PGE(2) inhibition of TGF-beta1-induced myofibroblast differentiation is Smad-independent but involves cell shape and adhesion-dependent signaling

Myofibroblasts are pathogenic in pulmonary fibrotic disease due to their exuberant production of matrix rich in collagen that interferes with gas exchange and the ability of these cells to contract and distort the alveolar space. Transforming growth factor-beta1 (TGF-beta1) is a well-known inducer of myofibroblast differentiation. TGF-beta1-induced transformation of fibroblasts to apoptosis-resistant myofibroblasts is adhesion-dependent and focal adhesion kinase (FAK)-mediated. Prostaglandin E(2) (PGE(2)) inhibits this differentiation via E prostanoid receptor 2 (EP2) signaling and cAMP elevation, but whether PGE(2) does so by interfering with TGF-beta1 signaling is unknown. Thus we examined the effects of PGE(2) in the presence and absence of TGF-beta1 stimulation on candidate signaling pathways in human lung fibroblasts. We now demonstrate that PGE(2) does not interfere with TGF-beta1-induced Smad phosphorylation or its translocation to the nucleus. Rather, PGE(2) has dramatic effects on cell shape and cytoskeletal architecture and disrupts the formation of appropriate focal adhesions. PGE(2) treatment diminishes TGF-beta1-induced phosphorylation of paxillin, STAT-3, and FAK and, in turn, limits activation of the protein kinase B (PKB/Akt) pathway. These alterations do not, however, result in increased apoptosis within the first 24 h of treatment. Interestingly, the effects of PGE(2) stimulation alone do not always mirror the effects of PGE(2) in the presence of TGF-beta1, indicating that the context for EP2 signaling is different in the presence of TGF-beta1. Taken together, our results demonstrate that PGE(2) has the potential to limit TGF-beta1-induced myofibroblast differentiation via adhesion-dependent, but Smad-independent, pathways.     

Effect of pyridoxine on the inhibition by dexamethasone of murine erythroleukemia cell differentiation

Glucocorticoids are known to inhibit the induced differentiation of murine erythroleukemia cells. I show here that the binding of glucocorticoid receptors to the intact nuclei of these cells is reduced by exogenous extracellular pyridoxine (1–4 mM). This reduction of glucocorticoid-receptor binding did not abrogate the inhibition by dexamethasone of the induced differentiation.     

4-Hydroxynonenal-induced MEL cell differentiation involves PKC activity translocation

4-Hydroxynonenal (HNE) is a highly reactive aldehyde, produced by cellular lipid peroxidation, able to inhibit proliferation and to induce differentiation in MEL cells at concentrations similar to those detected in several normal tissues. Inducer-mediated differentiation of murine erythroleukemia (MEL) cells is a multiple step process characterized by modulation of several genes as well as by a transient increase in the amount of membrane-associated protein kinase C (PKC) activity. Here we demonstrate that a rapid translocation of PKC activity from cytosol to the membranes occurs during the differentiation induced by HNE. When PKC is completely translocated by phorbol-12-myristate-13-acetate (TPA), the degree of HNE-induced MEL cells differentiation is highly decreased. However, if TPA is washed out from the culture medium before the exposition to the aldehyde, HNE gradually resumes its differentiative ability. The incubation of cells with a selective inhibitor of PKC activity, bisindolylmaleimide GF 109203X, partially prevents the HNE-induced differentiation in MEL cells. In conclusion, our results demonstrate that HNE-induced MEL cell differentiation is preceded by a rapid translocation of PKC activity, and that the inhibition of this phenomenon prevents the onset of terminal differentiation.     

Lung cancer-derived Dickkopf1 is associated with bone metastasis and the mechanism involves the inhibition of osteoblast differentiation

Wnt/β-catenin signaling and Dickkopf1 (DKK1) play important roles in the progression of lung cancer, which preferably metastasizes to skeleton. But the role of them in bone dissemination is poorly understood. This study aims to define the role of DKK1 in lung cancer bone metastases and investigate the underlying mechanism. Our results demonstrated that DKK1 over-expression was a frequent event in non-small-cell lung cancer (NSCLC) blood samples, and serous DKK1 level was much higher in bone metastatic NSCLC compared to non-bone metastatic NSCLC. We also found that conditioned medium from DKK1 over-expressing lung cancer cells inhibited the differentiation of osteoblast, determined by alkaline phosphatase activity and osteocalcin secretion, whereas the conditioned medium from DKK1 silencing lung cancer cells exhibited the opposite effects. Mechanistically, DKK1 reduced the level of β-catenin and RUNX2, as well as inhibiting the nuclear translocation of β-catenin. Taken together, these results suggested that lung cancer-produced DKK1 may be an important mechanistic link between NSCLC and bone metastases, and targeting DKK1 may be an effective method to treat bone metastase of NSCLC.     

Trophoblast giant-cell differentiation involves changes in cytoskeleton and cell motility

Trophoblast giant-cell differentiation is well-characterized at the molecular level, yet very little is known about how molecular changes affect the cellular functions of trophoblast in embryo implantation. We have found, using both explanted E7.5 mouse embryo ectoplacental cone and the rat choriocarcinoma (Rcho-1) cell line, that trophoblast differentiation is distinguished by dramatic changes in cytoarchitecture and cell behavior. Undifferentiated trophoblast cells contain little organized actin and few small, peripheral focal complexes and exhibit high membrane protrusive activity, while differentiated trophoblast giant cells contain prominent stress fibers, large internal as well as peripheral focal adhesions, and become immotile. The dramatic changes in cell behavior and cytoskeletal organization of giant cells correlate with changes in the activities of the Rho family of small GTPases and a decrease in tyrosine phosphorylation of focal adhesion kinase. Together, these data provide detailed insight into the cellular properties of trophoblast giant cells and suggest that giant-cell differentiation is characterized by a transition from a motile to a specialized epithelial phenotype. Furthermore, our data support a phagocytic erosion, rather than a migratory infiltration, mechanism for trophoblast giant-cell invasion of the uterine stroma.     

Effect of the antiglucocorticoid agent RU 38486 on the dexamethasone inhibition of Friend cell differentiation

Glucocorticoid hormones are known to inhibit the erythroid differentiation of Friend cells. The mechanism of action of these hormones has been questioned, and results suggesting an action not involving the nuclear binding of the receptors have been published. We have used the antiglucocorticoid RU 38486 to block the inhibitory effect of dexamethasone on the induced differentiation of Friend cells. Our results strongly suggest a glucocorticoid action involving the binding of classical receptors to the cell nucleus.     

Osteogenic differentiation of hypertrophic chondrocytes involves asymmetric cell divisions and apoptosis

We have investigated the early cellular events that take place during the change in lineage commitment from hypertrophic chondrocytes to osteoblast-like cells. We have induced this osteogenic differentiation by cutting through the hypertrophic cartilage of embryonic chick femurs and culturing the explants. Immunocytochemical characterization, [3H]thymidine pulse-chase labeling, in situ nick translation or end labeling of DNA breaks were combined with ultrastructural studies to characterize the changing pattern of differentiation. The first responses to the cutting, seen after 2 d, were upregulation of alkaline phosphatase activity, synthesis of type I collagen and single-stranded DNA breaks, probably indicating a metastable state. Associated with the change from chondrogenic to osteogenic commitment was an asymmetric cell division with diverging fates of the two daughter cells, where one daughter cell remained viable and the other one died. The available evidence suggests that the viable daughter cell then divided and generated osteogenic cells, while the other daughter cell died by apoptosis. The results suggest a new concept of how changes in lineage commitment of differentiated cells may occur. The concepts also reconcile previously opposing views of the fate of the hypertrophic chondrocyte.     

Confluence-induced cell cycle exit involves pre-mitotic CDK inhibition by p27Kip1 and cyclin D1 downregulation

Tissue homeostasis requires precise control of cell proliferation and arrest in response to environmental cues. In situation such as wound healing, injured cells are stimulated to divide, but as soon as confluence is reached proliferation must be blocked. Such reversible cell cycle exit occurs in G1, requires pRb family members, and is driven by p27Kip1-dependent Cdk inactivation. This implies that, while dividing, cells should simultaneously prepare the exit once mitosis is accomplished. For a long time, the decision to cycle or not was presumed to occur in G1, prior to the restriction point, beyond which the cells were bound to divide even in the absence of mitogens, before finally arresting after mitosis. However, more recent reports suggested that the commitment to cycle in response to serum occurs already in G2 phase and requires the Ras-dependent induction of cyclin D1, which promotes following G1/S transition. To test whether this hypothesis applies to arrest induced by contact inhibition, we used an in vitro wounding model where quiescent human dermal fibroblasts, stimulated to proliferate by mechanical injury, synchronously exit cell cycle after mitosis due to renewed confluence. We show that this exit is preceded by p27-dependent inhibition of cyclin A-Cdk1/2, cyclin D1 downregulation and reduced pre-mitotic pRb pocket protein phosphorylation. Over-expression of cyclin D1 but not p27 depletion reversed this phenotype and compromised confluence-driven cell cycle exit. Thus, a balance between cyclin D1 and p27 may provide sensitive responses to variations in proliferative cues operating throughout the cell cycle.     

EWI-2 regulates alpha3beta1 integrin-dependent cell functions on laminin-5

EWI-2, a cell surface immunoglobulin SF protein of unknown function, associates with tetraspanins CD9 and CD81 with high stoichiometry. Overexpression of EWI-2 in A431 epidermoid carcinoma cells did not alter cell adhesion or spreading on laminin-5, and had no effect on reaggregation of cells plated on collagen I (alpha2beta1 integrin ligand). However, on laminin-5 (alpha3beta1 integrin ligand), A431 cell reaggregation and motility functions were markedly impaired. Immunodepletion and reexpression experiments revealed that tetraspanins CD9 and CD81 physically link EWI-2 to alpha3beta1 integrin, but not to other integrins. CD81 also controlled EWI-2 maturation and cell surface localization. EWI-2 overexpression not only suppressed cell migration, but also redirected CD81 to cell filopodia and enhanced alpha3beta1-CD81 complex formation. In contrast, an EWI-2 chimeric mutant failed to suppress cell migration, redirect CD81 to filopodia, or enhance alpha3beta1-CD81 complex formation. These results show how laterally associated EWI-2 might regulate alpha3beta1 function in disease and development, and demonstrate how tetraspanin proteins can assemble multiple nontetraspanin proteins into functional complexes.     

In vitro models of intestinal epithelial cell differentiation

The intestinal epithelium is a particularly interesting tissue as (1) it is in a constant cell renewal from a stem cell pool located in the crypts which form, with the underlying fibroblasts, a stem cell niche and (2) the pluripotent stem cells give rise to four main cell types: enterocytes, mucus, endocrine, and Paneth cells. The mechanisms leading to the determination of phenotype commitment and cell-specific expressions are still poorly understood. Although transgenic mouse models are powerful tools for elucidating the molecular cascades implicated in these processes, cell culture approaches bring easy and elegant ways to study cellular behavior, cell interactions, and cell signaling pathways for example. In the present review, we will describe the major tissue culture technologies that allow differentiation of epithelial cells from undifferentiated embryonic or crypt cells. We will point to the necessity of the re-creation of a complex microenvironment that allows full differentiation process to occur. We will also summarize the characteristics and interesting properties of the cell lines established from human colorectal tumors.     

Analysis of nuclear degradation during lens cell differentiation

Lens cells demonstrate a terminal differentiation process with loss of their organelles including nuclei. Chromatin disappearance is characterised by the same changes as most apoptotic cells, i.e. condensation of chromatin and cleavage into high molecular weight fragments and oligonucleosomes. The endo-deoxyribonucleases (bicationic (Ca2+, Mg2+), mono-cationic (Ca2+ or Mg2+) and acidic non-cationic dependent nucleases) are present in lens fibre cells. Our results suggest that the acidic non-cationic nuclease (DNase II) plays a major role in chromatin cleavage. This nuclease, known to be lysosomal, is found in lens fibre nuclei and only an antibody directed against DNase II inhibits the acidic DNA cleavage of lens fibre nuclei. In addition, there must be another DNase implicated in the process which is not DNase I but appears to be a Ca2+, Mg2+ dependent molecule. Regulation of these DNase activities may be accomplished by the effect of post-translational modifications, acidic pH, mitochondrial release molecules, growth factors or oncogenes. Finally, fibre cells lose organelles without cytoplasmic elimination. The survival of these differentiated cells might be due to the action of survival factors such as FGF 1.     

p27(Kip1) is an inducer of intestinal epithelial cell differentiation

Constant renewal of the intestinal epitheliumis a highly coordinated process that has been subject to intenseinvestigation, but its regulatory mechanisms are still essentiallyunknown. In this study, we have demonstrated that forced expression ofthe cyclin-dependent kinase inhibitors (CKIs) p27^Kip1 andp21^Cip1/WAF1 in human intestinal epithelial cells led toexpression of differentiation markers at both the mRNA and proteinlevels. Cell differentiation was temporally dissociated from inhibitionof retinoblastoma protein phosphorylation and growth arrest, alreadyestablished 1 day after infection with recombinant adenoviruses.p27^Kip1 proved significantly more efficient thanp21^Cip1/WAF1 in induction of cell differentiation. Incontrast, forced expression of p16^INK4a resulted in growtharrest without induction of differentiation markers. These resultsimplicate both p27^Kip1 and p21^Cip1/WAF1 in thedifferentiation-timing process, but p21^Cip1/WAF1 may actindirectly by increasing p27^Kip1 levels. These results alsosuggest that induction of intestinal epithelial cell differentiation byCKIs is not related to their effects on the cell cycle and may involveinteractions with cellular components other than cyclins andcyclin-dependent kinases.