Characterization of cell fusion in an experimental mouse model of endometriosis†.

Cell fusion is involved in the development of some adult organs, is implicated in the pathogenesis of specific types of cancer, and is known to participate in repair/regeneration processes mediated by bone-marrow-derived cells (BMDCs). Endometriosis is a disease characterized by growth of functional endometrial tissue outside of the uterine cavity. Endometriosis shares some molecular properties with cancer and BMDCs home to endometriosis lesions in a mouse model. Our objective was to determine if cell fusion can occur in endometriosis and establish whether bone-marrow-derived cells participate in cell fusion events in lesions. We employed a Cre-Lox system to identify cell fusion events in a mouse model of endometriosis. Fused cells were detected in endometriotic lesions, albeit at a low frequency (∼1 in 400 cells), localized to the stromal compartment, and displayed restricted proliferation. Using 5-fluorouracil-based nongonadotoxic bone marrow transplantation model, we demonstrate that bone marrow cells represent a principal cell source for fusion events in lesions. Cell fusion progeny uniformly lacked expression of selected markers of hematopoietic, endothelial, and epithelial markers, though they expressed the mesenchymal/stromal markers Sca-1 and CD29. This study is the first to describe the phenomenon of cell fusion in endometriosis and points to a mesenchymal population derived from cell fusion events with limited proliferative activity, properties previously attributed to endometrial stem cells. Their putative role in the pathogenesis of the disease remains to be elucidated.

PTEN regulates the ubiquitin-dependent degradation of the CDK inhibitor p27(KIP1) through the ubiquitin E3 ligase SCF(SKP2).

The PTEN tumor suppressor acts as a phosphatase for phosphatidylinositol-3,4,5-trisphosphate (PIP3) [1, 2]. We have shown previously that PTEN negatively controls the G1/S cell cycle transition and regulates the levels of p27(KIP1), a CDK inhibitor [3, 4]. Recently, we and others have identified an ubiquitin E3 ligase, the SCF(SKP2) complex, that mediates p27 ubiquitin-dependent proteolysis [5-7]. Here we report that PTEN and the PI 3-kinase pathway regulate p27 protein stability. PTEN-deficiency in mouse embryonic stem (ES) cells causes a decrease of p27 levels with concomitant increase of SKP2, a key component of the SCF(SKP2) complex. Conversely, in human glioblastoma cells, ectopic PTEN expression leads to p27 accumulation, which is accompanied by a reduction of SKP2. We found that ectopic expression of SKP2 alone is sufficient to reverse PTEN-induced p27 accumulation, restore the kinase activity of cyclin E/CDK2, and partially overcome the PTEN-induced G1 cell cycle arrest. Consistently, recombinant SCF(SKP2) complex or SKP2 protein alone can rescue the defect in p27 ubiquitination in extracts prepared from cells treated with a PI 3-kinase inhibitor. Our findings suggest that SKP2 functions as a critical component in the PTEN/PI 3-kinase pathway for the regulation of p27(KIP1) and cell proliferation.

Induction of intracellular signalling by cyclic glycerophosphates and their deoxy analogues.

Cyclic glycerophosphates can be formed by enzymic degradation of phospholipids. They have only recently attracted attention, and their physiological function is still obscure. In this study, we have searched for signalling functions of the natural 1,3-cyclic and 1,2-cyclic glycerophosphates, their deoxy analogues, and the phenyl esters of the 1,3-cyclic phosphates. Linear sn-glycerol 3-phosphate and glycerol 2-phosphate served as the control compounds. Each of the six-membered ring cyclic phosphates tested induced rapid intracellular tyrosine phosphorylation in CHO and NIH-3T3 cells when applied extracellularly at a concentration of 0.5-4 microM. The phosphorylated intracellular proteins had molecular masses of approximately 35 kDa, approximately 45 kDa, 60-70 kDa and approximately 120 kDa. The five-membered ring cyclic phosphates had a similar effect, but at an external concentration of 2-10 microM, while sn-glycerol 3-phosphate and glycerol 2-phosphate had no effect. The six-membered cyclic phosphates also induced rapid threonine phosphorylation in CHO cells of approximately 18-kDa, approximately 35-kDa, and approximately 38-kDa proteins. Further experiments indicated that the cyclic phosphates partition rapidly into the cell cytosol where they activate kinases, including mitogen-activated protein kinase. When their intracellular level increases, dephosphorylation presumably takes place. This pattern may account for the signalling profile of cyclic phosphates and suggests that they may take part in processes associated with cell differentiation.

Genetic deletion of the Pten tumor suppressor gene promotes cell motility by activation of Rac1 and Cdc42 GTPases.

Pten (Phosphatase and tensin homolog deleted on chromosome 10) is a recently identified tumor suppressor gene which is deleted or mutated in a variety of primary human cancers and in three cancer predisposition syndromes [1]. Pten regulates apoptosis and cell cycle progression through its phosphatase activity on phosphatidylinositol (PI) 3,4,5-trisphosphate (PI(3,4,5)P(3)), a product of PI 3-kinase [2-5]. Pten has also been implicated in controlling cell migration [6], but the exact mechanism is not very clear. Using the isogenic Pten(+/+) and Pten(-/-) mouse fibroblast lines, here we show that Pten deficiency led to increased cell motility. Reintroducing the wild-type Pten, but not the catalytically inactive Pten C124S or lipid-phosphatase-deficient Pten G129E mutant, reduced the enhanced cell motility of Pten-deficient cells. Moreover, phosphorylation of the focal adhesion kinase p125(FAK) was not changed in Pten(-/-) cells. Instead, significant increases in the endogenous activities of Rac1 and Cdc42, two small GTPases involved in regulating the actin cytoskeleton [7], were observed in Pten(-/-) cells. Overexpression of dominant-negative mutant forms of Rac1 and Cdc42 reversed the cell migration phenotype of Pten(-/-) cells. Thus, our studies suggest that Pten negatively controls cell motility through its lipid phosphatase activity by down-regulating Rac1 and Cdc42.

Enhancement and inhibition of snake venom phosphodiesterase activity by lysophospholipids.

Lysophospholipids are liberated during venomous action. In this study we demonstrated that lysophosphatidyl choline (LPC) of various acyl chains enhances considerably the activity of snake venom phosphodiesterase (PDE). Lysophosphatidic acid (LPA) and its cyclic form (cLPA), on the other hand, were found to inhibit this enzyme in a non-competitive (LPA) or competitive (cLPA) manner. Both of these activities may contribute to the progression and subsidence of the poisoning profile. PDE from cellular origin was not substantially affected by any of the above lysophospholipids.