Mammalian Notch receptors contain 29–36 epidermal growth factor (EGF)-like repeats that may be modified by protein O-fucosyltransferase 1 (Pofut1), an essential component of the canonical Notch signaling pathway. The Drosophila orthologue Ofut1 is proposed to function as both a chaperone required for stable cell surface expression of Notch and a protein O-fucosyltransferase. Here we investigate these dual roles of Pofut1 in relation to endogenous Notch receptors of Chinese hamster ovary and murine embryonic stem (ES) cells. We show that fucosylation-deficient Lec13 Chinese hamster ovary cells have wild type levels of Pofut1 and cell surface Notch receptors. Nevertheless, they have reduced binding of Notch ligands and low levels of Delta1- and Jagged1-induced Notch signaling. Exogenous fucose but not galactose rescues both ligand binding and Notch signaling. Murine ES cells lacking Pofut1 also have wild type levels of cell surface Notch receptors. However, Pofut1–/– ES cells do not bind Notch ligands or exhibit Notch signaling. Although overexpression of fucosyltransferase-defective Pofut1 R245A in Pofut1–/– cells partially rescues ligand binding and Notch signaling, this effect is not specific. The same rescue is achieved by an unrelated...

Establishment of cell polarity is important for a wide range of biological processes, from asymmetric cell growth in budding yeast to neurite formation in neurons. In the yeast Saccharomyces cerevisiae, the small GTPase Cdc42 controls polarized actin organization and exocytosis toward the bud. Gic2, a Cdc42 effector, is targeted to the bud tip and plays an important role in early bud formation. The GTP-bound Cdc42 interacts with Gic2 through the Cdc42/Rac interactive binding domain located at the N terminus of Gic2 and activates Gic2 during bud emergence. Here we identify a polybasic region in Gic2 adjacent to the Cdc42/Rac interactive binding domain that directly interacts with phosphatidylinositol 4,5-bisphosphate in the plasma membrane. We demonstrate that this interaction is necessary for the polarized localization of Gic2 to the bud tip and is important for the function of Gic2 in cell polarization. We propose that phosphatidylinositol 4,5-bisphosphate and Cdc42 act in concert to regulate polarized localization and function of Gic2 during polarized cell growth in the budding yeast.

The homeodomain protein Distal-less-3 (Dlx3) plays a crucial role during embryonic development. This transcription factor is known to be essential for placental formation and to be involved in skin and skeletal organogenesis. In humans, a frameshift mutation in the coding sequence of the DLX3 gene results in an ectodermal dysplasia called Tricho-Dento-Osseous syndrome (TDO). The main features of this autosomal dominant disorder are defects in hair, teeth, and bone. To investigate the functional alterations caused by the mutated DLX3TDO isoform ex vivo, we used tetracycline-inducible osteoblastic and keratinocyte cell lines and calvarial derived osteoblasts in which the expression of DLX3WT and/or DLX3TDO could be regulated and monitored. Immunocytochemical analysis revealed that both DLX3WT and DLX3TDO recombinant proteins are targeted to the nucleus. However, as demonstrated by electrophoresis mobility shift assay, DLX3TDO is not able to bind to the canonical Dlx3 binding site. Furthermore, we demonstrate that the frameshifted C-terminal domain in DLX3TDO is accountable for the loss of DNA binding activity because the C-terminal domain in DLX3WT is not required for DNA binding activity. Although DLX3TDO alone cannot bind to a Dlx3 responsive element...

Isothiocyanates (ITCs) found in cruciferous vegetables, including benzyl-ITC (BITC), phenethyl-ITC (PEITC), and sulforaphane (SFN), inhibit carcinogenesis in animal models and induce apoptosis and cell cycle arrest in various cell types. The biochemical mechanisms of cell growth inhibition by ITCs are not fully understood. Our recent study showed that ITC binding to intracellular proteins may be an important initiating event for the induction of apoptosis. However, the specific protein target(s) and molecular mechanisms were not identified. In this study, two-dimensional gel electrophoresis of human lung cancer A549 cells treated with radiolabeled PEITC and SFN revealed that tubulin may be a major in vivo binding target for ITC. We examined whether binding to tubulin by ITCs could lead to cell growth arrest. The proliferation of A549 cells was significantly reduced by ITCs, with relative activities of BITC > PEITC > SFN. All three ITCs also induced mitotic arrest and apoptosis with the same order of activity. We found that ITCs disrupted microtubule polymerization in vitro and in vivo with the same order of potency. Mass spectrometry demonstrated that cysteines in tubulin were covalently modified by ITCs. Ellman assay results indicated that the modification levels follow the same order...

Here we identify a novel protein, named Parcs for pro-apoptotic protein required for cell survival, that is involved in both cell cycle progression and apoptosis. Parcs interacted with Apaf-1 by binding to the oligomerization domain of Apaf-1. Apaf-1-mediated activation of caspase-9 and caspase-3 was markedly decreased in a cytosolic fraction isolated from HeLa cells with reduced parcs expression. Interestingly, parcs deficiency blocked cell proliferation in non-tumorigenic cells but not in multiple tumor cell lines. In MCF-10A cells, parcs deficiency led to early G1 arrest. Conditional inactivation of parcs in genetically modified primary mouse embryonic fibroblasts using the Cre-LoxP system also resulted in the inhibition of cell proliferation. We conclude that Parcs may define a molecular checkpoint in the control of cell proliferation for normal cells that is lost in tumor cells.

Normal human prostate (NHP) epithelial cells undergo senescence in vitro and in vivo, but the underlying molecular mechanisms remain obscure. Here we show that the senescence of primary NHP cells, which are immunophenotyped as intermediate basal-like cells expressing progenitor cell markers CD44, α2β1, p63, hTERT, and CK5/CK18, involves loss of telomerase expression, up-regulation of p16, and activation of p53. Using genetically defined manipulations of these three signaling pathways, we show that p16 is the primary determinant of the NHP cell proliferative capacity and that hTERT is required for unlimited proliferative life span. Hence, suppression of p16 significantly extends NHP cell life span, but both p16 inhibition and hTERT are required to immortalize NHP cells. Importantly, immortalized NHP cells retain expression of most progenitor markers, demonstrate gene expression profiles characteristic of proliferating progenitor cells, and possess multilineage differentiation potential generating functional prostatic glands. Our studies shed important light on the molecular mechanisms regulating the proliferative life span of NHP progenitor cells.

Ankyrin-B targets ion channels and transporters in excitable cells. Dysfunction in ankyrin-B-based pathways results in defects in cardiac physiology. Despite a wealth of knowledge regarding the role of ankyrin-B for cardiac function, little is known regarding the mechanisms underlying ankyrin-B regulation. Moreover, the pathways underlying ankyrin-B targeting in heart are unclear. We report that alternative splicing regulates ankyrin-B localization and function in cardiomyocytes. Specifically, we identify a novel exon (exon 43′) in the ankyrin-B regulatory domain that mediates interaction with the Rho-GEF obscurin. Ankyrin-B transcripts harboring exon 43′ represent the primary cardiac isoform in human and mouse. We demonstrate that ankyrin-B and obscurin are co-localized at the M-line of myocytes and co-immunoprecipitate from heart. We define the structural requirements for ankyrin-B/obscurin interaction to two motifs in the ankyrin-B regulatory domain and demonstrate that both are critical for obscurin/ankyrin-B interaction. In addition, we demonstrate that interaction with obscurin is required for ankyrin-B M-line targeting. Specifically, both obscurin-binding motifs are required for the M-line targeting of a GFP-ankyrin-B regulatory domain. Moreover...

Hyaluronan, a widely distributed component of the extracellular matrix, exists in a high molecular weight (native) form and lower molecular weight form (HMW- and LMW-HA, respectively). These different forms of hyaluronan bind to CD44 but elicit distinct effects on cellular function. A striking example is the opposing effects of HMW- and LMW-HA on the proliferation of vascular smooth muscle cells; the binding of HMW-HA to CD44 inhibits cell cycle progression, whereas the binding of LMW-HA to CD44 stimulates cell cycle progression. We now report that cyclin D1 is the primary target of LMW-HA in human vascular smooth muscle cells, as it is for HMW-HA, and that the opposing cell cycle effects of these CD44 ligands result from differential regulation of signaling pathways to cyclin D1. HMW-HA binding to CD44 selectively inhibits the GTP loading of Rac and Rac-dependent signaling to the cyclin D1 gene, whereas LMW-HA binding to CD44 selectively stimulates ERK activation and ERK-dependent cyclin D1 gene expression. These data describe a novel mechanism of growth control in which a ligand-receptor system generates opposing effects on mitogenesis by differentially regulating signaling pathways to a common cell cycle target. They also emphasize how a seemingly subtle change in matrix composition can have a profound effect on cell proliferation.

Osterix/Sp7, a member of the Sp1 transcription factor family, plays an essential role in bone formation and osteoblastogenesis. Although Osterix has been shown to be induced by BMP2 in a mesenchymal cell line, the molecular basis of the regulation, expression and function of Osterix during osteoblast differentiation, is not fully understood. Thus we examined the role of BMP2 signaling in the regulation of Osterix using the mesenchymal cell lines C3H10T1/2 and C2C12. Osterix overexpression induced alkaline phosphatase activity and osteocalcin expression in C2C12 cells and stimulated calcification of murine primary osteoblasts. Considering that Runx2 overexpression induces Osterix, these results suggest that Osterix functions as downstream of Runx2. Surprisingly, BMP2 treatment induced Osterix expression and alkaline phosphatase activity in mesenchymal cells derived from Runx2-deficient mice. Furthermore, overexpression of Smad1 and Smad4 up-regulated Osterix expression, and an inhibitory Smad, Smad6, markedly suppressed BMP2-induced Osterix expression in the Runx2-deficient cells. Moreover, overexpression of a homeobox gene, Msx2, which is up-regulated by BMP2 and promotes osteoblastic differentiation, induced Osterix expression in the Runx2-deficient cells. Knockdown of Msx2 clearly inhibited induction of Osterix by BMP2 in the Runx2-deficient mesenchymal cells. Interestingly...

Krüppel-like factor 5 (KLF5), originally isolated as a regulator of phenotypic modulation of vascular smooth muscle cells, induces pathological cell growth and is expressed in the neointima. Although induction of KLF5 up-regulates growth factors like platelet-derived growth factor-A chain, how KLF5 actually contributes to vascular remodeling, notably its direct effects on cell proliferation, had been poorly clarified. To investigate the effects of KLF5 on neointimal formation, we at first performed adenoviral overexpression of KLF5 to rats subjected to carotid balloon injury. Neointimal formation and proliferating cell nuclear antigen-positive rate were significantly increased at 14 days after injury in the KLF5-treated animals. At the cellular level, overexpression of KLF5 also resulted in markedly increased cell proliferation and cell cycle progression. As a molecular mechanism, we showed that KLF5 directly bound to the promoter and up-regulated gene expression of cyclin D1, as well as showing specific transactivation of cyclins and cyclin-dependent kinase inhibitors in cardiovascular cells. Conversely, knockdown of KLF5 by RNA interference specifically down-regulated cyclin D1 and impaired vascular smooth muscle cell proliferation. Furthermore...

The NAD-dependent deacetylase SirT1 regulates factors involved in stress response and cell survival and is a potential drug target of activators and inhibitors. Determination of SirT1 function in tumor cells is important for its targeting in cancer therapy. We found that SirT1 knockdown by short hairpin RNA accelerates tumor xenograft formation by HCT116 cells, whereas SirT1 overexpression inhibits tumor formation. Furthermore, pharmacological inhibition of SirT1 stimulates cell proliferation under conditions of growth factor deprivation. Paradoxically, SirT1 inhibition also sensitizes cells to apoptosis by chemotherapy drugs. Immunohistochemical staining revealed high level SirT1 in normal colon mucosa and benign adenomas. SirT1 overexpression was observed in ∼25% of stage I/II/III colorectal adenocarcinomas but rarely found in advanced stage IV tumors. Furthermore, ∼30% of carcinomas showed lower than normal SirT1 expression. This pattern is consistent with SirT1 having pleiotropic effects during cancer development (anti-proliferation and anti-apoptotic). These results suggest a rationale for the use of SirT1 activators and inhibitors in the prevention and treatment of colon cancer.

Studies have attributed several functions to the Eaf family, including tumor suppression and eye development. Given the potential association between cancer and development, we set forth to explore Eaf1 and Eaf2/U19 activity in vertebrate embryogenesis, using zebrafish. In situ hybridization revealed similar eaf1 and eaf2/u19 expression patterns. Morpholino-mediated knockdown of either eaf1 or eaf2/u19 expression produced similar morphological changes that could be reversed by ectopic expression of target or reciprocal-target mRNA. However, combination of Eaf1 and Eaf2/U19 (Eafs)-morpholinos increased the severity of defects, suggesting that Eaf1 and Eaf2/U19 only share some functional redundancy. The Eafs knockdown phenotype resembled that of embryos with defects in convergence and extension movements. Indeed, knockdown caused expression pattern changes for convergence and extension movement markers, whereas cell tracing experiments using kaeda mRNA showed a correlation between Eafs knockdown and cell migration defects. Cardiac and pancreatic differentiation markers revealed that Eafs knockdown also disrupted midline convergence of heart and pancreatic organ precursors. Noncanonical Wnt signaling plays a key role in both convergence and extension movements and midline convergence of organ precursors. We found that Eaf1 and Eaf2/U19 maintained expression levels of wnt11 and wnt5. Moreover...

Mass spectrometry and immunoblot analysis of a rat brain fraction enriched in type-II postsynaptic densities and postsynaptic GABAergic markers showed enrichment in the protein septin 11. Septin 11 is expressed throughout the brain, being particularly high in the spiny branchlets of the Purkinje cells in the molecular layer of cerebellum and in the olfactory bulb. Immunofluorescence of cultured hippocampal neurons showed that 54 ± 4% of the GABAergic synapses and 25 ± 2% of the glutamatergic synapses had colocalizing septin 11 clusters. Similar colocalization numbers were found in the molecular layer of cerebellar sections. In cultured hippocampal neurons, septin 11 clusters were frequently present at the base of dendritic protrusions and at the bifurcation points of the dendritic branches. Electron microscopy immunocytochemistry of the rat brain cerebellum revealed the accumulation of septin 11 at the neck of dendritic spines, at the bifurcation of dendritic branches, and at some GABAergic synapses. Knocking down septin 11 in cultured hippocampal neurons with septin 11 small hairpin RNAs showed (i) reduced dendritic arborization; (ii) decreased density and increased length of dendritic protrusions; and (iii) decreased GABAergic synaptic contacts that these neurons receive. The results indicate that septin 11 plays important roles in the cytoarchitecture of neurons...

Mutations in Sizn1 (Zcchc12), a novel transcriptional co-activator in the BMP signaling pathway, are associated with X-linked mental retardation. Previously, we demonstrated that Sizn1 positively modulates the BMP signal by interacting with Smad family members and cAMP-responsive element-binding protein-binding protein. To further define the molecular basis of Sizn1 function, we have explored its subcellular localization and generated various deletion mutants to carry out domain analyses. Here, we report that Sizn1 localizes to promyelocytic leukemia protein nuclear bodies (PML-NBs). Sizn1 deletion mutants that disrupt the MA homologous domain or the middle region fail to target to the PML-NB. We show that two SUMO interaction motifs (SIMs) in Sizn1 can bind to SUMO and govern SUMO conjugation to Sizn1 in the absence of the consensus motif for SUMO attachment. Interestingly, the SIM mutant Sizn1 localizes to nuclear bodies, but not to PML-NBs. Thus, SIMs mediate the localization of Sizn1 to PML-NB. Interestingly, mutations in SIM sequences and deletion of the MA homologous domain also affected the transcriptional co-activation function of a Sizn1. Taken together, our data indicate that the SIMs in Sizn1 are required for its PML-NB localization and for the full transcriptional co-activation function in BMP signaling.

High grade gliomas such as glioblastoma multiforme express multiple members of the epithelial sodium channel (ENaC)/Degenerin family, characteristically displaying a basally active amiloride-sensitive cation current not seen in normal human astrocytes or lower grade gliomas. Using quantitative real time PCR, we have shown higher expression of ASIC1, αENaC, and γENaC in D54-MG human glioblastoma multiforme cells compared with primary human astrocytes. We hypothesize that this glioma current is mediated by a hybrid channel composed of a mixture of ENaC and acid-sensing ion channel (ASIC) subunits. To test this hypothesis we made dominant negative cDNAs for ASIC1, αENaC, γENaC, and δENaC. D54-MG cells transfected with the dominant negative constructs for ASIC1, αENaC, or γENaC showed reduced protein expression and a significant reduction in the amiloride-sensitive whole cell current as compared with untransfected D54-MG cells. Knocking down αENaC or γENaC also abolished the high PK+/PNa+ of D54-MG cells. Knocking down δENaC in D54-MG cells reduced δENaC protein expression but had no effect on either the whole cell current or K+ permeability. Using co-immunoprecipitation we show interactions between ASIC1, αENaC, and γENaC...

CUL4A and CUL4B, which are derived from the same ancestor, CUL4, encode scaffold proteins that organize cullin-RING ubiquitin ligase (E3) complexes. Recent genetic studies have shown that germ line mutation in CUL4B can cause mental retardation, short stature, and other abnormalities in humans. CUL4A was observed to be overexpressed in breast and hepatocellular cancers, although no germ line mutation in human CUL4A has been reported. Although CUL4A has been known to be involved in a number of cellular processes, including DNA repair and cell cycle regulation, little is known about whether CUL4B has similar functions. In this report, we tested the functional importance of CUL4B in cell proliferation and characterized the nuclear localization signal (NLS) that is essential for its function. We found that RNA interference silencing of CUL4B led to an inhibition of cell proliferation and a prolonged S phase, due to the overaccumulation of cyclin E, a substrate targeted by CUL4B for ubiquitination. We showed that, unlike CUL4A and other cullins that carry their NLS in their C termini, NLS in CUL4B is located in its N terminus, between amino acid 37 and 40, KKRK. This NLS could bind to importin α1, α3, and α5. NLS-deleted CUL4B was distributed in cytoplasm and failed to promote cell proliferation. Therefore...

Over the last few years, evidence has accumulated revealing the unexpected potential of committed mammalian cells to convert to a different phenotype via a process called transdifferentiation or adult cell reprogramming. These findings may have major practical implications because this process may facilitate the generation of functional autologous tissues that can be used for replacing malfunctioning organs. An instructive role for transcription factors in diverting the developmental fate of cells in adult tissues has been demonstrated when adult human liver cells were induced to transdifferentiate to the pancreatic endocrine lineage upon ectopic expression of the pancreatic master regulator PDX-1 (pancreatic and duodenal homeobox gene 1). Since organogenesis and lineage commitment are affected also by developmental signals generated in response to environmental triggers, we have now analyzed whether the hormone GLP-1 (glucogen-like peptide-1) documented to play a role in pancreatic beta cell differentiation, maturation, and survival, can also increase the efficiency of liver to pancreas transdifferentiation. We demonstrate that the GLP-1R agonist, exendin-4, significantly improves the efficiency of PDX-1-mediated transdifferentiation. Exendin-4 affects the transdifferentiation process at two distinct steps; it increases the proliferation of liver cells predisposed to transdifferentiated in response to PDX-1 and promotes the maturation of transdifferentiated cells along the pancreatic lineage. Liver cell reprogramming toward the pancreatic beta cell lineage has been suggested as a strategy for functional replacement of the ablated insulin-producing cells in diabetics. Understanding the cellular and molecular basis of the transdifferentiation process will allow us to increase the efficiency of the reprogramming process and optimize its therapeutic merit.

Plakoglobin and β-catenin are homologous armadillo repeat proteins found in adherens junctions, where they interact with the cytoplasmic domain of classical cadherins and with α-catenin. Plakoglobin, but normally not β-catenin, is also a structural constituent of desmosomes, where it binds to the cytoplasmic domains of the desmosomal cadherins, desmogleins and desmocollins. Here, we report structural, biophysical, and biochemical studies aimed at understanding the molecular basis of selective exclusion of β-catenin and α-catenin from desmosomes. The crystal structure of the plakoglobin armadillo domain bound to phosphorylated E-cadherin shows virtually identical interactions to those observed between β-catenin and E-cadherin. Trypsin sensitivity experiments indicate that the plakoglobin arm domain by itself is more flexible than that of β-catenin. Binding of plakoglobin and β-catenin to the intracellular regions of E-cadherin, desmoglein1, and desmocollin1 was measured by isothermal titration calorimetry. Plakoglobin and β-catenin bind strongly and with similar thermodynamic parameters to E-cadherin. In contrast, β-catenin binds to desmoglein-1 more weakly than does plakoglobin. β-Catenin and plakoglobin bind with similar weak affinities to desmocollin-1. Full affinity binding of desmoglein-1 requires sequences C-terminal to the region homologous to the catenin-binding domain of classical cadherins. Although pulldown assays suggest that the presence of N- and C-terminal β-catenin “tails” that flank the armadillo repeat region reduces the affinity for desmosomal cadherins...

The retinoblastoma (RB) tumor suppressor pathway is disrupted at high frequency in hepatocellular carcinoma. However, the mechanisms through which RB modulates physiological responses in the liver remain poorly defined. Despite the well established role of RB in cell cycle control, the deletion of RB had no impact on the kinetics of cell cycle entry or the restoration of quiescence during the course of liver regeneration. Although these findings indicated compensatory effects from the RB-related proteins p107 and p130, even the dual deletion of RB with p107 or p130 failed to deregulate hepatic proliferation. Furthermore, although these findings suggested a modest role for the RB-pathway in the context of proliferative control, RB loss had striking effects on response to the genotoxic hepatocarcinogen diethylnitrosamine. With diethylnitrosamine, RB deletion resulted in inappropriate cell cycle entry that facilitated secondary genetic damage and further uncoupling of DNA replication with mitotic entry. Analysis of the mechanism underlying the differential impact of RB status on liver biology revealed that, while liver regeneration is associated with the conventional induction of cyclin D1 expression, the RB-dependent cell cycle entry...

CD9 and CD81 are closely related tetraspanins that regulate cell motility and signaling by facilitating the organization of multimolecular membrane complexes, including integrins. We show that CD9 and CD81 are down-regulated in smoking-related inflammatory response of a macrophage line, RAW264.7. When functions of CD9 and CD81 were ablated with monoclonal antibody treatment, small interfering RNA transfection, or gene knock-out, macrophages were less motile and produced larger amounts of matrix metalloproteinase (MMP)-2 and MMP-9 than control cells in vitro. In line with this, CD9/CD81 double-knock-out mice spontaneously developed pulmonary emphysema, a major pathological component of chronic obstructive pulmonary disease (COPD). The mutant lung contained an increased number of alveolar macrophages with elevated activities of MMP-2 and MMP-9 and progressively displayed enlarged airspace and disruption of elastic fibers in the alveoli. Secretory cell metaplasia, a finding similar to goblet cell metaplasia in cigarette smokers, was also observed in the epithelium of terminal bronchioles. With aging, the double-knockout mice showed extrapulmonary phenotypes, including weight loss, kyphosis, and osteopenia. These results suggest that the tetraspanins CD9 and CD81 regulate cell motility and protease production of macrophages and that their dysfunction may underlie the progression of COPD.