RhoE controls myoblast alignment prior fusion through RhoA and ROCK.

Differentiation of skeletal myoblasts into multinucleated myotubes is a multi-step process orchestrated by several signaling pathways. The Rho small G protein family plays critical roles both during myogenesis induction and myoblast fusion. We report here that in C2C12 myoblasts, expression of RhoE, an atypical member of this family, increases until the onset of myoblast fusion before resuming its basal level once fusion has occurred. We show that RhoE accumulates in elongated, aligned myoblasts prior to fusion and that its expression is also increased during injury-induced skeletal muscle regeneration. Moreover, although RhoE is not required for myogenesis induction, it is essential for myoblast elongation and alignment before fusion and for M-cadherin expression and accumulation at the cell-cell contact sites. Myoblasts lacking RhoE present with defective p190RhoGAP activation and RhoA inhibition at the onset of myoblast fusion. RhoE interacts also with the RhoA effector Rho-associated kinase (ROCK)I whose activity must be downregulated to allow myoblast fusion. Consistently, we show that pharmacological inactivation of RhoA or ROCK restores myoblast fusion in RhoE-deficient myoblasts. RhoE physiological upregulation before myoblast fusion is responsible for the decrease in RhoA and ROCKI activities, which are required for the fusion process. Therefore, we conclude that RhoE is an essential regulator of myoblast fusion.

Degradation of B-Myb by ubiquitin-mediated proteolysis: involvement of the Cdc34-SCF(p45Skp2) pathway.

B-Myb, a highly conserved member of the Myb oncoprotein family, is a 110 kDa sequence-specific DNA binding protein expressed in virtually all proliferating cells. B-myb expression reaches its maximum at the G1/S phase boundary and during the S phase of the cell cycle. We have previously shown that B-Myb activity is cell cycle regulated and it is controlled by the antagonistic effects of cyclin D1 and A. Here we show that ectopic expression of cyclin A causes a pronounced reduction of B-Myb protein level. We provide evidence that in addition to triggering B-Myb activity an important effect of cyclin A is to facilitate multiple ubiquitination of B-Myb. The C-terminal domain of B-Myb is of key importance in mediating this effect of cyclin A. Contrary to full-length B-Myb, a C-terminal deletion mutant displays activity irrespective of cyclin A expression, does not undergo ubiquitination, and its half-life is not affected by cyclin A. Ectopic expression of either Cdc34 or the F-box protein p45Skp2, respectively the E2 and E3 components of a ubiquitination pathway that regulates the G1/S transition, accelerates degradation of B-Myb. We show that B-Myb physically and functionally interacts with components of the Cdc34-SCFp45Skp2 ubiquitin pathway and propose that B-Myb degradation may be required for controlling the correct alternation of events during progression through the cell division cycle. Oncogene (2000).

The interaction of TOGp with microtubules and tubulin.

TOGp is the human homolog of XMAP215, a Xenopus microtubule-associated protein that promotes rapid microtubule assembly at plus ends. These proteins are thought to be critical for microtubule assembly and/or mitotic spindle formation. To understand how TOGp interacts with the microtubule lattice, we cloned full-length TOGp and various truncations for expression in a reticulocyte lysate system. Based on microtubule co-pelleting assays, the microtubule binding domain is contained within a basic 600-amino acid region near the N terminus, with critical domains flanking a region homologous to the microtubule binding domain found in the related proteins Stu2p (S. cerevisiae) and Dis1 (S. pombe). Both full-length TOGp and the N-terminal fragment show enhanced binding to microtubule ends. Full-length TOGp also binds altered polymer lattice structures including parallel protofilament sheets, antiparallel protofilament sheets induced with zinc ions, and protofilament rings, suggesting that TOGp binds along the length of individual protofilaments. The C-terminal region of TOGp has a low affinity for microtubule polymer but binds tubulin dimer. We propose a model to explain the microtubule-stabilizing and/or assembly-promoting functions of the XMAP215/TOGp family of microtubule-associated proteins based on the binding properties we have identified.

The Xenopus XMAP215 and its human homologue TOG proteins interact with cyclin B1 to target p34cdc2 to microtubules during mitosis.

Cytoskeleton reorganization, leading to mitotic spindle formation, is an M-phase-specific event and is controlled by maturation promoting factor (MPF: p34cdc2-cyclinB1 complex). It has previously been demonstrated that the p34cdc2-cyclin B complex associates with mitotic spindle microtubules and that microtubule-associated proteins (MAPs), in particular MAP4, might be responsible for this interaction. In this study, we report that another ubiquitous MAP, TOG in human and its homologue in Xenopus XMAP215, associates also with p34cdc2 kinase and directs it to the microtubule cytoskeleton. Costaining of Xenopus cells with anti-TOGp and anti-cyclin B1 antibodies demonstrated colocalization in interphase cells and also with microtubules throughout the cell cycle. Cyclin B1, TOG/XMAP215, and p34cdc2 proteins were recovered in microtubule pellets isolated from Xenopus egg extracts and were eluted with the same ionic strength. Cosedimentation of cyclin B1 with in vitro polymerized microtubules was detected only in the presence of purified TOG protein. Using a recombinant C-terminal TOG fragment containing a Pro-rich region, we showed that this domain is sufficient to mediate cosedimentation of cyclin B1 with microtubules. Finally, we demonstrated interaction between TOG/XMAP215 and cyclin B1 by co-immunoprecipitation assays. As XMAP215 was shown to be the only identified assembly promoting MAP which increases the rapid turnover of microtubules, the TOG/XMAP215-cyclin B1 interaction may be important for regulation of microtubule dynamics at mitosis.

PCTAIRE-1: characterization, subcellular distribution, and cell cycle-dependent kinase activity.

PCTAIRE-1 is a member of the cyclin-dependent kinase (cdk) family whose function is unknown. We examined the pattern of PCTAIRE-1 protein expression in a number of normal and transformed cell lines of various origins and found that the kinase is ubiquitous. Indirect immunofluorescence indicated that PCTAIRE-1 exhibits cytoplasmic distribution throughout the cell cycle. Confocal microscopy showed that PCTAIRE-1 does not colocalize with components of the cytoskeleton or with the endoplasmic reticulum. We found that endogenous PCTAIRE-1 and ectopically expressed PCTAIRE-1 display kinase activity when myelin basic protein is used as an acceptor substrate. Similar to other members of the cyclin-dependent kinase family, PCTAIRE-1 seems to require binding to a regulatory subunit to display kinase activity. PCTAIRE-1 activity is cell cycle dependent and displays a peak in the S and G2 phases. We show that the low level of kinase activity observed until the onset of S phase correlates with elevated tyrosine phosphorylation of the molecule.

The TOGp protein is a new human microtubule-associated protein homologous to the Xenopus XMAP215.

We have recently identified a 6,449 bp cDNA, termed colonic, hepatic tumor over-expressed gene (ch-TOG), that is highly expressed in human tumors and brain. Its single open reading frame encodes a putative 218,000 Da polypeptide, TOGp. Antibodies generated against a bacterially expressed TOGp fragment specifically recognize a 218, 000 Da polypeptide in two human cell lines and in brain. Immunofluorescence microscopy using affinity-purified TOGp antibodies revealed that the distribution of TOGp was dependent upon the cell cycle. During interphase, TOGp was found concentrated in the perinuclear cytoplasm, where it co-localized with ER markers. In contrast anti-TOGp antibodies stained centrosomes and spindles in mitotic cells. TOGp co-sedimented with taxol-stabilized microtubules in vitro. Moreover, a TOGp enriched fraction promotes microtubule assembly both in solution and from nucleation centers. Finally, sequence comparison and immunologic cross-reaction suggest that TOGp is homologous to XMAP215, a previously described microtubule associated protein (MAP) from Xenopus eggs. These results suggest that TOGp is a MAP and that TOGp/XMAP215 may be necessary for microtubules rearrangements and spindle assembly in rapidly dividing cells.

Expression of the tumor over-expressed ch-TOG gene in human and baboon brain.

We recently reported on the over-expression of a new gene, colonic and hepatic tumor over-expressed gene (ch-TOG) in human hepatomas and colonic tumors. Although ubiquitous in healthy tissues, its expression was found to be particularly elevated in the brain. In the present study, we demonstrate the expression of ch-TOG mRNA in human brain tumors as well as in numerous areas of healthy human and baboon brain. This expression was analyzed by reverse transcription-polymerase chain reaction (RT-PCR), Northern blotting, RNAse protection assays and in situ hybridization. An interesting feature is the demonstration that, although ubiquitous in the brain, the ch-TOG gene is highly expressed in the Purkinje cell bodies of the human cerebellum.

Induction of CYP1A1 gene by benzimidazole derivatives during Caco-2 cell differentiation. Evidence for an aryl-hydrocarbon receptor-mediated mechanism.

The Caco-2 cell line, derived from a human colon adenocarcinoma, is unique in its property of spontaneously differentiating into a mature enterocyte cell type during its growth in culture. In this work, we compared the response of the CYP1A1 gene with the benzimidazole derivatives omeprazole and lansoprazole, and with the classical inducer beta-naphthoflavone in the Caco-2 cells at various culture stages. In addition, we characterized the Caco-2 aryl-hydrocarbon receptor. The protein-synthesis inhibitor cycloheximide led to a derepression of the CYP1A1 gene transcription, and to a superinduction when combined with either beta-naphthoflavone or benzimidazoles. Taking advantage of the spontaneous differentiation of Caco-2 cells in long-term cultures, we observed a difference in behavior between the classical inducer beta-naphthoflavone and the atypical inducer omeprazole. In the poorly differentiated cells, both compounds elicited comparable dose/response and rate of induction of CYP1A1 gene expression. In the fully differentiated cells, in contrast, the induction by omeprazole was only transient, whereas the response to beta-naphthoflavone was long lasting. The Caco-2 aryl-hydrocarbon receptor exhibited binding characteristics similar to those determined for human liver and other tissues. The induction of CYP1A1 transcription by benzimidazole derivatives in Caco-2 cells occurred with no direct binding of benzimidazole derivatives to the aryl-hydrocarbon receptor, as in human hepatocytes. However, transient transfection experiments clearly showed that the xenobiotic-responsive element enhancer, with which the activated aryl-hydrocarbon receptor interacts, could drive the induction of a heterologous promoter in the presence of benzimidazoles. Finally the presence of the activated aryl-hydrocarbon receptor in the nuclei of the Caco-2 cells exposed to these molecules was clearly demonstrated by gel-retardation experiments. These results question about the mechanism of ligand-independent activation of the aryl-hydrocarbon receptor and intracellular signaling, initiated by benzimidazole derivatives.

Characterization of the cDNA and pattern of expression of a new gene over-expressed in human hepatomas and colonic tumors.

A cDNA clone of 6.449 kb ch-TOG (for colonic and hepatic tumor over-expressed gene) initially selected from various human libraries and completed by 5' rapid amplification of cDNA ends (RACE) PCR is described. The original cDNA clone was extracted from an expression library constructed from a human tumoral brain. This library was screened with an antibody raised against the cytochrome P450tu that was shown to be over-expressed in chemically induced mouse hepatic tumors. Using this cDNA as a probe, a full-length cDNA was characterized. Its nucleotide sequence shows no significant similarity with any of the gene sequences collected in the various DNA data bases. The translation of the larger open reading frame leads to a putative protein of 1972 amino acids (molecular mass = 218453 Da). Hybridization analyses on Southern blot and on metaphase chromosomes indicate that this gene is present as a single copy in the genome and is localized on the short arm of chromosome 11. ch-TOG transcripts are present in several human tissues. Over-expression of ch-TOG in neoplastic liver and colon compared with the corresponding normal corresponding tissues is demonstrated. The level of the expression of ch-TOG transcripts was also studied in the various differentiation stages of the human colonic adenocarcinoma cell line Caco-2.

Thyroid hormone promotes transient nerve growth factor synthesis in rat cerebellar neuroblasts.

Primary cultures of cerebellum from 5-day-old rats indicated that proliferating neuroblasts synthesize and release nerve growth factor (NGF). Since NGF promotes DNA synthesis in these cells, our findings demonstrate that the early developing cerebellum is a suitable physiological model for studying the autocrine mitogenic action of NGF. Thyroid deficiency led to a greater reduction in the NGF content of the cerebellum than of the olfactory bulbs or hippocampus. Cerebellar NGF mRNA was also very sensitive to hormone deprivation. Physiological amounts of thyroid hormone stimulated both the mitotic activity and NGF production of cultured cerebellar neuroblasts. A lack of thyroid hormone is known to markedly alter cell formation in the cerebellum where postnatal neurogenesis is highly significant, in contrast to the olfactory bulbs and hippocampus. Taken together, these results suggest that the hormonal control of cell formation in the cerebellum is, at least partly, mediated by the autocrine mitogenic action of NGF. The thyroid hormone could temporally regulate the transient NGF synthesis by cerebellar neuroblasts directly and/or through its ontogenetic action, and hence all the NGF-dependent trophic effects.

Nerve growth factor enhances DNA synthesis in cultured cerebellar neuroblasts.

The cerebellar neuroblasts in primary cultures from five-day-old rats bore NGF receptor immunoreactivity, suggesting a potential responsive to NGF. At low plating density, NGF was found to enhance DNA synthesis in these cells in a dose-dependent manner. As these cells synthesize NGF, one possibility to account for the lack of response of neuroblasts plated at high density is that the amount of endogenous trophic agent produced in this culture condition is sufficient to ensure an optimal effect. The results demonstrate that premitotic neuroblasts in the CNS, as well postmitotic neurons, are responsive to NGF. At the early stage of its development, the cerebellum therefore appears to be a very good autocrine model of NGF action.