 21245411
gliogenesis; in utero electroporation; neurite outgrowth; neurogenesis; transfection
15.  p27Kip1 is required to maintain proliferative quiescence in the adult cochlea and pituitary 
Cell Cycle  2011;10(8):1237-1248.
Cell cycle inhibitors, such as the cyclin-dependent kinase (Cdk) inhibitor proteins and retinoblastoma (Rb) family members, control exit from the cell cycle during the development of a variety of terminally differentiated tissues. It is unclear whether sustained expression of these proteins is required to prevent cell cycle re-entry in quiescent and terminally differentiated cells. The organ of Corti (cochlear sensory epithelium) and pars intermedia (intermediate lobe of the pituitary) are two tissues that share the characteristic of ongoing cell division in mice lacking either the p27Kip1 Cdk inhibitor, Ink4 proteins or Rb. Here, we use tamoxifen-inducible mouse models to delete p27Kip1 in postnatal animals and show this is sufficient to induce proliferation in both the organ of Corti and pars intermedia. Thus, these tissues remain sensitive to the presence of p27Kip1 even after their developmental exit from the cell cycle. The neonatal cochlea displayed heightened sensitivity to changes in p27Kip1 expression, with a proliferative response higher than that of constitutive null mice. In adults, the proliferative response was reduced but was accompanied by increased cell survival. In contrast, re-establishment of normal p27Kip1 expression in animals with established pituitary tumors, in an inducible “knock-on” model, led to cessation of pituitary tumor growth, indicating the cells had maintained their susceptibility to p27-mediated growth suppression. Although restoration of p27Kip1 did not induce apoptosis, it did lead to resolution of pathological features and normalization of gene expression. Our data underscore the importance of p27Kip1 expression in the maintenance of cellular quiescence and terminal differentiation.
PMCID: PMC3230534  PMID: 21403466
proliferation; cell cycle; p27; Cdk inhibitor; auditory; cochlea; pituitary
16.  Tonic activity of Gα-gustducin regulates taste cell responsivity 
FEBS letters  2008;582(27):3783-3787.
The taste-selective G protein, α-gustducin (α-gus) is homologous to α-transducin and activates phosphodiesterase in vitro. α-Gustducin-knockout mice are compromised to bitter, sweet and umami taste stimuli, suggesting a central role in taste transduction. Here, we suggest a different role for Gα-gustducin. In taste buds of α-gustducin knockout mice, basal (unstimulated) cAMP levels are high compared to those of wild-type mice. Further, H-89, a cAMP-dependent Protein Kinase inhibitor, dramatically unmasks responses to the bitter tastant denatonium in gustducin-lineage cells of knockout mice. We propose that an important role of α-gustducin is to maintain cAMP levels tonically low to ensure adequate Ca2+ signaling.
PMCID: PMC2659647  PMID: 18930056
taste transduction; calcium signaling; phosphodiesterase; Protein Kinase A
17.  Regulation of Memory CD8 T-Cell Differentiation by Cyclin-Dependent Kinase Inhibitor p27Kip1▿ †  
Molecular and Cellular Biology  2010;30(21):5145-5159.
Induction of potent T-cell memory is the goal of vaccinations, but the molecular mechanisms that regulate the formation of memory CD8 T cells are not well understood. Despite the recognition that controls of cellular proliferation and apoptosis govern the number of memory T cells, the cell cycle regulatory mechanisms that control these key cellular processes in CD8 T cells during an immune response are poorly defined. Here, we have identified the cyclin-dependent kinase inhibitor p27Kip1 as a critical regulator of the CD8 T-cell homeostasis at all phases of the T-cell response to an acute viral infection in mice. By acting as a timer for cell cycle exit, p27Kip1 curtailed the programmed expansion of interleukin-2-producing memory precursors and markedly limited the magnitude and quality of CD8 T-cell memory. In the absence of p27Kip1, CD8 T cells showed superior recall responses shortly after vaccination with recombinant Listeria monocytogenes. Additionally, we show that p27Kip1 constrains proliferative renewal of memory CD8 T cells, especially of the effector memory subset. These findings provide critical insights into the cell cycle regulation of CD8 T-cell homeostasis and suggest that modulation of p27Kip1 could bolster vaccine-induced T-cell memory and protective immunity.
PMCID: PMC2953046  PMID: 20805358
18.  Expression of cyclin-dependent kinase inhibitor p27kip1 in malignant lymphomas. 
Journal of Korean Medical Science  2000;15(4):399-406.
p27kip1 is a cyclin-dependent kinase inhibitor that regulates progression from G1 into S phase. Aberrations in cell cycle control are often observed in tumors and might even be necessary in tumor development. Recent reports showed that low p27kip1 expression is associated with poor prognosis in several tumors and leukemia. To investigate the expression of p27kip1 in malignant lymphomas and elucidate the role of p27kip1 as a possible prognostic indicator, the authors performed an immunohistochemical staining of p27kip1 correlated with Ki-67 labelling index and clinical parameters. p27kip1 expression was reduced variably in most malignant lymphomas and inversely correlated with Ki-67 labelling index (p=0.0151). Regarding chemotherapeutic response, p271kip1 expression in the complete remission group showed statistically significant difference in expression compared to the progressive disease group (p=0.0021). There were significant differences in survival between cases with low and high p27kip1 expression (p=0.0071). In a multivariate Cox analysis, p27kip1 expression was independent prognostic factors as well as other known prognostic factors including age, grade, stage and chemotherapeutic response. In conclusion, the study suggests that reduced expression of p27kip1 protein may play a role in the pathogenesis and biologically aggressive behavior of malignant lymphomas.
PMCID: PMC3054650  PMID: 10983687
19.  T Cell-Intrinsic and Extrinsic Mechanisms of p27Kip1 in the Regulation of CD8 T Cell Memory 
Immunology and cell biology  2012;91(2):120-129.
CD8 T cells exhibit dynamic alterations in proliferation and apoptosis during various phases of the CD8 T cell response, but the mechanisms that regulate cellular proliferation from the standpoint of CD8 T cell memory are not well defined. The cyclin-dependent kinase inhibitor p27Kip1 functions as a negative regulator of the cell cycle in various cell types including T cells and it has been implicated in regulating cellular processes including differentiation, transcription and migration. Here, we investigated whether p27Kip1 regulates CD8 T cell memory by T cell-intrinsic or T cell-extrinsic mechanisms, by conditional ablation of p27Kip1 in T cells or non-T cells. Studies of T cell responses to an acute viral infection show that p27Kip1 negatively regulates the proliferation of CD8 T cells by T cell-intrinsic mechanisms. However, the enhanced proliferation of CD8 T cells induced by T cell-specific p27Kip1 deficiency minimally affects the primary expansion or the magnitude of CD8 T cell memory. Unexpectedly, p27Kip1 ablation in non-T cells markedly augmented the number of high quality memory CD8 T cells by enhancing the accumulation of memory precursor effector cells without increasing their proliferation. Further studies show that p27Kip1 deficiency in immunizing DCs fail to enhance CD8 T cell memory. Nevertheless, we have delineated the T cell-intrinsic, anti-proliferative activities of p27Kip1 in CD8 T cells from its role as a factor in non-T cells that restricts the development of CD8 T cell memory. These findings have implications in vaccine development and understanding the mechanisms that maintain T cell homeostasis.
PMCID: PMC3570738  PMID: 23207280
CD8 T cells; Cell cycle; Memory; p27Kip1; Proliferation
20.  Phosphorylation and Subcellular Localization of p27Kip1 Regulated by Hydrogen Peroxide Modulation in Cancer Cells 
PLoS ONE  2012;7(9):e44502.
The Cyclin-dependent kinase inhibitor 1B (p27Kip1) is a key protein in the decision between proliferation and cell cycle exit. Quiescent cells show nuclear p27Kip1, but this protein is exported to the cytoplasm in response to proliferating signals. We recently reported that catalase treatment increases the levels of p27Kip1 in vitro and in vivo in a murine model. In order to characterize and broaden these findings, we evaluated the regulation of p27Kip1 by hydrogen peroxide (H2O2) in human melanoma cells and melanocytes. We observed a high percentage of p27Kip1 positive nuclei in melanoma cells overexpressing or treated with exogenous catalase, while non-treated controls showed a cytoplasmic localization of p27Kip1. Then we studied the levels of p27Kip1 phosphorylated (p27p) at serine 10 (S10) and at threonine 198 (T198) because phosphorylation at these sites enables nuclear exportation of this protein, leading to accumulation and stabilization of p27pT198 in the cytoplasm. We demonstrated by western blot a decrease in p27pS10 and p27pT198 levels in response to H2O2 removal in melanoma cells, associated with nuclear p27Kip1. Melanocytes also exhibited nuclear p27Kip1 and lower levels of p27pS10 and p27pT198 than melanoma cells, which showed cytoplasmic p27Kip1. We also showed that the addition of H2O2 (0.1 µM) to melanoma cells arrested in G1 by serum starvation induces proliferation and increases the levels of p27pS10 and p27pT198 leading to cytoplasmic localization of p27Kip1. Nuclear localization and post-translational modifications of p27Kip1 were also demonstrated by catalase treatment of colorectal carcinoma and neuroblastoma cells, extending our findings to these other human cancer types. In conclusion, we showed in the present work that H2O2 scavenging prevents nuclear exportation of p27Kip1, allowing cell cycle arrest, suggesting that cancer cells take advantage of their intrinsic pro-oxidant state to favor cytoplasmic localization of p27Kip1.
PMCID: PMC3435274  PMID: 22970236
21.  Blockade of Protein Geranylgeranylation Inhibits Cdk2-Dependent p27Kip1 Phosphorylation on Thr187 and Accumulates p27Kip1 in the Nucleus: Implications for Breast Cancer Therapy▿ §  
Molecular and Cellular Biology  2009;29(8):2254-2263.
We describe the design of a potent and selective peptidomimetic inhibitor of geranylgeranyltransferase I (GGTI), GGTI-2418, and its methyl ester GGTI-2417, which increases the levels of the cyclin-dependent kinase (Cdk) inhibitor p27Kip1 and induces breast tumor regression in vivo. Experiments with p27Kip1 small interfering RNA in breast cancer cells and p27Kip1 null murine embryonic fibroblasts demonstrate that the ability of GGTI-2417 to induce cell death requires p27Kip1. GGTI-2417 inhibits the Cdk2-mediated phosphorylation of p27Kip1 at Thr187 and accumulates p27Kip1 in the nucleus. In nude mouse xenografts, GGTI-2418 suppresses the growth of human breast tumors. Furthermore, in ErbB2 transgenic mice, GGTI-2418 increases p27Kip1 and induces significant regression of breast tumors. We conclude that GGTIs' antitumor activity is, at least in part, due to inhibiting Cdk2-dependent p27Kip1 phosphorylation at Thr187 and accumulating nuclear p27Kip1. Thus, GGTI treatment might improve the poor prognosis of breast cancer patients with low nuclear p27Kip1 levels.
PMCID: PMC2663293  PMID: 19204084
22.  Taste cell formation does not require gustatory and somatosensory innervation 
Neuroscience letters  2010;471(3):189-194.
Dependency of taste buds and taste papillae on innervation has been debated for a long time. Previous research showed neurotrophins, Brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3), play an important role for the establishment of the lingual gustatory and somatosensory innervation. BDNF null mutant mice showed severe deficits in gustatory innervation and loss of taste buds while NT-3 null mutation reduced lingual somatosensory innervation to tongue papillae. These results proved BDNF or NT-3 null mutations affected different sensory modalities (i.e. gustatory and somatosensory, respectively). In this study, we analyzed taste bud development in BDNFxNT-3 double knockout mice to examine the relationship between taste bud development and gustatory/somatosentory innervation. Our results demonstrate that, at the initial stage, before nerve fibers reached the appropriate areas in the papilla, taste bud formation did not require innervation. However, at the synaptogenic stage, after nerve fibers ramified into the apical epithelium, innervation was required and played an essential role in the development of taste buds/papillae.
PMCID: PMC2830000  PMID: 20109530
gustation; neurotrophin; development; taste buds; BDNF; NT-3
23.  Defects in retinal pigment epithelium cell proliferation and retinal attachment in mutant mice with p27Kip1 gene ablation 
Molecular Vision  2007;13:273-286.
Little is known about the mechanisms that regulate cell cycle withdrawal of the retinal pigment epithelium (RPE) during development, or about the mechanisms maintaining epithelial cell quiescence in adult retinas. The present study examines the potential role of the negative cell cycle regulator p27Kip1 in controlling RPE proliferation, using mice with targeted ablation of the p27Kip1 gene.
Ocular tissues were obtained from wild-type and p27Kip1-null mice at several postnatal ages. Following aldehyde fixation, eyes were processed intact for JB-4 histology and electron microscopy. Alternatively, tissues were removed by manual or enzymatic dissection in order to obtain flat-mounts of the RPE attached to either the choroid-sclera or neural retina, respectively. Epithelial flat-mounts were either left unlabeled, in which case melanin pigment provided internal contrast, or labeled with Alexa Fluor 488-phalloidin and propidium iodide to visualize cell boundaries and nuclei, respectively.
Morphometric analysis using transverse plastic sections revealed a 96% increase in nuclear density and a 12% increase in thickness (apical to basal) for mutant vs. normal epithelia at postnatal day 35 (P35). These changes were not restricted to central or peripheral regions, and were uncorrelated with focal areas of dysplasia seen in the mutant neural retina. When similar tissues were viewed as flat-mounts, an observed 100% increase in nuclear density was accompanied by only a 46% enhancement in cellular density. This resulted in a larger proportion of multinucleated cells in the nullizygous RPE as compared with the wild-type epithelium (91 versus 47%). Such a pattern was achieved relatively early in development since, at P7 when the increase in RPE nuclear density was essentially complete, cellular density was augmented by only 39%. In addition to these proliferative changes, individual epithelial cells sometimes exhibited structural abnormalities, including an altered cortical actin cytoskeleton and displacement of nuclei from their normal central position. Surprisingly, while the RPE cells of null animals were similar ultrastructurally to those of the wild-type, interdigitation of their microvillous processes with outer segments was incomplete. Quantitative analysis revealed that such areas of detachment characterize, on average, 42% of the nullizygous retina, and that there is little correlation between detachment and neural retina dysplasia from one eye to another. Together with parallel evidence demonstrating a substantial decline in the apparent adhesiveness of mutant retinas relative to the normal tissue, the data is strongly indicative of an altered epithelium-photoreceptor interaction following gene ablation.
The absence of a functional p27Kip1 gene results in enhanced RPE nuclear division, without a commensurate increase in cell division. Although the mutant epithelium as a whole appears structurally normal, individual cells exhibit cytoskeletal changes and their interaction with the neural retina is compromised.
PMCID: PMC2633469  PMID: 17356514
24.  Lipopolysaccharide-induced inflammation attenuates taste progenitor cell proliferation and shortens the life span of taste bud cells 
BMC Neuroscience  2010;11:72.
The mammalian taste bud, a complex collection of taste sensory cells, supporting cells, and immature basal cells, is the structural unit for detecting taste stimuli in the oral cavity. Even though the cells of the taste bud undergo constant turnover, the structural homeostasis of the bud is maintained by balancing cell proliferation and cell death. Compared with nongustatory lingual epithelial cells, taste cells express higher levels of several inflammatory receptors and signalling proteins. Whether inflammation, an underlying condition in some diseases associated with taste disorders, interferes with taste cell renewal and turnover is unknown. Here we report the effects of lipopolysaccharide (LPS)-induced inflammation on taste progenitor cell proliferation and taste bud cell turnover in mouse taste tissues.
Intraperitoneal injection of LPS rapidly induced expression of several inflammatory cytokines, including tumor necrosis factor (TNF)-α, interferon (IFN)-γ, and interleukin (IL)-6, in mouse circumvallate and foliate papillae. TNF-α and IFN-γ immunoreactivities were preferentially localized to subsets of cells in taste buds. LPS-induced inflammation significantly reduced the number of 5-bromo-2'-deoxyuridine (BrdU)-labeled newborn taste bud cells 1-3 days after LPS injection, suggesting an inhibition of taste bud cell renewal. BrdU pulse-chase experiments showed that BrdU-labeled taste cells had a shorter average life span in LPS-treated mice than in controls. To investigate whether LPS inhibits taste cell renewal by suppressing taste progenitor cell proliferation, we studied the expression of Ki67, a cell proliferation marker. Quantitative real-time RT-PCR revealed that LPS markedly reduced Ki67 mRNA levels in circumvallate and foliate epithelia. Immunofluorescent staining using anti-Ki67 antibodies showed that LPS decreased the number of Ki67-positive cells in the basal regions surrounding circumvallate taste buds, the niche for taste progenitor cells. PCR array experiments showed that the expression of cyclin B2 and E2F1, two key cell cycle regulators, was markedly downregulated by LPS in the circumvallate and foliate epithelia.
Our results show that LPS-induced inflammation inhibits taste progenitor cell proliferation and interferes with taste cell renewal. LPS accelerates cell turnover and modestly shortens the average life span of taste cells. These effects of inflammation may contribute to the development of taste disorders associated with infections.
PMCID: PMC2898829  PMID: 20537148
25.  Distinct Developmental Roles of Cell Cycle Inhibitors p57Kip2 and p27Kip1 Distinguish Pituitary Progenitor Cell Cycle Exit from Cell Cycle Reentry of Differentiated Cells▿ † 
Molecular and Cellular Biology  2009;29(7):1895-1908.
Patterning and differentiation signals are often believed to drive the developmental program, including cell cycle exit of proliferating progenitors. Taking advantage of the spatial and temporal separation of proliferating and differentiated cells within the developing anterior pituitary gland, we investigated the control of cell proliferation during organogenesis. Thus, we identified a population of noncycling precursors that are uniquely marked by expression of the cell cycle inhibitor p57Kip2 and by cyclin E. In p57Kip2−/− mice, the developing pituitary is hyperplastic due to accumulation of proliferating progenitors, whereas overexpression of p57Kip2 leads to hypoplasia. p57Kip2-dependent cell cycle exit is not required for differentiation, and conversely, blockade of cell differentiation, as achieved in Tpit−/− pituitaries, does not prevent cell cycle exit but rather leads to accumulation of p57Kip2-positive precursors. Upon differentiation, p57Kip2 is replaced by p27Kip1. Accordingly, proliferating differentiated cells are readily detected in p27Kip1−/− pituitaries but not in wild-type or p57Kip2−/− pituitaries. Strikingly, all cells of p57Kip2−/−;p27Kip1−/− pituitaries are proliferative. Thus, during normal development, progenitor cell cycle exit is controlled by p57Kip2 followed by p27Kip1 in differentiated cells; these sequential actions, taken together with different pituitary outcomes of their loss of function, suggest hierarchical controls of the cell cycle that are independent of differentiation.
PMCID: PMC2655618  PMID: 19139274

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