Periostin, dentin matrix protein 1 and P2rx7 ion channel in human teeth and periodontal ligament.

The periostin is a matricellular protein present in the human periodontal ligament and human dental pulp-derived cells lines, that up-regulates the in vitro expression of some genes involved in the dentin mineralization, such as dentin matrix protein 1 and P2x7-ion channel receptor. Here we investigated the distribution of periostin in human teeth and periodontal ligaments, mapping in parallel the localization of dentin matrix protein 1 and P2x7-ion channel receptor to establish whether or not they are expressed in the same places as periostin. The periodontal ligament and the subodontoblastic layer of the dental pulp displayed strong periostin immunoreactivity, whereas dentin matrix protein 1 was detected in the periodontal ligament co-localized with periostin in the vicinity of the cement. The P2x7 ion channel receptor was regularly absent in both the periodontal ligament and dental tissues, but in some cases, it was observed in the odontoblasts. Present results demonstrate the occurrence of periostin in the healthy adult human tooth without co-localization with proteins involved in tooth mineralization, the expression of which it regulates. These results might serve as a baseline for future studies on pathological conditions.

Immunohistochemical localization of periostin in human gingiva.

The periostin is a matricellular protein expressed in collagen-rich tissues including some dental and periodontal tissues where it is regulated by mechanical forces, growth factors and cytokines. Interestingly the expression of this protein has been found modified in different gingival pathologies although the expression of periostin in normal human gingiva was never investigated. Here we used Western blot and double immunofluorescence coupled to laser-confocal microscopy to investigated the occurrence and distribution of periostin in different segments of the human gingival in healthy subjects. By Western blot a protein band with an estimated molecular mass of 94 kDa was observed. Periostin was localized at the epithelial-connective tissue junction, or among the fibers of the periodontal ligament, and never co-localized with cytokeratin or vimentin thus suggesting it is an extracellular protein. These results demonstrate the occurrence of periostin in adult human gingiva; its localization suggests a role in the bidirectional interactions between the connective tissue and the epithelial cells, and therefore in the physiopathological conditions in which these interactions are altered.

Higher sensitivity of Adamts12-deficient mice to tumor growth and angiogenesis.

ADAMTS (a disintegrin and metalloproteinase domain with thrombospondin motifs) constitute a family of endopeptidases related to matrix metalloproteinases. These proteases have been largely implicated in tissue remodeling and angiogenesis associated with physiological and pathological processes. To elucidate the in vivo functions of ADAMTS-12, we have generated a knockout mouse strain (Adamts12(-/-)) in which Adamts12 gene was deleted. The mutant mice had normal gestations and no apparent defects in growth, life span and fertility. By applying three different in vivo models of angiogenesis (malignant keratinocyte transplantation, Matrigel plug and aortic ring assays) to Adamts12(-/-) mice, we provide evidence for a protective effect of this host enzyme toward angiogenesis and cancer progression. In the absence of Adamts-12, both the angiogenic response and tumor invasion into host tissue were increased. Complementing results were obtained by using medium conditioned by cells overexpressing human ADAMTS-12, which inhibited vessel outgrowth in the aortic ring assay. This angioinhibitory effect of ADAMTS-12 was independent of its enzymatic activity as a mutated inactive form of the enzyme was similarly efficient in inhibiting endothelial cell sprouting in the aortic ring assay than the wild-type form. Altogether, our results show that ADAMTS-12 displays antiangiogenic properties and protect the host toward tumor progression.

Regulation of cyclin-Cdk activity in mammalian cells.

Cell cycle progression is driven by the coordinated regulation of the activities of cyclin-dependent kinases (Cdks). Of the several mechanisms known to regulate Cdk activity in response to external signals, regulation of cyclin gene expression, post-translational modification of Cdks by phosphorylation-dephosphorylation cascades, and the interaction of cyclin/Cdk complexes with protein inhibitors have been thoroughly studied. During recent years, much attention has also been given to mechanisms that regulate protein degradation by the ubiquitin/proteasome pathway, as well as to the regulation of subcellular localization of the proteins that comprise the intrinsic cell cycle clock. The purpose of the present review is to summarize the most important aspects of the various mechanisms implicated in cell cycle regulation.

c-Myc is necessary for DNA damage-induced apoptosis in the G(2) phase of the cell cycle.

The c-myc proto-oncogene encodes a transcription factor that participates in the regulation of cellular proliferation, differentiation, and apoptosis. Ectopic overexpression of c-Myc has been shown to sensitize cells to apoptosis. We report here that cells lacking c-Myc activity due to disruption of the c-myc gene by targeted homologous recombination are defective in DNA damage-initiated apoptosis in the G(2) phase of the cell cycle. The downstream effector of c-Myc is cyclin A, whose ectopic expression in c-myc(-/-) cells rescues the apoptosis defect. The kinetics of the G(2) response indicate that the induction of cyclin A and the concomitant activation of Cdk2 represent an early step during commitment to apoptosis. In contrast, expression of cyclins E and D1 does not rescue the apoptosis defect, and apoptotic processes in G(1) phase are not affected in c-myc(-/-) cells. These observations link DNA damage-induced apoptosis with cell cycle progression and implicate c-Myc in the functioning of a subset of these pathways.

Identification of CDK4 as a target of c-MYC.

The prototypic oncogene c-MYC encodes a transcription factor that can drive proliferation by promoting cell-cycle reentry. However, the mechanisms through which c-MYC achieves these effects have been unclear. Using serial analysis of gene expression, we have identified the cyclin-dependent kinase 4 (CDK4) gene as a transcriptional target of c-MYC. c-MYC induced a rapid increase in CDK4 mRNA levels through four highly conserved c-MYC binding sites within the CDK4 promoter. Cell-cycle progression is delayed in c-MYC-deficient RAT1 cells, and this delay was associated with a defect in CDK4 induction. Ectopic expression of CDK4 in these cells partially alleviated the growth defect. Thus, CDK4 provides a direct link between the oncogenic effects of c-MYC and cell-cycle regulation.

c-Myc regulates cyclin D-Cdk4 and -Cdk6 activity but affects cell cycle progression at multiple independent points.

c-myc is a cellular proto-oncogene associated with a variety of human cancers and is strongly implicated in the control of cellular proliferation, programmed cell death, and differentiation. We have previously reported the first isolation of a c-myc-null cell line. Loss of c-Myc causes a profound growth defect manifested by the lengthening of both the G1 and G2 phases of the cell cycle. To gain a clearer understanding of the role of c-Myc in cellular proliferation, we have performed a comprehensive analysis of the components that regulate cell cycle progression. The largest defect observed in c-myc-/- cells is a 12-fold reduction in the activity of cyclin D1-Cdk4 and -Cdk6 complexes during the G0-to-S transition. Downstream events, such as activation of cyclin E-Cdk2 and cyclin A-Cdk2 complexes, are delayed and reduced in magnitude. However, it is clear that c-Myc affects the cell cycle at multiple independent points, because restoration of the Cdk4 and -6 defect does not significantly increase growth rate. In exponentially cycling cells the absence of c-Myc reduces coordinately the activities of all cyclin-cyclin-dependent kinase complexes. An analysis of cyclin-dependent kinase complex regulators revealed increased expression of p27(KIP1) and decreased expression of Cdk7 in c-myc-/- cells. We propose that c-Myc functions as a crucial link in the coordinate adjustment of growth rate to environmental conditions.

Mysterious liaisons: the relationship between c-Myc and the cell cycle.

A large body of physiological evidence shows that either upregulation or downregulation of intracellular c-Myc activity has profound consequences on cell cycle progression. Recent work suggests that c-Myc may stimulate the activity of cyclin E/cyclin-dependent kinase 2 (Cdk2) complexes and antagonize the action of the Cdk inhibitor p27KIP1. Cyclin D/Cdk4/6 complexes have also been implicated as targets of c-Myc activity. However, in spite of considerable effort, the mechanisms by which c-Myc interacts with the intrinsic cyclin/Cdk cell cycle machinery remain undefined.

c-myc null cells misregulate cad and gadd45 but not other proposed c-Myc targets.

We report here that the expression of virtually all proposed c-Myc target genes is unchanged in cells containing a homozygous null deletion of c-myc. Two noteworthy exceptions are the gene cad, which has reduced log phase expression and serum induction in c-myc null cells, and the growth arrest gene gadd45, which is derepressed by c-myc knockout. Thus, cad and gadd45 are the only proposed targets of c-Myc that may contribute to the dramatic slow growth phenotype of c-myc null cells. Our results demonstrate that a loss-of-function approach is critical for the evaluation of potential c-Myc target genes.

Phenotypes of c-Myc-deficient rat fibroblasts isolated by targeted homologous recombination.

Rat fibroblast cell lines with targeted disruptions of both c-myc gene copies were constructed. Although c-myc null cells are viable, their growth is significantly impaired. The absence of detectable N-myc or L-myc expression indicates that Myc function is not absolutely essential for cell viability. The c-myc null phenotype is stable and can be reverted by introduction of a c-myc transgene. Exponentially growing c-myc null cells have the same cell size, rRNA, and total protein content as their c-myc +/+ parents, but the rates of RNA and protein accumulation as well as protein degradation are reduced. Both the G1 and G2 phases of the cell cycle are significantly lengthened, whereas the duration of S phase is unaffected. This is the first direct demonstration of a requirement for c-myc in G2. The G0-->S transition is synchronous, but S-phase entry is significantly delayed. The c-myc null cell lines reported here are a new experimental system in which to investigate the importance of putative c-Myc target genes and to identify novel downstream genes involved in cell cycle progression and apoptosis.

Specific in vitro guanylylation of a 43-kilodalton membrane-associated protein of Streptomyces coelicolor.

Incubation of [alpha-32P]GTP with cellular extracts or membranes of Streptomyces coelicolor labels a protein of 43 kDa, which was also labeled with [8,5'-3H]GTP but not with [alpha-32P]ATP or [gamma-32P]GTP. Radioactivity remained associated with this protein after boiling in 0.1 N NaOH, but it was dissociated after incubation in 0.1 N HCl or hydroxylamine. Chromatographic analysis of the HCl-dissociated compound showed that GMP was the covalently bound nucleotide. Furthermore, guanylylation appeared to be reversible and to take place by a pyrophosphorylytic mechanism. Guanylylation was more efficient at low temperatures. Several Streptomyces species showed a guanylylated protein with a similar molecular mass.