Non-confluent Cells Research Articles

Peroxisome Proliferator-activated Receptor (PPAR)-2 Controls Adipocyte Differentiation and Adipose Tissue Function through the Regulation of the Activity of the Retinoid X Receptor/PPARγ Heterodimer

Peroxisome Proliferator-activated Receptor (PPAR)-2 Controls Adipocyte Differentiation and Adipose Tissue Function through the Regulation of the Activity of the Retinoid X Receptor/PPARγ Heterodimer

The Confluence-dependent Interaction of Cytosolic Phospholipase A2-α with Annexin A1 Regulates Endothelial Cell Prostaglandin E2 Generation

The regulated generation of prostaglandins from endothelial cells is critical to vascular function. Here we identify a novel mechanism for the regulation of endothelial cell prostaglandin generation. Cytosolic phospholipase A(2)-alpha (cPLA(2)alpha) cleaves phospholipids in a Ca(2+)-dependent manner to yield free arachidonic acid and lysophospholipid. Arachidonic acid is then converted into prostaglandins by the action of cyclooxygenase enzymes and downstream synthases. By previously undefined mechanisms, nonconfluent endothelial cells generate greater levels of prostaglandins than confluent cells. Here we demonstrate that Ca(2+)-independent association of cPLA(2)alpha with the Golgi apparatus of confluent endothelial cells correlates with decreased prostaglandin synthesis. Golgi association blocks arachidonic acid release and prevents functional coupling between cPLA(2)alpha and COX-mediated prostaglandin synthesis. When inactivated at the Golgi apparatus of confluent endothelial cells, cPLA(2)alpha is associated with the phospholipid-binding protein annexin A1. Furthermore, the siRNA-mediated knockdown of endogenous annexin A1 significantly reverses the inhibitory effect of confluence on endothelial cell prostaglandin generation. Thus the confluence-dependent interaction of cPLA(2)alpha and annexin A1 at the Golgi acts as a novel molecular switch controlling cPLA(2)alpha activity and endothelial cell prostaglandin generation.

Phosphatidylethanol Accumulation Promotes Intestinal Hyperplasia by Inducing ZONAB-Mediated Cell Density Increase in Response to Chronic Ethanol Exposure

Chronic alcohol consumption is associated with increased risk of gastrointestinal cancer. High concentrations of ethanol trigger mucosal hyperregeneration, disrupt cell adhesion, and increase the sensitivity to carcinogens. Most of these effects are thought to be mediated by acetaldehyde, a genotoxic metabolite produced from ethanol by alcohol dehydrogenases. Here, we studied the role of low ethanol concentrations, more likely to mimic those found in the intestine in vivo, and used intestinal cells lacking alcohol dehydrogenase to identify the acetaldehyde-independent biological effects of ethanol. Under these conditions, ethanol did not stimulate the proliferation of nonconfluent cells, but significantly increased maximal cell density. Incorporation of phosphatidylethanol, produced from ethanol by phospholipase D, was instrumental to this effect. Phosphatidylethanol accumulation induced claudin-1 endocytosis and disrupted the claudin-1/ZO-1 association. The resulting nuclear translocation of ZONAB was shown to mediate the cell density increase in ethanol-treated cells. In vivo, incorporation of phosphatidylethanol and nuclear translocation of ZONAB correlated with increased proliferation in the colonic epithelium of ethanol-fed mice and in adenomas of chronic alcoholics. Our results show that phosphatidylethanol accumulation after chronic ethanol exposure disrupts signals that normally restrict proliferation in highly confluent intestinal cells, thus facilitating abnormal intestinal cell proliferation.

Mechanisms of induction of airway smooth muscle hyperplasia by transforming growth factor-β

Airway smooth muscle (ASM) hyperplasia is a characteristic feature of the asthmatic airway, but the underlying mechanisms that induce ASM hyperplasia remain unknown. Because transforming growth factor (TGF)-beta is a potent regulator of ASM cell proliferation, we determined its expression and mitogenic signaling pathways in ASM cells. We obtained ASM cells by laser capture microdissection of bronchial biopsies and found that ASM cells from asthmatic patients expressed TGF-beta1 mRNA and protein to a greater extent than nonasthmatic individuals using real-time RT-PCR and immunohistochemistry, respectively. TGF-beta1 stimulated the growth of nonconfluent and confluent ASM cells either in the presence or absence of serum in a time- and concentration-dependent manner. The mitogenic activity of TGF-beta1 on ASM cells was inhibited by selective inhibitors of TGF-beta receptor I kinase (SD-208), phosphatidylinositol 3-kinase (PI3K, LY-294002), ERK (PD-98059), JNK (SP-600125), and NF-kappaB (AS-602868). On the other hand, p38 MAPK inhibitor (SB-203580) augmented TGF-beta1-induced proliferation. To study role of the Smads, we transduced ASM cells with an adenovirus vector-expressing Smad4, Smad7, or dominant-negative Smad3 and found no involvement of these Smads in TGF-beta1-induced proliferation. Dexamethasone caused a dose-dependent inhibition in TGF-beta1-induced proliferation. Our findings suggest that TGF-beta1 may act in an autocrine fashion to induce ASM hyperplasia, mediated by its receptor and several kinases including PI3K, ERK, and JNK, whereas p38 MAPK is a negative regulator. NF-kappaB is also involved in the TGF-beta1 mitogenic signaling, but Smad pathway does not appear important.

Epidermal Growth Factor-, Transforming Growth Factor-β-, Retinoic Acid- and 1,25-Dihydroxyvitamin D3-Regulated Expression of the Novel Protein PTPIP51 in Keratinocytes

The novel protein PTPIP51 (protein tyrosine phosphatase-interacting protein 51), which has been found to interact with protein tyrosine phosphatases of the PTP1B/TcPTP subfamily, is expressed in all suprabasal layers of human epidermis. Hence, a human keratinocyte cell line (HaCaT) grown on culture slides was used as a simplified model system to study the influence of hormonal agents on the regulation of PTPIP51 expression. Results were obtained by immunocytochemistry and subsequent statistical analysis. Additionally, immunoblotting was performed to detect the possible occurrence of distinct molecular weight forms as described previously. Subcellular localization of PTPIP51 protein was analyzed by specific staining of cellular organelles. HaCaT cells were subjected to treatment with factors that are crucial for the regulation of proliferation and differentiation of keratinocytes in human epidermis: epidermal growth factor (EGF), transforming growth factor-β(TGF-β), retinoic acid (RA) and 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃]. Epidermal growth factor receptor (EGFR) expressed in HaCaT cells was inhibited by PD153035. Only about 35% of untreated HaCaT cells were immunoreactive for the PTPIP51 protein. Whereas cells treated with increasing concentrations of 1,25 (OH)₂D₃ showed a stepwise numerical increase of PTPIP51-positive cells, treatment with RA did not influence the number of PTPIP51-positive cells except when supraphysiological concentrations were applied. Concentration-dependent increase of cells stained positive for PTPIP51 was also observed when HaCaT cells were subjected to EGF treatment. Additional treatment of these cells with PD153035 led to a slight decrease in the fraction of PTPIP51-positive cells, which was not statistically significant. Immunoblotting results suggest a specific pattern of different molecular weight forms of PTPIP51 being expressed in HaCaT cells. Subcellular analysis revealed an association of the protein with mitochondria in nonconfluent cells, whereas confluent cells lack such correlation. The intracellular distribution of PTPIP51 resembled the localization of its interacting partner TcPTP. Furthermore, PTPIP51 was found to be present in both the nucleus and cytoplasm of HaCaT cells. In summary, the results indicate a possible association of PTPIP51 expression with differentiation as well as with apoptosis of keratinocytes.

Cell Confluence Regulates Hepatocyte Growth Factor-Stimulated Cell Morphogenesis in a β-Catenin-Dependent Manner

Following organ injury, morphogenic epithelial responses can vary depending on local cell density. In the present study, the role of cell confluence in determining the responsiveness of renal epithelial cells to the dedifferentiating morphogenic signals of hepatocyte growth factor (HGF) was examined. Increasing confluence resulted in a greater tendency of cells to organize into epithelial tubes and a significant decrease in migratory responsiveness to HGF. Analysis of downstream signaling revealed that the HGF receptor c-Met was equally activated in confluent and nonconfluent cells following HGF stimulation but that phosphoinositide 3-kinase-dependent activation of Akt and Rac were selectively diminished in confluent cells. In nonconfluent cells treated with HGF, the high level of Akt activation resulted in inhibitory phosphorylation of glycogen synthase kinase 3beta (GSK-3beta) and increased beta-catenin nuclear signaling. In contrast, confluent cells, in which HGF-stimulated Akt activation was diminished, displayed less inhibitory phosphorylation of GSK-3beta and less nuclear signaling by beta-catenin. Overexpression of beta-catenin (SA), which cannot be phosphorylated by GSK-3beta and targeted for ubiquitination, significantly increased migration in fully confluent cells. Thus, cells maintained at high confluence selectively downregulate signaling events such as Rac activation and beta-catenin-dependent transcription that would otherwise promote cell dedifferentiation and migration.

The Polycystin 1-C-terminal Fragment Stimulates ERK-dependent Spreading of Renal Epithelial Cells

Polycystin 1, the product of the PKD1 gene, is mutated in autosomal dominant polycystic kidney disease, a disease characterized by renal cyst formation and progressive renal failure. We show that expression of the C-terminal domain of human polycystin-1 (PKD1-CT) triggers spreading of isolated inner medullary collecting duct cells, a process mediated by Erk. As inner medullary collecting duct cells spread, PKD1-CT localizes to cell-extracellular matrix contacts, interacts with focal adhesion proteins Fak and paxillin, and stimulates Fak phosphorylation, paxillin phosphorylation, Fak-paxillin association, and formation of small focal complexes. PKD1-CT-mediated spreading requires membrane localization and the integrity of the C-terminal protein binding sites. We additionally show that Pkd1 null proximal tubule cells generated from Pkd1(flox/-):TSLargeT mice by in vitro Cre recombinase transfection demonstrate diminished spreading when compared with Pkd(flox/-) heterozygous parental cells. These findings suggest that membrane-bound PC1 has a central role in regulating morphogenic protein signaling at cell-matrix interfaces in non-confluent cells.

Cell density-dependent nuclear/cytoplasmic localization of NORPEG (RAI14) protein

NORPEG (RAI14), a developmentally regulated gene induced by retinoic acid, encodes a 980 amino acid (aa) residue protein containing six ankyrin repeats and a long coiled-coil domain [Kutty et al., J. Biol. Chem. 276 (2001), pp. 2831–2840]. We have expressed aa residues 1–287 of NORPEG and used the recombinant protein to produce an anti-NORPEG polyclonal antibody. Confocal immunofluorescence analysis showed that the subcellular localization of NORPEG in retinal pigment epithelial (ARPE-19) cells varies with cell density, with predominantly nuclear localization in nonconfluent cells, but a cytoplasmic localization, reminiscent of cytoskeleton, in confluent cultures. Interestingly, an evolutionarily conserved putative monopartite nuclear localization signal (P 270KKRKAP 276) was identified by analyzing the sequences of NORPEG and its orthologs. GFP-NORPEG (2–287 aa), a fusion protein containing this signal, was indeed localized to nuclei when expressed in ARPE-19 or COS-7 cells. Deletion and mutation analysis indicated that the identified nuclear localization sequence is indispensable for nuclear targeting.

The Effects of Morphology, Confluency, and Phenotype on Whole-Cell Mechanical Behavior

Emerging evidence indicates that cellular mechanical behavior can be altered by disease, drug treatment, and mechanical loading. To effectively investigate how disease and mechanical or biochemical treatments influence cellular mechanical behavior, it is imperative to determine the source of large inter-cell differences in whole-cell mechanical behavior within a single cell line. In this study, we used the atomic force microscope to investigate the effects of cell morphological parameters and confluency on whole-cell mechanical behavior for osteoblastic and fibroblastic cells. For nonconfluent cells, projected nucleus area, cell area, and cell aspect ratio were not correlated with mechanical behavior (p>or=0.46), as characterized by a parallel-spring recruitment model. However, measured force-deformation responses were statistically different between osteoblastic and fibroblastic cells (p<0.001) and between confluent and nonconfluent cells (p<0.001). Osteoblastic cells were 2.3-2.8 times stiffer than fibroblastic cells, and confluent cells were 1.5-1.8 times stiffer than nonconfluent cells. The results indicate that structural differences related to phenotype and confluency affect whole-cell mechanical behavior, while structural differences related to global morphology do not. This suggests that cytoskeleton structural parameters, such as filament density, filament crosslinking, and cell-cell and cell-matrix attachments, dominate inter-cell variability in whole-cell mechanical behavior.

A role for atm in E-cadherin-mediated contact inhibition in epithelial cells

Ataxia telangiectasia is a hereditary pleiomorphic syndrome caused by loss of Atm, a phosphoprotein involved in multiple signaling pathways. Here, we propose a novel role for atm in cultured epithelial cells, namely the regulation of cell growth by contact inhibition. We show that atm is upregulated in epithelial cells reaching confluence. Conditional expression of the PI 3-Kinase domain of atm in non-confluent Tac-2 epithelial cells increases the expression of the anti-proliferative gene Tis-21 and downregulates key cell cycle regulator genes, such as cyclins A, B1, B2, E and E2. Finally, we demonstrate that upregulation of atm, and thus Tis-21, in confluent Tac-2 cells can be inhibited by an E-cadherin antibody blocking specifically homophilic E-cadherin interactions between adjacent cell surfaces. Altogether, these results suggest that atm could participate in a molecular pathway linking extracellular signalling to cell cycle control and may help further clarify the role of Atm in epithelial cell biology and carcinogenesis.

HOXA1 Is Required for E-cadherin-dependent Anchorage-independent Survival of Human Mammary Carcinoma Cells

Forced expression of HOXA1 is sufficient to stimulate oncogenic transformation of immortalized human mammary epithelial cells and subsequent tumor formation. We report here that the expression and transcriptional activity of HOXA1 are increased in mammary carcinoma cells at full confluence. This confluence-dependent expression of HOXA1 was abrogated by incubation of cells with EGTA to produce loss of intercellular contact and rescued by extracellular addition of Ca2+. Increased HOXA1 expression at full confluence was prevented by an E-cadherin function-blocking antibody and attachment of non-confluent cells to a substrate by homophilic ligation of E-cadherin increased HOXA1 expression. E-cadherin-directed signaling increased HOXA1 expression through Rac1. Increased HOXA1 expression consequent to E-cadherin-activated signaling decreased apoptotic cell death and was required for E-cadherin-dependent anchorage-independent proliferation of human mammary carcinoma cells. HOXA1 is therefore a downstream effector of E-cadherin-directed signaling required for anchorage-independent proliferation of mammary carcinoma cells.

Rac1-null mouse embryonic fibroblasts are motile and respond to platelet-derived growth factor.

Previous studies of Rac1 in fibroblasts have used dominant negative constructs, which may have nonspecific effects. We used a conditional Rac1 allele to critically examine Rac1 function in mouse fibroblasts. Lack of Rac1 had dramatic effects on nonconfluent cells, which were elongated and had extensive blebbing, but no lamellipodia or ruffle formation. However, Rac1-null fibroblasts translocated using pseudopodia-like protrusions without lamellipodia, migrating toward a platelet-derived growth factor (PDGF) gradient as efficiently as their wild-type counterparts. Rac1-null fibroblasts closed wounds in vitro and spread on a fibronectin substrate, although at a slower rate than wild-type cells. However, Rac1-null cells were markedly impaired in proliferation, with a defect in G1 to S transition, although they were capable of surviving in culture for more than 2 wk. These results refine our understanding of the functions of Rac1, indicate that lamellipodia formation is not required for cell motility, and show that PDGF-induced chemotaxis can occur in the absence of both lamellipodia and Rac1.

Endothelial nitric oxide synthase is segregated from caveolin-1 and localizes to the leading edge of migrating cells

The enzyme endothelial Nitric Oxide Synthase (eNOS) is involved in key physiological and pathological processes, including cell motility and apoptosis. It is widely believed that at the cell surface eNOS is localized in caveolae, where caveolin-1 negatively regulates its activity, however, there are still uncertainties on its intracellular distribution. Here, we applied high resolution confocal microscopy to investigate the surface distribution of eNOS in transfected HeLa cells and in human umbilical vein endothelial cells (HUVEC) endogenously expressing the enzyme. In confluent and non-confluent HUVEC and HeLa cells, we failed to detect substantial colocalization between eNOS and caveolin-1 at the cell surface. Instead, in non-confluent cells, eNOS was concentrated in ruffles and at the leading edge of migrating cells, colocalizing with actin filaments and with the raft marker ganglioside GM1, and well segregated from caveolin-1, which was restricted to the posterior region of the cells. Treatments that disrupted microfilaments caused loss of eNOS from the cell surface and decreased Ca2+-stimulated activity, suggesting a role of the cytoskeleton in the localization and function of the enzyme. Our results provide a morphological correlate for the role of eNOS in cell migration and raise questions on the site of interaction between eNOS and caveolin-1.

Endothelial actin cytoskeleton remodeling during mechanostimulation with fluid shear stress

Fluid shear stress stimulation induces endothelial cells to elongate and align in the direction of applied flow. Using the complementary techniques of photoactivation of fluorescence and fluorescence recovery after photobleaching, we have characterized endothelial actin cytoskeleton dynamics during the alignment process in response to steady laminar fluid flow and have correlated these results to motility. Alignment requires 24 h of exposure to fluid flow, but the cells respond within minutes to flow and diminish their movement by 50%. Although movement slows, the actin filament turnover rate increases threefold and the percentage of total actin in the polymerized state decreases by 34%, accelerating actin filament remodeling in individual cells within a confluent endothelial monolayer subjected to flow to levels used by dispersed nonconfluent cells under static conditions for rapid movement. Temporally, the rapid decrease in filamentous actin shortly after flow stimulation is preceded by an increase in actin filament turnover, revealing that the earliest phase of the actin cytoskeletal response to shear stress is net cytoskeletal depolymerization. However, unlike static cells, in which cell motility correlates positively with the rate of filament turnover and negatively with the amount polymerized actin, the decoupling of enhanced motility from enhanced actin dynamics after shear stress stimulation supports the notion that actin remodeling under these conditions favors cytoskeletal remodeling for shape change over locomotion. Hours later, motility returned to pre-shear stress levels but actin remodeling remained highly dynamic in many cells after alignment, suggesting continual cell shape optimization. We conclude that shear stress initiates a cytoplasmic actin-remodeling response that is used for endothelial cell shape change instead of bulk cell translocation.

Dynamics of the Ins(1,4,5)P3 receptor during polarization of MDCK cells

The uneven distribution of the Ins(1,4,5)P3R [Ins(1,4,5)P3 receptor] within the ER (endoplasmic reticulum) membrane generates spatially complex Ca2+ signals. The ER is a dynamic network, which allows the rapid diffusion of membrane proteins from one part of the cell to another. However, little is known about the localization and the dynamics of the Ins(1,4,5)P3R in the ER of living cells. We have used a MDCK (Madin-Darby canine kidney) clone stably expressing the Ins(1,4,5)P3R1-GFP (where GFP stands for green fluorescent protein) to investigate the effect of cell polarity on the lateral mobility of the Ins(1,4,5)P3R. In non-confluent MDCK cells, the chimaera is homogeneously distributed throughout the ER and the nuclear envelope. FRAP (fluorescence recovery after photobleaching) experiments showed that the receptor can move freely in the ER with a diffusion constant (D=0.01 microm2/s) approx. ten times lower than other ER membrane proteins. In confluent polarized cells, two populations of receptor can be defined: one population is distributed in the cytoplasm and is mobile but with a slower diffusion constant (D=0.004 microm2/s) compared with non-confluent cells, whereas the other population is concentrated at the periphery of the cells and is apparently immobile. The observed differences in the mobility of the Ins(1,4,5)P3R are most probably due to its interactions with stable protein complexes that form at the periphery of the polarized cells.

Regulatory Factors on Parathyroid Hormone-related Peptide Production by Primary Culture of Lactating Rat Mammary Gland

Parathyroid hormone-related protein (PTHrP) is a major cause of humoral hypercalcemia of malignancy, but has also been widely found in fetal and adult non-neoplastic tissues. Lactating mammary gland has been shown to produce large amounts of PTHrP, and high levels of PTHrP have been measured in milk. We have examined the influences of several substances on the secretion of two different forms of PTHrP by primary cultures of mammary cells isolated from lactating rats to examine the regulatory mechanisms of PTHrP production by mammary cells. Primary cultures of mammary cells seeded at a density of 10(5) cells per 35 mm culture dish were grown on collagen gels. First, after cells were left 24 hours for attachment and incubated in 2 % FCS containing medium with for 12 hours, PTHrP (1 - 87) secretions were measured in conditioned medium with hormone supplementation for 1, 24 and 48 hours. Progesterone (10(-7) - 10(-5) mol/l) significantly suppressed PTHrP (1 - 87) secretion in a dose-dependent manner (p < 0.01), while 17beta-estradiol had no influence on PTHrP (1 - 87) secretion. Prolactin, a known stimulator of PTHrP expression in vivo, had no effect in this in vitro model. Second, PTHrP (1 - 34) secretion levels from confluent lactating mammary cells for 24 hours were evaluated. The same results were obtained in the case of PTHrP (1 - 87) secretion from non-confluent cells. Furthermore, dexamethasone (10(-6) mol/l) significantly suppressed PTHrP (1 - 34) secretion (p < 0.01). These results suggest that PTHrP production from the lactating mammary gland is suppressed by progesterone as well as dexamethasone. Progesterone dramatically falls after delivery, thus possibly accelerating PTHrP production by lactating mammary glands and resulting in considerable amounts of PTHrP secreted into the milk.

HFRS causing hantaviruses do not induce apoptosis in confluent Vero E6 and A‐549 cells

Hantaviruses are known to cause little or no cytopathic effect in vitro, but have been suggested to cause apoptosis. To determine whether different hantaviruses would induce apoptosis to varying degrees, confluent Vero E6 cells were infected with the hemorrhagic fever with renal syndrome (HFRS) causing viruses Hantaan, Dobrava, Saaremaa, and Puumala. However, no difference was found in the percentage of adherent cells, or of cells with condensed nuclei, between non-infected and virus-infected cells at 3, 6, 9, or 12 days after infection. Furthermore, no differences in the percentage of cells with inter-nucleosomal cleavage of DNA between uninfected and Hantaan infected cells could be detected using the TUNEL assay. Possibly, slightly more apoptotic cells, but never more than 5%, were detected after Hantaan infection of non-confluent cells as compared to the negative control. Earlier reported results that Tula hantavirus induces significant apoptosis on Vero E6 cells were also verified, suggesting that non-pathogenic hantaviruses might differ from HFRS-causing strains regarding induction of apoptosis. In conclusion, the results indicated that the HFRS-causing hantaviruses might induce a very low level of apoptosis in dividing cells, but not at all in confluent cells.

Novel ruthenium complex K 2[Ru(dmgly)Cl 4] · 2H 2O is toxic to C6 astrocytoma cell line, but not to primary rat astrocytes

A novel class of ruthenium (III) complexes of formulas K [ Ru ( sar ) 2 Cl 2 ] · 1 2 H 2 O and K 2[Ru(dmgly)Cl 4] · 2H 2O, containing bidentate chelates N-methylglycine (sarcosine, sar) or N, N-dimethylglycine (dmgly) and additional chloro ligands were synthesized. The complexes have been obtained by direct reaction of ruthenium(III) chloride with corresponding bidentate ligand followed by addition of base (KOH). These new complexes were characterized by elemental analysis, IR and electronic absorption spectroscopy. As astrocytomas, the most common of all brain tumors, are still very difficult to treat, we examined the influence of newly synthesized ruthenium-based complexes, as well as the earlier synthesized analogue platinum(IV) complexes [Pt(dmgly) 2Cl 2], [Pt(sar) 2Br 2] and [Pt(dmgly) 2Br 2], on rat astrocytoma C6 cells in vitro. Among these complexes only K 2[Ru(dmgly)Cl 4] · 2H 2O and [Pt(dmgly) 2Br 2] markedly inhibited the viability of non-confluent C6 cells. Furthermore, only complex K 2[Ru(dmgly)Cl 4] · 2H 2O was able to reduce viability in confluent C6 cultures. Importantly, this complex was not toxic to primary rat astrocytes or macrophages. Having in mind that appropriate chemotherapy should be effective against tumor cells without harming normal tissues, complex K 2[Ru(dmgly)Cl 4] · 2H 2O could be a promising agent for developing therapeutics against astrocytomas.

Cytosolic phospholipase A2-p11 interaction controls arachidonic acid release as a function of epithelial cell confluence.

Madin-Darby canine kidney type II cells were shown to release low amounts of AA (arachidonic acid) and prostaglandin E2 in response to various stimuli when analysed after cell confluence. In contrast, non-confluent Madin-Darby canine kidney type II cells released much higher amounts of AA and prostaglandin E2. In both stationary and non-confluent cells, AA was released by type IV cPLA2 (cytosolic phospholipase A2), as shown by the use of specific inhibitors and by analysis of the profile of fatty acids released. This confluence-dependent cPLA2 activation was not due to a difference in expression, or in phosphorylation of the enzyme, or in the amount of its substrate. To find out the mechanism by which cPLA2 activation may be regulated as a function of cell confluence, immunofluorescence and co-immunoprecipitation experiments were performed using cPLA2, p11, a natural inhibitor of the enzyme, and annexin II, the natural ligand of p11. These three proteins were expressed at a constant level, regardless of the cell confluence. In contrast, whereas annexin II and cPLA2 interacted at a constant rate, p11 and cPLA2 interacted more strongly in stationary cells, thus indicating that cPLA2 activation is regulated by its accessibility to p11, independent of their expression level. Our results indicate that, in epithelial cells, the cell confluence, i.e. the establishment of cell-cell contacts, rather than cell proliferation directly controls cPLA2 activation by changing the stoichiometry of p11/cPLA2 interaction.

Effect of cell confluence on ultraviolet light apoptotic responses in DNA repair deficient cells.

One of the major critical factors for cancer proneness is the cell response to DNA damage. In this work, we used human DNA repair deficient cell lines to investigate the responses to ultraviolet irradiation that lead to apoptosis, and the influence of maintaining the cells resting in confluent state. UV-induced apoptosis is prevented in photolyase-proficient HeLa cells when cyclobutane pyrimidine dimers (CPDs) are removed by photorepair. At the same time, we show recovery of RNA synthesis, thus indicating that blockage of RNA transcription may trigger apoptosis in human cells. On the other hand, confluent primary XPC and trichothiodystrophy (TTD)/XPD cell lines, related to xeroderma pigmentosum and trichothiodystrophy repair syndromes, had a reduced and delayed apoptosis when compared to non-confluent cells. In contrast, XPA cells were similarly sensitive in both the confluent and non-confluent growing state. The effect of cellular confluence on UV-mediated apoptosis in CSB cells, related to Cockayne's syndrome, was unclear. Thus, these results indicate that the induction of apoptosis by UV light may also be affected by DNA replication. In addition, they argue for the use of confluent primary cells in studies of induction of apoptosis by UV, a condition close to skin cells in vivo.